Tissue Engineering of Vascular Prosthetic Grafts
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llosadacTISSUE
ENGINEERING
INTELLIGENCE
UNIT 1
Tissue Engineering
of Vascular Prosthetic Grafts
Peter Zilla, M.D., Ph.D.
University of Cape Town
Cape Town, South Africa
Howard P. Greisler, M.D.
Loyola University
Maywood, Illinois, U.S.A.
R.G. LANDES
COMPANY
AUSTIN, TEXAS
U.S.A.
TISSUE ENGINEERING INTELLIGENCE UNIT
Tissue Engineering of Vascular Prosthetic Grafts
R.G. LANDES COMPANY
Austin, Texas, U.S.A.
Copyright © 1999 R.G. Landes Company
All rights reserved.
No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, includ-
ing photocopy, recording, or any information storage and retrieval system, without permission in writing from the
publisher.
Printed in the U.S.A.
Please address all inquiries to the Publishers:
R.G. Landes Company, 810 South Church Street, Georgetown, Texas, U.S.A. 78626
Phone: 512/ 863 7762; FAX: 512/ 863 0081
ISBN: 1-57059-549-6
Cover: “Endocytosis” by Thomas Maciag at <www.forestreetgallery.com> 1-877-874-8084.
The Editors and Publisher express our appreciation for permission to use this picture.
The Editors and Pulisher gratefully acknowledge the support of Medtronic.
Library of Congress Cataloging-in-Publication Data
Tissue engineering of prosthetic vasxcular grafts [edited by] Peter Zilla, Howard P. Greisler.
p. cm.--(Tissue engineering intelligence unit)
Includes bibliographical referecnces and index.
ISBN 1-57059-549-6 (alk. paper)
1. Blood vessel prosthesis. 2. Vascular grafts. 3. Biomedical materials. I. Zilla, P.P. (Peter Paul) II. Greisler,
Howard P. III. Series.
[DNLM: 1. Blood Vessel Prosthesis. 2. Blood Vessels--transplantation. 3. Cell Transplantation. 4. Endothelium,
Vascular--cytology. 5. Host vs Graft reaction--immunology. 6. Biocompatyible Materials. WG 170 T616 1999]
RD598.55.T57 1999
617.4’130592--dc21
DNLM/DLC 99-24890
for Library of Congress CIP
While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment
and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they
make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research,
equipment development, changes in governmental regulations and the rapid accumulation of information relating to the
biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.
CONTENTS
PART I
Bio-Inert Prostheses: Insufficient Healing
1. The Lack of Healing in Conventional Vascular Grafts ........................................................................................... 3
Lester Davids, Terri Dower, Peter Zilla
Introduction ..................................................................................................................................................................... 3
Midgraft Healing .............................................................................................................................................................. 4
ePTFE Grafts ..................................................................................................................................................................... 5
Dacron Grafts ................................................................................................................................................................... 9
Transanastomotic Healing ............................................................................................................................................. 15
Pannus Tissue ................................................................................................................................................................. 15
Transanastomotic Endothelialization ........................................................................................................................... 17
Porosity ........................................................................................................................................................................... 18
Fibrin ............................................................................................................................................................................... 26
Macrophages ................................................................................................................................................................... 30
Prosthetic Wall Vascularization ..................................................................................................................................... 34
Conclusion ...................................................................................................................................................................... 37
2. Noncompliance: The Silent Acceptance of a Villain ............................................................................................. 45
Alexander M. Seifalian, Alberto Giudiceandrea, Thomas Schmitz-Rixen, George Hamilton
Introduction ................................................................................................................................................................... 45
Historical Overview ........................................................................................................................................................ 45
Physical Properties of the Vessel Wall ........................................................................................................................... 46
Assessment of Compliance ............................................................................................................................................ 46
Synthetic and Biological Grafts ..................................................................................................................................... 49
Causes of Graft Failure ................................................................................................................................................... 50
Tubular Compliance ....................................................................................................................................................... 51
Anastomotic Compliance Mismatch ............................................................................................................................. 51
Signal Transduction Pathways and Flow ....................................................................................................................... 52
Development of a Graft with Better Compliance ......................................................................................................... 52
The Future ...................................................................................................................................................................... 55
PART II
Biolized Prostheses: Surface Healing
3. Endothelial Cell Seeding: A Review ...................................................................................................................... 61
Steven P. Schmidt, Gary L. Bowlin
Introduction ................................................................................................................................................................... 61
4. Surface Precoating in the 1980s:
A First Taste of Cell-Matrix Interactions .......................................................................................................... 69
J. Vincent Smyth, Michael G. Walker
Endothelial Cell Attachment to Prosthetic Grafts ........................................................................................................ 70
Cell Migration ................................................................................................................................................................ 72
Endothelial Cell Retention Under Flow Conditions .................................................................................................... 72
Seeding of Native Vascular Surfaces .............................................................................................................................. 73
Mechanism of Cell Adherence ....................................................................................................................................... 73
Nonendothelial Cell Lines .............................................................................................................................................. 75
Future Directions ........................................................................................................................................................... 75
5. Surface Precoating in the 1990s:
The Fine Tuning of Endothelial Cell Transplantation ..................................................................................... 79
Mark M. Samet, Victor V. Nikolaychik, Peter I. Lelkes
Introduction ................................................................................................................................................................... 79
Endothelialization: Past and Current Approaches ....................................................................................................... 80
Fine Tuning of Endothelialization via Surface Coating ............................................................................................... 80
Morphological Aspects of the Endothelialized Surfaces .............................................................................................. 84
Concluding Remarks ...................................................................................................................................................... 86
Microvascular Endothelial Cell Transplantation ..................................................................................91
6. Microvascular Endothelial Cell Transplantation: A Review ................................................................................ 93
Stuart K. Williams
Early Development of Endothelial Cell Transplantation Technology ........................................................................ 93
Cultured Endothelium for Transplantation ................................................................................................................. 94
Current Considerations in Endothelial Cell Transplantation ..................................................................................... 94
Markers for Isolated Endothelial Cells .......................................................................................................................... 95
Cell Deposition on Graft Surfaces: Seeding vs. Sodding .............................................................................................. 96
Animal Models of Endothelial Cell Transplantation. .................................................................................................. 97
Mechanisms Underlying the Formation of a Neointima in Tissue Engineered Vascular Grafts
Using Microvascular Endothelial Cell Transplantation ............................................................................................ 97
Future of Microvessel Endothelial Cell Transplantation ............................................................................................. 99
7. Morphological Aspects of Microvascular Cell Isolates ...................................................................................... 101
Manuela Vici
Introduction ................................................................................................................................................................. 101
Microvascular Cells ...................................................................................................................................................... 102
Electron Microscopic and Immunocytochemical Profiles of Microvascular Cell Isolates ...................................... 103
Discussion ..................................................................................................................................................................... 110
8. Functional Aspects of Microvascular Cell Isolates ............................................................................................. 115
M. Fittkau, Teddy Fischlein
Antiaggregatory Properties of Microvascular Endothelial Cells ............................................................................... 116
Anticoagulative Properties ........................................................................................................................................... 116
Profibrinolytic and Antifibrinolytic Properties ......................................................................................................... 117
Conclusion .................................................................................................................................................................... 119
9. Automated Seeding Devices ................................................................................................................................ 121
Dominic Dodd, J. Vincent Smyth, Michael G. Walker
Introduction ................................................................................................................................................................. 121
Early Seeding Techniques ............................................................................................................................................. 122
Optimal Conditions for Seeding ................................................................................................................................. 122
Choice of Seeding Technique ....................................................................................................................................... 123
Summary ....................................................................................................................................................................... 125
10. Healing Patterns Following Microvascular Seeding—
A Clinical Evaluation of Microvascular-Seeded A-V Access Grafts ............................................................... 127
Steven P. Schmidt, Sharon O. Meerbaum, Duane L. Donovan
Introduction ................................................................................................................................................................. 127
Materials and Methods ................................................................................................................................................ 128
Results ........................................................................................................................................................................... 129
Discussion ..................................................................................................................................................................... 129
11. Neointimal Hyperplasia in Small Diameter Prosthetic Vascular Grafts:
Influence of Endothelial Cell Seeding with Microvascular
Omental Cells in a Canine Model .................................................................................................................... 131
Miralem Pasic, Werner Müller-Glauser, Marko Turina
Introduction ................................................................................................................................................................. 131
Experimental Studies with Endothelial Cell Seeding at the University Hospital Zurich ......................................... 132
Endothelial Cells Reduce Late Neointimal Proliferation ........................................................................................... 134
Intimal and Neointimal Hyperplasia .......................................................................................................................... 137
Omental Microvascular Cells ...................................................................................................................................... 139
Limitations of Experimental Studies ........................................................................................................................... 140
12. Human Clinical Trials of Microvascular Endothelial Cell Sodding .................................................................. 143
Stuart K. Williams
History of EC Transplantation .................................................................................................................................... 143
Human Trial of Microvascular Endothelial Cell Transplantation ............................................................................. 144
Conclusions .................................................................................................................................................................. 147
Macrovascular Endothelial Cell Transplantation ................................................................................149
13. In Vitro Endothelialization:
Its Contribution Towards an Ideal Vascular Replacement ............................................................................. 151
Peter Zilla
Historical Perspective ................................................................................................................................................... 152
Current and Future Perspective ................................................................................................................................... 153
14. Serial Cultivation of Human Endothelial Cells .................................................................................................. 157
Caroline Gillis-Hægerstrand, Anders Hægerstrand
Introduction ................................................................................................................................................................. 157
Characteristics of the Endothelium ............................................................................................................................ 157
Sources of Endothelial Cells ........................................................................................................................................ 158
Culture Techniques for Human ECs ........................................................................................................................... 158
Does the Culture Technique Matter from a Clinical Perspective? ............................................................................. 159
Future Possibilities of Cultured ECs ........................................................................................................................... 160
15. Risk Factors for Autologous Endothelial Cell Cultures ..................................................................................... 163
Johann Meinhart, Manfred Deutsch, Peter Zilla
Introduction ................................................................................................................................................................. 163
Standard Cell Culture Technique for Autologous Endothelial Cell Cultures ........................................................... 163
Procedure Related Risk Factors ................................................................................................................................... 164
Patient Related Risk Factors ........................................................................................................................................ 165
Summary and Future Aspects ...................................................................................................................................... 166
16. Adhesion Molecule Expression Following In Vitro Lining ................................................................................ 171
Caroline Gillis-Hægerstrand
Introduction ................................................................................................................................................................. 171
Vascular Grafts .............................................................................................................................................................. 171
Autologous Grafts ......................................................................................................................................................... 171
Prosthetic Grafts ........................................................................................................................................................... 171
Glutaraldehyde-Tanned Grafts .................................................................................................................................... 172
Heart Valve Prostheses ................................................................................................................................................. 172
Inflammation ................................................................................................................................................................ 172
Leukocyte Adhesion Under Flow Conditions ............................................................................................................. 173
Endothelialization Reduces Monocyte Adhesion
to Xenogeneic Tissue in a Time Dependent Manner ............................................................................................... 174
Future Prospect: Antiadhesive Therapy? .................................................................................................................... 175
Concluding Remarks .................................................................................................................................................... 175
17. In Vitro Endothelialization Elicits Tissue Remodeling
Emulating Native Artery Structures ................................................................................................................ 179
Manfred Deutsch, Johann Meinhart, Peter Zilla
Case Report ................................................................................................................................................................... 180
Conclusions .................................................................................................................................................................. 183
18. In Vitro Endothelialization of Synthetic Vascular Grafts
in Long Term Clinical Use ................................................................................................................................ 189
Manfred Deutsch, Johann Meinhart, Peter Zilla
Introduction ................................................................................................................................................................. 189
Laboratory Procedure .................................................................................................................................................. 189
Surgical Procedure and Clinical Follow Up ................................................................................................................ 190
Randomized Clinical Study ......................................................................................................................................... 190
Clinical Routine Endothelialization ............................................................................................................................ 191
Conclusion .................................................................................................................................................................... 192
PART III
Biointeractive Prostheses: Complete Healing
Biological Components
Taming of Adverse Responses ...................................................................................................................... 197
Prevention of the Inflammatory Reaction ............................................................................................................ 197
19. Inflammatory Reaction: The Nemesis of Implants ............................................................................................ 197
James M. Anderson
Introduction ................................................................................................................................................................. 197
Inflammation and the Healing Response .................................................................................................................... 197
Foreign Body Reaction ................................................................................................................................................. 199
Macrophage Motility, Adhesion and Activation ......................................................................................................... 201
Foreign Body Giant Cell Formation ............................................................................................................................ 204
Future Perspectives on Inflammatory Responses
to Tissue Engineered Prosthetic Vascular Grafts ..................................................................................................... 205
Taming of Adverse Responses ...................................................................................................................... 207
Prevention of the Inflammatory Reaction ............................................................................................................ 207
20. Molecular Determinants of Acute Inflammatory Responses to Biomaterials ................................................. 207
Liping Tang, John W. Eaton
Introduction ................................................................................................................................................................. 207
Surface-Protein Interactions ........................................................................................................................................ 207
Fibrin(ogen) Is Necessary for Phagocyte Accumulation on Biomaterial Implants .................................................. 209
The Mechanism of Biomaterial-Mediated Inflammatory Responses ....................................................................... 212
Conclusions .................................................................................................................................................................. 214
Taming of Adverse Responses ...................................................................................................................... 219
Prevention of Fibrosis ............................................................................................................................................ 219
21. The Accumulation of Inflammatory Mediators:
A Target for the Prevention of Fibrosis ........................................................................................................... 219
John Zagorski, Sharon M. Wahl
Introduction ................................................................................................................................................................. 219
Inflammation ................................................................................................................................................................ 220
Regulation of Leukocyte Recruitment ........................................................................................................................ 220
Mitigation of Recruitment ........................................................................................................................................... 223
Summary and Perspectives .......................................................................................................................................... 224
Taming of Adverse Responses ...................................................................................................................... 229
Prevention of a Hyperplastic Intimal Response ................................................................................................... 229
22. Pathobiology of Hyperplastic Intimal Responses .............................................................................................. 229
Erik L. Owens, Alexander W. Clowes
Introduction ................................................................................................................................................................. 229
Arteries .......................................................................................................................................................................... 230
Grafts ............................................................................................................................................................................. 235
Summary ....................................................................................................................................................................... 236
Taming of Adverse Responses ...................................................................................................................... 241
Prevention of a Hyperplastic Intimal Response ................................................................................................... 241
23. Cell Cycle Interruption to Inhibit Intimal Hyperplasia .................................................................................... 241
Michael J. Mann, Ruediger C. Braun-Dullaeus, Victor J. Dzau
The Molecular and Cellular Biology of Neointimal Vascular Graft Disease............................................................. 242
Cell Cycle Progression: A Careful Orchestration ....................................................................................................... 243
Cell Cycle Arrest and Neointimal hyperplasia ............................................................................................................ 245
Genetic Engineering of Vein Graft Resistance to Atherosclerosis
via Cell Cycle Gene Blockade .................................................................................................................................... 246
Summary ....................................................................................................................................................................... 247
Facilitation of Healing.................................................................................................................................. 251
Chemotaxis ............................................................................................................................................................ 251
24. Signaling Mechanisms for Vascular Cell Migration ........................................................................................... 251
Ian Zachary
Introduction ................................................................................................................................................................. 251
Migration Factors ......................................................................................................................................................... 252
Future Perspectives ....................................................................................................................................................... 262
Facilitation of Healing.................................................................................................................................. 271
Chemotaxis ............................................................................................................................................................ 271
25. Adhesion Molecules: Potent Inducers of Endothelial Cell Chemotaxis ........................................................... 271
Zoltan Szekanecz, Alisa E. Koch
Introduction ................................................................................................................................................................. 271
The Role of Extracellular Matrix Macromolecule
in Endothelial Cell Motility During Vessel Formation ........................................................................................... 272
Adhesion Molecules in Endothelial Cell Migration and Angiogenesis ..................................................................... 272
Interactions of Soluble Mediators with Cellular Adhesion Molecules
and Extracellular Matrix Components During Endothelial Cell Recruitment
and Neovascularization ............................................................................................................................................. 273
The Relevance of Angiogenesis Studies in Vascular Surgery:
Aortic Aneurysms and Wound Healing .................................................................................................................... 274
A Regulatory Network in Sites of Endothelial Cell Migration:
Potential Target Promoting Graft Healing ............................................................................................................... 275
Facilitation of Healing.................................................................................................................................. 279
Angiogenesis ........................................................................................................................................................... 279
26. Angiogenesis in Tissues and Vascular Grafts ...................................................................................................... 279
Paula K. Shireman, Howard P. Greisler
Overview of Angiogenesis ............................................................................................................................................ 279
Angiogenesis in Vascular Grafts .................................................................................................................................. 282
Therapeutic Angiogenesis in Ischemic Tissues ........................................................................................................... 283
Future Directions ......................................................................................................................................................... 284
Facilitation of Healing.................................................................................................................................. 287
Angiogenesis ........................................................................................................................................................... 287
27. Polypeptide Growth Factors with a Collagen Binding Domain:
Their Potential for Tissue Repair and Organ Regeneration........................................................................... 287
Bo Han, Lynn L.H. Huang, David Cheung, Fabiola Cordoba, Marcel Nimni
Introduction ................................................................................................................................................................. 287
The Collagen Derived Matrix ...................................................................................................................................... 287
Interactions of Biosynthetic Matrices with Cells ....................................................................................................... 289
Modified Growth Factors: TGF-! With a Collagen Binding Domain ....................................................................... 291
Summary and Conclusion ........................................................................................................................................... 297
Facilitation of Healing.................................................................................................................................. 301
Angiogenesis ........................................................................................................................................................... 301
28. Fibroblast Growth Factors in Angiogenesis and Tissue Engineering ............................................................... 301
Karin A. Blumofe, Timothy J. Heilizer, Paula K. Shireman, Howard P. Greisler
Introduction ................................................................................................................................................................. 301
History .......................................................................................................................................................................... 301
Angiogenesis ................................................................................................................................................................. 302
FGF Characterization ................................................................................................................................................... 303
Structure ....................................................................................................................................................................... 303
FGF Secretion ............................................................................................................................................................... 303
Interaction with Heparin ............................................................................................................................................. 304
FGF and Vascular Grafts .............................................................................................................................................. 307
FGF and Ischemia ......................................................................................................................................................... 309
Conclusions .................................................................................................................................................................. 309
Facilitation of Healing .................................................................................................................................. 313
Matrix Degradation .............................................................................................................................................. 313
29. Role of Urokinase-Type Plasminogen Activator (uPA)
in In Vitro Angiogenesis in Fibrin Matrices .................................................................................................... 313
Pieter Koolwijk, Victor W.M. van Hinsbergh
Summary ....................................................................................................................................................................... 313
Introduction. ................................................................................................................................................................ 313
Components of the Plasmin/Plasminogen Activator System .................................................................................... 314
The Regulation of Plasminogen Activation and Pericellular Proteolysis
by Inflammatory and Angiogenic Mediators ........................................................................................................... 316
Interaction Between the uPA/Plasmin System and Matrix-Degrading Metalloproteases ....................................... 316
Involvement of uPA and uPA Receptor in Cell Migration
and Realignment of Endothelial Cells ...................................................................................................................... 317
Role of the Plasmin/Plasminogen Activator System
in the Formation of Endothelial Tubes in a Fibrin Matrix ...................................................................................... 318
Perspective .................................................................................................................................................................... 320
Facilitation of Healing .................................................................................................................................. 325
Matrix Modulation ................................................................................................................................................ 325
30. How Does Extracellular Matrix Control Capillary Morphogenesis? ................................................................ 325
Robert B. Vernon, E. Helene Sage
Angiogenesis: An Introduction .................................................................................................................................... 325
Models of Angiogenesis: The Essential Role of ECM ................................................................................................. 326
Interactions Between ECs and ECM that Mediate Angiogenesis .............................................................................. 330
Facilitation of Healing .................................................................................................................................. 343
Matrix Modulation ................................................................................................................................................ 343
! .......................................................................... 343
31. Collagen Matrices Attenuate Fibroblast Response to TGF-!
Richard R. Clark, John M. McPherson
Introduction ................................................................................................................................................................. 343
Results ........................................................................................................................................................................... 344
Discussion ..................................................................................................................................................................... 348
Facilitation of Healing .................................................................................................................................. 353
Matrix Modulation ................................................................................................................................................ 353
32. Extracellular Matrix Proteins Are Potent Agonists
of Human Smooth Muscle Cell Migration ...................................................................................................... 353
Terry L. Kaiura, K. Craig Kent
Introduction ................................................................................................................................................................. 353
Cell Migration .............................................................................................................................................................. 353
Extracellular Matrix Proteins ...................................................................................................................................... 354
Extracellular Matrix Protein and Migration ............................................................................................................... 355
Integrins ........................................................................................................................................................................ 356
Cell Signaling ................................................................................................................................................................ 357
Conclusion .................................................................................................................................................................... 359
Facilitation of Healing .................................................................................................................................. 363
Matrix Modulation ................................................................................................................................................ 363
33. Extracellular Matrix Effect on Endothelial Control
of Smooth Muscle Cell Migration and Matrix synthesis ............................................................................... 363
Richard J. Powell
Introduction ................................................................................................................................................................. 363
Model ............................................................................................................................................................................ 364
Effect of Matrix on Endothelial Cell Control of Smooth Muscle Cell Migration .................................................... 364
Matrix Effect on Endothelial Cell Control of Smooth Muscle Cell Matrix Synthesis .............................................. 366
Facilitation of Healing.................................................................................................................................. 371
Cell Entrapment from the Circulation .................................................................................................................. 371
34. Circulating Stem Cells: A Fourth Source for the Endothelialization
of Cardiovascular Implants ............................................................................................................................. 371
Willie R. Koen
Introduction ................................................................................................................................................................. 371
The History of Spontaneously Healing Surfaces ........................................................................................................ 371
Properties of Hematopoietic Stem Cells and Progenitor Cells .................................................................................. 372
The Location of Hematopoietic Progenitor Cells ...................................................................................................... 372
Recognition of Stem Cells ............................................................................................................................................ 373
Control of Stem Cells ................................................................................................................................................... 373
Speculative Application ................................................................................................................................................ 375
A Practical Vision for Future Cardiovascular Implants ............................................................................................. 376
Facilitation of Healing.................................................................................................................................. 379
Cell Entrapment from the Circulation .................................................................................................................. 379
35. Surface Population with Blood-Borne Cells ....................................................................................................... 379
William P. Hammond
Background ................................................................................................................................................................... 379
Previous Studies ........................................................................................................................................................... 380
Present Studies .............................................................................................................................................................. 383
Related Findings ........................................................................................................................................................... 383
Implications .................................................................................................................................................................. 384
Facilitation of Healing.................................................................................................................................. 387
Cell Entrapment from the Circulation .................................................................................................................. 387
36. Cellular Population of the Textured-Surface Left Ventricular Assist Devices
Leads to Sustained Activation of a Procoagulant and Proinflammatory Systemic Response ...................... 387
Talia B. Spanier, Ann Marie Schmidt, Mehmet C. Oz
Overview ....................................................................................................................................................................... 387
Thrombin Generation and Fibrinolysis ...................................................................................................................... 388
LVAD Surface Cellularization ...................................................................................................................................... 389
Cellular Activation ....................................................................................................................................................... 394
LVAD as Inflammatory/Immune Organ ..................................................................................................................... 395
NF-∀∀B Is a Marker of Cellular Activation in the LVAD Surface Milieu
and a Target for Anti-Inflammatory Intervention ................................................................................................... 398
Conclusion .................................................................................................................................................................... 400
Facilitation of Healing.................................................................................................................................................. 403
Transdifferentiation .............................................................................................................................................. 403
37. Transdifferentiation and the Vascular Wall ........................................................................................................ 403
William A. Beresford
Endothelial Cells ........................................................................................................................................................... 403
Fibroblasts ..................................................................................................................................................................... 406
Smooth Muscle Cells .................................................................................................................................................... 408
Pericytes ........................................................................................................................................................................ 410
Mast Cells ...................................................................................................................................................................... 410
Macrophages ................................................................................................................................................................. 410
Conclusions .................................................................................................................................................................. 410
Facilitation of Healing.................................................................................................................................. 417
Transdifferentiation .............................................................................................................................................. 417
38. Endothelial Cells Transformed from Fibroblasts During Angiogenesis ........................................................... 417
Takashi Fujiwara, Kazunori Kon
Introduction ................................................................................................................................................................. 417
Angiogenesis in Rabbit Ear Chamber ......................................................................................................................... 417
Discussion ..................................................................................................................................................................... 419
Concluding Remarks .................................................................................................................................................... 422
Facilitation of Healing .................................................................................................................................. 425
Transdifferentiation .............................................................................................................................................. 425
39. Mechanical Forces and Cell Differentiation ....................................................................................................... 425
Ira Mills, Bauer E. Sumpio
Introduction ................................................................................................................................................................. 425
PART III
Biointeractive Prostheses: Complete Healing
Engineering Components .....................................................................................................................439
Scaffold Engineering .................................................................................................................................... 441
Structural Designs ................................................................................................................................................. 441
40. An Integral Mathematical Approach
to Tissue Engineering of Vascular Grafts ........................................................................................................ 441
Greg R. Starke, A.S. Douglas, D.J. Conway
Introduction ................................................................................................................................................................. 441
Inflammation and the Healing Response .................................................................................................................... 441
The Mechanics of Arteries ........................................................................................................................................... 442
Material Laws for the Native Artery and Graft Materials .......................................................................................... 445
Finite Element Implementation .................................................................................................................................. 452
Conclusions .................................................................................................................................................................. 456
Scaffold Engineering .............................................................................................................................459
Material Aspects .................................................................................................................................................... 459
41. Bioinertness: An Outdated Principle .................................................................................................................. 459
David F. Williams
The Convention of Inertness ....................................................................................................................................... 459
The Impossibility of Inertness ..................................................................................................................................... 460
The Requirement for Controlled Reactivity ............................................................................................................... 461
The Essential Requirement of Reactivity in Tissue Engineering ............................................................................... 462
Conclusions .................................................................................................................................................................. 462
Scaffold Engineering .................................................................................................................................... 463
Material Aspects .................................................................................................................................................... 463
42. Bioinert Biomaterials: Are Their Properties Irreplaceable? ............................................................................... 463
Patrick T. Cahalan
Scaffold Engineering .................................................................................................................................... 469
Material Aspects .................................................................................................................................................... 469
43. Biostable Polymers as Durable Scaffolds
for Tissue Engineered Vascular Prostheses ..................................................................................................... 469
Arthur J. Coury
Introduction ................................................................................................................................................................. 469
Design Concept ............................................................................................................................................................ 469
Material Requirements and Limitations ..................................................................................................................... 469
Modes of Polymer Degradation .................................................................................................................................. 470
Polymers with Potential to Serve as Biostable Scaffolds ............................................................................................ 472
Concluding Comments ................................................................................................................................................ 477
Scaffold Engineering .................................................................................................................................... 481
Material Aspects .................................................................................................................................................... 481
44. Biophilic Polymers: What’s on the Horizon? ...................................................................................................... 481
Patrick T. Cahalan
Physical Methods .......................................................................................................................................................... 484
Chemical Methods ........................................................................................................................................................ 486
Summary ....................................................................................................................................................................... 487
Scaffold Engineering .................................................................................................................................... 489
Material Aspects .................................................................................................................................................... 489
45. Bioresorbable Grafts: A Counterintuitive Approach ......................................................................................... 489
David Fox, David A. Vorp, Howard P. Greisler
Overview ....................................................................................................................................................................... 489
Definitions .................................................................................................................................................................... 489
Introduction ................................................................................................................................................................. 489
Single Component Resorbable Grafts ......................................................................................................................... 490
Preventing Aneurysmal Dilatation .............................................................................................................................. 498
Resorbable Outer Wraps .............................................................................................................................................. 500
Biopolymers: Resorbable Prostheses of Biologic Origin ........................................................................................... 500
Conclusion .................................................................................................................................................................... 501
Scaffold Engineering .................................................................................................................................... 505
Material Aspects .................................................................................................................................................... 505
46. Biodegradable Materials ...................................................................................................................................... 505
K.J.L. Burg, S.W. Shalaby
Introduction ................................................................................................................................................................. 505
Criteria for Useful Tissue Engineering Materials ....................................................................................................... 505
Methods of Fabrication of Synthetic Polymeric Materials for Use in Tissue Engineering ...................................... 507
Biodegradable Polymers for Vascular Grafts—Past, Present, and Future ................................................................. 508
Newly Tailored Materials for Tissue Engineering ....................................................................................................... 510
Future Prospectives ...................................................................................................................................................... 510
Scaffold Engineering .................................................................................................................................... 513
Material Aspects .................................................................................................................................................... 513
47. The Influence of Porosity and Surface Roughness on Biocompatibility .......................................................... 513
J.M. Schakenraad, K.H. Lam
Introduction ................................................................................................................................................................. 513
Evaluation Models ........................................................................................................................................................ 514
Interpretation of Results .............................................................................................................................................. 525
Summary and Conclusions .......................................................................................................................................... 528
Scaffold Engineering .................................................................................................................................... 531
Surface Modification ............................................................................................................................................. 531
48. Microgroove Driven Tissue Ingrowth ................................................................................................................ 531
Edwin T. Den Braber, John A. Jansen
Implants, Tissue Engineering, and Biomaterials ........................................................................................................ 531
Surface Micropatterns Manipulating Cellular Behavior ............................................................................................ 532
Changed Cell Shape and Cellular Orientation ........................................................................................................... 533
Cell Attachment on Microtextured Surfaces ............................................................................................................... 534
Other Surface Topography Induced Cell Behavior Alterations In Vitro ................................................................... 538
Microtextured Surfaces In Vivo ................................................................................................................................... 538
The Hypotheses ............................................................................................................................................................ 540
Future Perspectives: Vascular Grafts and Microtextured Surfaces ............................................................................ 540
Scaffold Engineering .................................................................................................................................... 545
Surface Modification ............................................................................................................................................. 545
49. Surface Bonding of Heparin ................................................................................................................................ 545
Patrick T. Cahalan ......................................................................................................................................................................... 545
Scaffold Engineering .................................................................................................................................... 553
Surface Modification ............................................................................................................................................. 553
50. Covalent Grafting of RGD Peptides to Synthetic Surfaces ................................................................................ 553
Nina M.K. Lamba, S.L. Cooper
Introduction ................................................................................................................................................................. 553
Methods to Improve Endothelialization ..................................................................................................................... 553
Immobilization of RGD Peptides ................................................................................................................................ 554
Summary ....................................................................................................................................................................... 558
Matrix Engineering ...................................................................................................................................... 561
51. Hydrogels in Biological Control During Graft Healing ..................................................................................... 561
Jeffrey A. Hubbell
Introduction—Why Hydrogels? .................................................................................................................................. 561
Hydrogel Structure and Synthesis ............................................................................................................................... 562
In Situ Transformations ............................................................................................................................................... 563
Protein and Cell Interactions with Hydrogels ............................................................................................................ 564
Designed Cell Adhesiveness of Hydrogels .................................................................................................................. 566
Nonenzymatic Degradation of Hydrogels .................................................................................................................. 566
Enzymatic Degradation of Hydrogels ......................................................................................................................... 567
Controlled Release ........................................................................................................................................................ 567
Design Principles, Hydrogels for Biological Control During Graft Healing ............................................................ 568
Matrix Engineering ...................................................................................................................................... 571
52. In Vivo Synthesis of Organs
Using Collagen-GAG Copolymers ................................................................................................................... 571
Ioannis V. Yannas
Introduction ................................................................................................................................................................. 571
Summary of Evidence for In Vivo Synthesis of Organs ............................................................................................. 571
Methodological Principles of Induced Regeneration ................................................................................................. 572
Models of the Mechanism of Regeneration ................................................................................................................ 573
Conclusions .................................................................................................................................................................. 575
Matrix Engineering ...................................................................................................................................... 577
53. Artificial Extracellular Matrix Proteins for Graft Design ................................................................................. 577
Alyssa Panitch, David A. Tirrell
Introduction ................................................................................................................................................................. 577
Artificial Proteins ......................................................................................................................................................... 578
Artificial ECM Proteins ................................................................................................................................................ 578
Current and Future Directions .................................................................................................................................... 580
Matrix Engineering ...................................................................................................................................... 583
54. Cell-Extracellular Matrix Interactions Relevant
to Vascular Tissue Engineering ........................................................................................................................ 583
Stephen P. Massia
Introduction ................................................................................................................................................................. 583
Cell-Extracellular Surface Interactions at the Molecular Level ................................................................................. 584
Biomimetic Cell-Adhesive Biomaterials ..................................................................................................................... 586
Cell-ECM Interactions in Vascular Biology ................................................................................................................ 589
Modulating Cell-Extracellular Interactions for Vascular Tissue Engineering .......................................................... 591
Conclusion .................................................................................................................................................................... 593
Matrix Engineering ...................................................................................................................................... 599
55. Use of Hydroxypropylchitosan Acetate as a Carrier
for Growth Factor Release ................................................................................................................................ 599
Keiko Yamamura, Toshitaka Nabeshima, Tsunehisa Sakurai
Introduction ................................................................................................................................................................. 599
Use of HPCHA as a Carrier for bFGF ......................................................................................................................... 600
Influence of HPCHA on Release of bFGF ................................................................................................................... 600
Effect of bFGF-HPCHA on the Endothelialization of Grafts .................................................................................... 602
Concluding Remarks .................................................................................................................................................... 604
Cell Engineering ........................................................................................................................................... 605
56. Transplantation of Transduced Smooth Muscle Cells:
A Vehicle For Local and Systemic Gene Therapy ............................................................................................ 605
Randolph L. Geary, Alexander W. Clowes, Monika M. Clowes
Introduction ................................................................................................................................................................. 605
Smooth Muscle Cells as Targets for Gene Transfer .................................................................................................... 606
Vectors for SMC Gene Transfer. .................................................................................................................................. 606
Retroviral Vectors for Ex Vivo Arterial Gene Transfer ............................................................................................... 606
Retroviral Vectors for SMC Gene Transfer in Prosthetic Vascular Grafts ................................................................. 608
Index.............................................................................................................................................................................. 613
EDITORS
Peter Zilla, M.D., Ph.D.
Cardiovascular Research Unit
University of Cape Town Medical School
Cape Town, South Africa
Chapters 1, 13, 15, 17, 18
Howard P. Greisler, M.D.
Department of Vascular Surgery
Loyola University
Maywood, Illinois, U.S.A.
Chapters 26, 28, 45
CONTRIBUTORS
James M. Anderson, M.D., Ph.D. David Cheung
Institute of Pathology University of Southern California
University Hospital of Cleveland School of Medicine
Cleveland, Ohio, U.S.A. Childrens Hospital Los Angeles
Chapter 19 Division of Surgical Research
Los Angeles, California, U.S.A.
William A. Beresford Chapter 27
Department of Anatomy
Robert C. Byrd Health Science Center Richard R. Clark, M.D.
West Virginia University Department of Dermatology
School of Medicine State University of New York at Stony Brook
Morgantown, West Virginia, U.S.A. Stony Brook, New York, U.S.A.
Chapter 37 Chapter 31
Karin A. Blumofe Alexander W. Clowes, M.D.
Chapter 28 Department of Surgery
Division of Vascular Surgery
Gary L. Bowlin, Ph.D. University of Washington Medical School
Department of Biomedical Engineering Seattle, Washington, U.S.A.
University of Akron Chapters 22, 56
Akron, Ohio, U.S.A.
Chapter 3 Monika M. Clowes
Department of Surgery
Karen J.L. Burg, Ph.D. Division of Vascular Surgery
Department of General Surgery Research University of Washington Medical School
Carolinas Medical Center Seattle, Washington, U.S.A.
Charlotte, North Carolina, U.S.A. Chapter 56
Chapter 46
D.J. Conway, M.Sc.
Patrick T. Cahalan Department of Computation & Applied Mechanics
Intelligent Biocides University of Cape Town
Tewksbury, Massachusetts, U.S.A. Cape Town, South Africa
Chapters 42, 44, 49 Chapter 40
S.L. Cooper
Chapter 50
Fabiola Cordoba Ruediger C. Braun-Dullaeus
University of Southern California Department of Medicine
School of Medicine Brigham & Women’s Hospital
Childrens Hospital Los Angeles Harvard Medical School
Division of Surgical Research Boston, Massachusetts, U.S.A.
Los Angeles, California, U.S.A. Chapter 23
Chapter 27
Victor J. Dzau
Arthur J. Coury, Ph.D. Chairman, Department of Medicine
Focal, Inc. Brigham & Women’s Hospital
Lexington, Massachusetts, U.S.A. Harvard Medical School
Chapter 43 Boston, Massachusetts, U.S.A.
Chapter 23
Lester Davids, M.Sc.
Cardiovascular Research Unit John W Eaton, Ph.D.
University of Cape Town Medical School Department of Pediatrics
Cape Town, South Africa Baylor College of Medicine
Chapter 1 Houston, Texas, U.S.A.
Chapter 20
Edwin T. Den Braber, Ph.D.
Katolieke Universiteit Nijmegen Teddy Fischlein, M.D.
Faculty of Medical Sciences Johann Wolfgang Goethe-Universität Frankfurt
Dental School Klinik für Thorax-, Herz und Gefäßchirurgie
Nijmegen, The Netherlands Frankfurt, Germany
Chapter 48 Chapters 8, 18
Manfred Deutsch, M.D. M. Fittkau
Chirurgischen Abteilung Department of Thoracic and Cardiovascular Surgery
Krankenhaus Wien-Lainz J.W. Goethe University
Vienna, Austria Frankfurt/Main, Germany
Chapters 15, 17, 18 Chapter 8
Dominic Dodd, M.B.Ch.B., F.R.C.S. David Fox
Chapter 9 Chapter 45
Duane L. Donovan, M.D. Takashi Fujiwara, Ph.D.
Summa Health System Laboratory Animal Center
Akron City Hospital Ehime University
Akron, Ohio, U.S.A. School of Medicine
Chapter 10 Shingenobu Ehime, Japan
Chapter 38
A.S. Douglas
Mechanical Engineering Department Randolph L. Geary, M.D.
Johns Hopkins University Assistant Professor of Surgery
Baltimore, Maryland, U.S.A. & Comparative Medicine
Chapter 40 Bowman Gray School of Medicine
Medical Centre Boulevard
Terri Dower, B.Sc.(Hons.) Winston-Salem, North Carolina, U.S.A.
Cardiovascular Research Unit Chapter 56
University of Cape Town Medical School
Cape Town, South Africa Alberto Giudiceandrea
Chapter 1 Vascular Unit
University Department of Surgery
Royal Free Hospital and School of Medicine
London, U.K.
Chapter 2
Werner Müller-Glauser, Ph.D. John A. Jansen, DDS., Ph.D.
Department of Surgery Department of BioMaterials
Clinic for Cardiovascular Surgery Katolieke Universiteit Nijmegen
University Hospital Zurich Faculty of Medical Sciences
Zürich, Switzerland Dental School
Chapter 11 Nijmegen, The Netherlands
Chapter 48
Anders Haegerstrand, M.D., Ph.D.
Department of Neuroscience Terry L. Kaiura
Karolinska Institute Division of Vascular Surgery
Stockholm, Sweden Beth Israel Hospital
Chapter 14 Boston, Massachusetts, U.S.A.
Chapter 32
Caroline Gillis-Haegerstrand, M.D., Ph.D.
Department of Neuroscience K. Craig Kent, M.D.
Karolinska Institute Division of Vascular Surgery
Stockholm, Sweden New York Hospital/
Chapters 14, 16 Cornell University Medical Center
New York, New York, U.S.A.
George Hamilton, F.R.C.S. Chapter 32
Royal Free Hospital
London, U.K. Alisa E. Koch, M.D.
Chapter 2 Department of Medicine
Section of Arthritis and Connective Tissue Diseases
William P. Hammond, M.D. Northwestern University Medical School
The Hope Heart Institute Chicago, Illinois, U.S.A.
University of Washington Chapter 25
School of Medicine
Seattle, Washington, U.S.A. Willie R. Koen, M.B.Ch.B., F.C.S.
Chapter 35 Cardiovascular Research Unit
University of Cape Town Medical School
Bo Han Cape Town, South Africa
University of Southern California Chapter 34
School of Medicine
Childrens Hospital Los Angeles Kazunori Kon, Ph.D.
Division of Surgical Research Laboratory Animal Center
Los Angeles, California, U.S.A. School of Medicine
Chapter 27 Ehime University
Shingenobu Ehime, Japan
Timothy J. Heilizer Chapter 38
Chapter 28
Pieter Koolwijk, Ph.D.
Lynn L.H. Huang Gaubius Laboraroty
University of Southern California Leiden, The Netherlands
School of Medicine Chapter 29
Childrens Hospital Los Angeles
Division of Surgical Research K.H. Lam
Los Angeles, California, U.S.A. Department of Artificial Organs
Chapter 27 Division Biomaterials and Biocompatability
University of Groningen
Jeffrey A. Hubbell, Ph.D. Groningen, The Netherlands
Professor Of Biomedical Engineering Chapter 47
Institute of Biomedical Engineering
and Department of Materials
Swiss Federal Institute of Technology (EHT) Zürich
& University of Zürich
Zürich, Switzerland
Chapter 51
Nina M.K. Lamba, Ph.D. Victor V. Nikolaychik
Department of Chemical Engineering Laboratory of Cell Biology
University of Delaware Department of Medicine
Colburn Lab University of Wisconsin Medical School
Newark, Delaware, U.S.A. Milwaukee Clinical Campus
Chapter 50 Milwaukee, Wisconsin, U.S.A.
Chapter 5
Peter I. Lelkes, Ph.D.
Director, Laboratory of Cell Biology Marcel Nimni
University of Wisconsin Medical School University of Southern California
Milwaukee Clinical Campus School of Medicine
Sinai Samaritan Medical Center, Winter Research Childrens Hospital Los Angeles
Milwaukee, Wisconsin, USA Division of Surgical Research
Chapter 5 Los Angeles, California, U.S.A.
Chapter 27
John M. McPherson
Biotherapeutic Product Development Department Erik L. Owens, M.D.
Genzyme Corporation Department of Surgery
Framingham, Massachusetts, U.S.A. University of Washington Medical School
Chapter 31 Division of Vascular Surgery
Seattle, Washington, U.S.A.
Michael J. Mann Chapter 22
Department of Medicine
Brigham & Women’s Hospital Mehmet C. Oz, M.D.
Harvard Medical School Columbia University
Boston, Massachusetts, U.S.A. New York, New York, U.S.A.
Chapter 23 Chapter 36
Stephen P. Massia, Ph.D. Miralem Pasic, M.D., Ph.D.
Department Chemical, Bio, & Materials Engineering Deutsches Herzzentrum Berlin
Arizona State University Klinik für Herz-Thorax und Gefäßchirurgie
Tempe, Arizona, U.S.A. Berlin, Germany
Chapter 54 Chapter 11
Sharon O. Meerbaum Alyssa Panitch
Summa Health System Department of Polymer Science and Engineering
Akron City Hospital University of Massachusetts
Akron, Ohio, U.S.A. Amherst, Massachusetts, U.S.A.
Chapter 10 Chapter 53
Johann Meinhart, Ph.D. Richard J. Powell, M.D.
Krankenhaus Wien Lainz Dartmouth-Hitchcock Medical Center
Vienna, Austria Department of Surgery
Chapters 15, 17, 18 Lebanon, New Hampshire, U.S.A.
Chapter 33
Ira Mills
Department of Surgery Thomas Schmitz-Rixen
Yale University School of Medicine Zentrum fuer Chirurgie
New Haven, Connecticut, U.S.A. Johann-Wolfgang Goethe Universität
Chapter 39 Frankfurt-am-Main, Germany
Chapter 2
Toshitaka Nabeshima
Department of Neuropsychopharmacology
and Hospital Pharmacy
Nagoya University School of Medicine
Tsuruma-cho, Showa-ku, Nagoya, Japan
Chapter 55
E. Helene Sage, Ph.D. J.Vincent Smyth
University of Washington Department of Vascular Surgery
School of Medicine Manchester Royal Infirmary
Department of Biological Structure England
Seattle, Washington, U.S.A. Chapters 4, 9
Chapter 30
Talia B. Spanier, M.D.
Tsunehisa Sakurai Columbia University
First Department of Surgery New York, New York, U.S.A.
Nagoya University School of Medicine Chapter 36
Tsuruma-cho, Showa-ku, Nagoya, Japan
Chapter 55 Greg R. Starke, Ph.D.
Department of Computation & Applied Mechanics
Mark M. Samet University of Cape Town
Department of Medicine Cape Town, South Africa
Laboratory of Cell Biology Chapter 40
University of Wisconsin Medical School
Milwaukee Clinical Campus Bauer E. Sumpio, M.D., Ph.D.
Milwaukee, Wisconsin, U.S.A. Department of Surgery
Chapter 5 Yale University School of Medicine
New Haven, Connecticut, U.S.A.
J.M. Schakenraad Chapter 39
Department of Pathology
University Hospital Zoltan Szekanecz, M.D., Ph.D.
Rottendam Dijkzicht, The Netherlands Third Department of Medicine
Chapter 47 University Medical School of Debrecen
Hungary
Ann Marie Schmidt Chapter 25
Columbia University
New York, New York, U.S.A. Liping Tang
Chapter 36 Department of Pediatrics
Baylor College of Medicine
Steven P. Schmidt, PhD Houston, Texas, U.S.A.
Falor Centre for Vascular Studies Chapter 20
Akron City Hospital
Akron, Ohio, U.S.A. David A. Tirrell, M.D.
Chapters 3, 10 Materials Res. Science & Engineering Center
University of Massachusetts at Amherst
Alexander M. Seifalian Amherst, Massachusetts, U.S.A.
Vascular Unit Chapter 53
University Department of Surgery
Royal Free Hospital and School of Medicine Marko Turina, M.D.
London, U.K. Department of Surgery
Chapter 2 Clinic for Cardiovascular Surgery
University Hospital Zürich
S.W. Shalaby Zürich, Switzerland
Poly-Med, Inc. Chapter 11
Anderson, South Carolina, U.S.A.
Chapter 46 Victor W.M. Van Hinsbergh, Ph.D.
Gaubius Laboratory
Paula K. Shireman, M.D. Leiden, The Netherlands
Department of Vascular Surgery Chapter 29
Loyola University
Maywood, Illinois, U.S.A.
Chapters 26, 28
Robert B. Vernon, Ph.D. David F. Williams, Ph.D., D.Sc.
Department of Biological Structure Department of Clinical Engineering
University of Washington Faculty of Medicine
School of Medicine Royal Liverpool University Hospital
Seattle, Washington, U.S.A. Liverpool, U.K.
Chapter 30 Chapter 41
Manuela Vici, D.Sc. Stuart K. Williams, Ph.D.
Institute di Microscopica Elettronica Clinica University of Arizona
Universita di Bologna Department of Surgery
Policlinico S.Orsola Tucson, Arizona, U.S.A.
Bologna, Italy Chapters 6, 12
Chapter 7
Keiko Yamamura
David A. Vorp Department of Hospital Pharmacy
University of Pittsburgh Nagoya University School of Medicine
Pittsburgh, Pennsylvania Tsuruma-cho, Showa-ku, Nagoya, Japan
Chapter 45 Chapter 55
Sharon M. Wahl Ioannis V. Yannas
Chief, Oral Infection & Immunity Department of Mechanical Engineering
National Institute of Dental Health Massachusetts Institute of Technology
National Institutes of Health Cambridge, Massachusetts, U.S.A.
Laboratory of Immunology Chapter 52
Bethesda, Maryland, U.S.A.
Chapter 21 Ian Zachary
The Wolfson Institute for Biomedical Research
Michael G. Walker, M.D. University College London
Deparment of Vascular Surgery London, U.K.
Manchester Royal Infirmary Chapter 24
Manchester, U.K.
Chapters 4, 9 John Zagorski
Chief, Oral Infection & Immunity
National Institute of Dental Health
National Institutes of Health
Laboratory of Immunology
Bethesda, Maryland, U.S.A.
Chapter 21
PART I
Bio-Inert Prostheses: Insufficient Healing
CHAPTER 1
The Lack of Healing in Conventional Vascular Grafts
Lester Davids, Terri Dower, Peter Zilla
Introduction
S eldom has any prosthetic implant become the nemesis of so many manufacturers as small
diameter synthetic vascular grafts. In the era of microsphere-encapsulated cell transplants
and human gene therapy it is astonishing that we still do not fully understand why the in-
corporation of prosthetic cardiovascular grafts does not even remotely resemble healing.
Even if the postnatal organism has mostly forgotten the “restitutio ad integrum” healing of
the fetal period—the complete regeneration of highly complex tissue with its original cell
components—it is nevertheless capable of repairing every nonlethal injury other than chronic
infection with a quiescent scar tissue. In the case of synthetic implants, however, the tissue
response is a far cry from any concluded repair event. The majority of contemporary pros-
thetic vascular grafts are not only continually lacking typical components of a healthy native
artery such as a midgraft endothelium, contractile arrangements of smooth muscle cells,
functionally differentiated elastin formations and other specialized components of a vessel’s
extracellular matrix. Synthetic vascular grafts are also lacking the much simpler ability to
restore tissue integrity through a quiescent scar formation. Even after prolonged periods of
implantation, chronic inflammation dominates the interstices of these prostheses while the
luminal patency is endangered by thrombotic surface appositions or the uncontrolled pro-
liferation of a so-called “neointima”.
Today’s dilemma is certainly the consequence of a multitude of developments of the
past three decades. When modern cardiovascular surgery first set out to replace arteries with
synthetic grafts, a graft had simply to act as a nonleaking blood conduit. When it soon be-
came obvious that the surface thrombogenicity of these prostheses was significantly higher
than that of native vessels, this complication was seen as a shortcoming of imperfect materi-
als rather than the lack of active physiological functions. This material-centered era only
slowly gave way to the realization that the right cells need to be an integral part of a synthetic
substitute vessel. However, a full commitment to truly “healing” grafts was further delayed
by the industry’s fear of disproportionate delays for new products resulting from a new
dimension of complexity.
This historical retrospect makes it clear that the most consequential reason for the
scanty patchwork of data available today lies in the fundamentally different concepts of
graft designs in this earlier era. Instead of a holistic engineering approach which defined all
biological components separately before sensibly combining them, grafts were mostly manu-
factured on the basis of material choices and mechanical strength. Therefore, completed
products were investigated with the intention to fulfill regulatory requirements rather than
learn about principal mechanisms. As a consequence, a multitude of poorly defined variants
such as entirely different synthetics, a wide range of interstitial space dimensions and a variety
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
4 Tissue Engineering of Prosthetic Vascular Grafts
of animal models make it extremely difficult today to ex- ity of studies of the last three decades focused unintention-
tract significant information from decades of prosthetic graft ally on exactly this type of healing. By choosing animal mod-
research. At the same time, biology has only recently provided els which far exceed the human ability of transanastomotic
us with the necessary diagnostic means to analyze tissue re- healing, as well as graft lengths which were in average one
actions with regard to the identification of cell types as well tenth of those clinically used, midgraft healing under ex-
as secretion products. Thus, the emergence of basic biologi- perimental conditions occurred predominantly through
cal tools and engineering technologies in the recent past— anastomotic outgrowth rather than transmural ingrowth.
promising the development of implants which may eventu- Considering the fact, however, that prosthetic arterial graft
ally heal “ad integrum”—does not build on a comprehen- endothelialization in humans—mainly restricted to the anas-
sive knowledge regarding the shortcomings of previous tomotic region—often involves less than one thirtieth of the
concepts. entire surface, facilitated anastomotic outgrowth will most
Since the retrospective deduction of biological prin- likely continue to play a limited role.
ciples from data which had mostly been obtained with a dif- Therefore, in spite of the countless studies which in-
ferent intention will not be able to create a full understand- vestigated prosthetic arterial grafts, a description of midgraft
ing of the apparent absence of physiological healing patterns healing—clearly distinguishable from transanastomotic
in prosthetic vascular grafts, a systematic experimental ap- events—is the exception rather than the rule. Nevertheless,
proach will need to supplement the erratic experience of by putting the sporadic observations of all these studies into
the past thirty years. For any eventual conclusions preced- a framework, a surprisingly consistent pattern of healing
ing serious tissue engineering approaches of the future, well became apparent. A further confusion arising from the mul-
planned, biologically oriented in vivo and in vitro experi- titude of animal models used also proved to be less of a prob-
ments will need to be performed. In this chapter we will lem than expected. As with any other aspect of graft heal-
nevertheless attempt to retrospectively identify certain cri- ing, by and large midgraft healing also confirmed the previ-
teria which may have influenced the mitigated healing re- ous belief that it is not the healing pattern itself but its time
sponse of prosthetic vascular grafts in the past. In order to course which distinguishes the various animal models from
avoid dealing with an infinite number of variables, we have each other. However, there are healing aspects like the im-
concentrated our efforts on those two basic graft types which penetrable inner fibrin capsule of Dacron grafts in humans
have been dominating the field of synthetic vascular pros- which may well be based on principal differences between
theses for the past decades: Dacron and expanded the species rather than simply on a protracted time course
polytetrafluoroethylene. of common events. Nevertheless, these principal differences
may again reflect differences in the time course of common
Midgraft Healing sequences, as certain biological phenomena may only occur
No other aspect of prosthetic graft research missed at a certain point in time. Therefore, a solution to graft heal-
the point as fundamentally as did midgraft healing. In spite ing could well lie in the facilitation of the ideal timing of
of a unanimous agreement with regard to the limits of crucial biological events.
transanastomotic tissue outgrowth in man, the vast major-
Fig. 1.1. Schematic dia-
gram depicting the histo-
logical summary of
events over time for dif-
ferent experimental
models for low porosity
(< 30 ∝m) ePTFE. De-
tailed histological de-
scriptions can be found
in the text.
The Lack of Healing in Conventional Vascular Grafts 5
ePTFE Grafts During the initial 2 weeks of implantation, a fine-
In spite of the wide range of internodal distances which fibrillar layer of a fibrin coagulum of approximately 15 ∝m
were experimentally evaluated and the distinct characteris- thickness covers the graft surface.1,2 During the following
tics of each of those grafts, a certain cut off point emerged weeks the thickness of this thrombus does not further in-
beyond which tissue ingrowth has seemed to be principally crease,3,4 but its composition becomes more variable, reach-
possible. This cut off point was identified to lie somewhere ing from acellular loosely3,5 (Fig. 1.2) or highly compressed
between 30 ∝m and 45 ∝m of internodal distance. There- fibrin4,23 to platelet carpets (Fig. 1.3) with interspersed
fore, it became customary to refer to grafts with an intern- granulocytes.1,5 Interestingly, these dense layers of sometimes
odal distance of less than or equal to 30 ∝m as low porosity almost pure platelets often show a morphology of low grade
ePTFE grafts and to those of 45 ∝m or more as high porosity activation (Fig. 1.3) or even nonactivation.5 In spite of this
ePTFE grafts. lack of morphological signs of activation, these platelets al-
most seem to melt into a dense, amorphous matrix.5 Dur-
Low Porosity ePTFE Grafts ing the subsequent months4,6,7 the blood surface still remains
(< 30 mm Internodal Distance) covered by either unorganized, compacted fibrin or a more
In contrast to other prosthetic grafts, low porosity amorphous platelet rich material, but the thickness of this
ePTFE hardly shows any difference between animal models layer increases to about 80-290 ∝m.4,5,8 Even after prolonged
with regard to the time course of midgraft healing. observation periods of up to 3 years, midgrafts lack all forms
Fig. 1.2. Surface fibrin on ePTFE (30 ∝m
internodal distance) after 4 weeks of im-
plantation in the chacma baboon model.
Loosely covering fibrin meshwork on a
dense carpet of platelets adheres to a
compact, more amorphous thrombus in
the depth.
Fig. 1.3. Surface of ePTFE graft (30 ∝m
internodal distance) after 4 weeks of
implantation in the chacma baboon.
One can recognize the ePTFE structure
in the left lower corner. A relatively thin
layer of amorphous thrombus covers the
synthetic surface. Densely adherent
platelets which are mostly discoid in
shape seem to melt into the amorphous
protein layer.
6 Tissue Engineering of Prosthetic Vascular Grafts
of cellular coverage.6,9-11 However, anecdotal descriptions multinucleated giant cells13,18,19 and macrophages8-20 demar-
of multilayered cell islands in the central segments of grafts1,5 cating the outer graft surface against a well developed fibro-
add to the decades old controversy concerning the origin of elastic perigraft tissue,1,7 no persistent inflammatory reac-
intimal cells.12 tion is found around the graft.7 However, there is a distinct
Similar to surface healing, transmural tissue ingrowth difference between wrap-reinforced and nonreinforced grafts
remains incomplete. Until week 2 most of the interstices of with regard to the tissue interaction with the outside envi-
the grafts are devoid of any recognizable cellular material,1 ronment. In nonwrapped grafts, relatively few foreign body
with only scanty macrophages beginning to migrate into the giant cells (FBGC) adhere to the PTFE nodes on the outside
micropores.13 After 3 weeks the fibrin matrix in the graft surface, while single macrophages have infiltrated the inter-
wall has slowly become populated with inflammatory cells,2 stices. In wrapped grafts, the narrow structure of the wrap
but there is hardly any connective tissue ingrowth into the limits the infiltration of the interstices with cells. Therefore,
graft.13-17 In the chacma baboon model we find a typical the almost acellular fibrin matrix usually found in the cen-
triple layer picture, with macrophages invading from the ter of the prosthetic wall extends all the way to the outside
blood surface and the adventitia into an otherwise almost surface. This outside surface, in return, borders upon a dis-
acellular central fibrin matrix (Fig. 1.4). Apart from scanty tinct layer of macrophages and FBGC (Fig. 1.4). This indi-
Fig. 1.4. 30 ∝m ePTFE graft after 6
weeks in the chacma baboon. One can
clearly recognize the fine porous wrap
at the outer surface. A layer of foreign
body giant cells (FBGC) demarcates
the ePTFE from an otherwise well-
healed surrounding tissue. Loose and
scanty cell infiltrates from the blood
surface and the adventitial side simul-
taneously progress towards the acel-
lular central fibrin matrix, filling the
interstices.
Fig. 1.5. Schematic dia-
gram depicting the histo-
logical summary of events
over time for different
experimental models for
high porosity (>45 ∝m)
ePTFE. Detailed
histological descriptions
can be found in the text.
The Lack of Healing in Conventional Vascular Grafts 7
cates once more that the formation of FBGC may be signifi- a more amorphous protein matrix densely covered by plate-
cantly influenced by surface structures, because the mate- lets and a few white blood cells (Fig. 1.7). Overall, the thick-
rial is PTFE in both instances. In the case of the ePTFE wrap ness of the surface thrombus is moderate, because one can
it is difficult to say whether the higher density of FBGC is a recognize the nodal ePTFE structure throughout the graft
result of the finer porosity and structure of the PTFE or the (Fig. 1.8). Histologically, a homogenous fibrin layer covered
overall result of an impenetrable barrier plane compared to by a carpet of platelets enshrouds the ePTFE surfaces. Ex-
the relatively open spaced single nodes in unwrapped ePTFE. cept for very few occasional polymorphnuclear granulocytes,
Eventually, after 6 weeks of implantation, connective tissue this fibrin layer is practically acellular (Fig. 1.7). The pres-
cells occasionally begin to grow into the interstices of un- ence of a layer of macrophages underneath this fibrin ma-
wrapped prosthesis from outside.13,14,17,21-23 Six months af- trix—which is demarcated for the depth of the graft by a
ter implantation, however, connective tissue ingrowth from zone of equally acellular fibrin—indicates that the surface
outside still remains moderate and mostly limited to the fibrin has previously been permeable to inflammatory cells.
outer part of the graft wall,18-20,24-26 while the majority of In contrast to the senescent chacma baboon, high porosity
interstitial graft spaces continue to be primarily occupied ePTFE grafts lead to early and spontaneous
by fibrin and a mild inflammatory infiltrate in the proxim- endothelialization in juvenile recipients such as young yel-
ity of the surfaces.25 In contrast to the early days of implan- low baboons. Patches of endothelial cells30 and capillary ori-
tation, however, these cells lie in a very fine fibrillar extra- fices—approximately 100-500 ∝m apart31—appear on the
cellular meshwork which has replaced the solid acellular fi- blood surface as early as 1-2 weeks after implantation, lead-
brin. If present, this scanty and loose connective tissue con- ing to confluence shortly thereafter.30-35 Since the endothe-
tains hardly any capillaries.113,25 lium soon rests on a well developed layer of actin positive
cells3-33,35 which contains only a few macrophages,36 the fi-
High Porosity ePTFE Grafts brin matrix which initially covers the inner surface and pro-
( > 45 ∝m Internodal Distance) vides the outgrowth substrate for the endothelium repre-
In contrast to low porosity ePTFE grafts, there is a dis- sents a very transient matrix under these circumstances.
tinct difference in high porosity ePTFE grafts with regard to These actin positive cells appearing underneath the endot-
the time course of healing events in humans and different helium9,33 also exhibit the ultrastructural characteristics of
animal models. arterial smooth muscle cells.30 Although proliferating
Initially, the blood surface resembles that of low po- smooth muscle cells resemble wound fibroblasts, there is
rosity ePTFE grafts. It is covered by a thin fibrin layer which currently more evidence against fibroblasts trans-differen-
over time develops into a variable coagulum of fibrin, plate- tiating into smooth muscle cells19,37-40 than there is for it.
lets and erythrocytes.27 In dogs and other more senescent Therefore, it is likely that these cells are derived from
animal models, this stage lacking endothelial coverage per- pericytes accompanying EC.9,33 Over time the endothelium
sists well into the sixth week of implantation.27-29 In the se- and its subendothelial tissue layer develop into a stable
nescent chacma baboon, for instance, we find a six week pan- neointima20,31,35,41,42 with a higher density of actin positive
nus ingrowth of only 7.8 ± 3.5 mm reaching onto otherwise cells adjacent to the endothelium than to the graft sur-
nonendothelialized grafts (Fig. 1.6). The surface of these face.20,26,31,40,43-49 This intimal thickening is evenly distrib-
grafts is covered either by a relatively thin layer of smooth uted along the entire graft surface and not confined to the
fibrin containing white blood cells and a few platelets or by anastomotic region as in low porosity ePTFE grafts.30 It is
Fig. 1.6. Pannus outgrowth onto 60 ∝m
ePTFE after 6 weeks of implantation in
the chacma baboon. One can clearly see
the anastomotic outgrowth of the pan-
nus and the endothelium tapering off
onto an otherwise nonendothelialized
ePTFE surface.
8 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 1.7. Platelet-rich surface coverage of
the midsection of a 60 ∝m ePTFE graft
after 6 weeks of implantation in the
chacma baboon. One can faintly recog-
nize the ePTFE surface in the central up-
per part of the picture. The dense carpet
of platelets is again dominated by discoid
shaped thrombocytes which do not re-
semble the typical morphology of aggre-
gated platelets.
Fig. 1.8. 60 ∝m ePTFE after 6 weeks of
implantation the chacma baboon. The
surface thrombus is delicate and still al-
lows the underlying PTFE structure to
be recognized.
particularly noteworthy that these smooth muscle cells only macrophage products stimulate SMC proliferation and high
started to appear in the intima and began to proliferate after ones are inhibitory.69 Eventually the discovery of PDGF-A
endothelial cells had covered the luminal surface.30 With mRNA in the overlying endothelial cells resolved the issue
SMCs multiplying in the inner one third of the intima adja- of the source of SMC mitogens.114b This cytokine is not only
cent to an endothelium33 and without the concomitant pres- one of the strongest currently known proliferative agents
ence of platelets33 the situation is the opposite of trauma- for smooth muscle cells, but was previously already shown
tized arteries. Therefore, it is conceivable that endothelial in vitro to be secreted by endothelial cells vectorially into
cells themselves produce growth promoting substances for the abluminal basal compartment.228 Its strong presence also
smooth muscle cells. This is further supported by the fact explains the 5-100 times higher proliferative activity of SMCs
that after 3 months, SMCs are predominantly found in the and the 10-100 times higher one of endothelial cells50,51 in
subendothelial intima relatively distant from the macroph- grafts than in normal arteries. Nevertheless, the presence of
ages in the interstices.33 Apart from hinting at the involve- higher levels of PDGF and the proof of a higher mitotic ac-
ment of endothelial cells in intimal smooth muscle cell pro- tivity does not indicate whether this is an adaptive response
liferation, this observation of an increasing smooth muscle or a primary phenomenon. On the one hand one could ar-
cell proliferation with increasing distance from the macroph- gue that a high cycling rate may simply be the consequence
ages is inconsistent with the hypothesis that macrophages of an upregulated PDGF production. On the other hand,
are an important source of SMC mitogens,134,189 unless an however, it is also quite likely that a poor anchorage of en-
inverse gradient applies in which low concentrations of dothelial cells at this stage leads to mechanical detachment
The Lack of Healing in Conventional Vascular Grafts 9
and this in turn upregulates PDGF production as a repara- baboon, it takes 2-5 weeks and longer until sprouting capil-
tive response. The latter explanation is supported by the laries begin to reach into the macrophage dominated outer
morphological appearance of endothelial cells. Typical for one third to one half of the graft wall27-29 (Fig. 1.9). Dis-
endothelial surfaces affected by a distinct cell loss,5 these tinctly different from Dacron grafts, these microvessels re-
endothelial cells lack the regular spindle shaped pattern30 semble mature arterioles which are small in diameter (23.06
and are larger than those in arteries.30 This sequence of a ± 13.11 ∝m; personal observation) and regularly accompa-
higher cell loss preceding a higher mitotic activity, rather nied by at least one layer of smooth muscle cells. However,
than a higher cycling rate, being the reason for higher en- in spite of this initial vascularization, the histological pic-
dothelial cell shedding would coincide with the finding for ture of these implants remains dominated by a triple layer
in vitro endothelialized grafts that a distinct degree of en- appearance even after 6 weeks of implantation: While the
dothelial cell detachment occurs during early implantation blood and the adventitial part of the prostheses is infiltrated
in spite of continual endothelial integrity.5 However, since by inflammatory cells, the central zone of the prosthetic wall
the overall thickness of this neointima may also represent often remains filled with an almost acellular amorphous
an adaptation attempt of the tissue to flow conditions in an matrix (Fig. 1.10). Within this triple layered structure the
otherwise rigid graft, the PDGF upregulation in the surface cellularity of the outer third is regularly higher than that of
endothelium could as a third explanation also be a natural the inner third of the graft, although it does not change much
mechanism of macrovascular endothelial cell response to over time. Ham 56 positive macrophages continue to domi-
environmental changes such as flow. This would explain why nate over connective tissue cells,55 while foreign body giant
a many-fold thicker neointima is found under low flow con- cells are conspicuously absent.56 In spite of the scanty pres-
ditions than under high flow conditions.54 ence of connective tissue, the homogenous fibrin matrix ini-
When surface healing occurs so early and indepen- tially filling the interstices gradually gets replaced by a loose
dently of transanastomotic tissue ingrowth, the cell source transparent extracellular meshwork wherever inflammatory
for it can only be perigraft tissue which reaches the blood cell infiltrates reach into the interstices (Fig. 1.11). On the
surface through apt transmural ingrowth. However, since outside, high porosity ePTFE grafts are embedded into a
complete transmural ingrowth does not readily occur in moderately developed mature fibrous tissue which contains
other graft types, it is particularly interesting to see whether hardly any inflammatory cells. Only the direct interface be-
it differs substantially in high porosity ePTFE grafts com- tween adventitial tissue and graft occasionally shows a few
pared with other prostheses. single FBGCs.
Comparably to other grafts, the basically acellular ear-
liest interstitial fibrin matrix gets progressively populated Dacron Grafts
with macrophages and polymorphnuclear leukocytes28,33 In Dacron grafts the issue of porosity is slightly com-
during the initial period of graft implantation followed by plicated through the optional combination of the grafts with
capillary ingrowth. In the juvenile yellow baboon, these cap- a velour surface which differs from the basic texture.
illaries reach through the entire wall as early as after 2 weeks
of implantation. Although fibroblasts from the perigraft re- Low Porosity Dacron Grafts (Woven)
gion soon follow the capillaries,16 the majority of the graft Immediately after implantation a thin layer of fibrin,
interstices still remain populated with macrophages.33 In erythrocytes, white blood cells and platelets is deposited on
more senescent animal models like the dog or the chacma the blood surface of the prosthesis.40,57 During the first few
Fig. 1.9. Immunohistochemical staining
of #-actin in a 60 ∝m ePTFE graft after 6
weeks of implantation (chacma ba-
boon). It is obvious that those vessels
which were capable of penetrating into
the outer half of the graft all contain at
least one layer of smooth muscle cells.
10 Tissue Engineering of Prosthetic Vascular Grafts
hours to days this thrombus layer slowly starts thickening appear.58,59 These endothelial islands always rest on the com-
until it reaches a stable equilibrium of compacted fibrin.45 pacted fibrin rather than on the scanty islands of connec-
In dogs this stage is reached within 6 months,45 whereas in tive tissue.
humans it is observed after 1 1/2 years.223 Exceptionally, fi- Underneath this apparently impenetrable fibrin layer,
brous tissue begins to organize the basis of the fibrin cap- healing is most likely to take place; at least with regard to
sule in a few scanty areas of the graft.40,45 This process is perigraft tissue ingrowth, the initial events of graft incorpo-
observed after 4-8 weeks in dogs40,45 and after 1 1/2 years in ration are similar to ePTFE. A fibrin matrix fills the narrow
humans.58 Those few areas of a pseudoneointima where tis- prosthetic interstices within minutes of implantation,57 and
sue reaches the blood surface40,58,59 have a thickness which organizing tissue begins to invade the fibrin layer surround-
is comparable to that of the surrounding compacted fibrin ing the graft to form the outer fibrous tissue capsule.45,57 In
(400-500 ∝ m). 58 In humans these rare and localized the interface between this fibrous tissue and the Dacron yarns
neointimal spots extend over less than 1 mm and are col- a variable degree of foreign body giant cells begins to build
lagen rich, with increasing cellularity towards the graft sur- up.57 Subsequently, a few capillaries and fibroblasts start
face.47 The situation is similar with regard to EC. In areas growing into the tight interstitial spaces of the woven
which represent true midgraft regions—unaffected by anas- Dacron.40,57 This process is quite variable in its extent and
tomotic ingrowth—no endothelial cells are found within the time course. In dogs it can be seen as early as 2-3 weeks after
first 1-2 years.40,58,59 After prolonged implantation periods implantation,40 whereas in humans it may be observed after
of up to 11 years—which are naturally restricted to hu- 5 months11 but may also be absent after up to 18 years.47,58,59
mans—small islands of endothelial cells may occasionally However, even if tissue does occasionally grow through the
Fig. 1.10. 60 ∝m ePTFE graft without ex-
ternal wrap reinforcement. There is
hardly any inflammatory tissue demar-
cating the graft against the surrounding,
well-healed fibrous adventitial tissue.
After 6 weeks the central zone of the in-
terstitial spaces is still filled by an acel-
lular fibrin matrix. The outer and inner
third of the graft shows a loose infiltra-
tion with cells which previously de-
graded the acellular fibrin matrix. The
blood surface is covered by a thin irregu-
lar thrombus which is practically acel-
lular, not even containing inflammatory
cells.
Fig. 1.11. Knitted Dacron graft
(Vascutec) after 6 weeks of iliac interpo-
sition in the chacma baboon. Typically,
the Dacron structure is packed with for-
eign body giant cells and demarcated
from the surrounding adventitia by
granulation tissue which contains hugely
dilated capillary sinuses.
The Lack of Healing in Conventional Vascular Grafts 11
interstices of the prostheses, it does not break through the gions in particularly long grafts in which the inner fibrin
compacted fibrin of the inner capsule to reach the blood capsule appears to be both impenetrable for the ingrowing
surface43 even after decades of implantation. interstitial graft tissue43,44,48,59,60,71,72 and nonendothe-
lialized. 60,72,73 This lack of surface healing oc-
High Porosity Dacron Grafts (Knitted) curs43,44,48,59,60,71,72,74,75 although transmural tissue reaches
There is a distinct difference regarding the healing re- the compacted fibrin of the blood surface within 3-4 weeks
sponse to variations in porosity between Dacron grafts and in the calf,60 the yellow baboon35 and the dog,2,76 and 3-6
ePTFE grafts. In ePTFE grafts, the fibrin matrix may get rap- months in humans.48,60 However, in spite of this dominant
idly replaced by inflammatory and connective tissue if the healing deficit, complete tissue replacement of the inner fi-
porosity is increased. In Dacron grafts, the mighty inner fi- brin capsule may sporadically occur in the absence of a sur-
brin capsule mostly persists in both high and low porosity face endothelium and with a poorly differentiated fibroblast
grafts although interstitial healing is more accelerated in the matrix rather than with smooth muscle cells.61 Occasion-
higher porosity prostheses. ally, even complete endothelialization of very long prosthe-
In knitted dacron prostheses the initial surface net of ses is seen46,77 comparable to those in highly porous ePTFE
fibrin, white blood cells, erythrocytes and platelets48 increases grafts. In the yellow baboon, for instance, knitted Dacron
during the first week of implantation to a thickness of grafts may form complete endothelial linings, although not
100-120 ∝m.44,60 During the subsequent 5 weeks of implan- as readily and consistently as in 60 ∝m ePTFE.32,35,78,79 They
tation it appears as if the inner fibrin capsule has not yet initially show small islands of endothelium after 2 weeks,
reached an equilibrium and therefore holds a wide range of and in a minority of implants even confluence after
thickness: 100-500 ∝m in humans,60 30-210 ∝m in dogs2,61-66 4 weeks.32
and 290-300 ∝m in the yellow baboon.35,91 After a subse- However, in contrast to the case of high porosity
quent further insignificant increase,60-62 the thickness of the ePTFE grafts, it remains unclear whether this endothelium
internal capsule eventually reaches an equilibrium before is derived from transmural tissue ingrowth, facilitated
sixth months of implantation.61,62 Although a superficial transanastomotic outgrowth or fall-out healing.12,80 The lat-
screening of the literature suggests that it is only a matter of ter is a phenomenon predominantly observed in Dacron
time until this entire layer of compacted fibrin gets orga- grafts where—apparently independently of transmural tis-
nized by ingrowing tissue, deeper analysis makes it likely that sue ingrowth and with some delay—endothelial islands
this may well be a misinterpretation based on the confusion emerge on the surface of the compacted fibrin. These is-
of midgraft healing with anastomotic outgrowth. It is true lands are well separated from the transmurally ingrowing
that the vast majority of studies describe a mature endothe- fibrous tissue by a distinct layer of compacted fi-
lium and a well developed intimal smooth muscle layer, rest- brin.43,48,59,60,71 They either rest directly on the fibrin43,59,72
ing directly on the graft surface,2,67-69 but the graft length or on a few layers of actin positive cells with no other tissue
and implantation period in these studies make it almost cer- connection,59,71 an observation which can be made in dogs
tain that this mature neointimal tissue represents nothing after 2-6 months72 and in humans perhaps in every fourth
but extended anastomotic outgrowth.2,22,32,35,44,68-70 This graft after 1-11 years.43,59,71 However, although sporadic
suspicion is confirmed by descriptions of true midgraft re- endothelialization occasionally occurs in limited areas of the
Fig. 1.12. Schematic dia-
gram depicting the histo-
logical summary of
events over time for dif-
ferent experimental
models for woven
Dacron. Detailed histo-
logical descriptions can
be found in the text.
12 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 1.13. Schematic dia-
gram depicting the histo-
logical summary of events
over time for different ex-
perimental models for knit-
ted Dacron. Detailed histo-
logical descriptions can be
found in the text.
grafts, the majority of the surface remains covered by its basal lamina (Fig. 1.11). Those few capillaries which some-
fibrin only. times reach through the entire graft wall and reach the in-
If one focuses on the transmural tissue ingrowth it- ner capsule also remain free of smooth muscle cells.
self, it also begins with the infiltration of the surrounding However, in areas where transanastomotic ingrowth
outer fibrin matrix by granulation tissue.2,48 Initially it is has resulted in a mighty neointima covering the surface, one
primarily macrophages which begin to invade the intersti- occasionally finds true multilayered vessels with smooth
tial fibrin,14 populating the entire depth of the graft at the muscle cells underneath the endothelium.
end of the first month of implantation.60 Early giant cell In summary, the porosity of knitted, and to some ex-
formation surrounding the yarns and—if present—the ve- tent even woven, Dacron prostheses eventually allows a cer-
lour fibrils commences after 7 days and increases, predomi- tain degree of tissue ingrowth from outside. At the same time,
nantly in the outer half of the graft wall,62 during the however, it seems that the compacted inner fibrin capsule
following months.61,62 The subsequent ingrowth of blood on the blood surface of Dacron grafts represents an ingrowth
vessels and fibroblasts varies not only between the different barrier for transmural connective tissue regardless of
animal models but also within one and the same recipient whether the grafts are knitted or woven. Although this bar-
species. In dogs for instance, the interstitial tissue ingrowth rier may eventually be overcome by ingrowth of undiffer-
can either reach the inner fibrin capsule as early as 2-4 weeks entiated fibrous tissue, capillaries remain unconnected to
after implantation2,61,63,76 or be absent as late as after 2 the often poorly endothelialized blood surface.75 The pecu-
months.72,81 Similarly, clinical implants in humans show all liar coincidence of zones which are densely packed with for-
stages of ingrowth for up to three months.179 From then eign body giant cells and hugely dilated capillary sinuses in
onwards, ingrowing adventitial tissue reaches the internal the vicinity is particularly obvious in Dacron prostheses.
fibrous capsule without exception in all animal spe- Moreover, it is also interesting that the fibrin coverage on
cies.2,11,43,59,60,71,81,82 This tissue contains a moderate num- Dacron grafts seems to have a higher propensity to capture
ber of capillaries, fibroblasts and collagen2,11,43,59,71,82 and blood-borne cells than that on ePTFE grafts.80 In this con-
eventually extends along the inside surface of the graft.60,62,71 text it is also noteworthy that the resulting isolated endot-
However, a clear, layered structure of tissue elements still helial islands often rest on equally isolated smooth muscle
dominates the histological picture. The Dacron fibers ap- layers.43,59 However, in spite of the scientific excitement aris-
pear to demarcate themselves expressively against the sur- ing from this observation, the rare and late occurrence of
rounding tissue by a particularly distinct accumulation of the phenomenon makes it the least typical healing charac-
foreign body giant cells (FBGC). Between this outer layer of teristic of Dacron grafts.
the prosthetic wall which is packed with FBGC and the sur-
rounding connective tissue capsule with its longitudinally High Porosity Dacron Velour
aligned collagen bundles,59,60,81 hugely dilated capillary si- The rationale behind a filamentous coverage of Dacron
nuses are usually present during the early weeks of implan- fabric was the attempt to improve the healing between
tation. These sinuses typically consist of a single endothelial perigraft tissue and the prosthesis. Initially only used as an
cell layer which contains no smooth muscle cells or cell types external cover,60 it was soon also applied as an internal
other than macrophages and FBGC, which snuggle against cover.83 Interestingly, it was thought that a finer and more
The Lack of Healing in Conventional Vascular Grafts 13
even porosity would cover the coarser structure of the knit- contains white blood cells in deeper layers which one can
ted fabric underneath and thus achieve facilitated healing. still recognize from the surface (Fig. 1.15). Nevertheless, one
As is obvious from our porosity analysis (see “Porosity”) the also finds areas of densely packed fibrin, covered with mor-
opposite is true, and velour materials actually provide sig- phologically mostly unactivated platelets and white cells
nificantly wider ingrowth spaces than knitted or woven struc- comparable to ePTFE (Fig. 1.16). Very rarely does one see
tures. This wider porosity does result in a firmer tissue inte- vessel openings on the surface (Fig. 1.17). The relatively freely
gration on the outside,15,84 but reduces platelet survival and lying single fibers of the material are completely engulfed
leads to less pronounced pseudointimal development on the by conglomerates of foreign body giant cells, whereas the
inside.85 In order to study the healing pattern of entire ve- interstices are dominated by a loose fibrin matrix contain-
lour structures, we have fabricated graft tubes out of pure ing lymphocytes, polymorphonuclear granulocytes, macro-
velour material and studied the tissue reaction in the chacma phages and FBGCs. In spite of the wide open porosity, hardly
baboon. Similarly to previous results,86 the transanastomotic any fibroblasts or collagen are found in the interstices. The
outgrowth of a neointima is significantly less distinct than huge and dilated capillary sinuses, which were all localized
in nonveloured knitted Dacron, even after weeks of implan- between the outer layer of the graft and the surrounding
tation. The fibrin matrix itself often appears looser than on tissue in knitted prostheses, are equally present but scattered
ePTFE and, moreover, often covered with relatively densely throughout the meshwork of the grafts. These often enor-
adherent macrophages (Fig. 1.14). The loose fibrin often mously dilated capillary sacs are all factor VIII positive, and
Fig. 1.14. Blood surface of the midgraft
section of a Dacron velour tube (6 weeks,
chacma baboon). Typically, large areas of
the fibrin surface are covered with dense
layers of spreading macrophages.
Fig. 1.15. Same graft as Figure 1.10, but
showing a more loose meshwork of fi-
brin with inflammatory cells lying on the
surface and in the depth of the structure.
One can still recognize those white blood
cells which are lying close to the surface.
14 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 1.16. Midgraft section of a
Dacron velour tube covered by an
amorphous protein layer with an
adherent carpet of disk shaped
platelets. Occasionally, white blood
cells lie in lacunar spaces which ap-
pear to have been excavated from
the fibrin layer.
Fig. 1.17. Midgraft section of a
Dacron velour tube after 6 weeks of
implantation in the chacma ba-
boon. One very rarely sees capillary
openings surrounded by small is-
lands of endothelial cells, which
grow onto the otherwise endothe-
lial-free compacted fibrin surface.
Fig. 1.18. Histological cross-section
of a Dacron velour tube after 6
weeks of implantation in the
chacma baboon. The loose structure
allows capillary ingrowth at all lev-
els. As a consequence the dilated
capillary sinuses—which are other-
wise only found in the outer half of
knitted prostheses—are scattered
throughout the graft wall. The cap-
illary sinuses themselves consist of
factor viii positive endothelial cell
monolayers resting on basement
membranes surrounded purely by
inflammatory cells, which are pre-
dominantly foreign body giant cells.
The Lack of Healing in Conventional Vascular Grafts 15
equally as devoid of smooth muscle cells as knitted prosthe- region, the widely used term “neointima” in these studies
ses (Fig. 1.18). Occasionally, the simultaneous infiltration actually refers to anastomotic pannus tissue.
mode from inside and outside can still be detected after 6 However, in spite of the poorly defined criteria with
weeks, with a central cell free zone of fibrin wedged between regard to anastomotic tissue ingrowth and the scanty data
the inner and outer graft thirds, the outer being dominated available for some of these criteria, certain trends concern-
by foreign body giant cells. ing animal models, material and porosity still become
These observations indicate that in spite of wide open apparent.
interstitial spaces, there seems to be a distinct inhibition for
connective tissue ingrowth in Dacron scaffolds. It is even Pannus Tissue
more obvious from velour explants than from the narrower Within less than 2 days of implantation, a straighten-
structures of knitted Dacron that capillarization is rather ing and disruption of the internal elastic membrane of the
overshooting than underdeveloped. These widely dilated adjacent native artery occurs.87 Simultaneously, the smooth
endothelial sacs, however, neither reach the blood surface muscle cells of the inner one-third of the media begin to
nor attract concomitantly ingrowing smooth muscle cells. proliferate87 and migrate through the ruptured internal elas-
tic membrane into the intima. It is primarily this hyperplas-
Transanastomotic Healing tic intimal tissue which—together with the endothelium—
The assessment of anastomotic tissue ingrowth is an- migrates and proliferates onto the graft surface, thereby
other ambiguous task within the attempt to interpret the slowly replacing the inner fibrinous capsule from the anas-
healing of prosthetic vascular grafts. Although surface tomosis towards the midgraft. Typically, endothelial cells
endothelialization—which under experimental circum- precede this outgrowth,19,28,88 followed by connective tissue
stances overwhelmingly happens through transanastomotic cells. This means that the foremost edge of the outgrowing
ingrowth—was indirectly the focus of almost every single endothelium initially rests on the fibrin matrix of the inner
study in the past, there is hardly any other aspect of pros- capsule6,28,88 before a few layers of smooth muscle cells ap-
thetic graft healing which is so vaguely described. pear underneath.14,25,27,13 This edge of outgrowing endot-
When all studies included in this review are taken into helium often does not resemble a solidly migrating forma-
account, the average graft length is 10.8 ± 2.6 cm. This value tion of endothelial cells, but rather deeply meandered, tat-
includes the 11% of grafts which were longer than 10 cm. If tered and irregularly shaped islands and tongues of endot-
those grafts—which were substantially longer than the oth- helium (Fig. 1.19). Very often, the foremost edge consists of
ers (36.6 ± 2.4 cm)—are excluded, the mean length of the loosely lying endothelial cells, surrounded by platelet-cov-
remaining 89% of prostheses is only 5.5 ± 1.2 cm. With a ered fibrin (Fig. 1.20). Eventually, a multilayered connective
median implantation period of 91.8 ± 17.3 days in Dacron tissue, which increases in thickness over time, slowly builds
grafts and 60.0 ± 9.36 days in PTFE grafts, it does not come up between the graft and the blood surface.19 This tissue
as a surprise that transanastomotic ingrowth was frequently does not consist exclusively of secretory smooth muscle
completed before the prostheses were retrieved. This often cells69 but also of fibroblasts, collagen and a few macroph-
makes it impossible to determine retrospectively the point ages.30,40,52,55,61,62,68 After a few months a layered structure
in time when the surface coverage through transanastomotic develops with a hypercellular myofibroblast-dominated lu-
tissue ingrowth was completed. To create further uncertainty, minal aspect and a fibro-collagenous and glucosamino-
measurements of tissue ingrowth vary widely amongst those glycan-rich aspect near the graft surface.68,89 However, even
studies where surface endothelialization was not completed at that stage this subendothelial tissue lacks features of na-
prior to explantation, and which could thus be admitted to tive arteries like elastic fibers between the smooth muscle
a comparative ingrowth assessment. cell layers20 and an internal elastic membrane69 separating
Another point of confusion relates to the terms the endothelium from the bulk of smooth muscle cells. Al-
“neointima” and “pannus”. As we have argued under the topic together, the undifferentiated pannus near the anastomosis
“midgraft healing”, prior to the use of high porosity ePTFE of a prosthetic graft has not much in common with the or-
grafts neointimal tissue formation was mostly restricted to ganized intimal structures of a native vessel. Thus,
transanastomotic ingrowth, even if many of the investiga- transanastomotic tissue outgrowth remains a far cry from a
tors were not aware of it. As opposed to the mostly fibrin- self-terminating healing process leading to true neo-artery
ous “pseudoneointima” of midgraft regions, “neointima” structures on the inside of a prosthetic graft.
could almost synonymously be used as the term for In spite of the undifferentiated hyperplastic nature of
transanastomotically ingrowing tissue covering the blood this pannus tissue, however, there are various circumstances
surface of the graft. However, with truly transmurally de- which significantly affect at least its tissue composition and
rived neointimas observed in certain contemporary pros- thickness. Anatomical position, for instance,72,81 as well as
theses, it seems necessary to restrict the term “neointima” to the difference between distal and proximal anasto-
surface tissue which results from transmural midgraft heal- moses64,81,90 are known to influence the thickness of pan-
ing and to keep describing the anastomotic tissue ingrowth nus tissue. Moreover, tissue hyperplasia is also a means of
as “pannus”. As necessary as this restriction of the term compensating for the lack of compliance and contractility
“neointima” is, it eliminates further bits of assumed clarity. in prosthetic grafts. Therefore, variations in flow8,52 and shear
Since the majority of studies describe prostheses in which stress52 result in an adaptive reduction or increase in thick-
the anastomotic pannus has already reached the midgraft ness of the subendothelial tissue.
16 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 1.19. Typically meandered edge of
transanastomotically outgrowing en-
dothelium (30 ∝m ePTFE; chacma ba-
boon; 6 weeks).
Fig. 1.20. Loosely arranged endothe-
lial formation at the edge of trans-
anastomotically outgrowing pannus
tissue. All the cells are practically lack-
ing contact with the neighboring en-
dothelial cells and are surrounded by
a dense carpet of blood platelets
(Dacron velour, chacma baboon, 6
weeks).
Apart from these influences, it appears as if the thick- and transmural tissue ingrowth does not proceed into the
ness of the anastomotic pannus is to a certain degree also inner capsule of either at this stage, pannus outgrowth onto
predetermined by an almost template function of the initial the graft surface seems to be much faster in knitted
fibrinous inner capsule. Even at an early stage when trans- grafts,2,46,60,68,72,77,81 with their almost continual tissue layer
mural tissue ingrowth has hardly reached the inner fibrin- underneath the fibrin capsule compared with the scanty
ous capsule, pannus formation replicates the dimensions of transmural tissue ingrowth beneath the fibrin matrix of
the fibrin layer which it replaces. Two weeks after implanta- woven grafts.40 Similarly, a significantly faster transan-
tion for instance, the surface thrombus on ePTFE grafts mea- astomotic tissue outgrowth occurs in high porosity ePTFE
sures about one-eighth to one-fourth of that on Dacron grafts if interstitial tissue development is facilitated through
grafts.1,2,44,60 Similarly, the pannus replacing this thrombus external tissue wrapping. This facilitated outgrowth is ob-
in the anastomotic region not only measures one-eighth to served before transmural tissue ingrowth could account
one-fourth on ePTFE grafts compared with Dacron pros- for it.28
theses,2,35 but actually has the same thickness as the preex- As far as the cellular and matrix composition of the
isting fibrin layer.2,8,64 The alacrity, however, with which this pannus tissue is concerned, it appears as if the absence of a
pannus layer grows towards the midgraft region may well mature and confluent endothelium at the time of sub-en-
be accelerated by the presence of tissue underneath the in- dothelial tissue development may be one reason for its lack
ner fibrin capsule, as is the case with high porosity grafts. of differentiation. As early as 1975, Mansfield and Sauvage92
Although the fibrin layer on the blood surface of woven and described the prevention of undifferentiated pannus out-
knitted Dacron grafts is of equal thickness2,45,58,60-62,64-66,91 growth through in vitro endothelialization of grafts in calves.
The Lack of Healing in Conventional Vascular Grafts 17
Almost 20 years later clinical in vitro endothelialization dem- aiming at overcoming this limitation uses animal models
onstrated the same phenomenon: In endothelial lined grafts which characteristically show premature and rapid surface
in which the formation of a confluent endothelium precedes coverage with anastomotic endothelium. In the past this
that of the subendothelial tissue,5 smooth muscle cells rather rapid transanastomotic endothelialization was usually the
than the occasionally observed fibroblasts61 dominate a key parameter of investigations when primarily surface
mature matrix which even contains an internal elastic mem- endothelialization was compared between different pros-
brane.93 The use of purified cultured autologous endothe- thetic grafts. However, if one agrees that future grafts for
lial cells for the in vitro lining process makes it quite un- clinical use will require transmural tissue ingrowth as a main
likely that smooth muscle cell coseeding was the reason for cell source for surface endothelialization because of their
the development of such a well differentiated subendothe- extraordinary length, a better understanding of transan-
lial tissue rather than the interaction with the preexisting astomotic endothelialization is indeed necessary, but for a
endothelium. Since a tissue which lies beneath an internal different reason than before: To be able to clearly define for
elastic membrane qualifies per definition as neo-media each animal model a biological phenomenon which would
rather than hyperplastic neo-intima,93 such a differentiation otherwise continue to blur the distinction line between trans-
means a huge step towards true healing and therefore needs mural and transanastomotic tissue ingrowth.
to be understood with regard to the underlying biological
mechanisms. In another study, similar degrees of pannus Differences Between Animal Models
maturation were observed following endothelial cell seed- Alerted by the human situation of complete ingrowth
ing with mixed microvascular cells.61 In this case, fibroblasts stoppage, we can hardly assume that transanastomotic endo-
were initially coseeded with other cell types at similar pro- thelialization is a linearly progressing event in animal mod-
portions to those normally seen in anastomotic pannus tis- els. Therefore, extrapolation of pointwise observations to a
sue. Nevertheless, the subendothelial matrix—which ma- long term situation, or even to a constant daily outgrowth
tured after the endothelium reached confluence—consisted distance,2,94 seems obsolete, although it would have provided
of mature muscle cells and also elastin rather than the typi- valuable additional data for an ingrowth curve. Even in the
cal poorly differentiated collagen-rich pannus tissue.61 These yellow baboon with its vigorous angiogenic capacity, a simi-
observations support the presumption that the early pres- lar trend of ingrowth stoppage as in humans can be observed:
ence of an endothelium may influence the degree of differ- On 30 ∝m ePTFE prostheses—which do not allow transmu-
entiation of the subendothelial connective tissue of both pan- ral endothelialization—transanastomotic endothelial in-
nus and neo-intimal tissue. growth progressively slows down, to stop approximately 2 cm
from the anastomosis.95-97 Furthermore, high porosity
Transanastomotic Endothelialization ePTFE grafts, as well as a few knitted Dacron grafts, cannot
Endothelial outgrowth from the adjacent artery onto be admitted into this comparison because of complete trans-
the surface of a prosthetic vascular graft is another enigma mural surface endothelialization. Considering these exemp-
of prosthetic graft research. For unknown reasons, tions and the fact that only a minority of studies describe
transanastomotic endothelialization stops shortly after the ingrowth margins rather than percentages of surface cover-
anastomosis in humans.11,47,60 Yet, the experimental set-up age, it is difficult to compare accurately the various animal
Fig. 1.21. Transanastomotic endo-
thelial outgrowth on 30 ∝m ex-
panded polytetrafluoroethylene
(ePTFE) grafts in four experimen-
tal animal models. The curve was
plotted using pannus outgrowth
values in the dog model (top curve).
Points read off the main curve cor-
respond to humans, Papio ursinus/
chacma baboons, and Papio cyno-
cephalus/yellow baboons, as indi-
cated by connecting arrows. The
curve was drawn using Deltagraph
(for Macintosh).
18 Tissue Engineering of Prosthetic Vascular Grafts
models with regard to transanastomotic endothelial cell are usually also senescent at implantation,74 whereas pigs
outgrowth. Nevertheless, since most of the experiments in and calves can be expected to be juvenile. In the rat model,
the past were based on the canine model, a relatively com- the issue of dimensions complicates interpretation. A
plete chronology can be estimated for standard grafts in the transanastomotic endothelial outgrowth of approximately
dog. Single data obtained in other animal models can then 2.2 mm after 6 weeks13,16,17,88 in 30 ∝m ePTFE makes it im-
be related to the time course of surface healing in dogs. How- possible to relate these data to the 7.8-13.6 mm of other large
ever, the accuracy of even this narrowed appraisal decreases animal models.
rapidly after the 12th week of implantation when grafts in In summary, transanastomotic endothelialization is
most of the studies were fully endothelialized and thus can at least 7.5 times more pronounced in any animal model
not be used for the assessment of further transanastomotic than in man. Within the experimental set-up, however, the
endothelial cell outgrowth. senescent primate model of the chacma baboon at least
During the first 3 months of implantation promises to eventually reveal certain biological principles
transanastomotic endothelialization in dogs consecutively which make the human healing pattern of prosthetic grafts
covers a distance of approximately 2 cm in ePTFE so unique.
grafts1-3,10,21,86,98-102 and more than 3 cm in Dacron pros-
theses.2,60,62-64,66,72,76 These ingrowth distances also apply to Differences Between Grafts
the second most commonly used animal model, the yellow Since the endothelial margin represents the frontier
baboon (Papio cynocephalus).9,19,32,35,52,55,91 Taking into ac- of transanastomotically ingrowing tissue,19 it may well be a
count that the ingrowth margin of endothelium is often very key player in a synergistic interaction with sub-fibrinous tis-
irregular with endothelial tongues reaching far into the graft, sue. If one compares the two low porosity prostheses (30 ∝m
a minimal length of 15 cm in all animal models therefore ePTFE and woven Dacron), endothelial outgrowth seems
seems a reasonable choice for future 3 month studies. to be twice as high on ePTFE1-3,10,21,78,86,98-102 than on
In spite of this general guideline, there is still a dis- Dacron.40,43 This difference is not too surprising, because
tinct difference between the respective animal models with the mighty inner fibrinous capsule of Dacron seems to have
regard to transanastomotic ingrowth behavior. However, features of an ingrowth barrier for transmural capillar-
since the in vitro proliferative capacity of endothelial cells ies.43,44,48,59,60,71,72 One can imagine that the same biological
from humans and experimental animals hardly differs,103 reasons which inhibit capillary ingrowth from outside may
the distinctly prostrate transanastomotic outgrowth poten- inhibit endothelial outgrowth from the anastomosis. How-
tial of human endothelial cells must be a consequence of ever, although this fibrin matrix seems to be similar in high
the complex overall situation of the human model rather porosity2,35,44,60-66,91 and low porosity grafts,45,58 it allows
than a principal mitotic sluggishness of human endothelial better transanastomotic endothelial ingrowth under high po-
cells. Therefore, one should not downplay the importance rosity 40,46,47,60,68,72,81 than low porosity condi-
of differences between animal models. If one animal model tions.1,2,21,40,43,86,98-102 Since this observation only relates to
comes significantly closer to the human situation than an- grafts in which the transmural tissue ingrowth did not
other, it may well hold the key to understanding the biologi- traverse the fibrinous capsule and the time would have been
cal reasons for the human failure of graft healing. If too short for fallout endothelialization, the endothelium can
transanastomotic ingrowth values of different species on be assumed to be of transanastomotic origin. If however, a
standard ePTFE grafts are related to those of the dog, the main difference for transanastomotic endothelial ingrowth
coverage which is reached in humans after 56 weeks occurs on an otherwise not ideal surface seems to be a well devel-
in dogs after 3.5 weeks,1,2,21,98,86,100 in the yellow baboon af- oped tissue layer underneath the fibrin capsule, it is reason-
ter 5.6 weeks and in the chacma baboon after 7.6 weeks. Thus, able to speculate that this distant tissue layer may somehow
the transanastomotic outgrowth is 2.1 times slower in the affect surface endothelialization.
chacma baboon than in the dog. The difference between the
two primate species may well be due to the fact that the Porosity
chacma baboon represents a senescent model with a body Porosity was long known to be a determining factor
mass of up to 40 kg, whereas the yellow baboon is a juvenile behind the fate of prosthetic vascular grafts. Initial attempts
model which is on average 2 years old and weighs 10 kg. In to replace arteries with solid tubes of synthetic material soon
this context, it is interesting to note that the difference be- demonstrated that porosity is a prerequisite for graft pa-
tween the two kinds of baboons is much more pronounced tency.45,49 Subsequently, pioneers of prosthetic vascular
in transmural than transanastomotic ingrowth. Transmu- grafts60,71,74 unsuccessfully tried to achieve complete graft
ral capillarization leads to surface endothelialization of high healing through porosity. However, only with the advent of
porosity ePTFE grafts after 2 weeks in the yellow baboon, highly porous ePTFE grafts did it become apparent that com-
whereas perigraft tissue has usually not even grown into the plete surface healing of arterial prostheses was theoretically
outer two-thirds of these prostheses in the chacma baboon possible beyond a certain porosity, and this only in a very
(personal observation). juvenile animal model. In the endothelium-centered 1980s,
Other animal models like rats, calves104 and pigs105 are excitement arose primarily from the fact that higher poros-
either too different or not sufficiently documented. How- ity may allow surface endothelialization.30,32 However, if tis-
ever, pigs are usually large enough for vascular implants at a sue engineering of prosthetic vascular grafts ever intends to
young age, but dog puppies are too small. Therefore, dogs truly emulate functional arteries, the tandem of deep-seated
The Lack of Healing in Conventional Vascular Grafts 19
suspicion of the presence of smooth muscle cells and the water that passes through the material per unit time, assum-
narrow focus on endothelial cells will need to give way to a ing that the thickness of the membrane remains constant.
more comprehensive mode of tissue ingrowth. Neverthe- The resultant measurement has been misleadingly called
less, our current biological understanding validates the view “water porosity” by surgeons and manufacturers alike.
that the ability of a prosthetic graft to allow capillary in-
growth still holds the key to today’s grander picture. Porosity
Researchers were long puzzled as to why interstitial Porosity, in contrast, refers to the void spaces (pores)
graft spaces of particular prostheses were sufficient to allow within the boundaries of a solid material, compared to its
macrophages and other inflammatory cells to immigrate, total volume. Although one could argue that the voids within
whereas connective tissue cells of similar dimensions were a material will allow water to flow through them, numerous
often conspicuously absent.2,18,22,24 Today we understand pores may well be cul-de-sacs and thus fail to provide an
that certain of these connective tissue cells, like smooth open corridor from one side of the graft to the other. More-
muscle cells, mainly follow the ingrowing endothelial over, vascular prostheses are not designed to be rigid struc-
cells.106,107 Therefore, one important determining dimen- tures. They are compressible for ease of handling and can be
sion for complete graft healing is that which mechanically readily deformed by radial and longitudinal tensions as well
allows capillaries to sprout into the meshwork of a synthetic as by internal pressures, even in the relatively noncompliant
graft and still leave some space for accompanying cells. Since contemporary grafts. Such stresses not only change the di-
the average diameter of a capillary is ~10 ∝m,108-110 it would mensions of the prostheses, but also lead to a redistribution
seem as if the minimum area required for ingrowth of cap- of the yarns and fibers within the material. Naturally, the
illaries is at least 20-80 ∝m.2 The diameter of a functional change in textile structure by both circulatory stress and
vessel, i.e., an endothelium and at least one layer of smooth compression by ingrowing tissue itself affects the distribu-
muscle cells, is approximately 23.06 ± 13.11 ∝m in diameter tion, size and tortuosity of the channels that run from one
(personal observations). side of the graft to the other. Since the limiting factor for
The attempt to determine porosity requirements for tissue ingrowth is the bottleneck of the narrowest part of a
graft healing is no doubt complicated by material specific transmural space, it is further important to look at intersti-
characteristics and structural uniqueness. The complex three tial spaces throughout their three dimensional course.
dimensional structure of contemporary grafts results in a Unfortunately, hardly any information is available re-
wide range of voids between the synthetic surfaces, which garding the dimensions of porous structures in vascular
hardly allows a precise definition of ingrowth spaces. For grafts. In order to get a better insight into this critical
manufacturers, a way out of this dilemma was the introduc- parameter for tissue incorporation, we have evaluated in-
tion of water permeability as a means of defining porosity. terstitial space dimensions in a few typical contemporary
With this method, materials could be separated on the basis grafts, both before and after implantation. Moreover, we have
of low and high porosity. For example, materials such as investigated these prostheses regarding their true maximum
Dacron were distinguished not only on the basis of being interstitial ingrowth spaces and their resulting theoretical
knitted and woven, but further characterized as being of high ability to accommodate transmural microvessels.
porosity (1500 to 4000 ml/cm 2/min) or low porosity Microvessels were decided to be the dimension against which
(200-1000 ml/cm2/min).111 The same principle applied to measurements were compared, based on their central role
ePTFE on the basis of distances between the nodes of the in a variety of healing aspects from surface endothelialization
expanded material. However, although these values served to neo-media formation.
as useful parameters in characterizing the materials, they
were not helpful with regard to healing. The poorly defined Theoretical Considerations for Healing
space dimensions made it impossible to come to a conclu- of Vascular Grafts
sive answer concerning the role porosity plays in the mitiga- Although the process of expanding teflon material to
tion of tissue ingrowth into prosthetic vascular grafts. If one the typical nodes-and-fiber structure of ePTFE grafts
attempts to gain a better understanding of the relationship (Fig. 1.22A) allows for a wide range of internodal distances,
of porosity and healing, one needs to make a clear distinc- the resulting changes in ingrowth spaces are moderate. With
tion between porosity and permeability in order to avoid a well-defined anchor points and a rigid material like teflon,
confusion of terminology. It is important to understand that the distensibility of the fibrils is limited (Fig. 1.22B), thus
these terms are not synonymous, because a highly porous making disproportional increases in internodal distance
material may have a low permeability and vice versa. necessary to achieve increases in fibril length and hence an
increase of ingrowth dimensions of a few micrometers. To
Permeability try to correlate true ingrowth spaces with internodal dis-
The permeability of a material can be defined as its tances as defined by the manufacturers of ePTFE grafts is
ability to allow other substances (either gaseous, liquid or insufficient, therefore, if not combined with the assessment
solid) to pass through its pores or interstices. The custom- of fibril dimensions. Furthermore, in order to accurately de-
ary method for measuring permeability is using water and termine ingrowth spaces, one needs to assess these struc-
measuring the area and the applied hydrostatic pressure gra- tures throughout the entire thickness of the graft wall. In a
dient across a porous membrane. The permeability value is similar way, one needs to measure more than just bundle
therefore directly proportional to the measured volume of densities and fiber diameters in knitted (Fig. 1.24) and woven
20 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 1.22. Scanning electron micro-
graph of a 30 ∝ m expanded
polytetrafluoroethylene (ePTFE) graft
(Impra Inc., USA) showing the typi-
cal nodes-and-fibril structure. x750,
(top) inner surface and (bottom) outer
surface.
Dacron materials (Fig. 1.25) to obtain an accurate measure- grafts was based on the combined image analyses of histo-
ment of cell ingrowth spaces. These assessments are further logical cross-sections and scanning electron microscopy
complicated by the introduction of externally wrapped PTFE (SEM) of surface structures (see figure legends). Based on
grafts (Fig. 1.23) and veloured Dacron surfaces (Fig. 1.26).60 these measurements (Table 1.1), we attempted to reconstruct
These coverings are intended to prevent the grafts from an- the maximum ingrowth space dimensions throughout the
eurysmal dilatations and to increase the tissue bond between graft wall and draw conclusions about whether the graft
graft and perigraft tissues, with a subsequent better healing materials would sufficiently allow transmural ingrowth for
response.111 microvessels invading from the adventitia. In order to cal-
Taking all these factors into account, our approach culate ingrowth spaces, we measured a number of param-
toward the assessment of true ingrowth spaces in commer- eters related to the material characteristics (Table 1.1).
cially obtained both low porosity (< 30 ∝m) and high po- Scanning electron microscopic examination of the two
rosity (60 ∝m) ePTFE, Dacron velour and knitted Dacron different porosities of ePTFE used in this study gave a good
The Lack of Healing in Conventional Vascular Grafts 21
Fig. 1.23. Scanning electron micro-
graph of a 30 ∝ m expanded
polytetrafluoroethylene (ePTFE) graft
(Impra Inc., USA) showing external
circumferential wrap. The nodes-and-
fibril structures can be seen through
the wrap. x750, outer surface.
Fig. 1.24. Scanning electron micro-
graph of a knitted Dacron prosthesis
(Vascutek; water porosity = 1350 ml/
cm2/min), showing reasonably tightly
knitted bundles of fibers, x75.
indication of surface dimensions with respect to internodal the node-to-fibril structure and dimensions. Based on this,
distances, internodal spaces, mean fibril lengths and mean we measured internodal fibril lengths in 6 contemporary
interfibril space (Table 1.1). Internodal distance was mea- 30 ∝ m ePTFE grafts with varying internal diameters
sured from the center of one node to the center of another, (Table 1.2). Only the inner surface was measured, as all the
whereas internodal space width was defined as the actual grafts contained dense outer circumferential wraps some-
space between the margins of two adjacent nodes. No sig- times additionally reinforced with rings. The results showed
nificant difference was found between the inside and out- a reasonably consistent fibril length irrespective of internal
side surfaces for each respective graft. However, the intern- diameter (Table 1.2). The values of 24.28 ± 3.48 ∝m and
odal distance stated by the manufacturers did not precisely 20.69 ± 2.35 ∝m for the 6 and 10 mm I.D. grafts (W.L. Gore
match the internodal distance we measured. and Associates, Flagstaff, Ariz.) respectively, are in agreement
It could be argued that the internal diameter of ePTFE with earlier studies.1 However, although there were hardly
grafts may influence the manufacturing process and thus any differences in internodal distance between grafts of
22 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 1.25. Scanning electron micro-
graph of a woven Dacron prosthesis
(DeBakey; water porosity = 232 ml/
cm 2 /min), showing tightly woven,
well-defined bundles of fibers, x75.
Fig. 1.26. Scanning electron micro-
graph of the double veloured Dacron
prosthesis (Cooley; water porosity =
1660 ml/cm2/min), showing a com-
pletely wide open, random array of fi-
bers. Inset: Dacron felt (USCI) used in
our study, x75.
different inner diameter from the same manufacturer, there sumption of a worst scenario was that, beginning at the ad-
were distinct differences in internodal distance between com- ventitial surface, each successive layer of fibrils lies in the
parable grafts of different manufacturers. In 30 ∝m ePTFE center of the preceding layer, thereby creating the greatest
grafts from Impra (Table 1.1), a mean inner fibril length of possible obstruction. Therefore, this approach provides the
17.79 ± 5.62 ∝m was opposed by 23.57 ± 6.21 ∝m measured narrowest possible channel spaces for continual tissue in-
in W.L. Gore products (Table 1.2). growth from outside to inside.
In order to calculate the minimum continual trans- Figures 1.27 and 1.28 illustrate the theoretical com-
mural ingrowth channels through the wall of both ePTFE parison between the minimum continual transmural in-
grafts (Figs. 1.27 and 1.28), a mathematical approach was growth channel and the maximum available ingrowth space
chosen relating three dimensional structures of nodes and of a given layer throughout the walls of the 30 and 60 ∝m
fibers to factors such as the obstruction caused by intern- PTFE grafts. The reason for the discrepancy between the two
odal fibrils from one layer to the next. The underlying as- curves in the 30 ∝m PTFE (Fig. 1.27) is the fact that the
The Lack of Healing in Conventional Vascular Grafts 23
Table 1.1. Material characteristics of 5 mm internal diameter ePTFE grafts (Impra, Inc.)
Internodal Mean Mean Internodal Mean Fibril Lengths Mean
Distance as Internodal Space Width (mm) Interfibril
stated by the Distance (mm) Space
manufacturer (peak to (mm)
(mm)
30:INSIDE 24.12 ± 6.47 16.88 ± 5.49 17.79 ± 5.62 ND*
95% CI: 15.78-19.80 95% CI: 14.92-18.84
30:OUTSIDE 27.27 ± 13.19 14.49 ± 6.21 15.14 ± 6.12 5.11 ± 2.39
95% CI: 12.27-16.72 95% CI: 12.95-17.33
60:INSIDE 53.01 ± 20.44 42.41 ± 15.39 45.72 ± 16.22 ND*
95% CI: 37.23-47.58 95% CI: 40.27-51.17
60:OUTSIDE 47.94 ± 9.90 28.14 ± 6.36 30.80 ± 7.03 4.53 ± 0.76
95% CI: 25.99-30.28 95% CI: 28.43-33.16
Material characteristics of both low (30 ∝m) and high (60 ∝m) porosity 5 ∝m internal diameter (ID) ePTFE grafts (Impra, Inc.) as measured by
SEM anaylses. Counts were done over 5 random fields using a calibrated NIH image analyses system. Results are presented as mean ±SD
with 95% confidence intervals calculated around each mean vaule. n = 60. ND* = Not determined beacause cell ingrowth occurs through
the fibrils from the outside surface only.
Table 1.2. Comparative values of contemporary 30∝m ePTFE grafts (W.L. Gore and Associates)
30mm ePTFE Internal Diameter Mean Internodal Fibril Length ± SD (mm)
(mm) (Inner Surface)
3 21.12±5.56
5 23.57±6.21
6 24.28±3.48
10 20.69±2.35
15 32.09±5.10
22 26.17±2.43
Counts were done on 5 random fields on the inner surface of all the grafts using calibrated NIH image analysis software. The outer surface of
all the grafts was covered with a circumferentially dense wrap. The 5 and 15mm ID grafts were further reinforced with external rings. Results
are presented as mean±SD. A clear discrepancy exists between the 5mm ID internodal fibril lengths of the two manufactured grafts.
Fig. 1.27. Comparison of mean in-
growth space and the theoretically pre-
dicted minimum continual transmu-
ral ingrowth channels for 30 ∝ m
ePTFE. The ingrowth spaces (■) were
determined by image analysis on his-
tological cross-sections (n = 2) which
were subdivided into 10 consecutive
layers extending from the lumen to the
adventitia. In each layer, internodal
measurements were done in 8 random
fields using a Leica Q500MC image
analyzing system. Mean ingrowth
spaces are expressed as graphic points
for each measured layer (∝m). Vertical
lines denote ±1 S.D. For the theoreti-
cally calculated continual transmural
ingrowth channels (–∃–), internodal
spaces, fibril lengths and mean
interfibril spaces were measured using scanning electron microscopy. The results were plotted on a logarithmic scale representing
full wall thickness (2000 layers). Vertical lines denote ±1 S.D.
24 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 1.28. Comparison of mean in-
growth space and the theoretical pre-
diction of the minimum continual
transmural ingrowth channel for
60 ∝m ePTFE. The identical protocol
was followed as in Figure 1.27.
maximum space (Fig. 1.27, line marked with solid squares) in contrast, would be permitted by 100% of the transmural
has been worked out on the basis of the maximum ingrowth spaces.
space available between two fibrils without taking the inter-
action of the neighboring fibrils into account. These spaces Assessment of Ingrowth Spaces in Dacron Grafts
are therefore ingrowth spaces in isolation. If one compares If the task of assessing interstitial graft spaces is con-
the maximum ingrowth space available through the graft sidered difficult in ePTFE, it gets even more challenging when
(assuming that each histological layer equals 200 true layers Dacron prostheses are concerned. In the era prior to pro-
as previously established by SEM, resulting in a total of 2000 tein pretreatment of vascular prostheses, porosity was di-
layers throughout the entire wall thickness) to the theoreti- rectly proportional to bleeding. Therefore, textured grafts
cally available transmural ingrowth channel (Fig. 1.27, lined made of polyester braids aimed at providing surgeons with
marked by open diamonds), it becomes obvious that the an instantly hemostatic alternative. In contrast to ePTFE,
available space is in fact much less than it appears initially however, high or low porosity is not primarily achieved
on the basis of isolated layers of nodes and fibrils. Interest- through a variation of the same manufacturing process, but
ingly, the minimal ingrowth channel from outside for 30 ∝m rather through the different procedures of knitting and weav-
PTFE grafts progressively diminishes in size over the first 10 ing. Moreover, an additional variable is introduced by the
layers, as a result of the obstruction caused by the successive needle density used in the knitting process and by optional
layers of fibrils. Thereafter, the values remain consistent be- combination with a velour surface, which further influences
fore increasing again till layer 200. Throughout the rest of the size of the open spaces between the braids.
the graft, the predicted channel size would not accommo-
date capillaries, although the ingrowth space of each iso- Woven Dacron
lated layer of nodes and fibrils would remain theoretically Commercially, woven fabrics consist of two sets of
sufficient for capillary ingrowth. This calculation corre- yarns, the warp and the weft, which are interlaced at right
sponds with the histological observation that only a few cap- angles to each other (Fig. 1.25). Woven material is often pre-
illaries are found in the wall of nonwrapped 30 ∝m PTFE ferred by surgeons, as it is known for its high bursting
grafts, and those few are restricted to the outer third. strengths, low permeability to liquids, minimal tendency to
In 60 ∝m PTFE grafts (Fig. 1.28), the minimum di- deform under stress and less proneness to kinking. How-
mension of transmural ingrowth channels from outside also ever, the woven structure also has poor compliance, limited
progressively diminishes in size in the first 20 layers, after elongation, a tendency to fray and, above all, a very low po-
which the values become consistent throughout the rest of rosity. In spite of this low porosity, tissue ingrowth into the
the graft wall till layer 200. From then onwards the maxi- tight interstitial spaces of the fabric is occasionally ob-
mum diameter of a continual transmural ingrowth channel served.40,57
decreases again as a consequence of the narrowing of the However, since the available space does not appear to
internodal space. Despite the fact that the calculated chan- allow for cell infiltration in the manufactured configuration,
nel size represents the “worst” scenario in terms of ingrowth one must assume that cellular ingrowth is a consequence of
dimensions, as it nears the lumen it still provides sufficient a rearrangement of the relatively loosely lying interstitial fi-
space for ingrowing arterioles within the 5% confidence in- bers. Theoretically, therefore, the number of fibers, fiber di-
terval. Therefore, complete transmural vascularization would ameter and bundle area could be measured to obtain a rea-
still be possible in these grafts. Transmural capillarization, sonable measurement of ingrowth spaces. This information
The Lack of Healing in Conventional Vascular Grafts 25
would then be used to determine a packing factor for the Dacron. We therefore analyzed explanted histological sec-
material. The main shortfall of this approach, however, is tions for knitted as well as pure velour prostheses (Fig. 1.26)
that although the native graft material seems flexible and through ten layered areas from the adventitial surface to the
reorganization of fibers seems theoretically feasible, the in- lumen. The details of the measurements are described in
terstices of the graft upon implantation are soon filled with the figure legends. The commercial knitted graft (Vascutek)
a fibrin matrix which polymerizes into a fairly rigid struc- used in this study had a water porosity of 1350 ml/cm2/min,
ture, thus embedding the fibers and bundles and allowing falling into the lower range of porosities for knitted materi-
very little room for movement. It seems likely that the pro- als.15 On the basis of interfiber measurements through the
teolytic process required for this rearrangement needs a criti- graft wall, a graphical representation showing the mean val-
cal tissue presence with its associated degrading capacity, ues for maximum ingrowth spaces could be obtained
which in turn would need a higher initial porosity. This may (Fig. 1.29). In spite of the fact that this material lies in the
explain why single cells such as macrophages and fibroblasts lower range of porosity within knitted grafts, it was still sur-
are found near the surface of woven prostheses, but only prising to see how narrow the maximum ingrowth spaces
very occasionally capillaries.40,57 through the knitted graft were. When one compares the per-
centage distribution of spaces theoretically permissive for
Knitted Dacron capillaries and/or arterioles (Fig. 1.29B,C), however, the
According to the manufacturers,112 knitted construc- mean size of spaces within the first third of the graft wall
tions contain a set of yarns which are interlooped around (14.44 ± 3.74 ∝m, layers 7-10) would accommodate capil-
each other as opposed to interlaced (Fig. 1.24). The specific laries. In addition, albeit at a low percentage (5.16 ± 0.56%),
type and the compliance behavior of knitted fabrics depends the mean ingrowth space of 24.64 ± 1.75 ∝m would also al-
on the direction in which the yarns are interlooped. If the low a few arterioles to surpass this zone (Fig. 1.29C). To-
yarns lie predominantly in the lengthwise direction then the wards the middle of the graft (layers 4-6) the restriction for
fabric is “warp knit” whereas if they lie in the transverse di- capillaries becomes less pronounced (13.92 ± 2.68 ∝m), de-
rection, the fabric is “weft knit”. A significant difference be- fying the common sense expectation that cells would en-
tween these two types of knit is that weft knit fabrics will counter more tightly packed bundles in this midzone of the
unravel, whereas warp knits will not. This is an important prosthesis. The luminal third of the graft (layers 1-3) has
consideration for the surgeon, as the grafts are often cut at consistently narrow spacing, allowing capillary ingrowth
preimplantation for perfect abutting to the anastomoses. Un- only in 30.4 ± 0.38% with a mean ingrowth space of
doubtedly, once implanted, the difference is negligible. 12.34 ± 2.50 ∝m. None of the spaces in this zone allow arte-
Due to the difference in structure of Dacron grafts as riole ingrowth (Fig. 1.29C).
opposed to PTFE, a slightly different approach was adopted
to calculate ingrowth spaces. Surface measurements made Velour Surfaces
by SEM, and the histological assessment of wall thickness The term “velour” in a textile context refers to a thick-
and ingrowth space dimensions, are the same as in ePTFE. bodied fabric, either woven, knitted or nonwoven, that has a
However, while fibrils cause the main spatial limitation in smooth, soft surface by virtue of additional yarns. All com-
PTFE, fiber orientation and the arrangement of fiber bundles mercial velours, however, are knitted designs. They may be
throughout the graft thickness limit the spaces in knitted distinguished from the earlier knitted prostheses by their
Fig. 1.29. Graphic representation
of maximum ingrowth spaces for
knitted Dacron. Histological
cross-sections of explanted
samples were subdivided into 10
consecutive layers extending over
the full thickness of the wall from
lumen to adventitia. Interfiber
spaces were measured in 8 ran-
domly selected fields in each zone.
Results in (A) are expressed as
mean ± SD. A frequency distribu-
tion histogram was applied to the
results from (A) to obtain percent-
ages of ingrowth spaces of > 10 ∝m
and > 23 ∝m (B, C).
26 Tissue Engineering of Prosthetic Vascular Grafts
thicker fabric and greater porosity, as well as lower fabric for most of the contemporary vascular prostheses. It becomes
density and packing factor. To assess ingrowth space within clear that, although surface measurements of graft materi-
velour graft types has been very difficult, and most research- als give a good indication of ingrowth characteristics, addi-
ers have relied on methods such as water porosity. However, tional information such as interfiber distances and theoreti-
a few authors113 have attempted to measure its true dimen- cally predicted channel spaces throughout the graft wall are
sions by looking at pile height and the amount of piles in critical for assessing the degree of pure mechanical restric-
the veloured knit. The impetus for using velours stemmed tions for tissue ingrowth into prosthetic vascular grafts.
from a conviction that a rougher and more porous surface
both externally and internally would facilitate the develop- Fibrin
ment of neointimal and perigraft tissue. Prior to implantation, conventional nonpretreated
In order to assess the qualities of pure velour struc- grafts represent simply synthetic scaffolds. Therefore, all bio-
tures, we manufactured velour tubes made of Dacron ve- logical components which eventually fill the interstices of
lour felt (Fig. 1.26, inset). In sharp contrast to the seemingly these scaffolds are host derived. Within this process of host
restrictive interfiber spaces of the knitted material, the incorporation, proteinaceous ingrowth matrices naturally
Dacron velour showed mean ingrowth spaces ranging from precede infiltration with cellular components. Since the
30.02 ± 2.95 ∝m at the adventitia, to 36.88 ± 20.06 ∝m at the implantation of a vascular prostheses inflicts a surgical
lumen, with an overall mean ingrowth space of wound, this proteinaceous matrix is fibrinous. In low po-
27.97 ± 4.80 ∝m (Fig. 1.30A). These mean ingrowth spaces, rosity grafts, the surrounding fibrin coagulum derives en-
as in the case of the knitted grafts, were assessed on the basis tirely from iatrogenically injured vessels. The cut ends of
of histological measurements of interfiber distances through these vessels constrict within a few minutes and get sealed
ten consecutive areas of explanted samples (see figure leg- with aggregating blood platelets. As a result, the hematoma
end). Interestingly, although the mean space seems sufficient around the graft usually remains moderate. Within a few
for arteriole ingrowth, the wall profile (Fig. 1.30A) shows minutes tissue thromboplastin generates thrombin, which,
that the space becomes narrowed towards the central part together with platelet factor 3 (PF 3) released by the few
of the graft and may have a restrictive effect on ingrowth. entrapped platelets, leads to the transformation of fibrino-
The reason for this central restriction is the presence of nu- gen into fibrin. Particularly in nonpretreated porous grafts,
merous bundles in this area causing an overall smaller in- blood may also extravasate from the graft lumen to the out-
terfiber space. Despite this restriction, the calculated maxi- side. In this case the activation of the clotting cascade oc-
mum spaces would still allow the ingrowth of capillaries and curs through contact of factors XII, prekallikrein, XI and
arterioles at all levels. Percentage distribution profiles for high molecular weight kinogen114 and, again, through re-
both the capillaries and arterioles (Fig. 1.30B,C) show a simi- leased platelet PF 3. Although blood does not seep through
lar distribution pattern as the mean ingrowth space profile the wall of less porous grafts, plasma still clots within the
(Fig. 1.30A), with the majority of spaces of more than 23 ∝m synthetic meshwork through a similar mechanism. There-
(Fig. 1.30C) being in the outer and inner one-third of the fore, one can assume that the entire fibrinous matrix ini-
graft. tially surrounding the graft and filling its interstices is rep-
Taking all these results into account, it seems that the resentative of typical wound fibrin, which distinctly favors
dimensions of graft porosity may have been overestimated tissue ingrowth.
Fig. 1.30. Graphic repre-
sentation of ingrowth
spaces for Dacron velour.
An identical protocol was
followed as for Figure 1.29.
Results are expressed as
mean ± SD.
The Lack of Healing in Conventional Vascular Grafts 27
Fig. 1.31. Hypothetical sequence of cel-
lular ingrowth events in native fibrin.
The normal hemostatic process of clot
formation leads to a relatively low plate-
let number, fibrinogen and TGF-! con-
tent. The resulting low density of thick
fibrin fibers together with mild hypoxia
has a pro-angiogenic effect as it causes
an immediate upregulation within the
endothelial cell of integrins #v!3 and
#v!5, which facilitate adhesion to the
extracellular matrix, and the release of
proliferative and pro-migratory
cytokines such as vascular endothelial
growth factor (VEGF), platelet-derived
growth factor (PDGF) and basic fibro-
blast growth factor (bFGF). The
upregulation of the endothelial cell-spe-
cific integrins causes the release of serine
proteases, urokinase and tissue plasmi-
nogen activator (u/tPA). These proteases
in turn convert surrounding plasminogen to plasmin, which acts on the neighboring extracellular matrix to expose attachment
sites for the cell-specific integrins. This whole process eventually leads to revascularization and the maintenance of vessel integrity
at the wound site.
In contrast to this loose initial fibrin matrix, an en- the responsiveness of cells to PDGF.120 This is an important
tirely different fibrin clot may be continually generated on aspect of the triggering of angiogenesis at such an early stage.
the blood surface. While the interstitial and external clot- Furthermore, TGF-!—which is a chemotactically highly po-
ting environment is characterized by a small blood pool with tent cytokine for macrophages—is potentially active in fi-
limited availability of coagulation factors and platelets, brin,121 whereas it is inactivated in collagen matrices by
undiminishing amounts of fibrinogen and platelets are decorin.120 The relatively low platelet concentration in
present at the blood-contacting surface of the prosthesis. wound fibrin makes it likely that TGF-! augments macro-
Therefore, this consecutively increasing inner layer of throm- phage infiltration without inhibiting vessel ingrowth. This
bus obviously differs in both its mode of generation and its can be explained by the concentration dependence of TGF-!,
composition. which is only inhibitory for angiogenesis at high concentra-
tions 108 but chemotactic for macrophages in trace
Outer Fibrin Matrix amounts.122 This early fibrin matrix also facilitates protease
Since tissue ingrowth into prosthetic grafts occurs pri- upregulation and the resulting degradation of the fibrin clot
marily from outside, the outer fibrin clot surrounding the to enable tissue ingrowth. The initial events in this process
grafts serves as a provisional matrix for cell migration from are again regulated through an integrin-ligand mechanism.
the adjacent tissue. However, in order to understand pos- Ingrowing endothelial cells for instance, induce their pro-
sible adverse qualities of the inner fibrin matrix, one needs tease secretion through the binding of #5!1 to fibronectin
to understand the principal interactions occurring between and fibronectin fragments in the fibrin matrix.117,120 The
cells and the more natural wound-type fibrin on the out- relatively low number of platelets contributing to this initial
side of the graft. This blood clot filling the adventitial wound clot also makes it likely that the concentration of platelet-
space consists mainly of crosslinked fibrin, plasma derived thrombospondin (TSP1)—a potent inhibitor of pro-
fibronectin, vitronectin and thrombospondin (TSP),115 con- teases145—does not result in a mitigation of tissue ingrowth.
taining low levels of platelet derived cytokines like TGF-!
and PDGF. This fibronectin-rich fibrin matrix offers bind- Inner Fibrin Matrix
ing sites for integrins of ingrowing cells. For macrophages it Although the mechanism of ingrowth inhibition into
is primarily #5!1,116 whereas it is #v!3 for new blood ves- prosthetic vascular grafts may well be a multifactorial event,
sels.117 The integrin #v!5 has also been implicated in the for- it is conspicuous that the most impenetrable part of the graft
mation of new blood vessels.118 However, whether invasion wall in humans seems to be the inner fibrinous capsule. This
of endothelial cells into a fibrin matrix is #v!5-dependent phenomenon has been reported for more than 20 years in
remains to be established, as the role of this integrin points porous Dacron grafts.43,44,48,59,60,71,72,74,75 We have also ob-
to it interacting selectively with a vitronectin-rich matrix.119 served it in 60 ∝m ePTFE prostheses in the chacma baboon
The interaction between integrins and the matrix also where the impenetrability of the inner fibrin layer even ap-
influences other regulatory mechanisms. For instance, a plies to inflammatory cells. After a few weeks of implanta-
“stressed” fibrin matrix as a result of contraction increases tion, we found the fibrin coverage of the blood surface to be
28 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 1.32. Hypothetical sequence of
events in the inner fibrin capsule on
prosthetic grafts. Fibrin is continually de- Platelet Release
posited due to platelet and macrophage Reaction
accumulation and their subsequent de-
granulation. Platelets and red blood cells
become entrapped in a constantly in-
creasing, compacted fibrin layer. De-
granulation of the #-granules of the
platelets causes a microenvironmental Inactive TGF!
active TGF! Mo Tissue Factor
increase of inactive transformting
growth factor-beta which becomes se- PLASMIN
questered into the extracellular matrix in
a latent form. Endothelial cells at the PLASMINOGEN
outer surface of the graft material Migration
UPA/tPA # ! # !
upregulate u/tPA through an integrin- v 3 5 1 Proliferation
extracellular matrix mediated effect. Differentiation
These serine proteases convert blood-
borne plasminogen to plasmin, which in
turn activates the latent TGF-!. TGF-!
acts on the endothelial cells to initiate dif-
ferentiation with a concomitant decrease in migration and proliferation. This mechanism may therefore cause a premature cessa-
tion of angiogenesis and a reduction in transmural vascularization. Additionally, blood-borne macrophages express tissue factor
following adherence to biomaterials. This transmembrane protein initiates further fibrin deposition. Macrophages in the depth
may shed vesicles containing tissue factor activity which could further facilitate fibrin deposition on the surface.
almost completely acellular (Fig. 1.10). It is therefore tempt- affect the structure and/or composition of fibrin in such a
ing to ask whether it is possible to recognize obvious differ- way that it becomes hostile to tissue ingrowth?
ences in the composition or structure of the inner fibrin
capsule which may be responsible for this distinct ingrowth Fibrin Structure
inhibition. In pursuing this question it seems as if the mode It is a relatively recent discovery that fibrin structure
of fibrin generation on the inside of grafts already provides influences cell growth. At lower fiber density a fibrin matrix
significant clues with regard to growth inhibitory proper- strongly stimulates capillary morphogenesis,126 whereas it
ties. First of all, there is a uniqueness about this particular only stimulates endothelial migration without three dimen-
fibrin clot: Both clotting factors and platelets are not con- sional capillary formation at higher fiber density. On an ul-
sumed from a predefined quantity as is the case in a he- trastructural level, a similar inverse correlation between cell
matoma, but rather replenished on an ongoing basis. On growth and fiber density was found.127 The higher the den-
the one hand, a process of platelet-enrichment through sur- sity of fibrin fibers and the lower the average thickness of
face adherence results in an disproportionate density of these fiber bundles, the lower the penetrability for cells.127
thrombocytes which undergo activation through surface Since in vascular grafts cellular ingrowth seems more inhib-
contact and shear stress. As a consequence of this augmented ited in the inner than the outer fibrin matrix, it is therefore
platelet release reaction, high concentrations of #-granule of interest whether inside circumstances favor the genera-
products like platelet factor 3, fibrinogen and von Willebrand tion of a denser fibrin matrix with lower fiber bundle thick-
factor, as well as dense granule contents such as calcium, ness. It was recently demonstrated that an increase in fibrino-
continually sustain the coagulation process.123-125 On the gen concentration by a factor of 3 resulted in a decrease in
other hand, the rapid replenishment of blood plasma next fiber bundle thickness and an increase in fiber bundle den-
to the platelet carpet on the graft surface further guarantees sity, also by a factor of 3.127 Considering the high platelet
undiminishing concentrations of fibrinogen and other clot- density on the blood surface of grafts and the undiminishing
ting factors. Thus, one can assume that the fibrin generated pool of clotting factors, one can assume that the fibrinogen
on the inner surface of vascular prostheses has a high con- concentration inside this surface clot is higher than outside,
tent of fibrinogen. Since TGF-! is also liberated from #-gran- thus resulting in a fibrin matrix of higher fiber density and
ules, and its incorporation into fibrin matrices correlates with lower fiber diameter. The suspicion that higher fibrinogen
its preexisting concentration, this fibrinogen rich fibrin contents inhibit cellular ingrowth was further substantiated
matrix can also be expected to be particularly rich in TGF-!. by other studies using fibroblasts128 and endothelial cells.61,129
Based on these considerations, one needs to ask the initial
question differently: Can high fibrinogen concentrations as Cytokine Content
well as other #-granule release products like TGF-! and The high density of platelets adherent to the blood
thrombospondin and dense granule products like calcium surface, as well as their ongoing activation,5,130 suggest a high
The Lack of Healing in Conventional Vascular Grafts 29
concentration of platelet #-granule contents at the time of incorporation into the LAP-LTBP-TGF-! complex may well
coagulation. Amongst other substances, the #-granules of make it immunohistochemically unrecognizable.
platelets contain platelet derived growth factor (PDGF) and
transformting growth factor-beta (TGF-!).5 PDGF is a Inhibition of Matrix Degradation
dimeric growth factor which is mitogenic for endothelial Apart from directly inhibiting ingrowing endothelial
cells and smooth muscle cells. TGF-!, in contrast, is a po- cells, capillarization and tissue ingrowth into the inner fi-
tent inhibitor of cell proliferation, migration and protein- brin layer may also be indirectly inhibited through a par-
ase production in vascular endothelial cells.131 In view of ticularly high PAI-1 concentration in the matrix. Fibrinoly-
the suspected inhibitory effect of the inner fibrin matrix on sis of the fibrin matrix in normal wound healing is prima-
angiogenesis it is reasonable to hypothesize that particularly rily due to plasmin activation from its precursor molecule,
high levels of TGF-! contribute to the mitigated vessel out- plasminogen. In vivo, this conversion to plasmin is medi-
growth. However, the TGF-! content of human platelet #- ated by tissue plasminogen activator (tPA) and urokinase
granules exists exclusively in the !1 isoform, stored in a la- plasminogen activator (uPA), both of which are derived from
tent form in which the precursor peptide (the latency-asso- endothelial cells, macrophages and giant cells. The action of
ciated peptide LAP) remains noncovalently associated with these proteases is inhibited by PAI-1, secreted by endothe-
the mature 25 kDa TGF-!1 dimer.132 In addition, the latent lial cells and platelets. If one assumes that platelets play an
TGF-! complex from platelets has an additional protein, the disproportionate role in fibrin generation on the inside of
latent TGF-! binding protein (LTB-P) which is linked with the graft, one must also reckon with an disproportionately
a disulphide bond to the LAP.133 Since the activation of high level of platelet-derived PAI-1.139
TGF-! only occurs when the mature 25 kDa dimer is re-
leased from the LAP121 and the entire complex is incorpo- Angiogenesis Inhibitors
rated into the fibrin structure, fibrin degradation is a pre- Another platelet-derived protein which may detrimen-
requisite for liberating inhibitory TGF-! concentrations from tally affect capillarization of the inner fibrin capsule is the
the matrix. Coincidentally, the release of the active 25 kDa extracellular matrix molecule thrombospondin (TSP1).
TGF-! dimer needs the same proteases, such as plasmin, TSP1 is a large, multi-functional ECM glycoprotein that can
which are needed to degrade the fibrin matrix.134 There- influence endothelial cell function in vitro140 and angiogen-
fore, a likely explanation for the late inhibition of esis both in vitro and in vivo.141,142 The role of TSP1 in
capillarization through TGF-! would be the following sce- modulating endothelial cell function has been widely inves-
nario: Initially, endothelial cells from the adventitial region tigated. TSP1 was initially identified and characterized as a
of the vascular prosthesis are attracted into its interstices by protein released from the #-granules of platelets upon acti-
the PDGF and VEGF released by platelets and vation by thrombin. TSP1 is now known to also be produced
polymorphnuclear granulocytes.135,136 Since the fibrin ma- by many other cell types such as endothelial cells, fibroblasts,
trix represents a more or less normal wound matrix at this smooth muscle cells and monocytes/macrophages.
stage, cell receptors involved in initial migration and prolif- Thrombospondin occurs in two forms—soluble and
eration, such as the integrins #v!3 and #v!5, are expressed matrix-bound. The effect of the matrix-bound TSP1 is of-
together with the upregulation of serine proteases (uPA, tPA), ten different from that of the soluble form. Soluble TSP1,
enabling the matrix degradation required for migration. for instance, has been demonstrated to inhibit the prolifera-
Moreover, the interstices of the prostheses contain a mix- tion of endothelial cells143 while matrix-bound TSP1 appears
ture of macrophages and foreign body giant cells (FBGC) to be a permissive substrate for endothelial cell prolifera-
which are generators of large amounts of pro-angiogenic tion. Migratory, invading endothelial cells adhere to in-
cytokines such as bFGF (predominantly under hypoxic con- soluble, matrix-bound TSP1 while soluble TSP1 may satu-
ditions), and only minor amounts of inhibitory TGF-!.56 rate surface receptors and produce an anti-adhesive effect.144
Under those favorable circumstances, capillary ingrowth The anti-adhesive activity of soluble TSP1 may interfere with
slowly proceeds towards the inner fibrin layer with its higher the angiogenic process by preventing the cell-to-substrate
fibrinogen and TGF-! content, as well as its denser fibrin or cell-to-cell interactions necessary for endothelial cell mi-
structure. With increasing TGF-! concentrations as well as gration and capillary formation. Furthermore, recent reports
changing fibrin composition, angiogenesis slows down. The have revealed that TSP1 can also function as a protease in-
increase of TGF-! may be further augmented by macro- hibitor.145 These findings suggest that, apart from its direct
phages, which degrade fibrin matrices through the effect on cells, soluble TSP1 may also influence angiogen-
upregulation of endogenous serine proteases without con- esis by affecting ECM turnover and composition. There is
comitant release of plasminogen activator inhibitor (PAI-1). further evidence that TSP1 may inhibit the proteolytic en-
Therefore, matrix degradation by macrophages is much less zymes of the fibrinolytic pathway, including plasmin and
controlled than degradation by endothelial cells, where a urokinase plasminogen activator.146 The modulation of pro-
balanced, focalized proteolysis of the pericellular matrix tease activity therefore provides a potentially crucial role for
through the up or downregulation of proteases and PAI-1 TSP1 in regulating angiogenesis, as it seems to be a factor in
occurs.137,138 Eventually, both fibrin structure and liberated correcting the balance of protease activity, which is essential
and activated TGF-! amounts may be sufficient to bring in- for angiogenesis.
growing capillaries to a complete halt. A main challenge in Another important interaction of TSP1, particularly
substantiating this scenario will be the detection of high with respect to capillarization of vascular prostheses, is its
TGF-! amounts in the inner fibrous capsule, because the
30 Tissue Engineering of Prosthetic Vascular Grafts
interaction with transformting growth factor-beta (TGF-!). pression of tissue factor, a transmembrane protein.151 This
TSP1 has been shown to bind and activate secreted and factor is capable of initiating the extrinsic pathway of co-
ECM-sequested transformting growth factor-b.147 It follows agulation,152 leading to fibrin generation.153 In vitro studies
therefore that the interaction between soluble TSP1 and have shown that, in particular, the adherence to Dacron ini-
TGF-! may prove to be crucial in inhibiting cell growth and tiates more procoagulant activity than adherence to ePTFE.
proliferation. Even more interesting is the fact that TGF-! This seems to relate to the ability of Dacron to adhere more
and TSP1 are both found in the #-granules of platelets,148,149 monocytes than ePTFE,154 rather than to its surface charac-
increasing their presence in equal proportions in an teristics. Hence, the increased deposition of fibrin onto the
environment characterized by a significant platelet release luminal surface of Dacron grafts over time, as opposed to
reaction. Since thrombospondin released by platelets is not ePTFE, may relate to the initially higher adherence of mono-
incorporated into a secreted extracellular matrix, it would cytes to Dacron. In addition, macrophages are known to se-
provide another explanation for the inhibitory effect of a crete both Il-1! and TNF-# following adherence to synthetic
platelet-rich fibrin matrix, since the anti-proliferative effect materials. These cytokines also enhance tissue factor expres-
of TSP1 is determined by the soluble form of this sion,155 leading to a further induction of PCA and further
secreted protein. fibrin deposition. Since Dacron induces higher levels of se-
The one paradox that remains unresolved is the find- cretion of these cytokines than ePTFE, this may further fa-
ing that macrophages in wounds, as well as in other inflam- cilitate fibrin deposition on the Dacron surface. It would
matory settings, actively produce TSP1. The finding that the appear, therefore, that differences in the ability to mediate
macrophages are releasing a molecule that inhibits angio- monocyte adhesion and activation and so differentially
genesis is at first surprising. However, TSP1 release from upregulate procoagulant activity could partially explain the
macrophages may represent a “direct” control of angiogen- observed differences between the luminal fibrin layer of
esis, instead of an “indirect” control of angiogenesis through Dacron and ePTFE. In addition, tissue factor activity has
the protease activation of latent TGF-!. been associated with membrane vesicles that apparently are
shed concomitantly with procoagulant activity.156,157 Mac-
Fibrinogenic Effect of Macrophages rophages in the depth, therefore, could theoretically influ-
One of the most puzzling questions is why the inner ence fibrin deposition on the surface by shedding vesicles
fibrin capsule of Dacron grafts differs from that of ePTFE containing transmembrane tissue factor. The higher num-
grafts although the blood exposure of the material itself is ber of macrophages present in the depth of Dacron grafts as
only a transient initial event. From the first thin layer of fi- opposed to ePTFE grafts would, therefore, augment
brin which covers the synthetic material, one would expect coagulation.
no further difference between the surfaces with regards to In summary, although fibrin forms a pro-angiogenic
the thrombus formation. This, however, is not the case. If matrix and seems to be an “ideal” scaffold for wound heal-
the exposed surfaces themselves do not differ because of the ing, it may also present its Janus face under different cir-
capping effect of the fibrin, what are the most likely differ- cumstances. It seems likely that the unusual situation of
ences which could be responsible for the qualitative and ongoing surface thrombogenicity within the blood stream
quantitative composition of the inner fibrin capsule? One also produces an unusual fibrin matrix which is rich in fi-
such difference may well be the surface structure. Prior to brinogen and ingrowth inhibitory cytokines.
any cell accumulation, the relatively coarse surface of knit-
ted Dacron is covered by a thrombus of more than Macrophages
100 ∝m,44,60 whereas the fine-structured surface of ePTFE Macrophages dominate the tissue reaction against
lies under a thin fibrin coagulum of only 15 ∝m.1,2 However, prosthetic vascular implants more persistently than any other
the fate of crimped surfaces demonstrates that fibrin tends cell type. The deviation from a normal healing process, how-
to equalize surface unevenness. As a consequence, all graft ever, is not their early presence but the tenacity with which
surfaces will eventually have the same smooth fibrin cover- they reside in the graft structure even after years of implan-
age, irrespective of their structure. If there are no differences tation. Their physiological role in early phases of wound
between the initial surface fibrin, and the material itself is healing is opposed by the perseverance of a chronic inflam-
hidden underneath, the most likely explanation for an on- matory reaction to which they significantly contribute in
going difference in thrombogenicity would be the active par- later stages. Although it is difficult to answer the often re-
ticipation of cellular components in the coagulation pro- curring question of whether macrophages are friends or foes
cess. Since the predominant cell type at the time of the most in prosthetic graft healing, it seems that they are both: friends
significant difference in fibrin buildup between the Dacron at the beginning and foes at the end. Their active secretion
and ePTFE are inflammatory cells, and later on specifically of cytokines may act as a key chemotactic and mitogenic
the macrophages, one needs to answer the question of factor for capillary and connective tissue ingrowth in the
whether macrophages may be key players in the formation early phases of wound healing. Their continual presence,
of the inner fibrin capsule. In this context, it is interesting to however, may increasingly derail the delicate chronology of
note that macrophages are able to express procoagulant ac- cytokines required for the successful accomplishment of
tivities (PCA) through their adherence to surfaces.150 Adhe- healing and also result in material degradation. The diffi-
sion of macrophages to biomaterials causes activation and culty in identifying the key events responsible for this ad-
induction of this procoagulant activity by inducing the ex- verse development lies in the multitude of contributing fac-
The Lack of Healing in Conventional Vascular Grafts 31
tors. On the prosthetic side the different materials, struc- Following material contact, one of the most impor-
tures and microstructures of the graft are all capable of sepa- tant determinants in the chronic inflammatory reaction
rately influencing the foreign body reaction against the im- against synthetic vascular grafts is the biomaterial itself.
plant. From the biological standpoint, distinctly different Adherence, spreading and the secretion of inflammatory
environments characterize the luminal and the outer aspect mediators like TNF- #, 167-171 IL-1 ! 154,163,167,172-177 and
of grafts. IL-6167-169,171 have been extensively investigated on all ma-
The dynamics of macrophage infiltration can best be jor biosynthetics. Dacron, for instance, was shown to lead to
studied on ePTFE grafts, with their fairly even wall struc- a higher density of cells with a morphology indicative of
ture. In the first weeks of implantation one can typically activation than ePTFE,154 as well as to higher TNF-#,178
observe a sandwich structure of macrophage populations IL-6168 and Il-1! secretion.168,179 Not only does Dacron in-
in the graft wall with a relatively distinct accumulation of duce a greater response, but it seems to induce an earlier
Ham 56 and CD68 positive cells at the inside and outside response as well, with the IL-1 peak occurring on day 4 on
surface, separated by a cell-free fibrin matrix in be- Dacron but only on day 7 on all other biomaterials.177 Fur-
tween.13,18,54,28,33 This triple layered infiltration pattern thermore, material differences influence not only the ability
clearly proves that recruitment occurs from both sides. At of macrophages to secrete cytokines but also the expression
the luminal surface the extravasation of blood-borne cells of other markers of activation, for example, MHC-II,180
into the porosity of the material occurs simultaneously with Cd11b181 or TGF-!.29 Expanded PTFE and Dacron also dif-
insudation of blood proteins immediately following implan- ferentially induce alteration of expression of subunits of the
182
tation. At the adventitial surface, however, only a few mono- !2 integrins, which mediate both cell-substratum and cell-
cytes may extravasate during the initial bleeding phase prior cell interactions.
to hemostasis. Subsequently, macrophages have to get there However, there is mounting evidence that these effects
by adhering to capillary endothelial cells and transmigrat- are indirect, rather than directly material-mediated. The dif-
ing through basement membranes via the sequential action ferent abilities of the various materials to adsorb plasma
of a number of adhesion molecules on both the monocyte proteins, in particular, appear to be a main determinant be-
(leukocyte specific !2 integrins)158 and the endothelial cell hind the distinct macrophage reaction against certain
surface (ICAMs).159 biomaterials.183-185 On materials such as polyethylene tereph-
It follows, therefore, that macrophages, irrespective of thalate (PET), polytetrafluoroethylene (PTFE) and polyure-
whether they originate from the blood or from transmigra- thanes, the major blood proteins albumin, fibrinogen and
tion into the adventitial tissue, are faced with a similar task IgG are the predominant proteins adsorbed onto the mate-
to begin with, namely, to infiltrate a provisional fibrin ma- rial surface, with other blood proteins adsorbed to a lesser
trix probably not much different from that found in a nor- extent.183-185 Protein adsorption is not dependent, however,
mal wound. During infiltration, macrophage migration is on relative protein concentrations in the blood,184 but is
facilitated by a low level of basic uPA activity. This should determined primarily by the nature of hydrophobic inter-
also be sufficient to liberate and activate platelet derived actions between the material surface and hydrophobic do-
TGF-! from the fibrin matrix160 which was previously in- mains of the protein. Since most proteins have a net nega-
corporated during hemostasis-related fibrinogenesis.139 tive charge, secondary electrostatic interactions are also im-
Since TGF-! upregulates the expression of integrins on the portant in protein-material interactions and are governed
surface of the blood monocytes thereby promoting adhe- by the chemical nature of the material. The overall combi-
sion and migration,161 it acts as a strong chemotactic agent nation of hydrophobic and electrostatic interactions will have
to recruit macrophages to the inflammatory site.122 More- several effects on the process of protein adsorption. Firstly,
over, it also upregulates the uPA activity of macrophages162 different biomaterials adsorb different types and relative con-
to further facilitate migration. Macrophages are also capable centrations of proteins, with more hydrophobic materials
of binding and internalizing fibrin through the Mac-1 re- adsorbing more protein. It has also been shown that the
ceptor, which augments fibrinolysis by a plasmin-indepen- nature of the adsorbed proteins changes over time with vary-
dent mechanism.163 The degradation products of fibrinoly- ing rates of adsorption versus desorption on different ma-
sis, in return, also act as chemoattractants, recruiting still terials.183 Finally, material-dependent conformational
more macrophages into the implant site.164 In addition, traf- changes as a result of these interactions can be detected by
ficking of monocytes to inflammatory sites also involves the differences in elutability of proteins on different chemical
superfamily of chemoattractant cytokines (chemokines ) and structures.186,187 These changes in conformation differen-
their receptors.165 Specifically, the C-C chemokine MCP-1 tially expose hydrophilic domains to the aqueous environ-
has been demonstrated immunohistochemically to be ment, which may or may not contain sites capable of medi-
expressed by macrophages residing at the implant site as early ating cell adhesion. When comparing two hydrophobic sur-
as 48 h postimplantation. 166 This sequence of events faces of different chemical structure, therefore, although a
illustrates how this initial phase of healing is dominated by nonpolar hydrophobic material may adsorb more protein,
self-augmentation of macrophage chemotaxis. Until the the influence that interactions with polar groups on a chemi-
macrophage actually contacts the material surface, the cally different surface will have on the conformation of these
recruitment into the wound fibrin of the prosthetic graft adsorbed proteins will ultimately determine the differences
does not deviate from that taking place in normal in cellular interaction. This may explain why PTFE which,
wound healing. with a surface tension of 18.5 dynes/cm2,188 is certainly more
32 Tissue Engineering of Prosthetic Vascular Grafts
hydrophobic than Dacron (43 dynes/cm2)188 and should thus mitogenic for hypoxic endothelial cells through the release
adsorb more protein,189,190 mediates less cell adhesion. One of the FGFs. This suggests a paracrine mechanism in which
can imagine that the increased polarity of the carboxyl hypoxia stimulates macrophages to release mitogenic fac-
groups of PET compared to the apolar fluorine groups of tors for hypoxically preconditioned endothelial cells, thereby
PTFE affects the conformation of the adsorbed proteins to promoting angiogenesis. Additionally, TNF-#, which is also
the extent that Dacron binds more macrophages than upregulated in macrophages by hypoxia,198 is known to play
PTFE.154 a role in macrophage mediated angiogenesis, presumably
Of the predominant proteins absorbed, fibrinogen has through the activation and recruitment of further mono-
been shown to be a primary mediator of this adhesion as cytes.199 The role of TNF-# in angiogenesis is, however, con-
conformational changes on adsorption expose the P1 epi- troversial and seems to depend on the biological con-
tope which is recognized by the Mac-1 receptor (Cd11b/cd18, text. 200,201 These effects are further potentiated by
191-194
#m!2) of the macrophage. This receptor also binds to upregulation of MIP-1, a potent macrophage chemo-
adsorbed fragments of the complement component C3 attractant, during oxygen deprivation.198 Therefore, mac-
(C3bi),195 but studies in complement—depleted mice indi- rophages residing in the outer half of synthetic grafts to-
cate that this is not a major mechanism of monocyte adher- gether with VEGF-releasing infiltrating neutrophils136 are
ence.191-193 As we have previously noted, the luminal sur- likely to continue to contribute to a pro-angiogenic milieu
face of a vascular prosthesis is exposed to a practically un- at this stage. The low concentration of TGF-! released with
limited pool of fibrinogen derived from the circulating blood migration-associated degradation of the provisional wound
and released by platelets on activation. The insudation of fibrin should not be inhibitory for endothelial cell prolif-
this fibrinogen into the depth of the graft structure facili- eration at this stage.137
tates macrophage adhesion to the material. Thus, the supe- As the organization of the macrophages within the
rior healing pattern of identical implants in the subcutane- graft wall changes and transmural endothelial cell ingrowth
ous position, with limited exposure to circulating or plate- begins, the situation where the outer wall of the graft is domi-
let fibrinogen, may well be partly explained by this nated by single macrophages also slowly begins to change
phenomenon. by the increasing appearance of foreign body giant
Apart from the material effects on adhesion, the struc- cells.13,19,52,55,64 These may persist for the lifetime of the im-
ture of grafts certainly plays another important role with plant. As macrophage adhesion is determined by material
regard to the macrophage response. Increasing diameters of characteristics, so is macrophage fusion to form giant cells
synthetic fibers, for instance, result in increasing numbers dependent on material surface as well as on structure. It has
of adherent, nonspreading macrophages—a morphology been shown that relatively hydrophilic material surfaces in-
indicative of nonactivated cells.196 This effect may be due to duce more fusion than relatively hydrophobic surfaces.202-206
the associated changes in curvature. In addition to micro- Theoretical analyses show that the time-dependent foreign
structure, the overall porosity of the material will also have body giant cell formation is also influenced by the initial
important implications. Ultimately this will be constrained density of adherent macrophages.203,204 Therefore, any sur-
by two factors: high enough porosity to allow tissue ingrowth, face characteristic which influences the number of adherent
yet low enough to limit continuous insudation of fibrino- cells will also influence the extent of giant cell formation.
gen. It seems reasonable to expect a diminished chronic mac- Most conspicuous is the absence of foreign body giant cells
rophage response in grafts where a temporarily sealing in- (FBGCs) inside the wall structure of ePTFE grafts, compared
growth matrix limits the ongoing insudation of plasma and to Dacron and polyurethane. Although a rim of giant cells
hence limits monocyte adhesion to the material through fi- often demarcates these prostheses against the surrounding
brinogen adsorption. tissue19 and the interstices are sometimes packed with single
As the deposition of fibrin onto the graft surface con- macrophages, no fusion occurs. We have regularly observed
tinues, the inner macrophage zone increasingly becomes this phenomenon in both high porosity and low porosity
wedged between two almost acellular fibrin matrices, one ePTFE in the primate. Although the internodal distances of
in the graft center and one on the blood surface.2-4,7,60,66,72,82 a 60 ∝m ePTFE graft provide both wide localized ingrowth
As discussed under “Fibrin”, the most likely explanation for spaces of approximately 47.94 ± 9.90 ∝m and a significant
this mitigated macrophage recruitment from the blood adhesion area on the stretched fibrils, macrophages prefer a
seems to be the changing physical nature of the fibrin ma- solitary existence under these circumstances. Similarly, the
trix of the inner capsule due to different fiber structure and PTFE nodes—which are the adhesion site for foreign body
concentrations of fibrinogen. In the outer wall of the graft, giant cells on their smooth end on the exterior of the grafts—
however, a macrophage-dominated cell infiltrate continues remain free of giant cells inside the graft wall where numer-
to populate the outer one third to one half of the prosthetic ous fibrils subdivide their surface. The absence of giant cells
structure within 1-3 months.27,28 As the macrophage popu- on the nodal surface subdivided by numerous fibrils indi-
lation migrates into the depth of the graft structure, it must cates that giant cell formation may be suppressed by geo-
eventually overstep the maximum distance of a cell from a metrical space limitations. However, given the in vitro evi-
capillary and thus get into a mild hypoxic state. Macrophages dence that fewer monocytes adhere to ePTFE and it is less
exposed to hypoxia, however, are synthesizing and releasing inflammatory than Dacron,168,178,179,182 and that a more hy-
increased amounts of PDGF as well as aFGF and bFGF.197 drophobic surface decreases the propensity of single mac-
Medium conditioned by hypoxic macrophages in culture is rophages to fuse,202-204,206 the inflammatory potential of the
The Lack of Healing in Conventional Vascular Grafts 33
surface may also play a role in giant cell formation where a capillary sinuses and the presence of smooth muscle cells,
more chemically inert, more hydrophobic ePTFE surface re- seems to indicate that the giant cell contributes to the mis-
duces fusion. directing of the process of transmural capillary ingrowth.
Not only do mononuclear cells gradually fuse to form In situ hybridization of human explants has demonstrated
more giant cells in a material-dependent manner, but fur- that interstitial macrophages and foreign body giant cells
ther complexity is added by the changing nature of the are still secreting Il-1! even after numbers of years.213 Al-
mononuclear macrophage population over time. Macroph- though Il-1! is known to stimulate both smooth muscle cell
ages found in intimate contact with synthetic materials in proliferation and fibronectin deposition in atherosclerotic
situ were ED1 and MHCII positive, whereas ED2 positive plaque formation, 214 it has been suggested that this
macrophages were observed lying behind the ED1 positive interleukin can also inhibit vascular smooth muscle prolif-
cells.169 This indicates a pattern of distribution of not only eration in an autocrine fashion through nitric oxide
mono—versus multinuclear macrophages, but also imma- upregulation.215 The giant cells, therefore, may be respon-
ture (ED1 positive) versus mature (ED2 positive), and acti- sible for not only affecting capillarization but also inhibit-
vated (MHCII positive) versus nonactivated macrophages ing smooth muscle cell ingrowth through persistent Il-1!
within the implant site. Theoretical analysis shows that up secretion. In summary, the true character of the FBGC re-
to 5 weeks of implantation, FBGCs are in fact formed from mains elusive. On the one hand, their persistence at the im-
the fusion of a small percentage of the adherent macroph- plant site and their destructive role in biodegradation, as
ages which are already present on the third day of implanta- well as their possible role in the mitigation of vessel forma-
tion.203,204 This raises the issue of fusogenic potential of dif- tion, seem to label them as villains in incomplete graft heal-
ferent subsets of cells. Expression of cell surface receptors ing. Yet, the evidence supporting a role for a typically anti-
may be a prerequisite. For example, inhibitors of mannose inflammatory cytokine—IL-4—in stimulating giant cell for-
receptor activity have been shown to inhibit IL-4 induced mation is fairly convincing both in vitro and in vivo.99,208
fusion in vitro, suggesting that this receptor may play a role Given that inflammation associated with normal wound
in giant cell formation.207 Elaboration of certain cell surface healing is resolved because of the transient nature of the
markers in a material or environment dependent manner macrophage response, it would appear that the inability to
may be required for a higher propensity for giant cell remove the inflammatory stimulus (a nondegradable bio-
formation. material) confounds even the best intentions of these cells
If it is difficult to decide whether macrophages are ben- to mediate healing. The consequence is a chronic ongoing
eficial for graft healing or not, it is even more difficult to inflammation and the derailing of the normal sequence of
decide whether the presence of foreign body giant cells rep- healing events.
resents a state of pacification or a particularly detrimental In summary, two distinct zones develop in prosthetic
variant of chronic inflammation. One indication for the vascular grafts over time: one close to the blood surface which
FBGCs role as a mediator of pacification of inflammation becomes increasingly uninhabitable for macrophages, and
arises from the fact that their fusion can be induced by one in the outer portion of the graft which is densely packed
IL-4.99,208 IL-4 is secreted by Th2 lymphocytes and is known with macrophages and later on with foreign body giant cells.
to downregulate several macrophage inflammatory func- It is well documented that macrophages infiltrating a wound
tions, for example, the release of certain inflammatory me- environment stimulate the proliferation of endothelial cells
diators,209 the secretion of reactive oxygen intermediates210 and, hence, angiogenesis. However, the apparent lack of cor-
and collagenase production,211 to mention a few. In this con- relation between the number of resident macrophages in the
text it has been proposed that giant cells may represent a interstices of grafts and the extent of transmurally ingrow-
attempt to “mop-up” the deleterious effect of persistent ing vessels indicates that macrophages are not alone in af-
chronic foreign body reaction. In contrast, a detrimental role fecting endothelial ingrowth, but that other factors, for ex-
for the giant cell is suggested by the fact that material sur- ample, the nature of the ingrowth matrix, may affect the
face cracking occurs directly beneath adherent foreign body overall extent of capillarization. Aside from an often impen-
giant cells, implicating the secretory products of these cells etrable fibrin matrix, the protracted ingrowth of connective
in the degradation of biosynthetics.212 The presence of ac- tissue, specifically smooth muscle cells from outside, may
tin-containing adhesive structures202 indicates that the ad- well have to do with ongoing macrophage activation in the
hesion of the giant cell to the biomaterial forms a sealed presence of persistent material stimulus. The secretion of
compartment with a high local concentration of degradative inhibitory cytokines like Il-1! by both macrophages and for-
enzymes. It is also peculiar that the accumulation of giant eign body giant cells may be partly responsible. What is be-
cells in Dacron regularly coincides with the development of coming increasingly obvious is that a macrophage is not al-
huge convolutes of capillary sinuses in the vicinity. These ways a macrophage and that the contribution of each of these
widely dilated and irregularly shaped endothelial sacs are subsets of macrophage populations to the lack of healing of
principally devoid of any second cell layer (Figs. 1.11, 1.18). the vascular graft remains to be investigated. Overall, the
In contrast, 60 ∝m ePTFE, with its scanty presence of for- macrophage remains the Jekyll and Hyde of graft healing,
eign body giant cells, shows arterioles in its outer interstitial but as our insight into the mechanisms of healing grows so
spaces with one or more layers of smooth muscle cells. This does our understanding of which critical questions remain
histological observation, where the lack of giant cells corre- to be answered in the elucidation of the role of this
lates with the absence of malformed vessels in the form of enigmatous cell, namely:
34 Tissue Engineering of Prosthetic Vascular Grafts
1. If monocyte adhesion to biomaterials is prevented Apart from the favorable matrix environment, white blood
completely, do we compromise healing in any way; cells provide various pro-angiogenic stimuli at this stage.
2. To what extent does engineering tissue ingrowth Infiltrating neutrophils are a source of VEGF,136 and mac-
modulate the deleterious effect of the inflammatory rophages migrating into a presumably mildly hypoxic envi-
cell by promoting selective cellular infiltration be- ronment secrete acidic and basic FGF as well as PDGF.197
fore the development of chronic inflammation; and These cytokines are both chemotactic and mitogenic for
3. Can we identify secretory products of macrophages endothelial cells. Once again, integrin mediated binding to
or giant cells which specifically mediate the mitiga- the wound matrix is central to these responses, with both
tion of tissue ingrowth, either endothelial sprouts #v!3 and #v!5 involved in growth factor-stimulated angio-
or smooth muscle cells? genesis, although via different mechanisms:59 bFGF-induced
angiogenesis is mediated by the #v!3 integrin, whereas #v!5
Prosthetic Wall Vascularization integrin is crucial in VEGF-stimulated angiogenesis.
The “restitutio ad integrum” healing of a vascular pros- Although the initial phase of vascularization resembles
thesis—the formation of a neo-artery in place of a removed normal wound healing, the subsequent phase of centripetal
segment—would necessitate the formation of vasa vasorum vessel ingrowth through the interstices of the graft is already
in the graft wall, accompanied by the growth of a fully func- substantially slowed and often only reaches the outer half of
tional medium in an inflammation-free environment. The the graft wall.60,78 Considering the fact that capillarization
second best solution—the formation of granulation tissue of several millimeters of wound fibrin is normally accom-
with the resulting conclusion of the healing process through plished in humans within days, the time period of several
scar tissue—would at one stage also require extensive months required for achieving this ingrowth can only be
capillarization. Even if conventional synthetic grafts are lack- explained by a significant deviation from normal wound
ing the sophisticated biological information necessary for healing. One obvious difference between normal and graft
“ad integrum” healing, their porosity would in many cases healing is the nature of the chronic inflammatory response.
at least allow the ingrowth of granulation tissue. From our Specifically, the macrophage-derived giant cells which gradu-
daily surgical practice we are all familiar with the alacrity ally build up in the outer parts of the graft27,28,33,60,62 are
with which the formation of such granulation tissue and only formed in response to a foreign body. In high porosity
the subsequent stabilization through scar tissue is usually Dacron grafts, this foreign body reaction is not only associ-
accomplished. The tissue gap in technically the best of all ated with mitigated ingrowth but also the formation of
surgical wound closures is often more than one millimeter hugely dilated capillary sacs as opposed to regular vessels
wide, but capillarization is not only complete, but scar tis- (Figs. 1.11, 1.18). Moreover, in some models, the conspicu-
sue formation is already beginning, as early as after seven ous lack of smooth muscle cells, and therefore of vessels other
days. It is therefore surprising that an equally short distance than capillaries, indicates that not only angiogenesis but also
in the case of the wall of prosthetic vascular grafts does not musculogenesis is inhibited. Given these unique circum-
accomplish the same degree of full thickness capillarization stances, it should be possible to describe biological interac-
even after years of implantation. In order to further eluci- tions, which at this stage of graft healing, may be respon-
date this major shortcoming in graft healing. one needs to sible for the gradual mitigation of transmural ingrowth. His-
ask two key questions: tological analysis suggests that probably both matrix- and
1. Why does adventitial capillary sprouting into the cell-mediated phenomena are responsible for the inhibition
outer fibrinous capsule and later on into the inter- of angio- and musculogenesis.
stices of the prosthetic graft occur at such a slow pace As far as the matrix is concerned, we have already dis-
and eventually come to a halt; and cussed the possibility of a gradual transition in the nature
2. Why is anastomotic endothelial cell outgrowth so of the fibrin from the outer wound to a fibrinogen-rich blood
futile concerning both surface endothelialization and surface which may gradually slow down the capillarization
capillary sprouting into the depth? from outside to inside. In this regard, it is interesting that
The initial angiogenic response of the surrounding studies on matrix control of angiogenesis217,218 have ob-
tissue to a freshly implanted vascular prosthesis does not served that it is specifically capillary morphogenesis that
seem to differ from any other wound repair. This early phase depends on extracellular matrix (ECM).219 Especially, the
is typically characterized by infiltration into the wound fi- ability of endothelial cells to exert mechanical forces on the
brin of a relatively high proportion of polymorphonuclear surrounding matrix,217 and the subsequent development of
granulocytes, but also lymphocytes and a few macrophages. matrix networks218 to guide capillary sprout formation, are
Capillaries within the adventitia sprout into the freshly matrix determined. Physical and/or biochemical aspects of
formed fibrin matrix within days of injury.2,13,48,62,63,76,94 fibrin formation will, therefore, affect the malleability of the
During this formation of early granulation tissue, the ma- matrix and hence the susceptibility to cell mediated trac-
trix is probably inconspicuous with regard to fibrinogen tion and the realignment of matrix fibers. Hence, the sig-
concentration, fiber constitution and cytokine content and, nificance of the qualitative change in the matrix towards the
as such, is naturally pro-angiogenic. The sprouting endot- lumen may be inhibition of capillary ingrowth.
helial cells bind via #v!3 to the RGD site of the fibrin #-chain In addition to matrix mediated effects, two histologi-
and migrate into the pores of the graft. This also promotes cal observations hint at cellular events playing a role in the
survival of the angiogenic sprout by sustaining an anti- mitigation of vascularization. Firstly, there is the fact that
apoptotic response through inhibition of p53 activation.216 capillary sprouting increasingly comes to a halt towards the
The Lack of Healing in Conventional Vascular Grafts 35
acellular inner fibrinous capsule, while the macrophage- boon, for instance, capillary outgrowth is rapid and thus
dominated chronic inflammatory tissue, which is limited to completed before a changing environment can inhibit
the outer half of the graft, reaches a state of sufficient vascu- it.30,32,34 Under senescent circumstances as in humans78 and
larization.2,11,27-29,60 It would appear that, although hypoxic the chacma baboon, however, the protracted angiogenic
macrophages may initially be responsible for a pro-angio- potential of endothelial cells does not allow completion of
genic stimulus, the vascularization which has taken place in capillary ingrowth prior to the change in the biological en-
the meantime in the outer half of the graft re-establishes a vironment. This seems to explain why the same graft leads
normoxic environment. With an increasing oxygen tension to rapid spontaneous endothelialization in the one model
in the tissue, one would expect a downregulation of the se- but disproportionately fails to achieve this goal in another.
cretion of certain endothelial cell mitogens220,221 and hence While unfavorable matrix characteristics, continual
a downregulation of the angiogenesis. Moreover, the short- macrophage presence and host specific endothelial cell char-
lived neutrophil response may further deprive angiogenesis acteristics offer a possible explanation for the slowing down
of a potent stimulator—VEGF.56,136,222 of capillary ingrowth, the lack of musculogenesis within the
The second and perhaps most unique observation is graft wall is more challenging to explain. With potent tools
not that angiogenesis gradually slows down, but the fact that of tissue engineering available, one needs to overcome the
vessel formation is so derailed that the endothelial cells form deep seated suspicion of the presence of smooth muscle cells
sinuses as opposed to tubes. It seems likely that this sinus based on their role in intimal hyperplasia. This suspicion
formation may be due to the lack of appropriate intercellu- prevails despite reports that smooth muscle cell prolifera-
lar alignment and adhesion events, which are as critical to tion reaches an equilibrium on prosthetic vascular grafts,61,93
the process of angiogenesis as the migration and prolifera- depending partly on functional interactions with physiologi-
tion of endothelial cells.218 One hint of what the mecha- cal regulators like blood flow.54 However, the goal of even-
nism may be comes from an in vitro model of bFGF-in- tually emulating functional arteries through “smart” engi-
duced angiogenesis. It was observed that, in fibrin gels, stimu- neered scaffolds will necessarily need to assign the signifi-
lation of endothelial cells with bFGF alone resulted in large cant role of creating a “healthy” neo-media to smooth muscle
lumen sinuses similar to those seen in grafts, whereas the cells. It is therefore paramount to try to understand mecha-
addition of TGF-! at picogram concentrations reduced the nisms of ingrowth inhibitions for smooth muscle cells also
lumen dimensions.223 Addition of nanograms of TGF-! miti- and not only for endothelial cells. Therefore, one must see
gated lumen formation completely. Given these in vitro ob- two peculiar observations in prosthetic midgraft healing in
servations, it would appear that ultimately the 3-dimensional this context: the absence of smooth muscle cells in most parts
organization of capillary sprouts depends on a balance of of the graft wall2,16,26,27,94,224 and at the same time the pres-
proliferating versus differentiating stimuli, where an over- ence of smooth muscle cells underneath luminal endothe-
riding mitogenic stimulus (in this model, bFGF) results in lial cells,2,14,30-33,35,59,67,77,81 sometimes separated by acellu-
large sinuses but increasing concentrations of a differenti- lar fibrin59 from transmurally ingrowing connective tissue.
ating stimulus (TGF-!) arrests proliferation and so facili- With transdifferentiation of fibroblasts being only a
tates tube formation. Driven to the other extreme, an over- remote possibility, the presence of smooth muscle cells seems
riding differentiating stimulus arrests both proliferation and to be dependent on the differentiation of mural/mesenchy-
tube formation. If one concludes that the sinuses are the mal cells in the wake of angiogenesis.106,107 Embryonic data
result of overriding mitogenic signals in the outer zone of suggests that endothelial tube structures form first and di-
the graft wall, where could these signals originate? As previ- rect the subsequent recruitment of the surrounding cells—
ously mentioned, histologically we observe that sinus for- pericytes in small vessels and smooth muscle cells (SMC) in
mation is associated with the gradual buildup of giant cells. larger vessels. This promotion of mural cell proliferation and
The obvious association of these cells with the capillary si- migration is primarily achieved through the synthesis and
nuses indicates that they may be secreting an abundance of secretion of platelet derived growth factor-BB (PDGF-BB)
endothelial cell mitogens. Although macrophages themselves by endothelial cells, which upregulates smooth muscle cell
are known to stimulate endothelial proliferation,197,220 the expression of collagenase, #v!3 and PDGF-receptors. The
limited information available on the nature of secretory upregulation of these factors allows the mural cells to move
products of the foreign body giant cell makes it difficult to toward the endothelial cell, eventually establishing intercel-
define exactly what paracrine mechanism may be involved. lular contact through adherens junctions. This intimate con-
To further complicate the situation, the success of graft tact between the two cell types drives the expression and
wall vascularization appears to depend not only on the in- activation of TGF-!, resulting in a local high concentration
teraction of these pro- and anti-angiogenic influences of sufficient to inhibit endothelial cell migration and prolif-
inflammatory cells and matrix, but also on the interplay of eration225 as well as to promote differentiation of the
these two factors with the angiogenic vigor of host endo- pericytes/smooth muscle cells. This delicate cytokine bal-
thelial cells. The example of 60 ∝m ePTFE grafts in different ance between endothelial cells and smooth muscle cells re-
animal models illustrates best that there seems to be a net flects the interdependence of these two cell types, further
effect between the endothelial cell’s inherent ability to rap- emphasizing that smooth muscle cells require the presence
idly proliferate and migrate on the one hand and the gradual of endothelial cells for migrating into tissue.
build up of an environment which inhibits angiogenesis on If, on the one hand, capillaries eventually reach the
the other. In the juvenile animal model of the yellow ba- inner fibrinous capsule of prosthetic grafts and, on the other
36 Tissue Engineering of Prosthetic Vascular Grafts
hand, mesenchymal cell differentiation into smooth muscle a species high and the porosity favorable to allow ingrowth
cells normally “follows” the outgrowing endothelial sprouts, together with early sealing of the interstices against continual
why are capillaries often found in abundance but without fibrin/fibrinogen insudation, capillaries may reach across the
accompanying smooth muscle cells? Similarly to the inhibi- entire distance before the biological environment changes.
tion of capillarization, both cell-matrix and cell-cell inter- As a consequence, no premature maturation event would
actions seem to play a role in the inhibition of mus- prevent the endothelial cells from chemotactically attract-
culogenesis. As discussed under “Fibrin”, platelet activation ing mural cells to trail them. This scenario seems to explain
at the blood surface may result in relatively high TGF-! con- best why 60 ∝m ePTFE grafts in the juvenile yellow baboon
centrations in the nonfavorable fibrin matrix. Should show such a distinctly different healing pattern with regard
migration-associated degradation liberate this TGF-!, it may to smooth muscle cell ingrowth compared to other grafts
indeed prematurely inhibit proliferative responses of smooth and species. However, if the fibrinogenic potential of a graft
muscle cells and switch the phenotype of these cells to a rest- is relatively high and the angiogenic potential of the host
ing one. In this way, the biological signals encoding the matu- endothelial cell low, it is reasonable to imagine that the slowly
ration and eventually the arrest of a proliferative event may changing biological environment allows the first part of a
be prematurely transmitted to the smooth muscle cells as a chronological sequence but prevents the second one. As the
result of the changing fibrin environment over time. How- circumstances deteriorate to a point where the entire pro-
ever, as tempting as TGF-! may be as an explanation for the cess begins to halt, the first step—in this case, capillary
mitigation of further capillary ingrowth into the inner fibrin- sprouting—has already materialized while the subsequent
ous capsule, it does not explain the absence of smooth muscle step—the site-directed migration of mural cells—has not
cells in areas of dilated endothelial sinuses towards the ad- even commenced yet.
ventitial surface where the fibrin is assumed to be closer to When both endothelial cells and smooth muscle cells
normal wound fibrin both in structure and cytokine con- do reach the blood surface, additional large vessel signals
tent. Because these sinuses are associated with macrophages appear to play a role in their relationship. Flow conditions
and giant cell formation, one explanation may be the im- and compliance mismatch may result in an overriding stimu-
munohistochemical observation that macrophages and gi- lus for smooth muscle cell proliferation229 despite their di-
ant cells are secreting Il-1! even after years of implanta- rect contact with endothelial cells, which would normally
tion.213 This cytokine has been show to upregulate smooth terminate smooth muscle cell mitogenesis through the ef-
muscle cell nitric oxide production215 and could, as such, fect of TGF-!. It is well established that the endothelium
negatively regulate smooth muscle cell mitogenesis in areas transduces flow-related signals and directs smooth muscle
of macrophage infiltration, in contrast to its role in the cell proliferation when required. It is not surprising, there-
pathogenesis of atherosclerosis.214 More likely, however, is fore, that expression of the smooth muscle cell mitogen
an absence of PDGF-BB secretion by the endothelial cells, PDGF-AA has been detected in the endothelial cells of the
because they never reach maturation associated with vessel well developed intima of grafts implanted in the yellow ba-
formation. boon,52,226 while very little PDGF-B chain is detected. Con-
In addition, we have already discussed the role of a sidering the complexity of, for example, PDGF signaling
slowly changing matrix on the mitigation of full thickness where PDGF is a homo- or heterodimer of two different
wall capillarization. Such arrested endothelial cells may cease chains with three resulting isoforms, not to mention two
to send out chemotactic signals to smooth muscle cells in different receptors selectively binding all or only one of these
form of PDGF. Interestingly, although ingrowing capillaries isoforms, it is possible that these factors may combine to
in high porosity Dacron grafts were completely devoid of generate a wide variety of results and afford the cell a rela-
smooth muscle cells in the chacma baboon model, 60 ∝m tively tight control of the outcome.227,229 This would explain
ePTFE grafts in chacma and yellow baboons showed arteri- why PDGF-AA is present during intimal proliferation al-
oles with layers of actin positive smooth muscle cells though PDGF-BB normally directs smooth muscle cell mi-
(Fig. 1.9). This indicates that in those grafts in which gration and proliferation during angiogenesis.
capillarization was able to overcome ingrowth inhibition at If premature maturation signals are the reason for the
an early stage and proliferation of the endothelial cells was stoppage of transmural ingrowth of capillaries at the inside
not arrested, the smooth muscle cell component is normally of the inner fibrinous capsule, is it possible to apply the same
developed.2,30-33,35,46,77,81 This supports the hypothesis that interpretation to the ingrowth stoppage of anastomotic sur-
mitigation of capillarization influences smooth muscle cell face endothelium? At first thought, events only partially re-
migration. Moreover, it is noticeable that the combination semble those occurring in the graft wall. Since angiogenesis
of high porosity ePTFE with a juvenile and vigorously is a complex process including matrix degradation, migra-
endothelializing animal model achieves the most differenti- tion and proliferation, the earlier termination of endothe-
ated layers of smooth muscle cells.30-33,35 These smooth lial outgrowth in the wall matrix may be due to the inhibi-
muscle cells are observed in immediate proximity to endo- tion of either of these three. Since surface migration does
thelial cells within the initial 2-3 weeks of implantation when not require matrix degradation to the same extent, it must
biological circumstances seem to be more in favor of angio- be either proliferation or migration which is primarily af-
genesis, and hence smooth muscle cell recruitment, than at fected under these circumstances. Extrapolation of the num-
a later stage. Therefore, if the fibrinogenic potential of a graft ber of population doublings230 required to cover the trans-
is relatively low, the angiogenic vigor of endothelial cells of anastomotic ingrowth area clarifies that it is not the divid-
The Lack of Healing in Conventional Vascular Grafts 37
ing potential of the endothelial cell which is the limiting fac- widely held opinion, it would appear that in fact
tor of endothelial outgrowth. Therefore, inhibitory factors transanastomotic endothelialization progressively slows
other than an exhausted mitotic capacity must play a role. down in all models, although at different rates. One can
One explanation could be activated platelets that accumu- therefore not extrapolate short term observations to a long
late in and on the pseudointimal layer throughout the length term situation. Since the cessation of tissue outgrowth can-
of the graft. The complex signaling arising from platelet de- not be due to an exhausted mitotic potential of endothelial
granulation and the high local TGF-! concentration which cells, migration and proliferation appear to get actively ar-
results could affect endothelial cell proliferation at the sur- rested by changing biological circumstances. The fibrino-
face. However, there must be an additional explanation for gen and platelet-rich environment at the blood surface makes
transanastomotic ingrowth stoppage, because it is also seen the buildup of a dense and fine fibrillar fibrin capsule likely,
in grafts with minimal surface fibrin or platelet coverage. containing unusually high concentrations of TGF-!. Such a
The possible involvement of an additional factor becomes matrix seems theoretically capable of prematurely turning
apparent in Dacron grafts, where the outgrowth of endo- the outgrowing endothelium into a noncycling, resting inti-
thelium is more pronounced in a high porosity (knitted) mal tissue.
Dacron40,46,47,60,68,81,107 than in a low porosity (woven) Porosity has always been regarded as a crucial deter-
one.40,43 The difference between the two types of Dacron minant for tissue ingrowth. However, porosity alone is not
grafts obviously lies not in the composition of the fibrinous sufficient to quantify the available space for ingrowth. Even
pseudointima but in the ingrown tissue underlying this fi- in so-called high porosity ePTFE grafts for instance, full-
brin matrix. Could this ingrowing tissue in some way be thickness transmural ingrowth of microarterioles is only pos-
facilitating endothelial ingrowth from the anastomosis so sible in a marginal percentage of interstitial spaces. The graft
that the gradual cessation of transmural ingrowth becomes wall structure may provide the simplest level of inhibition,
reflected in a similar mitigation of outgrowth from the anas- and therefore this factor needs to be considered. The dimen-
tomoses? Paracrine signaling over such distances is known sions of the available ingrowth space in ePTFE grafts have
to occur during embryogenesis, so it is not beyond prob- not previously been assessed in order to determine the di-
ability that ingrowing tissue directs transanastomotic out- mensions of a transmural channel. The mathematical cal-
growth but the underlying mechanism has yet to be culations presented for these grafts in this chapter reveal that
determined. porosity was previously overestimated as a sole determinant
In summary, the most striking shortcomings of pros- for tissue ingrowth. Similarly, the measurements of interfi-
thetic wall vascularization, namely, the failure of the capil- ber spaces in Dacron grafts revealed a maximum space suf-
laries to reach the luminal surface as well as the absence of ficient for capillaries, and limited access to arteriolar in-
mature smooth muscle cells and hence arterioles, could be growth.
partly attributed to both the surrounding matrix and the On the biological level, a continual buildup of seem-
surrounding inflammatory cells. Graft healing therefore ingly adverse conditions eventually prevents the completion
seems to be a race between the endothelial cell’s angiogenic of transmural tissue ingrowth in most of the cases. In those
potential and the buildup of inhibitory influences. Since the instances in which microvessels successfully reach the blood
endothelial cell remains a relative constant in human im- surface, it appears that tissue ingrowth is completed more
plants it makes sense that graft designs should aim at, firstly, rapidly and hence before the environment turns inhibitory.
preventing the deposition of unfavorable, compacted fibrin When identifying inhibitory structures, the compact inner
and, secondly, attenuating the chronic inflammatory re- fibrin capsule and the outer zone, with massive accumula-
sponse. This could well be achieved by incorporating an in- tion of foreign body giant cells, appear to be the primary
growth matrix which not only temporarily seals the poros- culprits. The special composition of the internal fibrinous
ity of the graft wall to protein insudation but at the same capsule provides a reasonable explanation for the fact that it
time selectively promotes vascular cell infiltration while in- is almost impenetrable for capillaries. In contrast, the outer
hibiting the buildup of a prolonged macrophage response. graft zone which is packed with foreign body giant cells seems
rather to affect the quality of angiogenesis by derailing the
Conclusion delicate process of endothelial—and smooth muscle cell—
Healing of prosthetic vascular grafts implies transmu- sprouting. While unrivaled endothelial proliferation leads
ral tissue ingrowth. During the past three decades, however, to the formation of hugely dilated capillary sacs as opposed
the focus of research was almost entirely on transanastomotic to vessels, no smooth muscle cells follow the angiogenic ef-
rather than transmural ingrowth. In spite of this one-sided forts of the endothelial cells. This lack of musculogenesis
approach, an analytic effort combining historical data and occurring in the graft wall corresponds conspicuously with
personal experience with biological principles helped to the presence of foreign body giant cells. The presence of gi-
clarify some main issues involved in the mitigation of the ant cells is in turn a consequence of a pronounced macroph-
entire healing process of contemporary arterial prostheses. age attraction by the synthetic materials. This macrophage
The complete surface endothelialization through infiltration is augmented by fibrinogen insudation from the
transanastomotic ingrowth in animal models under shorter blood stream.
term experimental conditions often suggested that these If one tries to draw any conclusions from these obser-
models differ in principle from the human situation with vations for the tissue engineering of future grafts the fol-
its stoppage of endothelial ingrowth. In contrast to this lowing main guidelines appear to be crucial:
38 Tissue Engineering of Prosthetic Vascular Grafts
• In order to design future experiments with a view to- from sodden expanded polytetrafluorethylene grafts. J
wards transmural rather than transanastomotic Vasc Surg 1997; 25:187-197.
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thrombosed grafts in humans. Surgery 1983; 93: 809-817.
• Any structural design of a graft scaffold should make
12. Stumb MM, Jordan GL, DeBakey ME. Endothelium
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capable of accommodating at least arterioles in order Dacron hub. Amer J Path 1963; 43:361-368.
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CHAPTER 2
Noncompliance: The Silent Acceptance of a Villain
Alexander M. Seifalian, Alberto Giudiceandrea,
Thomas Schmitz-Rixen, George Hamilton
Introduction
A rterial occlusive disease is a problem of epidemic proportions in our aging society with
increasing need for vascular reconstructive surgery.1 The best results are achieved with
autologous vein, but because this is often not available, historically there has been extensive
research into production of a suitable substitute graft material.2 At present there are a hand-
ful of such vascular grafts that are either commercially available or under development, but
unfortunately no graft material has yet performed satisfactorily.3,4 Baird and Abbott’s hy-
pothesis of 1976 that a difference in circumferential compliance of a vascular graft and the
host artery is detrimental to graft performance was eventually experimentally verified in
1987.5,6 The nature of compliance mismatch is complex, as it is determined by the compli-
ance differences of the host artery, the anastomosis, and the graft itself. The hemodynamic
consequences of mismatch include increased impedance and decreased distal perfusion as
well as disturbed flow, turbulence and low shear stress rates.5 These changes could lead to
the development of myointimal hyperplasia around the anastomosis, finally resulting in
graft failure, particularly in small diameter vessels.7,8 Some efforts have been made over the
years to engineer prosthetic grafts with compliance similar to that found in human artery.9,10
To date these attempts have failed due to a variety of reasons, explaining our current accep-
tance of highly noncompliant and inferior graft materials. This chapter will give an over-
view of the history of compliance in vascular grafting, a discussion of the nature of compli-
ance and its measurement as a means of understanding the most recent attempts to engineer
a more compliant graft.
Historical Overview
Cardiovascular physiology started in 1628 with Harvey’s “De motu cordis et Sanguinis
in Animalibus” in which he describes the circulation.11 Fifty years later in 1676 Robert Hook
described the proportionality between stress and strain (“Ut Tensio Sic Vis” or, as the exten-
sion so the force) and the concept of elasticity of materials. Isaac Newton and his “Principia
Matematica” was the first to relate elasticity (hence compliance) to wave velocity. His for-
mula (equation 1) showed that velocity in a medium c is equal to the product of elasticity K
and the density of the flowing medium ∋.
(1)
Thomas Young (1773-1829) investigated this principle in detail, both theoretically
and experimentally, and gave his name to the elastic modulus. The Reverend Stephen Halea
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
46 Tissue Engineering of Prosthetic Vascular Grafts
in 1733 analyzed the role of a compliant vessel in maintain- The deformation undergone by this class of material
ing a continuous blood flow throughout the pulsation of a depends on the magnitude of the stress and on the rate at
cardiac cycle.12 The German translation of his work “He- which it is applied. Vessel compliance, the reciprocal value
mostatic”, introduced the important analogy in understand- of Young’s elastic modulus, is generally defined as the ratio
ing the physiological role of distensibility in arteries, namely of change in diameter over change in blood pressure
the Windkessel. This is an air reservoir which was fitted to (%/mm Hg x 10-2).20 It is defined by equation (2) where D
fire engines of the 18th century in order to smoothen out and P are diameter and pressure, and the d and s subscripts
the oscillations in flow of water due to intermittent pump- denote diastole and systole respectively.
ing, thus ensuring a constant flow to quench the fire.
Otto Frank in 1899 introduced a theoretical approach (2)
to explain the Windkessel effect on pressure waves.13 But it
was not until 1950 through the work of Wormersley and For convenience in comparing different vessels a stan-
McDonald that a rational mathematical approach to pulse dard reporting mean pressure of 100 mm Hg has been
flow and change in diameter over the cardiac cycle was adopted. When thickness is a major fraction of the diameter
reached which allowed comprehensive understanding of the of the vessel the distinction between inner and outer diam-
hemodynamics of arterial flow.14,15 eter becomes important. It must, therefore, be specified
Parallel to these physiological understandings, vascu- which diameter is used in calculating compliance. To com-
lar surgery evolved. The first great step which led to the foun- pute the absolute compliance the pressure should be re-
dation of vascular surgery was the pioneering work of Alexis corded at the exact point of diameter measurement, but this
Carrel in developing a technique for suturing blood vessels.16 is not always the case, especially when calculating compli-
This work published in 1902 lead to his Nobel Laureate in ance in a clinical setting.21,22
1912. The first use of a graft to replace an excised segment
of artery was reported by Goanes in Spain in 1906 when he Assessment of Compliance
bridged the defect with the popliteal vein of the patient.17 There are several strategies used to assess the elastic
Later, homologous grafts were used but these attempts were properties of vascular tissues. Classification of these meth-
frustrated by early graft degeneration and infections. In 1952 ods is to some extent arbitrary and these so-called different
appeared the first report of a synthetic graft made from methods share certain common principles. Two distinct
Vinyon-N, a new synthetic cloth used to manufacture ladies methods are commonly used, namely longitudinal compli-
undergarments.18 In 1954 Vinyon-N was formally intro- ance, which assesses elasticity of a selected length of the vas-
duced to clinical practice. Since then many different materi- cular system, and circumferential compliance. This latter
als have been introduced, with only Darcon and Teflon per- method is the most frequently used and assesses the com-
forming acceptably, such that virtually all modern prosthetic pliance of the cross sectional area of interest of the vessel or
grafts are made from these materials.19 It was not until 1976 graft.
that Baird and Abbott postulated compliance mismatch as a
major factor affecting graft patency.5,6 Since then a consid- Longitudinal Compliance
erable amount of research has elucidated the nature of com- This technique is based on monitoring of pulse wave
pliance mismatch, but as yet has not led to production of a velocity (PWV) of blood down a given arterial pathway.23,24
more compliant graft.9,10,20 The elastic component of the wall plays an important role
in determining the velocity of propagation of a pulse wave.
Physical Properties of the Vessel Wall The Moert-Kortensweg equation (3) describes this relation-
Knowledge of the elastic properties of the arterial wall ship. In this equation E is Elastic module, h is wall thickness,
is essential to study the dynamics of the arterial system. R is radius and ∋ is density of blood.
During systole, with an increase in pressure the arterial wall
circumference increases, with return during diastole to its (3)
previous dimension. This function of the vascular wall is of
crucial physiological importance and due to a combination The compliance C can be computed from the above
of elastic and viscous components inherent to arterial tissue. principle in equation 4.
Solid materials have elasticity to varying extents as an
inherent mechanical feature and this is defined as the ratio (4)
of applied stress to resultant strain. Young’s modulus ex-
presses this quality of elasticity and is measured in units of This approach is based on the assumption that (1/2R)
dyne/cm2. Solid materials which regain their original dimen- is small and that the vessel or grafts are filled with viscous
sions when the stress is withdrawn are perfectly elastic, while liquid. Experimental evaluation of this methodology reveals
those which retain the entire deformation are plastic. The an error of 16-24% in computation of compliance, thus re-
property of viscosity distinguishes a fluid from a solid. When quiring a correction factor.25
stress is applied to a fluid it will undergo viscous flow. The Since the pulse pressure and flow pulse propagates
vessel wall exhibits properties of both an elastic solid and a along a vessel with the same velocity, arterial compliance
viscous fluid and thus can be most properly described as can therefore be indirectly measured by observing the flow
being visco-elastic. pulse with Doppler ultrasound. Values for the time taken
for this pulse to travel a unit length of the arterial pathway,
Noncompliance: The Silent Acceptance of a Villain 47
in the absence of reflection from the periphery, can then be requires recording of systolic and diastolic pressures as well
applied to equation 4 to give the average arterial compli- as noninvasively recording diameter over cardiac cycle.
ance of a section of artery over which the pulse was mea- A wide spectrum of techniques have been used to
sured. This technique requires a simultaneous use of two measure the circumferential compliance in vitro as well as
Doppler devices.26 To further complicate this method, in- in the clinical setting, although less successfully. These meth-
creased PWV due to generalized stiffening of an artery or odologies include electrical, optical, ultrasound, magnetic
local plaque formation cannot be distinguished and ac- resonance imaging and digital angiography.
counted for. As a result, this approach has now been over-
taken by recent noninvasive accurate assessments of cross- Assessment of Compliance Using Electrical Techniques
sectional compliance. Measurement of changes in diameter by placement of
electrical resistance probes on blood vessels was first reported
Circumferential Compliance in 1960.27 The principle of this technique is based on place-
The most popular and acceptable method of comput- ment of transducers on the arterial wall with continuous
ing compliance is from measurement of changes in diam- changes in diameter being electrically produced. The device
eter over a cardiac cycle. which utilizes a miniature differential transformer measures
The first approach for assessment of elasticity, and so changes in external diameter of blood vessels and was used
compliance, is based on measurement of pressure and di- in particular to map the mechanical properties at several
ameter curves over the cardiac cycle. In quasistatic compli- sites along the aorta and some of its major branches.28 This
ance slow inflation and deflation of the blood vessels gener- methodology is highly invasive and effectively could only
ate a pressure diameter curve. The arterial wall, being an be used intraoperatively. In addition, the use of the device
anisotropic material, has a nonlinear elastic behavior. The imposes mechanical restraint on the vessel because of the
incremental elastic modulus is calculated using equation 5. need to apply calipers or coils. Since a typical change in di-
ameter of a blood vessel is in the order of about 5%, the
(5) degree of distortion and frictional force caused by these in-
struments led to acceptably high errors in measurements
Einc is the incremental elastic module, R is radius, o on small blood vessels. Furthermore these instruments mea-
and i subscripts denote inner and outer radius respectively, sure external diameter only, giving no information about
( is Poisson ratio, )R and )P are change in radius and pres- internal diameter movement, and all of this is can further
sure respectively. be compounded by problems in identifying exactly the limit
The second approach is based on assessment of ex- of the external wall in a native artery.
cursions of diameter and pressure during a cardiac cycle and
is known as dynamic compliance. Since strain relates not Optical Techniques
only to the magnitude of stress but also to the rate at which The main principle of this technique is application of
it is applied, it is logical to look at the elastic property in the light to measure diameter of the vessel. The vessel of inter-
physiological setting of pulsatile flow. A potential problem est is placed in a laser scanning system; this consists of a
in simultaneous recordings of changes in diameter and pres- laser light and photocell detectors. In operation the laser tube
sure is the phase lag between the pressure and diameter emits a light source which after passing through appropri-
curves, which is caused by the viscous component of the ate objects sweeps along a particular axis at a known veloc-
vascular wall. A complex elastic modulus has therefore been ity. The presence of an artery in the beam path prevents light
defined to formulate this concept. from reaching a photocell during a particular time. Know-
ing this time and sweep velocity calculations are made to
(6) compute the outside diameter of then artery in continuous
mode.29 This system has been applied to assess the compli-
ance of canine carotid artery, femoral artery and prosthetic
Where is the elastic compo- grafts in vitro by placing the vessels in pulsatile flow cir-
cuits.30 The technique has reported accuracy of detection in
nent and is the viscous component. lm is the order of 13 ∝m changes in outer diameter of the artery.29
The disadvantage of this system is that it is restricted to ex-
the average circumference and qm the average cross section perimental use only. In addition, it detects only changes in
area, P is the amplitude of the applied stress and Dl/l is the outer diameter and gives no information regarding inner
strain, w is the angular frequency, m is viscosity and f phase wall movement, which may be different, particularly in thick-
lag between the pressure and diameter curves. ened vessels.
Changes in diameter and pressure over cardiac cycle
can be measured at selected points on the vessel of interest, Ultrasonic Techniques
giving a more complete picture of the viscoelastic proper- Ultrasound has been the favored method of compli-
ties of the arterial wall. Recording of the pressure in vivo, ance measurement over the past two decades. There are two
however, requires insertion of an arterial catheter with its ways in which ultrasound has been applied to assess the com-
attendant possible complications. In the clinical setting, pliance of blood vessels. The first measures the PWV along
quasistatic compliance is more widely used. This technique a segment while the other is based on the local distention
48 Tissue Engineering of Prosthetic Vascular Grafts
wave form of a local artery (local compliance).20 This latter This technique is applicable clinically as well as experimen-
method requires assessment of diameter of the artery under tally for assessment of compliance. The disadvantage of MRI
investigation at the onset of a cardiac cycle, and thus disten- is that in vascular diseases such as atherosclerosis, focal le-
tion during the cardiac cycle under local pulse pressure. A sions are formed; thus the compliance at one site may be
radio frequency data acquisition system linked to a com- very different from that at another. Further problems may
puter is required and M-mode echo images of the blood ves- occur in follow up studies to monitor the course of the dis-
sel will be displayed. The anterior and posterior walls are ease or response to therapy, since relocation of the original
identified manually or automatically. The radio frequency site of measurement is very difficult. MRI, therefore, while
signals from the ultrasound M-mode output over a cardiac useful in demonstrating accurately pathological disease, has
cycle are digitized and relayed to a wall tracking system31 limitations in these measurements.
(see Fig. 2.1). From these signals end-diastolic and end-sys-
tolic intraluminal diameters can be easily determined and Digital X-ray Techniques
thus the maximum changes in diameter or distention for Angiography, or radiographic imaging of blood ves-
each beat. Blood pressure can be detected noninvasively and sels, has a well established role as the gold standard in the
compliance computed. diagnosis of vascular disease and is widely used in clinical
Ultrasound has inherent inaccuracies relating mainly practice for obtaining high quality vessel images.37,38 Digi-
to problems in recognizing the exact inner or outer diam- tal X-ray angiography has the necessary temporal and spa-
eter of the blood vessel.32,33 In vivo studies of the inter- and tial resolution for volume blood flow measurement and es-
intraobserver variability reveal errors of 5% in measuring timation of diameter over cardiac cycles. Diameter estimates
static diameter, and 10-15% in measuring pulsatile diam- are either based on accurate vessel edge location or by den-
eter changes.34 Increased angle of insonation by the trans- sitometry, i.e., by integrating image brightness perpendicu-
ducer in the longitudinal axis will falsely increase diameter lar to the axis of the vessel.39-41 The latter technique and prin-
and its changes. Due to the tortuous nature of some vessels ciple yield a number proportional to the vessel’s area inde-
in vivo it is difficult to be exactly perpendicular to the vessel pendent of lumen shape, in both healthy and diseased ves-
axis. The variability introduced by this problem in measur- sels. This technique has been used for estimation of compli-
ing diameter can affect the quality of results especially in ance as well as blood flow in the vessel of interest.42,43 Mea-
looking at small caliber vessels and consequently small surement of vascular diameter and volume blood flow in
changes over the cardiac cycle. This uncertainly is partially vessels which are tortuous and which do not lie parallel to
overcome by the use of intravascular ultrasound, which has the imaging plane relies on accurate computation of the 3-di-
the major drawback, however, of being invasive.35 mensional (3D) path length of the vessel, X-ray magnifica-
tion and the angle between the vessel axis and the X-ray
Magnetic Resonance Imaging Techniques beam. These factors can be computed from accurate 3D re-
Magnetic resonance imaging (MRI) is a noninvasive construction of vascular geometry.42,44,45 The authors and
imaging modality that is rapidly gaining clinical acceptance, collaborators have described a novel technique of measure-
although widespread introduction has been delayed by its ment of vessel cross sectional area, and hence diameter, us-
expense. MRI has been used directly to measure regional ing densitometric methods applied to standard intraarterial
aortic compliance as well as total cardiac aortic compliance.36 digital subtraction angiograms.41,43 The technique is based
Fig. 2.1. Displacement curves of the
anterior (ant) and posterior (pos)
walls of the common carotid artery
in healthy young male (age 25 years).
The bottom trace is the difference
between the displacements of both
arterial walls and represents the
change in arterial diameter during
the cardiac cycle. The first sign on the
distention tracing refers to the trig-
ger of the R-wave of the ECG (I), af-
ter which detection of end diastole
and peak systole (I) starts. Abbrevia-
tion: b, heart beat; dist, distention;
cca, common carotid artery. The data
is obtained using a wall tracking sys-
tem (Pie Medical, Masstricht, The
Netherlands).
Noncompliance: The Silent Acceptance of a Villain 49
on image densitometry in which the integral intensity of ization, X-ray angiography is still the modality of choice for
the contrast bolus is computed along a profile perpendicu- critical morphological vascular studies. That X-ray angiog-
lar to the projection of the vessel axis. As illustrated in Fig- raphy has not been widely used for measuring blood flow is
ure 2.2, the true cross-section A is related to the densito- due in part, we believe, to the use of inappropriate algo-
metric measure a by: rithms for processing the imaging digital data.46,47 The
method does, however, have great potential, especially when
(7) combined with lower dose digital subtraction angiography
and the new nonionic contrast agents.48 Mini-puncture
The X-ray magnification factor M and angle ∗ between needles and catheters have led to increased safety of the tech-
the vessel axis and the X-ray axis are computed from the 3D nique and the equipment and expertise is available in most
reconstruction of the vascular configuration from two views. centres.49
The densitometric calibration constant K relates the image
gray value to the mass of iodine integrated along the X-ray Synthetic and Biological Grafts
path from X-ray focus to image. These were obtained from Prosthetic grafts fare well when used in the aortic or
data generated from 3D reconstructions of biplanar X-ray aortofemoral position, but are much less successful for
angiographic data.42 Although it requires vascular catheter- bypasses below the inguinal ligament, with the patient’s own
Fig. 2.2. The true cross-section
is related to the integral of image
intensity along a profile perpen-
dicular to the projection of the
vessel axis. See text for details.
Table 2.1. Summary of results from literature for five years cumulative patency rates of infrainguinal reconstruction
with autogenous vein study
Research Workers No of Operative Primary graft Secondary Limb Patient
limbs mortality patency (%) graft patency (%) salvage (%) survival (%)
Taylor et al 199051 516 1 75 80 90 28
Berganini et al 199152 361 3 63 81 86 57
Donaldson et al 199253 440 2 72 83 84 66
50 Tissue Engineering of Prosthetic Vascular Grafts
saphenous vein giving the best long term patency rates.2,50 infrapopliteal reconstructions is dismal.62,63 PTFE grafting
Because of dismal patency rates, prosthetics are not used in into the popliteal artery at the knee shows a slightly better 5
coronary bypass grafting. Primary 5 year patency rates for year patency rate of 40%.61-63 Recently, better patency rates,
autogenous vein bypasses range between 63-75%, and sec- even to the tibial level, have been reported with the use of
ondary patency rates between 80-83%, leading to limb sal- PTFE bypass grafting onto an interposition vein collar or
vage rates which range from 84-92% (Table 2.1).51-53 These patch which is placed onto the distal artery. In part this may
are good clinical results but, unfortunately, in up to 40% of be due to improvement of the compliance match between
patients autogenous vein may not be available or be inad- the host artery and the graft.64
equate because of coexisting disease such as varicose veins Compliance mismatch has an important role in graft
or previous venous thrombosis. failure. Various research workers have shown that compli-
In these circumstances a prosthetic graft is the only ance of biological conduit is significantly greater than that
possible option.3 Since 1950, prosthetic grafts have been used of prosthetic material; this compares well with patency rates
in a variety of locations for arterial reconstruction. Knitted for the two different graft materials (Table 2.2).51,54,63 It ap-
and woven Dacron grafts provided favorable results in high pears that as compliance mismatch increases, patency de-
flow large caliber, vessels but performed poorly in more chal- creases; linear regression analysis of this data shows a highly
lenging small caliber low flow conditions characteristic of significant correlation for this association, as shown in Fig-
infrainguinal bypasses, particularly to the below knee arter- ure 2.3. The patency data for the different grafting materials
ies. To date the most successful prosthetic grafts in this situ- used in the femoro-popliteal position used in this study were
ation have been expanded polytetrafluoroethlylene (PTFE) obtained from several recent large studies and compliance
and the Dardik biograft or human umbilical vein tanned data from a study performed by Abbot et al in Bos-
with glutaraldehyde.54,55 The human umbilical vein graft was ton.51,54,55,63 Finally, prosthetic grafts have poor dynamic
introduced at roughly the same time as PTFE but never compliance profiles in comparison to arteries with no in-
gained broad acceptance, primarily because of a propensity crease in compliance occurring at low pressure, an impor-
for dilatation and aneurysm formation as early as 2 years tant physiological response to shock (Fig 2.4).20
after implantation, occurring in up to 57% of the
grafts.54,56,57 This phenomenon of graft dilatation is com- Causes of Graft Failure
mon to all biografts and is attributed to a combination of Causes of graft failure are complex and include tech-
deterioration of collagen crosslinks with time, and pro- nical failure, poor selection, compliance mismatch, primary
teolytic digestion by host enzymes almost certainly medi- thrombotic failure related to low flow environments, devel-
ated by an immune response.58 In vitro testing of 2 types of opment of intimal hyperplasia, late failure due to graft gen-
biografts, a bovine carotid and the human umbilical vein
graft, with long term perfusion with proteolytic enzymes,
demonstrated changes in the structural integrity of the graft. Table 2.2. Summary of results from literature relationship
This was detected by a degree in compliance, thus providing between compliance vs. patency rate.51,54,63
experimental data for the role of proteolytic digestion in
these grafts failures.59 Despite this, clinical experience with Graft type Compliance Patency %
the human umbilical vein gives a 5 year patency with ap- (%mm Hg x 10-2)
proximately 60% for femoral popliteal grafting with up to
80% in the above knee location.60,61 Thus these grafts per- Host artery 5.9 ± 0.5
form clinically well in the short term and have been recom- Saphenous vein 4.4 ± 0.8 75
mended for use in patients with a short life expectancy. Umbilical vein 3.7 ± 0.5 60
Results achieved with PTFE bypass grafting are less Bovine heterograft 2.6 ± 0.3 59
Dacron 1.9 ± 0.3 50
satisfactory particularly so onto the below knee tibial ves-
PTFE 1.6 ± 0.2 40
sels. On average the long term patency rates (> 3 years) in
Fig. 2.3. Data reported from com- 6
pliance of the various biological
Y = 0.09 x -2.0 ■ Umbilical vein
and prosthetic grafts vs. patency
(%/mm Hg x 10-2)
5 r2 = 0.877, p = 0.019 ● Bovine heterograft
rate.
Compliance
◆ Dacron
4 ▼ PTFE
▲ Saphenous vein
3
1
30 40 50 60 70 80
Patency (%)
Noncompliance: The Silent Acceptance of a Villain 51
Fig. 2.4. Compliance-pressure curve
comparing human superficial
femoral artery (●) with PTFE 6 mm
graft (❏). Note that an important
8 feature of human vessel is the in-
creased compliance found at lower
physiological pressures. This has the
important benefit of preserving
6 pulsatile energy in situations of
Compliance
shock, thus optimizing flow. This
dynamic change in compliance is
4 totally absent in PTFE and Dacron
grafts, and may be a further reason
for prosthetic graft thrombosis oc-
curring during hypotensive states.
2
0
40 60 80 100 120
Mean pressure (mm Hg)
eration and late failure due to progression of native vessel velocity gradients and turbulence. As a consequence of these
arterial sclerosis. More than one of these factors may be im- mechanical effects, vibratory weakening of the arterial wall
plicated in graft failure. Much research has been focused on has been postulated as a cause of endothelial damage, inti-
these various factors, with particular emphasis on reducing mal hyperplasia and even anastomotic aneurysm formation.
the thrombogenicity of grafts either by modifying the lumi-
nal surface or by endothelial cell seeding. Much less effort Anastomotic Compliance Mismatch
has been directed into the role of compliance in graft fail- Anastomotic compliance mismatch refers to the varia-
ure. Possibly, this is because of limitations in materials and tion in compliance occurring specifically at the anastomotic
material technology. There is much clinical data which em- site. The change in compliance from native artery and pros-
phasizes the importance of compliance mismatch and cer- thetic material is not monotonic because the creation of an
tainly it is established experimentally that current anastomosis always generates a focal decrease in diameter
noncompliant grafts owe their inferior performance to lack and a drop in compliance. This is partially determined by
of distensibility when compared to native arteries.6 It is most the elasticity, or rather rigidity, of the suture material, and
important to consider compliance mismatch as composed partly by the surgical technique, depending on whether a
of two major parts, namely tubular and anastomotic. continuous or interrupted anastomosis is used. Interrupted
anastomotic suture techniques give more compliant anas-
Tubular Compliance tomoses. There is also a paradoxical increase in compliance
Tubular compliance mismatch refers to the imparity of about 50% which occurs within a few millimeters on ei-
of elasticity between the prosthetic conduit and the native ther side of the suture line (Fig. 2.5). This characteristic is
artery. Essentially this results in an adverse hemodynamic known as the para-anastomotic hypercompliance zone
effect and reduced distal perfusion, as was previously dis- (PHZ).20,67,68 It has been hypothesized that PHZ may be re-
cussed. A compliant wall acts as an elastic reservoir absorb- sponsible for the intimal hyperplasia which characteristi-
ing energy during systole, which is released during diastole. cally develops at the same area as this hyper-compliance.
A rigid vessel wall consequently diminishes the pulsatile Thus mismatch of elastic properties around the anastomo-
component of the diastolic recoil, thus reducing the energy sis may act to promote intimal hyperplasia in at least 3 dif-
available for distal perfusion. ferent ways:
Impedance is the term given to resistance to pulsatile 1. Compliance mismatch between the artery and graft
flow. Change in impedance occurs at the interface between may lead to a region of excessive mechanical stress,
a compliant artery and noncompliant graft, and this results possibly resulting in wall injury, a major event in the
in propagation of less than 60% of the pulsatile energy.65 initiation of intimal hyperplasia;6,69,70
Optimal organ perfusion depends on pulsatile flow, and it 2. Cyclic stretching has a positive influence on replica-
has been shown that change from pulsatile to steady state tion of vascular smooth muscle cells and produc-
flow causes peripheral resistance to increase by 10%.66 Wave tion of extracellular matrix.70,71 Experimentally, it has
reflection of the graft artery interface also leads to increased been demonstrated that vascular smooth muscle cell
52 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 2.5. Para-anastomotic hyper-
compliant zone (PHZ). Typical
compliance profile of a continu-
ous anastomosis: Compliance vs.
distance. Anastomosis is at 0 mm.
start to produce extracellular matrix and to repli- finding of free calcium concentrations proportional to the
cate when they are subjected to high levels of disten- shear rate suggests an important role of calcium in the sig-
sion.72 Also, animal studies have confirmed that in- nal transduction pathway.81 Shear rate dependent release of
timal thickening is associated with increased growth factor is reflected by a varying expression of mul-
tangential stress;73 tiple growth factor-related genes when wall shear rates are
3. Flow studies show that a sudden increase in compli- changed.82 A possible mechanism underlying the increased
ance is associated with enhanced particle residence expression of growth factor genes may be a recently described
time, flow separation and stasis leading to low shear shear rate-induced transcription factor which may not only
stress conditions.74,75 Also, from animal studies it is regulate the expression of platelet derived growth factor
known that high levels of shear stress have a delete- (PDGF) genes but also of tissue plasminogen activator (tPA),
rious effect on endothelial cells but, most impor- intracellular adhesion molecule 1 (ICAM-1) and transform-
tantly, there is also mounting evidence that intimal ing growth factor-b1 (TGF-!1).83 A shear stress dependent
thickening occurs in areas of low shear rate. An ex- release of PDGE-! mRNA has also been observed in cul-
ample of this phenomenon in humans is of the sud- tured human umbilical vein cells.84 These findings begin to
den increase in compliance found in the carotid bulb illuminate the link between mechanical effects and changes
where the above hemodynamic changes occur. Since and the development of intimal hyperplasia in human ves-
this is a site with predilection for the occurrence of sels and bypass grafts.
atherosclerosis, one could postulate that increased
compliance with prolonged particle residence time, Development of a Graft
flow separation and low shear stress conditions can with Better Compliance
cause damage, allowing initiation of arteriosclerosis The ideal characteristics of a graft are biocom-
in man. patability, a nonthrombogenic surface, physical durability,
elastic or compliant properties similar to those of a native
Signal Transduction Pathways and Flow artery, resistance to infection and ease of implantation. Ad-
Flow is an important modulator of vascular structure vances in manufacture of biomaterials and tissue engineer-
and function, probably mediated through the effects of the ing are leading to developments in the manufacture of more
function on the endothelial cell.76,77 Pathways by which flow compliant grafts. What is not known, however, is the range
acts on the vessel wall are poorly defined, but an essential of compliance required to give the ideal match between the
role is attributed to the endothelial cell.78-80 Nitric oxide graft and the host artery. Arteries change with age with loss
(NO) plays an important role in this pathway, and it has of elastin and consequent reduction of elasticity and thus
been shown that specific potassium channel antagonists can compliance. When the ravages of arteriosclerosis with me-
block flow mediated vasodilatation and nitric oxide release, dial calcification occurring as a result of age are added to
suggesting that the signal transduction pathway underlying these effects, the compliance of the host artery in a patient
flow-mediated vasodilatation may be the activation of the requiring a bypass may be dramatically reduced (Fig. 2.6).20
potassium channel on the endothelial cell membrane.73 The Thus the aim should be to develop a graft which is pulsatile,
Noncompliance: The Silent Acceptance of a Villain 53
Fig. 2.6. Tendency of correlation of
compliance at mean pressure 100 mm
6 Hg with increasing age on human pa-
thology without calcification and ma-
jor plaque formation.
5
4
Compliance
0
30 40 50 60 70 80
Age (years)
Fig. 2.7. Compliance versus pres-
sure excursion of different
CronoFlex graft types (1-7).
but it may even be detrimental to have a graft which is too tion of artificial blood vessels with specific compliance char-
compliant. Study of typical compliances in aged and dis- acteristics (Fig. 2.7).10 In this process the graft material is
eased human arteries, coronary, femoral, popliteal and tibial, made using either a single shot method (Fig. 2.8)9 or a
is required before the ideal graft can be designed. prepolymer technique (Fig. 2.9).9 In a single shot method,
Development of more compliant grafts is currently all reactants are added simultaneously and the final struc-
occurring, either using biomaterials or by tissue engineer- ture is thus dependent on the relative reactivity of the for-
ing of vascular grafts. An example of developments in mulation. In the prepolymer method the ingredients are
biomaterials is the manufacture of grafts using polyurethane. added sequentially, allowing close engineering and design
Grafts have been made from this material for quite some of the final structure. Using a coagulation precipitation tech-
time and the major problem has been biodegradability and nique, porosity of the graft can be varied to form either mi-
subsequent aneurysm formation. A novel formulation of a cro- or macroporous structures. By varying the quantity and
polyurethane graft, namely CronoFlex (Poly Medica Indus- type of chemical groupings within the polyurethane polymer,
tries, Tarvin, Cheshire, UK) has recently allowed produc- it is possible to manipulate the level of crosslinking and thus
54 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 2.8. Production of polyurethane graft us-
ing single shot polymerization method. See
text for details.
Fig. 2.9. Production of polyurethane graft using prepolymer
polymerization method.
control mechanical properties. Thus a graft can be designed A further important area in design of such a biomate-
and modulated, varying not only the characteristics of the rial is in selection of optimal luminal characteristics. It is
polymer but also the degree of “sponginess” of the graft wall possible to design the lumen of the graft so that it is inher-
by modulating the size of the pores or bubbles in the wall of ently nonthrombogenic by incorporation, for example, of
the graft. This allows design of a graft to a specific compli- heparin or nonthrombogenic polymers. The alternative ap-
ance. This particular formulation of polyurethane grafts, proach is to make the lumen more attractive for endothelial
known colloquially as the “bubble graft,” has demonstrably cell adhesion and seeding. A simple example of such a meth-
higher compliance characteristics than others (Fig. 2.10).10 odology is the CronoFlex graft lumen, which is constituted
By modulation of the outer wall of the graft using various of many pits and valleys. Experimental evidence suggests
strategies to strengthen it, kinking as the graft crosses the that endothelial cells adhere better and are more resistant to
knee joint can be avoided. Extensive animal studies have the effects of flow shear stress because they can shelter within
shown no tendency for the CronoFlex graft to develop an- the pits, and proliferate upwards to endothelialize the graft
eurysms. (Fig. 2.11).85
Noncompliance: The Silent Acceptance of a Villain 55
Fig. 2.10. Scanning electrom micros-
copy cross-section of ChronoFlex graft.
(A) Nonreinforced (original magnifi-
cation x12.4).
Fig. 2.10.(B) Externally reinforced
(original magnification x110.5).
Tissue engineering holds much promise for the de- gin, which are said to be nonimmunogenic. Little data is
velopment of more compliant grafts. The relative roles of available as yet on the compliance characteristics of these
collagen, elastin and smooth muscles in the mechanical char- materials. Certainly this approach would hold the greatest
acteristics of an artery are now well understood. Experimen- promise of developing a graft with the necessary visco-elas-
tally, the development of arterial grafts using combinations tic properties comparable to those of human arteries.
of smooth muscle cells, collagen and endothelial cells is pos-
sible. Major problems persist in terms of the source of these The Future
cells. Ideally these should be from the patient, but certainly In the very near future it will be possible to move away
in peripheral vascular grafting the time and logistics involved from the use of the rigid materials, mostly Daron and PTFE,
would mitigate against the widespread use of this new tech- which are currently used in bypass grafting. Initially, pros-
nology. The important role of the immune response in even- thetic biomaterials with suitably designed and compatible
tual biodegradability means that such tissue engineered compliance characteristics will be used, with clinical trials
grafts made from nonautologus cells would require the use expected to begin within 2 years. The further addition of
of either immunosuppressive therapy, or cells of fetal ori- endothelial cell seeding to these more compliant grafts holds
56 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 2.11. Scanning electron micros-
copy (original magnification x660) of
fibronection coated PTFE (A, top)
and CPU (B, bottom) grafts after ex-
posure to flow for 6 h.
the promise of providing an off the shelf vascular conduit References
with performance approximating that of the human saphe- 1. Dormandy JA, Stock G. European consensus document
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52. Bergamini TM, Towne JB, Bandyk DF. Experience with mary artery smooth muscle. Lancet 1992; 340:878-9.
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Determinant factors of long term patency. J Vasc Surg endothelial potassium channel to relaese an endogenous
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53. Donaldson MC, Mannick JA, Whittemore AD. Causes of 88:1663-71.
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54. Dardik H, Ibrahim IM, Dardik I. Evaluation of glutaral- 1992; 25:297-310.
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56. Dardik H, Ibrahim IM, Sussman B. Biodegradation and 77. Hofstra L, Bergmans DC, Leunissen KM, Hoeks AP,
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57. Hasson JE, Newton WD, Waltman MD et al. Mural velocity on the development of intimal hyperplasia. Blood
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infrainguinal arterial reconstruction. J Vasc Surg 1986; dothelial-platelet-derived growth factor mRNA levels.
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PR. Haemodynamics of the interposition vein cuff. Br J Effect of prolonged pulsatile shear stress in vitro on en-
Surg 1986; 73:823-5. dothelial cell seeded PTFE and compliant polyurethane
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PART II
Biolized Prostheses: Surface Healing
CHAPTER 3
Endothelial Cell Seeding: A Review
Steven P. Schmidt, Gary L. Bowlin
Introduction
T he field of Tissue Engineering offers the promise of further elucidating and clarifying the
interactions between endothelial cells and smooth muscle cells. This understanding is
central to the creation of a successful small diameter vascular graft—a goal which has here-
tofore eluded researchers. Small diameter vascular grafts are defined as being less than 6 mm
in internal diameter. These grafts fail in patients from thrombosis and intimal hyperplasia,
processes which may, at least in part, originate from aberrant endothelial and smooth muscle
cell interactions. Clarification of the biology of these two cell types and their interactions is
critical in order to develop therapeutic solutions to the problems of small vessel prosthetic
grafting. These solutions will also undoubtedly involve further elucidation of the roles of
extracellular matrix in endothelial cell and smooth muscle cell biology, as well as of the
responsiveness of these cells to a variety of cytokines.
An endothelial cell lining does not naturally develop on the luminal surfaces of pros-
thetic vascular grafts in humans.1,2 This is in contrast to other animal species that have been
studied in which neoendothelialization of graft luminal surfaces occurs by endothelial cell
proliferation from perianastomotic artery, the microvessels of graft interstices, or circulat-
ing progenitor endothelial cells.3 Because endothelial cells release molecules that modulate
coagulation, platelet aggregation, leukocyte adhesion and vascular tone, the absence of these
cells lining prosthetic grafts, in combination with the inherent hostility of the biomaterial/
blood contacting surface, predisposes these grafts to platelet deposition leading to throm-
bosis. Although the precise mechanisms of the genesis of anastomotic intimal hyperplasia
are still being defined, endothelial cell/smooth muscle cell dysfunctions are thought to be
involved.
Early investigators in the field of small-diameter graft development sought to pro-
mote graft endothelialization by transplantation of autologous endothelial cells onto vascu-
lar grafts prior to their implantation—a process that became known as endothelial cell seed-
ing. The hypothesis underlying this research effort was really quite simple—that is, by pro-
moting the establishment of the patient’s own endothelial cells on the blood-contacting
surface of a vascular prosthesis, a “normal” endothelium-lined neointima would form on
the graft whose biology would counteract rheologic, physiologic, and biomaterial forces
promoting graft failure. In retrospect this simple hypothesis seems deceptively naive.
A review of the history of endothelial cell seeding research has been published which
details early animal and clinical studies.4 The results of these early animal studies were really
quite promising. This early optimism has been tempered, however, by clinical results that
have often been disappointing. It is clear that modifications of traditional methodologies to
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
62 Tissue Engineering of Prosthetic Vascular Grafts
promote endothelialization of vascular grafts are needed if ing and transplantation efficiencies in an appropriate time
endothelial cell transplantation is to be technically success- frame for clinical practicality. We will review briefly the his-
ful. What is even more important, however, is that academi- tory of these studies and then describe the novel technology
cians and physician researchers and investigators have grown of electrostatic cell seeding, which we feel offers a solution
to appreciate the complex biology of the cells forming the to many of the deficiencies of other approaches for trans-
vascular wall, and that technically successful transplantation planting cells onto vascular prostheses.
of endothelial cells does not necessarily presume a positive
outcome clinically in terms of vascular graft performance. Autologous Endothelial Cell Harvesting
The simple hypotheses of early endothelial cell seeding stud- Before endothelial cell seeding or transplantation can
ies have led to investigations providing enormous insights occur, autologous endothelial cells must be harvested from
into the biology of vascular wall cells, as well as stimulating an “appropriate, autologous” source. Sources of endothelial
suggestions for alternative therapeutic avenues that may uti- cells most often reported have been:
lize concepts and techniques of endothelial cell seeding. 1. Nonessential vessels (i.e., saphenous vein); and
The purpose of this chapter is to summarize the his- 2. Omental or subcutaneous adipose tissue (i.e., mi-
toric context and current base of knowledge regarding many crovascular endothelial cells).
of the technical issues relevant to endothelial cell seeding. From the nonessential vessels, endothelial cells have
Special reference will be made to recent exciting research been harvested by two techniques:
methodologies designed to promote endothelial cell attach- 1. Mechanical scraping;13 and
ment to prosthetic graft materials—a technology known as 2. Enzymatic digestion.14,15
electrostatic endothelial cell seeding. The wealth of animal Mechanical scraping uses an abrasive action to remove
studies has been reviewed elsewhere4 and will not be de- the endothelial cells from the vascular wall, which leads to
tailed here. Subsequent chapters in this text will summarize endothelial cell damage as well as the potential for signifi-
clinical outcomes. cant contamination with smooth muscle cells.12,13 The over-
all efficiency of endothelial cell harvest from autologous ves-
Historic Context sels by mechanical methods is < 75%.14 Enzymatic harvest-
The origin of the concept for the vascular prosthesis ing of cells from the vascular tissue requires collagenase or
is credited to Voorhees, Jaretski, and Blakemore, who pre- trypsin digestion to remove the endothelium.16-18 This en-
sented the first published hypothesis on synthetic tubes as zymatic digestion process can create damage to cellular pro-
replacements for natural blood vessel deficits in 1952.5 The teins, which in turn affects cell viability and attachment po-
first clinical applications of this hypothesis were published tential to a vascular prosthetic surface.19,20 The overall effi-
in 1954 and showed that the prosthetic tubes could be used ciency of endothelial cell harvest from autologous vessels
in the arterial setting.6 This publication prompted a race to using the enzymatic harvesting technique varies between
develop the best material for use as a prosthetic vascular graft. 80-100% of the available endothelial cells.14
The two designs of large diameter vascular grafts (> 6 mm Due to the limited availability of nonessential autolo-
I.D.) which are used successfully today clinically are made gous vessels in patients, the ready supply of endothelial cells
of Dacron or expanded polytetrafluoroethylene (ePTFE). harvested by either of the previously mentioned techniques
Patency rates for small diameter (< 6 mm I.D.) vascular pros- is necessarily limited. This limitation has prompted investi-
theses configured from these same materials are unaccept- gators to seek alternative sources which could supply a greater
able when utilized clinically, however, due to acute throm- number of endothelial cells. The alternative sources which
bus formation7,8 and chronic anastomotic hyperplasia9-11 have been most intensively studied include the mesothelium
which consequently prevent clinical usage. Malcolm Her- and the microvasculature, each of which offers the potential
ring, M.D., introduced the hypothesis underlying the tech- for derivation of large cell numbers. Microvascular endot-
nology of endothelial cell seeding in 1978 in an attempt to helial cells can be harvested from microvessels (arterioles,
increase the patency rate of small caliber prosthetic grafts. capillaries, and venules) found in adipose tissue.16,21,22 The
Herring proposed that if these small diameter vascular grafts overall efficiency of isolation of endothelial cells from
could be utilized successfully, they could be of enormous microvessels utilizing enzymatic techniques has been re-
clinical importance in peripheral and coronary bypass graft- ported to be approximately 84% ± 5%.23
ing procedures.12 Tissue culture of endothelial cells offers an alternative
Endothelial cell seeding since its inception has been approach to derive large numbers of cells for transplanta-
largely experimental, with significant technical success in tion from a small inoculum of harvested cells. The tissue
animal models. The technology has not been translated suc- culture approach was first reported by Graham et al.24 Jaffee
cessfully to any extent thus far published in human vascular et al17 initially reported that 92 h were required for an en-
surgery. Surgeons have long expressed concerns about many dothelial cell culture to double in number. However, im-
of the technical issues which must be overcome in order to provements in cell culturing techniques have reduced that
transplant endothelial cells successfully in a relevant time time to approximately 24 h using commercially available
frame for human surgery. Moreover, the long term benefits media (Clonetics Corporation. Unpublished data). Thus,
of a transplanted endothelial cell lining on graft patency in relatively large numbers of endothelial cells can be obtained
patients has not been demonstrated. from low derived inocula of cells in a time span of days to
Many research efforts have focused upon weeks in culture. A number of valid issues with tissue cul-
methodologic issues in an effort to maximize cell harvest- ture have been voiced, however. The exposure of endothe-
Endothelial Cell Seeding: A Review 63
lial cells in culture to an undefined serum (e.g., fetal bovine Hydrostatic seeding techniques use a pressure differ-
serum) as a part of the tissue culturing media presents the ential (internal pressure26,30 or external vacuum31) to force
opportunity for both genotypic and phenotypic modula- harvested endothelial cells resuspended in aliquots of hep-
tion.25 In addition, multiple passages of cells in culture and arinized autologous blood onto the luminal surface of
media changes create vulnerability to cultures becoming microporous graft materials. Experience has revealed at least
contaminated. three major limitations of the hydrostatic seeding techniques.
The first is that this technique is not adequate for fully hep-
Endothelial Cell Seeding Techniques arinized patients due to the graft porosity. In heparinized
Numerous methodologies for endothelial cell trans- patients, heparin interacts with the antithrombin III mol-
plantation onto prosthetic surfaces have been reported in ecule which inhibits the action of thrombin to form subse-
the literature. These investigations can be differentiated and quent clots within the graft pores, which is necessary to main-
summarized by virtue of the unique physical force utilized tain hemostasis.38 Secondly, immediately following the
in each transplantation process to seed endothelial cells to a preclotting technique, the surface may be rough and throm-
vascular prosthetic blood-contacting surface: bogenic.26 The third limitation of this seeding technique is
1. Gravitational;12,26-29 that, as previously mentioned, the endothelial cells adhere
2. Hydrostatic;30,31 and to the graft surface in a spheroid morphology (100% spher-
3. More recently, electrostatic.32 oid shaped) directly from the seeding suspension; thus an
The hypothesis for each of these approaches has been incubation period should be employed (> 2 h) for adhesion
that the transplantation of endothelial cells (initially low maturation prior to implantation.31
surface density, cells/area) on a graft luminal surface will As described above, many investigators have attempted
promote, through time, the development of a mature, physi- to increase the number of endothelial cells adhered to grafts
ologically appropriate, confluent graft luminal endothelial as well as the magnitude of surface adhesion (flattening) by
cell-lined neointima. placing adhesive proteins such as fibronectin on the graft
The most basic of the three seeding techniques that blood-contacting surface to act as a “glue”.39-48 Significant
have been tested utilizes gravitational forces to deliver en- research efforts have focused on glue formulations includ-
dothelial cells to a vascular prosthetic luminal surface. The ing fibronectin, extracellular matrix, collagen, laminin, fi-
basic concept has been to fill the graft with the harvested brin, fibroblast matrix, and plasma.27,40,41,43,45-50 The most
endothelial cells resuspended in tissue culture medium or commonly investigated “glue” has been fibronectin.
blood plasma. The filled graft is maintained horizontally and Fibronectin is an adhesive glycoprotein which is found in
rotated periodically or continuously over a prolonged seed- the basement membrane to which the endothelial cells are
ing time period. Biological glues have commonly been used attached in natural blood vessels. This glycoprotein is re-
with this technique to promote endothelial cell attachment quired to attach the endothelial cells to culture flasks in vitro
to the prosthetic material. One disadvantage of using a glue and it has thus made sense to try it as the primary “glue“ to
is that any nonendothelialized surface becomes more throm- enhance endothelial cell adhesion to other artificial surfaces.
bogenic due to the exposure of blood cells to the glue.8 The problem associated with using fibronectin or any other
A significant disadvantage of these early attempts at “glue” arises from the minimal number of endothelial cells
gravitational endothelial cell seeding was that the trans- which can be harvested relative to the total graft surface area
planted endothelial cells were in a spheroid morphology on to be covered, as well as the inefficiency of the seeding pro-
the graft surface upon completion of the transplantation cedures.51 Any nonendothelialized graft surface or subse-
procedure. Thus, the potential existed for a significant loss quent loss of endothelial cells from the surface upon im-
of endothelial cells from the graft surface when blood flow plantation renders the exposed, fibronectin-treated graft
was restored through the graft if the seeding time was short surface attractive to platelets, thus potentially promoting
(< 2 h) and the cells had minimal time to flatten and ma- thrombotic events that could lead to graft failure. 8
ture. Endothelial cell losses up to 95% in the initial 24 h Ramalanjaona et al40,52 showed that the number of endo-
postimplantation were observed due to this cellular mor- thelial cells adhered was increased and the loss of cells upon
phologic immaturity.33 To overcome this limitation of im- exposure to shear stress was reduced using a fibronectin
mature morphology, attempts were made to endothelialize “glue”. The problem these investigators encountered was that
the graft luminal surface by allowing adhesion to the graft the areas not endothelialized were thrombogenic, and ex-
with subsequent morphological maturation in tissue cul- perimental subjects required anticoagulant therapy to re-
ture (7-14 days in vitro).27,28,34-37 These efforts did in fact duce the complications due to thrombus formation.
result in minimal losses of endothelial cells from the grafts One of the most difficult of the technical issues re-
upon subsequent exposure to blood flow. However, as pre- lated to endothelial cell derivation from autologous sources
viously mentioned, the practicality of this approach remains and subsequent transplantation is the time required for these
of concern to surgeons and researchers. A long tissue cul- processes. It has been our experience that a minimum of
ture period may not be acceptable for a patient who needs 45-60 minutes is required for “immediate” seeding of en-
emergency bypass surgery. In addition, the questions of ge- dothelial cells onto a prosthetic graft being directly implanted
notypic and/or phenotypic changes in cultured endothelial in a patient. This minimum time frame does not allow for
cells as well as the potential for infection (cell culture con- cell flattening and maturation onto the graft prior to resto-
tamination) have been of major concern to investigators in ration of blood flow through the graft. Some investigators
this field. have suggested that a time greater than two hours from
64 Tissue Engineering of Prosthetic Vascular Grafts
harvest to transplantation would most definitely not be ac- these studies indicated that an increasingly positively charged
ceptable clinically, due to the chance of genotypic and/or surface leads to enhanced adhesion, spreading, and
phenotypic changes to the endothelial cells which may oc- magnitude of contact regions. The results on increasingly
cur while they are exposed to media containing undefined negatively charged substrates indicated the inverse, with
serum.25 In addition, it is obvious that longer time periods inhibited adhesion, reduced spreading, and reduced
required for endothelial cell seeding of the vascular pros- contact regions.
theses equates to longer periods of time for patients under
anesthesia, and thus the potential for complications. The Electrostatic Endothelial Cell Seeding
length of time and dosage of anesthesia and its safety are The only conclusion that can be drawn from evaluat-
difficult to generalize due to the fact that exposure compli- ing 15 years of research efforts related to cell seeding tech-
cation encompasses multiple drugs, techniques, anesthetists, niques is that few concrete technical advancements have been
and patients.53,54 made. We have recently proposed and tested a novel device
The necessity for an incubation period for attachment that we feel will be of significant value in improving the ef-
and morphological maturation of transplanted endothelial ficiency of transplanted cell attachment, as well as minimiz-
cells is related to the nature of the electrostatic interactions ing cellular losses upon implantation.32 The technique is
between the endothelial cells and the prosthetic graft mate- called electrostatic endothelial cell seeding. The electrostatic
rials. Endothelial cells are negatively charged.55,56 The clini- seeding technique has been evaluated in vitro using the pro-
cally successful vascular prosthetics (i.e., ePTFE) are highly totype apparatus shown in Figure 3.1. The key to this tech-
negatively charged. This negativity repels platelets which are nique is that it enhances endothelial cell adhesion by induc-
also negatively charged.57-61 Thus, the initial adherence of ing a temporary positive surface charge or a “temporary glue”
transplanted endothelial cells must overcome this negative- on the negatively charged ePTFE graft luminal surface. Fol-
negative charge repulsive force (long range) between cells lowing cell transplantation the ePTFE graft luminal surface
and graft material in order for the seeding procedure to be reverts to its original highly negatively charged surface. Thus,
successful. Experiments using platelets have demonstrated any nonendothelialized graft surfaces or any exposed graft
that the cellular adhesion of platelets on a negatively charged surfaces resultant from endothelial cell losses upon restora-
substrate is one order of magnitude less (ten times) than tion of blood flow remain nonthrombogenic due to the re-
expected by gravitational settling alone due to this electro- stored high negative surface charge of the graft material itself.
static, repulsive, interaction which must be overcome by a The basic issue underlying electrostatic endothelial cell
stochastic process.62-64 Similar short range repulsive inter- seeding is: “How can the surface potential of the graft be
actions alter cellular morphologies by preventing or slow- altered to attract endothelial cells without rendering the sur-
ing morphological maturation even when cells overcome the face thrombogenic?” The electrostatic endothelial cell seed-
repulsive interactions with the material and attach to the ing technique takes advantage of graft material properties
surface. This reality is further demonstrated by endothelial (dielectric material). When a dielectric material is placed
cell and fibroblast studies that have shown the dependence within a capacitor (electrostatic seeding apparatus), the elec-
of cell adherence on the substrate surface charge.65-70 These trons of the atoms and ions which make up the dielectric
studies used varying substrates with varying surface charges material (near surface) are attracted to the capacitor surface
to study cell adhesion, spreading, and contact regions which has accumulated the positive charge. The nuclei of
between the cells and the substrates. The overall results from the dielectric material (near surface) are attracted to the
Fig. 3.1. Prototype electrostatic endothelial
cell seeding apparatus. Features include: (a)
external conductor, (b) internal conductor, (c)
electric motor drive/pulley system, (d) filling d
apparatus, (e) voltage source, (f ) pillow
blocks, and (g) internal conductor end sup- b
ports.
a
f
Endothelial Cell Seeding: A Review 65
negatively charged surface. These small displacements, or necessary fibronectin (< 3 days) and basement membrane
polarizations, are what induce the surface charge on the graft collagen (< 1 week) to maintain cellular adhesion on the
luminal surface. It should be noted that the electrons in a graft for the long term.75-77 Preliminary in vivo studies
dielectric material are not free and the displacements of the (unpublished) using a canine femoral artery implantation
electrons are very slight. Also, the interior volume of the graft model suggests thromboresistance of the electrostatically
material, dielectric, remains unchanged, thus leaving a net seeded grafts.
charge of zero over the dielectric material.71,72
Several in vitro studies have been performed utilizing Genetically Engineering Endothelial Cells
this electrostatic seeding technique. When human umbili- for Seeding Vascular Prostheses
cal vein endothelial cells were transplanted onto 4 mm I.D. It has recently been suggested that genetically engi-
ePTFE using the electrostatic cell seeding technique, a com- neered endothelial cells could be transplanted, which might
plete nodal area coverage of morphologically mature (com- promote the reduction/prevention of anastomotic hyper-
pletely flattened) endothelial cells (73,540 cells/cm2) was plasia and enhance thromboresistance of small diameter
obtained in 16 minutes (+1.0 Volt applied to apparatus) with vascular prostheses. Additional theoretical therapeutic ap-
minimal cellular membrane damage or effect on endothe- plications using manipulated endothelial cells include the
lial cell viability.32 A section of electrostatically seeded ePTFE possibilities of gene replacement, correction, or augmenta-
is illustrated in the scanning electron micrograph in Fig- tion which may be effective in the treatment of atheroscle-
ure 3.2. These in vitro evaluations of the electrostatic seed- rosis. Localized drug delivery may also be feasible due to the
ing technique revealed no significant losses of endothelial direct contact of transplanted endothelial cells within the
cells upon exposure of the graft to a wall shear stress of blood stream.78,79
15 dynes/cm2 for up to 120 minutes immediately after seed- The initial use of transplanted, genetically modified
ing. The majority of endothelial cell loss (up to 30%) occurs endothelial cells is credited to Zweibel et al.80,81 Since that
within the first 30 minutes of implantation using traditional time, the study of endothelial cell transfection (incorpora-
techniques.33,40 Thus, the electrostatic seeding procedure is tion of foreign DNA) using retroviral and electroporation
superior to the gravitational and hydrostatic seeding proce- methodologies has demonstrated long term cell viability as
dures in terms of the seeding time required, magnitude of well as stable transfection using the reporter genes !-galac-
endothelial cell adhesion (attachment), and cellular reten- tosidase, chloramphenicol acetyltransferase, luciferase and
tion. It is also speculated that the actin microfilaments which human tissue plasminogen activator (tPA), growth hormone,
make up the endothelial cell cytoskeleton and possess an urokinase, and nitric oxide synthase genes.82-93 The efficien-
electrostatic potential will also be rearranged by the electro- cies of retroviral and electroporation transfection of endot-
static endothelial cell seeding technique to assist in the main- helial cells are 1-2% and 10%, respectively.85,89 Adenovirus-
tenance of endothelial cell adhesion (act as an anchoring mediated gene transfer is highly dose dependent and pro-
system), although this has not yet been demonstrated in our duces only transient transfection. 87 Transfection and
experiments.73,74 It is also hoped that the enhanced cell endothelial cell seeding are procedures each of which re-
attachment resultant from electrostatic endothelial cell seed- quires long time periods of laboratory work, which again
ing may allow the seeded endothelial cells to synthesize the raises a red flag among the pragmatists concerned with
Fig. 3.2. Scanning electron micrograph im-
mediately after the electrostatic seeding of hu-
man umbilical vein endothelial cells (+1.0
Volt applied for 16 minutes) on ePTFE
(GORE-TEX®; 30 ∝m internodal distance) il-
lustrating the complete, morphologically
mature coverage of the nodal areas (Magnifi-
cation x750).
66 Tissue Engineering of Prosthetic Vascular Grafts
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Conclusion 13. Herring M, Gardner A, Glover J. Seeding human arterial
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that potential has yet been adequately tested, as various tra-
cells. Surgery 1982; 91:550-9.
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Endothelial Cell Seeding: A Review 67
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CHAPTER 4
Surface Precoating in the 1980s:
A First Taste of Cell-Matrix Interactions
J. Vincent Smyth, Michael G. Walker
T echnical difficulties and the frequent lack of available saphenous vein for peripheral arte-
rial reconstruction resulted in the development of a variety of prosthetic materials from
which grafts could be constructed. Of these, polyester elastomer (Dacron) and expanded
polytetrafluorethylene (ePTFE) emerged as the most successful in clinical practice, and re-
main leaders in the field today. However, small caliber prosthetic grafts have consistently
shown lower patency rates, especially when passing across the knee joint, when compared
with autologous vein conduits. The understanding that the endothelial lining of vein grafts
was responsible for the improved patency rates achieved in peripheral bypass naturally led
to attempts to create such a lining on prosthetic grafts, when explants showed that no sig-
nificant endothelialization of such grafts occurs naturally in humans, contrary to the results
seen in animals where virtually complete endothelial linings develop on prosthetic grafts in
a matter of weeks.
Almost all of the work performed up to 1980 utilized animal tissue models such as
bovine, canine or murine endothelium, and extremely promising results had been repeat-
edly achieved both in vivo and in vitro in terms of reduction in graft thrombogenicity, resis-
tance to infection, accelerated re-endothelialization and improved patency.1-6 In order to
make the transition to clinical practice, however, it was clear that human cell lines would be
required, and human umbilical vein endothelial cells (Huvecs) were used as the principal
experimental model until Jarrell showed that human adult endothelial cells (HAECs) could
be harvested from saphenous veins and cultured.7 This work has now advanced so that the
acute enzymatic harvesting of autologous vein can be routinely achieved, with virtually com-
plete denudation of the donor vein segment and a large proportion of harvested cells re-
maining viable for culture. There is now extensive experience with both cell lines toward the
desired end point of successful graft seeding to the rapid development of an endothelial
monolayer that resists the shear stress of flow.8
Almost as soon as the successful culture of Huvecs on prosthetic graft material was
first reported, it was clear that human cell lines were more fragile and fastidious in their
requirements than the canine or bovine endothelial cultures that had principally been used
up to this point. The establishment of a culture was slower even under optimal conditions,
and when achieved these were less hardy and prone to failure from infection. Even so, suc-
cessful culture was reported on all the common graft materials, with typical appearances of
cobblestone morphology and cells expressing endothelium-specific markers such as vWF.
Although human cell lines showed slower cell division and spreading across the culture
surfaces than animal cell lines, even under the optimal experimental conditions of tissue
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
70 Tissue Engineering of Prosthetic Vascular Grafts
culture, the main reason for the delayed development of the grafts, up to 20 times as many cells as were required to form
monolayer was found to be low cell attachment to the graft. a monolayer would be needed for seeding, thus greatly in-
creasing the required cell numbers. Although this could be
Endothelial Cell Attachment accommodated by the use of preculture in the laboratory,18
to Prosthetic Grafts this method is not compatible with routine surgical prac-
The first seeding experiments were carried out in what tice and would necessitate a preliminary operation for vein
we would now classify as a static system, where the cells were harvest and carry risks of disease transmission or graft in-
introduced to the graft surface in a suspension of culture fection. To overcome the problem of limited cell harvests,
medium and allowed to settle on the graft over a period of the use of microvascular (capillary) endothelium19 and me-
time, then cultured in situ. Early work relied on qualitative sothelial20 cells have been proposed, but these only offer a
phase contrast microscopy of the seeded surface at intervals partial solution in terms of available cell numbers, as they
after seeding to assess degree of coverage. Later, the work of share many of the attachment and adhesion difficulties ex-
Sharefkin led to the use of the radio-isotope 111Indium as perienced with Huvecs and HAECs, and raise additional
an endothelial cell label,9 and attachment to grafts could be problems with tissue availability. As a consequence, to achieve
accurately quantified. It was discovered that prosthetic grafts rapid development of confluence in the clinical arena where
exhibit a uniformly poor surface for retention of seeded cells, such large cell numbers could not be obtained acutely using
typically around 4% for ePTFE,10,11 while cell adherence to established methods, either new sources of endothelial cells
plain Dacron was negligible because of its porosity, result- would be required or cell attachment significantly improved
ing in leakage of the seeding solution.12,13 Three approaches to permit successful seeding at subconfluent densities.
to the problem of rapidly developing a monolayer were It had been noticed that the results of human cell seed-
explored. ing work did not parallel those seen in the animal in vivo
The proportion of seeded cells that remained attached models of prosthetic graft seeding. Significantly better at-
to the graft could be increased by increasing the length of tachment was seen in the latter, and it was realized that the
time that the seeding suspension was exposed to the graft preclot typically used for the endothelial cell suspension
material.10,14-16 However, the seeding kinetics indicated that acted as a graft coating, improving cell adherence. By using
the rate of increase fell off after around 30 minutes, and that preclot or serum to coat the graft prior to seeding the en-
even with exposure times of over 60 minutes, the develop- dothelial cells, attachment rates could be dramatically im-
ment of a confluent monolayer was not significantly accel- proved to over 60% of seeded cells in a range of models.10,21
erated. Additionally, prolonged incubation times are incom- As a result, investigators began precoating the culture
patible with the constraints of the operating room. dishes and seeding surfaces with a variety of matrices before
Using more endothelial cells per graft area in the seed- the cells were exposed, and similar improvements in cell at-
ing solution, a higher seeding density, produced a greater tachment were seen11,22-27 (Figs. 4.1-4.3). Comparative stud-
number of cells attaching to the graft, but only in the same ies of a range of potential graft surface coatings were made.
proportion and consequently with an enormous waste of Because of Dacron’s inherent porosity, gelatin or collagen-
seeded cells.15,17 At established attachment rates to plain sealed grafts were frequently used, and this not surprisingly
Fig. 4.1. Cell attachment to pros-
thetic grafts as a function of graft
coating.
Surface Precoating in the 1980s: A First Taste of Cell-Matrix Interactions 71
Fig. 4.2. Electron micrograph of uncoated
prosthetic graft.
Fig. 4.3. Electron micrograph of
fibronectin-coated graft after endothe-
lial seeding.
improved initial cell adherence accordingly, as did all graft plasma show good rates of endothelial attachment, this is
coating matrices compared to the plain graft materials. thought to be due to their high FN content. Laminin and
Histological examination of the vascular endothelium collagens I, III and IV have not proved as effective as FN in
indicated that in the body it was supported by a protein- facilitating endothelial cell attachment. Gelatin-sealed
rich basement membrane layer, and that this layer was pro- Dacron grafts show good initial cell adherence, but the sta-
duced actively at least in part by the endothelium. Examina- bility of the gelatin crosslinking under physiological condi-
tion of endothelial cells in culture showed that the adherent tions is uncertain, as it degrades and may not provide a suit-
cells secreted a similar layer on artificial surfaces28,29 and that able matrix for long enough to allow the cells to establish
a principal component of the basement membrane was their own FN-rich basement membrane layer on the graft.
fibronectin (FN). Fibrin glue has been used successfully in some seeding work,
Following the work of Ramalanjoana21 and others,30-32 but requires fibrinolytic inhibition prior to use without of-
FN has become the most commonly used seeding matrix fering any advantages in terms of cell retention. In the labo-
because of efficacy and availability, giving consistently high ratory model, however, almost equivalent results to those
cell adherences in experiments using a variety of graft types, with FN have been achieved and, in retrospect, it is clear
including ePTFE and Dacron. Preclot, serum, plasma, that one reason for the success of the early animal seeding
laminin, gelatin, collagen and fibrin glue have also been in- experiments using prosthetic grafts had been the serendipi-
vestigated,10,27,33-36 the first three because they are readily tous use of FN-rich preclot as a readily available cell sus-
available in the clinical situation, and the remainder because pension to seed the graft, sealing the porous Dacron used in
they, along with fibronectin, are found as native constitu- this work.
ents of extracellular matrix. Although preclot, serum and
72 Tissue Engineering of Prosthetic Vascular Grafts
Many workers have investigated the attachment kinet- Following flattening, where incomplete confluence
ics of endothelial cells to FN-coated graft surfaces.10,15,27,37 results from seeding, the endothelial cells then migrate across
In the laboratory, the optimum incubation time for cell at- the seeding surface to produce a monolayer. By some elegant
tachment to a coating matrix appears to be 30 minutes with work using removable discs, the migration of saphenous vein
no significant benefit evident beyond this, though some endothelial cells following establishment of healthy cultures
workers have found as little as 15 minutes adequate. As with was measured on a variety of culture surfaces.41 These
uncoated grafts, improved cell attachment is also seen with showed that surface precoating not only improves initial
increasing the seeding density, up to a point whereafter the adherence, but also promotes cell migration and develop-
proportion of seeded cells attaching falls off, though the ment of confluence. Again, fibronectin was the most effec-
absolute number continues to increase. The kinetics of im- tive coating agent, with gelatin and extracellular matrix
proved cell adherence to FN-coated graft surfaces implied showing some advantage over uncoated culture flasks. In-
some specific binding mechanism, and this was confirmed terestingly, laminin, despite being a constituent of the cell
by the finding that endothelial cells express specific FN re- substratum in vivo, did not improve cell migration, imply-
ceptors on their cell membrane. ing that its role in the body is not primarily concerned with
Comparative studies of different grafts seem to sug- maintenance of endothelial monolayer integrity. In addition
gest that ePTFE is a better surface for matrix coating than to enhancement of cell-surface adhesion, seeded cells also
Dacron, although there is some disagreement between indi- proliferate more rapidly on precoated surfaces,22 and some
vidual studies. This may be related to the nature of the graft groups developed variations of the surface coatings specifi-
surface at the fiber level, with a flatter surface being offered cally designed to improve graft endothelialization by pro-
by the multinodal fibrillary construction than the woven or moting cell migration and proliferation.42 Coatings enriched
knitted Dacron. Minor refinements in terms of graft com- with growth factors such as FGF have been shown to en-
position, pore size, internodal distances or woven vs. knit- hance ingrowth through the graft interstices43 and the de-
ted Dacron, do not seem to affect the cell seeding kinetics velopment of an endothelial lining with a minimum of
significantly. On precoated grafts, the differences in endo- smooth muscle hyperplasia.44 The ability of small diameter
thelial cell adherence between graft materials is not striking vascular prostheses to adsorb and retain ECGF under flow
once the porosity of the Dacron is reduced by sealing the conditions has been clearly demonstrated45 though in the
graft with the surface coating, and this can be explained by rabbit model used, no differences in graft re-
the production of essentially similar attachment character- endothelialization was seen at 1 month. Although the com-
istics at the molecular level on the surfaces to which the bination of growth factor-enhanced graft coatings and hu-
seeded endothelial cells are exposed. man endothelial cell seeding would appear to be a promis-
With the establishment of a FN-rich coating as the ing avenue of investigation, there have been no reports of
optimal seeding surface, several workers have investigated such work.
the adsorption kinetics of FN onto prosthetic grafts,38,39
showing that the development of FN-graft binding is pro- Endothelial Cell Retention
portional to the FN concentration and the duration of in- Under Flow Conditions
cubation, though there may be minor differences between The advantages of precoated over plain grafts also ex-
graft materials. The optimum concentration of FN to use as tend to dynamic seeding systems, in which the seeded grafts
the seeding matrix has been investigated,40 and the relation- are exposed to the shear stress of flow, though different
ship between the concentration of FN graft coating and rates groups have achieved wide variation in results. A range of
of endothelial cell adherence approximates first order ki- levels of flow, and thus shear stresses, have been used, in-
netics to 50 ∝g/ml, with no benefit obtained by increasing cluding systems that approximate rates of flow seen in
this to 250 ∝g/ml, further evidence for specific FN-binding grafts.46,47 Electron microscopy of seeded grafts exposed to
sites on the cell surface. pulsatile flow systems show that a proportion of endothelial
loss occurs in areas where the underlying graft is exposed by
Cell Migration loss of surface coating,48 and that this is more marked with
It had been noted that following seeding, cells initially FN-coating than with preclot, which may account for the
attached to the graft in a rounded morphology, later devel- differences some workers have seen in levels of cell reten-
oping a more flattened phenotype responsible for the tion. The monolayer’s resistance to denudation under flow
‘cobblestone’ appearance of the mature endothelial mono- is a combination of the strength of the endothelial cell at-
layer.32 Increased duration of incubation was associated with tachment to the matrix, and of the matrix to the graft. With
progression of the cell morphology from rounded and poorly regard to the former, factors previously observed to improve
attached, to flattened and adherent, and this was generally cell adherence such as duration of incubation, and also de-
accepted to indicate the acclimatization of the seeded cell to gree of confluence and interval prior to commencement of
the seeding surface. The speedier development of an adher- flow, are also relevant to resisting denudation under flow.
ent monolayer following seeding onto prepared graft and Established endothelial monolayers formed by culture on
culture surfaces was thought to correspond with the more the graft resist flow better than acutely attached cells,46,49,50
rapid establishment of the flattened phenotype, as noted although previously noted problems with duration of incu-
microscopically.16,33 bation or graft preculture apply in terms of clinical applica-
bility. Cell loss appears to occur in a biphasic pattern,47,51
Surface Precoating in the 1980s: A First Taste of Cell-Matrix Interactions 73
with an initial rapid rate presumably corresponding to tachment and retention on autologous native surfaces might
loosely attached cells or areas of coating, which settles after be expected to be optimal.
a few minutes to a slower, more steady rate of loss (Fig. 4.4). Seeding after endothelial denudation by angioplasty
However, we have noted that within a flow system sudden balloon has been assessed in a rabbit iliac vein model52 us-
changes in the level of pulsatile shear stress result in a further ing a double balloon catheter-based system and has shown
period of rapid loss which then returns to the slower rate. that acute seeding with autologous cells on a relatively short
The bond between coating and graft has been less thor- time scale can restore a monolayer,53 which is maintained
oughly investigated, but seems to depend on the nature of on restoration of flow. This monolayer retains functional
the graft, with gelatin-coated Dacron performing signifi- integrity54 and may reduce neointimal hyperplasia.55 How-
cantly less well than ePTFE.38,40 Whether the FN-gelatin or ever, the relevance of these results to chronic arterial disease
the gelatin-Dacron interface is the important one is as yet in humans remains uncertain, especially with previous ex-
uncertain, but clinically may not be particularly important, perience of promising animal seeding studies. Endothelial
as ePTFE is the usual graft of choice in the infrainguinal cell seeding following endarterectomy in animals has also
region where it is expected that seeding will be most appli- given hope, demonstrating most of the advantages seen in
cable. Up to 75% of applied matrix may be lost in the first seeded prosthetic grafts.56-60
15 minutes after restoration of flow, but thereafter the graft- Work using human cell lines has demonstrated excel-
matrix bond appears stable within the time scale of the labo- lent cell attachment to both endarterectomy and vein
ratory model. Electron microscopy of coated grafts after angioplasty surfaces,61-63 and in the latter model flow stud-
exposure to flow shows clearly that this early loss occurs at ies have suggested cell retention kinetics similar to coated
least partly by detachment of regions of matrix from the grafts. Following endarterectomy the exposed surface is very
graft, presumably where the matrix-graft bond is less estab- similar to that on which the endothelium normally rests and
lished, with matrix solubility probably contributing more is high in FN, which probably accounts for the optimal at-
towards the later, slow rate of matrix loss. tachment rates seen, and HAECs seeded onto endarter-
ectomized vessels have been shown to be capable of form-
Seeding of Native Vascular Surfaces ing a monolayer,61 again retaining functional integrity.64
The difficulties in achieving an endothelial monolayer
on prosthetic grafts immediately after seeding, given the al- Mechanism of Cell Adherence
most inevitable shortfall in numbers of seeded cells, ethical As a result of the improvements in seeding efficiency
and technical problems with cell or graft preculture, and the produced by surface coatings, increasing interest has devel-
high losses from even FN-coated graft surfaces under flow, oped in the interactions between endothelial cells and the
have prompted workers to examine the seeding potential of matrix. The attachment and migration characteristics of the
native arterial surfaces. The use of autologous cells and na- endothelial cell are due to the presence of a wide range of
tive conduits avoids any potential immune or foreign body adhesion molecules and ligand-specific receptors on the cell
reaction which might result in cell loss, and seeding follow- membrane.65 Many of the receptors have multiple, overlap-
ing angioplasty or endarterectomy have both been consid- ping roles in different pathways, or respond to more than
ered. The advantages of seeding such regions are that the one signal molecule. Variation in ligand affinity and pat-
area for seeding is lower due to shorter segments being in- terns of receptor activation are common and the degree of
volved, thus increasing the seeding density without needing complexity of the intracellular responses to the extracellu-
an increase in the numbers of seeded cells, and that cell at- lar environment is only now becoming more fully
Fig. 4.4. Cell retention on seeded grafts under flow.
74 Tissue Engineering of Prosthetic Vascular Grafts
appreciated. Some receptors are endothelial cell specific, tory mediators, ICAM-2 and ICAM-3 are constitutively pro-
some are shared with other vascular cells such as platelets or duced, but all bind to circulating leukocytes. PECAM-1 is
leukocytes, and some also occur on other vessel wall cell types constitutively produced and binds platelets in addition.
including smooth muscle cells and fibroblasts. Because of Again, these molecules are principally involved in the en-
their differential actions, different classes of adhesion mol- dothelial response to inflammation, and these groups are
ecules are frequently asymmetrically arranged between the therefore not further considered.
luminal and abluminal cell membrane (Fig. 4.5). Cell-substratum interactions such as the endothelial
There are three main families of adhesion molecules cell adherence to prosthetic grafts and coatings are medi-
present on endothelial cells, though classification is amended ated by the integrin family of adhesion molecules,66 which
from time to time as new members are continually being are 10- to 100-fold more common on the cell membrane
identified and characterized, and many are known by more than selectins or immunoglobulin-like adhesion molecules,
than one name. The three major adhesion molecules fami- and are present principally on the abluminal surface.
lies consist of the selectins or leukocyte adhesion molecules Integrins are heterodimeric molecules, each subtype being
(ELAMs), responsible for initial capture of circulating leu- composed of a noncovalently bonded pair of # and ! sub-
kocytes in response to inflammatory mediators; the immu- units, each of which has multiple variants. There are several
noglobulin superfamily, also implicated in cell-cell interac- subclasses, distinguished by their ! subunit, though their
tions; and the integrins, involved in both cell-substratum specific ligand is to a large extent determined by the # sub-
and cell-cell interactions. unit. They bind with relatively low affinity to their ligands,
The selectins present on endothelial cells comprise two made up for in large part by their frequency of expression,
major groups; E-selectin or ELAM-1, and P-selectin or gran- allowing multiple weak bonds to the substratum rather than
ule membrane protein-140 (GMP-140). These are induced a few strong links, and thus facilitating cell spread and mi-
by inflammatory mediators and promote the adherence of gration without loss of firm adhesion, the ‘Velcro’ theory of
leukocytes to affected regions of endothelium via leukocyte cell attachment. Ligand binding is cation dependent, Ca2+
surface ligands such as fucose groups. They are located on or Mg2+ depending on the integrin, bound to specific sites
the luminal membrane surface and play little part in endo- on the # subunit.
thelium-substratum interactions. The immunoglobulin su- The VLA group, identified by a !1 subunit, includes
perfamily contains several classes, differing in the length and specific receptors found on the abluminal endothelial cell
composition of a string of immunoglobulin domains which membrane for fibronectin, laminin and collagen. A !2 sub-
lie on the luminal side of the endothelial cell membrane, unit identifies the leucam group which are found on circu-
attached to a peptide chain that extends through this to a lating inflammatory cells and bind to ICAMs on endothe-
cytoplasmic terminal carboxyl group. They include the in- lial cells. The cytoadhesin group of integrins, with a !3 sub-
tercellular adhesion molecules (ICAMs) -1, -2, and -3; vas- unit, includes low specificity, multiligand receptors binding
cular cell adhesion molecule-1 (VCAM-1); and platelet en- fibrinogen, fibronectin, vitronectin, thrombospondin,
dothelial cell adhesion molecule-1 (PECAM-1). ICAM-1 and laminin and vWF. Individual moieties with !4-!8 subunits
VCAM-1 are inducible in response to circulating inflamma- binding a variety of basement membrane proteins, includ-
ing those mentioned previously, have also been identified.
Fig. 4.5. Vascular endothelial cell
adhesion molecules.
Surface Precoating in the 1980s: A First Taste of Cell-Matrix Interactions 75
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of human collagen surfaces with human adult endothe- seeding of damaged native vascular surfaces: Prostacyclin
lial cells. Am J Surg 1985; 150:197-200. production. Eur J Vasc Surg 1992; 6:487-493.
35. Dalsing MC, Kevorkian M, Raper B et al. An experimen- 55. Thompson MM, Budd JS, Eady SL et al. The effect of
tal collagen-impregnated Dacron graft: Potential for en- transluminal endothelial seeding on myointimal hyperpla-
dothelial seeding. Ann Vasc Surg 1989; 3(2):127-133. sia following angioplasty. Eur J Vasc Surg 1994; 8:423-434.
36. Zilla P, Fasol R, Preiss P et al. Use of fibrin glue as a 56. Bush HL, Jakubowski JA, Curl GR et al. Luminal healing
substrate for in vitro endothelialization of PTFE vascular of arterial endarterectomy: Role of autogenous endothe-
grafts. Surgery 1989; 105:515-522. lial cell seeding. Surg Forum 1985; 36:446-450.
37. Mosquera DA, Goldman M. Endothelial cell seeding. Br J 57. Bush HL, Jakubowski JA, Sentissi JM et al. Neointimal
Surg 1991; 78:656-660. hyperplasia occurring after carotid endarterectomy in a
38. Carr HMH, Vohra R, Welch M et al. Fibronectin binding canine model: Effect of endothelial cell seeding vs
to gelatin-impregnated Dacron (Gelseal) prostheses. Artif perioperative aspirin. J Vasc Surg 1987; 5:118-125.
Organs 1992; 16:342-345. 58. Schneider PA, Hanson SR, Price TM et al. Confluent du-
rable endothelialisation of endarterectomised baboon aorta
Surface Precoating in the 1980s: A First Taste of Cell-Matrix Interactions 77
by early attachment of cultured endothelial cells. J Vasc 72. Eskin SG, Ives CL, Frangos JA et al. Cultured endothe-
Surg 1990; 11:365-372. lium: The response to flow. ASAIO J 1985; 8:109-112.
59. Krupski WC, Bass A, Anderson JS et al. Aspirin-indepen- 73. Kugiyama K, Sakamoto T, Misumi I et al. Transferable
dent antithrombotic effects of acutely attached cultured lipids in oxidized low-density lipoprotein stimulate plas-
endothelial cells on endarterectomised arteries. Surgery minogen activator inhibitor-1 and inhibit tissue-type plas-
1990; 108:283-291. minogen activator release from endothelial cells. Circ Res
60. Sterpetti AV, Schultz RD, Bailey RT. Endothelial cell seed- 1993; 73:335-343.
ing after carotid endarterectomy in a canine model re- 74. Yamamoto C, Kaji T, Sakamoto M et al. Effect of
duces platelet uptake. Eur J Vasc Surg 1992; 6:390-394. endothelin on the release of tissue plasminogen activator
61. Smyth JV, Rooney OB, Dodd PDF et al. Culture of hu- and plasminogen activator inhibitor-1 from cultured hu-
man endothelial cells on endarterectomy surfaces. Eur J man endothelial cells and interaction with thrombin.
Vasc Endovasc Surg 1995; 10:308-315. Thromb Res 1992; 67:619-624.
62. Walluscheck KP, Steinhoff G, Haverich A. Endothelial cell 75. Loskutoff DJ, Ny T, Sawdey M et al. Fibrinolytic system
seeding of native vascular surfaces. Eur J Vasc Endovasc of cultured endothelial cells; regulation by plasminogen
Surg 1996; 11:290-303. activator inhibitor. J Cell Biochem 1986; 32:273-280.
63. Thompson MM, Budd JS, Eady SL et al. Effect of seeding 76. van Hinsbergh VWM, Bertina RM, van Wijngaarden A et
time and density on endothelial cell attachment to dam- al. Activated protein c decreases plasminogen activator
aged vascular surfaces. Br J Surg 1993; 80:359-362. inhibitor activity in endothelial cell-conditioned medium.
64. Smyth JV, Rooney OB, Dodd PD et al. Production of plas- Blood 1985; 65:444-451.
minogen activator inhibitor-1 by human saphenous vein 77. van Hinsbergh VWM, Kooistra T, van den Berg EA et al.
endothelial cells seeded onto endarterectomy specimens Tumor necrosis factor increases the production of plas-
in vitro. J Vasc Surg 1997; 25:722-725. minogen activator inhibitor in human endothelial cells in
65. Mousa SA, Cheresh DA. Recent advances in cell adhesion vitro and in rats in vivo. Blood 1988; 72:1467-1473.
molecules and extracellular matrix proteins: Potential 78. Hanss M, Collen D. Secretion of tissue-type plasminogen
clinical implications. DDT 1997; 2:187-199. activator and plasminogen activator inhibitor by cultured
66. Extracellular matrix receptors on animal cells: The human endothelial cells: Modulation by thrombin, endo-
integrins. In: Alberts B, Bray D, Lewis J et al. Molecular toxin and histamine. J Lab Clin Med 1987; 109:97-104.
biology of the cell. 3rd ed. New York: Garland Publish- 79. Lundgren CH, Herring MB, Arnold MP et al. Fluid shear
ing, Inc, 1994:995-1000. disruption of cultured endothelium: The effect of cell spe-
67. Sterpetti AV, Crucina A, Napoli F et al. Growth factor cies, fibronectin crosslinking and supporting polymer.
release by smooth muscle cells is dependent on ASAIO Trans 1986; 32:334-338.
haemodynamic factors. Eur J Vasc Surg 1992; 6:636-638. 80. Ives CL, Eskin SG, McIntire LV et al. The importance of
68. Greisler HP, Henderson SC, Lam TM. Basic fibroblast cell origin and substrate in the kinetics of endothelial cell
growth factor production in vitro by macrophages exposed alignment in response to steady flow. ASAIO Trans 1983;
to Dacron and polyglactin 910. J Biomater Sci Polym Ed 29:269-274.
1993; 4:415-430. 81. Li JM, Menconi MJ, Wheeler HB et al. Precoating ePTFE
69. Greisler HP, Dennis JW, Endean ED et al. Macrophage/ grafts alters production of endothelial cell derived
biomaterial interactions: The stimulation of endo- thrombomodulators. J Vasc Surg 1992; 15:1010-1017.
thelialization. J Vasc Surg 1989; 9:588-593. 82. Christ G, Seiffert D, Hufnagl P et al. Type 1 plasminogen
70. Sumpio BE, Widmann MD, Ricotta J et al. Increased activator inhibitor synthesis of endothelial cells is
ambient pressure stimulates proliferation and morphologi- downregulated by smooth muscle cells. Blood 1993;
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1994; 158:133-139. 83. Scott-Burden T, Vanhoutte PM. Regulation of smooth
71. Iba T, Sumpio BE. Tissue plasminogen activator expres- muscle cell growth by endothelium-derived growth fac-
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Cell Transpl 1992; 1:43-50.
CHAPTER 5
Surface Precoating in the 1990s:
The Fine Tuning of Endothelial Cell Transplantation
Mark M. Samet, Victor V. Nikolaychik, Peter I. Lelkes
Introduction
E ach year 900,000 people in the USA alone suffer from arterial disorders requiring some
form of surgical intervention. Over half of these procedures involve peripheral recon-
struction using vascular grafts to bypass an occluded vessel segment. However, 60% of the
patients undergoing vascular surgery do not have a suitable vessel for grafting and, there-
fore, need synthetic grafts.1 In this realm, about 350,000 artificial vascular grafts are im-
planted annually, and the numbers are expected to rise rapidly in the future. In spite of the
increasing demand for vascular prostheses, the long term success of their use as arterial
substitutes is, at best, mixed.2,3 In artery bypasses, with bore sizes + 5 mm in diameter Dacron®
and ePTFE grafts demonstrate cumulative patency rates similar to those of the natural grafts.1
But, as small artery substitutes, these prostheses have performed poorly and, to date, no
vascular prosthesis of , 4 mm in diameter remains patent for more than a couple of months.1
The high failure rates of small diameter prosthetic grafts are believed to be intimately asso-
ciated with the poor healing and rejection process of the implanted conduit.3-7 One of the
key determinants in this “cause and effect” relationship is the problem of insufficient
hemocompatibility of the polymeric graft material.8
Insufficient hemocompatibility of the biomaterials is also a pressing issue in the field
of cardiac prostheses. Each year, end-stage heart diseases claim some 75,000 victims in the
USA alone. Although 28,000 of these patients qualify for heart transplants, the supply of
donated hearts is limited to approximately 2,000 hearts per year.9,10 Thus, the majority of
these patients has no realistic hope of long term survival, unless permanent total artificial
hearts (TAH) or ventricular assist devices (VAD) become available.
The vision of developing a permanent cardiac prosthesis is more than a half century
old. The first practical implementation of this idea was achieved in the 1950s by W.J. Kolff
and his collaborators, who kept a dog alive for 90 min after successfully replacing the native
heart with an artificial device.11 By the mid-1970s, the first generation of TAHs and VADs
was undergoing advanced clinical trials.11-14 These units were activated externally by pneu-
matic drivers and connected trough transcutaneous pressure lines. Besides restricting the
patient’s postoperative quality of life, these lines served as a major source for bacterial infec-
tion and initiated thromboembolic complications. The newest generation of cardiac pros-
theses, designed to be totally implantable, are powered by a compact electrical source.14 In
spite of significant mechanical and electronic improvements in the new designs, the perma-
nent or even temporary clinical use of cardiac prostheses as a bridge to recovery15 is still
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
80 Tissue Engineering of Prosthetic Vascular Grafts
thwarted by serious obstacles such as infections and throm- lium by mechanical forces.38,39 Many investigators, however,
boembolic complications which are manifested in stroke and turned to surface treatment with select extracellular matrix
multiple organ failures.16,17 As in the case of artificial vascu- (ECM) proteins or blood plasma proteins (“fibrin glue”),
lar grafts, many of the complications with cardiac prosthe- with or without supplements, as a means for improving cel-
ses can be traced back to the problem of inadequate lular performance in grafts.40,41 Our ongoing studies focus
hemocompatibility of the blood-contacting biomaterials in on surface coating as a means for optimizing endo-
these devices. Thus, it follows that if we can successfully over- thelialization.42,43 Specifically, our motivation is to form a
come the blood compatibility barrier, we shall markedly multifunctional protein complex that will perform as a
improve the clinical prospects of both artificial hearts and biointegrator between the living cells and the underlying
small diameter prosthetic grafts. polymeric scaffold in cardiac prostheses.
Endothelialization: Fine Tuning of Endothelialization
Past and Current Approaches via Surface Coating
The interaction of blood with “medical-grade” The best biointegrating interface for ECs is their na-
biomaterials commonly results in thrombus formation tive basement membrane which, in vivo, contributes to the
and/or thromboembolic complications. After decades of dis- maintenance of the architectural and functional integrity of
appointing attempts to passivate the synthetic blood-con- the endothelium.44 This basement membrane is comprised
tacting surfaces by chemical modifications or by generation of structural proteins such as collagens, as well as adhesive
of a pseudoneointima,15,18 the focus has turned towards es- proteins such as laminin (LM) and fibronectin (FN). These
tablishing on the synthetic blood-contacting surfaces and several other ECM proteins regulate, via RGD (argin-
“nature’s own blood-compatible container”,19 i.e., recon- ine-glycine-aspartic acid)-based integrin-mediated recep-
structing the endothelial cell (EC) lining of the vascular wall. tion, cell adhesion and spreading.45 In addition, many of
The rationale for covering the prosthetic luminal surface with these ECM molecules are also actively synthesized during
a viable layer of ECs, i.e., endothelialization, is rather simple: cell proliferation/migration.46 Using ECM-contained adhe-
in vivo, ECs present under normal physiologic conditions sive proteins, various researchers have employed FN, LM,
an anti-thrombogenic surface which is pivotal to the pre- or collagen iv (Col IV), or a combination thereof, as a sur-
vention of fibrin deposition and inhibition of platelet adhe- face coating to facilitate endothelialization of bio-
sion on the vessel wall. Thus, it has been reasoned that materials.40,47,48 Similarly, short RGD peptides, or their mim-
endothelialization of cardiovascular prostheses would reduce ics, have been covalently coupled to the PU backbone to
the thrombogenicity of these devices. In addition, adverse achieve the same goal.49
immunogenic responses from the host would be avoided by We revisited the effectiveness of select ECM-contained
transplanting autologous cells. Indeed, these principles were adhesive monoproteins to promote cells growth on PUs. For
successfully demonstrated more than two decades ago in this purpose we seeded bovine aortic ECs (BAEC) at a den-
artificial grafts.20 However, in 1975, the art of procuring and sity of 25,000 cells/cm2 onto PU substrates and monitored
culturing endothelial cells was in its infancy, and very little their growth for several days. As a control, we cultured BAECs
was known about EC-biomaterial compatibility. Since then, on tissue culture grade polystyrene (TC) in 24 well plates
together with significant advances in endothelial cell biol- (Costar, Cambridge, MA). The PU samples were made of
ogy, molecular biology, and biotechnology,21,22 a fairly thor- Chronoflex, and fitted into the wells of 24 well plates as pre-
ough understanding has been gained of the basic require- viously described.50 The surface of the PUs was either left
ments for successfully growing endothelial cells on the uncoated or was coated by passive adsorption for 1 h with
biomaterials used in cardiovascular prostheses: PTFE, ePTFE, FN (20 ∝g/ml, pH=7.4) or Col IV (300 ∝g/ml, pH=3.0) prior
Dacron®, and various brands of polyurethanes (PUs).23-26 to cell seeding. The results of this study are summarized in
In recent years, endothelialization of vascular graft surfaces Figure 5.1a. Each data point (mean ± standard deviation)
with autologous ECs may have solved several important represents the number of cells per cm2 from 3 independent
problems in cardiovascular surgery27-31 and paved the way experiments (6 wells per case). As expected, the cells grew
for the development of new areas, such as biohybrid organs32 poorly on plain (uncoated) PU, and their number by day
and gene therapy.33-36 Thus, at present, the question is no nine was significantly lower than in controls (0.92 ± 0.1 x
longer whether or not endothelialization is feasible, but, 105 vs. 1.96 ± 0.8 x 105, p < 0.05). Proliferation of BAEC
rather, how it can be optimized. improved significantly upon coating the PUs with either FN
Optimization, or fine tuning of endothelialization is or Col IV, but it did not match the rate of growth on TC.
a multifaceted task. It involves, but is not limited to, improve- Interestingly, there was no substantial difference in the rate
ment of endothelial cell attachment and growth on the syn- of growth between cells cultured on FN or Col IV coatings.
thetic surface, enhancement of cell adaptation to and sub- In fact, on day 9, the number of cells in both cases was al-
sequent endurance in a hemodynamic environment, and fi- most equal (1.77 ± 0.87 x 105 on FN vs. 1.69 ± 0.11 x 105 on
nally, promotion of the anti-coagulant repertoire of the en- Col IV, p=0.425). Thus, our data confirm the findings of
tire EC lining. Several researchers have used genetically others that biomaterials coated with ECM proteins are more
modified ECs as a vehicle for achieving these goals,23,33 while cytocompatible than their untreated counterparts.40,51,52
others focused on increasing the hydrophilicity of the syn- Moreover, our results also imply that in coating the poly-
thetic scaffold37 or preconditioning the cultured endothe- meric scaffold with only one kind of ECM adhesive protein,
Surface Precoating in the 1990s: The Fine Tuning of Endothelial Cell Transplantation 81
the particular type of this protein is of secondary impor- biointegrated proteinaceous meshwork at the PU surface
tance for ECs grown in a static environment. which would structurally mimic the topology of the suben-
Subsequently, we examined whether the effectiveness dothelial basement membrane and also provide space for
of a particular surface coating in promoting cell growth may the incorporation of the endogenous, EC-derived extracel-
depend on the method of protein immobilization onto the lular proteins. Such a provisional ECM meshwork might be
polymeric surface. To this end, we seeded BAEC at a density found in fibrin glue, the reaction product of fibrinogen and
of 25,000 cells/cm2 onto PUs coated by different means with thrombin.
ECM proteins, and monitored their growth for several days. In the past, fibrin glue was shown to be beneficial in
The PU samples consisted of 4 separate groups: supporting both EC attachment and growth on synthetic
Group #1: PU coated with FN by adsorption. The materials40,42,43,55-57 and for establishing a stable, shear-re-
starting concentration of FN was 20 ∝g/ml (pH=7.4). sistant monolayer on ePTFE grafts.58 To improve this coat-
After 1 h of incubation at 37°C, the final concentra- ing we substituted purified fibrinogen with plasma cryopre-
tion on the surface, as determined by the cipitate. Indeed, the results of those studies were encourag-
sulforhodamine B assay,53 was 20 ng/cm2. ing: After 4 days in culture the number of cells on fibrin-
Group #2: PU coated with FN by photoimmo- based coating was higher by almost 45% than on TC. Cell
bilization using the method of Clapper et al.54 The coverage was even higher when the fibrin-based coating was
final concentration of FN on the surface was as in supplemented with heparin and insulin.42 Based on these
group #1. findings, we assessed the efficacy of the fibrin-based coating
Group #3: PU coated with Col IV by adsorption. The to promote EC growth on the luminal surface of PU-cov-
starting concentration was 300 ∝g/ml at pH = 3.0. Af- ered “ersatz” ventricles, (T-25 tissue culture flasks61). For
ter 1 h of incubation at 37°C, the final concentration comparison purposes we also used “ersatz” ventricles cov-
on the surface was 50 ng/cm2. ered with either plain or FN-coated PU. In all cases the lu-
Group #4: PU coated with Col IV by photoimmo- minal surface of the PUs used (Biospan™)50 was smooth
bilization. The final concentration of Col IV on the and the coatings were prepared without supplements such
surface was as in group #3. as growth factors, heparin, or insulin.
As shown in Figure 5.1b, the cells grew equally well on The study was conducted in two sequential steps to
FN substrates, whether adsorbed or photoimmobilized. On realistically mimic a full scale endothelialization of the blood
the other hand, when Col IV-based substrates were used, sac of a VAD. First, the cells (BAECs) were seeded with rota-
cell growth was significantly better on PU coated by adsorp- tion for 3 h onto either precoated or plain PU surfaces.61
tion than by photoimmobilization (1.69 ± 0.11 x 105 vs. 1.33 Thereafter, the “ersatz” ventricles were removed from the ro-
± 0.11 x 105, p < 0.05). Thus, for select proteins some meth- tator and kept in an upright position, and the adherent ECs
ods of immobilization may indeed result in better substrates were allowed to spread and proliferate for several days. We
for cell growth than others. However, in our hands, no ECM- monitored cell growth every other day by Alamar Blue as-
derived, monoprotein coating of PU can match the condi- say.50 The results obtained are presented in Figure 5.2a. As
tions offered by a tissue culture-grade polystyrene. Clearly, expected, the lowest rate of growth was obtained on plain
a substrate comprised of two dimensionally assembled, ECM PU. On FN-coated PU the cells grew somewhat better, pre-
adhesive monoproteins does not provide for an adequate sumably because there were more cells attached immedi-
surface coating that can be used as a vehicle for optimizing ately after seeding and so they could interact more effec-
endothelialization of cardiovascular prostheses. Hence, more tively with one another. In the past, several studies had al-
sophisticated approaches are required to generate a ready shown that such interactions play an important role
Fig. 5.1. Cell growth on various sub-
strates under static conditions: (a)
Effect of select surface treatments on
cell proliferation. FN or Col IV were
immobilized onto PU samples by
adsorption as detailed in the text. (b)
Effect of protein immobilization
methods on cell densities. FN or Col
IV were immobilized onto PU
samples either by adsorption or
photoimmobilization as detailed in
the text. Cell number was
quantitated every other day by
Alamar Blue assay.
82 Tissue Engineering of Prosthetic Vascular Grafts
in cell proliferation in vitro.62,63 By contrast, cells seeded on (p = 0.24). On both CRY-coated PUs, the number of cells
plasma cryoprecipitate-made fibrin substrate (CRY) grew was about 1.5 times higher than on plain, textured PUs. These
rapidly and reached confluence by the seventh day in culture. data suggest that fibrin-based coating is indeed a preferred
Importantly, purified fibrinogen-made fibrin substrate substrate for optimizing EC growth on the luminal surface
(FBG) was not as effective as CRY in promoting cell growth; of cardiovascular prostheses.
even on the ninth day in culture the number of cells on FBG In addition to enhancing cell growth, increasing the
was still significantly lower than on CRY (2.86 ± 0.28 x 105 initial efficiency of cell attachment onto biomaterials is piv-
vs. 3.41 ± 0.25 x 105, p < 0.05). Since both coatings are otal for successfully establishing an EC monolayer in pros-
comprised of a fibrin-based matrix, it is likely that some of thetic devices. In the past, we and others have explored vari-
the intrinsic plasma proteins contained in CRY may have ous ways for increasing the affinity of synthetic surfaces to
contributed to the enhanced rate of cell growth on cells, for example, by glow discharge treatment,68 coating
this substrate. with substrates composed of select proteins,40,69,70 or both.61
Smooth-surface PUs have been used in cardiac pros- Indeed, when BAECs were seeded under static conditions
theses as a means of preventing platelet aggregation and onto various PU substrates, we saw a substantial improve-
thrombus formation on the blood-contacting surfaces. How- ment in attachment efficiency on treated surfaces vs. plain
ever, notwithstanding the improvements in PU manufac- PU (Fig. 5.3a). Our study also indicated that the most suit-
turing and surface processing, the microscopic appearance able surface for cell attachment was TC; CRY-coated PU was
of the luminal surface in these devices has never been com- second to the best (94.1 ± 3.23%, p < 0.05). In our hands,
pletely smooth, and also the hemocompatibility of the the performance of CRY (and FBG) coatings was depen-
smooth surfaces has not met the initial expectations. On the dent upon the concentration of fibrinogen employed. As
other hand, textured PU surfaces have been advocated as a demonstrated in Table 5.1, cell attachment efficacy of puri-
scaffold of choice for enhancing pseudoneointima forma- fied fibrinogen varied in a bell-shaped manner between con-
tion64,65 and for promoting endothelial cell attachment onto centration levels of 0-30 mg/ml. The best performance was
the luminal surface of cardiovascular devices.29,66,67 In adopt- obtained at 5 mg/ml but, for preparing a coating of control-
ing the latter concept for the endothelialization of VAD blood lable thickness on a textured surface, we used only 3.5 mg/ml.
sacs, we evaluated CRY enhancement of cell growth on tex- Optimal adherence of endothelial cells under static
tured PU. The experiments were conducted in the same conditions onto flat PU substrates or a TC surface does not
manner as above with smooth, uncoated PUs serving as con- necessarily translate into efficient attachment of cells dur-
trols. Figure 5.2b exemplifies our findings for plain and CRY- ing endothelialization under rotation of a complex geomet-
coated PUs after seven days in culture. BAECs proliferated ric structure such as a cardiovascular prosthesis. In our
much faster on the uncoated, textured PU than on its smooth hands, there are four equally important factors that deter-
counterpart, (2.17 ± 0.21 x 105 vs. 1.49 ± 0.21 x 105, p < 0.05). mine the efficiency of cell attachment under these unique
Again, the significant difference in the number of cells cov- conditions: number of cells in the inoculum, speed of rota-
ering the surfaces may have resulted, in part, from more ef- tion, duration of seeding and surface treatment by coating.
ficient attachment of cells on textured surface than on By fine tuning these parameters we were able to im-
smooth PU. Application of CRY coating masked the topo- prove the homogeneity of surface coverage, optimize the time
logical difference between the two types of surfaces and en- of residence/contact of cells with the substrate, and thus
hanced cell growth with the same effectiveness. Thus, the promote cell attachment. We established the following as
number of cells on both PUs was virtually the same at all optimal for lining our artificial ventricles: speed at 10 rota-
times; for example, on the seventh day in culture (Fig. 5.2b) tions/h, duration of seeding at 3 h, and inoculation density
we measured 3.24 ± 0.23 x 105 cells/cm2 on smooth PU+CRY at 6.0 x 104 cells/cm2. Utilizing these settings, we examined
vs. 3.56 ± 0.23 x 10 5 cells/cm 2 on textured PU+CRY the efficiency of attachment of cells that were seeded onto
Fig. 5.2. Cell growth on various substrates
in PU-covered “ersatz” ventricles after seed-
ing with rotation: (a) Effect of select sur-
face coatings on cell proliferation. (b) Cell
densities in cultures grown on smooth and
textured PUs, with or without CRY coat-
ing. “ersatz” ventricles, consisting of PU-
covered T-25 flasks, were seeded with rota-
tion for 3 h at an inoculation density of
60,000 cells/cm2. After stopping the seed-
ing, the adherent BAECs were allowed to
spread and proliferate under static condi-
tions. Cell number was assessed every other
day by Alamar Blue assay, and the cultures
were refed after completion of the assay.
Surface Precoating in the 1990s: The Fine Tuning of Endothelial Cell Transplantation 83
various substrates in smooth PU-covered “ersatz” ventricles. abundant, randomly dispersed cavities of varying dimen-
Plain TC served as controls. The results obtained are shown sions, ranging in size from 30-120 ∝m in diameter. Figure
in Figure 5.3b. In contrast to the findings under static con- 5.4b exemplifies attachment efficiencies on plain and CRY-
ditions, plain TC was the least suitable substrate for cell at- coated PUs. It is apparent from panel A that cell attachment
tachment under rotation, whereas CRY ranked the best onto an uncoated, textured surface was significantly better
(553.1 ± 21% over TC). FBG ranked second to the best (328.2 than onto plain smooth PU (134.8 ± 8% and 108.2 ± 6%,
± 13% over TC) and FN-based substrate was the third with respectively, compared to TC controls, p < 0.05). This im-
a considerably lower efficiency (257.3 ± 11% over TC). provement was likely due to the increase in cell residence
The topography of the luminal surface in most vascu- time at the site of contact and isolation from fluid currents,
lar grafts is primarily rough.71,72 Also, some of the TAHs and rather than resulting from enhancement in surface affinity.
VADs that are currently tested or developed, including the In the case of CRY-coated PUs, the fibrin meshwork com-
Heartmate® and the Milwaukee Heart™, have a rough lu- pletely filled the pores in the textured PU and masked the
minal surface. To gain a more factual representation of the topological differences between rough and smooth surfaces
attachment efficiencies during endothelialization of artifi- (Figs. 5.4 c,d). The resulting substrates had a finely patterned
cial devices, we repeated the above experiments using “er- luminal surface that was enriched in numerous RGD bind-
satz” ventricles covered with textured PU. Figure 5.4a pre- ing sites inherent to the component proteins. As shown in
sents a typical SEM photomicrograph of a textured surface panel B of Figure 5.4b, cells adhered readily onto both these
that was prepared, as previously reported,43 by sprinkling substrates, leading to similar attachment efficiencies of more
NaCl crystals (in this case the mean diameter was 32 ∝m) than 500% above control level. Thus, by using CRY coating,
over a wet PU cast. The entire surface appears corrugated by we completely eliminated the difference in attachment effi-
ciencies between smooth and rough PUs.
Having optimized cell attachment and growth on PUs,
we determined whether the CRY coating could also enhance
the retention of ECs in a monolayer that is exposed to dy-
namic conditions. For these in vitro studies, we used VAD
blood sacs which were fabricated from a commercially avail-
able PU (Biospan™) by the lost wax technique.43 For cell
seeding, the lumina of fully assembled VADs were filled with
125 ml of Air/CO2-presaturated culture medium contain-
ing 4.0 x 107 BAECs, mounted onto the rotating arm of the
seeding apparatus61 and rotated at 10 rotations/h for 3 h.
Following seeding, the VADs were placed, in an upright po-
sition, in a standard CO2 incubator. Cell proliferation was
measured every other day, and the cultures were refed after
conclusion of each Alamar Blue assay.50 Two days after
seeding, the monolayers in the blood sacs had reached
confluence. We then maintained them for eight additional
Table 5.1 Efficiency of cell attachment as a function of
fibrinogen concentration
Concentration of Efficiency of Cell
Fibrinogen (mg/ml) Attachment (%) a)
0.0 61.48 ± 4.87
0.5 72.13 ± 1.87
1.0 76.28 ± 3.14
2.0 83.56 ± 3.14
5.0 84.01 ± 2.91
10.0 83.72 ± 3.47
15.0 79.56 ± 2.43
Fig. 5.3. Efficiency of cell attachment onto various substrates:
20.0 76.44 ± 2.05
(a) under static conditions, and (b) after seeding with rotation 30.0 72.11 ± 1.52
of “ersatz” ventricles. BAECs were inoculated at a density of aThe study was conducted under static conditions and the number
60,000 cells/cm2. After 3 h of seeding, the cultures were washed
of cells recorded at each fibrinogen concentration was normalized
gently with medium to remove nonadherent cells, and assayed to the number of cells obtained on plain TC. The fibrinogen used in
by Alamar Blue assay to determine the number of cells attached this particular study was purified in the Hemostasis Research
to the surface. The resulting numbers were normalized to data Laboratory at Sinai Samaritan Medical Center, Milwaukee, WI, and
obtained on TC surface. was kindly donated by its director, Dr. M. Mosesson.
84 Tissue Engineering of Prosthetic Vascular Grafts
days under static conditions to allow the cells to synthesize, ing to pulsatile flow resulted in denudation of the monolay-
assemble, and mature their ECM on the underlying scaf- ers by more than 30%, but did not compromise the mono-
fold.73 Following a total of eleven days in a static environ- layers established on CRY coating. In fact, our results indi-
ment, the monolayers were exposed for 24 h to one of the cate that on CRY coating there was minimal, if any, cell loss
following dynamic conditions: due to exposure to the hemodynamic forces encountered in
1. Flexing of the blood sac wall at 1 Hz between systole a pumping artificial ventricle.
and diastole positions, with minimal flow of medium
inside the blood sac; Morphological Aspects
2. Perfusion of the blood sac in an assembled VAD with of the Endothelialized Surfaces
pulsatile flow at the mean rate of 500 ml/min (blood It has long been recognized that the morphology and
sac wall was maintained motionless during the whole function of vascular ECs, as well as the architecture of the
period); and subendothelial matrix, are modulated by hemodynamic
3. Pumping action of the VAD at 1 Hz, mean flow rate forces and by the tissue-specific microenvironment. In most
of 3.2 l/min, and ejection pressure of 150 mm Hg.
For all tests, the VADs were connected aseptically to a
mock loop consisting of a PVC venous reservoir, as previ-
ously described.43 The status of the monolayer lining the Table 5.2. Cell retention under dynamic conditions in
EC monolayers established on either FN or CRY coating
blood sacs was evaluated by the Alamar Blue assay before
and after each run. Our findings for FN and CRY-based coat-
ings are presented in Table 5.2. It is apparent that 24 h of % of cells remaining % of cells remaining
Dynamic Condition on FN coatinga on CRY coatinga
flexing did not dislodge cells from the scaffolds. In fact, the
rhythmic movement of the wall even increased cell density Flexing 120.7 ± 7.6 * 113.1 ± 3.3 *
markedly in all cultures by more than 13%. This observa- Perfusion 69.0 ± 7.1 * 91.6 ± 13.2
tion is in line with the findings previously reported by oth- Pumping 65.0 ± 16.0 * 95.2 ± 6.3
ers.74 On the other hand, exposure of ECs grown on FN coat- a
The data were normalized to the number of cells measured before
the test; * indicates significant difference, p<0.05.
Fig. 5.4. (a) A typical electron micro-
scopic (SEM) appearance of uncoated,
textured PU (magnification x160). The
average pore area, evaluated by com-
puter-aided image analysis was 9370 ±
12 ∝m2. (b) Efficiency of cell attachment
onto: A. uncoated PU, and B. CRY-coated
PU after seeding with rotation of “ersatz”
ventricles. Cell enumeration was per-
formed by Alamar Blue assay and the
resulting data were normalized to values
obtained for TC. (c,d) Typical SEM views
of CRY-coated, textured and smooth PU
surfaces, respectively. The specimens
were cut at a 30° inclination to reveal
both the coating and the underlying PU
scaffold (magnification x650).
Surface Precoating in the 1990s: The Fine Tuning of Endothelial Cell Transplantation 85
studies, these characteristics have primarily been addressed and/or assembly of the EC-derived ECM, or if it was an ar-
in the context of cellular response to mechanical forces.74-79 tifact of specimen preparation, is unclear. In any event, the
To gain a better insight into adaptation of the cells on subendothelial matrix in either case did not appear to
different surfaces, we focused on morphological aspects of provide sufficient mechanical stability and sturdiness for
the EC monolayer and the subendothelial region on plain supporting the cells during exposure to the dynamic condi-
and coated PUs in postconfluent, static cultures. After seven tions inside a beating ventricle. By contrast to the scanty ECM
days in culture, the cells were removed by treatment with on plain PUs, the subendothelial regions assembled on FN
detergent and the surface was examined by scanning and CRY coatings were more substantial, oftentimes span-
electron microscopy. ning the entire viewing areas (Figs. 5.5c and d, respectively).
A typical electron microscopic view of the subendo- On FN coated PU, (Fig. 5.5c), the network appeared planar
thelial region of cells grown on uncoated, smooth PU is pre- and in many sites it was much denser than on uncoated PUs.
sented in Figure 5.5a. The ECM, made of isolated regions of CRY alone formed a 3-dimensional and continuous
fibers that were interlaced by short fibrils, formed thin sheets meshwork atop the PU, composed of crosslinked fibrin and
of web-like structures of variable density. These regions only other highly organized adhesive plasma proteins such as
partially covered the PU surface and were connected to one fibronectin and vitronectin (Fig. 5.5d). When the cells grown
another by wider filamentous bands. The subendothelial on top this structure were removed, the remaining ECM was
region on uncoated, textured PU (Fig. 5.5b) appeared simi- comprised of thick bands of extremely dense structures,
lar to the ECM on smooth PU, except that oftentimes the closely apposed to the CRY meshwork, resembling the
long fibrillar structures bridged across cavities and recesses amorphous, natural basement membrane. Apparently the
within the surface. On both PUs, however, a large fraction architectural differences between the two coatings, as
of the surface area appeared devoid of any matrix-related manifested in their improved scaffolding capabilities and,
material. Whether this was due to nonuniform deposition ultimately, in their potential to minimize cell loss during
Fig. 5.5. SEM en face view of the subendothelial region after seven days of cell culturing under static conditions
on: (a) uncoated, smooth PU, (b) uncoated, textured PU, (c) FN-coated textured PU, and (d) CRY-coated
textured PU. BAECs were grown in blood sacs as described in the text. On seventh day, select samples of 1 x 1 cm2
were excised from the blood sacs and rinsed with PBS. The cells were removed from the surface by treatment
with 0.1% Triton X-100 and subsequent wash with 0.025 M NH4OH. The samples were rinsed with 0.1 M Na-
cacodylate buffer and, consecutively, with ddH2O. Following serial dehydration (30 sec each) by ethanol and
critical point drying, specimens from each sample were sputter coated with gold-palladium (~210 Å in thick-
ness) and viewed by Philips PW-6700 microscope (Eindhoven, The Netherlands) at a magnification of x2500).
86 Tissue Engineering of Prosthetic Vascular Grafts
exposure of the endothelialized surfaces to flow-induced Concluding Remarks
forces, played a considerable role in enhancing interactions Endothelialization of cardiovascular prostheses, as a
with EC-derived matrices. means of overcoming their inadequate hemocompatibility,
One of the conceivable advantages of the CRY coat- is a multifaceted task. It involves numerous aspects, notably:
ing over any other factitious substrates is its 3-dimensional 1. Procurement of autologous endothelial cells from the
meshwork architecture. Before endothelialization, and while prospective donor and their propagation into large
still naive, this unique structure consists of an array of criss- quantities;
crossed fibers which evenly cover the entire area (Fig. 5.6a). 2. Preparation of the biomaterial surface for cell seed-
The distance between adjacent fibers is not constant, and it ing and establishment of a stable, shear resistant
can be manipulated, as already reported elsewhere,42 in order monolayer atop the synthetic material; and
to optimize its ability to support the overlying cells. Indeed, 3. Manipulation of the ECs to express their
on a finely patterned meshwork, endothelial cells propagate athrombogenic and fibrinolytic repertoires during
readily across the entire surface and form a confluent mono- static culturing and after implantation into the host.
layer within 48 h after seeding. Seven days after seeding, the Each of these aspects is complex and encompasses
underlying ECM appears matured and well integrated into critical issues that require attention from a wide spectrum
the CRY meshwork (Fig. 5.6c). A subendothelial “basement of disciplines, such as biology, chemistry, bioengineering and
membrane” on top of the fibrillar, “provisional matrix” is medicine. In many instances the solutions to these prob-
clearly visible through a shrinkage gap which was artificially lems are not straightforward and call for additional research
created during sample preparation. When grown atop this and for the development of novel technologies. However,
subendothelial complex, the cells seem well differentiated more often than not, genuine progress is achieved through
and closely apposed next to each other (Fig. 5.6b). In a trans- innovative approaches. Indeed, in recent years we have wit-
verse view, the densely packed ECs appear as a continuous nessed promising developments with the use of genetically
chain, which relative to the thickness of the CRY coating, engineered endothelial cells23,33 and bioabsorbable materi-
looks very thin (Fig. 5.7a). Along the EC chain, cell to cell als6 in vascular prostheses. Also, the reconstitution of hier-
contact between neighboring cells is well established archically structured walls by ECs, smooth muscle cells, and
(Fig. 5.7b). fibroblasts80-82 provides a reason for cautious optimism. Yet
another promising approach, explored by us and others,40-42
is to precoat the biomaterial surface with a three dimen-
Fig. 5.6. Scanning electron microscopic view of: (a) CRY-meshwork before cell seeding (magnification x2500), (b) Confluent and
well-differentiated BAEC monolayer after 7 days in static culture (magnification x650), and (c) Shrinkage gap in the BAEC mono-
layer which was formed during specimen processing in order to provide a detailed insight into the subendothelial region; * indi-
cates a section of the extracellular matrix deposited and organized by BAEC (magnification x2500). On the seventh day, select
samples of 1 x 1 cm2 were excised from the blood sacs and rinsed with PBS. The samples were fixed for 30 min at room temperature
with 1% glutaraldehyde in 0.1 M Na-cacodylate buffer and postfixed with OsO4 for 25 min. Further processing followed the
procedure described in Figure 5.5.
Surface Precoating in the 1990s: The Fine Tuning of Endothelial Cell Transplantation 87
Fig. 5.7. A transverse view of an endothelialized,
cryoprecipitate coated surface seven days after
seeding. BAEC monolayer and CRY-coating were
gently peeled off from PU surface, and immedi-
ately immersed in Karnovsky’s fixative for 30 min
at room temperature. The specimens were subse-
quently postfixed in a 1% OsO4 solution for 40
min, dehydrated by using graded ethanol and, fi-
nally, embedded in SPURR. (a) Semithin sections
of 1 ∝m were cut from the specimens and stained
with toluidine blue for observation by light mi-
croscopy (magnification x100). (b) Ultrathin sec-
tions of 60-90 nm were mounted on copper grid
and examined in a Philips 400, electron microscope
(Eindhoven, The Netherlands) at a magnification
of x3000.
sional meshwork of cryoprecipitate-derived plasma proteins Currently, the focus of our work and that of others is
that serves as a biointegrator between the endothelial cell to further optimize the hemocompatibility of the blood-
monolayer and the underlying synthetic scaffold. In this contacting surfaces in cardiac prostheses by broadening the
chapter we have presented our experience with the plasma concept of “endothelialization”. Over the years, progress in
cryoprecipitate-produced fibrin meshwork as it relates to op- this area has been made largely by extending the idea of a
timizing endothelial cell attachment and growth, and to in- “passive endothelial cell lining”, as applied in the 1980s and
creasing resistance to denudation of the cellular monolayer before, to the concept of biointegration of the textured poly-
upon exposure to the dynamic conditions inside a beating meric scaffold, the 3-dimensional matrix and the “active”
cardiac prosthesis. interaction between the cells and their matrix. This integral
Our in vitro studies establish that denudation of approach requires the coordinated input of a
endothelialized prostheses can be minimized by culturing multidisciplinary team of scientists, and is only one of the
ECs on a 3-dimensional meshwork, rich in both structural many steps needed for the recreation of the entire vascular
and adhesive proteins, as well as by allowing the confluent wall structure lined by a nonthrombogenic cellular lining.
monolayer time for maturation. All these factors, viz, seed- In the past it has been assumed that this cellular lining should
ing of endothelial cells on the three dimensional “provisional be comprised only of endothelial cells, but recently there is
matrix” and subsequent establishment of the subendothe- some evidence that other cell types, either native or geneti-
lial ECM, as well as the functional maturation of the EC cally modified, can also be rendered nonthrombogenic when
monolayer, will likely play vital roles in averting denudation exposed to pulsatile blood flow.
of ECs by mechanical forces. Fine tuning of the surface coat- Reconstruction of the cardiac/vascular wall in vitro
ing will confer added physical stability to the neobasement requires a thorough understanding of how to coculture the
membrane (“shock absorber effect”) and promote functional diverse cell types comprising the entire vascular wall, e.g.,
contacts between the cells in the monolayer. However, once smooth muscle cells, fibroblasts and endothelial cells, in or-
in contact with blood/plasma, other factors may also be of der to achieve a hierarchic, structural and functional orga-
importance for the maintenance of an intact EC monolayer. nization resembling that of the adventitia, media and in-
For example, monolayer integrity may be challenged by vari- tima. Interestingly, preliminary data suggest that exposure
ous proteases, activated plasma proteins and/or circulating of such cocultures to hemodynamic forces such as cyclic
blood cells when the cell culture media is replaced by plasma strain and pulsatile flow will sort out the cells and initiate
or citrated blood during mock loop tests in vitro. Similar, their hierarchic arrangement. Unanswered, as yet, is the ques-
and even more demanding, challenges should also be ex- tion of whether the cells lining the blood-contacting sur-
pected during in vivo trials. faces are indeed functional, viz., whether the cellular lining
will indeed reduce the thrombogenicity of the devices. An
88 Tissue Engineering of Prosthetic Vascular Grafts
exciting approach is to use genetically modified cells, which totypes, policies, and patients. Washington DC: National
would accelerate the hierarchic organization within the ves- Academy Press, 1991:251-261.
sel wall and/or overexpress specific molecules that charac- 10. Marshall E. Artificial heart: The beat goes on. Science
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surface.
future of artificial hearts and of mankind. Artif Organs
Finally, in our opinion, the ultimate cardiovascular 1988; 12:89-111.
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ment organ. In the not too distant future we envisage that the human body to the first permanent implant of the
dramatic progress in cell and molecular biology, in concert Jarvik-7 total artificial heart. Trans Am Soc Artif Intern
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even entire organs. Some of these exciting procedures, as both temporary and permanent ventricular assist devices:
futuristic as they may sound, are currently being explored Experimental and clinical observations. Artif Organs 1989;
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bioprostheses will soon be surpassed by a new area of “as bolic and infectious complications of total artificial heart
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Acknowledgments Res 1987; 48:349-362.
The present study was supported by the Milwaukee 18. Yozu R, Golding LA, Jacobs G et al. Preclinical evalua-
Heart™ Research Foundation. We thank the entire engineer- tion of a biolized temporary ventricular assist device.
ing group of the Milwaukee Heart™ Project for providing Trans Am Soc Artif Intern Organs 1989; 35:556-558.
19. Gimbrone MA Jr. Vascular endothelium: Nature’s blood-
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endothelial cells subjected to cyclic strain in vitro. lar tissue composed of vascular cells and collagen for low-
Microvasc Res 1991; 42:245-254. pressure-loaded venous system. Cell Transplantation 1995;
75. Sumpio BE, Banes AJ, Buckley M, Johnson G Jr. Alter- 4:597-608.
ations in aortic endothelial cell morphology and
PART II
Biolized Prostheses: Surface Healing
Microvascular Endothelial Cell Transplantation
CHAPTER 6
Microvascular Endothelial Cell Transplantation: A Review
Stuart K. Williams
Early Development of Endothelial Cell Transplantation Technology
T he development of an endothelial cell lining on the lumenal surface of vascular implants
(e.g., peripheral vascular grafts, arteriovenous fistulas, coronary artery bypass grafts) has
been realized only with the understanding of the complex physiology of these cells. The
development and first clinical experiences with polymeric vascular grafts provided the ini-
tial evidence of the importance of a functional endothelium on the lumenal surface of blood
vessels. These synthetic blood vessels perform adequately in large diameter (> 6 mm) posi-
tions (e.g., abdominal aortic replacements); however, as the diameter of the synthetic grafts
needed became smaller, long term function was compromised. The best evidence for the
need of a lumenal endothelial cell lining is provided by the poor patency observed when
synthetic grafts with diameters less then 6 mm are used. These grafts exhibit extremely poor,
clinically unacceptable, long term patency, due predominantly to the formation of blood
clots, resulting in lost patency.
The bypass of occluded native vessels with diameters less then 6 mm has been limited
to the use of autologous native blood vessels. While these vessels have provided acceptable
long term patency, clinicians have realized the importance of maintaining the integrity of
the lumenal lining of endothelial cells.1-4 Autologous vessels provide superior long term
patency as compared to synthetic grafts, due not only to their compliant natural tissue char-
acteristics, but also to the antithrombogenic nature of the endothelial cell lining. Procedures
which disrupt the endothelium, such as angioplasty and atherectomy, provide additional
evidence for the need to maintain a functional endothelium.
The first report of the successful isolation and transplantation of endothelial cells was
a report by Dr. Malcolm Herring in 1978.5 His methods involved the use of a large vessel-
derived endothelium obtained by scraping the lumenal surface of vein segments. These cells
were subsequently transplanted onto the lumenal surface of polymeric graft materials, a
process which was termed seeding. The methods used for seeding include mixing the iso-
lated cells with autologous blood and subsequently using this blood-cell inoculum to preclot
porous grafts. The lumenal blood-contacting surface was observed to be predominantly
fibrin and red cells, with endothelial cells present predominantly within the resulting clot.
The hypothesis behind these studies was that seeded endothelial cells would migrate from
within the clot to the lumenal blood flow surface, proliferate and finally form a continuous
monolayer through the formation of typical interendothelial cell junctions. Subsequent work
by a large number of investigators, including work from the laboratories of Schmidt,6 Stanley7
and Graham,8 established the ability to accelerate the formation of monolayers on pros-
thetic grafts using large vessel endothelial cells as a source of cells for seeding.
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
94 Tissue Engineering of Prosthetic Vascular Grafts
Publication of these studies established the feasibility methods provided a means to produce large quantities of
of performing endothelial cell transplantation and resulted autologous endothelial cells by obtaining a small segment
in extensive work to optimize seeding technology. Numer- of a patient’s blood vessel, isolating and culturing the en-
ous questions were identified which needed to be addressed dothelium in heparin and endothelial cell growth supple-
before endothelial cell transplantation was expected to be ment (ECGS) and transplanting these cells at very high seed-
found clinically acceptable. These questions identified nu- ing densities onto prosthetic grafts.
merous issues related to the source of tissue for subsequent Large vessel-derived endothelial cell seeding at high
endothelial cell transplantation, the function of immediately density was achieved in vitro; however, additional questions
isolated endothelium and methods to improve the interac- have been raised, delaying wide spread clinical utilization of
tion of endothelium with prosthetic graft surfaces. Subse- this technique. Concerns related to the use of cultured en-
quent laboratory work has established the efficacy of intra- dothelium for cell transplantations are surmountable.
operative methods for endothelial cell isolation and deposi- Dr. Peter Zilla has performed a careful series of studies to
tion onto the lumenal surface of vascular grafts. address the major concerns raised about using cultured cells,
as well as providing evidence of the efficacy of this form of
Sources of Endothelium for Transplantation tissue engineering.14-16 Some of these questions included
As described above, Herring first reported the use of concerns relating to the derivation of cells from large vessel
venous-derived endothelium for subsequent transplantation. sources, which requires a separate surgical procedure, most
The yield of cells from a suitable piece of vein segment was likely from patients with an already compromised circula-
approximately 1 x 104 cells.9 Thus, based on the largest piece tory system. This increased risk of a separate vascular com-
of vein which could be obtained from a patient, only 10,000 plication had to be weighed against the need for endothelial
endothelial cells could be obtained. Under even the most cell transplantation. If cultured cells were used for trans-
ideal conditions, this cell number would provide a relatively plantation, the conditions of culture must be evaluated to
sparse coating of cells, requiring extensive cell proliferation determine the effect on subsequent cell function. Bovine
to achieve a monolayer. Kempzinski et al10 reported that even derived serum components such as viruses, and growth fac-
if cells could be isolated and deposited on the surface of tors such as heparin and ECGS, would be carried with trans-
polymeric grafts with optimal efficiency, a large number of planted cells into patients, requiring characterization of their
these cells would be lost from the lumenal surface of the effects. Considering that endothelial cells have an extremely
graft due to the effects of blood flow-induced shear on the low mitotic index, the stimulation of endothelial cell growth
lumenal surface. Endothelial cells seeded onto grafts with must be considered to determine how cell growth may alter
this technology would be required to undergo even more endothelial cell function. Finally, little data is available con-
extensive proliferation or, alternatively, a new source of en- cerning the appropriateness of transplanting vein-derived
dothelial cells obtained in large numbers must be found. endothelial cells into an arterial circulation with respect to
the ability of these cells to differentiate and function under
Cultured Endothelium for Transplantation arterial conditions. Nonetheless, this technique has been
Methods for the isolation and culture of nonhuman successfully implemented in a series of patients undergoing
endothelial cells were first reported in the 1920s, providing peripheral bypass surgery. Initial results indicate both the
methods for the expansion of endothelial cell numbers by safety and efficacy of large vessel endothelial cell
in vitro proliferation.11 However, the same methods used transplantation.16
for nonhuman endothelial cell proliferation had no stimu-
latory effect on human endothelial cell proliferation. Dur- Current Considerations in Endothelial Cell
ing the 1970s a method for human endothelial cell isola- Transplantation
tion, based on the pioneering work of Lewis in 1922, pro-
vided human endothelial cells in culture; however, these cells Use of Microvessel Endothelium for Tissue Engineering
exhibited minimal proliferative capacity. A number of en- of Vascular Grafts
dothelial cell growth factors were discovered that slightly Given the small numbers of cells available if autolo-
stimulated human endothelial cell proliferation. The major gous vein-derived endothelium are used without culture, and
breakthrough in human endothelial cell growth in culture concerns about culturing effects, investigators have been
was work performed by Dr. Susan Thornton, working with considering alternate sources of endothelial cells for trans-
Dr. Elliot Levine, which identified heparin as an integral plantation. In 1986 Jarrell and Williams17 reported methods
cofactor with endothelial cell growth factor for the stimula- for the isolation of autologous microvessel endothelial cells
tion of human umbilical vein endothelial cell growth in cul- from adipose tissue for use in cell transplantation. The source
ture.12 Jarrell and coworkers13 subsequently reported that of fat for this EC isolation was initially fat deposits associ-
heparin and ECGF would stimulate human adult endothe- ated with omentum. This fat is well vascularized, with a
lial cell growth in culture. For the first time, cells from hu- density of endothelial cells in excess of 106 endothelial cells
man artery and vein segments from numerous anatomical per gram of fat isolated. The need to place cells in culture to
positions could be grown in large quantities. Large num- increase cell number is obviated by the large amounts of
bers of human endothelia could be produced from single endothelium available per gram of omental associated fat.
endothelial cells, providing nearly unlimited supplies of However, access to omental-associated fat still would
human adult endothelium. These breakthroughs in culture
Microvascular Endothelial Cell Transplantation: A Review 95
necessitate a separate surgical procedure, with skin incision or trocar puncture to insert the liposuction
related complications. cannula. The liposuction cannulas used for this procedure
The use of omental-associated fat-derived endothe- have been continuously improved for plastic and cosmetic
lial cells was questioned by Visser et al,18 who reported that surgical applications.
omental tissue derived from humans contains predomi- The use of liposuction fat is also advantageous since
nantly mesothelial cells and not endothelium. This report the tissue is effectively minced during the liposuction re-
raised a significant controversy concerning the use of omen- moval process. Scanning electron microscopic evaluation of
tal-associated fat as a source of endothelial cells for trans- liposuction fat (Fig. 6.1) illustrates the morphological char-
plantation. Since this initial report by Visser et al, subsequent acteristics of this tissue. The predominant cell type present
reports have clarified the controversy and provided addi- at this magnification is adipocytes. Recently, a complete char-
tional options for obtaining microvessel endothelial cells.19 acterization of cells present in liposuction-derived fat was
First it was established that investigators have erroneously reported and established that the major cell type, other than
used the terms omentum and omental fat interchangeably. adipocytes, present in this tissue is endothelium.19 Prior to
These two tissues are histologically and anatomically dis- any cell dissociation procedures, human subcutaneous fat
tinct. While omentum as used by Visser et al is a vascular- contains in excess of 85% endothelium based on the total
ized tissue with an extensive number of mesothelial cells cells present per unit volume of fat. Adipocytes account for
present, omental-associated fat, as used by Jarrell and Will- approximately 12% of the cell population, again before any
iams, is composed of predominantly endothelial cells and tissue dissociation and cell isolation techniques are used.
adipocytes. This is not to say that use of omental-associated Therefore, following tissue dissociation and cell isolation,
fat avoids the possibility of mesothelial cells in the primary endothelial cells must account for at least 85% of the cells in
cell isolate. Omental fat deposits are covered by a thin layer the final inoculum.
of mesothelium, and a single cell population isolated from
omental-associated fat will contain a small number of me- Markers for Isolated Endothelial Cells
sothelial cells in addition to endothelium. Careful isolation One significant difficulty in establishing the identity
of omental-associated fat away from omentum will reduce of cells present in the primary isolate following tissue diges-
contamination by mesothelium. tion has been the relative lack of a dependable marker of
Alternate anatomic sources for microvascularized au- endothelial cells. While antibodies directed against von
tologous human fat for endothelial cell transplantation have Willebrand factor or factor VIII-related antigen (FVIIIrAg)
been reported since these original reports using omentum- have been used somewhat routinely for endothelial cell iden-
associated fat. A significant improvement was the identifi- tification, these markers do not unfailingly react with freshly
cation of subcutaneous fat as a source of endothelial cells isolated microvascular endothelial cells. First, upon primary
and the use of liposuction to derive this fat from patients.20 isolation and due in part to the action of proteolytic en-
A patient undergoing endothelial cell transplantation there- zymes, endothelial cells will release these proteins from cy-
fore does not need to undergo a laparotomy to remove omen- toplasmic granules, thus resulting in cells which exhibit nega-
tal fat or undergo a separate vascular procedure to remove a tive staining properties.21 Also confounding is the relative
segment of vein. A small amount (50 cc) of fat can be re- lack of Weibel-palade bodies in fat-derived microvessel en-
moved using a hand held syringe cannula device. This pro- dothelial cells. The cytoplasmic inclusions contain extremely
cedure requires less then five minutes and requires a small high concentrations of vWF, which subsequently stain
Fig. 6.1. Scanning electron micrograph of
human liposuction-derived subcutaneous fat,
illustrating the predominance of adipocytes.
96 Tissue Engineering of Prosthetic Vascular Grafts
extremely well in cell types such as vein-derived endothe- Cell Deposition on Graft Surfaces:
lium. Venous endothelial cells contain a significant number Seeding vs. Sodding
of Weibel-palade bodies, such that investigators who observe The availability of large quantities of autologous en-
vWF staining in microvessel endothelial cells correctly assess dothelial cells does not necessarily overcome another major
cells with limited reaction product as compared to venous concern related to endothelial cell transplantation. The depo-
endothelium. Often investigators will suggest that since the sition of endothelial cells onto the lumenal (blood flow)
bright punctate staining common with Weibel-palade bod- surface of vascular grafts is a critical step in the transplanta-
ies is not observed in microvascular endothelial cells, these tion process. Once placed as an interpositional graft, cells
cells must not be endothelium. On the contrary, microvas- on the lumenal surface must resist the flow of blood and
cular endothelium is phenotypically distinct from venous remain adherent to the polymeric surface during the matu-
or arterial endothelial cells and shows appropriate differ- ration of an endothelial cell monolayer. The original meth-
ences in markers such as vWF and FVIIIrAg. Upon primary ods used to transplant endothelial cells onto graft surfaces
culture of microvessel endothelial cells, the morphology of- were termed seeding procedures, since relatively low con-
ten takes on a fibroblastic appearance and, in concert with centrations of cells were placed in suspension in either whole
the relatively minimal reaction with vWF antibodies, inves- blood or plasma, and this suspension transferred to the in-
tigators will often conclude that the predominant cell type ner lining of vascular grafts. The endothelial cells in this in-
in fat is fibroblasts. What is actually being observed is the oculum were subsequently expected to proliferate and mi-
ability of endothelial cells to phenotypically differentiate grate to the blood flow surface, creating a contiguous lining
under different in vitro and or in vivo conditions. Interest- of cells. An alternate form of transplantation, described us-
ingly, a recent report suggests that fibroblasts can differenti- ing the term sodding, was subsequently suggested and evalu-
ate in vivo into endothelial cells. This phenotypic drift may ated in vitro as well as in preclinical animal and human stud-
be a common characteristic of pluripotent mesenchymal ies.24 Sodding describes a method of endothelial cell depo-
cells, with possible utilization in future cell transplantation sition wherein a concentration of cells which would pro-
technologies. vide a confluent density of cells on the lumenal graft surface
The methods for the isolation of endothelial cells from is forcibly deposited onto the graft lumenal surface. This
fat are essentially modifications of the methods first reported forced deposition is most easily carried out by establishing a
by Wagner in 1972 for the isolation of endothelial cells from pressure gradient across the flow surface of the graft whereby
rat epididymal fat pads.22 Human fat microvessel endothe- the cells are essentially filtered onto the surface of the graft.
lial cells are isolated by first digesting the fat with a pro- While previous methods used gravity to allow cell deposi-
teolytic enzyme mixture composed predominantly of colla- tion, sodding of cells using pressure deposition results in
genase and trypsin. The characteristics of this enzyme are near immediate association of cells with the lumenal sur-
critical for successful isolation of endothelial cells from fat.23 face. The time to prepare a tissue engineered vascular graft
Following collagenase digestion the slurry is centrifuged, for implantation, a period inclusive of tissue isolation, cell
resulting in the separation of buoyant adipocytes from more isolation and cell deposition, has effectively been reduced to
dense endothelium. The endothelial cell rich pellet is then less than 60 minutes.20
used for cell transplantation procedures.
Fig. 6.2. Endothelial cell monolayer estab-
lished on an ePTFE graft using autologous
microvascular endothelial cell transplanta-
tion. The graft was implanted as an
interpositional device in the carotid artery in
a canine model and explanted after 5 weeks.
Microvascular Endothelial Cell Transplantation: A Review 97
Animal Models of Endothelial formation of an endothelial cell lining, but questions have
Cell Transplantation been raised regarding whether the cells present on the lu-
The availability of methods for endothelial cell isola- menal surface of microvessel sodded grafts represent the
tion and culture, as well as methods to deposit cells onto same cells transplanted during the initial sodding procedure.
graft surfaces, has resulted in numerous investigations to This question has been addressed using a method to trace
evaluate the efficacy of endothelial cell transplantation.25-33 the fate of transplanted endothelial cells using a fluorescent
The earliest animal models were developed to evaluate the tagging method.35 A fluorescent dye is permanently incor-
ability to accelerate the formation of endothelial cell linings porated into the membranes of microvessel endothelial cells
on polymeric grafts. Most of these studies have been at the time they are transplanted onto the lumenal surface
performed in the canine model, due primarily to the accep- of vascular grafts. This dye has unique characteristics in that
tance of this animal model as a predictor of graft function it is long lived and provides a marker of the fate of trans-
in humans. Earliest studies established the ability to planted cells. If, however, fluorescently labeled cells dupli-
accelerate the formation of a continuous monolayer of cate, each daughter cell receives one half the fluorescence of
endothelial cells on the lumenal surface of vascular grafts the initial cell. For cells which undergo multiple duplica-
(Fig. 6.2). In general, monolayer formation is highly depen- tion, a process which could occur following cell transplan-
dent on the density of cells used to treat the graft surface. tation, the subsequent progeny would contain undetectable
When cells are transplanted at confluent densities or greater, amounts of fluorescence. Use of this technique in a canine
monolayer formation appears to be complete in under three model of microvessel endothelial cell sodding revealed that
weeks. Subconfluent densities require more extended peri- after 5 weeks the monolayer of endothelial cells on the flow
ods of time. surface exhibited extensive fluorescence, establishing their
identity as cells present in the original cell inoculum used
Mechanisms Underlying the Formation for sodding. Thus, following sodding, the microvessel en-
of a Neointima in Tissue Engineered Vascular dothelial cells need simply adhere to the polymer surface
Grafts Using Microvascular Endothelial Cell and to each other to form a confluent monolayer without
Transplantation the need to undergo extensive cell proliferation.
A number of preclinical animal studies have estab-
lished that, following the transplantation of microvessel en- Preclinical Animal Trials
dothelial cells onto synthetic grafts, the accelerated forma- of Endothelial Cell Transplantation
tion of an endothelial cell monolayer on the lumenal flow With the development of methods for the isolation of
surface is observed. The earliest time point evaluated is 4 endothelial cells from numerous tissue sources, including
days in a rat aortic graft model of microvessel endothelial macro- and microvascular sources, numerous animal stud-
cell transplantation.34 At this time a confluent layer of en- ies have been performed to evaluate the effects of endothe-
dothelial cells is observed. Studies in dogs have established lial cell transplantation on small diameter vascular graft pa-
that the endothelial cell lining observed in microvessel en- tency. These studies use small diameter grafts (< 6 mm),
dothelial cell sodded grafts remains stable for periods of up most often in the canine species. The predominance of ca-
to one year, the longest published animal implant data.33 nine models for these preclinical evaluations is based on the
Figure 6.3 illustrates the lining on a microvascular endothe- general acceptance of this model as being similar to humans
lial cell sodded ePTFE graft explanted after one year. This with respect to graft healing. While the canine species is com-
morphologic data establishes the ability to accelerate the monly used, techniques for graft placement (e.g., end to end,
Fig. 6.3. Scanning electron micrograph of a
sodded ePTFE carotid artery after one year.
98 Tissue Engineering of Prosthetic Vascular Grafts
end to side anastomoses) site of placement (e.g., femoral,
Chi-Square
carotid, aortofemoral), use and duration of antiplatelet/an-
2.64
2.07
0.25
0.96
8.98
24.7
2.64
0.7
6.4
*
ticoagulant drugs (e.g., asprinin, persantine) and the type
and use of control grafts (e.g., paired vs. nonpaired grafts)
varies significantly among investigators. The rationale for
95% Lower
Confidence
the choice of each variable is often based on the goals of
-0.20,0.65
-0.09,0.30
0.02,0.62
0.03,0.37
0.06,0.46
0.44,1.00
0.50,0.86
0.01,0.62
0.27,0.89
and Upper
Intervals
individual studies. Quite often decisions are based on the
need to evaluate healing characteristics of grafts as compared
to evaluation of patency. The use of paired grafts (e.g., ca-
rotid or femoral interpositional grafts) is most common,
since cell transplanted grafts can be compared to control
Increase in
Fractional
for Seeded
grafts in the same animal. Variability in animal to animal
Patency
Grafts
0.36
0.17
0.73
0.68
0.11
0.22
0.32
0.58
0.2
thrombotic state is partially accounted for in paired graft
models.
Due to the variability in animal models previously
used by investigators, comparison of data is complicated.
Table 6.1 illustrates a compilation of the results of published
Patent Clotted
Control Grafts
13
16
10
20
11
11
animal trials using 4 mm inner diameter polymer grafts in
8
4
preclinical studies of endothelial cell transplantation. These
studies were chosen for the ability to evaluate patency using
25
26
11
6
1
8
6
1
nonpaired statistical analysis within each study. The signifi-
cance of endothelial cell transplantation can be evaluated
first by assessing the fractional increase in patency between Table 6.1. Endothelial cell-seeded graft versus control graft canine studies using paired 4 mm diameter grafts
Endothelial Cell-Seeded
cell transplanted and control grafts. In this column a posi-
Clotted
tive fractional increase in patency indicates a benefit of cell
7
9
2
2
3
1
1
7
4
transplantation, a zero increase indicates no benefit and a
Grafts
negative fractional increase indicates cell transplantation has
Patent
a negative influence on graft patency. The 95% confidence
12
32
11
27
30
14
9
7
8
limits have been provided for each study to indicate vari-
ability. The major conclusion from this table is the consis-
tently observed positive benefit of endothelial cell transplan-
tation with respect to graft patency. Although not all studies
Antiplatelet
presented in this table demonstrated a statistically signifi-
Therapy
None
None
None
None
6 wk
4 wk
4 wk
2 wk
2 wk
cant improvement in patency with cell transplantation, treat-
ment of grafts with endothelial cells was the most common
predictor of improved patency. Most significantly, only two
reports provide evidence of statistically significant improve-
Duration
ment in graft patency as a direct result of endothelial cell
12 wk
13 wk
12 wk
16 wk
28 wk
10 wk
4 wk
4 wk
4 wk
transplantation.25-33 Both of these reports are unique in that
they treated the lumenal surface of grafts with a confluent
density of endothelial cells using the method known as sod-
ding. One of these two reports used autologous microvessel
endothelial cells as the source of cells for sodding. Thus, pre-
Dacron
Dacron
Dacron
Dacron
Dacron
ePTFE
ePTFE
ePTFE
ePTFE
Graft
clinical animal studies of endothelial cell transplantation
*For chi-square >3.84 for 1 degree of freedom, P<0.05.
have, until recently, not provided evidence of statistically sig-
nificant improvement in graft patency following endothe-
lial cell transplantation. On the other hand, deposition of
Femoral and carotid
endothelial cells at high densities and the use of microvessel
endothelial cell sodding has provided statistically significant
improvement in patency in preclinical animal studies.
Iliofemoral
Femoral
Carotid
Carotid
Carotid
Carotid
Carotid
Carotid
Microvascular Endothelial Cell Transplantation
Site
for Arteriovenous Fistulas and
Coronary Artery Bypass Grafts
Arteriovenous Fistulas for Dialysis
Tannenbaum32
Kempczinski10
The initial preclinical animal studies of endothelial cell
Investigator
Williams33
transplantation were consistently performed in models
Douville27
Schmidt28
Stanley31
Belden26
Shindo30
which sought to duplicate the environment typical of pe-
Allen25
Microvascular Endothelial Cell Transplantation: A Review 99
ripheral arterial interpositional grafts. The development of cellular vehicle for gene therapy.41 Endothelial cells reside at
a synthetic peripheral arterial graft is clearly of clinical im- the interface between blood and tissue and therefore main-
portance; however, new designs in synthetic grafts for use in tain a strategic position for the production and release of
other anatomical positions are also of great interest. In the therapeutic materials. Molecular biologists have begun to
USA, prosthetic grafts are presently the most common vas- take advantage of the extensive methodology established for
cular access created for hemodialysis.36 These grafts exhibit endothelial cell transplantation. Reports of genetic modifi-
a failure rate requiring intervention to restore patency at a cation of endothelial cells have exploded recently as the
mean of nine months after patency.37 The estimated annual attractiveness of this cell is realized. The use of genetically
cost to the USA health care system to maintain vascular ac- modified microvascular endothelial cells has moved beyond
cess for hemodialysis is believed to be in excess of $900 mil- prosthetic devices, and studies are ongoing to establish the
lion.37 The reason for the failure of these grafts is common use of these cells in almost every anatomic site under
to all synthetic grafts and includes the development of pre- consideration for gene therapies. Again, the availability of
dominantly distal anastomotic neointimal thickening and methods to easily isolate and transplant endothelial cells,
thrombosis due to the inherent thrombogenicity of poly- especially subcutaneous fat-derived microvessel endothelial
mer materials. Recent studies have begun to evaluate the use cells, provides an exciting new use for endothelial
of microvascular endothelial cell transplantation to improve cell transplantation.
AV graft function.38 These initial studies have demonstrated
that the healing of microvessel endothelial cell sodded grafts References
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12. Thornton SC, Mueller SN, Levine EM. Human endothe-
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the availability of operating room compatible methods for ing heparin. Science 1983; 222:623-624.
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H, Tsangaris S, Groscurth P. Use of fibrin glue as a sub- GR. Impact of endothelial cell seeding on long term pa-
strate for in vitro endothelialization of ePTFE vascular tency and subendothelial proliferation in a small-caliber
grafts. Surgery 1989; 105(4):515-522. highly porous polytetrafluoroethylene graft. J Vasc Surg
15. Zilla P, Preiss P, Groscurth P, Rosemeier F, Deutsch M, 1987; 5:544.
Odell J, Heidinger C, Fasol R, von Oppel U. In vitro-lined 28. Schmidt SP, Hunter TJ, Hirko M. Small diameter vascu-
endothelium: Initial integrity and ultrastructural events. lar prostheses: Two designs of PTFE and endothelial cell
Surgery 1994; 116(3):524-534. seeded and nonseeded Dacron. J Vasc Surg 1985;
16. Zilla P, Deutsch M, Meinhart J, Puschmann R, Eberl T, 2:292-297.
Minar E, Dudczak R, Lugmaier H, Schmidt P, Noszian I. 29. Shepard AD, Eldrup-Jorgensen J, Keough EM. Endothe-
Clinical in vitro endothelialization of femoropopliteal by- lial cell seeding of small caliber synthetic grafts in the
pass grafts: An actuarial follow-up over three years. J Vas- baboon. Surg 1986; 99:318.
cular Surgey 1994; 19(3):540-548. 30. Shindo S, Takagi A, Whittemore AD. Improved patency
17. Jarrell BE, Williams SK, Stokes G, Hubbard FA, Carabasi of collagen impregnated grafts after in vitro autogenous
RA, Koolpe E, Greener D, Pratt K, Moritz MJ, Radomski endothelial cell seeding. J Vasc Surg 1987; 6:325.
J, Speicher L. Use of freshly isolated capillary endothelial 31. Stanley JC, Burkel WE, Linbald B. Endothelial cell seed-
cells for the immediate establishment of a monolayer on ing of synthetic vascular prostheses. Acta Chir Scand Suppl
a vascular graft at surgery. Surg 1986; 100(2):392-399. 1985; 529:17-27.
18. Visser MJP, van Bockel JJ, van Muijen GNP, van 32. Tannenbaum G, Ahlborn T, Benvenisty A, Reemstma K,
Hinsbergh VWM. Cells derived from omental fat tissue Nowygrod R. High density seeding of cultured endothe-
and used for seeding vascular prostheses are not endot- lial cells leads to rapid coverage of PTFE grafts. Curr Surg
helial in origin: A study on the origin of epitheloid cells 1983; 222:623.
derived from human omentum. J Vasc Surg 1991; 33. Williams SK, Rose DG, Jarrell BE. Microvascular endot-
13:373-381. helial cell sodding of ePTFE vascular grafts: Improved
19. Williams SK, Wang TF, Castrillo R, Jarrell BE. Liposuction patency and stability of the cellular lining. J Biomed Mater
derived human fat used for vascular graft sodding con- Res 1994; 28:203-212.
tains endothelial cells and not mesothelial cells as the 34. Ahlswede KM, Williams SK. Microvascular endothelial cell
major cell type. J Vasc Surg 1994; 19:916-923. sodding of 1-mm expanded polytetrafluoroethylene vas-
20. Williams SK, Jarrell BE, Rose DG, Pontell J, Kapelan BA, cular grafts. Arteriosclerosis and Thrombosis 1994;
Park PK, Carter TL. Human microvessel endothelial cell 14:25-31.
isolation and vascular graft sodding in the operating room. 35. Williams SK, Kleinert LB, Rose D, McKenney S. Origin
Ann Vasc Surg 1989; 3(2):146-152. of endothelial cells that line expanded
21. Lenzi R, Alpinin G, Liu MH, Rand JH, Tavoloni N. Von polytetrafluoroethylene vascular grafts sodded with cells
willebrand factor antigen is not an accurate marker of rat from microvascularized fat. J Vasc Surg 1994; 19:594-604.
and guinea pig liver endothelial cells. Liver 1990; 36. Palder SB, Kirkman RL, Whittemore AD. Vascular access
10:372-379. for hemodialysis. Ann Surg 1986; 202:235-239.
22. Wagner RC, Matthews MA. The isolation and culture of 37. Windus DW. Permanent vascular access: A nephrologist’s
capillary endothelium from epididymal fat. Microvasc Res view. Am J Kid Dis 1993; 21(5):457-471.
1975; 10:286-297. 38. Williams SK, Jarrell BE, Kleinert LB. Endothelial cell trans-
23. Williams SK, McKenney S, Jarrell BE. Collagenase lot se- plantation onto polymeric arteriovenous grafts evaluated
lection and purification for adipose tissue digestion. Cell using a canine model. J Invest Surg 1994; 7:503-517.
Transplantation. 1995; 4:281-289. 39. Phillips M, Yamaguchi H, Miller V, Williams SK, Morris
24. Williams SK, Carter T, Park PK, Rose DG, Schneider T, JJ, Shaff HJ. Endothelial cell sodding of the Permaflow
Jarrell BE. Formation of a multilayer cellular lining on a prosthetic coronary artery bypass conduit. Annals of Thor
polyurethane vascular graft following endothelial cell sod- Surg 1998; in press.
ding. J Biomed Mat Res 1992; 26:103-117. 40. Arzouman D, Kleinert LB, Patula VB, Phillips M, Will-
25. Allen BT, Long JA, Clark RE, Sicard GA, Hopkins KY, iams SK, Copeland JG. Endothelial cell sodding of ePTFE
Welch MJ. Influence of endothelial cell seeding on plate- coronary artery bypass grafts. Abstract. American Heart
let deposition and patency in small-diameter Dacron ar- Association 70th Scientific Session.
terial grafts. J Vasc Surg 1984; 1:224-232. 41. Stopeck AT, Vahedian M, Williams SK. Transfer and ex-
26. Belden TA, Schmidt SP, Falkow LJ, Sharp WV. Endothe- pression of the interferon gamma gene in human endot-
lial cell seeding of small-diameter vascular grafts. Trans helial cells inhibits vascular smooth muscle cell growth
Am Soc Artif Intern Organs 1982; 28:173. in vitro. Cell Transplant 1997; 6(1):1-8.
CHAPTER 7
Morphological Aspects of Microvascular Cell Isolates
Manuela Vici
Introduction
T he lack of spontaneous endothelialization after implantation of small caliber vascular
grafts in humans has made the lining of the prosthetic surface with endothelial cells ad-
visable in order to reduce thrombogenicity and promote hemocompatibility. This has added
a promising new field to tissue engineering.
Numerous research areas are critical for the success of tissue engineering. Some are
related to the biological side, others to the engineering side. With regard to the former, issues
such as cellular differentiation and growth, as well as the influence of biological components
(extracellular matrix, growth factors etc.) on cell functions or the establishing of cell sources
and of cell preservation are likely to be crucial for the future of tissue engineering.1
Morphology is one of the aspects that can effectively contribute to adding informa-
tion about cell biology related to tissue engineering. It is well established that the level of
compatibility of a certain cell type with a particular substratum can be evidenced by study-
ing the morphological changes which occur when the cell surface interacts with the material
examined. Of course, the use of transmission and scanning electron microscopy permits the
collection of very detailed information. A clear example comes from the study of cell adhe-
sion to lenses in ophthalmology, where correlated transmission and scanning electron mi-
croscopy investigations have permitted the description of different steps in cellular adhe-
sion onto a particular substratum.2
Briefly, rounded cells always indicate the initial attachment to the substratum. This
initial interaction depends mostly on the ability of cells to form cytoplasmic extensions
which bring the cell close enough to the substratum to allow adhesion. This surface behav-
ior corresponds inside the cell to a reorganization of cytoskeletal elements, which produces
a flattening effect, first of the periphery and then, while adhesion is proceeding, of the cell
body. Filaments develop into a network, eventually forming specialized contacts between
the cell surface and the substratum. This surface and cytoskeletal modification continues
until the cell is completely spread over the substratum. On the other hand, a negative re-
sponse in cell adhesion can be described by a permanent rounded cellular shape with no
cytoskeletal modifications and consequently no interaction with the surface.
Other examples can describe the correlation between morphological aspects and cell
functionality. Nucleus morphology can always give information about cell viability. Cul-
tured or seeded cells with pyknotic nuclei testify that the environment in which they are
placed is totally unsuitable. Moreover, nuclei together with the organelle apparatus can in-
form about phenotypical changes, for example the change from a quiescent phenotype to-
ward a synthetic phenotype is always characterized by a remarkable increase of rough endo-
plasmic reticulum cisternae.
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
102 Tissue Engineering of Prosthetic Vascular Grafts
For all these reasons we believe morphological infor- method for the isolation of microvascular endothelium from
mation to be very important for studies involved in the tis- first trimester decidua.17
sue engineering of vascular grafts. Moreover, when identi-
fication or localization of certain cell types is also requested, Isolation of Microvascular Endothelial Cells
they can successfully be supplied by immunocytochemical Jarrel and Williams first documented how microvas-
investigations. cular endothelial cells can easily be recovered from enzy-
matically disrupted fat tissue.3
Microvascular Cells In our lab we developed a modification of the Jarrel
Microvessels are considered the alternative to vein as method starting from human subcutaneous fat tissue ex-
a source of endothelial cells for cell transplantation. There cised from the abdominal wall of patients undergoing sur-
are many tissues in humans which are abundant in capillar- gery for abdominal aneurysms.18 The fat tissue is carefully
ies and promptly available either surgically or by noninvasive minced and mixed with a 0.2% collagenase solution in an
methods. During years of experiments the protocols of the Erlenmayer flask and incubated under constant agitation for
isolation of microvascular endothelial cells have become 30 minutes at 37°C. After digestion, the floating adipocytes
increasingly fast and efficient—normally a large number of are discharged and the suspension centrifuged. The pellet is
viable endothelial cells is easily obtainable per gram of tissue. first washed in PBS-BSA and then filtered through a 120 ∝m
nylon filter; the suspension is gently layered onto 45% ster-
Sources of Microvascular Endothelial Cells ile Percoll and endothelial cells are recovered from the milky
Jarrel and Williams first started to harvest microvas- layer formed after centrifugation. Normally a variable num-
cular endothelial cells enzymatically for cell transplantation.3 ber of viable cells ranging from 2.5-8 x 105 is obtained per g
Initially they reported on methods established for the isola- of tissue. Recently we proposed an alternative method of
tion and culture of human adult microvessel endothelial cells microvascular endothelial cell isolation based on the use of
from different sources of human fat such as omental, peri- magnetic polystyrene beads (Dynabeads M-450) coated with
renal and subcutaneous fat. Then they focused on human anti-CD34 monoclonal antibody.19 After the mincing and
subcutaneous fat tissue as the most suitable source of en- collagenase digestion of the subcutaneous fat tissue, the cells
dothelial cells, as it is composed mainly of adipocytes richly filtered and suspended in PBS-BSA as previously described
perfused with microvascular endothelium and pericytes. are incubated with anti-CD34 coated Dynabeads
Human subcutaneous fat is easily obtainable either (Dynabeads M-450 CD34, Dynal) at a ratio of 10 beads to 1
surgically by patients undergoing abdominal surgery, (this endothelial cell for 30 minutes at 4°C under gentle mixing.
is what has been mainly experienced in our lab), or by Rosetted cells are positively collected by means of three wash
liposuction of fat deposits on the abdominal wall.4 cycles performed with a Dynal Magnetic particle concen-
Omentum and omentum-associated fat has also been trator (MPC). Rosetted cells are then resuspended in M199
utilized as a rich source of microvascular endothelial cells, plus 20% FCS containing 10 ∝l DetachaBead (Dynal) per
but recently it has been proved by Williams et al that be- 1 x 107 Dynabeads used for rosetting, and incubated for 1 h
cause of its anatomical location, the primary isolate from at room temperature with constant agitation. After incuba-
omentum often comprises not pure endothelium but a mix- tion cells are negatively recovered using the Dynal MPC.
ture of cells including endothelium, smooth muscle cells and Usually approximately 0.8 x 105 cells per g of fat tissue are
mesothelium.5 obtained.
Microvascular endothelial cells can also be harvested Recently, Chen et al proposed a simple new method
from other sources. Hewett et al developed a method for the for the isolation of microvascular endothelial cells avoiding
isolation and long term culture of human microvessel en- both chemical and mechanical injuries.20 They indicated that
dothelial cells from mammary adipose tissue obtained at when small pieces of lung or muscles of the chest wall of
breast reduction.6 Robinson et al reported on the seeding of rats are placed into a flask, erythrocytes and leukocytes leave
immortalized human dermal microvascular endothelial the tissues first, followed by vascular endothelial cells. Fi-
cells.7 Procedures for the isolation and culture of microvas- broblasts and other mixed cells grow after 72 hours of cul-
cular endothelium from neonatal foreskins have also been ture. When the tissues are discarded within 60 hours the flask
previously described by Sherer et al, Marks et al, and Karasak contains only microvascular endothelial cells and blood cells.
et al.8-10 Hewett et al isolated and cultured microvessel en- The latter can be cleared out after the cells are subcultured.
dothelial cells from lung tissue obtained from lung trans-
plant recipients.11 Haraldsen et al established a method for Microvascular Cell Isolates
the isolation and culture of intestinal microvascular endot- The purity of a microvascular isolate has always been
helial cells.12 Kacemi et al devised a method of isolation and a major concern when microvessel-derived endothelial cells
culture of endothelial cells from villous microvessels from are chosen for cell transplantation. The attempt to charac-
human placenta.13 Masek et al and Schweitzer et al described terize the cell population of a microvascular primary isolate
two simple methods for the isolation and culture of endo- from human fat tissue has lead to controversies. This is a
thelial cells from human bone marrow.14,15 McDouall et al consequence of the fact that the type of cells involved in a
isolated and cultured microvascular endothelial cells from microvascular primary isolate correlates directly with the
the human heart.16 Finally, Grimwood et al developed a novel source of microvessels chosen.
Morphological Aspects of Microvascular Cell Isolates 103
Omentum and omentum fat are rich in mesothelial rience) is that the possibility of collecting information such
cells; consequently the primary isolate comprises a hetero- as the appearance of subcellular components, or the pres-
geneous cell population including endothelial cells, pericytes, ence of intercellular connections, from a primary isolate, can
fibroblasts and mesothelial cells. Human subcutaneous fat give a realistic description of the biological status of the cells
is composed predominantly of adipocytes, pericytes and analyzed and can add an indispensable contribution when
microvascular endothelial cells (Fig. 7.1a,b), and thus the an accurate characterization of the cell types involved
primary isolate is expected to be more homogeneous. Re- is necessary.
cent studies are focusing on the fact that mesothelial cells For example, as already mentioned, the morphologi-
can also be utilized for the seeding of vascular prostheses, cal changes in rough endoplasmic reticulum, Golgi appara-
behaving very much like endothelial cells derived from large tus and mitochondria are correlated to the level of meta-
vessels when seeded.21 bolic activity of the cell; Weibel-palade bodies are undoubt-
Thus, depending on the aim of the study, the charac- edly considered a morphological marker for endothelial cells,
terization of the cell population of a microvascular primary while “fibronexus” is the morphological marker for
isolate can be very important. To do this we focused on myofibroblasts. Moreover, the fact that cells start to develop
morphological studies. Our conviction (derived from expe- intercellular junctions when placed in culture or when
seeded, often gives positive information about growth and
stability. Furthermore, the localization of molecules specifi-
cally present in certain cells by immunohistochemistry is
definitely helpful for the characterization of the different cell
types involved in a primary isolate and can also contribute
to the definition of molecular pathways signaling, when
present, metabolic alterations.
Electron Microscopic and
Immunocytochemical Profiles
of Microvascular Cell Isolates
To characterize the phenotype of the microvascular
cells recovered from human subcutaneous fat tissue precisely,
cell samples obtained at different steps in the isolation pro-
cedure have been studied by means of electron microscopy
as well as by immunohistochemical and immunoelectron
microscopic techniques using a wide variety of antibodies.
Transmission Electron Microscopy
All the samples processed for transmission electron
microscopy (TEM) analysis were first fixed in 2.5% cacody-
late-buffered glutaraldehyde, then postfixed with 1% OsO4,
dehydrated in a graded series of alcohols, and embedded in
araldite. Thin sections were obtained with a Reichert Omu3
ultramicrotome, counterstained with uranyl acetate and lead
citrate and examined under a Philips 400T transmission elec-
tron microscope.
With TEM, enzymatically harvested microvascular
endothelial cells (MECs) appear both as single cells and
multicell aggregates (Fig. 7.2). Generally MECs show oval
to round nuclei with clumps of condensed chromatin at the
nuclear periphery. Occasional nucleoli are also seen. Nuclear
irregularity is present in some cells. In particular, invagina-
tion of the nuclear envelope and deep clefts appear to be
responsible for the presence of cleaved and convoluted en-
dothelial nuclei. The bulky cytoplasm contains a small Golgi
apparatus, mitochondria, short strands of rough endoplas-
mic reticulum, and many intermediate filaments (Fig. 7.3A),
occasionally arranged to form characteristic fibrous bodies.
Moreover, small to moderate quantities of microcystic la-
Fig. 7.1. Human subcutaneous fat tissue from abdominal wall.
cunae, which appear as the remains of the primitive vascu-
(A), top. Characteristic appearance of an adipocyte. Lipid drop-
lar lumen and of micropinocytotic vescicles, can be clearly
let (asterisk); cytoplasm (arrow); nucleus (arrowhead). (TEM;
seen (Fig. 7.3A). Sometimes, clustered microvascular endot-
x11,600). (B), bottom. Microvascular endothelial cells. Weibel-
helial cells present tight junctions (Fig. 7.2), which appear
palade bodies are visible (arrows). (TEM; x9,000).
104 Tissue Engineering of Prosthetic Vascular Grafts
as sites of membrane fusion with adjacent electron-dense acteristic symmetrical clefts. The cytoplasm is rich in in-
“fuzzy” cytoplasm. Specific rod-shaped microtubulated bod- termediate filaments and microfilaments and very few or-
ies (the so-called Weibel-palade bodies) are occasionally seen ganelles are found. Microfilaments are usually disposed par-
but are evident in less than 5% of the cells examined, par- allel to the long axis of the cell, among which are interspersed
ticularly in the clustered elements (Fig. 7.2). Weibel-palade numerous focal densities. These are electron dense struc-
bodies are specifically found in the endothelium (Fig. 7.3B); tures scattered in the cytoplasm which are part, together with
their typical rod shape is contoured by a membrane con- the contractile filaments, of the contractile apparatus. Pre-
taining numerous parallel cylindrical tubules embedded in cisely three different kinds of filaments can be described:
an electron-dense matrix. Immunological studies have myosin filaments (18 nm in diameter); actin filaments
shown that Weibel-palade bodies are sites of storage of the (6-8 nm in diameter) and vimentin and desmin intermedi-
von Willebrand protein. ate filaments. In the subplasmalemmal site there are many
MECs isolated with the help of CD34-coated micropinocytotic vescicles and plasmalemmal introflexions
Dynabeads appear, prior to DetachaBeads application, with called “caveolae” which increase the plasmalemmal surface
1-6 beads bound to the plasma membrane (Fig. 7.4). After by about 25%. SMCs can form gap junctions and under spe-
bead detachment, MECs are present mainly as single cells, cific conditions can change into a “synthetic” phenotype and
morphologically homogeneous and well preserved. Weibel- become myofibroblasts.
palade bodies are occasionally observed in the cytoplasm Myofibroblasts are characterized by nuclear polymor-
together with other typical endothelial features previously phism, cytoplasmic extensions and abundant rough endo-
mentioned (intermediate filaments, micropinocytotic plasmic reticulum, Golgi apparatus and mitochondria. They
vescicles and microcystic lacunae).19 are connected to the extracellular matrix by the so-called
When other sources of microvascular cells are chosen, “fibronexus”, which are transmembrane complexes of intra-
the primary isolate can be more heterogeneous and other cellular microfilaments in apparent continuity with extra-
cell types can be described. Mesothelial cells are character- cellular fibronectin fibers. These cell to stroma attachment
istically identified by their numerous microvilli at the apical sites are more developed and numerous than in smooth
surface; they are heterogeneous in nature and measure up muscle cells, and three types of fibronexus can be described:
to 3 ∝m in length and 0.1 ∝m in diameter. In the cytoplasm plaquelike, tracklike and tandem associations. Within the
there are some pinocytotic vesicles, very few Golgi cytoplasm there are numerous bundles of microfilaments
membranes, scanty granular endoplasmic reticulum and (stress fibers) arranged parallel to the long axis of the cell,
many mitochondria. Junctions of all types can be found in with interspersed numerous dense bodies.
situ. Intermediate filaments are present, sometimes Fibroblasts have no basal lamina and display a slen-
prominent and often arranged in a perinuclear, circumfer- der fusiform and smoothly contoured nucleus; in the cyto-
ential distribution.22 plasm microfilaments are sometimes arranged in bundles
Smooth muscle cells (SMCs) are delimited by a basal beneath the plasma membrane and there is a well developed
lamina and present elongated and lobated nuclei with char- Golgi area and abundant dilated cisternae of rough endo-
Fig. 7.2. Multicell aggregate of iso-
lated microvascular endothelial
cells after Percoll step. Weibel-
palade bodies (arrows). Tight
junctions (arrowheads). (TEM;
x6,500).
Morphological Aspects of Microvascular Cell Isolates 105
Fig. 7.3. Percoll isolated microvascular
endothelial cells. (A), top. The predomi-
nant cytotype is characterized by
microcystic lacunae (arrow), micro-
pinocytotic vesicles (arrowheads) and
extensive intermediate filaments (aster-
isk). (TEM; x20,000). (B), bottom left.
Weibel-palade bodies at higher magnifi-
cation. (TEM; x26,000). With permission
from: Vici M, Pasquinelli G, Preda P et
al. Electron microscopic and immuno-
cytochemical profiles of human subcu-
taneous fat tissue microvascular endo-
thelial cells. Ann Vasc Surg, 1993;
7:541-548. (C), bottom right. Numerous
gold particles (arrowheads) bind CD34
antigen on the cell surface (TEM;
x12,000). With permission from: Vici M,
Pasquinelli G, Preda P et al. Electron
microscopic and immunocytochemical
profiles of human subcutaneous fat tis-
sue microvascular endothelial cells. Ann
Vasc Surg, 1993; 7:541-548.
plasmic reticulum. Cell contours are generally smooth or
display a few short cytoplasmic extensions.
Pericytes that are embedded within the basement
membrane of microvessels share with vascular SMCs the
#-smooth muscle actin content and also intermediate fila-
ment proteins. In fact, both cell types express vimentin or
vimentin and desmin. Pericytes form an integral part of the
microvascular wall.23
A few adipocytes can also be found in a primary mi-
crovascular isolate from enzymatically treated adipose fat
tissue. Mature adipocytes have characteristic features
(Fig. 7.1A): The nucleus is flattened against the cytoplasmic
membrane by a large lipid droplet, with only a thin, tenu-
ous rim of cytoplasm surrounding it. Adjacent to the cell
membrane are deposits of basement membrane. In situ cap-
illaries are closely opposed to the adipocyte membrane.24
106 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 7.4. Dynabeads isolated microvascular
endothelial cells. Immunomagnetic beads are
closely adherent to the plasma membrane.
(TEM; x3,400).
Scanning Electron Microscopy Immunophenotyping
Isolated MECs were seeded at a density of 1 x 105/cm2 The immunophenotype of a primary microvascular
on both uncoated and coated glass coverslips. As coating isolate can be studied using a wide variety of antibodies. One
materials we used poly-L-lysine and fibronectin; briefly, glass of the most commonly employed, when endothelial cells
coverslips were incubated with 1 mg/ml poly-L-lysine di- need to be identified, is the FVIII RA antibody. FVIII RA is
luted in PBS at room temperature for 1 hour, or with specifically expressed by endothelial cells. With immuno-
5 ∝g/cm2 fibronectin suspended in M199 and kept at 37°C histochemistry a positive staining gives a typical cytoplasm
for 2 h. The seeded coverslips were fixed with 2.5% glutaral- granularity, while at the immunoelectron microscopical level
dehyde in 0.1 M cacodylate buffer, postfixed with 1% OsO4, FVIII RA specifically stains Weibel-palade bodies and slightly
dehydrated in a graded series of alcohol and critical point stains the cell membrane.
dried. Mounted samples were coated with gold and exam- Other typical endothelial cell markers include: Ulex
ined in a Philips 505. europaeus type i lectin, which selectively binds to L-fucose
Most of the MECs show many slender microvilli, residues of the endothelial membrane—the binding between
bulbous protrusions and membrane folds, the overall im- the lectin and the endothelial surface can then be evidenced
pression being a flower-like appearance (Fig. 7.5A). The sur- with an antibody specific for the lectin itself;25 CD31, a mem-
face specializations probably begin at the original luminal brane glycoprotein that is the platelet endothelial cells ad-
aspect of the cell membrane. At the same site the extracellu- hesion molecule PECAM 1; 26 CD34, a surface
lar space appears by TEM to be compartmentalized into glycophosphoprotein expressed on hemopoietic stem and
microcystic lacunae by interdigiting cytoplasmic flaps. On progenitor cells, small-vessel endothelial cells, and embry-
the contrary, other elements exhibit a smoother surface onic fibroblasts. The function of CD34 is not yet clear. Re-
morphology, and scanning electron microscopy (SEM) oc- cent studies on the functions of this molecule indicate that
casionally reveals microvillus-like projections and irregular CD34 expressed on endothelial cells may play a role in leu-
surface corrugations. kocyte adhesion and “homing” during the inflammatory
Exposure of MECs to nonvascular surfaces induces process. A role for CD34 in adhesion has been theorized
rapid morphological changes. Isolated MECs lose their con- because, like other known adhesion molecules, CD34 is a
voluted appearance and become relatively spherical, extend- heavily glycosylated surface sialomucin.27
ing long, slender filopodia making tenuous contact with the PAL-E is a monoclonal antibody specific for endothe-
substrate. As the endothelial attachment proceeds, the in- lial cells. It markedly stains endothelium of capillaries, me-
teraction appears more tenacious. Endothelial cells seem to dium size and small veins, and venules. The antigenic deter-
sink into the surface and indent it with peripheral cytoplas- minant recognized by PAL-E is associated with endothelial
mic flaps (skirted cells) (Fig. 7.6A). In the late stage MECs vesicles.28
completely spread over the substrate, resulting in a fried egg EN4 was originally described as a monoclonal anti-
appearance. At the same time, round to stellate, as well as body that reacts specifically with human endothelial cells
elongated, spreading endothelial cells begin to appear and the reagent has not been assigned to any of the pres-
(Fig. 7.6B). ently known CD. Recently, Burgio et al have demonstrated
Morphological Aspects of Microvascular Cell Isolates 107
Fig. 7.5. Percoll isolated microvascular endothelial cells. (A), left. SEM of a multicell aggregate. (SEM; x4,200). With permission
from: Curti T, Pasquinelli G, Preda P et al. An ultrastructural and immunocytochemical analysis of human endothelial cell adhe-
sion on coated vascular grafts. Ann Vasc Surg, 1989; 3:351-363. (B), right. Immunoelectron microscopic demonstration of FVIII
RA. SEM reveals strong positivity at cell surface. (SEM; x8.500). With permission from: Curti T, Pasquinelli G, Preda P et al. An
ultrastructural and immunocytochemical analysis of human endothelial cell adhesion on coated vascular grafts. Ann Vasc Surg,
1989; 3:351-363.
that EN4 and CD31 monoclonal antibody recognize the ing of cytoplasmic and surface membrane antigens on cell
same 130 kDa antigen.29 smears at optical level. Cells seeded onto fibronectin-coated
Since a primary microvascular isolate can be com- coverslips were fixed in acetone-metanol (7:3 at -20°C for
posed of a mixture of cell types, it might also be useful to 10 minutes) and incubated in the primary antibody, then in
characterize most of them. Vascular smooth muscle cells anti-mouse immunoglobulin and finally in APAAP com-
specifically express vimentin and smooth muscle actin, while plexes. The reaction was revealed by adding the substrate
a low percentage is desmin positive. Cytokeratins such as containing naphtol AS phosphate and new fuchsin. For
CK8 and CK18 can be found in some SMCs. Pericytes also polyclonal antibodies, samples were treated with a bridge
express smooth muscle-specific actin. Nonproliferating me- antibody.
sothelial cells express both vimentin and a variety of Protein a-gold technique was performed on MECs
cytokeratins (CK7, CK8, CK18, CK19).22 Adipocytes express seeded on fibronectin-coated coverslips by incubating cells,
S-100 protein, while monocytes and macrophages can be after the primary antibody, with 18 nm protein a-gold par-
detected using antibodies against specific antigens such as ticles.31,32 The reaction was then silver enhanced. For mono-
150.95 protein and MAC 387. clonal antibodies a secondary antibody was employed.
We studied the immunophenotype of microvascular For immunoelectron microscopy, suspensions of
isolates from human subcutaneous fat tissue at different steps MECs were fixed in 3% paraformaldehyde, then incubated
in the isolation procedure with the help of different immu- with the primary antibody and afterwards with an appro-
nocytochemical techniques using monoclonal and priate dilution of 18 nm protein a-gold complexes. Finally,
polyclonal antibodies. Immunofluorescence was performed MECs were postfixed in 1.7% glutaraldehyde and embed-
by first seeding the isolate onto fibronectin-coated cover- ded in araldite. Immunolabeling specificity was always
slips for 1 hour at 37°C, and then fixing the cells with cold checked by omitting the incubation with the specific pri-
acetone and incubating them with the primary antisera. A mary antibody or replacing the primary antibody with an
second incubation with a fluorescein-conjugated anti-rab- unrelated antibody.
bit or anti-mouse Ig followed. Coverslips were then mounted Our investigation was performed employing the fol-
and observed under a photomicroscope equipped with an lowing antibodies. As endothelial cell markers we used: a
epifluorescent condenser. polyclonal antibody against Factor viii RA; a polyclonal an-
Immunoenzymatic labeling was also performed on tibody specific for the Ulex europaeus type i lectin previ-
MECs cells using the alkaline phosphatase monoclonal ously bound to the endothelial membrane; a monoclonal
antialkaline phosphatase (APAAP) procedure.30 The APAAP antibody against CD31 antigen and QB-End 10, a mono-
technique is particularly suitable for clear and specific stain- clonal antibody specific for the CD34 antigen. Antibodies
108 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 7.6. Microvascular endothelial
cells—substrate interaction. (A), top.
SEM reveals different stages of adhesion.
Round, surface convoluted cell (arrow);
skirted-cell (arrowhead). (SEM; x2,300).
With permission from: Curti T,
Pasquinelli G, Preda P et al. An ultra-
structural and immunocytochemical
analysis of human endothelial cell adhe-
sion on coated vascular grafts. Ann Vasc
Surg 1989; 3:351-363. (B), bottom. Dif-
ferent stages of adhesion on fibronectin-
coated glass. (SEM; x600). With permis-
sion from: Curti T, Pasquinelli G, Preda
P et al. An ultrastructural and immuno-
cytochemical analysis of human endo-
thelial cell adhesion on coated vascular
grafts. Ann Vasc Surg 1989; 3:351-363.
directed against cytoskeletal proteins (actin, smooth muscle With immunohistochemistry we concluded that en-
actin, desmin and vimentin), monocyte-macrophage spe- zymatically harvested MECs did not contain adipocytes and
cific antigens (MAC 387, anti-protein 150.95) and S-100 pericytes, since immunostaining showed protein S-100 and
protein, which is present in adipocytes, were also used. desmin to be virtually nonexistent. Furthermore, the pres-
Following collagenase digestion approximately 90% ence of pericytes was ruled out by a negative response for
of MECs stained for factor VIII, as well as for CD31, CD34, smooth muscle-specific actin. As demonstrated by
and Ulex. After the Percoll step, cell isolate weakly expressed immunostaining for monocyte-macrophage specific anti-
factor VIII (5%) (Fig. 7.7A). Furthermore, 18% of the iso- gen (150.95 protein and MAC387), the presence of mono-
lated cells stained for CD31 (Fig. 7.7B) and 5% were labeled nuclear blood cells was negligible (1-2%).
with Ulex europaeus. Remarkably these endothelial-specific On the other hand, our results on MECs harvested
antigens were found mostly in cell clumps. Interestingly, from human abdominal subcutaneous fat tissue (which as
CD34 was detected in 90% of the harvested cells compris- previously demonstrated show ultrastructural features typi-
ing the primary isolate irrespective of the separation step cal of endothelium), using common endothelial cell mark-
(Figs. 7.7C and 7.3C). Only 1-2% of cells expressed mono- ers (FVIII RA, Ulex europaeus, CD31) have apparently led
cyte-macrophage specific antigens, 150.95 protein and MAC to some discrepancies that we related to factor VIII release
387. Immunostaining for desmin and S-100 protein was al- and surface antigen rearrangements occurring in vitro dur-
most negative. Smooth muscle actin was expressed in ing procedures of cell isolation, mainly at the Percoll step.
few cells. Interestingly, immunoelectron microscopic demonstration
Morphological Aspects of Microvascular Cell Isolates 109
Fig. 7.7. Immunophototyping of isolated
microvascular endothelial cells. (A), top.
Percoll isolated microvascular endothelial
cells. Factor VIII is expressed only in clus-
tered cells. (APAAP technique; x170).
With permission from: Vici M, Pasquinelli
G, Preda P et al. Electron microscopic and
immunocytochemical profiles of human
subcutaneous fat tissue microvascular
endothelial cells. Ann Vasc Surg 1993;
7:541-548. (B), center. Percoll isolated mi-
crovascular endothelial cells. CD31 anti-
gen is expressed by clustered endothelial
cells and occasional single elements (ar-
rowhead). (APAAP techniques; x170).
With permission from: Vici M, Pasquinelli
G, Preda P et al. Electron microscopic and
immunocytochemical profiles of human
subcutaneous fat tissue microvascular
endothelial cells. Ann Vasc Surg 1993;
7:541-548. (C), bottom. Percoll isolated
microvascular endothelial cells. CD34 an-
tigen is present on approximately 90% of
cells. (Protein a-gold-silver stain; x350).
With permission from: Vici M, Pasquinelli
G, Preda P et al. Electron microscopic and
immunocytochemial profiles of human
subcutaneous fat tissue microvascular
endothelial cells. Ann Vasc Surg 1993;
7:541-548.
110 Tissue Engineering of Prosthetic Vascular Grafts
of factor VIII RA by SEM reveals a moderate to strong posi- to mesodermally derived tissue. The fact that after collage-
tivity at the cell surface (Fig. 7.5B). A possible adverse effect nase isolation the cells expressing vWF and EN4 were fewer
of Percoll on cell function has also been observed by DOT than those observed in intact fat suggests that a portion of
immunoassay. Precisely, DOT immunoassay documented an cells lose vWF and EN4 during the isolation procedure.
intense factor VIII delivery in the washing solutions follow- Stansby et al attempted to characterize microvascular
ing Percoll density gradient, while CD34 was always nega- endothelial cells from human omental tissue using both
tive. Notably, in MECs obtained with the Dynabeads proce- endothelial-specific markers, (namely anti-factor VIII, EN4,
dure, where the Percoll step was omitted, factor VIII RA was PAL-E and QB-End/40) and monoclonals against antigens
expressed by the vast majority of cells (Fig. 7.7D) and DOT specific of cells of connective tissue origin (Thy-1 and
immunoassay of the washing buffers collected after CDw44).34 The authors found that the microvascular cells
Dynabeads application was virtually negative excluding any they harvested from omentum are extremely heterogeneous
loss of factor VIII during the isolation steps. Only CD34 was with respect to their antigenic expression and are probably
constantly expressed by MECs during and after the isola- initially a mixture of several different cell types: only occa-
tion by Percoll density gradient. We concluded that QB- sional cells are positive for factor VIII RA, Thy-1 and CDw44
End/10 monoclonal antibody, which recognizes the highly glycoprotein. No positivity for EN4, PAL-E and QB-End/40
resistant CD34 molecule on the surface of endothelial cells, was reported. After culture it seems likely that one cell type
is particularly well suited for characterizing enzymatically predominates, as the cultures are then homogeneous both
harvested MECs.33 morphologically and immunophenotypically. The majority
Also, Williams et al observed a cellular response to the of cells are then positive for factor VIII RA, Thy-1 and Cdw44
isolation procedure. They studied the cell population of glycoprotein. A small proportion (< 5%) is also positive for
liposuction-derived human fat before and after collagenase the endothelial markers EN4, PAL-E and QB-End/40. The
dispersion with the help of immunocytochemistry and scan- authors suggest that Thy-1 and CDw44 glycoprotein posi-
ning electron microscopy.5 Samples of liposuction-derived tive cells, which were shown to take up acetylated ldl and
human fat have been processed for the presence of FVIII produce prostacyclin, might be pericytes.
RA-von Willebrand factor, #-smooth muscle cell actin, Also, Meerbaum et al performed an immunohis-
cytokeratin (CK18) and EN4. The same study was performed tochemical investigation on the primary isolate enzymati-
on the cells recovered after collagenase dispersion of the cally harvested from subcutaneous fat tissue obtained by
liposuction fat. 86.1% of the cells in intact liposuction-de- liposuction and used for limited clinical trials.35 They found
rived fat expressed vWF, 5.7% were positive for #-smooth 55.8% of cells positive for factor VIII while 42.5% were re-
muscle cell actin, 1% expressed the mesothelial cell-related active to muscle-specific actin. 9.4% of cells reacted with
antigen cytokeratine peptide 18 and 89.6% of the cells in monocyte-macrophage specific markers. They speculated on
intact fat resulted positive for EN4. After collagenase diges- the possibility that contaminating cells might have been
tion of the fat and centrifugal separation of adipocytes from pericytes. They also observed that Percoll separation nega-
vascular and stromal cells, the expression of vWF, #-smooth tively affects cell attachment and growth in culture.
muscle cell actin, and cytokeratin was 77.5%, 5.8%, and 2.1%
respectively. 74.6% of the isolated cells expressed EN4. Discussion
This study demonstrated that the major cell compo- Since our unit was founded in 1962, it has been de-
nent present in liposuction-derived fat, before and after col- voted to the elaboration of a subcellular approach to clini-
lagenase digestion, has been characterized as endothelium, cal manifestation. As soon as the Vascular Surgery unit of
while only a minor fraction is composed of cells common the hospital where we were placed decided to start looking
Fig. 7.7. Immunophototyping of isolated mi-
crovascular endothelial cells. (D). Dynabeads
isolated microvascular endothelial cells. Almost
all the cells are positive for factor VIII. (APAAP
technique; x500).
Morphological Aspects of Microvascular Cell Isolates 111
into the endothelial cell seeding of vascular grafts, it asked One example comes from our own experience. Our
for our participation, finding our experience immediately attempt to characterize microvascular endothelial cells
important to initiate this challenging new task. immunohistochemically yielded some discrepancies. After
Undoubtedly a successful morphological approach Percoll density gradient isolation, we found that typical
correlates well with experience, both for technical skill, which markers (factor VIII, Ulex europaeus, and CD31) were ex-
is always a guarantee for precision and efficiency, and for pressed by only a small percentage of cells. Immuno-
the ability to collect and interpret the results obtained. For histochemically we concluded that microvascular endothe-
example, each lab which focuses on ultrastructure has elabo- lial cells harvested from human subcutaneous fat tissue did
rated its own protocols, and it is always very important to not contain contaminating stromal cells such as adipocytes
learn, when observing samples, how to distinguish artifacts and pericytes, and the presence of mononuclear blood cells
from the “real” structure. was negligible. Moreover, harvested cells showed ultrastruc-
Although the optimization of these aspects could ap- tural features typical of endothelium. Subcellular criteria
pear time consuming, the study of the morphology of single included residual lumina, loosely textured intermediate fila-
cells or of a tissue can give important information about ments, micropinocytotic vesicles, and Weibel-palade bod-
their biology and function. Electron microscopy techniques ies. Interestingly, factor VIII, CD31 and Ulex europaeus al-
represent powerful tools which provide very accurate infor- most exclusively stained cell aggregates. Likewise, TEM dem-
mation about cell ultrastructure and, thanks to combined onstrated Weibel-palade bodies in clustered endothelial cells
immunocytochemistry, about cell biology (related to anti- only, whereas single cells, which also showed the subcellular
gen expression, synthesis of specific molecules, etc.). We features of endothelium, lacked these highly specific endo-
believe that, for all these reasons, morphology can make an thelial organelles.
important contribution to the development of the tissue We hypothesized an adverse effect of Percoll on
engineering of vascular grafts. endothelial cell function. Our hypothesis was supported by
As already reported, submicroscopic studies have been the finding of factor VIII in the washing solutions after
very important in defining the processes involved in the ad- Percoll separation, documented by DOT immunoassay and
hesion of cells to a surface. This information can success- by the detection of factor VIII by immunoSEM on the sur-
fully be applied to the study of vascular cell seeding onto face of some of the cells, which could represent a possible
prosthetic grafts. With the scanning electron microscope we step in the delivery of factor VIII in the washing solutions
can easily follow the different steps of adhesion of the en- following the Percoll treatment. Interestingly, when using
dothelial cells seeded on the vascular substratum that we alternative methods where the Percoll step is omitted the
are testing. First of all, we can perceive a qualitative response: majority of the harvested endothelial cells stain for factor
No interaction with the substratum, as revealed by a steady, VIII.19 Accordingly, the decreased expression of CD31 and
rounded cellular shape, is informative of an unsuitable sur- other membrane glycoproteins, or part of them, could be a
face—cells do not like that substratum. At an intermediate consequence of surface antigen rearrangements also induced
level the endothelial cell has already started to interact with by the Percoll step.
the vascular graft and the cell surface is modifying; endo- Here we have reported on other authors who also per-
thelial cells now appear as skirted cells. If the interaction formed correlated morphological and immunohistochemi-
proceeds, meaning that the vascular substratum is particu- cal studies in order to determine the cellular composition of
larly suitable for the cells, their surface modifies further, be- a microvascular primary isolate; in some cases these studies
coming completely flattened. Depending on the number of have also led to evaluation of the influences of the isolation
cells seeded, they can eventually reach each other and, by procedures chosen over the cell function.
forming intercellular junctions, start to initiate a monolayer. Being correlated to the potential of technological ap-
We can explain all these surface modifications by TEM as paratus, the “limits” of morphological studies are subject to
cytoskeletal rearrangements. constant modification. Computer science, together with in-
TEM has been very helpful for the characterization of novative technology, has permitted the development of very
microvascular primary isolates. We already know that the elaborate microscopes, adding a new perspective to the study
cell types involved in a primary microvascular isolate vary of morphology. Confocal microscopy is an advanced tech-
with the source chosen. Different sources placed in anatomi- nique used to produce multidimensional cellular and sub-
cally different sites, clearly comprise, besides microvessels, cellular structural images. It provides high resolution im-
other cell types. Whether a microvascular isolate contains ages and its optical sectioning ability allows images to be
pure endothelium or not can be of primary importance obtained from different depths within a thick tissue speci-
during clinical trials. TEM with the help of immunocy- men, avoiding processing and sectioning procedures. For this
tochemistry can give a very accurate description of the cell reason, confocal microscopy has made it possible to view
types involved in a microvascular isolate. Ultrastructurally biological tissues under more physiological conditions than
there are morphological markers that can, without doubt, was previously possible. Commonly, its biological applica-
allow different cell types to be recognized. This is supported tion addresses the localization of immunofluorescently la-
by immunocytochemistry which localizes molecules specifi- beled proteins in cell culture or within excised blocks of
cally synthetized by certain cells or groups of cells. More- tissue. Because of its noninvasive optical sectioning capa-
over, together they can also permit the identification of al- bility, it is also ideally suited to the study of living cells in
terations of the biological status. situ and of tissue in intact living animals.36
112 Tissue Engineering of Prosthetic Vascular Grafts
Confocal microscopy immunofluorescence staining ported by very sophisticated machines and, as a consequence,
allows a better resolution than that with traditional immu- their potential is continuously improving. As we have seen,
nofluorescence. It would therefore be possible to perform they have already been successfully employed in the tissue
very detailed in situ or in vivo studies of adhesion molecules engineering of prosthetic vascular grafts, and it is becoming
or other cell products in cultures or, in the near future, in clearer that their future development will be of great value
implanted grafts. The scanning resolution of the confocal in the evolution of this field.
microscope has also remarkably improved on that of the tra-
ditional SEM. More precise evaluations about surface infor- Acknowledgments
mation such as cell lining, for example, can be simultaneously I would like to thank Prof. Massimo Derenzini for
supported, thanks to the optical sectioning ability, by supporting me and giving me the free run of the archives of
“deeper” information regarding thickenings and their bio- the Institute, and Prof. Massimo D’Addato of the Vascular
logical compounds. Understandably, confocal microscopy Surgery Unit, Director of the Center for the Study of Vascu-
is considered to be one of the techniques that in the near lar Prostheses, and his staff for their collaboration. I am also
future will enormously improve the understanding of bio- extremely grateful to Dr. Paola Preda and Dr. Gianandrea
logical events. Pasquinelli for their invaluable assistance and to Mr. Walther
The constant development of technologies for the Mantovani for his indispensable technical skill. Finally,
quantitative analysis of images has made image analysis the thanks to Patrizia and Lorenzo for their patience.
most suitable tool for quantitative studies on cell seeding,
or whenever a precise definition of the number of cells (or References
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CHAPTER 8
Functional Aspects of Microvascular Cell Isolates
M. Fittkau, Teddy Fischlein
B ecause of the limited availability of autologous venous endothelium for transplantation
onto vascular implants, microvascular cells from human subcutaneous fat tissue were
first used for the coating of synthetic vascular prostheses in 1986.1,2 Subcutaneous fat tissue
is easily accessible, usually available in sufficient amounts and very well vascularized.3 In
comparison to the method used for macrovascular endothelial cell harvest, however, the
isolation of microvascular endothelium from adipose tissue represents a fundamentally dif-
ferent approach because the enzymatic detachment of endothelial cells cannot be achieved
by perfusion of the vascular system. It requires a complete dissociation of the tissue, fol-
lowed by gradual separation of capillary segments and endothelial cells from lipids and
cellular and noncellular connective tissue components. In particular, the complete removal
of other, rapidly proliferating cells such as fibroblasts and pericytes represents a core prob-
lem when isolating microvascular endothelial cells.
In cooperation with the Physiological Institute of the University of Munich (LMU)
we developed a method for the isolation of pure microvascular endothelial cells (Fig. 8.1).
Following enzymatic detachment, the endothelial cells are centrifuged over a density gradi-
ent and thus separated from other cell types. Approximately one week after seeding, the
remaining contaminating cells are eliminated using a combination of specific antibodies
and complement.4 In this procedure mixed cell cultures are incubated with diluted human
serum and polyclonal antibodes. The latter are intednded to eliminate alcalic phosphatase
positive contaminating cell types (i.e., pericytes). After 2 h the contaminating cells are rinsed
off the cultures with ECs remaining in the flask. Subsequently, microvascular endothelial
cells can be examined under the phase contrast and fluorescent microscope after incorpora-
tion of 3,3'-dioctodecylindocarbocyamine (DiI) acetyl-LDL (Fig. 8.2). Like macrophages,
ECs internalize Ac-LDL via a specific receptor, in contrast to fibroblasts and smooth muscle
cells.
For the purpose of transplantation onto synthetic vascular prostheses, the question of
to what extent microvascular endothelium differs from its macrovascular counterpart with
regard to its significant metabolic functions remains of fundamental importance. In this
context, priority is placed on the antiaggregatory (release of NO and prostacyclin, loss of
adenine nucleotide), anticoagulative (release of antithrombin III and protein S, activation
of protein C), profribrinolytic (release of plasminogen activator) and antifibrinolytic activ-
ity (plasminogen activator inhibitor) as shown in Figure 8.3.
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
116 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 8.1. Primary mixed mi-
crovascular culture with cen-
tral endothelial clone sur-
rounded by pericytes.
Antiaggregatory Properties of Microvascular fect.10 In addition, NO functions as a strong vasodilator,
Endothelial cells immunomodulator and neurotransmitter. The release of NO
is locally continued and triggered by activated thrombocytes
Prostacyclin (PGI2) and various mediators (thrombin, adenine nucleotides,
PGI2 activates the thrombocytes’ adenylate cyclase by bradykinin, thromboxane, histamine, etc.). In comparison
adhesion to their guanosine-nucleotide receptors. The re- to macrovascular cells, cardiac microvascular endothelial
sulting increase of intracellular cyclic adenosine monophos- cells (rat) showed a low basic release of NO. However, when
phate (cAMP) causes platelet inhibition.5 While this takes stimulated with a combination of inflammation mediators,
place, prostacyclin signals a relaxation of the vascular smooth a significant increase of NO release could be detected within
muscle cells and blocks interaction between the endothelial 24 h through induction of NO synthetase II.12
cells and the monocytes. Among the stimulants of PGI2 syn-
thesis are thrombin, histamine, bradykinin and—possibly Adenine Nucleotide Metabolism
to an even higher degree—shear stress.5 In microvascular An enzyme expressed on the endothelial cell surface
endothelial cells of various tissue types in both humans and dephosphorylates the strongly proaggregatory platelet re-
animals, comparable values of basal PGI2 release were found lease product adenosine diphosphate (ADP) to adenosine
to those in macrovascular endothelial cells.5-9 However, sig- monophosphate (AMP). Subsequently, AMP is further
nificant differences were found in terms of the secretion ca- catabolized to adenosine, an endothelium-independent va-
pacity and responsiveness in relation to various stimulants. sodilator. Intracellularly, yet another degradation takes place,
Histamine and thrombin for instance—when applied to mi- the final product of which is uric acid in the case of mi-
crovascular endothelial cells—resulted in a weaker increase crovascular cells, while macrovascular cells—due to their lack
in PGI2 production6,7 than found in venous endothelial cells, of xanthine dehydrogenases—catabolize no further than to
whereas bradykinin caused a stronger PGI2 stimulation in hypoxanthine.12
microvascular than in macrovascular EC.8 Arachidonic acid
as a nonspecific precursor in the prostaglandin synthesis Anticoagulative Properties
caused a significant increase in the prostacyclin release in all
cases.6-9 After transplantation onto cardiovascular implants Thrombomodulin
such as synthetic ePTE-vascular prostheses7,8 or detoxified Endothelial cells express the membrane protein
biological cardiac valve protheses,9 this centrally important thrombomodulin, which catalyzes the thrombin activation
antiaggregatory function was fully retained. of protein C. The anticoagulative effect of the thus activated
protein C is a result of the neutralization of the activated
Nitric Oxide (NO) factors V and VIII, as well as of the inhibition of plasmino-
NO diffuses into thrombocytes and causes an increase gen-activator inhibitor (PAI). Thrombomodulin is expressed
of cyclic guanosine monophosphate (cGMP). This leads to by endothelial cells throughout the vasculature. However, it
an inhibition of platelet activation, adhesion and aggrega- reaches a particular high density in microvascular cells of
tion. With respect to the inhibition of adherence and aggre- the lung.13 In vitro measurements in omental microvascu-
gation of thrombocytes as well as the dissolution of primary lar endothelial cells showed thrombomodulin activity with
aggregates, NO and PGI2 exert a significant synergetic ef- the transformation of protein C in homogenized cells to be
Functional Aspects of Microvascular Cell Isolates 117
Fig. 8.2. Light-microscopy
photographs of microvascular
endothelial cell isolates from
human fat tissue: (A, top)
Phase contrast print, (B, bot-
tom). After incorporation of
Dil-marked Ac-LDL. (Prof. S.
Nees, Phys. Inst. Univ. of
Munich.)
approximately 6-fold higher than in confluent cell cultures.14 for passaging of cultured cells, causes the destruction of those
In human macrovascular endothelial cells (saphenous vein), proteoglycans.
thrombomodulin activation was seen even after transplan-
tation onto ePTFE vascular prostheses.15 Profibrinolytic and Antifibrinolytic Properties
Heaprin Sulfate Plasminogen Activators (PA, Tissue Type PA
Heaprin sulfate, which is also expressed on the cell’s and Urokinase Type PA) and Plasminogen Activator
surface, inactivates thrombin as a cofactor of anti- Inhibitor (PAI)
thrombin III as well as the activated forms of the coagula- The endothelium represents the most important site
tion factors IX, X and XII. By quantifying the acceleration of production for tissue-type plasminogen activator (tPA).
of the thrombin-antithrombin complex formation, a signifi- In combination with plasminogen activator inhibitor (PAI
cant production of heaprin sulfate by microvascular endot- type I), which is also synthetisized by endothelial cells, the
helial cells from fat tissue (epididymal fat pads, mouse) was endothelium significantly contributes to the intravasal
shown.16 Enzymatic treatment, for instance trypsinization fibrinolytic activity, which requires, however, a finely tuned
regulating control mechanism.
118 Tissue Engineering of Prosthetic Vascular Grafts
Plasmin
Fig. 8.3. Antithrombotic properties of the endothelium.
tPA showed inconsistent results. When the tPA production of
Thrombin and shear stress are the most important omental endothelial cells was compared with that of human
physiological stimuli for synthesis and release of tPA. In vitro, umbilical vein endothelial cells, it was three times higher in
endothelial cells from different blood vessels and flow areas one study17 but 100 times higher in another.21
show significant differences with regard to their fibrinolytic
potential. The largest production of tPA was measured in uPA
endothelial cells from the vena cava. When compared to In primary cultures of human endothelial cells, the
microvascular (foreskin) as well as macrovascular aortic and presence of urokinase type plasminogen activator (uPA)
umbilical endothelial cells, the tPA production in vena cava could not be proven. uPA activity could, however, be proven
endothelial cells was 4- and 20-fold higher, respectively.17-21 in subcultures of human endothelial cells of various
In contrast to microvascular endothelial cells from macrovascular and microvascular sites.17
foreskin, endothelial cells from the omentum (human)
Functional Aspects of Microvascular Cell Isolates 119
Plasminogen Activator Inhibitor (PAI) 12. Des Rosiers C, Nees S, Gerlach E. Purine metabolism in
PAI is the physiological inhibitor of the plasminogen cultured aortic and coronary endothelial cells. Biochem
activator. Among the stimuli for its release are inflamma- Cell Biol 1989; 67:8-15.
tion, hormones and cytokines. Its molar concentration in 13. Carley WW, Niedbala MJ, Gerritsen ME. Isolation, culti-
vation and partial charaterization of microvascular endot-
plasma is higher than that of tPA. As a potent regulator of
helium derived from human lung. Am J Respir Cell Mol
the fibrinolytic endothelial function, PAI is produced by both Biol 1992; 7:620-630.
macrovascular and microvascular endothelial cells.21-30 14. Anders E, Alles J, Delvos U, Pötzsch B et al. Microvascu-
lar endothelial cells from human omentum tissue. Modi-
Conclusion fied method for long-term cultivation and new aspects of
In summary, the metabolic functional performance characterization. Microvasc Res 1987; 34:239-249.
of microvascular endothelial cells corresponds to that of their 15. Gillis-Haegerstrand C, Frebelius S, Haegerstrand A,
macrovascular counterparts. Differences are mainly found Swedenborg J. Cultured human endothelial cells seeded
in the quantity of the synthetic activities. However, it ap- on expanded polytetrafluoroethylene support thrombin-
pears that following transplantation of microvascular en- mediated activation of protein C. J Vasc Surg 1996;
dothelial cells, a certain adaptation to the new environment 24(2):226-234.
16. Marcum JA, Rosenberg RD. Heparinlike molecules with
will be necessary. Even when mixed cultures were seeded in
anticoagulant activity are synthesized by cultured endot-
the course of animal experiments (dog), a regulation and helial cells. Biochemical and Biophysical Research Com-
adaptation to the physiological requirements could be ob- munications 1985; 126 No 1:365-372.
served.31 Whether the same observations will apply to hu- 17. Van Hinsbergh VWM. Regulation of the synthesis and
mans is questionable. Due to the lack of systematic exami- secretion of plasminogen activators by endothelial cells.
nation following clinical studies, relevant results cannot as Haemostasis 1988; 18:307-327.
yet be supplied. 18. Rijken DC, Van Hinsbergh VWM, Sens EHC.
Quantitation of tissue-type plasminogen activator in hu-
References man endothelial cell cultures by use of an enzyme immu-
1. Jarell BE, Williams SK, Stokes G et al. Use of freshly iso- noassay. Thromb Res 1984; 33:145-153.
lated capillary endothelial cells for the immediate estab- 19. Van Hinsbergh VWM, Binnema D, Scheffer MA,
lishment of a monolayer on a vascular graft at surgery. Sprengers ED et al. Production of plasminogen activators
Surgery 1986; 100:392-399. and inhibitor by serially propagated endothelial cells from
2. Radomski JS, Jarrel BE, Williams SK et al. Initial adher- adult human blood vessels. Arteriosclerosis 1987;
ence of human capillary endothelial cells to Dacron. J Surg 7:389-400.
Res 1987; 42:133-140. 20. Van Hinsbergh VWM, Sprengers ED, Kooistra T. Effect
3. Weiss. Cell tissue biology New York: Urban & of thrombin on the production of plasminogen activators
Schwarzenberg. 1988:191-205. and PA inhibitor-1 by human foreskin microvascular en-
4. Fittkau M, Fischlein T, Reichart B, Juchem G, Nees S. dothelial cells. Thromb. Haemostasis 1987; 57:148-153.
Autologe Endothelialisierung menschlicher Gefä !% 21. Speiser W, Anders E, Preissner KT, Wagner O et al. Dif-
prothesen. Z Kard 1994; 83, Supp:1:38. ferences in coagulant and fibrinolytic activities of cultured
5. Wu KK, Thiagarajan P. Role of endothelium in throm- human endothelial cells derived from omental tissue
bosis and hemostasis. Annu Rev Med 1996; 47:315-331. microvessels and umbilical veins. Blood 1987; 69:964-967.
6. Stansby G, Shukla N, Hamilton G, Jeremy J. Comparison 22. Booyse FM, Osikowicz G, Feder S, Scheinbuks J. Isola-
of prostanoid synthesis in cultured human vascular en- tion and characterization of a urokinase-type plasmino-
dothelial cells derived from omentum and umbilical vein. gen activator (Mr = 54,000) from cultured human endot-
Eur J Vasc Surg 1991; 5:501-506. helial cells indistinguishable from urokinase. J Biol Chem
7. Budd JS, Allen K, Hartley J, Walsh A et al. Prostacyclin 1984; 259:7198-7205.
production from seeded prosthetic vascular grafts. Br J 23. Booyse FM, Scheinbuks J, Radek J, Osikowicz G et al.
Surg 1992; 79:1151-1153. Immunological identification and comparison of plasmi-
8. Sterpetti AV, Schultz RD, Hunter WJ et al. Comparison nogen activator forms in cultured normal human endot-
of two techniques to isolate microvascular endothelial cells helial cells and smooth muscle cells. Thromb Res 1981;
from the omentum. J Surg Res 1990; 48:101-106. 24:495-504.
9. Fischlein T, Lehner G, Lante W, Fittkau M et al. 24. Loskutoff DJ, Van Mourik JA, Erickson LA, Lawrence D.
Endothelialization of cardiac valve bioprotheses. Artif Detection of an unusually stable fibrinolytic inhibitor pro-
Organs 1994; 17 No 6:345-352. duced by bovine endothelial cells. Proc Natn Acad Sci
10. Radomski MW, Palmer PMJ, Moncada S. The anti-aggre- USA 1983; 80:2956-2960.
gating properties of vascular endothelium interactions 25. Emeis JJ, Van Hinsbergh VWM, Verheijen JH, Wijngaards
between prostacyclin and nitric oxide. Br J Pharmacol G. Inhibition of tissue-type plasminogen activator by con-
1987; 92:639-642. ditioned medium from cultured human and porcine vas-
11. Ungureanu-Longrois D, Balligand JL, Okada I et al. Con- cular endothelial cells. Biochem Biophys Res Commun
tractile responsiveness of ventricular myocytes to isopro- 1983; 110:392-398.
terenol is regulated by induction of nitric oxide synthase 26. Philips M, Juul A, Thorsen S. Human endothelial cells
activity in cardiac microvascular endothelial cells in het- produce a plasminogen activator inhibitor and a tissue-
erotypic primary culture. Circ Res 1995; 77 No 3:486-493. type plasminogen activator-inhibitor complex. Biochem
Biophys Acta 1984; 802:99-110.
120 Tissue Engineering of Prosthetic Vascular Grafts
27. Van Hinsbergh VWM, Bertina RM, Van Wijngaarden A, 30. Sprengers ED, Verheijen JH, Van Hinsbergh VWM, Emeis
Van Tilburg NH et al. Activated protein C decreases JJ. Evidence for the presence of two different fibrinolytic
plasminogen activator-inhibitor activity in endothelial cell- inhibitors in human endothelial cells conditioned me-
conditioned medium. Blood 1985; 65:444-451. dium. Biochem Biophys Acta 1984; 801:163-170.
28. Van Mourik JA, Lawrence DA, Loskutoff DJ. Purification 31. Baitella-Eberle G, Groscurth P, Zilla P, Lachat M et al.
of an inhibitor of plasminogen activator (antiactivator) Long-term results of tissue development and cell differ-
synthesized by endothelial cells. J Biol Chem 1984; entiation on Dacron prostheses seeded with microvascu-
259:14914-14921. lar cells in dogs. J Vasc Surg 1993; 18 (6):1019-1028.
29. Dosne AM, Dupuy E, Bodevin E. Production of a fibrin-
olytic inhibitor by cultured endothelial cells derived from
human umbilical vein. Thromb Res 1978; 12:377-387.
CHAPTER 9
Automated Seeding Devices
Dominic Dodd, J. Vincent Smyth, Michael G. Walker
Introduction
A lmost as soon as the application of endothelial seeding to enhance graft patency was
recognized, groups started to develop techniques and equipment to facilitate the process.
The requirements of an ideal seeding system for clinical work include simplicity, efficiency
and speed. These considerations must be applied to the delivery of a high yield of viable
endothelial cells using a readily available tissue source. The maintenance of sterility to avoid
introduction of infective agents; effective seeding onto the graft while retaining the func-
tional integrity of seeded cells; and the rapid development of monolayer within a time-
frame suitable for operating room use are also fundamental. The search for a reproducible,
inexpensive, low-tech, high efficacy technique has been a tortuous and convoluted one in
which the solution to each problem has led to further questions.
In the design of a fully automated seeding device, several key steps must be consid-
ered. The automated seeding process commences with tissue reception. A closed system in
which sterility is maintained is essential. The tissue receiver may be combined with a me-
chanical homogenization process to speed subsequent cell separation by enzymatic diges-
tion. The aim of this stage is to acquire large numbers of endothelial cells (ECs) with rela-
tively few contaminating cell types. Subcutaneous adipose tissue is a ready source of mi-
crovascular endothelial cells and is easily harvested in sufficient quantities from all but the
thinnest of patients. Once ECs have been isolated, maximal graft-cell attachment is needed.
The constraints of the operating room mean that this must be achieved in a relatively short
time. Graft surface coatings clearly have a major role in determining the efficiency of this
stage. Finally, the seeded graft must be presented to the operating team in a package that will
maintain its sterility and preserve the viability of the seeded EC lining. Ideally, a fully auto-
mated system should optimize cell harvest and seeding efficiency; however, a compromise
between these aims and the duration of the procedure must be met. Completion of the
seeding process in approximately one hour is a realistic time scale in a busy operating room.
Much of this time will be taken by cell digestion and separation, leaving little time for cell
attachment or final rinsing.
If vascular graft seeding is to be widely adopted in clinical practice, a reliable, easily
used system is required that can be accommodated in a conventional operating room. It
should be recognized that surgeons’ enthusiasm for the introduction of a complex and time
consuming process into the operating room is limited. Unless the process is automated,
vascular graft seeding will remain a “laboratory” technique, confined to very few surgical
centers. Therefore, in addition to the development of graft materials and coatings for opti-
mal seeding performance, automation of the seeding process must be achieved. This aspect
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
122 Tissue Engineering of Prosthetic Vascular Grafts
of seeding technology is both a biological and engineering onto a fibronectin precoated graft and incubated for at least
challenge. Perhaps the difficulties in successfully achieving 30 minutes.7 To produce a complete monolayer on a graft
this goal are reflected in the relatively small number of pub- would thus require an initial harvest of 1.5 x
lications of techniques and designs investigated. 105−.−d−l−(% Seeding Efficiency) cells, where d=inside diam-
eter and l = length, of the graft. Our own work with imme-
Early Seeding Techniques diately harvested saphenous vein endothelial cells has dem-
At the beginning of the 1980s, seeding methodology onstrated that a seeding efficiency of 30% can be reliably
relied on mechanical harvesting of the endothelial cells by achieved,8 and thus for a standard 30 cm, 6 mm internal
metal pledget from vein segments.1 As well as being labori- diameter graft suitable for a femoropopliteal graft, an initial
ous, the yield of viable cells was relatively low due to harvest harvest of 28 x 106 cells is required. A 60 cm long, 4 mm
trauma. In addition, the low specificity of mechanical har- internal diameter graft such as might be used for femorotibial
vesting resulted in frequent contamination of the endothe- bypass would require 38 x 106 cells. This quantity of endot-
lial culture with fibroblasts and smooth muscle cells. This helial cells can not be derived from autologous saphenous
technique sufficed for laboratory work and was successful vein, since such cell harvest would require a vein segment of
using animal cell lines, but a major step forward was made equivalent size, which would normally be used as the pre-
with the adoption of enzymatic harvesting, leading to the ferred conduit. Microvascular endothelial cells have been
now routine collagenase incubation-based method.2 This proposed as an alternative cell type,9,10 with harvests from
rapidly superseded mechanical harvesting as a straightfor- both omentum and subcutaneous adipose tissue being used
ward method which allowed rapid procurement of pure for seeding experimentally. However, considerably more
endothelial cells, and the promise of seeding prosthetic grafts experimental data have been gained with macrovascular
in terms of patency, resistance to infection and reduced endothelial cells to suggest inhibition of smooth muscle cell
neointimal hyperplasia began to be realized in a range of proliferation in vivo, and acceptable results of retention
experimental laboratory and in vivo models. However, only under arterial levels of shear stress.
animal cell lines had as yet been used extensively and the
next step was clearly to extend this work to clinical practice. Graft Material Considerations
Up to the early 1980s, work with human cell lines had The large majority of EC seeding studies have been
been restricted to umbilical vein endothelium, HUVECs, carried out using commercially available graft materials that
because of difficulties with maintaining adult endothelial were not specifically designed for cell attachment. Indeed,
cell lines, HAECs, in culture. The next breakthrough in en- considerable materials technology has been applied to make
dothelial seeding was the successful culture of HAECs by the luminal surface of these graft materials hydrophobic by
Jarrell, with the recognition of the need for EDGF and hep- negatively-charged fluoropolymer lining and consequently
arin in the culture medium.3 Despite significant problems unsuitable for direct seeding.
with the more fragile human cell cultures and the disap- There has been continued interest over the last de-
pointing results of cell attachment studies detailed in an ear- cade on choice of graft coating materials for optimal cell
lier chapter, initial protocols using harvest, culture and seed- attachment. It is now recognized that the extracellular ma-
ing stages demonstrated that at least some benefit might be trix plays an integral role in cellular function and the choice
realized. In clinical practice, however, the time constraints of graft coating material will influence not only cell attach-
of the operating room meant that cell harvesting would need ment but also cellular activity. Soluble components of the
to be carried out as a preliminary step, or a more rapid tech- extracellular matrix such as fibronectin, collagen and laminin
nique of producing an endothelial monolayer was needed, have all been used to achieve comparable cell attachment
or clinical seeding would be carried out at subconfluent den- rates.11,12 Recently, synthetic EC binding site peptides con-
sities. Subconfluent seeding, whilst effective in the labora- taining arginine-glycine-aspartic acid (RGD) have been used
tory, has been shown to be less able to resist the shear stress to precoat ePTFE, with a significant increase in EC attach-
of flow than an intact monolayer,4 and has been largely aban- ment and retention compared with fibronectin.13 The use
doned. In those clinical studies that have been carried out, of RGD peptides is attractive commercially, as they are simple
the seeding densities achieved were probably subconfluent, to produce and cheaper to use than more complex protein
and did not result in measurable reduction in coatings. EC attachment through focal adhesions is RGD
thrombogenicity measured by labeled platelet scintigraphy,5 dependent; if no RGD sequences are detected in extracellu-
though there was some indirect evidence from serum mark- lar matrix, apoptosis occurs.
ers of a reduction in platelet adhesion and activation.6 In contrast, Sipehia reported the use of gaseous
“plasma” surface modification to enhance cellular attach-
Optimal Conditions for Seeding ment and replication.14 He reported the addition of amino
groups to the surface of a PTFE membrane by anhydrous
Cellular Considerations ammonia in a gaseous plasma increased bovine arterial EC
In order to achieve the maximum coverage from a lim- attachment from 36 to 92% after 96 h in culture.
ited source of cells for seeding, optimal conditions for seed- This suggests that appropriate surface engineering of
ing were extensively investigated. Work with both expanded the prosthetic material to be seeded may have as large an
polytetrafluoroethylene (ePTFE) and polyester graft mate- influence on the rapid development of a healthy neointima
rials suggests the use of at least 150,000 cells/cm2, seeded as cell delivery and modification itself.
Automated Seeding Devices 123
Cell Delivery Considerations Although this approach allows the use of
In addition to selection of optimal seeding cell iso- macrovascular endothelial cells with only modest require-
lates and graft surface characteristics, the delivery of the seed- ments for initial cell number, the patient is required to un-
ing population to the graft has been investigated. Several dergo two surgical procedures, which means it is not com-
approaches to achieve rapid, even coating of the graft lu- patible with revascularization in cases of critical ischemia,
men have been reported. These include graft rotation,15 arguably those most in need of endothelialized grafts. Fur-
vacuum16 or pressure seeding.17 These latter two are similar thermore, the use of cell culture may increase the risk of
in concept and rely on the use of a porous graft material to pathogen transmission, both to and from the patient, un-
facilitate pressure gradient filtering of cells, either singly or less strict safety controls are applied.
in clumps. This latter technique is described as graft
sodding.18 Microvascular Cells and Graft Sodding
Rotational systems represent basic automated seed- Adipose tissue contains large numbers of microvas-
ing devices. In those designs previously described the graft cular endothelial cells lining its capillaries. Jarrell et al first
is held horizontally in a scaffold and filled with EC suspended proposed its use a an alternative EC source in 1986.23 In
in culture medium or heparinized blood.19 The graft is particular the greater omentum offers a readily available
clamped at its ends and rotated in its longitudinal axis so source of adipose tissue. The use of omentum as a cell source
that gravity assists even seeding. led to some debate as to the true nature of the cells har-
The duration allowed for cell attachment has also been vested, some workers demonstrating the presence of me-
studied. Increased resistance to shear stress has been dem- sothelial cells24 and others microvascular EC25,26 as the pri-
onstrated by Budd et al.20 However, we found that initial mary cell isolate. In reality the exact harvest conditions are
cell attachment did not increase significantly on coated grafts likely to influence predominant cell population. In any case,
after 30 minutes8 and, given the time constraints in clinical peritoneal mesothelial cells (PMC) have been shown to form
practice, do not feel prolonged seeding time is justified. confluent monolayers on vascular graft material in culture27
and have demonstrated anti-thrombotic activity, produc-
Choice of Seeding Technique ing prostacyclin,28 and as such may be considered as an al-
ternative to EC for seeding.
Single Stage Seeding
This approach is the most attractive form of auto- Liposuction
mated graft seeding. In a single operative session the patient’s Williams and Jarrell, having championed the use of
endothelial cells are retrieved and seeded onto the graft, adipose microvascular cells, most recently advocated the use
which is then implanted. This avoids the need for cell cul- of liposuction to harvest subcutaneous tissue.29 They have
ture, and reduces operative time overall. It is, however, prob- shown that before cell isolation 77% of cells harvested were
ably the most technically demanding. To adopt this approach, of microvascular origin.
adequate cell numbers must be harvested to cover the graft
surface. The most simple devices include the development Vein Fragment Sodding
of harvesting kits to provide endothelial cell suspensions for Noishiki et al first reported the use of diced fragments
seeding commercially available grafts, vein holding cham- of canine venous tissue seeded into a porous Dacron graft.30
bers to facilitate enzymatic harvest and graft chambers for They used a sodding technique to force cell clumps under
culture in which the seeded graft can be maintained in a pressure into the graft interstices. By this technique they
culture cabinet and rotated mechanically to allow even coat- sealed the graft with a heterogeneous cell population, but
ing of the graft surface with endothelial cells. demonstrated in culture the development of an endothelial
intimal lining.
Two Stage Seeding
Cultured two stage seeding is an alternative approach Autoseeding
that has been successfully applied in clinical trials by Leseche In our own laboratory, we use a single stage, highly
et al21 and Zilla et al.22 This approach follows more closely automated seeding machine similar in concept to the tech-
the standard laboratory practice of harvesting ECs from nique devised by Jarrell and Williams, and to the heteroge-
autologous vein, which are then grown to confluence and neous sodding technique of Noishiki. This machine is a de-
passaged in standard tissue culture flasks until adequate cell velopment prototype similar to that proposed by Pasic et
numbers are available for seeding. Cultured ECs are then al31,32 and built by Sulzer AG (Winterthur, Switzerland)
seeded onto the graft, which is maintained in a specifically (Fig. 9.1). By using this method, the initial porosity of our
constructed culture flask in which culture medium can be graft is reduced to such an extent that there is no need for
circulated through the lumen of the graft. The graft is incu- preclotting prior to implantation, and we use surgically ex-
bated until confluence is deemed to have been reached on cised wound margin adipose tissue as a source of tissue. In
the luminal surface, at which time the graft is then implanted. the technique originally described by Noishiki,17 the tissue
This period of incubation allows basement membrane to be was finely diced prior to sodding, but we have incorporated
laid down, which significantly enhances shear stress resis- an enzymatic digestion phase into our process to try to op-
tance, but prolonged culture and repeated passage may pro- timize viable cell numbers by reducing mechanical cell
duce an adverse effect on phenotype. damage.
124 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 9.1. Schematic diagram of automated
seeding device incorporating a cell
separation phase.
To seed a typical peripheral vascular graft using our cell monolayer of typical endothelial appearance
device, the operator introduces 100-300 g adipose tissue into (Figs. 9.2A,B). Full characterization of the monolayer has
a tissue receiver and starts the seeding program, which is been complicated by the apparent loss of many cell surface
controlled by the machine’s central processing unit. The adi- immunomarkers with the digestion process. Indeed, we have
pose tissue is minced and forced through a metallic mesh by been unable to demonstrate the presence of tPA, vWF, CD34,
an air-driven piston into a tissue digestion flask containing CD31, eNOS or bFGF in our specimens.
300 ml of buffered saline at 37°C. Into this is added 1,000 IU We have used the seeding system for in vivo experi-
collagenase II per gram of adipose tissue and digestion con- mentation using a porcine model. Subcutaneous adipose
tinues with constant agitation for 40 minutes. After this time tissue was harvested from immature Yucatan miniswine and
the digested fat forms a slurry which is coarsely filtered and a segment of 6 mm internal diameter graft implanted im-
seeded under pressure into the interstices of a specially pre- mediately after processing into the infrarenal aorta. After
pared woven polyester graft. The graft has been precoated four weeks, the graft was explanted and compared with
with bovine gelatin and retains a porosity of approximately nonseeded control grafts, also implanted for four weeks. In
1500 ml/cm2/min. After seeding, this porosity is reduced to our study there was no detectable loss in early patency fol-
almost zero at arterial pressure levels. The process takes 50 lowing seeding with a mixed cell population. The presence
to 55 minutes to complete and the graft can be removed of potentially thrombogenic tissue debris in the graft inter-
from the machine and handed to the operating team in a stices did not have a detrimental effect in the immediate
sterile blister pack. This device allows for a fully automated postimplantation phase. However, at four weeks the seeded
seeding process to be undertaken in the operating room samples, whilst remaining patent, demonstrated neointimal
without excessively prolonging the surgical procedure, or hyperplasia, with marked SMC proliferation. We surmise
requiring on-site laboratory facilities or highly skilled cell that, despite endothelial cover, neointimal hyperplasia oc-
culture technicians. Its secondary advantages are the reduc- curs and that this may be due to seeding of SMC which are
tion in risk of pathogen transfer and the avoidance of pre- stimulated in the process. To overcome this problem we pro-
liminary cell harvesting surgery. The main disadvantage with posed the introduction of a cell separation process to mini-
our particular machine is that a heterogeneous cell popula- mize the seeding of nonendothelial cells. The most promis-
tion is initially seeded onto the graft surface. This popula- ing way to include EC purification in an automated seeding
tion is difficult to fully characterize, but is anticipated to in- device would be to use immunomagnetic bead separation.33
clude vascular smooth muscle cells, fibroblasts, adipocytes, Beads coated with antibodies to the EC surface antigen CD34
and pericytes. If omentum is used, then PMC may also be are already commercially available and are approved for clini-
present, although the digestion conditions in the machine cal use in hemopoietic stem cell separation (Baxter
are considerably more rigorous than those normally used Healthcare Ltd, Thetford, UK). However, there is a time pen-
for PMC harvest from omentum and it is likely that most alty incurred with this approach. To offset this, work is in
PMC would be digested. progress to determine the feasibility of replacing enzymatic
In vitro experiments using human subcutaneous adi- tissue digestion with a mechanical homogenization process
pose tissue and omentum and subsequent graft culture in to reduce the overall procedure time. We estimate that a
our laboratory have confirmed the rapid development of a highly selected EC suspension can be seeded onto a pros-
Automated Seeding Devices 125
Fig. 9.2. (A, top) Environmental scanning
electron micrograph of a porous polyester
graft, immediately after human omental tis-
sue seeding. Original magnification x340.
(B, bottom) A similar graft after 24 h of in
vitro culture. Original magnification x390.
thesis in under two hours. While this time scale may be un- References
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of patients for whom this technology is required are worth nique for seeding vascular grafts with autogenous endot-
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2. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture
Summary of human endothelial cells derived from umbilical veins.
J Clin Invest 1973; 52:2745-56.
Although our understanding of vascular biology, and
3. Jarrell BE, Levine E, Shapiro S, Williams SK, Carabasi RA,
in particular the elucidation of many cell-matrix signaling Mueller S, Thornton S. Human adult endothelial cell
interactions, have led to dramatic breakthroughs in the labo- growth in culture. J Vasc Surg 1984; 1:757-64.
ratory, endothelial cell seeding of vascular prosthetic grafts 4. Miyata T, Conte MS, Trudell LA, Mason D, Whittemore
is not widely regarded as clinically useful. Until an automated AD, Birinyi LK. Delayed exposure to pulsatile shearstress
harvesting and delivery system is fully developed, it remains improves retention of human saphenous vein endothelial
unlikely that this will change. cells on seeded ePTFE grafts. J Surg Res 1991; 50:485-93.
It is hoped that the development of new surface treat- 5. Jensen N, Lindblad B, Bergqvist D. Endothelial cell seeded
ments will lead to greatly increased rates of seeded cell ad- dacron aortobifurcated grafts: Platelet deposition and
herence, retention and replication. In addition the role of long-term follow-up. J Cardiovasc Surg 1994; 35:425-9.
6. Ortenwall P, Wadenvik H, Kutti J, Risberg B. Endothelial
genetically modified EC with enhanced antithrombotic ac-
cell seeding reduces thrombogenicity of Dacron grafts in
tivity is just now being recognized. It is possible that humans. J Vasc Surg 1990; 11:403-410.
immunomodulated, cryopreserved bioprosthetic grafts will 7. Vohra R, Thomson GJL, Carr HMH, Sharma, Walker MG.
represent the next generation of small diameter arterial by- Comparison of different vascular prostheses and matrices
pass prostheses. in realtion to endothelial seeding. Br J Surg 1991;
78:417-20.
126 Tissue Engineering of Prosthetic Vascular Grafts
8. Carr HMH, Vohra R, Sharma H, Smyth JV, Rooney OB, ing using endothelial cell seeded PTFE grafts: Five-year
Dodd PD, Walker MG. Endothelial cell seeding kinetics clinical experience. Ann Vasc Surg 1995; 9:S15-23.
under chronic flow in prosthetic grafts. Ann Vasc Surg 22. Zilla P, Deutsch M, Meinhart J, Puschmann R, Eberl T,
1996; 10:469-75. Minar E, Dudczak R, Lugmaier H, Schmidt P, Noszian I,
9. Stansby G, Shukla N, Fuller B, Hamilton G. Seeding of Fischlein T. Clinical in vitro endothelialization of
human microvascular endothelial cells onto femoropopliteal bypass grafts: An actuarial follow-up over
polytetrafluoroethylene graft material. Br J Surg 1991; three years. J Vasc Surg 1994; 19:540-8.
78:1189-92. 23. Jarrell BE, Williams SK, Stokes G, Hubbard FA, Carabasi
10. Schmidt SP,Monajjem N, Evancho MM, Pippert TR, RA, Koolpe E, Greene D, Pratt K, Moritz MJ, Radomski
Sharp WV. Microvascular endothelial cell seeding of J, Speicher L. Use of freshly isolated capillary endothelial
small-diameter Dacron vascular grafts. J Invest Surg 1988; cells for the immediate establishment of a monolayer on
1:35-44. a vascular graft at surgery. Surgery 1986; 100:392-9.
11. Thomson GJ, Vohra RK, Carr HMH, Walker MG. Adult 24. Visser MJ, van Bockel JH, van Muijen GN, van Hinsbergh
human endothelial cell seeding using expanded VW. Cells derived from omental fat tissue are not endot-
poltytetrafluoroethylene vascular grafts: A comparison of helial in origin. A study on the origin of epitheloid cells
four substrates. Surgery 1991; 109:20-7. deirved from omentum. J Vasc Surg 1991; 13:373-81.
12. Koveker GB, Graham LM, Burkel WE, Sell R, Wakefield 25. Williams SK, Kleinert LB, Rose D, McKenney S. Origin
TW, Dietrich K, Stanley JC. Extracellular matrix prepara- of endothelial cells that line expanded
tion of expanded polytetrafluoroethylene grafts seeded polytetrafluoroethylene vascular grafts sodded with cells
with endothelial cells: Influence on early platelet deposi- from microvascularized fat. J Vasc Surg 1994; 19:594-604.
tion, cellular growth and luminal prostacyclin release. 26. Vici M, Pasquinelli G, Preda P, Martinelli GN, Gibellini
Surgery 1991; 109:313-9. D, Freyrie A, Curti T, D’Addato M. Electron microscopic
13. Walluscheck KP, Steinhoff G, Kelm S, Haverich A. Im- and immunocytochemical profiles of human subcutane-
proved endothelial cell attachment on ePTFE vascular ous fat tissue microvascular endothelial cells. Ann Vasc
grafts pretreated with synthetic RGD-containing peptides. Surg 1993; 7:541-8.
Eur J Vasc Endovasc Surg 1996; 12:321-30. 27. Pronk A, Hoynck van Papendrecht AAGM, Leguit P,
14. Sipehia R. The enhanced attachment and growth of Verbrugh HA, Verkooyen RPAJ, van Vroonhoven TJMV.
endothelial cells on anhydrous ammonia gaseous plasma Mesothelial cell adherence to vascular prostheses and their
modified surfaces of polystyrene and subsequent growth in vitro. Cell Transplantation 1994;
poly(tetrafluoroethylene). Biomater Artif Cells Artif Or- 3:41-8.
gans 1990; 18:437-46. 28. Nicholson LJ, Clarke JMF, Pittilo RM, Machin SJ, Woolf
15. Gerlach J, Kreusel KM, Schauwecker HH, Bucherl ES. En- N. The mesothelial cell as a non-thrombogenic surface.
dothelial cell seeding on PTFE vascular prostheses using Thromb Haemostas 1984; 52:102-4.
a standardized seeding technique. Artif Organs 1989; 29. Williams SK, Wang TF, Castrillo R, Jarrell BE.
12:270-5. Liposuction-derived human fat used for vascular graft
16. van Wachem PB, Stronck JW, Koers-Zuideveld R, Dijk F, sodding contains endothelial and not mesothelial cells as
Wildevuur CR. Vacuum cell seeding: A new method for the main cell type. J Vasc Surg 1994; 19:916-23.
the fast application of an evenly distributed cell layer on 30. Noishiki Y, Yamane Y, Tomizawa Y, Okoshi T, Satoh S,
porous vascular grafts. Biomaterials 1990; 11:602-6. Wildevuur CR. Endothelialization of vascular prostheses
17. Nosihiki Y, Yamane Y, Tomizawa Y. Sealing of highly by transplantation of venous tissue fragments. ASAIO
porous fabric vascular prostheses by adipose connective Trans 1990; 36:346-8.
tissue fragments instead of preclotting with fresh blood. J 31. Pasic M, Muller-Glauser W, von Segesser LK, Lachat M,
Invest Surg 1993; 6:231-40. Mihaljevic T, Turina M. Superior late patency of small-
18. Williams SK, Jarrell BE, Rose DG, Pontell J, Kapelan BA, diameter Dacron grafts seeded with omental microvascu-
Park PK, Carter TL. Human microvessel endothelial cell lar cells: An experimental study. Ann Thorac Surg 1994;
isolation and vascular graft sodding in the operating room. 58:677-83.
Ann Vasc Surg 1989; 3:146-52. 32. Pasic M, Muller-Glauser W, Odermatt B, Lachat M, Siefert
19. Mazzutocelli JP, Roudiere JL, Bernex F, Bertrand P, B, Turina M. Seeding with omental cells prevents late
Leandri J, Loisance D. A new device for endothelial cell neointimal hyperplasia in small-diameter Dacron grafts.
seeding of a small-caliber vascular prosthesis. Artif Or- Circulation 1995; 92:2605-16.
gans 1993; 17:787-90. 33. Jackson CJ, Garbett PK, Nissen B, Schrieber L. Binding
20. Budd JS, Allen KE, Bell PRF. Effects of two methods of of human endothelium to Ulex europaeus I-coated
endothelial cell seeding on cell retention during blood Dynabeads: Application to the isolation of microvascular
flow. Br J Surg 1991; 78:878-82. endothelium. J Cell Sci 1990; 96:257-62.
21. Leseche G, Ohan J, Bouttier S, Palombi T, Bertrand P,
Andreassian B. Above-knee femoropopliteal bypass graft-
CHAPTER 10
Healing Patterns Following Microvascular Seeding—
A Clinical Evaluation of Microvascular-Seeded
A-V Access Grafts
Steven P. Schmidt, Sharon O. Meerbaum, Duane L. Donovan
Introduction
T he need for a more successful artificial bypass graft to replace small and medium-diam
eter blood vessels remains an important issue in vascular surgery. Approximately 600,000
patients require arterial reconstructive surgeries in the United States annually, including
20,000 femoropopliteal bypasses. In addition, each year approximately 90,000 new patients
with end stage renal disease (ESRD) present to vascular surgeons for access-related surgeries
for chronic hemodialysis. Surgeons prefer to use autologous native vessels as bypass con-
duits. For peripheral revascularizations, the collective experiences of vascular surgeons have
been that long term performances of bypass grafts constructed from native veins are supe-
rior to those using other biologic or synthetic alternatives. Likewise, autogenous arterio-
venous (AV) fistulas of the Brescia-Ciminio type are the hemodialysis access procedures
that theoretically provide the longest term trouble-free patency compared to synthetic graft
alternatives. Nevertheless, limitations of the use of native veins as bypass grafts have been
described. Less than 15% of the entire dialysis population have well-functioning native AV
fistulas. Eighty percent of dialysis patients receive polytetrafluoroethylene (PTFE) vascular
grafts for access, which is accompanied by the morbidity attendant with implantation of
these grafts. When used as peripheral vascular replacements, one-quarter to one-third of
saphenous vein bypass grafts deteriorate, as assessed by histology, in the relatively higher
flows and pressures of the arterial circulation. It is common, therefore, for patients with no
usable native veins available for grafting (or remaining veins of poor quality) to present to
vascular surgeons for repeated revascularization procedures. The use of synthetic vascular
grafts is imperative in these patients.
The long term performances of commercially available synthetic vascular prosthetics
are poor. Primary patency of PTFE grafts used for AV access is approximately one year;1 the
patency rate for PTFE grafts implanted for femorodistal reconstruction is approximately
20% at three years.2 Failure of vascular grafts at short times postoperatively has been gener-
ally attributed to the combination of low flow hemodynamics plus the inherent
thrombogenicities of the graft materials. Late graft failure most frequently occurs from anas-
tomotic neointimal hyperplasia (NIH) and consequent narrowings at the outflow tract. In
synthetic grafts used for hemodialysis access, midgraft hyperplasia adjacent to frequently
used needle puncture sites may also cause failure. The etiology of NIH remains to be clarified.
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
128 Tissue Engineering of Prosthetic Vascular Grafts
One of the early assumptions for the superiority of ferences between seeded and nonseeded graft patencies in
autogenous vascular grafts compared to synthetic grafts was this study and the extent of endothelialization of seeded
the presence of an intact endothelium on the luminal sur- grafts was minimal. Ortenwall et al7,8 reported reduced plate-
faces of the native grafts. Under in vivo conditions of nor- let deposition on both seeded Dacron and polytetra-
mal circulatory physiology, endothelial cells (ECs) are the fluoroethylene grafts implanted in patients. Kadletz et al9
most nonthrombogenic cells known. The blood-contacting also observed little or no platelet accumulation on PTFE
(luminal) surfaces of synthetic vascular grafts implanted in prostheses that had been endothelialized in vitro prior to
animals endothelialize, in time, along their entire lengths implantation in patients. Herring et al10 compared the per-
and typically achieve long term patencies. This endo- formances of 66 seeded PTFE grafts with 53 autologous vein
thelialization occurs, however, only in the anastomotic re- grafts and concluded that at 30 months vein graft patency
gions of human vascular grafts and is limited at most to was superior and that seeding did not improve patency in
2-3 cm of ingrowth. below the knee bypasses. In contrast, Leseche and his col-
Although the issues remain debatable, it has generally leagues11 reported good clinical results when endothelial cell-
been accepted that humans do not heal synthetic bypass seeded PTFE grafts were implanted as above-knee
grafts with an endothelial cell lining as other animals do. As femoropopliteal bypass grafts. These investigators utilized a
a consequence of this failure to heal, in addition to other two stage seeding technique in which a period of in vitro
clinical issues, it is generally regarded as not feasible to uti- incubation was utilized to increase the number of available
lize small caliber vascular grafts (< 6 mm I.D.) clinically in autologous endothelial cells for transplantation onto the
patients, as these grafts fail quickly from accumulated throm- graft.
bus and/or neointimal hyperplasia. The bottom line in this discussion is that the efficacy
The logical assumption of many researchers has been of endothelial cell seeding in improving prosthetic graft pa-
that synthetic graft performances in humans may be en- tency has not been resolved. In fact, proving that graft per-
hanced and patencies extended by promoting formance is indeed enhanced as a result of endothelial cell
endothelialization of the luminal surfaces of these synthetic seeding is complicated in and of itself. Among investigators
grafts. One method of creating such an endothelialized sur- there has been minimal consensus related to many techni-
face is transplantation of autogenous ECs onto the blood- cal issues. One such issue has been the definition of the spe-
contacting graft surface. A new research technology has thus cific tissue source of endothelial cells for transplantation.
evolved called “endothelial cell seeding (ECS)” to investi- Our laboratory program of endothelial cell transplantation
gate this possibility. The underlying logic supporting ECS has focused upon the use of microvascular endothelial cells
investigations is that the presence of transplanted endothe- (MVEC) derived from enzymatic processing of subcutane-
lium mimics the anti-thrombogenic intima of native ves- ous fat. We have been impressed with the relative numbers
sels. In addition, it has been suggested that rapid of endothelial cells that can be derived from fat sources com-
endothelialization in anastomotic regions may normalize pared with the limited endothelial cell harvests typically
aberrant flow patterns, which are thought to contribute to available from autologous blood vessels. Because subcuta-
the genesis of intimal hyperplastic lesions.3 neous fat from which MVEC can be derived is available in
Animal studies have shown that endothelialization of virtually all patients presenting for vascular surgery, we ini-
prosthetic grafts can be encouraged by transplanting endo- tiated a clinical trial of endothelial cell seeding in patients
thelial cells into or onto the graft at the time of implanta- requiring PTFE grafts for A-V access. The study compared
tion. These endothelial cell-seeded grafts typically have im- the histological appearances of biopsies derived from MVEC-
proved patencies versus nonseeded controls when evaluated seeded and nonseeded ePTFE arteriovenous fistulas im-
in animals. To date, however, the prospect from animal stud- planted in patients for blood access for renal dialysis.
ies of successful long term performances of endothelial cell-
seeded grafts in humans has not been realized in the clinical Materials and Methods
trials that have been reported, which are few in number when
compared with the numbers of reported animal studies. AV Fistula Study
The first true clinical trial of endothelial cell seeding Following informed consent, 9 patients (3 males and
in patients was reported by Herring et al.4 Seeded endothe- 6 females; mean age 59.8 years) enrolled in the AV fistula
lial cells were derived by mechanical methods from autolo- study. All of these patients had renal disease requiring long
gous veins and transplanted onto Dacron grafts, which were term hemodialysis. In this study patients were randomized
subsequently implanted in patients as femoropopliteal re- to receive either microvessel cell “seeded” ePTFE grafts or
constructions. Analyses revealed no differences in patencies “nonseeded” ePTFE grafts as AV fistulas. The purpose of the
between seeded and nonseeded grafts; smokers had espe- study was to compare the histological appearances of bi-
cially poor graft patencies. Herring and his colleagues5 were opsy samples of each type of graft at scheduled
able to confirm endothelialization of the luminal graft sur- postimplantation times including 1 month, 3 months and
face from a seeded graft retrieved from a patient via histo- 1 year after surgery. The details of the experimental
logical analysis. Zilla et al6 also reported early clinical data methologies are reported elsewhere.12 An average of 1.1 x 107
from a series of patients who underwent femoropopliteal microvessel cells were transplanted onto the seeded grafts.
bypass using endothelial cell-seeded expanded Records of the length of graft implanted were not kept, so it
polytetrafluoroethylene (ePTFE) grafts. There were no dif-
Healing Patterns Following Microvascular Cell Seeding 129
is impossible to estimate the density of the cells which were seeded graft showed significant concentric fibrous plaque
transplanted onto the graft. formation with a high level of collagenation in the region of
the thickening closest to the lumen. Large vessels could be
Results seen within the fibrous thickening (Fig. 10.1).
There were no operative complications attendant upon The nonseeded graft which was biopsied at one month
derivation of subcutaneous fat. The patients tolerated all postimplantation revealed a luminal surface with exposed
operative procedures well. Harvesting the microvessel cells graft material and some adherant red blood cells. No endo-
added one hour to the total operative time for each patient. thelial cells were seen or identified by factor VIII staining on
A complicating issue in this study was the refusal of the luminal surface of the nonseeded grafts. At 16 months a
patients to comply with the biopsy schedule. One patient biopsied section of a nonseeded graft revealed irregular ar-
agreed to a one month biopsy only. One additional patient eas of intimal fibrous thickening; however, there was no in-
agreed to biopsies at one and three months. A third patient flammatory response or calcification, and the flow-contact-
presented for biopsies at one month and 16 months. And ing surface of the nonseeded graft remained thin.
one patient was biopsied at one month, three months, 20
months and 24 months. Only two patients agreed to one Discussion
year postimplantation biopsies. Therefore, the data from this Ten years of research efforts have investigated the po-
study must be interpreted in light of only limited tential efficacy of ECS in enhancing vascular graft perfor-
postimplantation biopsy samples. mances in in vitro benchtop laboratory models of seeding,
The seeded grafts from biopsied patients at one month animal implants, and preliminary clinical studies. In our
postimplantation revealed few red blood cells and no accu- opinion, however, the definitive studies have yet to be per-
mulating thrombus. The intimal lining consisted of areas of formed and reported that support or refute ECS as a mo-
single cells in thickness to areas with beginning organiza- dality of clinical significance to the vascular surgeon. The
tion of fibrinous material. factor VIII analysis confirmed that research described in this report was intended to accom-
the neointima was lined by endothelial cells. plish that goal. The research was performed in patients re-
Interestingly, the neointimas of seeded grafts obtained quiring placement of synthetic PTFE grafts for access for
at subsequent postimplantation periods were thicker than hemodialysis. We felt that data derived from the analysis of
the neointimas at one month. The thickening consisted of endothelial cell-seeded grafts in hemodialysis patients would
concentric intimal fibrous material, which appeared as a offer insight into the potential efficacy of endothelial cell
mixture of myofibroblasts and fibroblasts with prominent seeding for other synthetic vascular graft applications as well.
collagen deposition. No significant inflammatory cells were Unfortunately patient compliance with this protocol
found. These thickened intimas were also lined by factor VIII and the post operative biopsy schedule was minimal, at best,
positive endothelial cells. At 20 months the biopsied seeded which was disappointing. This was especially disappointing
graft revealed significant concentric intimal thickening, with because in the seeded samples that were analyzed the intima
the beginning of calcification of the intimal plaque. There was greatly thickened compared to the nonseeded grafts. This
were no accumulations of foamy macrophages or choles- would be an important observation if it could be repeated
terol clefts. The luminal surface was smooth and no thrombi in a relatively large number of patients.
were observed. At 24 months, the biopsy sample from a
Fig. 10.1. Neointima of microvessel cell-
seeded ePTFE graft, biopsy at 24 months
postimplantation. See text for descrip-
tion.
130 Tissue Engineering of Prosthetic Vascular Grafts
It is interesting to speculate on the etiology of the in- 5. Herring M, Baughman S, Glover J. Endothelium devel-
timal thickening of the seeded grafts analyzed in this study. ops on seeded human arterial prosthesis: A brief clinical
Certainly the AV fistula is an aberrant hemodynamic envi- note. J Vasc Surg 1985; 2:727-30.
ronment, in which high wall shear stresses may continually 6. Zilla P, Fasol R, Deutsch M et al. Endothelial seeding of
PTFE vascular grafts in humans: A preliminary report. J
perturb transplanted endothelial cells, resulting in a con-
Vasc Surg 1987; 6:535-41.
tinual state of activation. If this is in fact the case, then en- 7. Ortenwall P, Wadenvik H, Risberg B. Reduced platelet
dothelial cell seeding will not enhance graft performance— deposition on seeded versus unseeded segments of ex-
it will only exacerbate the inevitable failure due to intimal panded polytetrafluoroethylene grafts: Clinical observa-
hyperplasia. However, one must caution again that the re- tions after a 6-month follow-up. J Vasc Surg 1989;
sults described herein are from only a limited number of 10:374-380.
patients in the unusual hemodynamic environment of the 8. Ortenwall P, Wadenvik H, Kutti J et al. Endothelial cell
AV fistula, and may not be representative of the fate of seeded seeding reduces thrombogenicity of Dacron grafts in hu-
grafts implanted elsewhere within the vascular tree. mans. J Vasc Surg 1990; 11:403-410.
In summary, we recommend that a well-orchestrated 9. Kadletz M, Magometschnigg H, Minar E et al. Implanta-
study of endothelial cell seeding in AV fistulas be conducted tion of in vitro endothelialized polytetrafluoroethylene
grafts in human beings. A preliminary report. J Thorac
to clarify this issue, with sufficient numbers of patients such
Cardio Surg 1992; 104:736-742.
that the impact of endothelial cell seeding on the perfor- 10. Herring M, Smith J, Dalsing M et al. Endothelial seeding
mance of synthetic AV fistula can be clarified. Transplanta- of polytetrafluoroethylene femoral popliteal bypasses: The
tion of microvessel-derived endothelial cells resulted in fac- failure of low-density seeding to improve patency. J Vasc
tor VIII positive graft sections, but “graft healing” resulted Surg 1994; 20:650-655.
in thickened neointimas which jeopardized blood flow 11. Meerbaum S, Sharp W, Schmidt S. Lower extremity
through the graft. revascularization with polytetrafluoroethylene grafts
seeded with microvascular endothelial cells. In: Zilla P,
References Fasol R, Callow A, eds. Applied Cardiovascular Biology
1. Bell D, Rosenthal J. Arteriovenous graft life in chronic 1990-91. Int Soc Appl Cardiovasc Biol. Basel: Karger,
hemodialysis. Arch Surg 1988; 123:1169. 1992; 2:107-119.
2. Bergen J, Veith F, Bernhard V et al. Randomization of 12. Schmidt S, Meerbaum S, Anderson J et al. Evaluation of
autogenous vein and polytetrafluoroethylene grafts in expanded polytetrafluoroethylene arteriovenous access
femoro-distal reconstructions. Surg 1982; 92:921. grafts onto which microvessel-derived cells were trans-
3. Eguchi H, Okadome K, Mii S et al. Significance of the planted to “improve” graft performace: Preliminary re-
endothelial lining in prevention of intimal thickening of sults. Ann Vasc Surg 1998; 12:405-411.
autogenous vein grafts in dogs. J Surg Res 1991; 50:179.
4. Herring M, Gardner A, Glover J. Seeding human arterial
prostheses with mechanically derived endothelium. The
detrimental effect of smoking. J Vasc Sur 1984; 1:279-89.
CHAPTER 11
Neointimal Hyperplasia in Small Diameter Prosthetic
Vascular Grafts: Influence of Endothelial Cell Seeding
with Microvascular Omental Cells in a Canine Model
Miralem Pasic, Werner Müller-Glauser, Marko Turina
Introduction
Ideal Vascular Graft
T he ideal vascular graft should be biocompatible, resistant to infection, nonthrombogenic,
easy to suture, and most important, durable. Unfortunately, none of the existing pros-
thetic vascular grafts lives up to all of these criteria. Therefore, the early patency of pros-
thetic grafts depends on a multitude of secondary factors such as the creation of a techni-
cally satisfactory anastomosis, the degree of interaction of blood components with the vessel
wall, and an adequate distal run-off. Long-term patency is mainly dependent on the pro-
gression of proximal and distal arterial disease, and the healing of the anastomosis. The
interaction between blood components, mostly platelets and leukocytes, and the vessel wall
is particularly complex in areas of injury or at suture lines. Overall, the lack of an endothelial
surface is one of the most important variables causing the relatively poor patency of small
diameter synthetic bypass grafts when compared to autologous saphenous veins, although
other factors such as length, caliber, flow, and kink resistance may also affect the patency of
small diameter arterial prostheses.1
Intimal Hyperplasia
Intimal hyperplasia is a characteristic fibromuscular cellular response to vascular in-
jury during vascular reconstruction2,3 and represents—apart from surface thrombogenicity—
the second most important reason for prosthetic graft failure. All forms of vascular recon-
struction cause both injury and a wound healing response,4 resulting in different degrees of
intimal or neointimal proliferation after reconstruction. Neointimal hyperplasia, particu-
larly pronounced in the area of distal anastomoses, accounts for more than 20% of late
failures of infrainguinal prosthetic graft revascularizations.5-8,13 On gross examination, inti-
mal hyperplasia is a white, firm, fibrous lesion.9,10 Histologically, it is characterized by cellu-
lar proliferation and accumulation of extracellular matrix material, occurring as a result of
excessive proliferation and accumulation of smooth muscle cells.11 The formation of
neointimal hyperplasia occurring after implantation of prosthetic vascular grafts is, how-
ever, not identical to intimal proliferation after endarterectomy or angioplasty, in that it is
primarily due to chronic inflammatory responses associated with implanted synthetic grafts.12
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
132 Tissue Engineering of Prosthetic Vascular Grafts
In contrast to occlusions after angioplasty or atherectomy, dothelium. One explanatory finding is that PDGF is also
which tend to appear in the first 6 months,14,15 the stenos- produced in endothelialized grafts.25 For weeks after graft
ing intimal lesions of autologous or prosthetic grafts usu- insertion in dogs, PDGF production of ePTFE grafts seeded
ally appear later.5,13 with autologous venous endothelial cells is even greater than
in nonseeded controls; it correlates positively with endo-
Pathogenesis of Anastomotic Hyperplasia thelial cell coverage and inversely with platelet deposition.25
The pathogenesis of anastomotic hyperplasia, which Although endothelial cells play an important role in
includes the interaction of the various cells and the mito- preventing or inhibiting neointimal hyperplasia by releas-
gens and cytokines they produce, and regulation of cellular ing inhibitors of smooth muscle cells, 35-37 incomplete
growth is not fully understood. Cell migration and prolif- endothelialization seen at the anastomotic areas in
eration during intimal thickening appear to be regulated by nonseeded grafts may induce smooth muscle cell prolifera-
factors from the blood, particularly platelets and leukocytes, tion beneath the endothelium38,39 due to platelet-derived
as well as by interaction with adjacent vascular wall cells.16 growth factor, and other mitogen production by perturbed
Operative manipulation and hemodynamic factors such as endothelial cells.40-42 In contrast to a vein graft, in a seeded
high and low flow velocities, high and low wall shear stress,17 prosthesis there is only a subendothelial layer, without the
and mechanical compliance mismatch 18 also influence medial layer. This might be a reason for early suppression of
smooth muscle cell proliferation and intimal thickening. A smooth muscle cell proliferation after completion of the
possible explanation for the smooth muscle cell prolifera- endothelial layer, which does not occur in vein bypass. The
tion beneath the pannus endothelium in prosthetic grafts is influence of complete endothelialization of a prosthetic graft
the production of smooth muscle cell mitogens by perturbed on the development of late neointimal hyperplasia is un-
anastomotic endothelial cells.19 These endothelial cells are known.
known to produce platelet-derived growth factor (PDGF) The complexity of the pathways leading to formation
and other mitogens.20,21 Furthermore, endothelial cells are of neointimal hyperplasia suggests that one agent alone will
not only actively involved in the regulation of smooth muscle likely not be entirely effective in its prevention. This multi-
cell migration and proliferation during pathologic changes factorial cause seems to be responsible for failed single medi-
such as intimal hyperplasia, but also during normal pro- cal therapies aiming at the prevention of anastomotic
cesses such as vessel development, depending on their state hyperplasia. By achieving a confluent endothelium in a
of activation. It is unknown whether endothelial cells on seeded graft, however, one possible factor for neointimal
implanted prosthetic grafts become permanently activated anastomotic hyperplasia—the nonendothelialized luminal
or if they return to the “normal” inactive state.19 It is, how- surface of the graft—might be excluded, or might be
ever, known that a recovered endothelial layer after arterial converted into an active factor for prevention of late
injury or after endothelial cell lining of a prosthetic graft neointimal hyperplasia.
only decreases or slows the process of intimal proliferation,
without preventing it completely. Experimental Studies with Endothelial Cell
Seeding at the University Hospital Zurich
Endothelial Cells and Neointimal Hyperplasia At the University Hospital Zurich we performed sev-
Endothelial cells possess both inhibitional and prolif- eral studies whose main aim was to test whether complete
erative properties with regard to smooth muscle cell prolif- endothelial-like cell coverage of a small vascular graft—
eration. On the one hand, the degree of myointimal hyper- seeded with omentally derived cells—is capable of reducing
plasia caused by arterial damage is less in areas which are or even preventing late neointimal proliferation and anas-
covered by regenerating endothelium.22,23 This may be due tomotic hyperplasia.43-46 In one study44 we performed long-
to synthesis of heparin proteoglycans.24 Neointimal smooth term tests for up to 1 year in 24 mongrel dogs (weight
muscle cells may even become nonproliferative when the 20-30 kg; age 1-4 years). The dogs were cared for according
overlying endothelial layer is re-established. Therefore, the to the European Convention on Animal Care. All experi-
confluent endothelial cells in a seeded graft may synthesize ments were reviewed and approved by the Ethics Commit-
active substances which inhibit proliferation of smooth tee of the University Hospital Zurich. Anesthesia was induced
muscle cells by releasing inhibitors of smooth muscle cells with sodium thiopental (20 mg/kg IV); the dogs were intu-
such as heparan sulfate,24,26 endothelium-derived relaxing bated and ventilated with an oxygen, nitrous oxide and ha-
factor (EDRF),27-30 and prostacyclin.31,32 Grafts, whether lothane mixture. Intraoperatively the animals received an
seeded or nonseeded, produce prostacyclin (PGI2) in small infusion of lactated Ringer’s solution at the rate of 10 ml/kg
amounts,28,30,33 but seeded grafts have a significantly higher per h during the operation.
production of prostacyclin than the nonseeded ones; it is,
nevertheless, lower than that of the native arteries.27-30 In an Surgical Technique
in vitro model of vascular injury, endothelial cell seeding A small supraumbilical laparotomy was made, and as
significantly increases both basal and stimulated PGI2 release much of the omentum as possible was removed and placed
from damaged vein segments.34 On the other hand, incom- in a sterile 150 ml plate containing 60 ml of HEPES buff-
plete late suppression of smooth muscle cell proliferation ered saline. The omentum was further processed in the cell
may cause late intimal and medial thickening at anastomotic laboratory by the cell-laboratory staff, while the surgical staff
areas despite regeneration of a morphologically intact en- closed the abdominal incision and performed exposure of
Neointimal Hyperplasia in Small Diameter Prosthetic Vascular Grafts 133
the arteries for the implantation. Starch-free gloves were used Cell Identification
throughout the study to avoid the cytotoxic effect of glove An aliquot of approximately 0.5 ml of the cell suspen-
powder.47 A midline neck incision was made and both com- sion was removed for quantification of harvested cells. Cell
mon carotid arteries were dissected free. A 6 cm segment of numbers were assessed by fluorometric deoxyribonucleic
each artery was isolated between the vascular bulldog clamps acid measurements of cell pellets.50 Cell identification re-
and excised, and then both ends were beveled to approxi- lied on specific perinuclear uptake of acetylated low density
mately 45 degrees. A 4 mm internal diameter uncrimped lipoprotein,51 immunohistochemical staining for von
Dacron graft (Sulzer Brother, Ltd., Winterthur, Switzerland) Willebrand factor52 and the determination of cell viability
with an average water porosity of 700 ml/min per cm2 at in culture.53,54 Viability was defined as the number of at-
120 mmHg was simultaneously preclotted with autologous tached cells counted after 18 h in the cell culture, and was
blood and seeded. Immediately thereafter they were im- attributed to those cells which resisted rinsing with PBS. After
planted into the right carotid artery. The oblique end-to- washing, the cell culture was trypsinized and the number of
end anastomoses were performed with a continuous 7-0 cells was calculated by fluorometric deoxyribonucleic acid
polypropylene suture. Paired anatomic sites (the common measurements on cell pellets.50 The density of viable cells
carotid arteries) were used throughout the study; each dog per cm2 of a seeded graft was estimated by the ratio of the
received one seeded (inserted into the right carotid artery) number of viable cells to the seeded graft surface.
and one nonseeded graft (inserted into the left carotid
artery). Graft Surveillance and Explantation
Graft patency was assessed by angiography at 1, 5, 12,
Medication 26, and 52 weeks after surgery. The angiographic catheter
All animals received prophylactic cephalosporine an- was introduced through the common femoral artery and
tibiotics (cefalexinum monohydratum, 1200 mg/day) and than forwarded into the common carotid artery. The
analgesics (buprenorphine chloride 0.9 mg/day) for 4 days angiographic examination was performed by manual injec-
postoperatively, as well as dipyridamole (Dip, 75 mg/day) tion of 10 ml of diluted (1:1 with 0.9% sodium chloride)
and acetylsalicylic acid (ASA, 325 mg/day) orally, beginning nonionic angiographic dye (Iopromidum Ultravist 370,
on 1 and 4 days before surgery, respectively. The antiplatelet Schering, Berlin, Germany).
medication (ASA) was continued for 4 weeks postoperatively. The prostheses were explanted after 5 weeks (n=6
dogs), 12 weeks (n=6 dogs), 26 weeks (n=6 dogs) and 52
Microvascular Endothelial Cell Harvesting weeks (n=6 dogs). After transfemoral angiography, the grafts
The cell harvesting procedure followed the modified were dissected free and then removed with the adjacent 3 cm
method of Schmidt et al.48 The omentum (28.8 g ± 7.3 g of proximal and distal carotid artery. Grafts were histologi-
SD) was rinsed with HBS-2, trimmed, minced with two scal- cally investigated with light microscopy after staining with
pels into very small pieces, and aspirated into a 10 ml pi- hematoxylin-eosin. For scanning electron microscopy, grafts
pette; the tissue was then transferred into a 50 ml Erlenmayer were processed according to the method of Schrotter.55 Im-
flask containing 1500 U/ml collagenase type II (Sigma munohistochemical staining for von Willebrand factor was
Chemical Co., St. Louis, MO, USA). The ratio was 1 g of performed on formalin-fixed paraffin embedded tissue. Sec-
omental tissue to 2 ml of collagenase. The suspension of tions were predigested for 10 min with 0.1% pronase E
omental tissue and collagenase was incubated for 40 min- (Merck, Dreieich, Germany) at 37°C. After blocking endog-
utes in a 37°C shaking water bath at 100 rotations per minute. enous endoperoxidase with methanol-H2O2, rabbit antise-
The digested tissue was homogenized by repetitive pipetting, rum against von Willebrand factor (factor VIII-related an-
transferred into 15 ml tube, and centrifuged twice at 100G tigen) was applied. Rabbit antibodies were detected using
for 5 minutes. The supernatant contained mainly adipocytes the ABC/peroxidase method with diaminobenzidine as a
and the collagenase solution. The cell pellet was resuspended substrate of the color reaction.52 Sections were counter-
in 10 ml PBS, filtered through a 250 micron pore size poly- stained with hemalum. (All immunological reagents were
ester mesh (Bolting Cloth Weaving, Zurich, Switzerland) to from DAKOA/S, Glostrup, Denmark). The maximum thick-
remove nondigested large tissue fragments, and then washed ness of the neointimal tissue within 10 mm of the suture
twice with HBS-2. Finally, a suspension of the endothelial lines, as well as of the central part of the graft, were mea-
cells (2 ml) was added to 15 ml of autologous plasma and sured on hematoxylin-eosin stained cross section by means
centrifuged at 45G for 20 minutes onto the prosthesis using of a computer-assisted automated image analysis system
a rotation device (Sulzer Brothers, Ltd., Winterthur, Swit- (Video planimeter, Zeiss Inc., Switzerland).
zerland). After implantation, the seeded grafts demonstrated Late neointimal hyperplasia was assumed if a signifi-
clusters of cells adhering to the Dacron fibers on the pros- cant difference in neointimal thickness was found when
thetic lumen embedded in red thrombus, preventing the comparing:
wash-out and subsequent loss of the seeded cells.49 Cell seed- 1. The mean neointimal thickness of certain parts of
ing density was 1.75 (0.77 to 2.72, 95% confidence inter- the grafts with the mean neointimal thicknesses of
vals) x 106 cells per square centimeter of graft surface, rang- the other parts at the same time point (e.g., the mean
ing from 0.37 x 106 to 4.54 x 106 cells. value of the neointimal thicknesses of all the distal
parts of all grafts explanted at three months after
134 Tissue Engineering of Prosthetic Vascular Grafts
implantation vs. the mean value of the proximal or 100%, 86%, 49%, 40%, and 13% for nonseeded grafts, re-
central parts of the same group of grafts, etc.); or spectively (Fig. 11.1).
2. The difference between the mean neointimal thick-
nesses of the same parts of the grafts at different time Angiographic Differences of Seeded and Nonseeded
points (e.g., the mean value of the neointimal thick- Grafts
nesses of all the distal parts of all grafts explanted at We found obvious angiographic and histological dif-
three months after implantation vs. the mean value ferences between the seeded and the nonseeded grafts with
of the neointimal thicknesses of all the distal parts regard to neointimal hyperplasia, except when the nonseeded
of all grafts explanted at six months after implanta- grafts were also completely covered with endothelial-like
tion, etc.). cells. According to the fine structural details of the grafts
seen on angiography and later confirmed by histological
Statistics examination, thrombotic processes and neointimal hyper-
Patency data determined by serial angiographic stud- plasia were confirmed as the two main causes of graft occlu-
ies were tabulated and presented in the life table format by sion. Early graft occlusions were obviously caused by throm-
the method of Cutler and Ederer.56 Event-free proportions botic processes, because these grafts showed smooth anas-
at 1, 5, 12, 26, and 52 weeks postoperatively are reported tomotic regions with intraluminal clots on angiography.
with 95% confidence limits. The data are presented as means Contrary to these early findings, progression of late
with 95% nonparametric confidence intervals. Differences neointimal proliferation, predominantly in the anastomotic
in mean values for neointimal thicknesses at different time regions, was the dominant finding in grafts with late occlu-
points and between different locations were assessed by re- sion. The first changes in terms of neointimal hyperplasia
peated ANOVA measures; simple regression was used to were noted by angiographic studies performed at 3 months
evaluate the development of the neointimal thickness dur- after graft insertion, with a trend to progression with time.
ing the time at the same location. A value of p < 0.05 was However, this anastomotic stenosis was not observed in the
considered to be significant. two nonseeded grafts which were totally covered with en-
dothelial-like cells.
Endothelial Cells Reduce Late Neointimal
Proliferation Histological Findings
Based on the combination of three highly conclusive Histological studies of the seeded grafts revealed a
parameters (patency rate, angiographic findings, histologi- neointima with luminal endothelial-like lining and highly
cal examinations), our study strongly supported the hypoth- organized subendothelial layers in the absence of anasto-
esis that complete coverage with endothelial-like cells of the motic neointimal hyperplasia (Fig. 11.2). The subintimal
luminal side of a small vascular graft seeded with omentally- tissue was covered by a luminal monolayer of coherent epi-
derived cells reduces or even prevents late neointimal pro- thelial-like, flat cells expressing von Willebrand factor
liferation and anastomotic hyperplasia. (Fig. 11.3). Microscopic examinations confirmed that
neointimal hyperplasia had caused the anastomotic steno-
Improved Patency of Seeded Grafts sis seen by angiography. Furthermore, histological exami-
The actuarial patency rates at 1, 5, 12, 26, and 52 weeks nations revealed that the stenotic lesions seen on angiogra-
were 100%, 95%, 95%, 95%, and 95% for seeded grafts, and phy in the central parts were predominantly caused by a
thrombotic process, although it was difficult to distinguish
Fig. 11.1. Actuarial patency rate of seeded and
nonseeded grafts. All experimental animals received
antiplatelet medication for 4 weeks postoperatively.
C.L., Confidence limits.
Neointimal Hyperplasia in Small Diameter Prosthetic Vascular Grafts 135
Fig. 11.2. Immunohistochemical staining for
von Willebrand factor of the proximal anas-
tomotic region of a seeded Dacron graft ex-
planted at 1 year showing the monolayer na-
ture of the cells covering the graft (arrowheads
indicate the black-stained nuclei) (magnifi-
cation x700). A=the common carotid artery,
N=neointima.
Fig. 11.3. Immunohis-
tochemical staining for
von Willebrand factor of
the longitudinal sections
of the central region of a
seeded graft 6 months af-
ter implantation (magnifi-
cation x1000). The highly
organized intima is cov-
ered by a luminal mono-
layer of coherent, flat epi-
thelial-like cells (arrow-
heads indicate the black-
stained nuclei) expressing
von Willebrand factor
(brown reaction product).
hyperplasia from organized thrombus in some specimens large areas covered with coagulum consisting of fibrin, plate-
of the occluded nonseeded grafts. lets, and occasional leukocytes interspersed with regions of
spontaneous coverage by endothelial-like cells.
Spontaneous Endothelialization
In contrast to the confluent endothelial lining of Neointimal Tissue Thicknesses
seeded grafts, the nonseeded grafts showed a varying degree In endothelial seeded grafts, the measurements of the
of spontaneous coverage with endothelial-like cells maximum thicknesses of the neointimal tissue at the anas-
(Fig. 11.4). The extent of this process was time dependent; tomoses as well as in the central parts of the seeded small
two patent nonseeded grafts, for instance, were completely diameter Dacron grafts showed neither late neointimal pro-
covered by endothelial-like cells after one year. However, liferation nor anastomotic hyperplasia. The mean neointimal
these grafts did not show any neointimal hyperplasia, and tissue thickness of the seeded grafts increased during the
in that resembled seeded grafts. In the other patent initial 26 weeks and then stabilized or slightly regressed af-
nonseeded grafts, scanning electron microscopy revealed ter this period (Fig. 11.5). Simple regression analysis showed
136 Tissue Engineering of Prosthetic Vascular Grafts
progressive reduction of the mean neointimal thickness raphy for routine long-term surveillance, because this type
(-0.5 mm per week). However, this tissue regression was sta- of angiography provided fine anatomical details enabling
tistically not significant. There was no difference when com- us to identify structural changes in grafts.5 Angiograms of
paring any specific part among the different grafts at any the seeded grafts showed a smooth intimal surface without
time point, nor in comparison with two nonseeded grafts luminal narrowing, whereas angiographic examinations of
which were totally covered with endothelial-like cells. most nonseeded grafts demonstrated different degrees of
Repeated ANOVA measures revealed no significant differ- luminal stenosis at the anastomotic regions in the late phase
ence among mean neointimal thicknesses of seeded grafts of implantation.
at different time points (p=0.51) or different locations Thus, the main factors for occlusion of prosthetic small
(Greenhouse-Geisser corrected p=0.16). diameter vascular grafts are increased graft surface
thrombogenicity and technical error in the early postopera-
Graft Occlusion tive course, and progression of the atherosclerotic disease
In summary, both the angiographic studies and his- and neointimal hyperplasia in the late phase.57 In our com-
tological examinations revealed that neointimal prolifera- parative study, technical errors such as incorrectly performed
tion and anastomotic hyperplasia were the main factors lead- anastomoses were excluded by postoperative angiography;
ing to late occlusion of the nonseeded grafts. To obtain fine atherosclerotic process as a possible cause for late occlusion
assessment of our implants, we used conventional angiog- was also excluded by the use of young animals. Therefore,
Fig. 11.4. Macroscopic appearance of
the luminal surface of a nonseeded
graft (upper position) and a seeded
graft (lower position) explanted 6
months after surgery. The nonseeded
graft exhibits spontaneous
endothelialization in the central por-
tion and anastomotic neointimal in-
growth. The seeded graft is throm-
bus free.
Fig. 11.5. Neointimal tissue thickness
of seeded 4 mm I.D. uncrimped
Dacron grafts explanted between 5
and 52 weeks after implantation.
C.L., Confidence limits.
Neointimal Hyperplasia in Small Diameter Prosthetic Vascular Grafts 137
thrombotic graft occlusion was found to be the main cause endothelial-like cells of a synthetic vascular graft following
of early failure. In all explants, the cause of occlusion was seeding with pure endothelial cells or with a mixture of dif-
determined after considering the results of all diagnostic ferent cells from omentum is an unknown process, too. It
methods (angiographic, histological and macroscopic find- probably requires cell duplication, although it has been
ings), never relying on one single method only. Since grafts shown that endothelial cells remain on the graft surface af-
were explanted at the dates assigned by preoperative ran- ter cell transplantation.59 Due to the similarity of the scan-
domization and not immediately after occlusion was noted, ning microscopic findings between the seeded grafts and the
it was sometimes difficult to distinguish hyperplasia from spontaneously endothelialized regions in nonseeded grafts,
organized thrombus in occluded control grafts. Once throm- it can be speculated that these two different processes prob-
bosis of a graft has occurred, the thrombus rapidly becomes ably involve similar mechanisms leading to surface cover-
organized and fibrotic, such that it is difficult to distinguish age with endothelial-like cells and ultimately to a
from pre-existing hyperplasia. The technique of quantita- thrombogenicity of the graft surface.
tive histologic analysis of neointimal hyperplasia used in our
study permits accurate measurement only in grafts that are Intimal and Neointimal Hyperplasia
patent, so that progression can be observed. However, the It should be emphasized that the term “late neointimal
progressive course of hyperplasia can only be compared be- hyperplasia” does not refer to the absence of any neointimal
tween different animals. Therefore, we performed the two tissue on the luminal side of a seeded prosthetic vascular
types of comparisons described above: firstly, simultaneous graft. It is a well established fact that seeding with omental
measurements and comparisons of the different locations cells produces a neointima on these grafts. Therefore, we
at the same time point and secondly, measurement and com- defined the term “late neointimal hyperplasia” in our study
parisons of the same locations on different specimens at dif- as a significant increase of the neointimal thickness in cer-
ferent times. tain parts of the seeded grafts (proximal, central, distal) as
compared with the other parts at the same time point, or
The Role of Complete Endothelial Coverage the difference between the neointimal thicknesses of the
The second important observation of our study is that same parts of the grafts at different points in time.
complete coverage of the prosthetic surface with endothe- Neointimal hyperplasia in a nonseeded graft is a pro-
lial-like cells per se, regardless of by which method this has cess of smooth muscle cell proliferation which occurs at both
been achieved, was the single most important factor for pre- the proximal and distal anastomoses between a prosthetic
vention of late neointimal hyperplasia and the subsequent nonseeded graft and the adjacent artery. However, it occurs
improvement of graft patency. This conclusion is further to a significantly greater degree at the distal anastomosis than
supported by the fact that late neointimal hyperplasia was the proximal one.60 In our study, we found no difference in
found in nonseeded grafts which were only partly covered neointimal thickness between any parts (proximal, distal,
by endothelial-like cells, but not found in the nonseeded or central) of the seeded grafts at any time point. This makes
grafts which were totally covered with endothelial-like cells late neointimal hyperplasia an unlikely event in seeded pros-
through spontaneous outgrowth. theses because one would expect:
We further conclude that an omental seeding tech- 1. Changing intimal thickness in predefined graft ar-
nique may reduce or prevent late neointimal hyperplasia via eas over time; and
the mechanism of complete luminal coverage with endo- 2. Differences in intimal thickness within one and the
thelial-like cells. However, it should be emphasized that the same graft at the same time point, particularly when
improved overall patency rate of the seeded grafts can only comparing anastomotic regions with midgraft areas.
be partly attributed to the reduction or prevention of late The regular angiographic follow-up and microscopic
neointimal hyperplasia, while other mechanisms such as examination revealed that there was a significant difference
reduced thrombogenicity due to luminal surface coverage between the seeded and the nonseeded grafts with reference
with endothelial-like cells also contribute substantially. to neointimal hyperplasia. The fact that no difference be-
The third important observation of our study is that tween the neointimal thicknesses was found when compar-
we found no scanning electron microscopic differences be- ing the two nonseeded grafts which were patent at 1 year
tween seeded grafts and the regions of the nonseeded grafts (and were totally covered with endothelial-like cells) and the
which were covered with endothelial-like cells. Although seeded grafts does not speak against the hypothesis but rather
spontaneous endothelialization of nonseeded grafts is usu- sheds more light on the impact of complete endotheliali-
ally seen in animals, the precise origin of luminal endothe- zation on neointimal proliferation.
lial-like cells in both endothelialized seeded and nonseeded Intimal hyperplasia (intimal changes in a venous or
grafts remains unclear. The findings of endothelialized anas- arterial graft or native artery) and neointimal hyperplasia
tomotic regions as well as islands of endothelium through- (de novo generation of an intima on a synthetic graft sur-
out the grafts indicate that spontaneous endothelialization face which was previously lacking an intima) are a charac-
can occur by several mechanisms, such as migration of en- teristic fibromuscular cellular response to vascular injury
dothelial cells from the host artery towards the prosthesis, during vascular reconstruction.61 It is characterized by cel-
endothelial cell ingrowth from capillaries originating out- lular proliferation and accumulation of extracellular matrix
side the graft and passing through the interstices of the fab- material which occur as a result of excessive proliferation of
ric, or from circulating blood cells.58 However, coverage with smooth muscle cells.62 All forms of vascular reconstruction
138 Tissue Engineering of Prosthetic Vascular Grafts
cause injury and a wound healing response,4 leading to inti- binding of growth factors can trigger cytoplasmic signal
mal or neointimal proliferation after reconstruction with transduction pathways.77 These regulatory mechanisms of
vein or prosthetic grafts63,64 as well as in a host artery after specific cell behavior are not well understood. Thus, analy-
endarterectomy, atherectomy, or coronary angioplasty.65-68 sis of biological activity of growth factors on smooth muscle
However, formation of neointimal hyperplasia occurring cell growth might be useful for antagonizing pathological
after implantation of prosthetic vascular grafts is not smooth muscle cell migration and proliferation.75,76,82
identical to intimal proliferation after endarterectomy or an- The complexity of the pathways leading to formation
gioplasty, largely due to chronic inflammatory responses of neointimal hyperplasia suggests that one agent alone will
associated with implanted synthetic grafts. Early after im- likely not be entirely effective for its prevention. This multi-
plantation of a prosthetic graft, leukocytes adhere to the graft factorial cause is responsible for failed single medical thera-
surface, followed by monocyte infiltration and formation pies for preventing anastomotic hyperplasia. Since growth
of foreign body giant cells. The latter characterize the chronic factors are the main stimuli for smooth muscle cell migra-
inflammatory or foreign body response to vascular prosthe- tion and proliferation, several pharmacological approaches
ses.69 Neointimal hyperplasia in a synthetic graft had already to smooth muscle cell hyperplasia might be useful.83 Differ-
been recognized in the early phase of peripheral vascular ent agents, such as anti-inflammatories,84 antihypertensive
surgery.8,70 It occurs both in Dacron and polytetrafluoro- drugs,85,86 fish oil,87 prostaglandin analogues,88 #1-adrener-
ethylene grafts71,72 as early as 3 months and as late as 1 year gic blocking agents,89 and anticoagulants90 have been shown
after implantation.7,8,70 In contrast to occlusions after to suppress the process of intimal hyperplasia in animal stud-
angioplasty or atherectomy, which tend to appear in the first ies. The role of anti-platelet therapy in the prevention of
6 months,73 the stenosing intimal lesions of autologous or intimal hyperplasia after arterial reconstruction remains
prosthetic grafts usually appear later after surgery.5,6 Anas- controversial.91,92 Human trials have demonstrated that as-
tomotic hyperplasia involves both the proximal and distal pirin and dipyridamole significantly improve patency of
anastomoses but it has a predilection for the distal anasto- prosthetic, but not saphenous vein, femoropopliteal by-
mosis, with a tendency to progress with time.72 Neointimal passes;93,94 they are particularly beneficial when started be-
hyperplasia in the area of the distal anastomoses accounts fore operation94 or within 24 h of operation.95 However,
for 20%-50% of late lower extremity bypass graft failures.6-8 clinical trials of anti-platelet agents suggest that platelet in-
The regulation of cell migration, proliferation, and hibition alone does not prevent anastomotic intimal
growth during intimal thickening appear to be regulated by hyperplasia.96
factors from the blood, particularly platelets and leukocytes, The present study showed that establishing an endo-
and the interaction of vascular wall cells and grafts them- thelial-like cell coverage on the luminal surface of small di-
selves.16 Operative manipulation and hemodynamic factors ameter prosthetic grafts plays an important role in reduc-
such as high and low flow velocities, high and low wall shear tion or prevention of late neointimal and anastomotic hy-
stress,17 as well as compliance and diameter mismatch be- perplasia. Anastomotic neointimal hyperplasia was sup-
tween graft and host artery,18 also influence smooth muscle pressed in those grafts that were totally covered with endo-
cell proliferation and intimal thickening. thelial-like cells, regardless of whether they had been seeded
It has been proposed that growth factors such as plate- or not. In contrast, occluded control grafts showed clearly
let-derived growth factor and basic fibroblast growth factor visible anastomotic tissue ingrowth in the late phase. There-
released from activated platelets, leukocytes, altered smooth fore, seeding with omental cells may play an important role
muscle cells and endothelial cells may be involved in the in preventing or inhibiting late smooth muscle cell prolif-
smooth muscle cell migration and proliferation after vascu- eration by total coverage of the prosthetic luminal surface
lar injury.74,75 Basic fibroblast growth factor is a prototype with endothelial-like cells. The characteristic of endothelial
of the family of heparin-binding growth factors that regu- cells to possess both inhibitory and proliferative properties
late a variety of cellular responses including cell growth, on smooth muscle cells may thus play an important role in
morphogenesis and differentiation of various cell types, in- pathogenesis of neointimal hyperplasia.97,98 A possible ex-
cluding smooth muscle cells.76 Fibroblast growth factors planation for the smooth muscle cell proliferation beneath
deliver their signals to cells by binding to a dual receptor endothelium in the nonseeded grafts is the production of
system which activates the bound growth factor before its smooth muscle cell mitogens by perturbed endothelial
delivery to the signal-transducer receptors.77,78 Signaling of cells.69 By reaching early luminal confluence of endothelial-
growth and differentiation involves multiple pathways.79 At like cells in a seeded graft, one possible factor for neointimal
least two families of receptors bind basic fibroblast growth anastomotic hyperplasia, namely nonendothelialized lumi-
factor and mediate its response: tyrosine kinase-containing nal surface of the graft, might be excluded, or might be con-
fibroblast growth factor receptors,77 and heparan sulfate verted into an active factor for prevention of late neointimal
proteoglycans.80 Both are known to undergo internalization hyperplasia. Although endothelialized surfaces will not al-
by different pathways.79 Heparan sulfate proteoglycans are ter all factors potentially contributing to anastomotic inti-
obligate partners in binding of basic fibroblast growth fac- mal hyperplasia, they seem to reduce the deposition and
tors to their receptors; fibroblast growth factors do not bind activation of circulating blood elements, including platelets,
to fibroblast growth factor receptors unless heparan sulfate leukocytes, and components of the coagulation and comple-
or its analog, heparin, is present.81 The activation of intrin- ment system. They furthermore seem to decrease the release
sic tyrosine kinase function of receptor tyrosine kinases upon of mitogen factors for smooth muscle cells and prevent
Neointimal Hyperplasia in Small Diameter Prosthetic Vascular Grafts 139
thrombus formation by generation of prostacyclin, immunostaining seen in some of our specimens in the up-
antithrombotic factors and plasminogen activators. per layers of the intima could be caused by previous platelet
deposition and therefore previous thrombosis, which was
Omental Microvascular Cells supposedly prevented by the seeding with omental cells. The
A large number of cells can be harvested from the other methods for identification of these cells, and their dif-
omentum. The cells derived from omental suspension com- ferentiation from mesothelial cells, are the use of endothe-
prise a mixture of microvascular endothelial cells and me- lial cell-specific monoclonal antibodies, such as antibody
sothelial cells, with a few other cell types.48,99 Overall, the BMA 120,113 and the characterization of different biologi-
contamination of a seeding suspension with nonendothelial cal functions typical for these cells.112 Moreover, endothe-
cells depends on the harvesting method. A purer endothe- lial cells express thrombomodulin, a cofactor for activation
lial cell yield can be achieved by lowering the separation den- of protein c, intracellularly; thus they stimulate the activa-
sity during the harvesting procedure, but this leads to a re- tion of protein c by thrombin. 114,115 Furthermore,
duced number of all cells available for seeding. Wang et al100 thrombomodulin has been identified immunologically on
estimated that 10% or less of the total cell number of har- endothelial cells of veins, arteries, and capillaries as well as
vested omentum cells were nonendothelial in origin. Kern on the endothelium of lymphatics and on syncytio-
et al101 obtained a completely pure microvascular endothe- trophoblasts.116
lial cell population harvested from omental tissue with no Comparing cells derived from human omental fat tis-
growth of other cell types in serially passaged cultures, but sue with those of human umbilical vein endothelial cells,
the endothelial cell yield was greatly reduced, at only 103 Visser et al103 concluded that the cells from omental tissue
endothelial cells per gram of omental tissue. were not endothelial but mesothelial in nature. Their state-
ment was based on the observations that cells isolated from
Origin of the Omental Cells human omental tissue did not stain with endothelial spe-
The identification of the cells derived from omentum cific antibodies EN4 and PAL-E; these cells contained abun-
poses a problem and there is no consensus in the literature dant cytokeratins 8 and 18, which were absent in endothe-
regarding their identity and origin.102,103 It is sometimes dif- lial cells.107 The presence of cytokeratins 8 and 18, as deter-
ficult to establish the identity of isolated endothelial cells, mined with the monoclonal antibodies M20 and M9, was
especially their distinction from mesothelial cells,104 because abundant in the cells derived from omentum but not in the
both types of cells are present in omental tissue. Like endo- human umbilical vein endothelial cells.103 Vimenten could
thelial cells, mesothelial cells produce prostacyclin105,106 and be detected (monoclonal antibody V9) in both types of cells,
possess fibrinolytic activity. These cells produce large whereas desmin (monoclonal antibody D33) was present
amounts of tissue-type plasminogen activator in vitro, to- only in omentally derived cells. A faint and diffuse staining
gether with type 1 and 2 plasminogen activator inhibitor.107 of von Willebrand factor was seen in cells from omentum,
Moreover, when seeded onto vascular prostheses these cells whereas microvascular endothelial cells from subcutaneous
acquire a confluent monolayer lining with no adherent plate- fat displayed this factor as indistinct granular structures.107
lets or amorphous material within one month after sur- In contrast to the human umbilical vein endothelial cells,
gery.108,109 In tissue culture, both types of cells show similar which stained with both anti-von Willebrand factor anti-
growth patterns under light microscopy.102,104,105 However, bodies, omentally derived cells stained for von Willebrand
it has been shown that in a culture of mixed cells derived factor only if the antibodies were polyclonal. Moreover, scan-
from omentum, endothelial cells were rapidly displaced by ning electron microscopy revealed that cultured cells derived
mesothelial cells, resulting in a pure culture of mesothelial from omentum contain numerous surface microvilli,
cells.103 Similarly, in a tissue culture of endothelial cells and whereas human umbilical vein endothelial cells did not. All
fibroblasts, endothelial cells are suppressed by fibroblasts that of these findings suggest that the cells derived from omen-
proliferate to form a confluent layer.110 In our study, we did tum were a mixture of predominantly mesothelial cells and
not seed the grafts either with omentum cells immediately a low number of microvascular endothelial cells.103 In con-
after harvesting or with cultured cells. In this way, the sup- trast to these findings, Stansby et al117 were not able to prove
pressive effect of mesothelial cells on endothelial cells in that the omental cells were mesothelial in origin. They con-
culture could be excluded in our study. cluded that human omental microvascular endothelial cells
Although the origin of the omentally derived cells has were pericytic in nature.117 Hernando et al105 proved the
remained controversial, its endothelial nature has usually nonendothelial origin of the cells derived from human
been proven by morphological and functional criteria. The omentum, demonstrating that these cells showed positivity
most widely methods used for identification of endothelial for monoclonal antibodies specific for endothelial cells (anti-
cells are the demonstration of von Willebrand factor by im- CD34 QBEND10), antibodies to intermediate filaments
munofluorescence staining,111 and the uptake of diacetylated (anti-vimentin and anti-desmin) and anti-smooth muscle
low-density lipoprotein. 51 The incorporation of cell antibodies (anti-actin and anti-total actin).
[35S]methionine into von Willebrand factor demonstrates Endothelial cells from different organs show various
the ability of the cultured microvascular endothelial cells differences in tissue culture and in vivo; human endothelial
from human omental tissue to synthesize von Willebrand cells harvested from saphenous or umbilical vein, for in-
factor,112 proving the identity of the cultured cells as endo- stance, exhibit distinctive features in transmission electron
thelial. However, it could be speculated that the extensive microscopic examination if cultivated on precoated PTFE
140 Tissue Engineering of Prosthetic Vascular Grafts
grafts.118 Microvascular endothelial cells migrate more slowly References
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2. Chervu A, Moore WS. An overview of intimal hyperpla-
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Willebrand factor than human umbilical vein endothelial vascular surgery: the intimal hyperplastic response. Ann
cells,121 with weaker fluorescence in the cytoplasm than large Thorac Surg 1997; 63:582-591.
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from hyperplastic lesions of prosthetic bypass grafts: Pro-
Limitations of Experimental Studies duction of a platelet-derived growth factor-like mitogen
Our experimental model, using optimal conditions and expression of a gene for a platelet-derived growth
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10:157-167.
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Y, Inokuchi K. Effect of wall shear stress on intimal thick-
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32. Clagett GP, Burkel WE, Sharefkin JB, Ford JW, Hufnagel J Surg Res 1986; 41:463-472.
H, Vinter DW, Kahn RH, Graham LM, Stanley JC, 48. Schmidt SP, Monajjem N, Evancho MM, Pippert TR,
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minal production of prostacyclin, platelet adherence, and land: The University of Zurich; 1994:79-82.
fibrinogen accumulation. J Vasc Surg 1988; 7:600-605. 50. Anderson JS, Price TM, Hanson SR, Harker LA. In vitro
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36. Castellot JJ, Addonizio ML, Rosenberg R, Karnovsky MJ. dase complex (ABC) in immunoperoxidase techniques: A
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purified canine venous endothelial cells for experimental behavior of a dacron arterial substitute: Clinical, roent-
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54. Zilla P, Siedler S, Fasol R, Sharefkin JB. Reduced repro- 71. Selman SH, Rhodes RS, Anderson JM, DePalma RG,
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CHAPTER 12
Human Clinical Trials of Microvascular Endothelial
Cell Sodding
Stuart K. Williams
History of EC Transplantation
W hile advances in clinical vascular surgery have resulted in significant progress in the
treatment of vascular diseases, a significant frustration has been the inability to sustain
the patency of small diameter (< 6 mm) synthetic vascular grafts. A specific example of the
poor performance of small diameter synthetic grafts is in peripheral bypass procedures.
When compared to saphenous vein autologous grafts, synthetic grafts constructed of either
ePTFE or Dacron exhibit clinically poor outcomes. The clinical experience with synthetic
grafts for coronary artery bypass grafting is even more disappointing, with only rare ex-
amples of successful use of synthetic grafts for coronary revascularization.
The importance of a stable endothelial cell lining on natural blood vessels toward
maintenance of an anti-thrombogenic, metabolically active surface, and as a barrier con-
trolling the selective transport of cellular and plasma protein constituents of blood, has
been well established.1,2 The importance of establishing an endothelial cell lining on syn-
thetic grafts has also been recognized by numerous research laboratories.3,4 Numerous tech-
niques have been proposed toward the accelerated formation of an endothelial cell lining on
synthetic grafts. These techniques generally fall into the two categories of either accelerated
spontaneous endothelialization or accelerated endothelialization through cell transplanta-
tion. Spontaneous endothelialization has been explored through several mechanisms, in-
cluding accelerated pannus ingrowth and stimulated migration of vascular elements di-
rectly though the wall of the graft.5 The efficiency of spontaneous endothelialization is highly
variable, with significant species to species variation. More extensive work has been per-
formed evaluating the use of endothelial cell transplantation toward accelerated
endothelialization of synthetic vascular grafts.6
Numerous variables have been explored during the development of techniques for
endothelial cell transplantation, including source of endothelial cells for transplantation,
the need to culture cells prior to transplantation, methods for cell deposition and graft sur-
face modifications to improve cell-polymer interactions. Each of these variables has been
shown to be critical to the development of operating room compatible methods for endo-
thelial cell transplantation. Prior to the initiation of human clinical trials, endothelial cell
transplantation methods were evaluated in several preclinical animal models where both
the safety of the methods was established and, finally, the effects of endothelial cell trans-
plantation on graft patency were confirmed. This latter point has been critical to support
the hypothesis that the accelerated formation of an endothelial cell monolayer on synthetic
vascular grafts following endothelial cell transplantation will improve graft patency.
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
144 Tissue Engineering of Prosthetic Vascular Grafts
Following extensive evaluation of different variables sue culture medium type 199E. This solution was pushed
for endothelial cell transplantation, our laboratory estab- through the interstices of the graft using a pressure gradient
lished methods for the use of autologous endothelial cell equal to 5 psi. Grafts were prewet for at least 30 minutes.
transplantation using adipose tissue-derived microvascular
endothelial cells. Preclinical animal studies established that Cell Deposition
endothelial cells could be isolated from fat and transplanted A suspension of endothelial cells was prepared in
rapidly onto the lumenal surface of synthetic grafts (both medium 199E at a density to provide 2 x 105 cells/cm2 graft
ePTFE and Dacron), and that this form of graft treatment lumenal surface area. The solution was placed into the graft
resulted in a statistically significant improvement in graft and a pressure gradient created across the wall of the graft
patency. The development of these methods was not with- of 5 psi, resulting in the flow of fluid through the graft and
out controversy. Several investigators questioned whether concomitant deposition of endothelial cells. We term this
adipose tissue even contained endothelium, suggesting the process pressure sodding of cells.
cells isolated from fat were mesothelium and not endothe-
lium.7 Extensive evaluation of the cellular components iso- Time to Graft Implantation
lated from adipose tissue following its dissociation using col- Immediately after pressure sodding the grafts were
lagenase established the major contribution of endothelium brought to the operating field and implanted.
to the total cellular makeup of adipose tissue.8 But more As part of this clinical evaluation of endothelial cell
important was the fact that microvascular endothelium transplantation using autologous microvessel endothelial
could be rapidly transplanted onto synthetic vascular grafts, cells, a specialized endothelial cell isolation and cell deposi-
resulting in the accelerated formation of an endothelial cell tion kit was developed. This kit and its use is diagrammed
monolayer and improved patency in the grafts once im- in Figure 12.1. The performance of this kit was modeled af-
planted. A complete discussion of the results of endothelial ter the endothelial cell sodding procedure first developed
cell transplantation studies in preclinical animal models is and used during preclinical animal trails and subsequently
discussed in an accompanying chapter. used during human trials (Fig. 12.2).
Human Trial of Microvascular Endothelial Cell Patients and Methods
Transplantation The protocol for this study received approval from the
Based on the success of preclinical animal studies, an Human Subjects Committee/Institutional Review Board of
evaluation of endothelial cell transplantation in patients re- Thomas Jefferson University, Philadelphia, PA and the Uni-
quiring peripheral bypass grafts was planned. We used the versity of Arizona Health Sciences Center, Tucson AZ. Pa-
term sodding to describe our method of endothelial cell tients in need of a peripheral bypass graft were eligible for
transplantation, since it involved the placement of large inclusion in the study based on the inclusion/exclusion cri-
numbers of endothelial cells directly onto the lumenal sur- teria outlined in Table 12.1. Just prior to surgery all patients
face of synthetic grafts. Several variables were considered and were phlebotomized of 150 cc of blood to obtain serum for
evaluated prior to establishment of the final human sod- sodding the prosthesis.
ding trial protocol. The abdomen and the site of graft placement was
prepped and draped. To obtain adipose tissue for microvessel
Trial Variables endothelial cell isolation, liposuction of subcutaneous fat
Source of Endothelial Cells
During previous studies we evaluated methods for iso-
lating endothelial cells from both large vessel (vein) and
Table 12.1. Microvascular endothelial cell sodding trial:
microvascular sources (fat). Several sources of fat were con-
inclusion and exclusion criteria
sidered, including omental-associated fat, perirenal fat and
abdominal wall fat, as well as methods to remove the fat,
Inclusion Criteria:
including direct surgical excision and liposuction removal.
Following extensive evaluation we identified liposuction-
Patients with atherosclerotic peripheral vascular disease
derived fat from the abdominal wall as having the most resulting in tissue loss. Patients with claudication alone are not
optimal cellular characteristics for subsequent sodding. included in study.
While these cells could be subsequently cultured, we deter- >18 years of age
mined the cell yield from patients was more than sufficient Adequate fat for liposuction
(>1 x 10 6 cells/g fat) to produce enough cells for our No acceptable vein
sodding methods. Life expectancy >1 year
Informed consent
Graft Type and Pretreatment Exclusion Criteria:
Clinically used ePTFE vascular grafts with nominal
30 micron internodal distance were used for all implants. Serious heart or pulmonary disease
To improve cell deposition all grafts were prewet with a so- Serious coagulopathy
Uncontrolled hypertension
lution containing 1 part serum (autologous) and 6 parts tis-
Detection of positive serology for viral infection
Human Clinical Trials of Microvascular Endothelial Cell Sodding 145
Fig. 12.1. Endothelial cell isolation and
transplantation kit design.
Fig. 12.2. Process of
endothelial cell isolation
and transplantation.
from the abdominal wall was performed with a liposuction Overview of Patients
cannula and specially designed 60 cc syringe. Approximately All patients were referred to participating vascular
50 g of fat was removed from each patient and processed for surgeons for amputation.
MVEC isolation. The completely assembled kit, being used Pt. 01. 52 yr old M, diabetic, coronary artery and re-
during a human endothelial cell sodding procedure, is shown nal artery disease, rest pain, severe left lower ischemia, his-
in Figure 12.3. The isolation procedure as outlined above tory of failed bypass graft in leg to receive sodded graft. Re-
resulted in ePTFE grafts with approximately 2 x 105 cells/cm2 ceived ileo-peroneal sodded bypass graft.
graft lumenal surface area. Grafts were immediately im- Pt. 02. 82 yr old M, history of failed bypass grafts in
planted in patients. study leg. Rest pain. Received ileo-peroneal sodded
bypass graft.
146 Tissue Engineering of Prosthetic Vascular Grafts
Pt. 03. 71 yr old F, diabetic with history of coronary Pt. 10. 83 yr old M, diabetic, hypertensive with his-
artery and peripheral vascular disease. Previously received tory of failed bypass grafts in study leg. Received femoral-
several bypass grafts including in situ vein graft. Received tibial sodded bypass graft.
femoral-tibial sodded bypass graft. Pt. 11. 57 yr old M diabetic with hypertension. His-
Pt. 04 69 yr old F, history of failed bypass grafts and tory of peripheral and cardiac bypass grafts including failed
angioplasty. Received ileo-tibial sodded bypass graft. autologous peripheral graft in study leg. Received femoral-
Pt. 05. 75 yr old F, diabetic with hypertension. History tibial sodded bypass graft.
of failed bypass grafts. Received femoral-tibial sodded by-
pass graft. Patient Follow Up
Pt. 06. 74 yr old M. History of cardiovascular disease All patients received routine follow up evaluation of
including CABG, peripheral vascular, renal artery proce- graft function, including color flow Doppler and duplex
dures. Presented with intermittent claudication. Received imaging. Of the original 11 patients, seven were available
femoral-popliteal sodded bypass graft. for follow up at four years while four expired prior to this
Pt. 07. 72 yr old F, diabetic with hypertension. History time due to nongraft related causes. The cumulative patency
of failed bypass grafts including previous posterior tibial in of the study grafts is illustrated in Figure 12.4. As expected
situ bypass graft in study leg which failed after 7 months. of the patient population in this trial, several patients have
Received femoral-posterior tibial sodded bypass graft. experienced other vascular complications including cardiac
Pt. 08. 74 yr old M, history of peripheral and cardiac arrhythmias, myocardial infarctions, nonstudy leg periph-
disease. Received ilio-peroneal sodded bypass graft. eral vascular disease progression, renal disease and pulmo-
Pt. 09. 75 yr old M, received ilio-tibioperoneal sodded nary embolism. Several patients have also undergone treat-
bypass graft. ment for cancer. Follow up evaluation of patients did not
Fig. 12.3. Assembled endothelial cell isolation and
sodding device in use in the operating room.
Fig. 12.4. Cumulative patency of microvascular
endothelial cell sodded grafts in human periph-
eral vascular graft trial.
Human Clinical Trials of Microvascular Endothelial Cell Sodding 147
provide information predictive of subsequent graft failure. 2. Richardson JV, Wright CB, Hiratzka LF. The role of en-
One patient exhibited a mid-graft concentric intimal thick- dothelium in the patency of small venous substitutes. J
ening just below the knee approximately 9 months after graft Surg Res 1980; 28:556-562.
implantation. This thickening had disappeared at subsequent 3. Zilla P, Preiss P, Groscurth P, Rosemeier F, Deutsch M,
Odell J, Heidinger C, Fasol R, von Oppel U. In vitro-lined
graft follow-up.
endothelium: Initial integrity and ultrastructural events.
Surg 1994; 116(3):524-534.
Conclusions 4. Zilla P, Deutsch M, Meinhart J, Puschmann R, Eberl T,
This trial was designed as an initial safety study to Minar E, Dudczak R, Lugmaier H, Schmidt P, Noszian I.
evaluate the ability to isolate endothelial cells from Clinical in vitro endothelialization of femoropopliteal by-
liposuction fat and transplant these cells onto ePTFE grafts. pass grafts: An actuarial follow-up over three years. J Vasc
The initial four year follow up data is quite encouraging and Surg 1994; 19(3):540-548.
suggests endothelial cell transplantation can be performed 5. Clowes AW. Graft endothelialization: The role of angio-
using an automated kit. The patient population selected for genic mechanisms. J Vasc Surg. 1991; 13:734-736.
this trial represents individuals with significant vascular dis- 6. Williams SK. Endothelial cell transplantation. Cell Trans
ease. The lack of significant limb loss in this patient popula- 1995; 4:401-410.
7. Visser MJP, van Bockel JJ, van Muijen GNP, van
tion during the first three years follow up is equally encour-
Hinsbergh VWM. Cells derived fromomental fat tissue and
aging. We conclude that an expanded trial of endothelial cell used for seeding vascular prostheses are not endothelial
transplantation using autologous endothelial cells derived in origin: A study on the origin of epitheloid cells de-
from liposuction derived fat is warranted. rived from human omentum. J Vasc Surg 1991;
13:373-381.
References 8. Williams SK, Wang TF, Castrillo R, Jarrell BE. Liposuction
1. Quist WC, Haudenshield CC, Logerfo FW. Qualitative derived human fat used for vascular graft sodding con-
microscopy of implanted vein grafts: Effects of graft in- tains endothelial cells and notmesothelial cells as the ma-
tegrity on morphological fate. J Thorac Cardiovasc Surg jor cell type. J Vasc Surg 1994; 19:916-923.
1992; 103:671-677.
Macrovascular Endothelial Cell Transplantation
CHAPTER 13
In Vitro Endothelialization: Its Contribution Towards
an Ideal Vascular Replacement
Peter Zilla
E very era in medicine has been driven by one particular discipline which recognized an
exciting new development occurring outside its own sphere as an opportunity for a quan-
tum leap. Although this initial phase of integrating an unfamiliar dimension into a tradi-
tional medical dominion was seldom blessed with clinical success, retrospectively this era is
always perceived as the grand epoch of the particular discipline. For instance, the integra-
tion of technical achievements into a discipline like surgery, previously considered to be
more a skillful art than anything else, made cardiac surgery possible. Those pioneering car-
diac surgeons who dared to let a mechanical pump take over the heart’s function, thus al-
lowing procedures on the open heart, were initially viewed with suspicion by their peers.
Typically, it was not a single technical aspect which was absorbed into this young discipline
but rather a basic openness towards the integration of mechanical and electrical contrap-
tions into the circulatory system, a part of the body which was considered to be untouchable
since the days of Theodor Billroth. It seems also typical that the enthusiasm of such a pio-
neering phase eventually draws the newly discovered, unrelated support discipline further
into its sphere of influence. It was not a coincidence that Earl Bakken developed the first
pacemaker in Minneapolis in the wake of the emerging new discipline of cardiac surgery,
which was plagued by postoperative rhythm problems in this early phase. Retrospectively,
the quantum leap which this pinnacle of cardiac surgery initiated for other medical profes-
sions remains unchallenged.
In vascular surgery, the late 1970s and early 1980s may well, retrospectively, turn out
to have been a similarly fateful period for medicine. However, in contrast to cardiac surgery
twenty years before, it was not the world of sophisticated mechanical devices and electronics
which were integrated into medicine but the amazing world of biology. Symptomatically,
naive amateurism and polarization characterize the early days of this era. Amateurism, be-
cause the first to test the water were surgeons and not biologists. This is the trademark of all
developments of this kind in medicine: Brave and alert medical doctors initially apply dis-
coveries from an alien field to medicine themselves. On the one hand, this effervescent breed
with their urge for knowledge has all the ingredients to overcome initial thresholds and
obstacles. On the other hand, it is often a surgical discipline within the medical profession,
with its desire for clear cut and simple solutions, which drives such initiatives. As a conse-
quence, this surgical mentality may endanger its own achievements when necessary com-
plexity falls victim to impatience. In the case of the awaking biological awareness of the late
1970s, it was indeed surgeons again who were behind it. Almost predictably, they caused
their own setback as they had in previous years with heart transplantation by not handing
the newborn over to scientists in time. In the case of heart transplantation, immune and
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
152 Tissue Engineering of Prosthetic Vascular Grafts
drug research rather than the fury of an ongoing surgical openly admitted, each champion of a particular approach
push could have saved 10 years. The eventual breakthrough towards graft endothelialization therefore hoped for com-
of the cyclosporin era fought an uphill battle against the parable fame. Another aspect of those days in the 1960s was
prejudices arising from the surgical desire for a quick fix that their pioneers demonstrated that almost everything
during the preceding 13 years. In the case of the incorpora- which previously seemed unresolvable became feasible
tion of biology into prosthetic vascular surgery, it was the through the application of new materials and mechanically
endless striving of surgeons to succeed with a simple and determined technologies. Naturally, the emergence of a new
instantly applicable, single-staged procedure right from the dimension like biology caused friction. As a result, at all con-
beginning which almost meant the death knell for the en- ferences in the 1980s, the believers in existing technologies
tire idea 10 years later. A further tragedy which repeats itself asked angrily after each talk on endothelial seeding whether
in the early phases of “quantum leap” eras lies in the typical the speakers were aware that graft patencies depended pri-
negative feedback amongst the alien partners which soon marily on surgical skills. Therefore, they recommended that
follows the initial enthusiasm. Surgeons often feel intimi- the presenter spend his time improving those skills rather
dated by the thought of scientific complexity and scientists than wasting it in the laboratory. The fact that this latter
shy away from the “gung-ho” approach of surgeons. As con- group of surgeons still represents the majority within our
tradictory as it may sound, in the end it is often the perse- ranks proves how successful the previous quantum leap was
verance of a few dedicated surgeons who continue their in getting its new standards generally accepted. In his book
mission regardless, accepting a higher level of complexity. The Structure of Scientific Revolutions, Thomas Kuhn1 ex-
This perseverance allows an idea to survive long enough for plains the mechanisms involved in such paradigm shifts.
its final breakthrough. In heart transplantation, Norman Stephen Hawking2 brought it to the point by arguing that
Shumway fulfilled this role. In the attempt to incorporate people are very reluctant to give up a theory in which they
biological principles into prosthetic vascular surgery, in vitro have invested a lot of time and effort. This theory defining
endothelialization may, in retrospect, have played a similar concepts and procedures is the accepted paradigm, which is
role. If the next decade of research succeeds with tissue en- recognized by all scientists working in that field. If, however,
gineering of physiologically functional vascular prostheses, unexpected developments result in increasing inconsistency
the credit for opening a new era in medicine will undoubt- with the prevailing paradigm, a tense situation ensues
edly be deserved by the early pioneers of single staged en- amongst the scientists. At that stage the majority initially
dothelial cell seeding. However, the role which Norman questions the accuracy of the observations. If that fails, they
Shumway played in heart transplantation—to prevent the try to modify the existing theory in an ad hoc manner. Even-
flickering flame from being extinguished—may well be tually, the old paradigm becomes creaking and ugly and a
granted to those who continuously propagated the complex new one is accepted which explains all the awkward obser-
but eventually successful variant of in vitro vations in an elegant and natural manner. Quantum leaps
endothelialization, to clinically prove the benefit of a prin- in medicine are certainly not such revolutionary shifts in
ciple and thus provide sufficient incentive for today’s efforts scientific paradigms, but their principles and their conse-
towards a broadly acceptable breakthrough. quences are similar. The angry discussant, for instance, ques-
tioning the purpose of merging vascular prosthetic research
Historical Perspective with biology, is not an isolated phenomenon but rather a
If the amateurism of the pioneering years of endothe- typical veteran who may have actively contributed to
lial seeding, with its lack of involvement of basic scientists, yesterday’s paradigm shift. This again has many parallels in
contributed to a delay of today’s drive towards an integrated truly revolutionary paradigm shifts in science. Albert
approach to cardiovascular tissue engineering, one can at Einstein, for instance, who caused a paradigm shift in phys-
least explain this shortcoming by the fundamental differ- ics with his special theory of relativity in 1905 was himself,
ence between the worlds of surgeons and biologists. This many years later, one of the major antagonists to the next
does not apply to the polarization which plagued all of us paradigm shift by resisting the acceptance of quantum me-
who were involved in efforts to “biolize” prosthetic grafts chanics. Surgeons are thus in good company with regard to
from within our own discipline. This polarization was two- the resistance to paradigm shifts. However, the 13 year delay
fold: On the one hand, each different approach to endothe- in achieving this goal in heart transplantation, and the 20
lial seeding was almost religiously upheld by the respective year delay in accepting biology in cardiovascular surgery,
groups which stood for it. On the other hand, a fiercely makes it clear that we are dealing with a particularly conser-
fought confrontation of principal values led to a schism vative discipline.
which continues to divide the surgical community today. Having tried to understand the driving forces behind
Both polarizations may be explained in terms of the previ- the twofold polarization which characterized the past 20
ous quantum leap era, under whose spell the majority of years of attempts to create a biologically functional vascular
cardiovascular surgeons still stood. One aspect of the pre- prosthesis, it seems easier to explain the concrete develop-
ceding grand era of cardiovascular pioneering was that it ments of those two decades as well as today’s situation. The
created heroes as never before. Each facet of the overall quan- internally dividing question, for instance, regarding the prin-
tum leap was associated with a big name, whether it was cipal approach to the endothelialization of a prosthetic sur-
Lillihey, Kirklin, De Bakey, Barnard or Cooley. Even if not face, namely the acceptance of initial complexity versus
In Vitro Endothelialization: Its Contribution Towards an Ideal Vascular Replacement 153
a priori simplicity, was not an issue at the beginning when Current and Future Perspective
no pressure of expectation was exerted by the surgical com- Today’s cumulative experience with clinical endothe-
munity. In the early to mid 1970s, the entire initiative to lial cell lining covers almost a decade and comprises more
surface endothelialization was driven by attempts to culture than 200 patients.52-60 It has clearly demonstrated that in
endothelial cells on synthetic surfaces prior to implanta- vitro endothelialized synthetic prostheses are equal or better
tion.3,4 Pioneers of endothelial seeding, like the groups of than saphenous vein grafts with regard to patency in all
Jim Stanley and Linda Graham were among those who first anatomical positions and any clinical stage other than
cultured autologous endothelial cells on vascular graft sur- stage IV.52-60
faces.5,6 Only subsequently in the mid-1980s, when surgeons Although ePTFE grafts with 30 ∝m internodal distance,
saw the opportunity of implementing their discoveries which are noncompliant and do not allow transmural tis-
through main commercial players, did the focus shift almost sue ingrowth, were used for these studies, the autologous
entirely to single stage procedures. Ironically, when the great- surface endothelium alone was not only sufficient to sig-
est enthusiasm for endothelial seeding seized the surgical nificantly improve patency rates, but also led to the devel-
community at the beginning of the second half of the 1980s, opment of a neomedia between an internal elastic mem-
the death sentence was already sealed through both brane underneath the endothelium and the ePTFE surface.59
premature clinical trials7-11 and “commercial kits” for clini- This observation indicates that the previous fear of “con-
cal single staged procedures. What happened after the ma- taminating” smooth muscle cells was unfounded. Further-
jority of disappointed vascular surgeons turned away from more, it not only supports the current holistic approach to-
this idea really resembled the above mentioned late devel- wards tissue engineered prosthetic vascular grafts which aim
opments in heart transplantation. With the hype of the early at fully functional neoarteries, but also removes the last rem-
1980s over, those who continued were prepared to accept nants of the previous polarization between mixed microvas-
both a long and arduous route of homework prior to imple- cular mass seeding and macrovascular in vitro lining.
mentation and the knowledge that their contribution would The tools which biology offers today often seem like a
be a quiet one. dream to those of us who believed in the idea of integrating
Attitude-wise back to square one, the remaining biology into prosthetic grafts, long before vascular biology
groups focused on the main weakness of graft had entered its maturity. At the same time we also see that
endothelialization, the low cellular inoculum. Mass harvest these tools require a commitment of all parties to a many-
methods for microvascular cells,12-16 as well as mass culture fold higher level of complexity than the one from which most
procedures for macrovascular endothelial cells,17-19 were sci- of us had shied away in the early days of endothelial seed-
entifically refined. One of the main reasons for a more re- ing. Last but not least, we as surgeons are still the majority
laxed approach in this second half of the 1980s was the con- shareholders in this venture, although the scientists will soon
viction that a principle rather than a particular solution take over the supervisory board. This is a necessary and cor-
needed to be proven. At the onset of the era of vascular biol- rect development which should have happened many years
ogy, nobody challenges that a biologically functional pros- ago. However, even if the scientists do take over the lead, in
thesis would eventually come as a product from the shelf, the end there will still be three partners: the industry, the
integrating all encoded signals for spontaneous healing. surgeons and the scientists. The industry will need strongly
Nevertheless, such an undertaking needs a critical mass convincing data to voluntarily accept the replacement of a
which can more easily be achieved on the basis of at least simple and profitable product by a complex and expensive
one proven principle. Although important principles of to- one. The surgeons will need the final painful push to accept
day like intramural contractility, compliance and cellular the shift of a paradigm and the biologists will need both the
quiescence could not yet be tackled, we all focused on the true commitment of the other two parties and a strong
proof that graft endothelialization alone can already dra- motivation with regard to the broad application of their dis-
matically improve synthetic graft performance, even if the coveries before they will wholeheartedly join hands. This may
old prosthetic scaffolds of yesterday are used. In a step by well be the last challenge for the veteran surgeons on board.
step approach, adherence and shear stress resistance of cul- Convincing clinical studies with a method which signifi-
tured endothelial cells on various protein matrices were as- cantly improves graft performance will eventually break the
certained20-37 prior to in vivo experiments ranging from ca- resistance against a paradigm shift amongst our peers and
nine implants20 to primate experiments38,39 and preclinical thus prepare the way for the acceptance of tissue engineer-
primate studies.40 The success of those implants was con- ing. Acceptance of a complex approach by surgeons will
vincing enough to apply the principle to very small diam- mellow the scientists who still see us as “gung-ho” cowboys.
eter grafts41 as well as to bioprosthetic surfaces42-47 and to And finally, a joint front of scientists and surgeons will even-
heart valves.48-51 Eventually, the step into clinical trials had tually force the industry to see beyond today’s profits. Last
to be taken. However, in contrast to the clinical studies with but not least, one needs to keep in mind that even a “Los
single staged endothelial seeding, the situation regarding Alamos” approach to tissue engineering will take many years
clinical trails with in vitro lining was distinctly different: By for the development and many years for clinical trials. This
having accepted the disadvantages of a complex procedure, consideration certainly upgrades clinical in vitro
we had eliminated most of the uncertainties prior to com- endothelialization to a procedure which could benefit an
mencing the trials. uncountable number of patients over many years to come.
154 Tissue Engineering of Prosthetic Vascular Grafts
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prosthetic grafts and vein grafts. ASAIO Journal 1997;
43:M515-M521.
CHAPTER 14
Serial Cultivation of Human Endothelial Cells
Caroline Gillis-Hægerstrand, Anders Hægerstrand
Introduction
U nderstanding of the tremendous capabilities of the endothelial cell has grown consider
ably during the last decades. The use of cell culture techniques has been instrumental in
this development. The single most important breakthrough for endothelial cell (EC) cul-
ture, and still probably the most used technique, was published by Jaffe and coworkers in
1973. They described how to isolate and propagate human umbilical vein endothelial cells
(HUVECs).1 Since then several modifications and improvements have been published,2,3
with the fundamental principle remaining unchanged, i.e., the incubation of a vessel, ideally
clamped at both ends, with a collagenase containing solution, and the subsequent plating
and cultivation in a protein-coated tissue culture flask, the cells being fed with a medium
enriched by relatively large amounts of serum and/or growth factors. Some basic ideas about
the fundamental functions of ECs, e.g., the production of arachidonic acid derivatives, the
expression of adhesion molecules, the tight interactions with leukocyte or smooth muscle
cells and the synthesis of nitric oxide (NO), have been derived from the use of cultured ECs,
mainly HUVECs. It should be remembered that ECs in culture are something very different
than ECs in their natural environment. Compared to many, if not most, other cell types, it is
harder to mimic in vitro what the natural circumstances are under which the ECs operate.
This must be remembered when interpretations of EC functions are made. The in vivo cor-
relate remains the only true measure of the predictive value of in vitro findings.
The idea of culturing endothelial cells for clinical purposes is not new, but only a
limited number of studies have actually been performed. In this review we will discuss some
basic techniques and principles for the culture of human endothelial cells and some of the
future possibilities.
Characteristics of the Endothelium
The total area of ECs in a human body is indeed huge, approximately 5000 m2.4 The
ECs in situ are known to have a relatively low frequency of mitotic figures. The replication
rate of ECs ranges from one EC per 102-104 cells/24 h5 to one EC per 10 cells/24 h depending
on in which part of the body the endothelium is located. In areas with increased hemody-
namic force, the replication rate is higher.6 This phenomenon seems to have an in vitro
correlate in that cells cultured under pressure higher than ambient, e.g., 40-100 mm Hg,
show a higher frequency of divisions.7 ECs in vitro act similarly to ECs in vivo in that they
proliferate to form a monolayer and their growth is restricted by contact inhibition.8 How-
ever, even a seemingly confluent layer of ECs with established physical contacts can become
more densely populated under the right conditions, but one EC does not seem to grow on
top of another EC.
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
158 Tissue Engineering of Prosthetic Vascular Grafts
There are differences in morphology, growth rate and types during culture, e.g., fibroblasts, and the long term cul-
protein synthesis between cultured arterial and venous ECs,9 tivation itself to reach the desired amount of cells make EC
as well as between ECs derived from capillaries, arterioles culture for clinical purposes a complicated task.
and venules.10 Venous ECs are larger, thinner and more pleo-
morphic than ECs deriving from aorta. ECs derived from Culture Techniques for Human ECs
veins are considered more difficult to establish in culture A lot can be said about general requirements for cul-
and they grow at a lower growth rate.9 These differences are ture of human cells. Some relatively general components
believed to be due to the different requirements by the he- used during culture of human ECs are summarized in
modynamic environment. Whether this has an impact on Figure 14.1.
utility for transplantation purposes is not known. ECs are continuously exposed to blood, which is
ECs from different sizes and types of blood vessels known to contain growth stimulating factors, although many
show well known differences in metabolic properties, espe- at low concentrations. In our work we have been focusing
cially in their response to vasoactive mediators.11 The physi- on human serum as a source for stimulating growth and
ological significance of this heterogeneity is not clear, but attachment on the prosthetic material, whereas most inves-
may be related to local variations in the function the ECs tigators have used serum containing medium supplemented
serve, for example in arterial versus venous circulation.12 with growth factors and heparin. Serum free alternatives are
When ECs from one source are compared, there is also a commercially available, but these are often quite expensive.
vast difference in basal secretion of, e.g., PGI2 and plasmi- Such media are typically better to introduce after an initial
nogen/plasminogen inhibitors in cultures of ECs from dif- culture period using medium which contains serum. The
ferent patients.13-15 basal medium in most compositions, irrespective of serum
A characteristic that is unique for endothelial cells, dependence or independence, normally contains a relatively
regardless of their origin, is the presence of Weibel-palade high concentration of glucose, and basal media such as M199
bodies.16 In granules inside the Weibel-palade bodies, and MEM are often used. It is generally accepted that some
P-selectin and von Willebrand factor (vWF) are colocalized. matrix component precoated on the tissue culture treated
Other characteristics of the ECs, though not unique, are plastic is beneficial. Most often, as also originally described
prostacyclin production (PGI2),17 angiotensin converting by Jaffe et al, denaturated bovine collagen, i.e., gelatin, can
enzyme (ACE) activity and positive staining for the surface be used. The method by which it is prepared also seems to
antigen PECAM (CD31).18 In most cases, investigators char- fit with current guidelines on the use of bovine/xenogeneic
acterize the ECs by staining with vWF, by showing the pres- material for tissue culture. Fibronectin in concentrations of
ence of Weibel-palade bodies or by showing uptake of acety- approximately 1-10 ∝g per square centimeter is often rec-
lated low density lipoprotein (LDL), the latter being another ommended, but it should be remembered that concentra-
characteristic not restricted to ECs. tion dependent functional alterations can be expected.
Laminin, collagen type I or IV and extracellular matrix
Sources of Endothelial Cells (ECM) have also been used. Shortly after seeding, within
Several sources for isolating ECs during an operation 6 h, ECs deposit matrix components, e.g., laminin and col-
to immediately seed a vascular graft have been demonstrated, lagen type IV, on the plastic, and thereby influence their own
including freshly isolated cells from veins and fat.19-25 Tech- attachment.
niques for isolation and culture of ECs from human tissues
other than large vessels or fatty tissue have been de- Addition of Serum and Growth Factors
scribed.26-29 Others have successfully isolated ECs from Human serum (HS) and fetal calf serum (FCS) pre-
omental tissue,30 although it has been questioned whether pared for cell culture purposes contain growth factors from
they are truly endothelial.31 It is known that mesothelial cells plasma and growth factors released from platelets upon their
share several properties with ECs, especially those related to aggregation. It should be remembered that ECs grow con-
antithrombotic and fibrinolytic functions.32-34 Subcutane- siderably more slowly in blood plasma without platelets or
ous fat is probably a better source of true ECs as compared coagulation factors, which is quite often referred to as se-
to omental fat.25 Sources of ECs for effective and reproduc- rum. Cell culture serum must be prepared according to spe-
ible cultivation are fewer, especially when the cells are in- cific protocols. Serum contains several factors which pro-
tended for clinical purposes. Typically, veins such as the great mote proliferation, platelet derived growth factor (PDGF),
saphenous, basilic, cephalic and external jugular have been epidermal growth factor (EGF), transforming growth fac-
used.35-38 Cultured arterial ECs have not yet been used for tor ! (TGF-!) and insulin-like growth factors (IGFs).39 Fi-
clinical purposes to our knowledge, although that may po- broblast growth factors (FGFs) play many roles in cell
tentially be beneficial, since the purpose is most often to re- growth, differentiation and survival,40 and are well estab-
place nude or denuded tissue in an arterial position. lished growth factors for ECs. Both basic FGF and acidic
In spite of an increasing number of techniques, EC FGF are often used in recombinant form to stimulate EC
research is often hampered by the lack of sufficient num- growth. Endothelial cell growth factor (ECGF), a precursor
bers of ECs of an adequate origin. Even if vessels or tissue form of acidic FGF (AFGF), is a potent mitogen for most
rich in ECs become available, the isolation and cultivation ECs.39,40 A rat brain extract, known as endothelial cell growth
is not easy. The quality and short term storage of the tissue supplement (ECGS), containing different forms of FGFs, is
derived from the patient, the contamination of other cell also commonly used42,43 to improve cell proliferation, and
Serial Cultivation of Human Endothelial Cells 159
was the most frequent additive to EC culture a decade ago, both growth and maintenance of the epitheloid morphol-
before purified and recombinant factors were made com- ogy in human microvascular ECs.26,59,61,62 HUVECs have
mercially available at reasonable prices. also been shown to increase in cell number after stimula-
tion with CT and endogenous peptides which stimulate
Heparin and Endothelial Proliferation cAMP formation.63 Using a medium without cAMP-stimu-
The fact that heparin enhances the stimulatory effect latory compounds, Watkins and coworkers have described
of ECGF on cell proliferation in vitro is well established.43,44 aberrant cell morphologies after repeated passages using
Both heparan sulfate and heparin act by stabilizing growth trypsin.64 In our experience using cAMP-stimulatory fac-
factor activity, partly by preventing proteolytic degradation tors in the medium, the number of cells appearing aberrant,
of the growth factor.45,46 The binding and mitogenic activ- e.g., large and multinuclear, constitutes no more than one-
ity of bFGF are to some degree dependent on heparin-like fiftieth of the cells even after 8-9 passages. This may be due
molecules on the cell surface, or in their absence, on exog- to continuous cAMP stimulation or to the high proportion
enously added heparin-like molecules.47 It has recently been of serum. Cyclic AMP formation has also been shown to
shown that heparin is an FGF-independent activating ligand modulate EC migration,65,66 inhibit hypoxia-induced in-
for FGF receptor 4 (FGFR4) on cultured transfected myo- crease in EC permeability,67 enhance EC thromboresistance68
blasts, fibroblasts and lymphoid cells.48 In our work it was and to amplify agonist-induced release of EDRF.69 Both pro-
shown that heparin alone increased growth of adult human tective and indirect mechanisms may thus be of importance
ECs when heparin was added to a medium containing as for the growth promoting effects of CT/IBMX and db-cAMP.
much as 40% HS. An explanation for these results in com- We have shown that HS is more efficient than FCS in
bination with 40% serum49 is that they may be due to the stimulating growth of HSVECs and that heparin, bFGF and
presence of FGFR4 on ECs or that heparin stabilizes growth cAMP-elevating compounds, together with relatively high
factors in serum. Different growth factors may act differ- concentrations of serum, are efficient in increasing the num-
ently on cultured ECs from various vascular beds. While ber of HSVECs in culture.49 The differences in ability of the
bFGF stimulates proliferation of most ECs, afgf and PDGF different sera and other factors to enhance cell proliferation
have been shown to have no effect on large vessel may depend on the cell type being studied. In our case, it
endothelium but to stimulate microvascular cell growth.50 may be speculated that HS contains factors which are espe-
Bovine microvascular ECs have been shown to express a dif- cially suited for human cells and especially ECs. Human se-
ferent set of growth factor receptors than bovine arterial rum carries the advantage of potential autologous use, which
ECs.51 Interestingly, ECs are known to synthesize both bFGF may increase safety for the patient if the purpose of the cul-
and PDGF, which may act as autocrine and paracrine ture is for transplantation.
growth factors.52-54
Does the Culture Technique Matter
Cyclic AMP and Endothelial Cell Proliferation from a Clinical Perspective?
The addition of purified cholera toxin (CT) to the EC Zilla and coworkers have demonstrated a cell culture
culture medium leads to an increase in intracellular cyclic technique where low density seeding of first passage ECs can
adenosine monophosphate (cAMP) synthesis. The phos- yield 14 x 106 ECs in 26 days.36 The method involves in situ
phodiesterase inhibitor IBMX decreases cAMP degradation, administration of collagenase and basically no passaging of
and both thereby act to increase the intracellular cAMP level. the cells. This may have the advantage of increasing the to-
Cyclic AMP is an ubiquitous messenger which is involved in tal yield and avoidance of exposing the cells to trypsin or
many cellular processes, including changes in morphology, EDTA, which may have negative effects on the cells. In addi-
proliferation55,56 and release of vasoactive substances in re- tion to possible morphological changes, functional changes
sponse to stimulation. 57 When the membrane-bound of the cells after passaging using trypsin, e.g., altered pat-
adenylyl cyclase is activated, ATP is enzymatically converted tern of receptor expression, have also been indicated.70
to cyclic AMP, which leads to activation of protein kinase A In the study which has been used for clinical purposes,
(PKA). These events have been shown to influence cell pro- the medium contained serum, ECGS and heparin. Interest-
liferation,55 and the role of cyclic AMP as a regulator of cell ingly, heparin and ECGS have been shown to supress
proliferation has been studied extensively. Negative control prostacyclin production.44
was demonstrated as early as in the 1970s. Today, however, The choice of culture technique may actually lead to
accumulating data have shown that increase in cAMP levels selection of a selectivley responsive subpopulation of ECs
is also associated with stimulatory effects on several cell types, that will then constitute the majority of the cells available
including epithelial cells.55,58,59 It has also been shown that for transplantation. Direct comparisons of cells from one
activation of cAMP may enhance receptor binding of growth single donor cultured using different methods have not been
factors and progression of the resultant signals.60 performed. Partly differing functions of the cells on the graft,
Db-cAMP is a synthetic cAMP analog which can pen- and thus the clinical result, may become a result of the choice
etrate cell membranes. In our studies cholera toxin, IBMX of culture technique. On the other hand, one may speculate
and db-cAMP have all been shown to increase the prolifera- that the ECs will become functionally normalized once
tion of ECs derived from the great saphenous vein brought back a more natural environment. Nothing but large
(HSVECs).14,49 Karasek and coworkers have shown that com- controlled studies will reveal whether the culture technique
binations of HS and cAMP stimulation are beneficial for will affect the clinical results. Other aspects, e.g., safety and
160 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 14.1. Illustration of typical/pos-
sible media and components used for
serial cultivations of human ECs.
PDGFs: platelet-derived growth fac-
tors, IGFs: insulin like growth factors,
bFGF: basic fibroblast growth factor,
ECGF/S: endothelial cell growth fac-
tor/supplement, CT: cholera toxin,
IBMX: isobutyl methylxanthine,
ECM: extracellular matrix.
rate of successful cultures using a specific technique, will 2. Gimbrone M, Cotran R, Folkman J. Human vascular en-
probably override such speculations. dothelial cells in culture. Growth and DNA synthesis. J
Cell Biol 1974; 60:673-684.
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viruses often carry the same connotation, and most likely
cells. J Cell Physiol 1994; 158(1:133-139.
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morphology and proliferation rate. J Cell Physiol 1981; 72. Newman KD, Nguyen N, Dichek DA. Enhancement of the
106:253-58. fibrinolytic activity of sheep endothelial cells by retroviral
60. Olashaw NE, Pledger WJ. Cellular mechanisms regulat- vector-mediated gene transfer. Blood 1991; 77(3):533-41.
ing proliferation. Advances in second messenger and phos- 73. Lee SW, Kahn ML, Dichek DA. Optimization of retroviral
phoprotein research. 1988; 22:139-173. vector-mediated gene transfer into endothelial cells in
61. Kramer R, Fuh G-M, Karachek M. Type IV collagen syn- vitro. Circ Res 1992; 71(6):1508-17.
thesis by cultured human microvascular endothelial cells 74. Dichek DA, Nussbaum O, Degen SJ, Anderson WF. Ge-
and its deposition into the subendothelial basement mem- netically engineered endothelial cells remain adherent and
brane. Biochemistry 1985; 24:7423-30. viable after stent deployment and exposure to flow in
62. Tuder RM, Karasek MA, Bensch KG. Cyclic adenosine vitro. Circ Res 1992; 70(2):348-54.
monophosphate levels and the function of skin microvas- 75. Moldovan F, Bennani H, Fiet J, Cussenot O, Dumas J,
cular endothelial cells. J Cell Physiol 1990; 142(2):272-283. Darbord C, Soliman HR. Establishment of permanent
63. Haegerstrand A, Dalsgaard C-J, Jonzon B, Larsson O, human endothelial cells achieved by transfection with
Nilsson J. Calcitonin gene-related peptide stimulates pro- Large T antigen that retain typical phenotypical and func-
liferation of human endothelial cells. Proc Natl Acad Sci tional characteristics. In vitro Cell Dev Biol Anim 1996;
1990; 87(9):3299-3303. 32(1):16-23.
CHAPTER 15
Risk Factors for Autologous Endothelial Cell Cultures
Johann Meinhart, Manfred Deutsch, Peter Zilla
Introduction
T he history of cell culture started over a hundred years ago, when Wilhelm Roux showed
in 1885 that embryonic cells can survive outside the animal body. Since then the knowl-
edge of how to culture cells in vitro has steadily increased. Today, cell cultures are a powerful
tool to address many fundamental questions in biology and medicine. Recent advances have
made it possible to utilize cultured human cells for therapeutic use. Cultured cells have been
used to reconstruct skin, bone and many other tissues, including the endothelium. As a
consequence, endothelial cells can be used clinically for the physiological masking of syn-
thetic vascular grafts in order to reduce their thrombotic risk. Several studies were performed
in past years with different methods1-7 to achieve endothelialization of otherwise nonhealing
grafts. The presence of endothelial cells on synthetic vascular grafts reportedly diminishes
the thromboembolic risk8,9 and it seems to inhibit the formation of anastomotic hyperpla-
sia.10 Additionally, endothelial cell-covered grafts are probably less prone to graft infection.11
One method of actively facilitating graft endothelialization is in vitro lining with cultured
autologous endothelial cells.12-14 This technique could prove its effectiveness in long term
clinical applications.15-19 However, despite optimized culture and harvesting procedures,
cultures may fail to grow. Since reproducibility of viable autologous cell cultures is a prereq-
uisite for routine clinical use, we have investigated the influence of certain patient-related
risk factors on the growth capability of autologous endothelial cells.
Standard Cell Culture Technique for Autologous Endothelial
Cell Cultures
Optimal cell harvesting and culture conditions for human vascular endothelial cells
have been established and described accurately in previous publications20-22 and also reviewed
in this volume (see chapter 14). For autologous in vitro endothelialization, a short segment
of a subdermal vein (mainly jugular and cephalic) is taken under local anesthesia. The vessel
is cannulated in situ and filled with collagenase. Primary cells are plated in one T12 culture
flask. At a well defined stage of preconfluence (90% surface coverage; p index equal 0.9),
primary cultures are passaged to two T162 culture flasks. Culture medium is renewed twice
a week by replacing half of the volume with fresh culture medium, consisting of medium
199 with Earls’s salts, supplemented with 180 ∝g/ml preservative-free heparin, 10 ng/ml bFGF,
10 ∝g/ml and 20% autologous serum. For autologous serum preparation, 250 ml blood is
taken from patients and allowed to clot over night at 4°. For both the assessment of 90%
confluence in the primary culture and the continual quantification of cells in the two T162
flasks, a microgrid technique is routinely applied.23 This method provides a partial coverage
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
164 Tissue Engineering of Prosthetic Vascular Grafts
index (“p” index) of a given cell culture area ranging from 0 tion, by their typical morphological appearance. However,
(no cells) to 1.0 (confluent cell culture). Thus, the p index as more information on clinical long term results emerges,
semiquantitatively reflects the growth capacity of primary smooth muscle cells and fibroblasts appear in a different
endothelial cells in culture. light, as they play a substantial role in vessel wall forma-
Apart from the evaluation of endothelial cell growth, tion.18,25 In future, one will probably tend to have a mixture
cultures are also morphologically assessed. Vacuolization and of vascular cells seeded onto the graft surface, which would
cell shape are classified on a scale ranging from 0 (typical de facto mean a convergence of culture and mass harvest
polygonal) to 5 (dendritic) for cell shape and from 0 (no procedures.
vacuoles) to 5 (predominantly vacuoles) for vacuolization. After successful growth of primary cultures, trypsin is
The morphology index (“m”) is therefore m=0 for ideal the standard enzyme for the detachment of cells from the
polygonal, nonvacuolized endothelial cells and m=10 for surface of culture flasks in order to passage them into larger
maximally vacuolized, highly dendritic but still viable cells. containers. Trypsinization, especially of primary human
Continuous light microscopic assessment of morpho- endothelial cell cultures, should be performed very carefully.
logical appearance and quantification of cultures provides a When using 37°C 0.05% trypsin-0.02% EDTA solution, in-
good estimation of cell viability and proliferative capacity. cubation time should not exceed 2 minutes.23
Thus, mitigation of cell growth can be detected at an early For endothelial cell cultures, different media like
stage. The most frequent problems with autologous RPMI, MEM or medium 199 have been used by different
endothelial cell cultures will be described in the authors, with satisfactory results. Medium 199 was originally
following chapters. formulated for the growth of chick fibroblasts, but with Earl’s
salts is now widely used for endothelial cell culture. Liquid
Procedure Related Risk Factors media have a limited shelf life due to the decay of several
compounds over time, even at 4°C. This is especially true
Surgical Vein Harvest for L-glutamine. Complete medium should therefore not
Naturally, autologous human venous endothelial cell be kept at 37°C for a prolonged time, since this will result in
cultures need a sufficient vein segment as a cell source. This pH and osmolality change. Moreover, free radical forma-
vein should be easily accessible for surgical excision in order tion might occur, which would result in severe cell damage.
to keep dissection time short and, therefore, mechanical Substitution of the culture medium with growth fac-
damage low. To further reduce premature mechanical de- tors is necessary to reach optimal proliferation, even when
tachment of endothelial cells, a no touch technique for vein serum is supplemented. A multitude of growth factors for
harvesting is in use.23 In brief, after careful touch-free dis- endothelial cells, like PDGF, EGF and FGFs have been iden-
section, the vessel is cannulated in situ with two free-flow tified and have been described in numerous publications.
vessel cannulas with attached 3-way taps. In order to remove For standard endothelial cell culture, bFGF is the most
remaining blood, the vein is flushed with culture medium frequently used growth factor. The importance of FGF ex-
in a pulsatile manner and filled with a collagenase solution. ceeds mere acceleration of proliferation, as total FGF depri-
The solution is kept under minor pressure within the vein vation in the culture medium also leads to apoptosis of
by closing the 3-way taps. After excision, the cannulated and endothelial cells.26
collagenase-filled vein segment is transferred to the labora- Supplementation with antibiotics might be necessary,
tory in a thermos container at 37°C for further incubation. but they should be used with great care, due to their poten-
If cannulation takes too long, the endothelium might be tial cytotoxicity. Microbial contamination can occur from
damaged by air drying. Thus, if cannulation is difficult due time to time. Serious infections with yeasts, molds and bac-
to a small vessel diameter or other circumstances, the vein teria are macroscopically visible in cultures. However, chroni-
should be flushed intermittently with medium. All manipu- cally infected cultures can be inconspicuous, often only dis-
lations with the vein should be done gently, wearing starch tinguished by decreased cell proliferation. Therefore, micro-
free gloves, since glove powder has been found to be cyto- biological tests should be routinely performed for clinically
toxic for endothelial cell cultures.24 used cultures. Decreased proliferation capacity without sub-
stantial morphological deterioration might also be an indi-
Cell Culture cation for an infection with mycroplasms. Several test kits
A 15 minute incubation time with a 0.1% collagenase are available for mycoplasm detection. In contrast, the de-
solution guarantees a maximal yield of endothelial cells. tection of viruses in cell cultures is more difficult than that
Since all proteolytic enzymes are potentially cytotoxic to cells of bacteria or fungi. Detection methods include coculture
and commercially available collagenases vary in activity, it systems, transmission electron microscopy and immunof-
might be necessary to test each vial separately. Since the ini- luorescence. Some viruses cause cytological changes like cell
tial goal of in vitro endothelialization was to use only pure rounding, vacuolization or syncytium formation. Since these
endothelial cell cultures for the lining of synthetic vascular morphological changes are often similar to other deterio-
prostheses, we tried to avoid the concomitant harvesting of rating influences like cell aging or increased lipid content of
undesirable cells like fibroblasts and smooth muscle cells by serum, the existence of viruses should be kept in mind.27
fine tuning the enzymatic digestion time and enzymatic ac- Standard cell culture flasks made of polystyrene are
tivity. These supposedly “contaminating” cells can be de- surface treated to promote adhesion and proliferation of
tected by immunofluorescence or, in a later stage of cultiva- attachment dependent cells. Endothelial cells cultured in
Risk Factors for Autologous Endothelial Cell Cultures 165
20% autologous serum readily adhere to polystyrene with enrolled in our endothelialization of vascular grafts program
subsequent spreading and growth. This might be facilitated already had quite a long history of vascular disease before
by serum vitronectin and serum fibronectin.28 The role of they had to undergo surgical reconstruction for peripheral
fibronectin as a promoter of cell migration and tight an- arterial occlusion. They were treated conservatively and
chorage of endothelial cells to many biomaterials has been many of them were already operated for other vascular dis-
described extensively.29-31 Therefore, polystyrene culture eases like coronary heart disease or occlusion of the iliac ar-
dishes are frequently precoated with fibronectin, but other tery. A substantial number had already undergone PTA (per-
adhesion substances like collagen or gelatin are also used. cutaneous transluminal angioplasty) before they had to be
The quality of practically all reagents varies from sup- treated surgically. The majority of patients had one or more
plier to supplier. Additionally, quality might even be different risk factors for atherosclerosis. Therefore, these patients
between batches from the same supplier. These facts should formed a quite homogenous group with respect to age, risk
be kept in mind when encountering cell culture problems. factors and clinical stage of disease. Despite this fact, we
Defective machines, like centrifuges, rotation devices and found differences with regard to the in vitro growth behav-
incubators, might also lead to culture problems and should ior of the endothelial cells of these patients. Thirty-four per-
be mentioned briefly here. Above all, incubators should be cent of all cultures showed impaired growth capacity. Cells
checked for accurate temperature, humidity and CO2. In- displayed strong vacuolization and were partially dendritic
cubators might also be a source of repeated contaminations. (Fig. 15.1), a morphological appearance which was previ-
ously shown to lead to imminent cell death.16 When we
Patient Related Risk Factors measured a multitude of serum parameters we found all
Influences of age or genetic constitution on cell vi- three main lipid constituents, triglycerides, cholesterol and
ability and proliferative capacity are well known. In the car- lipoprotein A, significantly (p < 0,05) elevated in sera of pa-
diovascular system, age-associated alterations in vascular cell tients whose cell cultures initially failed to grow (Table 15.1).
function lead to vessel wall thickening, increased dilation Numerous studies have previously shown that hyperlipi-
and aberrant distribution of smooth muscle cells. It is be- demia causes endothelial changes. This includes increased
lieved that these changes play a fundamental role in the on- endocytotic activity34 inhibition of prostacyclin produc-
set of atherosclerosis.33 Besides these intrinsic factors, ac- tion,35 increased monocyte-endothelial cell adhesion,36 dis-
quired factors may alter cellular functions as well. Patients ruption of the barrier function, 37 apoptosis 38 and
Fig. 15.1. Day 3 culture of a patient’s
endothelial cells cultured in medium
containing 20% autologous serum with
high levels of serum lipids. Cells exhibit
strong vacuolation and have a dendritic
appearance.
Table 15.1. Content of cholesterol, triglycerides, lipoprotein A and glucose in sera of patients with normal growth
characteristics of cells compared to sera of patients with initial growth failure of cells and to nonrecovering cell
cultures after serum change.
mg/dl ± SD cholesterol triglycerides lipoprotein glucose
normal growth 220.1±51.1 250.9±202.0 22.2±26.6 139.1±76.3
initial growth failure 262.0±56.8 421.4±298.0 35.9±28.3 108.25±66.25
nonrecovering cultures 222.8±57.5 265.0±166.1 56.25±16.7 205.0±97.1
166 Tissue Engineering of Prosthetic Vascular Grafts
cytotoxicity.39 From experimentally induced rabbit athero- that reversibility applied especially to elevated triglyceride
sclerotic aortas, an increased number of multinucleated gi- and cholesterol levels. The fact that those cultures which ir-
ant endothelial cells have been harvested and cultured,40 a reversibly failed to grow had unproportionally increased lev-
finding quite similar to our own observation. Moreover, li- els of lipoprotein A might be an indication that this serum
poprotein A has previously been found to be an indepen- lipid has a particularly damaging effect on endothelial cells.
dent risk factor for atherosclerosis.41
Apart from hyperlipidemia, diabetes is another major Summary and Future Aspects
risk factor for pathological changes of the vasculature. On The successful cultivation of autologous endothelial
the cellular basis it has been shown that endothelial cells cells is endangered by both procedure dependent and pa-
cultured in the presence of high glucose levels have a re- tient dependent risk factors. Reasons for failing cultures due
duced proliferative capacity. Accelerated endothelial cell to procedure-related problems are mechanical damage dur-
death42 is believed to be due to facilitated apoptosis,43 but ing vein dissection, contaminations and inadequate reagents.
may also have osmotic causes. In this context it was interest- However, the most frequent problems with autologous en-
ing that we found higher glucose levels in the serum of pa- dothelial cell cultures are related to patient factors. Most of
tients with normally growing cultures than in sera of pa- the patients who have to undergo bypass surgery have a
tients whose cultures initially failed to grow. Therefore, multitude of major risk factors. Numerous clinical and ex-
moderately increased glucose does not seem to affect viabil- perimental studies have revealed smoking, diabetes and
ity and growth capacity of primary endothelial cell cultures. hyperlipidemia to be major independent risk factors for ath-
The third well established risk factor for the vascula- erosclerosis. The additive detrimental effect of these risk fac-
ture is smoking tobacco. Its negative influence on endothe- tors on the vasculature has been further documented.47-49
lial cells has previously been described.44 Particularly, a lower Our data seem to reflect these findings in vitro. We found
harvest efficiency and a suppressed reproductive capacity significantly different contents of cholesterol, triglycerides,
has been reported for autologous endothelial cell cultures lipoprotein A and glucose in the sera of patients with initial
from smokers.45 This is in accordance with our finding that growth failure as compared to sera of patients with normal
the mean number of harvested cells from smokers was sig- primary growth. However, by replacing the patient’s own
nificantly lower in smokers than nonsmokers. However, since serum as a culture supplement with pooled serum from
the subsequent proliferative behavior of endothelial cells did healthy young donors, the majority of cultures recovered,
not significantly differ, our observation supports the previ- regaining their normal morphological appearance and full
ous interpretation that a considerably thicker basement proliferative capacity. Thus, the success rate of autologous
membrane may be responsible for a stronger endothelial cell endothelial cells cultured for the endothelialization of syn-
attachment in smokers.46 thetic vascular grafts was increased from the previous
However, in contrast to other risk factors influencing 73-95%. This improvement, together with a further simpli-
the successful growth of autologous endothelial cells, the fication of the seeding process, makes in vitro
damaging effects of hyperlipidemia can be reversed. When endothelialization even more clinically practicable. The ben-
serum is changed from autologous to pooled homologous eficial long term results in peripheral revascularization and
serum in cultures with morphological signs of growth fail- the first promising data for clinically used endothelialized
ure, all cultures, with a few exceptions, regain their prolif- synthetic coronary bypass grafts50 provide encouraging ar-
erative capacity together with a typical morphological ap- guments for a wider application.
pearance (Figs. 15.2 and 15.3). It was also interesting to see
Fig. 15.2. Same culture as in Figure 15.1,
but 48 h after change from autologous
serum to low-lipid pooled homologous
serum. The vacuolation has almost com-
pletely disappeared.
Risk Factors for Autologous Endothelial Cell Cultures 167
Fig. 15.3. Double y-curve of p indexes
(squares) and m indexes (circles) of pri-
mary cultures with initial growth failure.
Serum change led to an increase in prolif-
erative capacity with a decrease of altered
morphological appearance at the same
time.
It is quite obvious and desirable that tissue engineer- 2. Graham LM, Burkel WE, Ford JW et al. Immediate seed-
ing is directing its effort towards fascinating goals like high ing of enzymatically derived endothelium in dacron vas-
tech, ready to implant grafts with features of a native artery. cular grafts. Arch Surg 1980; 115:289-94.
However, the fact that a substantial number of patients will 3. Zilla P, Fasol R, Deutsch M et al. Endothelial cell seeding
of polytetrafluoroethylene vascular grafts in humans: A
have an impaired endothelium, not able to regenerate or
preliminary report. J Vasc Surg 1987; 6:535-41.
fulfill necessary physiological functions, should be consid- 4. Meerbaum SO, Sharp WV, Schmidt SP. Lower extremity
ered when discussing these future approaches. Until the suc- revascularization with polytetrafluoroethylene grafts
cessful introduction of a fully engineered vascular prosthesis seeded with microvascular endothelial cells. In Zilla P,
which emulates native arteries structurally and functionally, Fasol R, Callow A, eds. Applied Cardiovascular biology
in vitro endothelialization will remain a feasible method of 1990-1991. Basel: Karger, 1992:107-119.
improving the performance of synthetic vascular grafts. 5. Williams SK, Rose DG, Jarrell BE. Microvascular endo-
Centers of excellence could provide endothelialized grafts thelial cell sodding of ePTFE vascular grafts: Improved
to a number of neighboring hospitals. This prospect is even patency and stability of the cellular lining. J Biomed Mat
more exciting in view of the possibility to genetically modify Res 1994; 28:203-212.
endothelial cells.51 Moreover, endothelialization could be 6. Pasic M, Müller-Glauser W, Odermatt B et al. Seeding
with omental cells prevents late intimal hyperplasia in
used as a generalized hemocompatible drug delivery system
small-diameter Dacron grafts. Circulation 1995;
which enables the creation of individual delivery schemes 92:2605-16.
for each single patient. Vascular grafts and endothelialization 7. Greisler HP, Cziperle DJ, Kim DU et al. Enhanced
could also be used for the development of various tissue endothelialization of expanded polytetrafluoroethylene
engineered organs. Different cell types, allogenic or grafts by fibroblast growth factor pretreatment. Surgery
xenogenic, could be seeded on the graft surface. These cells 1992; 112:244-54.
would be subsequently covered confluently by autologous 8. Örtenwall P, Wadenvik H, Risberg B. Reduced platelet
endothelial cells, in order to inhibit the immune response. deposition on seeded versus unseeded segments of ex-
Thus some fascinating prospectives still inhere panded polytetrafluoroethylene grafts: Clinical observa-
to endothelialization. tions after a 6-month follow-up. J Vasc Surg 1989;
10:374-380.
9. Örtenwal P, Wadenvik H, Kutti J. Endothelial cell seed-
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CHAPTER 16
Adhesion Molecule Expression Following In Vitro Lining
Caroline Gillis-Hægerstrand
Introduction
T he possibility that human ECs can be seeded on vascular prosthetic grafts to create a
“look-alike” to the natural blood vessel is intriguing. The new field of tissue engineering
has awakened an interest in many scientists from different disciplines which makes the re-
search area fascinating. Important breakthroughs have greatly enhanced our understanding
of the functions of the human endothelial cell. Today it is well accepted that the endothe-
lium is indeed a very active “organ”. The endothelium is crucial in maintenance of hemosta-
sis, coagulation and fibrinolysis, as well as in the regulation of vascular tonus, inflammation,
immune reactions, angiogenesis and vascular permeability.1-6
Long term patency of artificial vascular grafts for hemodialysis access and for bypass
or interposition in small caliber arteries is limited due to neointimal hyperplasia and associ-
ated graft thrombosis. If these grafts could be seeded with well functioning, adherent ECs
which are not activated and expresses adhesion molecules to which leukocytes can adhere,
these problems could partly be overcome. The same reasoning is valid for biological heart
valves.
In this chapter, I will try to cover aspects of how the endothelium interacts with circu-
lating leukocytes and how an endothelial lining of artificial, cell free materials may effect the
adherence of leukocytes.
Vascular Grafts
There are different kinds of grafts to be used for different purposes in vascular surgery.
Autologous Grafts
Autologous vessels are still the preferred vascular substitute. They are superior to all
other available alternatives, although it has been demonstrated that surgical preparation
partly denudes the vessels and damages the endothelium.7 A complete re-endothelialization
after implantation of these grafts has been shown.8 The obvious drawback to using autolo-
gous vessels is the limited supply. Another problem is intimal hyperplasia, which is thought
to be initiated when the endothelium is damaged during the operation. Hyperplasia is a
major reason for re-operation or repeated PTCA procedures after open heart surgery and
peripheral vascular surgery.
Prosthetic Grafts
One feature that distinctly distinguishes the cardiovascular prosthetic graft from its
natural counterpart is its lack of a confluent endothelium. The development of the ideal
prosthetic vascular graft has been one of the major goals of vascular surgery since the first
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
172 Tissue Engineering of Prosthetic Vascular Grafts
grafts were used over 40 years ago. The aim has been to Inflammation
achieve a completely endothelialized prosthetic graft where Inflammation is the reaction of living tissues to all
the cells not only remain when the blood flow is restored, forms of injury. It involves vascular, neurologic, humoral
but also retain their wide range of functions. Synthetic grafts and cellular responses at the site of injury. The adherence of
have acceptable patency rates when used in areas of high neutrophils and monocytes to the vascular endothelium is a
blood flow and good run-off to replace vessels of an inner hallmark of acute inflammation. At sites of inflammation,
diameter greater than 5-6 mm.9 When used in low flow ar- vascular ECs undergo functional and morphological changes
eas, the expectations regarding patency are not currently collectively referred to as endothelial activation. 19,20
being fulfilled. The two types of synthetic vascular grafts that Cytokines such as IL-121 and TNF-#22 induce functional
have gained the widest acceptance are Dacron and ePTFE. changes in human ECs in vitro, including increased adhe-
Glutaraldehyde-tanned homologous grafts are another al- siveness for leukocytes.23 In vivo models have also shown
ternative but their use is under debate. that cytokines induce changes corresponding to endothelial
activation when introduced to tissues.24 When stimulated
Dacron with histamine, thrombin or superoxides the ECs rapidly
The manufacturing process allows defined variation express P-selectin for approximately 20 min. When activated
of porosity. The pores are larger in Dacron grafts than in by cytokines such as IL-1 or TNF-#, the endothelium ex-
ePTFE grafts. After Dacron graft implantation, platelet ad- presses an array of inducible adhesion molecules, e.g.,
hesion and aggregation occurs, followed by fibrin forma- P-selectin, E-selectin, VCAM-1 and ICAM-1 (Fig. 16.1).
tion. Then there is a gradual ingrowth of fibroblasts and The improvement of cardiovascular prosthetic mate-
capillaries, and a deposition of matrix proteins. A fibrous rials, to reduce thrombus formation and inflammatory/im-
tissue is formed on the inner lining of the graft as capillaries munological reactions, is a technological as well as a bio-
grow from the exterior to the interior. logical challenge. By mediating adhesion and by expressing
adhesion molecules to which leukocytes adhere, ECs play a
ePTFE key role in inflammatory reactions. The process of leuko-
ePTFE grafts are made by controlled expansion of the cyte adherence, activation and migration involves an inter-
material by heating. The initial thrombus formation on the play between ECs, leukocyte activation and local cytokine
inner surface of ePTFE grafts when platelets adhere and ag- activity. An activated leukocyte is likely to adhere to the en-
gregate is similar to that in Dacron grafts. However, the dothelium even if the endothelium itself is expressing a “nor-
porosity is much less than that in Dacron grafts, which pre- mal” range of adhesion-promoting molecules on its surface.
vents ingrowth of fibrous tissue and capillaries. No or little A firm adhesion and transmigration into the subendothe-
spontaneous ingrowth of ECs from anastomoses occurs lial tissue requires an interplay between the leukocyte and
in man.10 the ECs, where activation and structural changes occur in
the endothelium. Although the neutrophil is considered
Glutaraldehyde-Tanned Grafts more reactive and has been most extensively studied in this
Glutaraldehyde treatment is implemented to reduce respect, the monocyte expresses a similar range of adhesion
antigenicity, sterilize the graft and stabilize the tissue by molecules as do neutrophils and respond to similar stimuli.
crosslinking of collagen.11 Two of the side effects of this treat- T lymphocytes are likely to play an important role in the
ment are that calcification of the tissue is promoted and a body´s reaction to a foreign material. Less is known about T
spontaneous ingrowth of ECs is prohibited.12,13 The hostile lymphocyte-endothelial interactions.
environment to any adhering or ingrowing cell type due to Endothelial cells express three families of adhesion
toxic residues of glutaraldehyde may delay an inflammatory/ molecules which interact with leukocytes: selectins, immu-
foreign body reaction to the graft. However, inflammatory noglobulins and integrins.
cells, macrophages and T cells in the tissue at explantation
have been described by several groups.14 Selectins
Selectins are cell surface glycoproteins found on the
Heart Valve Prostheses endothelium, leukocytes and platelets. Selectins binds to
Mechanical heart valves, or heart valve bioprostheses, carbohydrate rich domains on white blood cells. Platelet
similar to the native are frequently used in cardiac surgery. selectin (P-selectin) is found in granules of the ECs, known
Patients receiving the former, most often containing one or as Weibel-palade bodies. Upon stimulation with, e.g., throm-
more pyrolytic carbon disks attached to a sewing ring, need bin, P-selectin is rapidly mobilized to the cell surface of ECs
life long anticoagulant medication to prevent thromboem- as well as platelets.25 The expression is short lived, declining
bolic events, which is associated with a higher risk of bleed- substantially within a few minutes.
ing disorders. The degenerative process of a bioprosthetic Endothelial cell selectin (E-selectin) expression is
graft has a multifactorial etiology. Inflammatory,15 mechani- largely restricted to activated ECs.26 Cultured ECs express
cal,16 and metabolic processes17,18 as well as the glutaralde- E-selectin after exposure to interleukin-1 (IL-1), tumor ne-
hyde per se,12,13 have been discussed as contributing factors. crosis factor alpha (TNF-#) or lipopolysacharide (LPS).23,27
These bioprostheses are slowly deteriorated due a complex Maximal surface expression is observed at 4-6 h, followed
reaction involving stepwise calcification, resulting in poor by a decline. The basal levels are usually reached 24-48 h
hemodynamic performance. after stimulation.
Adhesion Molecule Expression Following In Vitro Lining 173
Fig. 16.1. Induction and peak expression of endothelial cell adhesion molecules. H2O2 = superoxide, IL = interleukin, TNF =
tumor necrosis factor, LPS = lipopolysaccharicde (endotoxin), IFN = interferon, ICAM = intercellular adhesion molecule, VCAM
= vascular cell adhesion molecule, MHC = major histomcompatibility complex. Please note that ICAM-2 and LFA-3 are constitu-
tively expressed and that MHC class I is both constitutively expressed and inducible.
Ig Superfamily surface of other cells, e.g., neutrophils and monocytes.36
Unlike E-selectin expression, the elevated expression CD31 binding is homophilic and is thought to participate
of the Ig superfamily is not transient but is, rather, sustained in the EC-EC interaction.37 CD31 antibodies have been
over several days. The Ig superfamily binds to integrins on shown to inhibit granulocyte transmigration, suggesting that
the surface of the white blood cells. Intercellular adhesion neutrophil-EC CD31 binding is important for the inflam-
molecule-1 (ICAM-1) is expressed on ECs after 8-12 h of matory process.38
stimulation with IL-1 or TNF-#.28 ICAM-2 expression on
ECs does not appear to be regulated by cytokines.29 Integrins
ICAM-3 is a ligand for the leukocyte integrins LFA-1 The integrins are heterodimers consisting of
(Cd11a/cd18) and #d!2.30,31 ICAM-3 is constitutively ex- noncovalently associated # and ! subunits which mediate
pressed at high levels by all resting leukocytes, such as mono- both cell-substratum and cell-cell adhesion. They are im-
cytes, lymphocytes and neutrophils, and antigen presenting portant in cell adhesion, platelet adhesion and cell migra-
cells. It shows a pattern clearly distinct from ICAM-1 and tion. Integrin ligands, e.g., RGD (arginine-glycine-aspartic
ICAM-2. ICAM-3 also seems to be inducible on vascular acid) sequences on extracellular matrix proteins, are crucial
endothelium in certain disease states, especially lymphomas for attachment of ECs to natural or other surfaces.
and myelomas.32 The ECs may actively participate in specific immune
Vascular cell adhesion molecule-1 (VCAM-1) is also reactions by expressing the ABO antigen system. They ex-
upregulated by stimulation of IL-1 or TNF-#, with maximal press major histocompatibility complex (MHC) anti-
activity reached after 8-12 h.33 The expression of immuno- gens.39,40 MHC class I is expressed constitutively by ECs and
globulin superfamily adhesion molecules occurs more slowly class II (HLA-DR) can be induced by interferon-&.41 Once
and is prolonged in comparison to E-selectin, as stated ear- ECs express the MHC class II antigen, they can be consid-
lier. The expression of these adhesion molecules on ECs char- ered to act as antigen presenting cells.
acterizes a later phase of inflammation, usually when leuko- Furthermore, activated neutrophils often release oxy-
cytes adhere to the vessel wall. In cardiac transplant rejec- gen free radicals and other short-lived substances that acti-
tion the correlation between endothelial expression of vate the endothelial cells to release platelt activating factor
VCAM-1 and infiltration of T lymphocytes is strong.34 Con- (PAF) and interleukin-8 (IL-8).42 Platelet selectin (P-selectin)
sequently, the ECs play an important role in transplant re- and PAF, coexpressed on the surfaces of activated ECs, pre-
jection. In the experimental situation, this function is em- sumably perpetuate the neutrophil-EC interaction.
phasized by the reduced rate of allograft rejection described
when the native endothelium was removed.35 Leukocyte Adhesion Under Flow Conditions
CD31 is another member of the immunoglobulin su- One of the most important aspects of leukocyte ex-
perfamily. It is expressed on the EC surface as well as on the travasation is that it is a multistep process. This process is
174 Tissue Engineering of Prosthetic Vascular Grafts
divided into several steps: initial contact, primary adhesion ied. The human monocytic cell line U937 was used for
(often referred to as rolling), activation, secondary adhesion adhesion studies. It was shown that monocyte adhesion to
and transmigration. Figure 16.2 shows schematically the nonendothelialized PA was significantly higher than adhe-
several steps involved in neutrophil extravasation in acute sion to endothelialized specimens on days 3 and 7. The spon-
inflammation. Following initial contact, leukocytes often roll taneous monocyte adhesion to EC-seeded porcine aorta was
slowly along the endothelium. Rolling is believed to keep highest 1 day after seeding, declined on day 3 and remained
the cells in close enough contact with the endothelium so low on the 7th day. When the endothelialized tissue was
that they can be stimulated by substances in the local envi- stimulated with IL-1 ! , the adhesion of monocytes
ronment and engage additional binding mechanisms.43 Roll- increased significantly.
ing requires a definite adhesive interaction, as was first dem- Human saphenous vein ECs (HSVECs) seeded on xe-
onstrated by the observation that neuotrophils roll much nogeneic vascular tissue were shown to express E-selectin,
more slowly than predicted for cells tumbling in the fluid ICAM-1, VCAM-1 and MHC class II in a time-dependent
stream adjacent to the vessel wall without any adhesion.44,45 manner. This expression pattern correlates well with the
Stimulated neutrophils adhere to the microvasculature. This adhesion of monocytes. It was found that the expression of
is referred to as firm adhesion. Once firm adhesion has been adhesion molecules and MHC class II was similar on viable
established, the leukocytes migrate to the intercellular junc- and nonviable tissue, as well as on ECs seeded on gelatin-
tions, where they can diapedese.46 coated culture plastic. However, it was also shown that non-
There is a great interest in in vitro endotheli- specific mouse IgG binding to the HSVECs was higher one
alization of cardiovascular prosthetic materials to achieve a day after seeding, although not as high as the adhesion mol-
confluent and flow-resistant vascular lining.14,47-51 But, to ecule expression. A significant increase in cellular adhesion
my knowledge, nothing has been published regarding ad- molecule expression and in MHC class II expression was
hesion molecule expression on EC surface when seeded on observed after stimulation with IL-1! or IFN-&. The non-
prosthetic grafts and exposed to shear stress. More has been specific binding was not affected after cytokine stimulation.
written about neutrophil-endothelial cell interactions. Although mechanical de-endothelialization is a relatively
Quantitative studies of neutrophil adhesion under flow con- imprecise technique, porcine aorta was used as a biological
ditions have shown that overall adhesion decreases with in- model tissue, mainly because of its availability. Furthermore,
creased flow and wall shear stress.52,44 It has been shown the matrix proteins exposed after scraping are reasonably
that the leukocyte adherence is optimal in the postcapillary representative of the matrix on a bioprosthetic graft.
venular wall, where it is about 1-4 dyn per cm2. It decreases The human monocytic cell line U937 is a relatively
rapidly at higher shear stresses. However, there are some re- immature clone derived from a human histiocytoma. How-
sults suggesting that integrin mediated adhesion in the ab- ever, the U937 cells present leukocyte surface integrins to
sence of selectins appears to be effective only at wall shear allow adequate attachment to ECs.56 To our knowledge, the
stresses well below 1 dyn cm2.53,54 expression of U937 integrins, and their role in adhesion to
subendothelial tissue, is not well characterized. However, it
Endothelialization Reduces Monocyte is likely that “normal” integrins are present on U937 and
Adhesion to Xenogeneic Tissue in a Time contribute to the increased binding to de-endothelialized
Dependent Manner collagenous tissue as compared to endothelialized tissue.57
Our group published a paper in 1995 wherein we The time dependent manner in which the adhesion mol-
investigated monocyte adhesion to de-endothelialized and ecule expression declines, which was found in this study, is
re-endothelialized porcine aorta (PA) before and after interesting for several reasons. It indicates that perhaps one
cytokine stimulation.55 In addition, the expression of a should wait 3-7 days before operating an endothelialized
selection of adhesion molecules on the EC surface before graft in a patient. This notion is supported by other find-
and after cytokine stimulation after 1, 3 and 7 days were stud- ings such as that the maturation of the cytoskeleton may be
Fig. 16.2. Schematic
illustration of adhesion
molecule dependent
leukocyte interaction
with endothelial cells.
Initial rolling is a carbo-
hydrate dependent
event, whereas sticking
and firm adhesion is de-
pendent on Ig-super-
family adhesion mol-
ecules on the endothe-
lium binding to integrins on the leukocyte. The diapedesis is at least partly dependent on the homologous binding of the autorecepetor
CD31 on both the endothelium and the leukocytes. See text.
Adhesion Molecule Expression Following In Vitro Lining 175
more pronounced after 7-9 days.58 Furthermore, it has been and organs for transplantation and various gene therapy
shown that ECs exposed to chronic shear stress in vitro, ap- techniques. Because of the integrated systems, and analysis
plied in a stepwise fashion over several days, are induced to required to understand intracellular functions and intercel-
become tightly adherent to the substratum and exhibit more lular interactions, this development should be done in close
differentiated features.59 Most likely this assay also gives a cooperation with researchers with training in biology
better cell retention in seeded grafts after the blood flow has and bioengineers.
been restored. The human body is a marvelous feat of engineering.
There was no difference in the expression of EC adhe- Many vital systems continuously interact with each other in
sion molecules in cells seeded on viable and nonviable tis- ways that are simultaneously extremely complex yet beauti-
sue specimens. This can be explained by the fact that no fully simple. Our understanding at the molecular level has
immunocompetent cells were present. Apparently, the vi- made major advances in the last decade. Because these in-
able smooth muscle cells, fibroblasts or matrix proteins teractions occur dynamically and are subject to various lev-
which were communicating with or exposed to the human els of control, much of our increase in knowledge has been
ECs, did not influence the ECs’ expression of adhesion mol- possible because of the awareness that real advances require
ecules in this model. cross-disciplinary teams of bioengineers and biological sci-
Interestingly, the high levels of monocyte adhesion and entists. This interaction has been and will be extremely ben-
nonspecific binding of antibodies on the first day after seed- eficial in vascular biology.
ing were also true for ECs seeded on gelatin. This may sug-
gest a nonspecfic activation of ECs by passaging. Acknowledgments
In conclusion, there are several findings suggesting that I wish to thank Associate Professor Anders
the endothelium is more ready to cope with and survive the Hægerstrand for fruitful comments on the manuscript. This
conditions in vivo if they are allowed to mature on the graft study was supported by grants from The Swedish Heart Lung
before transplantation. These findings include the adhesion Foundation, The Memorial Foundation of Carl Jeppsson and
molecule expression, at least in vitro. R & E Lundström.
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CHAPTER 17
In Vitro Endothelialization Elicits Tissue Remodeling
Emulating Native Artery Structures
Manfred Deutsch, Johann Meinhart, Peter Zilla
I n humans, the retrieval of samples from cardiovascular implants is a rare event. Therefore,
it is almost impossible to evaluate the long term healing pattern of cardiovascular pros-
theses. At the same time, animal experiments do not offer a satisfying solution either. The
significantly shorter length of the prostheses and an incomparably short implantation time
make transanastomotic tissue ingrowth their predominant healing mode. Therefore, there
is hardly an opportunity to histologically study blood surfaces of clinical implants which are
not affected by transanastomotic ingrowth. This is particularly frustrating in an exciting
field such as clinical in vitro endothelialization of arterial grafts, in which the patency results
are highly encouraging but the proof for the ongoing existence of functional intimal tissue
has only sporadically been provided.
When the techniques for in vitro lining were refined in the mid 1980s, the seeding
community was haunted by the fear of so called “contaminating” smooth muscle cells. With
the strong emphasis on intimal hyperplasia of those days, the smooth muscle cell was the
declared enemy. As a result, much of the research effort went into purification of endothelial
cell cultures.1,2
Preclinical primate experiments3 confirmed that a mature and functional endothe-
lium continued to form a confluent cell coverage after weeks of implantation. Moreover, the
opportunity to obtain a specimen of a clinically in vitro lined ePTFE graft also showed that
a single layer of pure endothelial cells confluently covered the protein precoated ePTFE sur-
face of the graft 5 weeks after implantation.4 Nevertheless, fears of fibrinolytic degradation
of the underlying matrix, with the concomitant detachment of the endothelium, were justi-
fied in view of the lack of long term specimens. Furthermore, in native arteries endothelial
cells closely interact with their underlying smooth muscle cells in an adaptive attempt to
standardize flow and shear stress conditions.5 Therefore, the presence of a monolayer rest-
ing almost directly on the synthetic graft structure seemed more and more unphysiological,
and perhaps even unsustainable. However, clinical long term studies increasingly indicated
that a functional endothelium continued to cover the graft surface, even after years of im-
plantation. Eventually, we got the chance to morphologically analyze a significant piece of
explanted graft 41 months after clinical in vitro lining. Since the healing pattern of a certain
prosthetic implant does not principally differ from case to case, the extrapolation of a case
report to a principal healing response seems justified, particularly in view of the fact that a
representative number of samples cannot be expected in the clinical setup.
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
180 Tissue Engineering of Prosthetic Vascular Grafts
Case Report6 endothelium was still confluent, but the cells were flatter,
A 69 year old man who had undergone a bilateral with better pronounced marginal overlapping.
femoro-popliteal bypass graft procedure with in vitro Forty-one months later at the time of revision, the
endothelialized ePTFE grafts was readmitted 41 months later angiographic appearance of the stenosed area resembled a
for graft revision. The original indication for an in vitro lined native arteriosclerotic lesion. A 21 cm long graft segment
graft was the fact that both saphenous veins had already been was subsequently replaced by an equally in vitro
taken due to previous coronary artery bypass grafting. The endothelialized piece of ePTFE graft. The distance of the
reason for readmission and revision of one graft was the affected segment to the anastomoses was approximately
recurrence of severe claudication and a deterioration of the 15 cm on both sides. On gross examination of the explant,
ankle-brachial index from 0.89-0.59. Furthermore, angiog- the inner surface of the entire specimen was smooth and
raphy demonstrated a significant stenosis in the midsegment gray-white, showing multiple yellow and white plaques again
of the affected graft. resembling native atherosclerosis. Histologically, the inner
The lining procedure with cultured autologous venous surface was completely covered by a single layer of flattened
endothelial cells has been extensively described elsewhere.7-10 cells which were strongly positive for factor VIII, CD31 and
Endothelial cells were harvested by the in situ no-touch tech- CD34 (Fig. 17.1). Between this intima and the graft, a con-
nique previously reported.1 Routine immunohistochemical tinuous tissue layer of 1.21 ± 0.19 mm thickness was de-
screening of the preseeding mass cultures with factor VIII monstrable (Fig. 17.2) showing all histological and immu-
confirmed the purity of endothelial cells. The precoating of nohistochemical criteria of an arterial tunica media. It con-
the ePTFE structure prior to lining was done with a tained numerous actin (Fig.17.3) and desmin-positive cells
fibrinolytically inhibited fibrin glue.3,4,6-10 On light micros- resembling smooth muscle cells. A well developed internal
copy of freshly lined grafts and of preimplantation controls, elastic membrane could regularly be found underneath a
a confluent endothelial cell layer rested on a homogenous collagen iv-positive basement membrane of the endothe-
and evenly covering protein matrix. The postseeding matu- lium (Fig. 17.4). In atheromatous areas the internal elastic
ration of the confluent endothelium on the fibrin matrix membrane was disintegrated to various degrees (Fig. 17.4).
did not result in a diminished thickness of this adhesion The outside of the graft was surrounded by a rim of
matrix (7.3 ∝m versus 7.0 ∝m). By scanning electron mi- histiocytes with multiple multinucleated giant cells. The
croscopy, the freshly lined graft surface showed a completely prosthesis itself did not show any capillary ingrowth. In ad-
covering, confluent endothelium at the end of the lining dition, multiple fatty streaks, fibrous plaques and athero-
procedure. Nine days later, at the time of implantation, the mas (indistinguishable from conventional atherosclerotic
Fig. 17.1. Confluent
monolayer of CD34
positive endothelial
cells resting on a well
developed, concen-
trically aligned neo-
media. The structure
of the expanded
PTFE graft is clearly
visible underneath
(APAP technique;
x200).
In Vitro Endothelialization Elicits Tissue Remodeling Emulating Native Artery Structures 181
Fig. 17.2. Transverse section of in vitro
endothelialized ePTFE graft 41 months
after implantation. Typical extraprosthetic
foreign body reaction on the outside along
the low porosity PTFE wrap of the pros-
thesis. The inside surface of the graft is cov-
ered by cell rich, regularly aligned tissue
(H.E.; x40).
Fig. 17.3. Immunohistochemical proof of
the presence of actin in the cells of the neo-
media. Negative staining of the cell mono-
layer at the surface (APAP technique;
x200).
lesions) could be seen. Only in these areas, KP-1 and PG-M1 in diameter. These protrusions were only partially covered
positive foamy macrophages were found (Fig. 17.5). A by intact endothelium, whose cells were polygonal rather
neovascularization of the intima by small capillaries growing than aligned with the blood stream (Fig. 17.7). In contrast
in from the luminal surface of the graft was only found in to the unaffected graft areas, highly polarized leukocytes were
fibrous plaques, but not in the unaffected parts of the graft. adherent. Occasionally, only lamellopodia of these leuko-
On scanning electron microscopy, the entire graft seg- cytes were visible, indicating subendothelial leukocyte mi-
ment was covered by a mature and confluent endothelium gration. Between these endothelial remnants the subendo-
whose cells were mostly elongated parallel to the bloodstream thelial matrix was openly exposed (Fig. 17.7).
(Fig. 17.6). These endothelial cells showed numerous mi- Ultrastructurally, transmission electron microscopy
crovilli and pronounced marginal overlapping. The entire demonstrated a well differentiated endothelium with nu-
surface was free of thrombotic adherences, either fibrin or merous Weibel-Pallade bodies in those areas which were not
platelets. Areas with macroscopically visible plaques were affected by arteriosclerotic changes (Fig. 17.8). Confluence
characterized by surface protrusions of approximately 1 mm was maintained through complex intercellular contacts. In
182 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 17.4. Border area between non-
diseased part of in vitro lined ePTFE graft
and atheromatous lesion. The well defined
internal elastic membrane (left) becomes
disintegrated (right), together with
increasing intimal thickening (Orcein;
x200).
Fig. 17.5. Intimal plaque protruding into the lu-
men of the in vitro endothelialized prosthesis.
CD68 positive foamy macrophages are only found
in this area, in the depth of the neo-media close to
the ePTFE graft (APAP technique; x200).
In Vitro Endothelialization Elicits Tissue Remodeling Emulating Native Artery Structures 183
Fig. 17.6. Typical scanning electron
microscopic appearance of the mature
endothelium which covered the entire
21 cm long graft segment. The elon-
gated endothelial cells show numerous
short microvilli and pronounced mar-
ginal overlapping (x4,000).
Fig.17.7. Surface morphology of a macro-
scopically visible atherosclerotic plaque.
The endothelium is partly denuded and
characterized by polygonal rather than lon-
gitudinally aligned cells. Occasionally, ad-
herent blood cells are seen on freely ex-
posed subendothelial matrix (x530).
immediate proximity to the endothelial cells a delicate and proximity of a plaque, these cells contained strikingly large
noncontinuous basal membrane was found. The endothe- lipid vacuoles (Fig. 17.10).
lium and its basal membrane rested on a dense network of
collagen fibrils. The subendothelial matrix was character- Conclusions
ized by mature myofibroblasts embedded in densely packed In vitro endothelialization of small diameter vascular
collagen strands (Fig. 17.9). Although these cells had well prostheses has significantly improved prosthetic graft pa-
differentiated peripheral actin filaments they also showed tency.9-12 Due to the difficulty in obtaining specimens from
characteristics of secretory cells: a well developed Golgi com- human implants, it could only be assumed that a persistent
plex and often a pronounced endoplasmic reticulum. In the endothelium was the underlying reason for this better
184 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 17.8. Transmission electron micro-
graph of unaffected part of in vitro
endothelialized graft. A well differentiated
endothelium rests on a collagen-rich sub-
endothelial matrix. The cytoplasm shows
numerous Weibel-Pallade bodies (0) and
normal organelles. Complex intercellular
contact (1) and incompletely visible basal
lamina ()) (TEM; x33,000).
Fig. 17.9. Mature myofibroblasts from the
depth of the neo-media with perinuclear or-
ganelles. These cells typically contain a well
differentiated actin filament system at the
periphery and are embedded in a collagen-
rich extracellular matrix (TEM; x47,000).
In Vitro Endothelialization Elicits Tissue Remodeling Emulating Native Artery Structures 185
Fig. 17.10. Myofibroblast from the ath-
erosclerotic plaque area with huge lipid
vacuoles in the center. Note the periph-
eral actin filament bundles, as well as the
membrane-bound dense bodies which
are characteristic of myofibroblasts
(TEM; x42,000).
performance. Although the short term integrity of the lined Coseeding During the Initial Endothelialization
endothelium has previously been proved in a 5 week ex- If the progenitor cells of the neo-media were seeded
plant,4 long term developments remained speculative. With together with the endothelial cells, they must have remained
the opportunity to investigate a 41 month implant we were undetectable during cultivation. Even if the assessment of
eventually able to substantiate the clinical success with mor- purity of endothelial cell cultures was based on phenotypic
phological evidence. We could show that in vitro lined grafts characteristics, connective tissue cells can usually be easily
maintain an intact endothelial coverage even after 3 1/2 years detected. Smooth muscle cells and fibroblasts lack the con-
of implantation. Unexpected, however, was the finding that tact inhibition of endothelial cells and therefore tend to over-
a neomedia had developed between the prosthesis and the grow each other in a typical elongated, spindle shape. Since
endothelium throughout the entire length of the graft. The our lining technique only uses first passage mass cultures,1
cells of this neomedia contained actin filaments and were endothelial cells do not show signs of phenotypic senes-
separated from the intima by a true internal elastic mem- cence.14 This renders them even more the typical cobble-
brane which distinguished them from a neointima. Since stone monolayer in their morphology. Furthermore, first
untreated ePTFE grafts do not develop a tissue layer on their passage mass cultures necessitate prolonged cultivation in
luminal side, it is evident that this neomedia was enabled or one and the same flask,1 which usually allows connective
caused by the in vitro lining procedure. Considering the fact tissue cells to develop their typical shape. Therefore, only a
that cell ingrowth on conventional grafts is limited to the very insignificant number of connective tissue cells might
immediate perianastomotic region,13 the most eminent have been inoculated. To achieve an evenly distributed neo-
question is: How did these neomedia cells get there? media with such a low inoculum in a graft of 50-60 cm
The three principal options are: length, cells would need to migrate over long distances. Thus,
1. Through coseeding during the initial endo- even if the neo-media originates from initially coseeded
thelialization process; smooth muscle cells, cell migration would remain a signifi-
2. Subendothelial migration from the adjacent artery; cant phenomenon. Alternatively, a higher inoculum might
or have been achieved by cells which barely differ phenotypi-
3. Colonization from the blood. cally from endothelial cells and were therefore not detected
in the initial culture. This would be feasible because the vas-
cular wall intima contains a stem cell population which is
186 Tissue Engineering of Prosthetic Vascular Grafts
epitheloid in culture but still represents a subpopulation of exactly this concern. In order to clarify this issue we have
smooth muscle cells. However, such a scenario would mean observed tissue development following mixed microvascu-
that two screening methods failed, the immunohistochemi- lar seeding over a long-term period.29 The major outcome
cal staining because of the small sample size and the pheno- of this study was the concurrence of two events: the matu-
typic because of the presence of a rare subpopulation of ration of the subendothelial cell matrix from fibroblastoid
smooth muscle cells. towards myocytoid and the accomplishment of an equilib-
rium of cell proliferation. After the long lasting disputes of
Subendothelial Migration from the Adjacent Artery the 1980s over the method of choice for actively achieving
If the progenitor cells of the neo-media were not graft endothelialization, it is exciting to see that two meth-
seeded but migrated from the anastomotic region, the ques- ods as diverse as in vitro lining and mixed microvascular
tion is: Why was this possible? For reasons not fully under- seeding eventually lead to an identical result: the healing of
stood yet, connective tissue ingrowth onto synthetic vascu- a prosthetic graft accompanied by initial tissue proliferation,
lar prostheses stops shortly after the anastomosis.13 One ex- cell differentiation and endothelialization. This realization
planation may lie in an inhibitory fibrin layer on the blood further underlines the current redefinition of standpoints
surface16 which gradually builds up. If, however, an endot- in prosthetic graft research. In contrast to previous beliefs,
helial cell population of the surface was possible through host tissue appears to be capable of physiologically incor-
apt transmural ingrowth before this change in biological porating synthetic grafts, provided it happens prior to a
surface environment, smooth muscle cells grow well on the buildup of adverse conditions.16 Therefore, the manipula-
same synthetic surface on which they ceased to proliferate tion of the chronology of events may well hold the key to
before. Therefore, it seems as if the presence of a PDGF- ingrowth facilitation. What seems unchallenged is the fact
producing endothelium17 makes the difference with regard that early endothelialization facilitates the subsequent
to the presence of a subendothelial smooth muscle cell popu- musculogenesis within the graft wall. All nonbiological as-
lation. Although quiescent endothelial cells barely produce pects like compliance or porosity—as important as they
any PDGF, a significant PDGF production occurs in regen- might be—were previously overemphasized16 although they
erating endothelium.18,19 In spite of continual integrity of will need to be an integral part of an entirely engineered
the lined endothelium we have seen a significant regenera- arterial graft. Nevertheless, it is quite possible that these
tion in the primate model.3 Others have also found a 8- to nonbiological aspects are the limiting factors of currently
10-fold increase in endothelial replication on ePTFE available methods of graft endothelialization: Both non-
prostheses20 accompanied by a significant production of compliance and low porosity might well be responsible for
growth factors.21 the lack of complete differentiation of myofibroblasts to
myocytes and for the early appearance of true arterioscle-
Colonization from the Blood rotic lesions in endothelialized prosthetic grafts. The most
Finally, as a third option, circulating multipotent stem striking observation, however, was that newly formed vessel
cells must be considered. All four cell types, fibroblasts, structures can emulate a native artery even to the extent that
myocytes, endothelial cells and blood borne cells, are em- they participate in the generalized arteriosclerotic disease
bryologically of the same origin. Since they represent vari- of the arterial tree.
ous differentiations of one common stem cell, transforma-
tion within this cell family appears to be a reasonable op- References
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scribed.22-24 Our finding that white blood cells were not only microgrid follow-up and low density plating provide first
adherent, but also clearly migrated underneath the endot- passage endothelial cell mass cultures for in vitro lining.
helium, might be explained not only with an inflammatory J Vasc Surg 1990; 12:180-9.
2. Haegerstrand A, Gillis C, Bengtsson L. Serial cultivation
reaction, but might well fit into the multipotent blood cell
of adult human endothelium from the great saphenous
theory, which is currently best suited to explain isolated is- vein. J Vasc Surg 1992; 16:280-285.
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Whichever theory will eventually explain the forma- Odell J, Heidinger C, Fasol R, Von Oppell U. In vitro
tion of a differentiated neo-media on a 55 cm long pros- lined endotheliaum: Initial integrity and ultrastructural
thetic graft, distinct migration and intermesenchymal trans- events. Surgery 1994; 16:524-534.
formation of cells appear to be pivotal events in this process. 4. Fischlein T, Zilla P, Meinhart J, Puschmann R, Vesely M,
Apart from explaining the origin of this neo-media, Eberl T, Balon R, Deutsch M. In vitro endotheliaization
another question seems to be of imminent practical impor- of a meso-systemic shunt—A clinical case report. J Vasc
tance: Does this neo-media formation represent a patho- Surg 1994; 19:549-554.
5. Kaufman BR, De Luca DJ, Folsom DL, Mansell SL,
logical process in the sense of a nonself-limiting hyperplas-
Gorman ML, Fox PL, Graham LM. Elevated platelet-de-
tic cell proliferation or rather a self-limiting mechanism of rived growth factor production by aortic grafts implanted
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cular seeding seems to provide an explanatory analogy. When 15:806-816.
Park et al28 presented a case report on a seeded human pros- 6. Deutsch M, Meinhart J, Vesely M, Fischlein T, Groscurth
thesis, an unusually thick subendothelial cell layer had raised P, Von Oppell U, Zilla P. In vitro endothelialization of
In Vitro Endothelialization Elicits Tissue Remodeling Emulating Native Artery Structures 187
expanded polytetrafluoroethylene grafts: A clinical case 18. Kraiss LW, Raines EW, Wilcox JN, Seifert RA, Barraett
report after 41 months of implantation. J Vasc Surg 1997; BT, Kirkman TR, Hart CE, Bowen-Pope DF, Ross R,
25:757-763. Clowes AW. Regional expression of the platelet-derived
7. Zilla P, Fasol R, Preiss P, Kadletz M, Deutsch M, Schima growth factor and its receptors in a primate graft model
H, Tsangaris S, Groscurth P. Use of fibrin glue as a sub- of vessel wall assembly. J Clin Invest 1993; 92:338-348.
strate for in vitro endothelialization of PTFE vascular 19. Golden MA, Au YPT, Kirkman TR, Wilcox JN, Raines
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8. Müller-Glauser W, Zilla P, Lachat M, Bisang B, Rieser F, activity and mRNA expression in healing vascular grafts
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CHAPTER 18
In Vitro Endothelialization of Synthetic Vascular Grafts
in Long Term Clinical Use
Manfred Deutsch, Teddy Fischlein, Johann Meinhart, Peter Zilla
Introduction
P rimary patency of synthetic vascular grafts varies between 30% and 55%, whereas it is
between 68% and 85% for autologous reversed saphenous vein grafts after 5 years of
implantation.1-3 Apart from technical errors and anastomotic intimal hyperplasia, surface
thrombogenicity appears to be a major reason for the limited performance of synthetic con-
duits. The tendency of surgical disciplines towards ready to use products led to efforts aim-
ing at improved synthetic materials, delaying the overdue adoption of biological principles.
When endothelial seeding was eventually introduced as a first attempt to overcome the prob-
lem of graft thrombogenicity with biological tools,4 an elaborate approach soon surren-
dered to the clinicians’ demand for a single staged procedure. Predictably, the first studies in
humans were controversial and mostly disappointing. The most likely reason for the failure
of these approaches was an insufficient seeding density which did not allow the formation
of a sufficient endothelial coverage. One way to overcome a low cell inoculum is by immedi-
ate harvesting of large numbers of cells like microvascular cells from adipose tissue.5-7 The
alternative method of actively facilitating graft endothelialization is in vitro lining with cul-
tured autologous endothelial cells.8-10 After the preclinical proof of this concept in the non-
human primate model,10 our group performed a randomized clinical study involving 49
patients between 1989 and 1991. Based on the encouraging three year results11 and on the
histological proof of an endothelium on a 5 week explant12 and a central graft segment after
41 months of implantation,13 we commenced a clinical program of routine implantation of
in vitro endothelialized grafts.
Laboratory Procedure
In a step by step approach, reliable culture techniques for autologous endothelial cells,8
as well as precoating14 and lining techniques15 which achieved shear stress resistance of the
endothelium,16 were developed. In brief, in vitro endothelialization is a two staged proce-
dure. It needs a laboratory stage before graft implantation in order to provide a high cell
number, sufficient for a complete cellular lining on the luminal graft surface. Therefore, a
small segment of a patient’s subdermal vein (mostly jugular or cephalic) is used as the cell
source and taken out under local anesthesia. After careful, touch-free dissection, the vessel is
cannulated in situ with two free-flow vessel cannulas with attached 3-way taps. In order to
remove remaining blood, the vein is flushed with culture medium in a pulsatile manner. It is
subsequently filled with a 0.1% solution of collagenase. The solution is kept under minor
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
190 Tissue Engineering of Prosthetic Vascular Grafts
pressure within the vein after closing the 3-way taps. Fol- cell-free areas. The whole in vitro procedure from vein exci-
lowing excision, the cannulated and collagenase-filled vein sion to graft implanation took 36.9 days in our first clinical
segment is transferred to the laboratory in a container filled study and could be reduced to 30.3 days in the second phase
with prewarmed phosphate buffered saline. An enzyme in- of clinical routine application. Since most procedures are
cubation time of 15 minutes with collagenase guarantees a elective, this time span appears to be tolerable for a great
maximal yield of endothelial cells without contaminating number of patients.
fibroblasts or smooth muscle cells. After centrifugation of
the cell suspension, the pellet is resuspended in complete Surgical Procedure and Clinical Follow Up
culture medium. Complete culture medium consists of Me- Patients were admitted to the study when their saphe-
dium 199 supplemented with preservative-free heparin, nous veins were unavailable, unsuitable or had to be spared
10 ng/ml bFGF, 50 ∝g/ml gentamycin and 20% autologous for other surgical procedures like aortocoronary bypass graft-
serum. For autologous serum preparation, 250 ml blood was ing. All patients received the same anti-aggregatory treat-
previously taken from each patient and allowed to clot over- ment, namely oral dipyridamole, 75 mg/day, and oral acetyl
night at 4°C in order to guarantee a maximum release of salicylic acid, 330 mg/day, beginning two days prior to im-
growth factors into the serum. Cells are subsequently plated plantation and continuing throughout the observation pe-
in 25 cm2 culture flasks. The culture medium is renewed riod. For endothelialized graft implantation, only minor
twice weekly by replacing half the culture medium with fresh changes were necessary as compared to standard surgical
complete medium. Having reached 90% confluency, primary procedure. Graft implantation was performed as previously
cultures are passaged to two 162 cm2 culture flasks. After described,18 with the exception that the culture medium was
having reached the required cell number, the autologuous kept inside the graft without clamping the prosthesis. Pa-
cells are seeded onto the graft surface by using a rotation tients were anesthetized with a continuous epidural block
device which facilitates an even cell distribution under con- using Bupivacaine-Hydrochloride. Thin-walled reinforced
trolled conditions (Fig. 18.1). However, prior to this, grafts PTFE (6 mm inner diameter) was used as graft material in
are preclotted with a special dilution of clinically approved both phases of implantation.
and fibrinolytically inhibited fibrin glue in order to provide All patients underwent a tight postoperative follow up.
an adequate matrix for endothelial cells. In our pilot clinical During the first year of our randomized clinical study we
study, we further pretreated grafts with virologically tested assessed the resistance of the endothelium by means of 111In-
fibronectin, to increase adhesion and shear stress resistance dium labeling. Patency was determined by clinical evalua-
of endothelial cells. However, in our second phase of clini- tion as well as by Duplex sonography. Angiography was rou-
cal routine application of in vitro endothelialization, no clini- tinely performed on an annual basis and in patients with
cally approved fibronectin was commercially available. In suspected graft thrombosis. This follow up also applied to
order to control the success of the seeding procedure, speci- clinical routine in vitro endothelialization, with the excep-
mens for scanning electron microscopy were taken imme- tion of 111Indium labeling of platelets.
diately after rotation and shortly before implantation
(Fig. 18.2). At the time of implantation, the majority of grafts Randomized Clinical Study
show a confluent endothelial cell lining. Only a few grafts Between June 1989 and December 1991, the clinical
occasionally show a preconfluent endothelium or even small benefit of graft endothelialization was evaluated in a ran-
Fig. 18.1. New rotation device
for quick in vitro
endothelialization of vascular
grafts, consisting of a motor
unit and a control unit. Grafts
are placed in sterile filter pro-
tected glass tubes and
mounted on the motor unit,
which can be easily placed in
standard CO2 incubators. Ro-
tation speed and time can be
regulated continuously.
In Vitro Endothelialization of Synthetic Vascular Grafts in Long Term Clinical Use 191
Fig. 18.2. Scanning electron micrograph of
an in vitro endothelialized graft surface
shortly before implantation. The whole
surface is covered confluently with endo-
thelial cells.
domized control study comprising 49 patients. After a 1:2 Clinical Routine Endothelialization
randomization pattern, 33 patients were assigned to the These favorable results encouraged us to transfer in
endothelialized group and 16 to the control group. Growth vitro endothelialization to clinical routine in vascular sur-
failure of cell cultures eliminated 9 patients of the gery. So far, 93 in vitro lined femoropopliteal bypass grafts
endothelialized group. The remaining 24 patients received have been implanted in 82 patients. The primary patency
27 endothelialized grafts. Patients were monitored for more rate for all endothelialized femoropopliteal reconstructions
than 7 years. 111Indium labeling revealed a significant re- was 68% after 4 years; this is equivalent to that of reversed
duction of surface thrombogenicity of lined grafts. Through- saphenous vein grafts. Above-knee procedures were done in
out the first year of phase 1 of our endothelialization pro- 71, and below-knee procedures in 22 cases. Analysis of pa-
gram, the difference in platelet activation index (PAI) be- tency clearly revealed that endothelialization especially ex-
tween the endothelialized group and the control was sig- erts its beneficial effect in the below-knee position. Primary
nificant (p < 0.05). Although it was most obvious 9 days patency was 61% in above-knee grafts but was 82% in be-
post implantation, the difference remained significant even low-knee grafts (Fig. 18.3). Secondary patency for all
after one year. In the endothelialized group the PAI (platelet femoropopliteal reconstructions was 94% and limb salvage
activation index) was -2.5 ± 9.4 at 9 days, -4.4 ± 13.5 at 3 rate was 99%, since only one limb had to be amputated
months -0.7 ± 7.1 at 6 months and 7.0 ± 15.2 at 12 months. within the entire observation period.
In contrast, the control group’s values were as high as 37.1 ± However, although the patency rate for in vitro lined
at 9 days, 27.8 ± 22.8 at 3 months, 37.2 ± 22.9 at 6 months ePTFE grafts was comparable to that of vein grafts during
and 29.0 ± 17.4 at 12 months. both phases of our clinical endothelialization program, the
After 3 years a Kaplan-meier analysis showed a pri- primary patency rate of phase 2 was lower than that of phase
mary patency rate of 84.7% for endothelialized grafts and 1. This difference may have several causes:
55.4% for control grafts. After 5 years, it was 73.8% for the 1. Phase 1—as an initial randomized trial—included
endothelialized group and 31.5% for controls.11 After 7 years, considerably fewer patients than phase 2;
primary patency for control grafts dropped to zero, whereas 2. The follow up time of phase 2 was shorter and im-
that for endothelialized grafts remained high, at 73.8%.18 plantations were conducted as clinical routine pro-
One patient who underwent bilateral in vitro endothelialized cedures, including substantially more redo opera-
grafting had a unilateral stenosis after 41 months. We de- tions than phase 1;
cided to remove the stenosed portion of the graft and to 3. Grafts of phase 2 had not been precoated with
implant a newly endothelialized graft. This explant proved fibronectin, a potent adhesion molecule, due to the
the persistence of the transplanted endothelium. Except for unavailability of a clinically approved product. Al-
the immediate atherosclerotic plaque area, a confluent en- though the fibrin glue used for precoating contains
dothelium covered the entire length of the 21 cm long speci- fibronectin and endothelial cells deposit their own
men. Endothelial cells were mostly elongated and orientated fibronectin-rich extracellular matrix with time, ini-
parallel to the bloodstream. They were situated on a tial cell adherence may well have been diminished
continuos tissue layer, which showed many morphologic with a lower density of binding sites. When HFN-
criteria of an arterial tunica media13 (see also chapter 17). pretreated grafts were compared with untreated ones,
192 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 18.3. Patency rate of routinely
endothelialized femoropopliteal grafts ac-
cording to the distal anastomoses. Below-
knee grafts (n=22; top line) have a signifi-
cantly higher patency rate than above-knee
grafts (n=71; bottom line) after 4 years of
implantation.
the difference was indistinct (77.8% versus 72.6%; clinical tool and the motivation for intensified further at-
p = 0.3). However, this difference was more pro- tempts searching for an equally effective or even better prod-
nounced when above-knee grafts were compared uct from the shelf.
(80.0% versus 68.9%, p = 0.17) after 3.5 years of im- With this goal in mind, we were one of the few groups
plantation. Although the differences between which did not abandon the idea of surface endothelialization
fibronectin pretreated or nonpretreated grafts were of vascular grafts in the mid-1980s. We decided to rectify
not statistically significant (which might be due to procedural weaknesses before declaring endothelial cell
the different number of observed grafts), the curve transplantation inefficient. Since a low cell inoculum and
may suggest that nonfibronectin treated grafts have an immature cytoskeleton seemed to be the main culprits
a higher failure risk within the first year of implan- for the failure of single-staged venous seeding, we elected to
tation, a fact probably especially true in the above- defy the surgical aversion towards cell culture procedures
knee position with its high flow, high shear stress by confluently in vitro endothelializing prosthetic grafts with
condition. As a solution, pretreatment of the pro- autologous mass cultures of endothelial cells.
tein matrix with synthetic or recombinant RGD Eight years of experience with clinical in vitro endo-
(arginine-glycine-aspartic acid)-containing mol- thelialization not only provided us with patency data which
ecules could be considered. eventually verified the assumption that surface endo-
Our overall 7 year experience with endothelialized thelialization improves the performance of synthetic grafts.
femoropopliteal ePTFE grafts shows a patency of 66.0%. It also helped us to diminish three major concerns linked to
When pretreated with HFN the seven year patency was in vitro lining: infection, failure rate of cultures and the proof
72.1%. In the above-knee group the 7 year patency was even of a persisting endothelium after graft implantation. In 120
as high as 75.8%20 (Fig. 18.4). in vitro endothelialized ePTFE grafts which have been im-
planted altogether since the start of our in vitro endo-
Conclusion thelialization program, only one graft showed late infection
At the end of this century, biological awareness is in a patient with IDDM and lymph fistula. This graft needed
steadily rising among a new generation of cardiovascular to be removed and amputation could be avoided. However,
surgeons. Today’s research no longer focuses solely on sur- routine microbiological examination of this graft has been
face endothelialization, but rather on the understanding of performed before implantation and was found to be nega-
complex events that might eventually lead to real graft heal- tive for infections. One lined graft which was diagnosed to
ing. Nevertheless, biological events facilitating spontaneous be contaminated during the cultivation period was discarded
graft healing are not yet fully understood and it might take and remained the only occurrence of its kind. The analysis
many more years until such a prosthesis is available for clini- of risk factors for culture conditions enabled us to bring the
cal implantation. Until then, an active method of graft failure rate for autologous endothelial cell cultures down
endothelialization which was shown to significantly improve from 27.3-5% (see chapter 15). Considering the fact that
patency rates could provide us with both an indispensable this two staged procedure provides 93% of patients with an
In Vitro Endothelialization of Synthetic Vascular Grafts in Long Term Clinical Use 193
Fig. 18.4. Kaplan-Meier survivorship
function for patency rates of all
endothelialized above-knee femoro-
popliteal grafts grouped according to
fibronectin pretreatment. After 7
years, the patency rate was 75.8% for
the fibronectin pretreated grafts
(n=36).
arterial prosthesis which equals a vein graft, and that the 6. Jarrell B, Williams S, Stokes G, Hubbard A, Carabasi A,
remaining 7% end up with the prosthesis which they would Koolpe E, Greener D, Pratt K, Moritz M, Radomski J,
have had without this procedure, one can accept the low Speicher L. Use of freshly isolated capillary endothelial
probability of growth failure without ethical concerns. Fi- cells for the immediate establishment of a monolayer on
a vascular graft at surgery. Surgery 1986; 100:392-399.
nally, the proof of the existence of a confluently covering
7. Baitell-Eberle G, Groscurth P, Zilla P, Lachat M, Müller-
endothelium 5 weeks and 41 months after in vitro lining Glauser W, Schneider J, Neudecker A, von Segesser L,
dissipated concerns regarding the continual presence of an Dardel E, Turina M. Long term results of tissue develop-
endothelium after implantation. In summary, eight years of ment and cell differentiation on dacron prostheses seeded
clinical in vitro endothelialization were able to prove two with microvascular cells in dogs. 1993; 18:1019-1028.
main hypotheses: Autologous endothelial cell lining signifi- 8. Zilla P, Fasol R, Dudeck U, Siedler S, Preiss P, Fischlein
cantly improves the patency of prosthetic small diameter T, Müller-Glauser W, Baitella G,Sanan D, Odell J, Reichart
vascular grafts, and a cell culture-dependent procedure can B. In situ cannulation, microgrid follow-up and low den-
be carried over into clinical routine. sity plating provide first passage endothelial cell mass cul-
tures for in vitro lining. Vasc Surg 1990; 12:180-189.
References 9. Zilla P, Fasol R, Preiss P, Kadletz M, Deutsch M, Schima
1. Veith FJ, Gupta SK, Ascer E et al. Six year prospective H, Tsangaris S, Groscurth P. Use of fibrin glue as a sub-
multicenter randomized comparison of autologous saphe- strate for in vitro endothelialization of PTFE vascular
nous vein and expanded polytetrafluoroethylene grafts in grafts. Surgery 1989; 105:515-522.
infrainguinal arterial reconstructions. J Vasc Surg 1986; 10. Zilla P, Preiss P, Groscurth P, Rösemeier F, Deutsch M,
3:104-14. Odell J, Heidinger C, Fasol R, von Oppell U. In vitro-
2. Cacciatore R, Inderbitzi R, Stirnemann P. Five years ex- lined endothelium: Initial integrity and ultrastructural
perience with infra-inguinal arterial reconstructions: A events. Surgery 1994; 116:524-534.
comparison of venous with PTFE bypass. VASA 1992; 11. Zilla P, Deutsch M, Meinhart J, Puschmann R, Eberl T,
21:171-176. Minar E, Dudczak R, Lugmaier H, Schmidt P, Noscian I,
3. Quiñones-Baldrich WJ, Prego AA, Ucelay-Gomez R, Fischlein T. Clinical in vitro endothelialization of
Freischlag JA, Ahn SS, Baker JD, Machleder HI, Moor WS. femoropopliteal bypass graft: An actuarial follow-up over
Long-term results of infrainguinal revascularization with three years. Vasc Surg 1994; 19:540-548.
polytetrafluorethylene: A ten-year experience. J Vasc Surg 12. Fischlein T, Zilla P, Meinhart J, Puschmann R, Vesely M,
1992; 16:209-217. Eberl T, Balon R, Deutsch M. In vitro endothelialization
4. Herring M, Gardner A, Glover J. A single-staged technique of a mesosystemic shunt: A clinical case report. Vasc Surg
for seeding vascular grafts with autogenous endothelium. 1994; 19:549-554.
Surgery 1978; 84:498-504. 13. Deutsch M, Meinhart J, Vesely M, Groscurth P, Von
5. Vici M, Pasquinelli G, Preda P, Martinelli G, Gibellini D, Oppell U, Zilla P. In vitro endothelialization of ePTFE
Freyrie A, Curti T, D’Addato M. Electron microscopic and grafts: A clinical case report after 41 months of implanta-
immunocytochemical profiles of human sucutaneous fat tion. Vasc Surg 1997; 25:757-763.
tissue microvascular endothelial cells. Ann Vasc Surg 1993; 14. Kaehler J, Zilla P, Fasol R, Deutsch M, Kadletz M.
7:541-548. Precoating substrate and surface configuration determine
194 Tissue Engineering of Prosthetic Vascular Grafts
adherence and spreading of seeded endothelial cells on 17. Zilla P, Fasol R, Deutsch M, Fischlein T, Minar E,
polytetrafluoroethylene grafts. J Vasc Surg 1989; 9:535-541. Hammerle A, Krupicka O, Kadletz M. Endothelial cell
15. Zilla P, Fasol R, Preiss P, Kadletz M, Deutsch M, Schima seeding of polytetrafluoroethylene vascular grafts in hu-
H, Tsangaris S, Groscurth P. Use of fibrin glue as a sub- mans: A preliminary report. J Vasc Surg 1987; 6:535-541.
strate for in vitro endothelialization of PTFE vascular 18. Meinhart J, Deutsch M, Zilla P. Eight years of clinical
grafts. Surgery 1989; 105:515-522. endothelial cell transplantation. Closing the gap between
16. Franke RP, Graefe M, Schnittler H, Seiffge D, Mittermayer prosthetic grafts and vein grafts. ASAIO J 1997;
C. Induction of human vascular endothelial stress fibres 43:M515-M521.
by fluid shear stress. Nature 1984; 307:648-649.
PART III
Biointeractive Prostheses: Complete Healing
Biological Components
Taming of Adverse Responses
Prevention of the Inflammatory Reaction
CHAPTER 19
Inflammatory Reaction: The Nemesis of Implants
James M. Anderson
Introduction
N emesis is the Greek goddess of retributive justice or vengeance. Thus, the term “nemesis”
has been used to identify one that inflicts retribution or vengeance. Alternatively, nem-
esis has been defined as a source of harm or destruction. Following the implantation of a
medical device, biomaterial or prosthesis, the inflammatory reaction is the first host defense
system which interacts with the medical device, biomaterial or prosthesis, and may be the
source of harm or destruction to the implant or may result in untoward inflammatory and
healing responses which lead to failure of the device in its intended function. In considering
the inflammatory response to prosthetic vascular grafts, the inflammatory response is a com-
plex series of cellular and humoral interactions which are time-dependent and lead to the
healing response. Given these perspectives, the purpose of this chapter is to review the in-
flammatory response to prosthetic vascular grafts and provide a current state of the art
perspective on the inflammatory response to prosthetic vascular grafts and the potential for
inflammatory response interactions with tissue-engineered prosthetic vascular grafts.
Inflammation and the Healing Response
Inflammation is generally defined as the reaction of vascularized living tissue to local
injury.1-3 With porous vascular grafts, i.e., Dacron or ePTFE, a dual perspective of the injury
which initiates the inflammatory and wound healing responses must be considered. The
surgical procedure itself causes local injury to vascularized living tissue, and inflammation
is initiated by injury to soft tissue or muscle. This form of injury produces a response to the
outer or periadventitial surface of the vascular graft. The second response which initiates
injury is the blood-material interaction which follows implantation and re-establishment of
blood flow of the vascular graft. Blood-material interactions and the inflammatory response
are intimately linked and, in fact, early responses to injury involve mainly blood and the
vasculature. The interaction of blood and its components with the luminal surface of vascu-
lar grafts must also be considered as an initiator of the inflammatory response. Some types
of porous vascular grafts with relatively high porosity must be rendered impermeable prior
to implantation by a process called preclotting. Preclotting of vascular grafts with blood and
its humoral and cellular components can be expected to initiate the inflammatory response
to the prosthesis immediately prior to surgical implantation of the prosthesis.
Following the implantation of a vascular graft, a series of local events occur. These
local events (Table 19.1) are described as the tissue response continuum or, more commonly,
the inflammatory and healing responses. In the 1950s and ’60s, woven, knitted and velour
types of fabrics of various polymers were evaluated or utilized as vascular grafts. Initially,
vascular grafts were conceptually considered to be scaffolds over which the vasculature would
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
198 Tissue Engineering of Prosthetic Vascular Grafts
recapitulate itself, with the formation of intimal, medial and While little is known regarding the acute and chronic
adventitial components of the vessel wall and the inflammatory responses to the luminal surfaces of vascular
reestablishment of an endothelial lining which separated grafts, some understanding of the series of events following
blood from vascular wall components with their attendant injury in the inflammatory and wound healing responses to
adverse blood-tissue interactions.4,5 This concept has not the porous periadventitial surface of vascular grafts is ap-
been realized in humans to date, although use of highly po- preciated. Acute inflammation is of relatively short dura-
rous ePTFE vascular grafts have produced endothelial lin- tion, lasting from minutes to days, and is characterized by
ings in baboons. In lower species, i.e., rabbits, pigs, dogs and the exudation fluid and plasma proteins and the immigra-
others, the re-endothelialization process does occur on an tion of leukocytes, predominantly neutrophils. Neutrophils
inner capsule (pseudointima) and thus constitutes a are short lived and disintegrate and disappear after 24-48 h,
neointima in these species. depending on the form and topography of the vascular graft.
Immediately following injury, changes occur in vas- Highly porous prostheses may have a longer lived neutro-
cular flow, caliber and permeability. Fluid, proteins and blood phil response, as these cells migrate into the porous body of
cells escape from the vascular system into the injured tissue the vascular graft. This is especially true with loosely knit-
in a process called exudation. Following changes in the vas- ted and velour Dacron vascular grafts and has not been a
cular system, which also include changes induced in blood prominent observation with ePTFE grafts.
and its components, cellular events occur and characterize The accumulation of leukocytes, in particular neutro-
the inflammatory response. Although injury initiates the phils and monocytes, is the most important feature of the
inflammatory response, chemicals and agents released from inflammatory reaction. Leukocytes accumulate through a
plasma, cells and injured tissue, as well as blood, mediate series of processes including margination, adhesion,
the response to vascular grafts. integration, phagocytosis, and extracellular release of
Figure 19.1 illustrates the temporal variation in the leukocyte products. Leukocyte immigration is controlled in
tissue response continuum, i.e., the acute inflammatory re- part by chemotaxis, which is the unidirectional migration
sponse, chronic inflammatory response, granulation tissue of cells along the chemical gradient. A wide variety of exog-
development and foreign body reaction to implanted vas- enous and endogenous substances have been identified as
cular grafts. The intensity and time variables are dependent chemotactic agents. Important to the immigration is move-
upon the extent of injury created in the implantation and ment of leukocytes, as is the presence of specific receptors
the size, shape and topography, and chemical and physical for chemotactic agents on the cell membranes of leukocytes.
properties of the biomaterial as well as the porosity of the These and other receptors may also play a role in the activa-
vascular graft. tion of leukocytes.
Blood protein adsorption to the luminal,
periadventitial and interstitial surfaces of porous vascular
graft biomaterials is a significant early response which is
Table 19.1. Sequence of local events following poorly understood. The protein adsorption phenomenon
implantation occurs prior to any cellular interaction. Prior to the immi-
gration of leukocytes to and within the vascular graft, blood
Injury protein has coated the surfaces of the graft material and thus
Acute inflammation presents a protein-modified surface to cellular interactions.
Chronic inflammation
The physical and chemical characteristics of the vascular
Granulation tissue
Foreign body reaction graft material itself may play little role in subsequent cellu-
Fibrosis lar and humoral responses. Obviously, preclotting of the
Fig. 19.1. The temporal variation in the
tissue response continuum.
Inflammatory Reaction: The Nemesis of Implants 199
vascular graft accomplishes the protein precoating in an ex- studies with human blood and vascular grafts. Heparin was
cessive fashion.6 used as the anticoagulant for these studies; this anticoagu-
To better understand the early blood protein adsorp- lant permits complement activation with subsequent leu-
tion onto vascular graft materials, we have utilized a surface kocyte activation. The utilization of citrate, EDTA or EGTA
radioimmunoassy technique and an immunogold labeling will deplete or bind calcium ions significantly, thus inhibiting
technique with scanning electron microscopy to character- complement activation by the calcium-dependent pathway.
ize human blood protein adsorption onto Dacron and ePTFE With highly porous grafts, i.e., knits and velours, acute
vascular grafts in an in vitro human blood recirculation sys- and chronic inflammatory cell infiltration into the interstices
tem.7-10 The detection of the various proteins deposited on of the vascular grafts occurs. The time dependent nature of
the surfaces of the Dacron, ePTFE and control materials fell these responses and the extent of their duration within the
into two groups: Fibrinogen, IgG and albumin were found interstices of the vascular grafts are dependent upon the
in the highest amounts; Hageman factor (factor XII), porosity and structure of the vascular grafts. As described
factor VIII/vWF, fibronectin and hemoglobin were found earlier, preclotting of Dacron vascular grafts provides a bar-
in moderate amounts. Of the proteins assayed, IgG showed rier to this response and little is known regarding inflam-
the greatest degree of adsorption and affinity to all of the matory cell infiltration into preclotted vascular grafts.
materials tested and particularly to Dacron and ePTFE. Af- In the early 1980s, Meadox Medicals provided a col-
ter exposure of the tested materials for five minutes, IgG ac- lagen coated Dacron vascular graft, Hemashield with
counted for approximately 25% of the radioactivity bound Microvel, in which a thin layer of collagen was present on
to Dacron and to ePTFE. In the recirculation system, sig- the luminal and adventitial surfaces as well as within the
nificant protein adsorption had occurred within 5 minutes interstices of the vascular graft. This simple concept, origi-
of blood contact and did not significantly increase out to nally considered to replace preclotting of the Dacron vascu-
180 minutes. Fibrinogen adsorption to Dacron and ePTFE lar grafts, also had a significant inhibition of the inflamma-
decreased as the time of exposure to blood increased. tory cell infiltrate in the early response. The collagen coat-
Hageman factor (factor XII) significantly decreased on ing converted a highly porous material into a nonporous
Dacron as the time of exposure increased, but its adsorption material which developed its porosity in vivo by the bio-
to other materials, i.e., ePTFE and silica-free PDMS, degradation of the collagen coating. The biodegradation of
remained constant over the 180 minute time period. the collagen coating occurs within 4-12 weeks, and thus po-
Adsorption of IgG to Dacron decreased with time, but IgG rosity develops after the inflammatory responses and dur-
adsorption to ePTFE and PDMS did not change over ing the granulation tissue and fibrosis phases of the healing
180 minutes. response. As the porosity develops, macrophages and fibro-
These studies demonstrate that early adsorption of blasts present at the graft/tissue interface migrate into the
blood proteins which are adhesion receptor ligands vascular graft to produce the foreign body reaction and fi-
(fibronectin, fibrinogen), opsonins which can facilitate brosis without having to contend with the detritus of the
complement activation (IgG), and significant coagulation inflammatory reaction.12 The collagen coated Dacron grafts
cascade proteins (Hageman factor) adsorb quickly and are an early example of vascular graft tissue engineering.
readily to the luminal surfaces, where they may participate
in subsequent inflammatory, coagulation and throm- Foreign Body Reaction
botic processes. The foreign body reaction consisting of macrophages
Our human blood recirculation system has been also and foreign body giant cells develops at the periadventitial
utilized to investigate vascular graft-associated complement tissue-material interface coincidentally with the development
activation and leukocyte adhesion.11 Materials tested were of granulation tissue, with its attendant fibroblast prolifera-
ePTFE, crimped Dacron Bionit (C.R. Bard) and preclotted tion and neovascularization, i.e., capillary formation. At the
Dacron Bionit. A decrease in the total blood leukocyte con- same time, smooth muscle cells and fibroblasts from the wall
centration with perfusion time was seen for all materials of the native artery migrate and proliferate along the sur-
tested, and increasing leukocyte adhesion to the graft sur- face of the vascular graft to produce a pseudointima. En-
face was observed by scanning electron microscopy. The most dothelial cells also migrate and proliferate from the native
dramatic decrease in leukocyte concentration was observed artery at the anastomosis, but in humans their migration
for the interaction of heparinized whole blood with Dacron. into the vascular graft is limited in extent as compared to
This was due to the selective decrease in neutrophils and animal models where endothelialization is extensive. This
monocytes, and was correlated with an increase in both leu- lack of endothelialization of the pseudointimal surface of
kocyte adhesiveness and complement activation, as mea- the vascular graft in humans has remained a challenge and a
sured by C5a elevation. Minimal complement activation or profound problem with human vascular grafts. Of special
leukocyte adhesion was observed for ePTFE and the silicone note is the apparent lack of inflammatory cell interaction at
rubber control. Interestingly, Dacron Bionit exhibited the the luminal surface of human vascular grafts.13 Given the
highest C5a levels when compared to Dacron Bionit which potential for complement activation and leukocyte adhe-
had been preclotted in whole blood. Dacron Bionit sion, one might have expected early monocyte adhesion and
preclotted in platelet-poor plasma gave C5a levels which were differentiation into macrophages which can fuse into for-
slightly greater than ePTFE which was comparable to the eign body giant cells at the luminal surfaces of vascular graft
silicone rubber control. These studies demonstrated the sig- materials. Studies of retrieved human vascular grafts, how-
nificance of utilizing an appropriate anticoagulant in in vitro ever, suggest that this sequence of events does not occur, as
200 Tissue Engineering of Prosthetic Vascular Grafts
foreign body giant cells are not identified at the material- cretion of extracellular matrix materials, and endothelial cell
blood, material-blood protein or material-pseudointimal proliferation, which can affect the hemostatic, thrombotic
interface on the luminal surface of ePTFE vascular grafts. balance, and smooth muscle cell proliferation which may
With Dacron, FBGCs at the luminal surface of the graft participate in anastomotic or pseudointimal hyperplasia.
material are seen following the healing response and This hypothesis developed from two aspects of our research.
neovascularization within the graft interstices. Studies in our First, the consistent and all-inclusive finding that macro-
laboratory have suggested that leukocyte adhesion is depen- phages and foreign body giant cells were present at the in-
dent on both complement C3 and fibronectin deposition, terface of clinically retrieved human vascular grafts regard-
as well as being sensitive to the levels of shear stress in flow- less of implant time pointed toward the importance of the
ing blood.14 High levels of shear stress and/or turbulence, as macrophage and foreign body giant cells in tissue-material
well as low levels of complement C3 and fibronectin depo- interactions. Secondly, our in vivo cage implant studies with
sition, may result in minimal to no leukocyte adhesion, and a wide variety of vascular graft materials, including polyether
thus macrophage development and foreign body giant cell polyurethanes, demonstrated the early—as soon as three
formation at the luminal surfaces is limited. days—adhesion of monocytes/macrophages on the surfaces
The histological evaluation of retrieved human vas- of the materials.
cular grafts shows the presence of macrophages and foreign The second area of research was the first demonstra-
body giant cells interacting with the prosthetic material tion that adherent activated macrophages and foreign body
within the graft interstices and at the periadventitial sur- giant cells on polyether polyurethane surfaces were respon-
face. The foreign body reaction with macrophages and for- sible for the biodegradation of these materials.17,18 This find-
eign body giant cells has been demonstrated as early as sev- ing explained the earlier clinical studies of polyurethane deg-
eral weeks following implantation. In vascular grafts which radation in cardiac pacemaker leads, and also explains the
have been implanted for over two decades, these cells are biodegradation of polyether polyurethanes when they are
still present at the tissue-material interface. As the foreign utilized as vascular graft materials. Numerous academic and
body reaction appears to be virtually an all-inclusive inter- industrial scientists have attempted to utilize polyether poly-
action at soft tissue-material interfaces, we have focused our urethanes as porous and nonporous vascular grafts, but long
attention on the potential roles played by macrophages and term implant studies indicate biodegradation with attendant
foreign body giant cells not only in modulating the wound dilatation of the vascular graft or loss of integrity of the wall
healing response but also their potential participation in fail- of the polyether polyurethane vascular graft. These studies,
ure mechanisms of human vascular grafts, i.e., anastomotic in part, have led to the development of more stable polyure-
hyperplasia and stenosing pseudointimal hyperplasia. thanes by the incorporation of more efficient antioxidants,
In 1984, we began to address the hypothesis that ad- substitution of the degradable polyether soft segments by
herent macrophages which were activated released chemi- more stable soft segment prepolymers such as
cal mediators, in particular cytokines, which in an autocrine, polydimethylsiloxane or polycarbonates, and the chemical
paracrine and endocrine fashion modulated the formation modification of polyether polyurethanes by so-called
of the foreign body reaction at the tissue-material interface “end-capping,” where more stable moieties are bonded to
and the development of the fibrous capsule.3,15,16 Figure 19.2 the chain ends to produce polymers which are inherently
illustrates this early concept, in which activated macrophages less biodegradable.19
on protein-coated biomedical polymer surfaces produce and These new materials are in the early stages of
secrete various cytokines and growth factors which can development and testing for application as vascular graft ma-
modulate platelet activity, fibroblast proliferation and se- terials, and it is anticipated that the desirable elastomeric
Fig. 19.2. The tissue/implant inter-
face with protein adsorption,
macrophage adhesion and activa-
tion, cytokine and growth factor
production, and cellular synthesis
and proliferation.
Inflammatory Reaction: The Nemesis of Implants 201
and biocompatible properties of the polyurethanes will prove more indicative of the inflammatory reaction at the tissue/
to be efficacious and not undergo inflammatory material interface. Studies under these conditions demon-
cell-mediated biodegradation when utilized in various strated that Dacron and polyethylene exhibited a high reac-
vascular applications. tivity as measured by IL-1 release, ePTFE was intermediate
and Biomer and polydimethylsiloxane were low in their abili-
Macrophage Motility, Adhesion and Activation ties to activate macrophages and release interleukin-1.24 To
The accumulation of macrophages in vascular graft examine the paracrine effect of interleukin-1 on fibroblast
implant sites is achieved through selective cellular and hu- proliferation, we studied the effect of supernatants produced
moral mechanisms that both attract and immobilize mac- by the in vitro interaction of monocytes/macrophages on
rophages and macrophage precursors where their functional various biomedical polymers including Dacron and ePTFE
activity can be directed at resolution and/or repair of tissue. and subsequently measured the fibroblast-stimulating po-
Monocytes contact stimulatory factors within blood vessels tential for these supernatants. For Dacron and ePTFE, the
around the vascular graft and adhere to endothelial cells lin- fibroblast stimulatory potential was equivalent, but the
ing the vessels. These monocytes then pass through the ves- interleukin-1 activity for Dacron was much higher than that
sel wall and into the tissue undergoing transformation in exhibited for ePTFE. Neutralization experiments utilizing
response to the stimuli and differentiate into macrophages. specific antibodies to interleukin-1 in our experiments dem-
Monocyte and macrophage movement within the tissue to- onstrated the potential production and paracrine effects of
ward the site of injury is controlled and directed by chemo- other cytokines and growth factors which may also be pro-
taxis or chemokinesis. Chemokinesis is the accelerated ran- duced in the macrophage activation and secretory process.
dom locomotion in response to, and the speed of turning of Table 19.3 illustrates some monocyte/macrophage
the cells toward, chemical stimuli, whereas chemotaxis is the molecules important in modulation of inflammation and
highly directed movement of cells along a chemical gradi- wound healing with vascular graft materials. The potential
ent. Table 19.2 shows important agents which mediate role for the molecules is shown; these molecules may be ei-
chemotaxis and are integral features of the inflammatory ther secreted to produce an autocrine, paracrine or endo-
process. These agents can both sustain and control the se- crine effect or may be cell membrane associated, such as
verity of the inflammatory response. In addition to dead or important adhesion molecules and receptors which may
dying cells, chemotactic stimuli include fragments derived participate in the adhesion of monocytes/macrophages to
from the activation of complement protein, mediators of protein-coated vascular graft materials or in cell-cell inter-
the kinin, clotting and fibrinolytic systems, and products actions important in the inflammatory and wound healing
produced by leukocytes themselves. processes. Table 19.4 provides a partial list of chemo-
Our early studies on human monocyte/macrophage attractants and growth modulators produced by macro-
activation and interleukin-1 generation by biomedical poly- phages. This table is limited to only those agents thought to
mers were carried out in in vitro cell culture systems where be important in the tissue-material interaction in the in-
the interleukin-1 production was assayed by a biological flammatory response to vascular graft materials. It is note-
thymocyte proliferation biological assay.20-26 In the mid to worthy that interleukin-1 and tissue growth factor-! (TGF-!)
late 1980s, a paucity of reagents was available to study spe- may function as growth agonists or growth antagonists. The
cific cytokines and growth factors produced by macrophages schizophrenic nature of cytokines and growth factors must
and foreign body giant cells in contact with vascular graft be considered when attempting to explain the autocrine,
materials. Later, when radioimmunoassays and ELISAs be- paracrine or endocrine activity of cytokines and growth
came available for interleukin-1 quantitation, these were factors.
incorporated into our studies. It is noteworthy that biomedi- Greisler and colleagues have utilized monocyte/mac-
cal polymers have a very low potential for activating mac- rophage molecules released during the inflammatory and
rophages with the release of interleukin-1. In our studies, wound healing processes to modulate cellular proliferation
we utilized lipopolysaccharide to additionally stimulate the in the wound healing and neointimal response to vascular
adherent monocytes/macrophages, as we believe that this is graft materials.27 In in vitro studies, fibroblast growth fac-
tor type I (FGF-I) and heparin placed into suspensions of
fibrinogen polymerized by addition of thrombin (fibrin
glue) promoted endothelial cell proliferation while suppress-
ing smooth muscle cell proliferation. These studies demon-
Table 19.2. Major mediators of macrophage chemotaxis
strated the potential for controlled release systems coupled
Complement Components
with vascular graft materials which could cellularly engineer
Lymphokines the wound healing response with vascular grafts. Prelimi-
Fibronectin Fragments nary in vivo experiments utilizing growth factor release from
Leukotriene B4 vascular grafts in canines demonstrated this concept and
Interleukins (IL-1, IL-8) identified the potential for the direct control of inflamma-
Endothelial Cell Products tory and healing responses directly by cytokines and growth
Monocyte Chemotactic Peptide 1 (MCP-1) factors.28 From a tissue engineering perspective, this is a
Thrombin
Fibrinopeptides
promising avenue of exploration which requires
additional effort.
202 Tissue Engineering of Prosthetic Vascular Grafts
Table 19.3. Monocyte/macrophage molecules important in the modulation of inflammation and wound healing
Molecule Expression Role
Interleukin-1 (IL-1) Receptor Secreted Increased Adhesion Molecule Expression, Smooth Muscle Cell
Migration and Proliferation, Inhibit Endothelial Cell Proliferation
Interleukin-1 Receptor Secreted Decreases Interleukin-1 Response
Antagonist (IL-1Ra)
Interleukin-6 (IL-6) Secreted Activation of Coagulation, Smooth Muscle Cell Secondary Gene
Induction
Interleukin-8 (IL-8) Secreted Recruitment of Lymphocytes and Endothelial Cells
Monocyte Chemoattractant Secreted Monocyte Chemoattractant, Activation of !2 Integrin Receptors
Protein-1 (MCP-1)
Monocyte Colony-Stimulating Secreted Monocyte Chemoattraction, Proliferation, and Survival
Factor (M-CSF)
Granulocyte/Monocyte Colony Secreted Chemoattractant for Monocytes
Stimulating Factor (GM-CSF)
Tumor Necrosis Factor-# (TNF-#) Secreted Increase Endothelial Cell Adhesion Molecule Expression, Monocyte
Chemoattractant, Smooth Muscle Cell Proliferation, Inhibits Endothelial
Cell Proliferation
Tissue Growth Factor-! (TGF-!) Secreted Monocyte Chemoattractant, Increases Smooth Muscle Cell Proliferation
Inhibits Endothelial Cell Proliferation
Interferon-& (INF-&) Secreted Inhibits Endothelial Cell and Smooth Muscle Cell Proliferation
Osteopontin Secreted Cell Adhesion and Migration, Neovessel Formation, and Calcification
SPARC, Osteonectin Secreted Cell Migration, Proliferation, and Neovessel Formation
Thrombospondin Secreted Cell Migration, Neovessel Formation
Oxidized Low Density Secreted Increases Monocyte Chemotaxis and Macrophage Secretion of IL-1 and
TNF-#
Lipoprotein (oxLDL) Increases VCAM-1 Expression on Endothelium
basic Fibroblast Growth Secreted Endothelial Cell Chemoattractant, Increases Endothelial Cell and
Factor (bFGF) Smooth Muscle Cell Proliferation
Vascular Endothelium Growth Secreted Increases Endothelial Cell Proliferation
Factor (VEGF)
Insulin-like growth factor Secreted Smooth Muscle Cell Chemoattractant, Increases Smooth Muscle
(IGF-1) Cell Proliferation
acidic Fibroblast Growth Secreted Endothelial Cell Chemoattraction and Proliferation
Factor (aFGF)
Heparin-Binding Epidermal Secreted Smooth Muscle Cell Chemoattraction and Proliferation
Growth Factor (HB-EGF)
Platelet-Derived Growth Secreted Smooth Muscle Cell Chemoattraction and Proliferation
Factor (PDGF)
Vascular Adhesion Molecule-1 Cell-Associated Monocyte/Macrophage Activation and Retention
(VCAM-1)
Intercellular Cell Adhesion Cell-Associated Monocyte/Macrophage Activation and Retention
Molecule (ICAM-1)
Complement C3bi Receptor Cell-Associated Thrombosis and Macrophage Adhesion
Thrombin Receptor Cell-Associated Monocyte/Macrophage Activation
To complement our in vitro human monocyte/mac- The luminal surface of the vascular graft specimens
rophage activation and cytokine secretion studies, we have showed no to some endothelium at the blood-pseudointima
continued to evaluate retrieved human vascular grafts and interface. The pseudointima (inner capsule) was predomi-
our latest effort involves cytokine and growth factor analy- nantly acellular but isolated areas of fibroblasts and smooth
sis utilizing immunohistochemistry and steady state mRNA muscle cells were identified. In situ hybridization studies
analysis with in situ hybridization techniques. Cytokines and identified IL-1#, IL-1!, TNF-#, TGF-!, and PDGF-A and -B
growth factors/inhibitors studied included interleukin-1# in the pseudointima. The pseudointima-graft interface
(IL-1#), interleukin-1! (IL-1!), tumor necrosis factor-# showed the presence of macrophages and foreign body gi-
(TNF-#), transformtin growth factor-!1 (TGF-!1), and ant cells adjacent to the ePTFE graft nodes and fibrils and
platelet derived growth factor A and B (PDGF-A and -B). Dacron fibers and fiber bundles. These components of the
For analysis, five contiguous zones or areas on or within re- foreign body reaction were localized at the synthetic graft
trieved vascular grafts were evaluated: luminal surface, fiber interface but not in areas distant from the luminal sur-
pseudointima (inner capsule), pseudointima-graft interface face of the graft. The interstitial network of the synthetic
(including the luminal portion of the graft), periadventitia- vascular grafts contained isolated fibroblasts. In situ hybrid-
graft interface (including the outer portion of the graft), and ization and immunohistochemistry analysis identified the
periadventitia (outer capsule). presence of IL-1#, IL-1!, TNF-#, TGF-!, and PDGF-A and
Inflammatory Reaction: The Nemesis of Implants 203
Table 19.4. Chemoattractants and growth modulators produced by macrophages
Chemoattractants Growth Agonists Growth Antagonists
Granulocyte/Monocyte- Granulocyte/Monocyte- Interferon-& (IFN-&)
Colony Stimulating Factor (GM-CSF) Colony Stimulating Factor (GM-CSF)
Monocyte-Colony Stimulating Monocyte-Colony Stimulating Interleukin-1 (IL-1)
Factor (M-CSF) Factor (M-CSF)
Basic Fibroblast Growth Factor (bFGF) Heparin-Binding Epidermal Growth Tissue Growth Factor-! (TGF-!)
Factor (HB-EGF)
Monocyte Chemotactic Protein-1 (MCP-1) Insulin-like Growth Factor (IGF-1)
Tissue Growth Factor-! (TGF-!) Vascular Endothelial Growth Factor (VEGF)
Platelet Derived Growth Factor (PDGF) Basic Fibroblast Growth Factor (bFGF)
Vascular Endothelial Growth Factor Interleukin-1 (IL-1)
(VEGF)
Oxidized Low Density Lipoprotein Tumor Necrosis Factor-# (TNF-#)
(oxLDL)
Tissue Growth Factor-# (TGF-#)
Tissue Growth Factor-! (TGF-!)
Platelet Derived Growth Factor (PDGF)
-B in the pseudointima-graft interface. The graft-peri- pressed by macrophages and foreign body giant cells. Steady
adventitial tissue interface exhibited a foreign body reaction state TNF-# mRNA expression was variable, with moderate
with the presence of macrophages and foreign body giant amounts of mRNA and protein being associated with mac-
cells localized at the synthetic graft surface. This interface rophages and foreign body giant cells.
also exhibited the same cytokines and growth factors iden- TGF-!1 mRNA expression was low compared to IL-1#
tified at the pseudointima-graft interface by immunohis- and IL-1! mRNA. Likewise, protein levels of TGF-! as de-
tochemical and in situ hybridization techniques. The termined by immunohistochemistry were also lower than
periadventitial fibrous capsule (outer capsule) showed a well- IL-1# and IL-1!. Fibroblast-like cells in the pseudointima
healed fibrous capsule with fibroblasts and capillaries within and periadventitial fibrous (outer) capsule, and macroph-
a type I collagen matrix. These histopathologic features were ages and foreign body giant cells at the graft-tissue inter-
similar for all types of retrieved human vascular grafts. In faces, were the predominant cells that expressed TGF-!1
situ hybridization and immunohistochemical analysis mRNA. The expression of steady state PDGF-A and -B
showed the presence of IL-1#, IL-1!, TNF-#, TGF-!, and mRNAs was examined, and the relative levels were similar.
PDGF-A and -B within the outer capsule. Relatively low levels were identified, and immunohis-
Overall, for IL-1#, high levels of expression of steady tochemical analysis indicated no or little protein.
state IL-1# mRNA were identified by in situ hybridization, Our preliminary study focused on the utilization of
and protein expression as determined by immunohis- mRNA for transcriptional analysis and cell localization in
tochemistry was also high. Higher levels of steady state IL-1# combination with protein expression by immunohistochem-
mRNA were present in the periadventitial fibrous (outer) istry as an indicator of translational events. Detection of
capsule relative to the pseudointima, the pseudointima-graft IL-1# and -! mRNAs and protein suggests that IL-1 may act
interface and the periadventitia-graft interface. Fibroblast- as a regulatory protein in the induction of intimal/anasto-
like cells were the predominant cell type in the outer cap- motic hyperplasia. In addition, the presence of IL-1# mRNA
sule and expressed higher levels of IL-1# mRNA, as com- in fibroblast-like cells and myofibroblast-like cells suggests
pared to other cell types including smooth muscle cells and that it may function as a paracrine messenger, stimulating
endothelial cells in the capillaries. In the pseudointima, fi- fibroblasts and myofibroblasts to proliferate and differenti-
broblast-like cells expressed high levels of IL-1# mRNA, ate. Activated fibroblasts also produce a number of soluble
whereas smooth muscle cells did not appear to express IL-1# mediators including bFGF, PDGF and IL-1 which can acti-
mRNA. In contrast to the pseudointima and the vate other inflammatory cells in addition to smooth muscle
periadventitial fibrous capsule, macrophages and foreign cells. Although not as strong as IL-1# mRNA, steady state
body giant cells in the pseudointima-graft and IL-1! mRNA was identified and may stimulate smooth
periadventitia-graft interfaces were predominant cell types muscle cell proliferation in a paracrine fashion.
that expressed IL-1# mRNA and protein. Relative levels of Transformtin growth factor-!1 (TGF-!1) was identi-
IL-1! mRNA and protein detected in Dacron and ePTFE fied at low levels of steady state mRNA expression in macro-
vascular graft sections were similar to that of the IL-1# phages and foreign body giant cells. TGF-!1 is an important
mRNA detected. Overall, the periadventitial fibrous (outer) regulator of the duration of the inflammatory response and
capsule expressed higher levels of steady state IL-1! mRNA also is a potent inhibitor of cell proliferation, as well as a
relative to the other areas. However, with the ePTFE vascu- stimulator of connective tissue formation by cells. The en-
lar grafts, very low levels of IL-1! mRNA were found in all hanced activity of macrophages and foreign body giant cells
of the areas studied. IL-1! mRNA was predominantly ex- was exhibited by the detection of steady state TNF-# mRNA.
204 Tissue Engineering of Prosthetic Vascular Grafts
TNF-# is known to enhance the phagocytic activity of macro- fibronectin, for which monocytes also express multiple types
phages and foreign body giant cells. Our in situ hybridiza- of receptors.
tion study found low levels of steady state mRNAs for Because of experimental complexities in dealing with
PDGF-A and -B in macrophages and foreign body giant cells, the three dimensional structures of Dacron and ePTFE, we
and very little to no protein was detected by immuno- initiated our studies utilizing chemically-modified polysty-
histochemistry. rene surfaces to explore the role of surface chemistry in
The detection of various cytokine mRNAs and pro- monocyte adhesion, macrophage phenotypic expression and
tein implicates cytokine-mediated interactions of cellular foreign body giant cell formation.29 Human monocyte in
components in the ePTFE and Dacron vascular grafts. The vitro adhesion with fluorinated, siliconized, nitrogenated and
paracrine interaction mediated by IL-1 in macrophages/ oxygenated surfaces were reduced by 50-100% when comple-
foreign body giant cells and fibroblasts/myofibroblasts with ment component C3-depleted serum was used for adsorp-
vascular smooth muscle cells is strongly suggested by tion. The fluorinated surfaces exhibited the greatest inhibi-
these findings. tion of monocyte adhesion with C3-depleted serum. Mono-
cyte adhesion was restored on all surfaces when C3-depleted
Foreign Body Giant Cell Formation serum was replenished with purified C3. Monocyte adhe-
As part of our ongoing investigation into the role of sion to serum-adsorbed surfaces was inhibited by mono-
the monocyte/macrophage in biocompatibility, a major goal clonal antibodies to the leukocyte integrin beta subunit,
has been to identify the adhesion and fusion mechanisms CD18, and partially inhibited by a monoclonal antibody to
that initiate and promote the observed in vivo morphologic the alpha subunit Cd11b. These findings suggest adhesive
progression of monocyte to macrophage to foreign body interactions between adsorbed C3bi, the hemolytically in-
giant cell on biomaterials and vascular graft materials. Ad- active form of the C3b fragment, and the leukocyte integrin
herent monocyte-derived macrophages and foreign body Cd11b/cd18. Additional studies demonstrated that adsorbed
giant cells (FBGCs), formed by macrophage fusion, are fibrinogen reduced the effectiveness of these inhibiting
prominent and persistent cell types on implanted vascular monoclonal antibodies, indicating that alternative adhesion
grafts and, through their numerous secretory capacities, are mechanisms may operate, depending on the critical adhe-
believed to exert multiple and complex influences on the sion-mediating components adsorbed onto the different
inflammatory response at the implant site and on the surfaces.
biocompatibility of vascular grafts. Although monocyte ad- Recent studies in our laboratory have implicated IL-4
hesion to implanted vascular grafts is critical to and IL-13 cytokines in the in vitro formation of foreign body
biocompatibility outcome, as it initiates macrophage devel- giant cells derived from human monocytes/macro-
opment and FBGC formation, it is unknown how mono- phages.30-33 These studies have resulted in the development
cytes recognize biomaterials and how surface properties of an in vitro culture system that may now be utilized for
might influence this event. testing various materials for their ability to participate in
Material surface property-dependent blood protein foreign body giant cell formation. When IL-4 or IL-13 are
adsorption occurs immediately upon surgical implantation added to human monocyte cultures at day 3, foreign body
of a vascular graft, and it is the protein-modified biomate- giant cells containing as many as 100 nuclei and measuring
rial that inflammatory cells subsequently encounter. Mono- 1.0 mm2 in surface area are formed on various modified sur-
cytes express receptors for various blood components, but faces. Critical to these experiments was the addition of the
they recognize naturally occurring foreign surfaces by re- cytokine at day 3 of the culture and not at day 0 of the cul-
ceptors for opsonins such as fragments of complement com- ture. Simultaneous addition of the cytokine and the cells at
ponent C3. Because complement activation by biomaterials the beginning of the culture does not lead to foreign body
has been well documented, we investigated monocyte inter- giant cell formation and may result in the lack of mono-
actions with foreign surfaces. Exposure to blood during vas- cyte/macrophage adhesion to the substrate. This sequential
cular graft implantation may permit extensive opsonization addition of the cytokine points out the importance of
with the labile fragment C3b and the rapid conversion of temporal variations which may occur in addressing cytokine/
C3b to its hemolytically inactive but nevertheless opsonic cell interactions.
and more stable form, C3bi. C3b is bound by the Cd35 re- IL-4 and IL-13 are lymphokines secreted predomi-
ceptor, but C3bi is recognized by distinct receptors, nantly by Th2 lymphocytes in humans. Thus, lymphocytes
Cd11b/cd18 and Cd11c/cd18. Fibrinogen, a major plasma present transiently in the early chronic inflammatory phase
protein that adsorbs to vascular grafts, is another described of the inflammatory response may participate in the for-
ligand for these molecules, which together with Cd11a/cd18 eign body reaction through facilitation of the formation of
constitutes a subfamily of integrins that is restricted to leu- foreign body giant cells. IL-4 and IL-13 downregulate the
kocytes. Studies with monoclonal antibodies to their com- secretion of proinflammatory cytokines such as IL-1, IL-6
mon !2 subunit (CD18) and distinct # chains (Cd11a,b,c) and TNF-#; growth factors such as granulocyte-macrophage
have implicated Cd11a/cd18 in cell-cell adhesive interactions CSF and granulocyte CSF; and chemokines such as IL-8 and
and Cd11b/cd18 and Cd11c/cd18 in multiple phagocytic cell macrophage inflammatory protein-1#. Fc&R expression, re-
responses. Other potential adhesion-mediating proteins that active oxygen intermediate secretion and cytotoxic activi-
adsorb to biomaterials include IgG, which may interact with ties are also inhibited by these cytokines. Conversely, IL-4
monocytes via receptors to its Fc constant region, and and IL-13 upregulate monocyte/macrophage production of
Inflammatory Reaction: The Nemesis of Implants 205
IL-1R antagonist, antigen-presenting capacity and cell sur- The second major barrier that must be confronted is
face expression of several adhesion molecules including the highly complex and time-dependent nature of the cel-
Cd11b/cd18 and Cd11c/cd18. Although IL-4 and IL-13 nega- lular and humoral components of the inflammatory re-
tively regulate several cytotoxic and inflammatory functions sponses. Our approach has been to utilize human material
of monocytes/macrophages, they are not generally viewed whenever possible, but this limits one to in vitro experiments.
as downregulators of monocyte/macrophage activity. As humans cannot ethically be used in preliminary experi-
The role for IL-4 in foreign body giant cell formation ments to test the safety of devices, animal models must be
in vivo has been demonstrated in a rat cage implant system utilized. The question resulting from the use of animal
with polyurethane.31 Utilization of both recombinant IL-4 models is the equivalency of, and similarity and differences
and anti-IL-4 injected into the cage demonstrates the en- between, the animal model and the response in humans. Not
hancement and inhibition of foreign body giant cell forma- only must the various components of the inflammatory
tion on the polyurethane surface, respectively. The in vivo response be considered, but also the extent and duration of
studies also point out the lack of effect by IL-4 in facilitating the components of the inflammatory response and the
macrophage adhesion to the polyurethane surface. Thus, the time-dependent nature, i.e., kinetics, of the inflammatory
IL-4 appears to facilitate foreign body giant cell formation responses and their components. These factors may or may
through mechanisms not related to the surface density of not vary when comparing animal model responses to
adherent macrophages. human responses.
Complementary experiments with IL-4-induced These barriers and lack of knowledge of inflamma-
macrophage fusion in foreign body giant cell development tory responses offer new challenges that must be met in the
has shown the prevention of macrophage fusion and the for- research and development of tissue-engineered prosthetic
mation of foreign body giant cells by the use of inhibitors of vascular grafts. New paradigms must be developed with new
mannose receptor activity. # -Mannan and synthetic perspectives based on new information, and older para-
neoglycoprotein conjugates inhibit the fusion of macro- digms, while comfortable to some investigators, may not
phages to form foreign body giant cells. Inhibition of mac- adequately and appropriately address the significant scien-
rophage activity in the formation of foreign body giant cells tific issues that are necessary in the development of tissue-
may be facilitated by blocking inhibitors which participate engineered prosthetic vascular grafts.
in the macrophage fusion process. This offers a pathway by A third major barrier facing us in the development of
which the foreign body reaction may be controlled. tissue-engineered prosthetic vascular grafts is the adequate
and appropriate evaluation of new concepts or products
Future Perspectives on Inflammatory from a safety and efficacy perspective. Unique devices will
Responses to Tissue Engineered Prosthetic require unique testing and evaluation protocols. Appropri-
Vascular Grafts ate rationale and justification must be provided for the in-
The future development of tissue engineered pros- clusion or omission of test methods, which will be based on
thetic vascular grafts requires new knowledge and informa- the unique characteristics of the tissue-engineered prosthetic
tion on normal and directed healing mechanisms, biologi- vascular graft under consideration. New materials and de-
cal signals and signal mechanisms, and delivery and pheno- vices designed for deliberate and specific interactions with
typic expression of cells in and on tissue-engineered pros- blood and tissue components must be based on those delib-
thetic vascular grafts. Research efforts in the tissue engineer- erate and specific interactions, which are a component of
ing area must be directed towards developing new informa- the design of the tissue engineered prosthetic vascular graft.
tion, i.e., biologically-based design criteria, and an expan- In the development of tests for tissue-engineered prosthetic
sion of our current knowledge base regarding these processes vascular grafts, it is important that we emphasize the com-
and mechanisms. There is no doubt that we are exception- plex and interactive milieu of the cellular and humoral com-
ally limited in our knowledge base as it applies to the re- ponents that interact or are anticipated to interact with the
search and development of tissue-engineered prosthetic vas- tissue-engineered prosthetic vascular graft. We must appre-
cular grafts. In considering the early responses to implanted ciate the interactive environment provided by the numer-
tissue engineered prosthetic vascular grafts, emphasis must ous humoral enzyme systems (kinin, fibrinolytic, coagula-
be placed on developing an understanding of the inflam- tion and complement), formed elements (platelets), and cells
matory response interactions with the tissue-engineered (polymorphonuclear leukocytes, monocytes, lymphocytes
prosthetic vascular graft with its unique characteristics. As and eosinophils) which provide for positive (activating) and
such, the evaluation must take into consideration the unique negative (inhibiting) interactions. Failure to appreciate these
function and application of the tissue-engineered prosthetic types of interactions and feedback control mechanisms may
vascular graft. A major hurdle to this effort is the apparent lead to false interpretations of results from single or isolated
species differences which exist with prosthetic vascular grafts test methods. Furthermore, emphasis must be placed on the
and in particular the process of endothelialization which quantitative determination of parameters in the biological
occurs in primates and other mammals, but not in human response evaluation. As our knowledge base for the research
prosthetic vascular grafts. Coupled with this problem is the and development of new tissue-engineered prosthetic vas-
lack of knowledge regarding the phenotypic expression of cular grafts increases, enhanced integration of disciplines
cellular components in the inflammatory response in dif- affecting not only the basic research but also the develop-
ferent species. ment of test methods will be necessary.
206 Tissue Engineering of Prosthetic Vascular Grafts
It is our perspective that the inflammatory response and Ratner B, eds. Polymers as Biomaterials. New
to prosthetic vascular grafts and tissue-engineered prosthetic York:Plenum Press, 1984:209-223.
vascular grafts is not necessarily a nemesis. In the past, we 17. Zhao Q, Agger MP, Fitzpatrick M et al. Cellular interac-
have tended to label the inflammatory response as a nem- tions with biomaterials: in vivo cracking of prestressed
Pellethane 2363-80A. J Biomed Mater Res 1990;
esis, i.e., source of harm or destruction, based upon our in-
24:621-637.
complete knowledge of the inflammatory and wound heal- 18. Zhao Q, Topham N, Anderson JM et al. Foreign-body
ing responses. With the expansion of our knowledge base of giant cells and polyurethane biostability: in vivo correla-
inflammatory cell interactions with prosthetic vascular grafts tion of cell adhesion and surface cracking. J Biomed Mater
and tissue-engineered prosthetic vascular grafts, we can an- Res 1991; 25:177-183.
ticipate that the new and expanded knowledge base of in- 19. Mathur AB, Collier TO, Kao WJ et al. In vivo
flammatory responses and interactions will be utilized to biocompatibility and biostability of modified polyure-
modify inflammatory cell responses to these new devices, thanes. J Biomed Mater Res 1997; 36:246-257.
and possibly convert a foe into a friend or ally. 20. Miller KM, Anderson JM. Human monocyte/macrophage
activation and interleukin 1 generation by biomedical
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13. Anderson JM, Abbuhl MF, Hering T, Johnston KH. Im- Differential lymphokine regulation of macrophage fusion
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healing response of human vascular grafts. ASAIO J 1985; cells. Amer J Pathol 1995; 147:1487-1499.
8:79-85. 31. Kao WJ, McNally AK, Hiltner A, Anderson JM. Role for
14. Kao WJ, Sapatnekar S, Hiltner A, Anderson JM. Comple- interleukin-4 in foreign-body giant cell formation on a
ment-mediated leukocyte adhesion on poly(etherurethane poly(etherurethane urea) in vivo. J Biomed Mater Res
ureas) under shear stress in vitro. J Biomed Mater Res 1995; 29:1267-1276.
1996; 32:99-109. 32. McNally AK, DeFife KM, Anderson JM. Interleukin-4-in-
15. Hering TM, Marchant RE and Anderson JM. Type V col- duced macrophage fusion is prevented by inhibitors of
lagen during granulation tissue development. Experimen- mannose receptor activity. Am J Pathol 1996; 149:975-985.
tal and Molecular Pathology 1983; 39:219-229. 33. DeFife KM, McNally AK, Colton E, Anderson JM.
16. Marchant RE, Miller KM, Hiltner A, Anderson JM. Se- Interleukin-13 induces human monocyte/macrophage fu-
lected aspects of cell and molecular biology of in vivo sion and macrophage mannose receptor expression. J
biocompatibility. In: Shalaby S, Hoffman A, Horbett T Immunol 1997; 158:3385-3390.
Taming of Adverse Responses
Prevention of the Inflammatory Reaction
CHAPTER 20
Molecular Determinants of
Acute Inflammatory Responses to Biomaterials
Liping Tang, John W. Eaton
Introduction
D espite the fact that most biomaterials are inert, nontoxic and nonimmunogenic, bioma-
terial implants often cause adverse reactions. Typically, shortly after implantation, bio-
material surfaces attract a layer of phagocytic cells (especially, macrophages/monocytes and
neutrophils)1-4 and fibroblast-like cells.5 This very much resembles the acute inflammatory
response seen with many other types of foreign bodies and, to an extent, with focal bacterial
infections. The acute inflammatory responses to tissue contact biomaterials are very often
followed by chronic inflammation6-8 and the appearance of fibrotic tissue surrounding many
types of implants.7,9-18 Chronic inflammation and fibrosis are also associated with the deg-
radation and failure of many types of implants, including pacemaker leads,19-21 mammary
prostheses,22,23 temporomandibular24,25 and other joint implants.26
At first glance, these inflammatory responses to inert, nonimmunogenic and non-
toxic materials are difficult to understand. Why should such unreactive and so-called
“biocompatible” materials cause recruitment and evident activation of phagocytes? The fol-
lowing represents a brief summary of our present understanding of the inflammatory re-
sponses to implanted biomaterials.
Surface-Protein Interactions
Because biomaterial surfaces spontaneously and rapidly acquire a layer of host pro-
teins upon contact with blood or proteinaceous body fluids, it is generally believed that
these absorbed proteins likely influence, or absolutely dictate, the ensuing adverse
responses.27-30 Because this protein adsorption occurs much faster than the arrival of cells
on the foreign surface, host cells almost certainly interact with surface-adsorbed proteins,
rather than with the material itself.29-31 Therefore, the types and states of adsorbed proteins
are probably critical determinants of biocompatibility.30,32-35
The composition of adsorbed proteins varies with different biomaterials.29,36 Unfor-
tunately, due in part to the bewildering variety of model materials and protein types which
have been used to investigate protein adsorption by biomaterials, the general principles gov-
erning surface-protein interactions under real life conditions are by no means clear. Gener-
ally speaking, albumin, fibrinogen and immunoglobulin G (IgG) predominate on many
types of blood-contact biomaterials, including Dacron® (a woven form of polyester tereph-
thalate; hereafter, ‘PET’), expanded polytetrafluoroethylene, polydimethylsiloxane (‘silastic’),
polyurethanes and polyethylene.29,36,37 Especially on hydrophobic surfaces such as these
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler
©1999 R.G. Landes Company.
208 Tissue Engineering of Prosthetic Vascular Grafts
(which are typical of the majority of implanted biomaterials), dant protein on biomaterial surfaces, can “passivate”
the adsorbed proteins may undergo conformational changes biomaterials, blunting both inflammatory and thrombogenic
over a period of hours, becoming tightly adherent.38-41 These responses.1,33,53,58 We therefore initially hypothesized that
conformational changes of adsorbed proteins have been IgG, which is also adsorbed on biomaterial surfaces in large
detected using many different techniques, including sodium amounts,29,31,36,53 might be the crucial mediator of phago-
dodecyl sulfate (SDS) elution,42,43 scanning angle reflecto- cyte responses to biomaterial implants.
metry,44 scanning force microscopy,45 attenuated total re-
flectance Fourier transform infrared spectroscopy39 and dif- Adsorbed IGg Is Not Required for Phagocyte
ferential scanning calorimetry.46 It has been assumed that, Accumulation on Biomaterial Implants
upon binding to hydrophobic surfaces, many proteins tend The possible involvement of adsorbed IgG in acute
to unfold47 and, within a short period, undergo surface “de- inflammatory responses to biomaterial implants was sup-
naturation” (perhaps via loss of the normal sphere of hy- ported by several observations. First, immunoglobulins are
dration encouraged by contact with the hydrophobic sur- important in the opsonization of foreign particles, which
face).48 The resulting conformational changes of adsorbed makes them more readily ingested by phagocytes.59 Second,
protein may expose hidden epitope(s) which, in turn, help both surface-bound and heat-denatured IgG and IgA are
initiate adverse reactions such as coagulation and inflam- potent activators of human neutrophils, causing the release
mation.49-52 However, a direct link between protein confor- of lysosomal enzymes (lysozyme, !-glucuronidase and
mational changes and these adverse responses has not been myeloperoxidase) and stimulation of phagocyte oxidative
established. metabolism. 60-62 Finally, in our experimental animal
implantation model we found that IgG-coated surfaces ac-
Which Adsorbed Host Protein(s) Might Be Important cumulated as many inflammatory cells (both neutrophils
in Mediating Inflammatory Responses to Biomaterial and macrophages/monocytes) as did uncoated or plasma-
Implants? coated surfaces.53
In order to investigate the mechanisms of biomate- However, further experimental evidence indicates that
rial-mediated inflammatory responses, we developed a adsorbed immunoglobulins are not essential to acute inflam-
simple animal implantation model. Biomaterial films are cut matory responses to implants. We placed implants in mice
into 1.2 cm diameter disks. After repetitive washes with etha- with severe combined immunodeficiency (SCID) which have
nol, the disks are precoated with various types of proteins extremely low plasma IgG levels (< 1 ∝g/ml vs. 4 mg/ml in
and then implanted in the mouse peritoneal cavity for normal, otherwise syngeneic, animals). In SCID mice, the
16-18 h (in this model, the time of maximal phagocyte ac- inflammatory response to uncoated PET disks as evidenced
cumulation).53 The surface associated activities of myelo- by the recruitment of both neutrophils (Fig. 20.1) and
peroxidase (MPO) and nonspecific esterase (NSE) are mea- macrophages (not shown) is almost as great as in immuno-
sured to estimate the numbers of adherent neutrophils and competent animals. The modest decrement in neutrophils
macrophages/monocytes, respectively.53,54 Although a num- and macrophage recruitment in SCID vs. normal mice prob-
ber of other investigators have used subcutaneous implants ably reflects a general defect in phagocyte mobilization, be-
or cage implants (e.g., refs. 2,3,19,20), these experimental cause this difference is apparent on both uncoated and
systems have some shortcomings compared to intraperito- IgG-coated surfaces.
neal implants.7 The peritoneal cavity provides a good site
for studying inflammatory responses because reactions elic- Complement Activation Is Not Required for Phagocyte
ited by the polymer are clearly defined with minimal par- Accumulation on Biomaterial Implants
ticipation of the coagulation system.7,53-57 Furthermore, it A few blood-contact biomaterials may trigger comple-
appears that at least the majority of phagocytic cells found ment activation and (during, e.g., hemodialysis or
adherent to explants are, indeed, recruited de novo and by leukapheresis) transient neutropenia, suggesting that
the implant itself. Thus, mice subjected to sham surgery show complement activation might contribute to the inflamma-
only a small increment in numbers of phagocytes in perito- tory effects of blood-contact biomaterials (e.g., see refs. 32,
neal lavage fluids compared to implant-bearing animals. 63-67). This hypothesis is well supported by the known ef-
As indicated above, albumin, IgG, fibrinogen and (to fects of activated complement components. For example,
a lesser extent) complement are the most abundant proteins surface-induced complement activation will generate C3a
in plasma and are found to predominate on many blood- and C5a fragments,68,69 which are known to mediate granu-
contact and tissue-contact biomaterials, including polyeth- locyte aggregation,67 chemotaxis,70 and adhesiveness to en-
ylene terephthalate, expanded polytetrafluoroethylene, poly- dothelial cells.71 In addition, complement activation by some
dimethylsiloxane, polyurethanes and polyethylene.29,36,37,53,54 polymeric materials has been shown in vitro to cause
Especially on hydrophobic surfaces, these adsorbed proteins phagocyte adhesion.65,72 By extrapolation, one might antici-
tend to assume an altered conformation and become tightly pate that similar events might be involved in acute inflam-
adherent.38-41,53,54 Taking advantage of this property, we were matory responses to tissue contact biomaterials. However,
able to determine the possible role of various plasma pro- complement depleted mice (pretreated with cobra venom
teins—precoated on implant surfaces—in initiating bioma- factor) exhibit normal recruitment of inflammatory cells to
terial-mediated inflammatory responses. In agreement with implant surfaces.53 Consequently, it appears that although
many previous reports, we find that albumin, the most abun- activated complement components may powerfully
Molecular Determinants of Acute Inflammatory Responses to Biomaterials 209
Fig. 20.1. Numbers of surface-
associated neutrophils (PMN)
on clean PET implants and PET
implants precoated with either
immunoglobulin G (IgG) or hu-
man albumin. The implants were
placed either in control Balb/c
mice or congeneic animals with
severe combined immunodefi-
ciency (SCID) and extremely low
levels of circulating IgG. Follow-
ing 16 h implantation, the mate-
rial was removed and numbers of
PMN estimated by analyses of
surface-associated myeloper-
oxidase. Calculated numbers of
PMN from explants in Balb/c
mice: 166,000 ± 74,000/cm2 on
uncoated implants (numbers
similar to those recovered from
plasma-coated implants),
153,000 ± 31,000/cm2 on IgG-
coated implants and 41,000 ±
11,000/cm2 on albumin-coated implants. In SCID mice: 225,000 ± 94,000/cm2 on uncoated implants (similar values obtained for
plasma-coated implants), 150,000 ± 36,000/cm2 on IgG-coated implants and 24,000 ± 19,000/cm2 on albumin-coated implants.
Vertical lines denote ± 1 SD (n = 4 in all cases). In both control and SCID animals, values for albumin-coated material differ
significantly from both IgG-precoated implants and uncoated implants at p<0.05 (Student’s ‘t’ test, two tailed). (Data from ref. 53.)
stimulate phagocytes, complement activation is not impor- both neutrophils and macrophages. In order to exclude the
tant in acute inflammatory responses to biomaterial implants possible involvement of activated coagulation factors, simi-
(or, at least to implants of hydrophobic polymers such as lar studies were done using afibrinogenemic plasma in which
polyethylene terephthalate, polyninyl chloride or fibrinogen was undetectable. PET coated with afibrino-
polyether urethane). genemic plasma attracted very few phagocytes (roughly equal
to the numbers which accumulate on albumin-coated ma-
Fibrin(ogen) Is Necessary for Phagocyte terial). However, when physiologic levels (3 mg/ml) of pu-
Accumulation on Biomaterial Implants rified fibrinogen were restored to this afibrinogenemic
The foregoing results indicate that, insofar as our ex- plasma, PET disks incubated in this mixture prompted nor-
perimental murine model is analogous to the human cir- mal recruitment of phagocytes.54
cumstance, neither complement activation nor adsorbed IgG Most importantly, the requirement for fibrinogen ad-
is a necessary mediator of acute inflammatory reactions to sorption to the surface of PET implants would appear to
implanted materials such as Dacron (PET). Together, albu- hold in vivo as well. Mice having undetectable plasma fi-
min, IgG and fibrinogen may comprise > 80% of adsorbed brinogen were produced by repetitive injections of
surface proteins, at least on certain polymers.29,36,73,74 Con- ancrod.75-80 Ancrod infusion causes severe hypofibrino-
sequently, it seemed that fibrinogen—also present in large genemia and hypoplasminogenemia, but does not disturb
amounts on implant surfaces36—might be the host protein the number or turnover of platelets or the levels of other
most important in triggering inflammatory reactions. coagulation factors and plasma proteins (including
This hypothesis was supported by a surprising obser- fibronectin).76,78,81,82 Ancrod-treated mice (having undetect-
vation: Serum coated surfaces, like albumin coated surfaces, able fibrinogen at the time of implantation) show almost
fail to prompt inflammatory responses.54 The major differ- no neutrophil accumulation on untreated disks, but do so if
ence between plasma and serum is that plasma, but not se- the disks are precoated with purified murine fibrinogen
rum, contains fibrinogen. We therefore reconstituted serum (Fig. 20.2). 54 The same dependence on adsorbed
by adding a physiological concentration of fibrinogen and fibrin(ogen) was also observed in similar experiments em-
used this to coat experimental implants. Disks precoated with ploying subcutaneous, rather than intraperitoneal, implants.
fibrinogen-repleted serum attract as many phagocytes as do Finally, we should note that although some studies have
disks coated with fresh, minimally heparinized plasma. In shown that fibronectin can serve as a bridge for phagocyte-
addition, fibrinogen alone is sufficient to trigger inflamma- surface fibrin(ogen) interactions,83,84 our preliminary ex-
tory responses; PET disks preincubated with purified hu- periments have revealed no difference in in vitro phagocyte
man (or murine) fibrinogen also attract large numbers of adherence to materials coated with normal plasma vs.
210 Tissue Engineering of Prosthetic Vascular Grafts
fibronectin-depleted plasma (Tang et al, unpublished ob- epitope(s) responsible for phagocyte accumulation, plasmin
servations). This may, however, reflect the absence of expo- degradation fragments of fibrinogen were generated and
sure of these phagocytes to factors which, e.g., upregulate used to coat implant surfaces (Fig. 20.3). Implants precoated
cell adherence molecules (vide infra). with the plasmin degradation fragment D100
(MW = 105 kDa), but not E50 (MW = 50 kDa), accumulate
Which Portion of Fibrinogen Is Critical large numbers of phagocytes (both neutrophils and mac-
in Mediating Phagocyte Adhesion? rophages/monocytes), as many as fibrinogen-coated disks.
The foregoing results provide strong support for the To determine more precisely the specific portion of fibrino-
proposition that the spontaneous adsorption of host fibrino- gen responsible, D100 was digested to D80 (MW = 80 kDa)
gen to the surfaces of implanted PET is a necessary prece- and further to D30 (MW = 30 kDa). Both D80 and D30 are
dent to the subsequent inflammatory and fibrotic processes. fully active in fostering the in vivo accumulation of both
We reasoned that the adsorbed fibrinogen probably displayed neutrophils and macrophages/monocytes.85 This suggests
one or more epitopes normally occult in the soluble protein that at least one necessary motif for phagocyte accumula-
but exposed by surface-mediated “denaturation” of the pro- tion is within the fibrinogen D30 fragment.
tein. In order to determine the location of the hypothetical
Fig. 20.2. Numbers of surface-associ-
ated phagocytes on clean (‘uncoated’)
PET implants and PET implants
precoated with either fibrinogen or
human albumin. The implants were
placed either in (A) control Balb/c
mice or (B) mice pretreated with
ancrod to produce a state of hypo- or
afibrinogenemia at the time of implant
placement. Following 16 h implanta-
tion, the material was removed and the
numbers of PMN estimated by analy-
ses of surface-associated myelo-
peroxidase while the numbers of
monocytes/macrophages were as-
sessed by assay of nonspecific esterase.
Calculated numbers of PMN from ex-
plants in control mice: 84,000 ±
32,000/cm2 on uncoated implants,
46,000 ± 25,000/cm2 on fibrinogen-
coated implants and 100 ± 500/cm2 on
albumin-coated implants. In ancrod-
treated mice: 12,000 ± 6,000/cm2 on
uncoated implants, 49,000 ±
25,000/cm2 on fibrinogen-coated im-
plants and 2,000 ± 2,000/cm2 on al-
bumin-coated implants. Vertical lines
denote ± 1 SD (n = 6 in all cases). In
both control and ancrod-treated ani-
mals, values for albumin-coated ma-
terial differ significantly from both fi-
brinogen-precoated implants and un-
coated implants at p < 0.01, and val-
ues for uncoated materials in control
vs. ancrod-treated mice differ signifi-
cantly at p < 0.01 (Student’s ‘t’ test, two
tailed). (Data from ref. 54.)
Molecular Determinants of Acute Inflammatory Responses to Biomaterials 211
Altieri and colleagues87 had earlier found that one seg- How Does Adsorbed Fibrinogen Become
ment of D30, &190-202 (hereafter abbreviated as ‘P1’), is criti- Pro-Inflammatory?
cally important in mediating phagocyte-fibrinogen interac- The preceding results indicate that fibrinogen
tions. Thus, we thought that this particular short sequence &190-202 is the most important factor in mediating the ac-
might be a crucial determinant of phagocyte interactions cumulation of phagocytes on fibrinogen-bearing implant
with adsorbed fibrinogen on implant surfaces. To test this, surfaces. However, it remains unclear how the simple ad-
P1 and a P1-based scrambled peptide were synthesized and sorption of fibrinogen to implant surfaces might modify the
covalently linked to human albumin (which has been used protein, converting it into a pro-inflammatory signal. It is
widely as a carrier to enhance cellular responses to pep- very likely that the pro-inflammatory nature of adsorbed
tides88-90). Indeed, implant surfaces precoated with P1 pep- fibrinogen reflects the occurrence of a conformational
tide, but not with the scrambled peptide, induce substantial change, probably also reflected by the progressive resistance
phagocyte accumulation, roughly equivalent to that caused of adsorbed fibrinogen to elution by detergents.38 For
by fibrinogen-coated surfaces (Fig. 20.4).
Fig. 20.3. Schematic drawing of
the various domains of a single
fibrinogen molecule showing
the three (#, ! and &) chain types,
two major plasmin cleavage
domains (D and E) and the site
of the evidently important 13
amino acid epitope P1 at
&190-202. Redrawn from ref. 86,
with permission from the
author and publishers.
Fig. 20.4. Phagocyte accumulation
on the surfaces of PET disks
precoated with human fibrinogen, a
covalent adduct between a synthe-
sized version of & 190-202 (P1)
(‘P1:albumin’), a covalent adduct
between a synthetic 13 amino acid
peptide containing the same amino
acids in random sequence
(‘scrambled albumin’) or unmodi-
fied albumin. Values shown repre-
sent the numbers of neutrophils
(PMN; cross-hatched bars) and
monocytes/macrophages (MØ; ver-
tical striped bars) on the surfaces of
explants following 16 h implantation
in Balb/c mice. Vertical lines denote
± 1 SD (n = 6 in all cases). Estimated
numbers of PMN: 120,000 ±
51,000/cm2 on fibrinogen-coated
disks, 115,000 ± 64,000/cm 2 on
‘P1:albumin’-coated disks, 400 ±
900/cm2 on ‘scrambled: albumin’-
coated disks and 2,100 ± 2,500/cm2 on albumin-coated disks. Calculated numbers of monocytes/macrophages: 221,900 ± 76,000/cm2
on fibrinogen-coated disks, 393,000 ± 128,000/cm2 on ‘P1:albumin’-coated disks, 9,100 ± 7,300/cm2 on ‘scrambled: albumin’-
coated disks and 7,300 ± 5,500/cm2 on albumin-coated disks. Values obtained for fibrinogen vs. P1:albumin-coated surfaces do
not differ, whereas both of these differ at p < 0.001 when compared to either albumin or ‘scrambled albumin’-coated surfaces.
(Data from ref. 85.)
212 Tissue Engineering of Prosthetic Vascular Grafts
example, after PET film has been incubated with purified also occur naturally in the normal course of hemostasis and
fibrinogen for 4 h, more than 60% of adsorbed protein is wound resolution. The presence of fibrin has long been rec-
resistant to elution by sodium dodecyl sulfate.53 The con- ognized as coeval with inflammatory and fibrotic responses,
formational changes of adsorbed fibrinogen also have been and fibrin deposition is typical of both acute and chronic
recently probed by differential scanning calorimetry inflammatory processes.92-95 Furthermore, patients who are
(DSC),46 based on the assumption that denaturation of hypo- or afibrinogenemic are known to have abnormal in-
adsorbed protein will prevent some or all further thermal flammatory and wound healing responses. Therefore, it may
transitions. The DSC thermograms of native bovine fibrino- be that the body interprets the presence of fibrin as an indi-
gen have two transition peaks at 55°C and 96°C, reflecting cation that vascular barriers have been breached and that
denaturation of the D and E domains of fibrinogen, respec- invasion by microorganisms might have occurred. In this
tively. After prolonged interaction with low temperature iso- light, the attraction of phagocytic cells—important in both
tropic carbon (which has a highly hydrophobic surface), the host defense and in the ultimate dissolution of the coagu-
transition peaks for both domains are diminished, but the lum—makes perfect biological sense.
peak for the fibrinogen D domain is reduced more than that
of the E domain. This suggests that the D domain may be The Mechanism of Biomaterial-Mediated
more susceptible to surface induced denaturation than the Inflammatory Responses
E domain. Because the P1 epitope is within the D domain Although the above may represent some small progress
and the D domain is very susceptible to conformational in understanding the importance of surface protein adsorp-
changes, we now believe that the interaction between fibrino- tion in the response to biomaterial implants, there remain
gen and surfaces mediates the conformational changes of many mysteries concerning the mechanisms involved in the
the D fragment causing the exposure of P1. As a result, oth- overall inflammatory response. The evolution of biomate-
erwise inert and unreactive surfaces displaying this epitope rial-mediated inflammatory responses may be somewhat
are recognized by phagocytes. The exposure of normally arbitrarily divided into three consecutive events (Fig. 20.5):
hidden epitopes as a result of adsorption to polymeric sur- 1. Phagocyte transmigration through the endothelial
faces has some precedent; upon adsorption to plastic tissue barrier;
culture surfaces (which, having been plasma discharge 2. Chemotaxis toward the implants; and, finally,
treated, are not precisely analogous to most hydrophobic 3. Adherence to the biomaterial.
implant surfaces), fibrinogen changes conformation and A fuller understanding of these processes—discussed
exposes multiple receptor-induced binding sites (RIBS) below in reverse order of occurrence—may help in the de-
&112-119 and A#95-98.91 velopment of strategies to moderate biomaterial-mediated
Thus, we now believe that surface fibrin(ogen) un- inflammatory responses and associated tissue responses.
dergoes changes in conformation, becoming fibrin-like and
that this anomalous fibrin(ogen) is critical in the attraction Phagocyte Adherence to Biomaterial Implants
and adherence of phagocytic cells to implant surfaces. The In order to interact with implant surfaces, receptors
ensuing responses of phagocytic cells to this product may on cell surfaces must interact with adsorbed fibrinogen. We
Fig. 20.5. Hypothetical (and simpli-
fied) sequence of events important
in acute inflammatory responses to
implanted biomaterials.
Molecular Determinants of Acute Inflammatory Responses to Biomaterials 213
presently think that the phagocyte integrin Mac-1 (Cd11b/ intraperitoneal surface phagocytic cells are obviously pref-
cd18) is required for this interaction. This is partly because erentially accumulated on the implant. We have assumed
earlier investigators had shown that the critical fibrin(ogen) that certain chemokines might participate in attracting the
epitope, P1, is recognized by Mac-1.87 As a partial test of incoming phagocytic cells toward implant surfaces. A num-
this hypothesis, we employed recombinant neutrophil in- ber of chemokines have been reported to foster the recruit-
hibitory factor (hereafter, ‘NIF’), which blocks Cd11b-de- ment of inflammatory cells in a variety of inflammatory dis-
pendent binding of Mac-1 to fibrinogen (which presum- eases.102-105 In searching for possible candidates among many
ably occurs through the P1 epitope) without affecting other potential chemokines we employed reverse transcript poly-
Mac-1 integrin functions such as binding to coagulation fac- merase chain reaction to estimate levels of chemokine mRNA
tor X.96,97 Indeed, when mice were given repetitive injec- produced by implant adherent cells. We observed high lev-
tions of NIF before and during implantation, phagocytes els of mRNA production for two particular chemokines:
failed to accumulate on fibrinogen-coated surfaces. Further macrophage inflammatory protein-1# (MIP-1#) and mono-
support for the importance of Mac-1 in these phagocyte- cyte chemoattractant protein-1 (MCP-1), both of which
implant interactions derives from recent experiments in appear to be involved in phagocyte-implant interactions.
animals lacking this integrin. In Cd11b knockout (KO) and Neutralizing antibodies to both MIP-1# and MCP-1,
CD18 KO mice, the ‘normal’ accumulation of phagocytes but not to MIP-1! and MIP-2, block 70-90% of the accu-
on implant surfaces fails to occur. Interestingly, this impair- mulation of both macrophages and neutrophils on implant
ment of phagocyte accumulation is not caused by decreased surfaces. However, the numbers of recruited phagocytes do
phagocyte recruitment; the numbers of macrophages and not differ amongst control and treated groups.106 Therefore,
neutrophils in peritoneal lavage fluids following implanta- it is likely that the reduction in numbers of adherent phago-
tion in both types of KO animals are roughly equivalent to cytes is caused by impairment of directed phagocyte chemo-
those in wild type controls.98 taxis (although other interpretations are possible at the
Thus, the Mac-1 integrin appears to be crucial in the moment). Indeed, these results are in accord with prior stud-
adhesion of inflammatory cells to implant surfaces. How- ies indicating that MIP-1# and MCP-1 contribute to inflam-
ever, unstimulated phagocytes normally express only low matory and immune tissue responses via promotion of
levels of surface Mac-1 and most of this is in the ‘low-affin- chemotaxis and activation of both phagocytes and T lym-
ity’ state. This raises the question of what factors might phocytes.107-112 Because many different types of cells are able
prompt the apparent upregulation of this phagocyte integrin. to generate either MIP-1# and MCP-1,109,113,114 it is still not
Earlier studies indicated that TNF-# is a potent stimulator clear which cell type is responsible for the production of
of the expression of Mac-1 on cell surfaces.99-101 Further- MIP-1# and MCP-1 in the course of biomaterial-mediated
more, by ELISA assays, we have found that TNF-# is present inflammatory responses.
in peritoneal lavage fluids from implant-bearing mice within Thus, in interim summary, we are gaining some idea
four hours after implantation (unpublished results). This of the mechanisms involved in implant surface-protein
suggested the possibility that TNF-# might be critical in interactions and in phagocyte chemotaxis towards, and
upregulating Mac-1 integrin expression on incoming adherence to, tissue contact biomaterials. However, the
phagocytes. In at least partial support of this possibility, we processes involved in the critical initial step—phagocyte
find that administration of neutralizing anti-TNF-# anti- transmigration through the endothelial barrier—
bodies to implant-bearing mice blocks phagocyte accumu- remain undefined.
lation on implant surfaces. Furthermore, as was the case with
the Mac-1 KO mice described above, we find that neutraliz- Phagocyte Transmigration Through the Endothelial
ing antibodies against TNF-# do not affect the extent of Barrier
phagocyte recruitment to the site of the implant because the For a number of reasons, we hypothesized that the
numbers of recruited cells are roughly equivalent in treated release of histamine might be important for the recruitment
and control animals.98 of inflammatory cells to implantation sites. The most direct
Therefore, the adhesion of inflammatory cells to im- evidence favoring this idea was the observation made by us
plant surfaces appears to depend on three separable events: and others115 that the tissue surrounding biomaterial im-
1. Precedent adherence of fibrin(ogen) accompanied plants is often hyperemic and edematous, features of a clas-
by exposure of the P1 epitope; sical histaminic response. In order to test the possible in-
2. The release of TNF-# in response to implantation; volvement of histamine, we administered the histamine re-
and ceptor antagonists pyrilamine (an H1 receptor antagonist)
3. Consequent TNF-#-mediated upregulation of Mac-1 and famotidine (an H2 receptor antagonist) to animals re-
on incoming phagocytes. ceiving implants. When given separately, neither receptor
antagonist significantly diminishes the accumulation of in-
Phagocyte Chemotaxis Toward Implant Surfaces flammatory cells on implant surfaces. However, combined
The accumulation of phagocytes on implant surfaces treatment of implant recipients with both H1 and H2 re-
is clearly not a random event. In the peritoneum of an ani- ceptor antagonists significantly reduces the numbers of
mal implanted ~16 h previously, ~20% of total neutrophils phagocytes on implant surfaces and the numbers of mac-
and ~50% of total macrophages are found on the implant rophages and neutrophils recruited de novo to the perito-
surface (as opposed to being recovered in peritoneal lavage). neal cavity (down to values of 50% and 40% of untreated
Inasmuch as the implant represents only ~2% of the total
214 Tissue Engineering of Prosthetic Vascular Grafts
controls, respectively).116 We tentatively conclude that his- 1. Implants (and blood-contact devices) made of hy-
tamine enhances the transmigration of phagocytes through drophobic polymers almost instantly acquire a layer
the endothelial barrier via both H1 and H2 receptors. of spontaneously adsorbed host protein;
Mast cells are the most likely source of the histamine 2. The adsorbed protein gradually ‘denatures’;
which may be involved (although macrophages also are re- 3. The presence of the implant is sensed by the host
ported to produce and release histamine).117-119 Indeed, the either through mechanical properties of the mate-
peritoneal space contains relatively large numbers of mast rial itself or through the presence of these ‘denatured’
cells (2-5% of total lavage cells)8,120 and mast cells have the surface proteins;
largest amounts of stored histamine, which can be released 4. Guardian cells—especially mast cells—are activated,
in a short period of time. The importance of mast cells in releasing histamine and, perhaps, other pro-inflam-
the pathogenesis of other types of inflammatory responses matory products;
has been investigated by others using mast cell deficient mice 5. The resultant histaminic response promotes the dia-
(WBB6F1-Wv/W).121-123 In part, the results indicate that pedesis of phagocytic cells across the endothelial
mast cell degranulation is important in neutrophil recruit- barrier;
ment,121 macrophage activation,124 IL-1 production by 6. Acting on the instructions of particular cytokines (es-
monocytes and macrophages,125 fibroblast proliferation and pecially MIP-1#, MCP-1 and TNF-#) the incoming
collagen accumulation,126 fibrin deposition,123 and, not sur- phagocytes migrate towards the implant whilst si-
prisingly, the Arthus reaction.123 In fact, several years ago, multaneously upregulating surface adherence mol-
Christenson and coworkers8 made the prescient suggestion ecules such as Mac-1;
that mast cells might play a key role in the inflammatory 7. Upon encountering the surface of the implant, the
responses to intraperitoneal implants. phagocytic cells bind to the proteinaceous coat of
We therefore tested the possible importance of mast the implant, a binding facilitated by interactions be-
cells in phagocyte recruitment using mast cell deficient tween Mac-1 and a small 13 amino acid epitope dis-
mice.116 After intraperitoneal implantation for 16 h, far fewer played by adsorbed fibrinogen.
PMN (< 50% of normal) and macrophages/monocytes And then? Clearly, a great deal more work will be re-
(< 30% of normal) accumulated on implant surfaces in mast quired to unravel the intricacies involved in the above steps
cell deficient mice than in their congenic controls. Further- and, as importantly, the nature of subsequent responses
more, as was the case with normal animals treated with com- which may eventuate in chronic inflammation and fibrosis.
bined histamine receptor antagonists, the reduction of the Nonetheless, this knowledge, once won, should eventually
numbers of adherent phagocytes is accompanied by greatly enable the purposeful engineering of materials with vastly
decreased recruitment of phagocytes to the peritoneum. improved biocompatibility.
Therefore, the results obtained with two different models in
which histamine-dependent signaling events are disrupted— References
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Taming of Adverse Responses
Prevention of Fibrosis
CHAPTER 21
The Accumulation of Inflammatory Mediators:
A Target for the Prevention of Fibrosis
John Zagorski, Sharon M. Wahl
Introduction
T he primary purpose of the mammalian immune response is to eliminate foreign objects
from the body. Most typically, immune surveillance is directed against pathogenic mi-
croorganisms that have entered the body via the respiratory or gastrointestinal tracts or
through inadvertent ruptures in epithelial tissues. Circulating leukocytes are rapidly recruited
from the circulation in response to infection and accumulate at the afflicted site. There,
leukocytes release a variety of microbicidal compounds including reactive oxygen interme-
diates (ROIs) such as superoxide anion, hydroxyl radicals and hydrogen peroxide which are
toxic to bacteria.1-5 These cells also play an important early role in the resolution of the
trauma that usually accompanies infection. Wounds or ruptures in epidermal or epithelial
tissue must be repaired to restore the integrity of the tissue. However, prior to repair, the
debris associated with the wound must be sequestered and cleared. Leukocytes are highly
secretory cells and, when activated, release a variety of strong proteolytic enzymes including
elastase,6-8 collagenase9-10 and members of the cathepsin family.11-13 Digestion of the tissue
debris during wound clearance is essential for remodeling of the wound site and its subse-
quent re-epithelialization and repair.
Killing of bacteria by neutrophils via chemical secretion and by phagocytosis consti-
tutes an early phase of the host inflammatory response. Subsequent migration of mono-
cytes and lymphocytes adds to the host response. Monocytes not only produce microbicidal
molecules and proteases, but are also prodigious producers of pro-inflammatory cytokines
and growth factors including interleukin-1 (IL-1) and tumor necrosis factor (TNF),
transformtin growth factor-b (TGF-!), members of the fibroblast growth factor (FGF) fam-
ily and numerous chemotactic factors including members of the chemokine superfamily.14-16
Indeed, the pivotal role of monocytes in wound repair is emphasized by the observations
that wound repair proceeds when other cell types are eliminated from experimental ani-
mals, but is retarded when monocytes are selectively removed.17-21 Monocytes thus play a
dual role in wound resolution by secreting inflammatory mediators that act directly at the
wound site to promote repair and regrowth of the surrounding tissue, but also are the key
cell type in orchestrating recruitment of additional cells, both myeloid and stromal. It is
therefore not surprising that dysregulation of the repair process, the subject of this review, is
in large part a consequence of impaired monocyte function.
Expansion of the inflammatory response can be a dangerous process. While extensive
recruitment of immune cells to a site of inflammation is intended to promote elimination
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler
©1999 R.G. Landes Company.
220 Tissue Engineering of Prosthetic Vascular Grafts
of microorganisms or antigen removal and subsequent tis- vated endothelial cells while E- and P-selectins, expressed
sue healing, a prolonged response invariably begins to dam- on the surfaces of activated endothelial cells, bind similar
age the inflamed tissue.22 Thus, chronic inflammation is al- ligands on circulating leukocytes37-39 (Fig. 21.1). Leukocyte
most always deleterious, and may in fact be viewed as a pa- rolling on activated endothelia most likely allows the leuko-
thology distinct from the transient and generally benefi- cytes time to survey the local environment for additional
cial acute inflammatory response.23 Expansion of the im- pro-inflammatory stimuli without requiring firm adhesive
mune response is mediated in large part by cytokine net- interactions that might commit the leukocyte to remaining
works which have evolved to regulate inflammation, with at that site. These additional stimuli may in large part be
numerous pro-inflammatory proteins functioning to pro- contributed by locally expressed chemoattractants, prima-
mote, and equally numerous immune-suppressive proteins rily members of the chemokine superfamily.40-43 Exposure
acting to attenuate, the inflammatory response. So long as a of rolling leukocytes to sufficiently high concentrations of
proper balance of pro- and anti-inflammatory mediators is chemoattractants causes a marked alteration in the adhe-
maintained within traumatized tissue, normal clearance of sive interactions between leukocytes and endothelial cells.
pathogens and tissue repair proceed. Dysregulation of the Selectin-mediated adhesion is dramatically reduced, in part,
cytokine balance, by various mechanisms, is at the core of by proteolytic shedding of L-selectin from the leukocyte
persistent, chronic inflammation, leading to the seemingly surface.44 However, prolonged expression of endothelial P-
contradictory results of tissue destruction and selectin has been observed.45
hyperproliferation (fibrosis).24-26 Concurrent with loss of selectin-mediated adhesion,
The grafting of an artificial prosthesis within normal leukocytes begin the second step in vascular emigration,
tissue, the deliberate placement of a foreign object within a adherence to endothelium via tight integrin-mediated in-
body, may be considered as several distinct and deliberate teractions.46-49 The three leukocyte integrins, LFA-1, Mac-1
pathological insults. First, the surgery involved is a controlled and p150,95, are heterodimeric proteins containing a shared
wound which must be managed in such a way as to maxi- !2 subunit (CD18) and one of three different # subunits, #L,
mize normal wound healing. Second, some degree of infec- (Cd11a), #m (Cd11b) or #x (CD11c). Among the multiple
tion often accompanies the grafting procedure and an acute other integrins present on leukocytes, !1 integrins can inter-
inflammatory response is then inevitable. Lastly, and most act with both endothelial cells and extracellular matrix mol-
importantly, the emplacement of a prosthetic device into a ecules.46 For example, #4!1 (VLA-4) receptors bind to re-
host may be interpreted by the host immune system as an gions of fibronectin, but also recognize VCAM, the corre-
immense “pathogen”, and may initiate chronic rejection at- sponding ligand on endothelial cells. Integrin-mediated leu-
tempts by effector cells of the innate and/or adaptive im- kocyte-endothelial adhesion is, at least in part, directly in-
mune system. Ideally, the process of grafting should not in- creased by the actions of chemoattractants on leukocytes.48,49
terfere with the normal acute inflammatory response, but This helps to insure that as leukocytes lose selectin-medi-
also should not evoke such a prolonged and unresolvable ated adhesive interactions in the intimate proximity of en-
stimulus that a chronic response spins out of control. As dothelial cells, a second type of adhesion is rapidly initiated.
such, it is critical that the prosthesis be constructed in such Endothelial ligands for the leukocyte integrins include the
a manner that it evokes as little recognition by the immune ICAM family of immunoglobulin-like adhesion molecules
system as possible. In particular, the biomaterials used in and VCAM.50-51 These molecules are differentially regulated
the construction of the prosthesis should be inert in the sense on the endothelial cell surface and are upregulated by such
that they do not promote activation of immune cells.27-28 In pro-inflammatory mediators as IL-1, TNF-# and IFN-&.
this review we will attempt to review the mechanisms by Integrin-ICAM and -VCAM adhesive interactions serve to
which inflammatory responses become prolonged and at- secure the leukocytes at the inflammatory site and also trans-
tempt to identify pro-inflammatory mediators which may duce signals which are necessary for migration through the
be attractive targets for mitigating some of the side effects endothelial monolayer and underlying basement membrane,
of prostheses and preventing fibrosis. the final step in emigration (diapedesis)52,53 (Fig. 21.1).
Inflammation Regulation of Leukocyte Recruitment
Emigration of circulating cells from the vasculature The long established paradigm that neutrophils are
to the tissues is a hallmark feature of inflammation. The recruited early in inflammation while mononuclear lympho-
mechanism of this emigration has been proposed to occur cytes and monocytes are recruited later strongly suggests that
via a three step model (Fig. 21.1).29 In the initial step, circu- chemotactic signals unique for each cell type are generated
lating leukocytes adhere transiently to activated endothelial near the inflammatory site in a temporally restricted man-
cells in the proximity of the emerging inflammatory site. ner.54 However, until relatively recently, cell type specific
When this process is observed by intravital microscopy, leu- chemotactic factors had not been identified. The classic
kocytes are seen to adhere to endothelial surfaces, roll along chemoattractants, complement fragments, leukotrienes and
the surface, detach and then re-attach, sometimes within a bacterial N-formyl peptides, as well as TGF-!, are chemo-
single microscope field.30-32 Rolling is mediated by comple- tactic for a wide variety of leukocytes and are not selective
mentary adhesion molecules expressed on the leukocytes and chemoattractants for promoting the accumulation of spe-
the activated endothelial cells, including members of the cific cell populations at any stage of inflammation.55,56
selectin family.33-36 L-selectin is expressed on the surface of Rather, it is more likely that these factors serve as pleiotro-
rolling leukocytes and binds carbohydrate ligands on acti- pic distress signals, initiating the recruitment of a spectrum
Inflammatory Mediators: A Target for the Prevention of Fibrosis 221
Fig. 21.1. Sequential events in re-
cruitment and activation of circu-
lating leukocytes at sites of inflam-
mation. In response to inflamma-
tory signals, leukocytes in the blood
vessel adhere and roll through
selectin-mediated attachment, and
then via a firmer integrin-ligand in-
teraction commence diapedesis
from the vessel into the tissue.
Within the inflammatory site, the
neutrophils (PMN), monocytes
(M2) and T lymphocytes (T) are ac-
tivated to generate additional
chemoattractants and cytokines to
augment the immune response. In
turn, cytokines and growth factors
promote fibroblast recruitment,
proliferation and extracellular ma-
trix generation essential for wound
healing. Excess cellular infiltration
and production of these molecules
eventuates in tissue fibrosis and
pathogenesis. Adapted from
Zagorski and Wahl, Encyclopedia of
Immunology, 1998.113
of immune cells from the vasculature. It is clear, however, humans, the CC gene cluster is located on chromosome 17,
that mechanisms do exist for the recruitment of specific cell while the CXC cluster is located on human chromosome 4.43
populations, since this has been observed histologically in It seems likely that the various members of each family arose
many pathological conditions.57-59 through recombination-mediated gene duplication of single
Within the past decade, a new group of related ancestral CC and CXC progenitors, a hypothesis with inter-
polypeptides, the chemokines, has emerged as a major source esting implications concerning the evolution of the mam-
of chemotactic activity responsible for promoting immune malian inflammatory response.
cell recruitment in numerous inflammatory pathologies.40-43 Members of the CXC chemokine family include IL-8,
Chemokines are a large superfamily of related proteins char- MIG, NAP-2, IP-10, ENA-78, and the three GRO/MIP-2 pro-
acterized by their close similarities in size and protein ter- teins (GRO#, !, and &). The CC chemokines are a somewhat
tiary structure. This superfamily draws its name from the more diverse family and include MCP-1, -2, -3, -4 and -5,
most important characteristic of the individual proteins, Rantes, I-309, Eotaxin-1 and -2, and MIP-1 #, ! and &
namely, that they are chemotactic cytokines. An additional (Fig. 21.2). The advent of high throughput DNA sequenc-
hallmark of the chemokine superfamily is the presence in ing continues to add putative chemokines to the databases
each protein of four positionally conserved cysteine residues. far faster than they can be characterized for function, and
The organization of the cysteines allows a convenient and the list above is only partial. While most of the current rep-
functionally significant subdivision of the proteins into two ertoire of chemokines were first isolated as cDNAs, either
major chemokine families (Fig. 21.2). In the !-chemokines, from specific or subtracted library screens or from random
these first two cysteines are adjacent (“CC”) while in the sequencing, many of the chemokine proteins were first pu-
#-chemokines the first two cysteines are separated by a single rified by traditional biochemical methods based on their ac-
apparently nonconserved amino acid (“CXC”). The genes tivities.60-63 For two very recent, but rapidly outdated, com-
for CC and CXC chemokines are arranged in clusters and in pilations of chemokines and chemokine ESTs (expressed
222 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 21.2. Chemokine receptors
and their ligands. Selective ex-
pression of chemokine recep-
tors on polymorphonuclear
neutrophils (PMN) and mono-
nuclear cells (MNL) and the
chemokines which specifically
interact with these receptors.
Adapted from Zagorski and
Wahl, Encyclopedia of Immu-
nology, 1998.113
sequence tags) the reader is referred to Wells and Peitsch42 cells that have migrated through the membrane and become
and Baggiolini et al.43 adherent to its basal surface, or by recovering cells floating
In general, CXC chemokines are chemotactic for neu- in the basal chamber medium.75
trophils while CC chemokines are active on mononuclear Although an oversimplification of what happens in
cells, predominantly monocytes and T lymphocytes and, for vivo, this assay has provided the foundation for understand-
some chemokines, basophils and eosinophils.64-69 It is this ing inflammatory cell recruitment. However, it has long been
cell type selectivity that separates the chemokines from the recognized that one of the properties shared by chemokines
more “traditional” chemoattractants such as complement (and many other proteins) is their affinity for charged mac-
fragments, leukotrienes and bacterial fMLP and that has romolecules. Most chemokines are positively charged pro-
stimulated research on them. In fact, some chemokines have teins and bind with high affinity to negatively charged bio-
exquisite cell type specificity as evidenced by the activities logical macromolecules such as heparin.43 This physical
of the CC chemokines, MIP-1# and MIP-1!. Both of these property of chemokines makes it unlikely that chemokines
proteins attract monocytes but also selectively attract dif- are always found in a “soluble” form at pro-inflammatory
ferent populations of T cells, with MIP-1# active on CD8 sites in vivo. Rather, it is becoming more obvious that
positive cells but MIP-1! active on CD4 positive T cells.70,71 chemokines in vivo may be bound by extracellular matrix
In contrast, the recently reported CC chemokine DC-CK1 (ECM) components and on the surfaces of cells, where they
is chemotactic for naive, CD45RA positive T cells,72 unlike are “presented” to passing leukocytes.76 This phenomenon,
other T cell active chemokines like MIP-1# and MIP-1!, the migration of cells along a gradient of immobilized
which only recruit activated T lymphocytes. The specifici- chemoattractants, is commonly referred to as haptotaxis. The
ties exhibited by chemokines in in vitro assays support the soluble phase chemotaxis induced by chemokines in vitro
numerous observations demonstrating cell type specific re- may only be an approximation of the haptotaxis that is ac-
cruitment in vivo, and offer them as strong candidates for tually generated by solid phase gradients in vivo.
mediators in the associated pathologies. It is possible that The likely physiological significance of chemokine
the unique combination of specificity and redundancy adherence on tissue ECM has important implications for
within the chemokine superfamily may allow therapeutic prostheses. A priori, polymers used for the construction of
inhibition of single chemokines to be efficacious in treating prosthetic devices dictate the electrostatic charge of the pros-
human inflammatory disease without simultaneously sup- thesis in vivo.27 Studies have demonstrated that charged
pressing immune surveillance. polymers are differentially coated with plasma proteins when
Chemotactic activities are usually measured in in vitro implanted into animals and that the kinetics of protein
assays. While many devices are currently used, most are deri- deposition do not necessarily reflect the relative plasma
vations of the Boyden chamber.73,74 However, the physical abundance of the adhering proteins.27,77 Thus, implanted
nature of chemotactic gradients in vitro and in vivo may be polymers are not necessarily preferentially coated with the
very different, leading to some profound physiological con- most abundant serum proteins, namely, albumin
sequences. In Boyden chamber type assays, leukocytes in an and immunoglobulins.
upper chamber are separated from the putative chemo- Circulating or locally expressed proteins with high
attractant in a lower chamber by a polycarbonate membrane affinity for a synthetic matrix may preferentially deposit on
containing 3 ∝m-8 ∝m diameter pores. The membrane mim- the matrix even if they are present at relatively low concen-
ics an endothelial cell layer, separating the leukocytes from trations. Simplistically, since many biopolymers are nega-
the source of chemoattractant and minimizing random cell tively charged, and most chemokines are positively charged,
migration. Cells that migrate in response to the chemo- it is reasonable to speculate that an implanted polymer may
attractant are usually quantitated by staining and counting become coated with significant amounts of chemokine pro-
Inflammatory Mediators: A Target for the Prevention of Fibrosis 223
tein, either directly or indirectly via secondary interactions. response with the generation of IL-4 and IL-10, which can
This effect could easily generate very high and persistent local dampen immune functions.80 Thus, glucocorticoids act as
haptotactic gradients, resulting in sustained leukocyte re- genetic switches, turning off or on entire sets of genes which
cruitment from adjacent microvascular endothelia. Worse control inflammation81,82 and cellular proliferation.83 Un-
still, the very high densities of adherent chemotactic pro- fortunately, chronic use of glucocorticoids is associated with
teins that may decorate implanted biopolymers are likely to severe side effects,84 including antifibrotic effects which may
strongly activate freshly recruited cells (see below). Since the impair wound healing.
biopolymer “matrix” to which the chemokines may become The other major category of drugs, the NSAIDs, in-
immobilized is not likely to turn over, implanted biopoly- hibit the cyclo-oxygenase pathway to reduce biosynthesis of
mers may become “sinks” for concentrating chemotactic fac- prostaglandins, with resulting anti-inflammatory, and an-
tors, resulting in indefinite leukocyte recruitment. It is this algesic, consequences.85,86 Though useful for the treatment
persistence of leukocyte recruitment and activation which of minor inflammatory disease or as part of a combination
results in overproduction of cytokines and growth factors, therapy, NSAIDs such as aspirin, indomethacin, aceta-
leading to chronic inflammatory diseases and fibrosis minophen, and ibuprofen, are relatively weak, and when used
(Fig. 21.1). Although these growth factors are essential for at high doses can have toxic gastrointestinal and renal side
recruitment of fibroblasts, fibroblast proliferation, and ma- effects.87
trix synthesis in the induction of tissue repair, excess quan- There is no doubt that powerful new anti-inflamma-
tities of such factors in the microenvironment of a chronic tory agents are needed which combine the potency of glu-
inflammatory lesion are causative in pathologic fibrotic cocorticoids without the negative side effects. Some such
sequelae. products are now emerging as a consequence of the suc-
cesses of molecular biology and biotechnology over the past
Mitigation of Recruitment two decades. The systematic dissection of the mammalian
Termination of the inflammatory response is as im- immune system has led to the identification of many pro-
portant as its initiation, and the persistence of chronic in- and anti-inflammatory cytokines with clinical potential. In
flammatory states may best be considered as a failure to addition, the numerous adhesion molecules that are a pre-
modulate the immune response rather than its uncontrolled requisite for both normal and aberrant migration of leuko-
initiation. It is notable in this respect that most chemo- cytes between lymphoid and systemic circulatory pathways
attractants have inherent self-limiting properties. In general, and between the circulation and tissues make attractive tar-
the chemotactic dose-response curve for most chemo- gets for synthetic inhibitors.
attractants is bell shaped, and as protein concentration in- An obvious approach for suppressing inflammation
creases beyond a maximum effective concentration, chemo- is by inhibiting leukocyte recruitment to the inflammatory
tactic activity decreases. However, at these higher concen- site. The “three step” model leading to leukocyte diapedesis
trations, chemoattractants tend to activate their target cells, discussed earlier offers several options for intervention. It
inducing in neutrophils, for example, degranulation, oxida- might be possible to inhibit the rolling of cells along acti-
tive burst and increased adhesiveness. Chemotactic dose vated endothelia, thus preventing any possibility of subse-
dependence may automatically trigger cells responding to a quent adhesion and chemotaxis. Synthetic analogs of the
local chemotactic gradient to cease migration near the in- carbohydrate ligand for L-selectin have shown promise in
flammatory site and commence their effector functions preclinical models of acute inflammation.88-90 However,
(Fig. 21.1). Combining chemotactic and activating functions selectins are crucial for normal recirculation of leukocytes
into single molecules may ensure a smooth transition from between the lymphoid and systemic circulations, and anti-
one phenotype to another as leukocytes migrate up the selectin drugs may adversely perturb this process. Integrins
chemokine gradient to their intended destination. This po- are also excellent candidates as targets for inhibitors. Reduc-
tential adaptive advantage is, however, offset by the conse- ing the strong adhesion between leukocytes and endothelial
quence that local conditions supporting leukocyte recruit- cells necessary for diapedesis should limit cellular recruit-
ment and activation may become self-sustaining. ment into inflamed tissue, and many studies have supported
There exists in the current pharmacopia of medicine this concept in vivo.46,91,92 In this regard, synthetic peptides
a wide variety of drugs used for the control of inflamma- derived from the A chain of fibronectin have been shown to
tion. The most commonly used and effective anti- not only block integrin-dependent adhesion,91,92 but also to
inflammatories can probably be assigned to one of two cat- interfere with matrix augmented cytokine signal transduc-
egories, glucocorticosteroids and nonsteroidal anti-inflam- tion and cellular activation. 93 These promising anti-
matory drugs (NSAIDs).78 Steroids, such as prednisolone inflammatory peptides have proven beneficial in animal
and dexamethasone, are very powerful anti-inflammatory models of arthritis, autoimmunity91,92,94,95 and liver fibrosis
agents that exert their effects through regulation of gene (Wahl SM, unpublished data).
expression. Many pro-inflammatory cytokine genes contain Another possibility for suppressing cellular recruit-
glucocorticoid responsive elements within their promoters ment might be to target chemotactic factors themselves, in
which contribute to the regulation of their expression.79 particular by identifying chemoattractant receptor antago-
Exogenous administration of high levels of steroids as thera- nists. If a specific chemokine can be identified as the major
peutics suppresses gene expression of these cytokines, with chemoattractant associated with a given disease, antagonists
the obvious result of limiting accumulation of the proteins of that chemokine might demonstrate efficacy without sig-
themselves. Glucocorticoids can also promote a Th2 cytokine nificant adverse effects or immune suppression. The latter
224 Tissue Engineering of Prosthetic Vascular Grafts
hypothesis derives from the obvious redundancy among shown promise in animal studies. An MCP-1ra has been used
members of the chemokine superfamily, and assumes that to successfully ameliorate experimental arthritis in mice,107
any systemic immune defect caused by specific inhibition and a CINCra (cytokine-induced neutrophil chemo-
of one chemokine will be obscured by the remaining, over- attractant, a rat CXC chemokine) reduced peritoneal inflam-
lapping activities of other chemokines. mation induced by injection of zymosan.106 The specificity
The specificity of chemokines for their respective tar- that chemokine receptor antagonists predictably inherit from
get cells is determined by the relative distribution of their normal counterparts might initiate a new paradigm
chemokine receptors expressed by the various populations for anti-inflammatory selectivity, which is desirable for any
of myeloid cells. Not surprisingly, there exist separate re- new generation of drugs. The need for specificity is exem-
ceptors for the two families of chemokines, CXC receptors plified by observations that similar CXC chemokines such
and CC receptors.96-98 Interestingly, the chemokine recep- as IL-8 (ELR-containing) and IP-10 (non-ELR) exert op-
tors display a wide range of specificities for the chemokines posing influences in idiopathic pulmonary fibrosis, with IL-8
themselves, with some receptors binding a single ligand and promoting, and IP-10 suppressing, angiogenic activity.108
others binding many ligands within a chemokine family. A simpler, but perhaps less obvious approach toward
When all the data are combined, they reveal the existence of antagonizing chemoattractants derives from the spatial re-
networks of redundant ligands and receptors for all types of strictions necessary for generating gradients. In vivo, endo-
myeloid cells, suggesting that recruitment of specific cell thelial layers and basement membranes serve a barrier func-
types in inflammation is dependent on temporal and spa- tion between leukocyte and chemoattractant to generate a
tial sequestration of chemokine expression. chemotactic gradient. With leukocytes circulating in the
Two neutrophil receptors have been identified for CXC vasculature and chemoattractants originating in the tissues,
chemokines which have very different properties (Fig. 21.2). the gradient flows inward towards the blood. It follows that
Both receptors were originally characterized as receptors for by intentionally increasing the concentration of chemo-
IL-8 and were thus named type A and type B IL-8 receptors. attractant in the blood, local gradients associated with in-
However, the type B receptor binds several other CXC flammatory sites should collapse. Simply put, administer-
chemokines besides IL-8, including GRO#, ! and &, NAP-2 ing chemokines or other factors intravenously should have
and ENA-78. In contrast to the type B receptor, the type A a paradoxically anti-inflammatory effect. Some data support
IL-8 receptor is selective for IL-8 and binds other CXC- this concept. Systemic administration of the potent
chemokines with approximately 100-fold lower affinity. chemoattractant TGF-! has been shown to reduce arthritis
Binding of CXC-chemokines to both IL-8 receptors is me- in rats, whereas direct tissue injection of the cytokine into
diated largely, but not entirely, by the important sequence joints aggravated the disease.109,110 Similarly, systemic ad-
motif “ELR” which is located immediately upstream of the ministration of the CC chemokine MCP-1 protected mice
signature CXC domain of these chemokines. Significantly, from lethal endotoxemia, although the mechanism of ac-
the three CXC chemokines that do not bind the type B re- tion was clearly complex, since the treatment notably altered
ceptor, IP-10, MIG and PF4, all lack the ELR domain. plasma TNF-#, IL-10 and IL-12 levels as well.111 Thus, by
The family of CC chemokine receptors is far more blocking the recruitment of leukocytes from the circulation
complicated than that for the two IL-8 receptors. At least to tissue sites of persistent infection, injury and/or implan-
eight receptors for CC chemokines have been identified on tation, it may be possible to attenuate the cascade of events
various mononuclear leukocytes and are designated CCR1 responsible for chronic inflammation, tissue damage and
through CCR8.96-102 Each of the receptors is expressed on fibrotic repair. Once fibropathogenesis has developed, it be-
the surfaces of specific mononuclear cells and binds a spe- comes more difficult to effectively intervene.
cific subset of the CC chemokines, as shown in Figure 21.2,
although the receptors CCR6 and CCR7 and their ligands Summary and Perspectives
are very poorly characterized. Alone among the well char- Technology often outstrips our ability to understand
acterized CC chemokine receptors, CCR8 is apparently and adequately control it. Medical technology, and in par-
unique in having only one well defined ligand, the T lym- ticular surgery, is no exception. For instance, the techniques
phocyte chemoattractant I-309.102 of whole organ transplant were successfully developed dur-
The development of chemokine receptor antagonists ing the 1950s and 1960s, and are now common. However,
(ra) is at the earliest research stage. Structure-function analy- the barrier to successful transplantation was not the physi-
sis of CXC and CC chemokines has revealed that the N-ter- cal act of moving the organ from donor to recipient, but
mini of both families of chemokines are crucial for both convincing the recipient’s immune system to accept, or more
receptor binding and signal transduction.43 Interestingly, precisely ignore, the foreign tissue. It was the advent of im-
chemokine mutants containing N-terminal truncations have munosuppressive anti-rejection drugs like cyclosporin A that
been described which have the desired properties of high finally allowed the full potential of surgical transplants to
affinity receptor binding but negligible receptor activa- be realized.112 Yet, though we can sometimes fool the im-
tion.103-106 These chemokine receptor antagonists generally mune system into accepting our interventions, our level of
retain the receptor type specificities of their normal coun- control remains incomplete.
terparts, suggesting that they might selectively block a lim- The discovery and characterization of an ever increas-
ited number of chemokines in vivo. The potential efficacy ing number of cytokines and their receptors offers the pos-
of chemokine receptor antagonists in treating human dis- sibility that new drugs will be developed that can exercise
ease has yet to be demonstrated, but such proteins have more selective control over inflammation than is currently
Inflammatory Mediators: A Target for the Prevention of Fibrosis 225
possible. Such selectivity almost certainly offers the addi- 18. Hugo NE, Thompson LW, Zook EG et al. Effect of
tional possibility that future therapeutics will have more lim- chronic anemia on the tensile strength of healing wounds.
ited adverse effects to complement their efficacy. While it is Surgery 1969; 66:741-745.
unlikely that we will be able to completely discount drug 19. Simpson DM, Ross R. The neutrophilic leukocyte in
wound repair. A study with antineutrophil serum. J Clin
toxicity in humans, the increasing selectivity of anti-inflam-
Invest 1972; 51:2009-2023.
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Taming of Adverse Responses
Prevention of a Hyperplastic Intimal Response
CHAPTER 22
Pathobiology of Hyperplastic Intimal Responses
Erik L. Owens, Alexander W. Clowes
Introduction
U nderstanding the pathologic response of blood vessels to injury remains a primary re
search focus of the vascular surgeon and vascular biologist. In addition to learning more
about the primary atherosclerotic lesion, the ultimate goal of this effort is to favorably influ-
ence the healing process after intervention, thus allowing the re-establishment of a widely
patent lumen not narrowed by thrombus, intimal hyperplasia, or vasoconstriction.
This hyperplastic healing response has been established as a cause for angioplasty and
vascular graft failure.1-3 Although the available interventional modalities (stents, endovascular
grafts, etc.) and the anatomic locations in which they are used (cerebral angioplasty, coro-
nary stenting, etc.) continue to grow rapidly, no clinically proven method exists for prevent-
ing the late failure of these treatments by intimal hyperplasia.
In the peripheral vascular surgery literature, restenosis is a clinical term commonly
used to describe the loss of more than 50% of luminal diameter in a vessel or graft after
intervention.4 It was previously thought that intimal hyperplasia was the principal cause of
restenosis. We now believe that it is just one of several contributory components. Other
mechanisms responsible for restenosis include vasoconstriction and accumulation of mural
thrombus. Understanding the relationship between intimal hyperplasia and restenosis is
crucial to properly interpreting results from pertinent clinical trials and animal models.
Clinical Relevance
Percutaneous transluminal coronary angioplasty (PTCA) has an excellent primary
success rate (about 90-95%). However, approximately 30% of dilated coronary arteries de-
velop marked restenosis by 6 months.5,6 Coronary vein grafts have a 10 year patency of 50%.7,8
Infra-inguinal saphenous vein grafts have a 24% failure rate at 4 years.9 Polytetra-
fluoroethylene (PTFE) grafts in the same position have a 46% failure rate at 4 years.9 Renal
artery stenting appears to have a restenosis rate of about 44% at 1 year.10 These observations
are just a sampling of the known clinical data related to restenosis and graft failure after
therapeutic intervention. The failure of a vascular graft placed during reconstruction or the
development of restenosis in an artery that has previously undergone angioplasty (or stenting)
exposes the patient to the risk of significant morbidity and mortality. Repeat intervention or
vascular reconstruction is often the only remedy for the situation, further increasing the risk
for the patient. From an economic viewpoint, considerable costs are incurred as a result of
repeat vascular intervention. If one considers the frequency with which vascular procedures
are currently performed and the incidence of graft failure or arterial restenosis after inter-
vention, it becomes clear why such intense interest exists in understanding intimal hyperplasia.
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
230 Tissue Engineering of Prosthetic Vascular Grafts
The development of methods to successfully control inti- Models of Intimal Hyperplasia
mal hyperplasia promises both improved patient outcomes Most of what we know about intimal hyperplasia is
and overall cost effectiveness of our vascular interventions. derived from animal studies. The majority of this informa-
tion has been obtained from models in which the hyper-
Mechanisms of Restenosis in Human Vessels plastic healing response is observed after injuring normal
A clearer picture of the mechanisms of stenosis and vessels, usually with balloon dilation. The injury response
restenosis in humans is emerging. Restenosis is most likely depends on the type of vessel injured (artery (normal or
related to several factors, including the type of intervention diseased, elastic or muscular) or vein graft (new or old))
performed and the relevant risk factors for each patient. Until and the severity of the injury (vessel dilation, valvulotomy,
recently, our knowledge about restenosis in humans was arterial angioplasty, stenting, atherectomy, or endarterec-
largely based on the examination of pathologic specimens tomy). It is unlikely that each of these maneuvers invokes
at the time of reoperation. New technology that generates the same degree, or even type, of injury response.
morphologic images in a non- or minimally-invasive man- Intimal hyperplasia in grafts may be a very distinct
ner has allowed serial observations of restenotic lesions in form of healing. Vein grafts start with a normal cellular ar-
vessels, grafts, and stents. chitecture which we know then undergoes adaptive changes
After carotid endarterectomy, hemodynamically sig- upon exposure to the arterial circulation. Prosthetic grafts,
nificant restenosis detected by duplex ultrasound occurs in however, have only an acellular, fibrous scaffolding on which
about 20% of patients, although only a small percentage of a neointima forms.
these patients require reoperation.11 The endarterectomy Several studies performed in animals have suggested
specimens removed during reoperations within the first two that certain drugs prevent intimal hyperplasia. However,
years of the primary procedure have the histologic appear- when similar studies have been performed in human clini-
ance of intimal hyperplasia.12 Lesions removed after two cal trials, no reduction in restenosis was observed. This may
years resemble atherosclerotic plaque, not intimal hyperpla- be a result of the previously mentioned inability to equate
sia.13 Evidence exists that some lesions regress with time.11 intimal hyperplasia with restenosis. However, species-spe-
Autogenous saphenous vein grafts placed during lower cific responses also appear to exist and offer one more po-
extremity revascularization have been extensively studied. tential limitation of the current animal model data. 20,21,22
Early failure of these grafts (within 0-30 days of operation) It is clear that intimal hyperplasia is a complex pro-
is commonly related to either technical defects or inadequate cess that occurs in many different clinical settings and is in-
runoff. Hemodynamically significant stenoses requiring in- fluenced by a myriad of factors. Animal models have, how-
tervention occur in 30% of these grafts within two years and ever, identified common threads in the basic pathophysi-
are caused by intimal hyperplasia.2 Lesions in grafts failing ologic responses to vascular injury. In vitro studies per-
later than two years after placement, designated as long term formed in numerous vascular biology laboratories have also
failures, appear to result from a combination of other fac- contributed greatly to our current understanding. Success-
tors, including intimal hyperplasia, progression of the ful application of this knowledge to the human clinical set-
patient’s underlying disease, and atherosclerosis.14 ting remains the daunting challenge.
Intraluminal, tubular, slotted stents placed in the coro-
nary circulation inhibit the acute recoil and chronic remod- Arteries
eling of the vessel wall. Theoretically, restenosis in this set- Intimal hyperplasia develops after many types of in-
ting should result primarily from tissue proliferation. Stud- jury, including the removal of adventitia (which causes is-
ies using intravascular ultrasound (IVUS) have confirmed chemia in the media due to loss of blood supply from ad-
that in-stent stenosis is solely a result of intimal hyperpla- ventitial capillaries), electrical injury, external clamping,
sia.15,16 While the clinical applications for vascular stents are blunt trauma, chemical or endotoxin infusion, as well as lu-
still being investigated, it appears that stent restenosis serves minal damage by passage of a dilated balloon catheter.23 Most
as a pure model for investigating strategies to inhibit inti- of these forms of injury have limited clinical relevance.
mal hyperplasia. The severity of injury is important in determining the
When atheroma form, arteries can dilate to preserve ultimate pathophysiologic response.24-27 A classification sys-
arterial lumen size, a phenomenon called remodeling. Evi- tem based upon the immediate histologic effect of the injury
dence of remodeling was first reported in monkeys17 and might be considered.4
then in human coronary and carotid arteries.18 Remodeling Type I injuries result in no significant loss of the vessel’s
has been demonstrated in a hypercholesterolemic rabbit basic cellular architecture. There may be a slight change of
model,19 and may occur after angioplasty in human vessels. the endothelial architecture and associated cellular adhesion.
The ability of an artery or graft to remodel in response to The fatty streak, an early atherosclerotic lesion, can be
intimal thickening may determine whether graft stenosis or thought of as a type I injury. Hemodynamic factors and flow
restenosis after angioplasty in humans will occur. disturbances represent type I injuries because they produce,
Other processes may play significant roles in human at most, only a modification of the established
restenosis but are not easily studied in animal models. Such cellular architecture.
processes include vasospasm, dissection, plaque instability Type II injuries involve loss of the endothelial layer.
or rupture, and thrombosis. The internal elastic lamina (IEL) remains intact, and there
is little or no damage to the media. These injuries occur
Pathobiology of Hyperplastic Intimal Responses 231
during simple arterial catheterization, endovascular proce- model form the basis for the proposed three phase para-
dures, or vein graft preparation. A rat model of a type II digm for the development of intimal hyperplasia.
injury uses gentle filament endothelial denudation of the Table 22.1 summarizes many of the putative regula-
carotid artery.24 In areas of endothelial loss, platelets may tory factors and their proposed role in intimal lesion for-
adhere and begin to form a thrombus. mation or inhibition.
Type III injuries are inflicted upon countless human
vessels each day in the form of balloon angioplasty, endart- First Phase: Proliferation
erectomy, atherectomy, and other forms of surgical repair The first cellular event after injury is the proliferation
or reconstruction. A type III injury results in transmural of a proportion of medial smooth muscle cells (SMCs).
damage. The endothelium is removed. The IEL is often dis- Medial SMCs in a normal artery proliferate at a very low
rupted, and a significant portion of the medial cells are level (0.06% per day). As soon as 24 hours after injury,
killed.24,28 An inflammatory response is initiated within the 10-30% of the medial SMCs are induced to enter the growth
vessel wall. Upon loss of the endothelium, platelets deposit cycle.29,30 The proportion of proliferating cells remains high
and thrombus forms. for several days but by 4 weeks returns to baseline levels.
Early observations of the events surrounding vascular in-
Molecular Mechanisms of Intimal Hyperplasia jury resulted in the “reaction to injury” hypothesis which
after Injury implicated platelets in the development of intimal hyper-
Much has been learned about the basic pathophysiol- plasia. 31,32 This theory was based upon three
ogy of intimal hyperplasia from the response of a rat ca- key observations:
rotid artery to a type III injury. The model is a modification 1. Platelets appear on the luminal surface of injured
of a method described by Baumgartner.28 A small Fogarty vessels immediately;
embolectomy catheter is passed into the common carotid 2. Platelets deposit #%granules which contain numer-
artery via the external carotid artery. The balloon is inflated ous growth factors for cultured SMCs including
and pulled through the common carotid artery, denuding platelet derived growth factor (PDGF), transform-
the endothelium and damaging the underlying tissue. Pro- ing growth factor (TGF)-!, and epidermal growth
gressive luminal thrombosis does not typically occur. Inti- factor; and
mal thickening occurs within 2 weeks and reaches a maxi- 3. Thrombocytopenic animals experience less intimal
mum at about 3 months. Luminal area declines transiently, thickening after balloon injury.33-35
partly because of intimal thickening and partly because the While overall intimal thickening is reduced in throm-
injured vessel contracts. The histologic and molecular bocytopenic animals, the first wave of SMC proliferation is
changes that have been documented after injury in this rat unaltered.35 Additionally, balloon-injured vessels (a type III
Table 22.1. Molecular mechanisms of intimal hyperplasia after arterial injury
Potential Stimulatory Factors Proposed Role in Lesion Formation
Basic fibroblast growth factor (bFGF) Medial SMC proliferation
Platelet-derived growth factor (PDGF) SMC migration/ECM formation
Angiotensin ii (AII) Medial and intimal SMC proliferation/SMC
migration
Transforming growth factor (TGF)-! Intimal SMC proliferation/matrix formation
urokinase-like plasminogen activator (uPA) SMC migration
tissue-type plasminogen activator (tPA) SMC migration
Metalloproteinases (MMP)-2 and -9 SMC migration
Integrins SMC migration
Thrombin Promotes platelet aggregation/medial SMC
proliferation
Endothelin Medial SMC proliferation/produces
vasoconstriction
Insulin-like growth factor (IGF)-! Medial SMC proliferation
Catecholamines Intimal and medial SMC proliferation
Cytokines Intimal SMC proliferation
Potential Inhibitory Factors Proposed Role in Lesion Inhibition
Nitric oxide (NO) Blocks platelet aggregation/SMC proliferation and
migration/produces vasorelaxation
Prostacyclin (PGI2) Blocks platelet aggregation/produces vasorelaxation
Heparin-like molecules Limits thrombosis/intimal SMC proliferation/SMC
migration
Tissue inhibitor of metalloproteinases (TIMP)-1 SMC migration
Plasminogen activator inhibitor (PAI)-1 SMC migration
232 Tissue Engineering of Prosthetic Vascular Grafts
injury) develop more medial SMC proliferation and inti- isoforms of PDGF, PDGF-BB, and stimulation of the
mal thickening than filament-injured vessels (a type II in- PDGF-! receptor as the major migratory signal. While PDGF
jury).35 In both of these models, the endothelial layer is re- was initially isolated from platelets, all vascular wall cells in-
moved and platelets adhere to the exposed subendothelium, cluding endothelial cells, SMCs, and macrophages can ex-
releasing their cytoplasmic granules. Thus, platelet-related press this factor. PDGF is a strong mitogen in vitro, but its
effects in the two models should be similar. The fact that main effect in vivo appears to be on migration. (For a re-
more injury is inflicted upon the medial layer in the balloon view on PDGF, see ref. 60.)
injury model suggests that the specific stimulus for In vitro work in our lab has demonstrated a strong
proliferation is related to the degree of injury in this area. migratory response of baboon SMCs to PDGF-BB but not
The most logical conclusion is that a nonplatelet derived to PDGF-AA.61 Of the three possible isoforms, AA, BB, and
factor released by injury is primarily responsible for the AB, PDGF-BB is the predominant isoform in platelet #-gran-
initial wave of medial SMC proliferation and that the platelet ules in rat, pig, and baboon. Human platelets contain all three
factors must affect a portion of the process other isoforms in equal amounts.62 Since PDGF-BB is able to bind
than proliferation. to both the # and the ! form of the PDGF receptor and
Lindner and Reidy demonstrated that the initial stimu- PDGF-AA is only able to bind to the # receptor,60 this
lus for proliferation is most likely basic fibroblast growth strongly suggests that activation of the ! receptor plays a
factor (bFGF).36-38 In vitro, bFGF is a strong mitogen and is leading role in SMC migration.
released from disrupted cultured SMCs.39,40 Endothelial cells, When a polyclonal antibody to human PDGF is ad-
SMCs, and extracellular matrix all store various amounts of ministered to rats undergoing balloon injury, intimal thick-
bFGF.39 In the extracellular matrix (ECM), bFGF is stored ening is inhibited by 40%.63 Smooth muscle proliferation is
bound to heparan sulfate proteoglycans. Treatment with not affected. When the recombinant form of PDGF-BB is
heparin infusion has been shown to blunt the proliferative administered intravenously or intraarterially, SMC migra-
response, presumably by displacing the bFGF from the ma- tion is markedly stimulated and intimal thickening is in-
trix.41 Proliferating SMCs express the relevant receptor to creased.64 SMC proliferation in the media is only slightly
bFGF.38 SMC proliferation is significantly enhanced by the increased. In our lab, an antibody to the PDGF receptor-!
administration of bFGF and antibodies to bFGF reduce SMC (PDGFR-!) administered to baboons subjected to balloon
proliferation by approximately 80%.36,37,42,43 injury of the saphenous artery or PTFE graft bypass signifi-
The renin-angiotensin system may also play a role in cantly reduces intimal hyperplasia (unpublished results).
the early injury response.44 In vitro, angiotensin ii is a mito- Similar results have been obtained using specific antibodies
gen for SMCs.45 Additionally, AII induces the expression of to the PDGFR-! in baboons and in rats.62 Sirois et al have
other known mitogens.46-48 In vivo, AII receptor antagonists recently shown using immunohistochemical analysis that
and angiotensin converting enzyme (ACE) inhibitors deliv- both PDGF receptors (PDGFR-# and -!) are present in the
ered after injury each reduce SMC proliferation and inhibit normal artery wall and are markedly upregulated after in-
intimal thickening.49 SMC proliferation and intimal thick- jury. 65 Treatment with antisense oligonucleotides to
ening are enhanced by infusion of AII.50,51 PDGFR-! significantly decreases PDGFR-! expression and
Other growth factors might have roles in this early inhibits intimal thickening.
proliferative stage of lesion development including PDGF,52 The roles of PDGF-A chain and the PDGFR%# are not
thrombin,53 endothelin,54-56 insulin-like growth factor-1 yet clear. Animal studies using neutralizing antibodies to
(IGF-1),57 and catecholamines.58 The evidence for their in- PDGF-A have shown no observable effect during injury.62
volvement is, however, not as strong as that for bFGF or AII. In a Boyden chamber used to measure migration, activation
of the PDGFR-# by either PDGF-AA or -BB generates an
Second Phase: Migration inhibitory signal for baboon SMC migration.61 Analysis of
As early as 4 days after injury, SMCs begin to appear human fetal, normal adult, and atherosclerotic aortas sug-
in the intima.59 This results from migration of cells across gests a maintenance role for PDGFR-#, since levels of
the IEL, since normal rat intima contains no SMCs. This PDGF-A message ribonucleic acid (mRNA) by reverse tran-
process continues for up to one month. Only about 50% of scription-polymerase chain reaction (RT-PCR) were 100
migrating cells continue to proliferate.28, 30 times higher in normal vessels than either developing (fe-
Two distinct events must take place for cells to traverse tal) or diseased (atherosclerotic) vessels.66 The idea that
the IEL and enter the forming intima. First, there must be a PDGF-A is a “survival factor” has also been proposed.67-69
chemoattractant or some form of signal for the SMCs to In order for the SMCs to break their attachments with
initiate the journey. Secondly, the cells must be freed from the extracellular matrix, proteases are required. The act of
their surrounding matrix. Multiple factors are certainly detachment itself may serve to further activate the migra-
involved in the migratory process. Some may have dual tory process.35 Plasmin is the main fibrinolytic enzyme in
roles as both chemoattractants and as initiators of the body and is also able to degrade most matrix compo-
matrix detachment. nents. It is derived from plasminogen through the action of
As previously mentioned, platelet factors might play urokinase-like plasminogen activator (uPA) and tissue-type
a role in intimal thickening, perhaps by influencing SMC plasminogen activator (tPA).70 Urokinase-like plasminogen
migration. Platelet #-granules are known to contain PDGF. activator (uPA) is activated within 2-3 hours after injury.71,72
Most of the evidence now seems to point to one of the Tissue-type plasminogen activator (tPA) appears several days
Pathobiology of Hyperplastic Intimal Responses 233
later, around the same time as SMCs are seen entering the The role of SMC migration in the injured atheroscle-
intima.73 Tranexamic acid, a plasmin inhibitor, inhibits SMC rotic lesion is unclear, since SMCs are already present in dis-
migration after injury.74 Knockout mice studies reveal that eased human arteries. It does appear, however, from analy-
deletion of uPA reduces intimal thickening after injury. De- sis of atherectomy specimens, that migration might be the
letion of tPA has no effect and deletion of the naturally oc- predominant mode of intimal formation in human lesions.88
curring plasminogen activator inhibitor (PAI)-1 increases
thickening.75 We have observed that overexpression of PAI- Third Phase: Intimal Expansion
1 in the rat carotid inhibits intimal hyperplasia (unpublished The lesion continues to expand by SMC migration and
results). intimal proliferation. These cells also synthesize and secrete
A second class of enzymes also linked to SMC migra- matrix proteins. The largest contribution to lesion forma-
tion is the matrix metalloproteinases (MMP). The MMPs tion is made by the deposition of these matrix proteins in-
are a group of proteases found in the ECM. They digest col- cluding elastin, collagen, and proteoglycan.89 It would seem
lagen, elastin, fibronectin, laminin, and proteoglycans.76 Plas- logical that some of the factors present in early lesion devel-
min activates MMPs.77 These proteinases have been observed opment might play a role in intimal expansion. Some of the
in stimulated SMCs in culture and in SMCs from injured aforementioned factors, especially TGF-! and PDGF, are
arteries and atherosclerotic plaques. Hours after injury, known to stimulate ECM production.90 Unlike the earlier
MMP-9 (gelatinase b) appears and remains elevated for ap- stages, where a single factor appears to control activity, con-
proximately one week. Several days later, at about the same trol of the later expansion phase appears multifactorial in
time as tPA appears, MMP-2 (gelatinase a), which is present nature. No one stimulatory factor has yet been shown to
in normal artery walls, increases.72 Conditioned media con- predominate.
taining tissue inhibitor of metalloproteinases (TIMP)-1 (a Regarding lesion expansion, studies seem to indicate
natural inhibitor of MMP) completely inhibits the PDGF-BB that bFGF is unlikely to be involved in regulating intimal
induced migration of SMCs in vitro, a process that can be SMC proliferation. Antibodies to bFGF do not affect SMC
overcome by treatment with an antibody to TIMP-1. Local proliferation when administered 5 days after balloon injury
overexpression of TIMP-1 suppresses intimal thickening in of the rat carotid.91 Furthermore, no convincing evidence
the injured rat carotid model.78 In an explant model using exists that PDGF plays a role. PDGF-BB infusion weeks af-
baboon tissue developed by Kenagy, antibodies to urokinase, ter injury does produce a modest but measurable increase
tPA, and MMP-2 and -9 each inhibit migration, while addi- in intimal SMC proliferation.63 However, PDGF-B has not
tion of plasminogen increases migration.22 been detected to any measurable extent in the forming in-
Detachment of the SMCs from their extracellular tima, only in the endothelium near the wound edge.92 Rather,
matrix might be further regulated by integrins, a family of PDGF-A ligand and PDGFR-! predominate in the intima
transmembrane proteins that link the cytoskeleton to the during this time period.52 Since PDGF-A ligand can only
ECM and to adhesion molecules on other cells. Integrins bind to PDGFR-#, it appears unlikely that PDGF plays a sig-
are heterodimers made up of two noncovalently linked sub- nificant role in this phase.
units, designated # and !. Blockade of the integrin receptor As discussed previously, evidence does indicate that
#v!3 reduces PDGF-induced SMC migration in vitro and the angiotensin system may play a role in SMC proliferation
inhibits intimal hyperplasia in a rabbit carotid injury after injury. A possible role in SMC migration49 and intimal
model.79 Blockade of another receptor, #IIb!3, appears to expansion has been demonstrated as well.23 Angiotensin ii
prevent platelet aggregation after injury.80 Human clinical may influence intimal growth indirectly through either its
trials using a monoclonal antibody fragment (c7E3 Fab) that interactions with other growth factors (AII stimulates
blocks both the #v!3 and the #IIb!3 receptors as a result of PDGF-A and TGF-! production) or its stimulation of the
their common !3 component decreases the frequency of is- sympathetic nervous system.50 Catecholamines have been
chemic events after PTCA and reduces restenosis by 26%.81 shown to be SMC mitogens in vitro. Angiotensin ii admin-
Whether this is the result of the inhibitory effect on platelet istration produces a vigorous increase in intimal SMC pro-
aggregation or SMC migration or a combination of the two liferation and lesion area, even when infused at a time when
is unknown. the rat carotid lesion is already well developed.23 Infusion of
New proteins or proteins that are normally present in ACE inhibitors and angiotensin receptor blockers inhibit
only small quantities begin to appear in abundance in the intimal thickening after balloon injury in the rat indepen-
matrix of the newly formed intima of the injured rat ca- dent of blood pressure effects.36-38 Cilazapril, an ACE in-
rotid. It is thought that many of these proteins, including hibitor, as well as other ACE inhibitors, and Dup 753, a spe-
osteopontin,82 versican,83 tenascin,84 thrombospondin,85 and cific angiotensin ii receptor antagonist, block intimal thick-
splice products of fibronectin, may play a role in the adhe- ening in rats after balloon injury.93 A human clinical trial
sion of cells in the injured vascular wall. Peptides with the using Cilazipril during coronary angioplasty, however, has
amino acid sequence arginine-glycine-aspartic acid (RGD) shown no effect on either restenosis or angiographic out-
interfere with integrin binding.86 RGD peptides inhibit mi- come.94,95
gration of SMCs in vitro and reduce intima formation in Messenger RNAs for insulin-like growth factor (IGF)-1
the rat.79 Integrins that bind these peptides have been dem- and TGF-! have been detected in the newly formed intima
onstrated in the injured rat carotid and are also abundant in and could play a role in regulating cell proliferation or ex-
normal human and atherosclerotic human arteries.87 tracellular matrix production.96,97
234 Tissue Engineering of Prosthetic Vascular Grafts
After about 3 months, intimal thickening stops. The In areas that remain devoid of endothelium, the lu-
final composition of the intima, once it achieves a resting minal SMCs may assume some endothelial functions, in-
state, is generally 80% ECM by volume.98 What determines cluding the production of inhibitory factors. For example,
the endpoint at which growth ceases? Most probably, the NO is generated via the inducible form of nitric oxide syn-
lesion reaches the point at which either the stimulatory sig- thase (iNOS) and prostacyclin (PGI2) via cyclooxygenase
nals subside or the inhibitory signals develop strongly (COX).117,118 Smooth muscle cell iNOS is upregulated in
enough to counter the stimulatory signals, or both. areas of injury.117 The administration of a NO synthase in-
Evidence exists that endothelial cells play a role in this hibitor (N-omega-nitro-L-arginine methylester (L-NAME))
aspect of lesion growth. In the rat carotid injury model, an accelerates intimal formation after balloon injury, most likely
endothelial surface is formed by the ingrowth of cells from by inhibiting NO production by iNOS since very little cNOS
uninjured areas proximal and distal to the zone of denuda- is present once the endothelium is removed.119
tion. The central portion remains uncovered indefinitely, and Hemodynamic factors also influence lesion formation.
the growing edge appears to stop 1 cm to 1.2 cm from the Increases in blood flow after balloon injury appear to have a
untraumatized zone.99,100 Smooth muscle cell proliferation protective effect. In a rat carotid injury model, intimal hy-
stops sooner (approximately 2 weeks) in areas covered by a perplasia is reduced in rats that undergo contralateral com-
new endothelial layer. After about 4 weeks, SMC growth re- mon carotid artery ligation (high flow) when compared to
turns to baseline even in areas left devoid of endothelium.89 rats that undergo ipsilateral internal carotid artery ligation
SMC proliferation may slow as a result of growth inhibitor (low flow).120 Since SMC proliferation is not different be-
release by the endothelial cells. tween the two groups of animals, this implies an alteration
Cultured endothelial cells are known to secrete hep- in SMC migration as the possible mechanism.
arin-like heparan sulfates.101 The growth inhibitory prop- Could the ultimate control mechanism in lesion de-
erties of this family of molecules are well known.102,103 He- velopment be related to flow (shear stress)? Endothelial cells
parin has also been shown to alter the composition of the serve as the primary sensor for flow related phenomena
ECM.104 Nitric oxide (NO) is another inhibitory factor pro- through shear stress receptors.121 Activation of these path-
duced by endothelial cells. Nitric oxide here is produced via ways may cause changes in the secretion of growth or in-
constitutive nitric oxide synthase or cNOS, the form of NOS hibitory factors. It has been well documented that increas-
unique to endothelial cells.105 In vitro data suggest that NO ing shear stress increases NO production by endothelial cells
is a potent inhibitor of cell proliferation and migration.106,107 in vitro122 and in vivo.123 In areas of endothelial denuda-
In vivo, NO may control lesion development through both tion, SMCs have been shown to undergo phenotypic changes
its vasoactive properties and its inhibitory effects on other to render themselves more endothelial-like.124 As the lesion
growth factor pathways. Since endothelial cells serve as the size increases, luminal area decreases, resulting in increased
major source of NO, loss of endothelium during injury re- shear stress (assuming constant blood flow and no signifi-
sults in the loss of an important SMC growth inhibitor. Once cant change in vessel contraction or dilation). There is some
endothelial regeneration occurs, the pace of intimal hyper- evidence that the vessel itself dilates in an attempt to com-
plasia and SMC proliferation may slow simply by restoring pensate for the loss of luminal area.18 This response is, how-
the inhibitory NO signal. In both a rat and rabbit balloon ever, probably limited. During the early period of luminal
injury model, L-arginine added to the drinking water in- dilation (relatively low shear stress) and loss of endothelium,
creases NO production and reduces intimal hyperpla- NO production is low. Lack of a strong inhibitory signal al-
sia.108,109 In vivo gene transfer with a plasmid containing a lows continued SMC growth. However, as the luminal area
cDNA encoding for cNOS or an adenovirus containing iNOS decreases, the shear stress begins to increase. As the shear
after rat carotid artery injury restores NO to levels seen in stress approaches some critical level and endothelial cells
normal, untreated vessels and inhibits intimal formation by begin to repopulate the area, NO production occurs, inhib-
70%.110,111 Rats kept in an atmosphere of 80 ppm NO after iting further growth of the lesion. The artery wall’s stimula-
balloon injury also develop significantly less intimal hyper- tory and inhibitory factors once again become balanced and
plasia at both 1 and 2 weeks after injury.112 A single, locally the system returns to an equilibrium that more closely re-
delivered treatment of bovine serum albumin modified to sembles the quiescent artery wall.
carry multiple NO groups inhibits intimal hyperplasia after As we know, this is not the end of the story. Many
balloon injury in a rabbit model in a dose dependent man- lesions continue to grow, ultimately causing significant com-
ner.113 Administration of this molecule decreases platelet promise of the lumen. Most probably, these advanced le-
adhesion and SMC migration in association with elevations sions result from a persistent imbalance of the growth and
in platelet and vascular guanosine 3’,5’-cyclic monophos- inhibitory factors in favor of continued lesion growth. As
phate (cGMP) content, the result of guanylate cyclase acti- an example, endothelial regrowth in an injured area might
vation by NO. Aside from its inhibition of SMC prolifera- not be complete, resulting in the absence of inhibitory sig-
tion and migration, NO also affects vascular smooth muscle nals. It should be noted, however, that endothelial cells also
relaxation,114 platelet inhibition,63 leukocyte adhesion,115 have the ability to produce stimulatory growth factors. This
and vascular permeability.116 It is apparent that NO inhibits suggests that their role in lesion formation is variable de-
lesion formation after arterial injury. Whether this is a re- pending upon their local environment. Additionally, the le-
sult of one specific effect or a combination of several is not sion may create focal turbulence in the flowstream, result-
yet known. ing in persistent growth factor stimulation via the previously
Pathobiology of Hyperplastic Intimal Responses 235
mentioned mechanoreceptors. In humans, atherogenic risk Preexisting venous disease might account for some degree
factors also contribute to altered healing after injury. of lesion formation at nontraumatized sites.134,135 At later
As has been noted before, there is an inflammatory times, vein grafts fail due to the development of frank ath-
response after injury. The role of leukocytes in lesion for- erosclerotic lesions. This is particularly prevalent in the
mation is not entirely clear. It is known, however, that T lym- aortocoronary bypass grafts.136 It is noteworthy that when
phocytes are a major component of human atherosclerotic the internal mammary artery, a conduit which requires no
plaque.125 T lymphocytes are seen in the rat carotid artery adaptation to the arterial circulation, is used for coronary
injury model. The most significant evidence that they play a grafting, a much lower failure rate occurs.7 Renewed inter-
role here is the fact that they express &%interferon, a known est also exists in using the radial artery as a coronary artery
inhibitor of SMC growth. Most of the nondividing SMCs bypass conduit in hopes of achieving similar results. Previ-
seen in the rat intima express the class II major histo- ously, its propensity to spasm and the increased likelihood
compatibility antigen Ia, which is known to be induced only of subclinical atheroma discouraged its use. Recent studies
by &%interferon.126 This raises the possibility that other seem to indicate that if appropriate intraoperative steps are
cytokines, including interleukin-1, might be active in taken to inhibit radial artery vasoreactivity, long term pa-
the wall. tency is excellent.137 The improved success achieved with
arterial conduits suggests that the adaptive process that oc-
Atherosclerotic Artery Injury curs in vein grafts might make them more susceptible to
The arteries of cholesterol-fed rabbits and larger spe- lesion formation.
cies develop atherosclerosis. To accelerate the atherosclerotic Regardless of the actual technique employed (in situ
process and generate lesions that resemble advanced human or reversed), vein graft injury undoubtedly occurs during
disease, investigators have combined cholesterol feeding with reconstruction, even in the best of hands. The immediate,
balloon injury. In the rabbit, these lesions cause stenosis and histologic effects of this injury do not differ significantly from
become restenotic after a second injury (Gruntzig that of arterial injury. Several differences have, however, been
angioplasty). Unfortunately, these lesions are distinct from observed despite this similar starting point. Re-
human lesions, typically consisting of a concentric foam cell endothelialization is fast and complete in injured veins.138
infiltration of the intima and media. Additionally, SMC proliferation appears to take place even
Lesions produced in nonhuman primate models more after endothelial coverage is restored. This is in contrast to
closely resemble those of humans.127,128 These animals first the artery, where endothelial coverage appears to inhibit in-
develop a fatty streak with infiltrates of lipid-filled macro- timal SMC proliferation.28 The time course of wall thicken-
phages and later proceed to develop intermediate and ad- ing differs as well. Maximal arterial wall intima formation
vanced lesions similar to those seen in humans. Balloon in- occurs at 2 weeks. Vein graft thickening continues for up to
jury of diseased, but not stenotic, iliac arteries in hyper- 12 weeks.138 What is responsible for these differences?
cholesterolemic monkeys demonstrate a repair response in- One apparent difference is that vein graft endothe-
volving intimal hyperplasia, recoil, and remodelling.129 These lium appears to possess altered physiologic properties from
three mechanisms are felt to form the basis of the restenotic that of the arterial endothelium. Vein graft endothelium has
pathway in humans. While this model is not characterized impaired vasomotor properties139 and increased permeabil-
by a high degree of stenosis or restenosis, the basic mecha- ity.140 In addition, the arterial circulation exposes the thin
nisms resemble the known human mechanisms more closely venous wall to a continuous type I hemodynamic injury.
than those seen in other animal models.12 This model has While not as dramatic as arterial balloon dilation, this in-
been used especially to characterize remodeling, the process jury provides a persistent stimulus for wall thickening. When
by which the arterial lumen size is preserved by vessel dila- the vein dilates immediately upon exposure to the high pres-
tion during atheroma formation. sure arterial circulation, two phenomena occur. First, the
wall of the vein is exposed to a dramatically higher shear
Grafts stress. Shear stress responsive elements present on the en-
dothelial surface are thought to play a role in the activation
Vein of certain stimulatory pathways. The ultimate result is SMC
Autologous vein is the most widely used conduit in proliferation and migration toward the lumen, possibly
both coronary and peripheral bypass surgery. Vein grafts through some of the same mechanisms that have been char-
develop intimal hyperplasia upon exposure to the arterial acterized in the rat injury model. Second, the increased lu-
circulation.130 During reconstruction, vigorous distention minal blood pressure has been shown to invoke medial thick-
of a vein graft already in spasm or passage of a valvulotome ening, primarily through a response to increased wall
damages the endothelium and the graft wall. However, these stress.141,142 It is not yet clear which of these mechanisms is
defects are relatively minor and are repaired within a few primarily responsible for wall thickening in vein grafts. What
days.131 Restenosis due to intimal hyperplasia occurs in about is clear, however, is that hemodynamic factors play an im-
10% of peripheral vein grafts and usually during the time portant role in vein graft patency.14
period 6-24 months after reconstruction. Intimal thicken-
ing is most pronounced at anastomoses, valve cusps, and Prosthetic
clamp injury sites.132 It is also known to occur in vein grafts The prosthetic graft is a man-made, acellular, fibrous
placed using the in situ technique, despite the fact that this scaffolding upon which the body constructs a modified ves-
technique is thought to be less traumatic to the vessel.133 sel wall. The anastomoses appear to be the areas of maximal
236 Tissue Engineering of Prosthetic Vascular Grafts
intimal hyperplasia as a result of surgical trauma and the the # and ! receptor are increased, but not to a significant
presence of flow disturbance. It is thought that human pros- level. We have recently completed a study using monoclonal
thetic grafts heal via two main mechanisms, capillary in- human/mouse chimeric antibodies to both the PDGFR-#
growth through the graft wall, depending upon the charac- and -! receptor in this model. The # receptor antibody had
teristics of the graft, and growth along the luminal surface no effect, possibly because circulating serum concentrations
from each anastomosis.143 New evidence in a canine model did not achieve desirable levels. Adequate levels of the ! re-
supports fallout endothelialization from the blood stream ceptor antibody were achieved and the intimal thickening
as another possible mode of graft healing.144 No current normally observed after “flow switch” was significantly in-
evidence exists that prosthetic grafts inserted into the hu- hibited in this group of animals (unpublished results). This
man circulation are able to develop an endothelialized in- lends further credence to the theory that PDGF-BB and the
tima along the entire flow surface. Rather, endothelial cells ! receptor play a significant role in intimal development. It
manage to populate the lumen in just the few centimeters is unclear, however, why the PDGF-B ligand has not been
near the anastomoses.145 The remainder of the graft is lined detectable at higher levels in the developing intimal lesion.
by thrombus containing fibrin, platelets, and leukocytes. One possible explanation is that it is being produced
In our experience with prosthetic PTFE grafts placed primarily by the endothelial cells in small but sufficient
in baboons, the development of an endothelial layer is de- quantities and serves mainly as a powerful chemoattractant
pendent upon porosity.146 In low porosity grafts (10 and to attract SMCs towards the lumen. While other factors may
30 ∝m internodal distance), luminal endothelial coverage is participate in the development of intimal hyperplasia in
limited to small areas near the anastomoses. In high poros- PTFE grafts, we feel that the flow-related effects and the
ity grafts, (60 and 90 ∝m), luminal endothelial coverage is resultant production of PDGF and NO play significant roles.
complete. The 90 ∝m grafts, for unknown reasons, develop Further studies are needed to better characterize
areas of endothelial loss. The 60 ∝m grafts perform opti- this relationship.
mally regarding endothelial coverage and intimal layer sta-
bility. Similar results have been obtained in dogs.147 Summary
Once an endothelial layer is present, intimal thicken- Vascular injury can induce intimal hyperplasia. The
ing forms. It is perplexing that in the rat carotid injury model, ultimate response depends upon the severity of the injury
the endothelial layer serves to inhibit intimal thickening, and the change in the overall balance of stimulatory and
while in the prosthetic grafts, intimal thickening occurs only inhibitory factors at the site of injury. Based upon animal
once endothelium is present. This observation appears to models, the essential components of this exuberant healing
confirm the variable role of endothelial cells in the develop- response are SMC proliferation, SMC migration, and extra-
ment of intimal hyperplasia. cellular matrix deposition. SMC proliferation after injury is
Our laboratory has extensively characterized the heal- predominantly stimulated by bFGF released mostly from
ing of prosthetic grafts in baboons. Since there is no signifi- damaged cells but also from the vessel wall matrix. PDGF-BB,
cant thrombotic response, it is unlikely that the growth fac- deposited at the site of injury by platelets and released by
tors responsible for intimal formation are derived from plate- cells in the vessel wall, serves as the primary stimulus for
lets. Endothelial cells, however, can produce growth factors, SMC migration. Release of SMCs from their surrounding
among them, PDGF. Golden observed that healing aortoiliac matrix is accomplished by plasmin and MMPs. Cellular ad-
PTFE grafts removed after 2, 8, and 12 weeks and perfused hesion receptors or integrins are also involved in this pro-
ex vivo released more mitogenic factors than control cess. Control of matrix production is not yet as well defined.
arteries.148 This activity was blocked by an antibody to PDGF. In addition to causing the release and production of stimu-
Furthermore, PDGF-A mRNA and protein levels were latory factors, vascular injury also transiently reduces the
detectable in the graft intima by both Northern analysis and availability of important inhibitory factors such as NO. In-
in situ hybridization. SMC proliferation was seen by timal hyperplasia occurring in atherosclerotic arteries, vein
thymidine labeling in the same region (inner third of the grafts, and prosthetic grafts is not as well understood as in
intima) as PDGF mRNA expression. PDGF-B was the rat carotid artery injury model, although similarities in
nearly undetectable.149 the basic pathophysiology most likely exist. The challenge
As discussed earlier, flow-related effects are seen in vein for the future is to further define the human response in a
graft healing. Studies have also demonstrated flow-depen- systematic manner and to successfully apply this knowledge
dent formation of intima in PTFE grafts in baboons.150 towards improving the effectiveness of our vascular inter-
Grafts placed in the aorto-iliac position under high flow ventions.
conditions created by placement of a distal fistula form
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Taming of Adverse Responses
Prevention of a Hyperplastic Intimal Response
CHAPTER 23
Cell Cycle Interruption to Inhibit Intimal Hyperplasia
Michael J. Mann, Ruediger C. Braun-Dullaeus, Victor J. Dzau
N eointimal hyperplasia is the hallmark of occlusive vascular graft disease. It is largely re
sponsible for the primary failures of up to 30% of infrainguinal grafts within two years,
and it is believed to form the substrate for the accelerated atherosclerosis that is linked to the
failures of 30-50% of coronary bypass grafts.1-3 Migration and proliferation of medial vas-
cular smooth muscle cells (VSMC) provide the bulk of the neointimal cell mass, and
antiproliferative regimens have therefore been sought as a means to curb this process.4-7
Targeting the molecular machinery that regulates cell cycle progression has proven to be
among the most potent of such antiproliferative strategies in experimental models of
neointimal disease, as this network of kinases and transcription factors offers a final com-
mon pathway to the myriad signaling cascades known to trigger the activation and prolif-
eration of vascular cells.
Recent studies suggest that the cell’s ability to enter and progress through the cell cycle
at a critical point during the onset of vascular remodeling is an essential element in the
pathogenesis of vascular graft disease.8,9 Data suggest that the activation of quiescent vascu-
lar cells results not only in cell division, but also in the abnormal expression of adhesion
molecules and cytokines that facilitate cell migration, enhance the inflammatory response
and perpetuate a cycle of vascular cell dysfunction. The results of these investigations fur-
ther indicate that manipulation of cell cycle regulatory events can induce alternative path-
ways of vessel wall adaptation to the traumatic and hemodynamic stresses associated with
bypass grafting. For example, cell cycle arrest has been shown to tip the balance away from
neointima formation and toward other, more salutary, pathways of graft remodeling that
result in a vessel wall architecture, hemodynamic stability and resistance to accelerated ath-
erosclerosis reminiscent of the native artery.
The process of cell cycle regulation has been dissected in recent years through in vitro
studies in numerous cell types. These studies have revealed an intricate system of checks and
balances that is initiated upon stimulation of quiescent cells to enter the proliferative cycle
of DNA replication and cell division. This system involves the regulation of specialized cell
cycle proteins on many levels: the initiation of new mRNA transcription, the translation and
accumulation of proteins at or above threshold levels, and the activation or inactivation of
these proteins through phosphorylation, dephosphorylation and protein-protein complex
formation. Proliferative diseases have been linked to alteration in these patterns of genetic
and biochemical regulation of cell biology. The elucidation of these pathways may therefore
yield the sophisticated tools necessary for clinicians to overcome pathobiological processes
that have until now proven elusive to traditional therapeutic approaches.
The role of cell cycle regulation in neointima formation has been most extensively
explored in models of arterial balloon injury. Recently, studies in our laboratory and others
have documented analogous events in the development of neointimal hyperplasia in
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
242 Tissue Engineering of Prosthetic Vascular Grafts
autologous vein grafts.8,10-12 We have therefore developed liferation in vitro, including basic fibroblast growth factor
strategies designed to alter patterns of vascular cell gene ex- (bFGF) and platelet derived growth factor (PDGF),18,19 and
pression associated with the onset of this abnormal accu- these factors have been identified in the vessel wall in vivo
mulation of intimal cells. We initially hypothesized that vein in arterial and venous models of neointimal hyperplasia, as
graft failures linked to neointima formation and/or acceler- well.20,21 Increased platelet adherence and degranulation and
ated atherosclerosis were the downstream result of pheno- release from injured vascular cells were initially thought to
typic changes in vascular cells. We subsequently observed be the primary sources of PDGF and bFGF, respectively, al-
that genetic blockade of cell cycle progression during a criti- though it is now recognized that vascular cells themselves
cal phase of vein graft response to the surgical, hemodynamic initiate the autocrine and paracrine production of these fac-
and inflammatory insults of grafting results in a shift of graft tors after arterial injury and vein grafting.18,19
biology toward a more adaptive remodeling process charac- Proliferation among medial VSMC has been docu-
terized by medial hypertrophy and stabilization of endo- mented within 24-48 hours after balloon injury, and migra-
thelial function. tion of VSMC across the internal elastic lamina is established
by days 3 and 4.17,22 Early neointimal cells have demonstrated
The Molecular and Cellular Biology rates of bromo-deoxyuridine incorporation, indicative of
of Neointimal Vascular Graft Disease DNA synthesis, as high as 50%, and this rapid proliferation
Despite progress in the development of artificial vas- establishes approximately 90% of the neointimal cell mass
cular prostheses, autologous vein grafts continue to have within two weeks of injury.22 Cell cycle progression and cell
higher long term patency rates of approximately 70% and division subsequently slows, while further increases in
50% at three and five years, respectively.1,2 Vein graft occlu- neointimal thickness result primarily from continued ex-
sions, however, have been linked to neointimal hyperplasia, tracellular matrix production.23 In addition to PDGF and
and “activated” VSMC are known to express an array of pro- bFGF, other factors, such as transformtin growth factor-!
teins that can contribute to a pro-atherogenic environment (TGF-!), angiotensin II and insulin-like growth factor-1
in the graft wall. These upregulated molecules include (IGF-1), are also expressed in the vessel wall after injury, and
cytokines such as interleukins -1, -2 and -6, and tumor ne- may play important roles in determining the extent of le-
crosis factor (TNF), adhesion molecules such as intercellu- sion formation.24-26
lar adhesion molecule-1 (ICAM-1) and chemoattractants A dramatic increase in VSMC DNA synthesis has also
such as monocyte chemoattractant protein-1 (MCP-1).12-15 been documented during the first week after operation in
In addition to midgraft thrombosis, anastomotic stenosis experimental vein grafts.8,10 In a study of rabbit jugular to
contributes significantly to the failures of artificial graft con- carotid interposition grafts, this proliferative rate returned
duits, and this anastomotic disease is similarly character- to a low level by postoperative weeks 2-4, by which time a
ized by a neointimal proliferative response stemming from thick, highly cellular neointimal layer had been formed.
the adjacent artery.16 Inhibition of neointimal hyperplasia Zwolack and colleagues further demonstrated that
has therefore become a focal point for research aimed at neointimal thickening proceeded over the subsequent 4-8
improving the long term success of surgical revascularization weeks, primarily via increased extracellular matrix produc-
(Fig. 23.1). tion, and that the neointimal layer then remained morpho-
The molecular and cellular biology of neointimal hy- metrically stable up to 12 months, establishing a pattern of
perplasia have been best characterized in animal models of cellular kinetics parallel to those observed after rat carotid
arterial injury that mimic the vessel’s response to balloon balloon injury. Francis et al27 succeeded in demonstrating
angioplasty.17 A primary process of neointimal hyperplasia the release of PDGF in porcine vein grafts at 1 and 4 weeks
is the activation of medial VSMC, and their subsequent pro- after surgery, while Hoch et al12 established the time course
liferation and migration across the internal elastic lamina. of both TGF-!1 and PDGF-A expression by rat vein graft
Numerous factors have been found to stimulate VSMC pro- cells and the relationship of this growth factor expression to
Fig. 23.1. Vascular graft neointimal hyper-
plasia. Proliferation and migration of
medial vascular smooth muscle cells
(VSMC) leads to development of a thick
layer of abnormal cells on the lumenal
aspect of the internal elastic lamina of an
experimental vein graft (A), and migra-
tion of cells from the adjacent artery leads
to a similar VSMC layer on the lumenal
aspect of a PTFE graft placed in the rab-
bit aorta (B).
Cell Cycle Interruption to Inhibit Neoinitmal Hyperplasia 243
the development of graft neointimal hyperplasia. Further therefore likely to be unsuccessful. There is a final common
studies have since documented the elaboration of cytokines pathway, however, onto which these various proliferative
such as IL-1!, TNF and MCP-1 in the vein graft wall during stimuli and their secondary messengers converge: the mo-
the first 1-4 weeks after operation;14,28 these proteins may lecular and genetic machinery controlling entry into and
play roles in the further “activation” of vascular cells, and progression through the cell cycle (Fig. 23.2). Scientists now
may enhance the infiltration of the vessel wall by pro-in- have a growing understanding of the complex regulation of
flammatory cells. the movement of cells from quiescence (G0-phase) into the
Neointimal growth also occurs in vascular prostheses phases of:
made of artificial materials. Ingrowth of endothelial and 1. Preparation for chromosomal duplication
VSMC from the anastomotic ends of experimental grafts (G1-phase);
comprised of either Dacron or polytetrafluoroethylene 2. DNA synthesis (S-phase);
(PTFE) progresses along the length of the graft to varying 3. Preparation for cell division (G2-phase); and finally
degrees, depending upon the species and material. 29 4. Mitosis (M-phase), and opportunities for targeted
Neointimal thickness, however, is always greatest near the intervention in these processes have therefore
anastomosis, and this heightened perianastomotic thicken- become available.
ing correlates to the clinical phenomenon of anastomotic
stenosis in artificial grafts.30 Increased porosity of the graft Cell Cycle Progression:
material also allows direct ingrowth of capillaries through A Careful Orchestration
the graft wall in animal models, with subsequent accelera- The precision with which cellular growth and prolif-
tion of neointima formation in the grafts’ midportions.31 eration are governed during normal development and heal-
As in the injured artery and the healing vein graft, prosthetic ing has necessitated the development of an elaborate system
vascular graft neointima formation tends to reach a point of checkpoints and regulatory events in the molecular con-
of stable equilibrium, after which cellular proliferation ceases trol of cell cycle progression (Fig. 23.3). Although a number
to contribute to neointimal thickness.30 Several animal mod- of details have been found to vary slightly in different cell
els, including dog and baboon, exhibit complete coverage types and in different species, a remarkably conserved pat-
of Dacron and high porosity PTFE grafts with neointimal tern of gene upregulation and enzymatic activity has been
and endothelial linings, although neointima formation in observed during the movement of cells through the various
human grafts is generally limited to the perianastomotic re- stages of new DNA synthesis and cellular division. In this
gion.29 Clowes and associates have documented the produc- context, several classes of molecules have been described as
tion of both PDGF and TGF-! in the walls of prosthetic vas- playing well defined roles in the management of cell cycle
cular grafts, by both inflammatory and proliferating progression, and studies have confirmed many of these roles
vascular cells.32 in VSMC in vitro.
A wide array of growth factors, such as PDGF, bFGF One of the first responses of cells to stimulation by
and IGF, have all been shown to stimulate the VSMC prolif- growth factors or other mitogens is the upregulated expres-
eration that is a necessary part of neointimal hyperplasia. sion of a series of protooncogenes, also known as immedi-
Strategies to inhibit this proliferative process via blockade ate early genes. Among these genes, c-fos and c-myc are
of any one or two of these redundant molecular targets are known to be important not only for entry of cells into the
Fig. 23.2. Multiple growth factors
and hormones are known to
stimulate VSMC proliferation
both in vitro and in vivo. Their
multiple secondary messenger sys-
tems, however, converge on a final
common pathway: the cell cycle
regulatory machinery and the co-
ordinated upregulation of cell
cycle regulatory genes.
244 Tissue Engineering of Prosthetic Vascular Grafts
cell cycle, but for successful progression through all four protooncogenes such as c-myc, genes encoding enzymes such
stages as well.33,34 The protein products of these genes act as as dihydrofolate reductase, and those for cell cycle regula-
transcription factors that increase the expression of other tory proteins such as Cdk2 and cyclin E. Hyperphos-
molecules (in particular the cyclin-dependent kinases de- phorylation of Rb and activation of E2F comprises the re-
scribed below) whose activities are required to trigger cell striction point R in the cell cycle, beyond which the cell is
cycle switches. The product of another protooncogene, committed to DNA synthesis and after which the cell cycle
c-myb, acts similarly to upregulate cell cycle proteins, whereas can be completed without further stimulation from exter-
the ras gene encodes a membrane-bound, guanine nucle- nal growth factors.39
otide-binding protein that couples signals from surface ty- After DNA replication has taken place in S-phase, an-
rosine kinase growth factor receptors to cytoplasmic second- other cyclin-Cdk complex, that of cyclin B and Cdk1 (also
ary messenger systems.35 known as cell division cycle 2 kinase or cdc2 kinase) is re-
The cyclin-dependent kinases (Cdks) form another sponsible for final progression of the cell through the G2/M
class of molecules that directly regulate the progression of transition. This complex, the mitosis promoting factor
cells through the cell cycle. Other proteins, termed cyclins, (MPF), requires phosphorylation by CAK and dephospho-
act as cofactors for the Cdks, and the binding of specific rylation by cdc25 phosphatase for activation.41,42 An example
cyclins to specific Cdks leads to formation of active holoen- of the coordination of cell cycle events is reflected in the fact
zymes. Both cyclins and Cdks are expressed only at low lev- that c-myc protein not only contributes to the upregulation
els in quiescent cells, and are upregulated at various inter- of Cdk1 protein levels, but also to induction of cdc25 phos-
vals after stimulation of the cell into cell cycle progression. phatase and thereby to Cdk1 activation.43 Activated MPF
Once upregulated, Cdk expression remains high as long as initiates mitosis, and also triggers a proteosomal pathway
the cell continues to progress through rounds of the cell cycle, involving ubiquination that not only facilitates anaphase,
whereas cyclin protein levels fluctuate at different points but also leads to destruction of cyclin B itself in a negative
through cell cycle progression. Early in the G1-phase, cyclin feedback loop.44
D and Cdk4 are upregulated and form a complex together In addition to cell cycle promoting factors, a number
with another cell cycle regulatory protein, proliferating cell of natural inhibitors of cell cycle progression are known to
nuclear antigen (PCNA). Cdk4 further requires phospho- play important roles in the management of cell cycle pro-
rylation by a Cdk activating kinase (CAK) to complete its gression. One group of these molecules is the cyclin-depen-
activation. Cdk2, which forms a holoenzyme with cyclin E, dent kinase inhibitors (CKIs). These inhibitors bind to cyclin/
is the other primary G1-phase Cdk, and it is responsible, Cdk complexes and block their enzymatic activation. In sev-
along with cyclin D/Cdk4, for hyperphosphorylation of the eral cell types, including VSMC, the CKI p27Kip1 is expressed
retinoblastoma gene product Rb.36-38 at high levels during G0-phase, and a drop in this protein
In quiescent cells, Rb exists in its hypophosphorylated level is seen during entry into G1-phase.45 This drop is be-
form, pRb, and resides in a complex with cyclin A, Cdk2 lieved to be necessary for effective phosphorylation of pRb
and the transcription factor E2F. Hyperphosphorylation to at the R point. Interestingly, another CKI, p21Cip1, is ex-
the ppRb state results in a release of E2F from this com- pressed only after cell cycle entry has been initiated, and it is
plex.39 E2F then transactivates a number of other genes believed to play a role in controlling cell cycle progression.45
whose products are essential for progression through the The tumor suppressor p53 acts as a transcription factor to
S-, G2- and M-phases of the cell cycle.40 These genes include inhibit cell cycle progression under certain conditions.46 Beta
Fig. 23.3. The cell cycle. The
upregulated expression and coor-
dinated activation of cyclin-de-
pendent kinases (Cdks) is required
at various points during cell cycle
progression. These enzymes are
regulated by inhibitory proteins
such as p27 and p21. At the R
point, the hypophosphorylated
retinoblastoma gene product
(pRb) is hyperphosphorylated by
G1 Cdks, releasing the transcrip-
tion factor E2F, which in turn
transactivates up to a dozen genes
that encode proteins required for
completion of the cell cycle.
(See text.)
Cell Cycle Interruption to Inhibit Neoinitmal Hyperplasia 245
particle and ultraviolet light irradiation, for example, lead The first report of in vivo intervention in vascular cell
to a p53-mediated upregulation of p21Cip1 expression, cycle gene expression was made by Simons et al,56 and in-
thereby preventing progression to S-phase and DNA repli- volved antisense ODN inhibition of c-myb expression after
cation in the context of radiation-induced DNA damage.47 rat carotid balloon injury. A porcine arterial balloon injury
In the presence of free E2F protein, p53 has also been linked model was subsequently used to demonstrate a similar re-
to induction of programmed cell death, or apoptosis, pro- duction in neointimal lesion size after treatment of the ves-
viding a mechanism for prevention of propagation of dam- sel wall with antisense ODN against c-myc.57 Whereas single
aged DNA in cells that have passed the R point of the cell antisense ODN targeting PCNA, Cdk1 or Cdk2 have also
cycle.48 The homeobox proteins, such as GAX, are another been shown to reduce neointimal hyperplasia after balloon
class of natural cell cycle inhibitors, and are believed to help injury, the simultaneous inhibition of a combination of cell
maintain differentiated cells in a quiescent state.49 cycle genes, such as Cdk1 and cyclin B or Cdk1 and PCNA,
was found to have a more profound, nearly complete inhi-
Cell Cycle Arrest and Neointimal Hyperplasia bition of neointima formation in a rat carotid injury model
The molecular and genetic basis of cell cycle regula- that lasted up to 8 weeks after injury.54,58 Blockade of the
tion has also been explored in the context of the intact ves- latter combination of genes using ribozymes has also suc-
sel wall during the proliferative response to vascular injury. cessfully inhibited neointimal hyperplasia in a similar arte-
Increases in mRNA levels of the protooncogenes c-myc, c-fos, rial injury model.59
and c-jun have been documented within hours after arterial The synergy observed with inhibition of multiple cell
balloon injury, whereas protein levels of the cell cycle in- cycle regulatory genes led to studies of in vivo inhibition of
hibitors GAX and p27Kip1 are rapidly downregulated.50-52 the transcription factor E2F using decoy ODN transfection
Expression of the “immediate early” genes c-fos and c-jun after carotid injury.60 Fusigenic liposomes comprised of coat
has also been demonstrated in human saphenous vein seg- proteins from inactivated hemagglutinating virus of Japan
ments within hours after harvest, even without re-implan- (HVJ) particles and neutral lipids were used to enhance de-
tation as bypass grafts.53 Expression of c-myc peaks a sec- livery of the decoy ODN. Free E2F is responsible for the
ond time 7 days after arterial injury, when VSMC prolifera- transactivation of up to a dozen cell cycle regulatory pro-
tive activity has been shown to reach its highest levels.50 The teins, including c-myc, Cdk1 and PCNA, and its blockade
upregulation of immediate early genes is followed in turn with the single decoy ODN proved as effective as combina-
by increases in both the mRNA and protein levels of cyclins tions of antisense ODN directed against multiple cell cycle
and Cdks in the arterial wall, within 1-4 days after injury.54,55 gene targets.
Cell cycle gene activation has further been documented in The transfer of genes encoding several bioengineered
medial and neointimal VSMC via the immunohistochemi- or naturally occurring cell cycle inhibitory proteins has also
cal staining of increased levels of PCNA in this time period succeeded in reducing neointimal hyperplasia after arterial
after injury, and increased PCNA and Cdk1 protein levels balloon injury. Overexpression of either p21Cip1 or GAX, for
have similarly been measured in autologous rabbit vein grafts example, has been shown to reduce neointimal hyperplasia
at 4 days after surgery.8 in injured porcine and rabbit arteries, respectively, and
Understanding the control of cell cycle progression at neointima formation has similarly been reduced via trans-
the level of cell cycle regulatory gene expression has led to a duction of porcine femoral arteries with a nonphos-
number of gene-based approaches at inhibiting VSMC pro- phorylatable mutant of Rb and of rat carotid arteries with a
liferation after vascular injury. Genetic manipulation can dominant negative mutant of Ras.61-64 Cell cycle interrup-
involve either the blockade of target genes using oligonucle- tion via gene transfer, however, is likely to require a very
otide transfection, or the introduction of genes using lipids high transduction efficiency to achieve a clinically signifi-
or viral vectors. Antisense oligodeoxynucleotides (ODN) are cant effect on VSMC proliferation and neointimal growth,
short chains of single stranded DNA that bear an antisense, since cell cycle progression will be blocked only in trans-
or Watson-Crick complementary, sequence to that of a re- duced cells. The studies described all used replication-defi-
gion of the target gene mRNA. After delivery to a target cell, cient, recombinant adenoviral vectors to achieve transgene
the ODN bind to this mRNA and prevent its translation into delivery to the target vessels. Although these recombinant
protein. Several mechanisms of antisense activity have been adenoviruses are the most widely studied and efficient vec-
proposed; the most commonly accepted, however, involves tors yet described in vascular tissue, transgene expression is
the destruction of DNA-RNA hybrids via RNAse H. An al- generally limited to approximately 30-50% of cells in an ar-
ternative gene blockade strategy utilizes double-stranded terial wall, and only after some form of vessel wall injury.63,65
“decoy” ODN that bear a consensus binding site for a target Furthermore, infection with these vectors is associated with
transcription factor protein (Fig. 23.4). The factor becomes a prominent inflammatory reaction, transient gene expres-
bound by the excess of decoy ODN, and is prevented from sion and the hazard of possible viral mutation. Nonviral
interacting with its binding site in the promoter region of a means of manipulating cell cycle gene expression may there-
gene under its control. Upregulation of target gene transcrip- fore provide a more practical opportunity at the present time
tion is therefore inhibited. Ribozymes, chains of RNA that for influencing vascular proliferative disease in both human
catalyze cleavage of mRNA in a sequence-specific manner, arteries and vein grafts.
offer another alternative for gene blockade.
246 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 23.4. The transcription factor “decoy”
approach. The transcription factor E2F is
bound to a regulatory complex and is inac-
tive in quiescent cells (A). Upon activation,
E2F is released and is free to interact with
the consensus binding site in promoters of
target genes (B). “Decoy” ODN bearing the
consensus binding site compete for tran-
scription factor binding and prevent gene
transactivation (C).
Genetic Engineering of Vein Graft Resistance significant neointimal hyperplasia, these genetically engi-
to Atherosclerosis via Cell Cycle Gene Blockade neered grafts resisted the accelerated, diet-induced deposi-
Neointimal hyperplasia plays a particularly important tion of foam cells and generation of atherosclerotic plaque
role in the pathogenesis of autologous vein graft failure, not observed in control grafts (Fig. 23.5).
only through direct encroachment upon the vessel lumen, Vein graft susceptibility to accelerated atherosclerosis
but also because it acts as a substrate for accelerated graft is associated with endothelial dysfunction similar to that seen
atherosclerosis. Neointima formation is a well characterized in early arterial atherosclerosis.66 In addition to blockade of
response to multiple forms of vascular injury, ranging from VSMC proliferation during the immediate postoperative
gentle denudation to desiccation to balloon distention. The period, inhibition of cell cycle regulatory gene expression
ischemic, mechanical and inflammatory injuries associated had a long term stabilizing effect on vein graft endothelial
with vein graft harvest and implantation are therefore likely function.9 This phenotypic alteration was documented via
to provide a strong stimulus for neointima formation. The enhanced endothelial cell nitric oxide synthase activity and
authors have hypothesized that blockade of gene expression maintenance of normal jugular vasoreactivity, as well as
necessary for cell cycle progression can prevent the initia- through reduced superoxide anion generation, adhesion
tion of neointimal hyperplasia as a response to these mul- molecule expression and monocyte binding to graft
tiple factors and stimuli. Furthermore, once graft healing endothelium. This preservation of endothelial function
has progressed and the stimulation from these injuries has may represent either a direct effect of cell cycle gene
subsided, the graft can respond to the hemodynamic impe- blockade in endothelial cells, or it may reflect a change in
tus for wall thickening via vascular hypertrophy and medial the influence normally exerted by underlying abnormal
thickening. Stabilization of vascular cell phenotype and func- neointimal VSMC.
tion during the postoperative period of graft healing may Transfection of the vein graft wall with the single agent
therefore allow vein graft adaptation to the hemodynamic E2F decoy ODN has subsequently been shown to similarly
stresses of the arterial circulation without incurring increased influence vein graft biology and prevent both neointimal
susceptibility to graft disease and failure associated with disease and atherosclerosis.67 This observation has been ex-
neointimal hyperplasia. tended to six months after operation, at which time E2F
This hypothesis was tested in a series of experiments decoy ODN-treated grafts remain free of significant
utilizing the delivery of ODN to cells of rabbit jugular veins neointima formation and resist diet-induced atherosclero-
at the time of grafting into the carotid artery. A combina- sis. Studies have also documented that ODN can be deliv-
tion of antisense ODN against both Cdk1 and PCNA inhib- ered efficiently to human saphenous vein segments ex vivo,
ited neointimal hyperplasia by 90%, and this inhibition per- and in an organ culture system these ODN have inhibited
sisted for up to 10 weeks after grafting.8 By week 6, however, target gene function in a sequence specific manner. These
adaptive graft wall thickening approached levels seen in findings have served as the basis for initiation of a large scale
untreated grafts largely through hypertrophy of the medial human clinical trial to test the efficacy of intraoperative vein
layer. This thickening translated into an effective reduction graft engineering via E2F decoy ODN transfection in pre-
in tangential wall stress. Most importantly, in the absence of venting infrainguinal bypass graft failures.68 This study rep-
Cell Cycle Interruption to Inhibit Neoinitmal Hyperplasia 247
Fig. 23.5. Inhibition of neointimal hyperplasia and
resistance to accelerated atherosclerosis in vein
grafts transfected intraoperatively with ODN that
block cell cycle regulatory gene upregulation
(C,D). Plaque in a control graft is comprised
largely of foam cells (A) and demonstrated posi-
tive immunohistochemical staining for macroph-
ages (B). From Mann et al. Proc Natl Acad Sci USA
1995; 92:4502-4506.
resents the first attempt at integrating a gene therapy strat- derway that may establish the practical, long term benefits
egy into a routine cardiovascular surgical procedure, and that a one time intervention in cell cycle regulation may have
the first large scale testing of our ability to influence cell cycle for vascular graft compatibility and adaptation. The results
biology and its role in human vascular pathogenesis. of these basic science and clinical studies will likely yield
vascular biologists, bioengineers and clinicians new tools to
Summary aid in the design of vascular prostheses with enhanced du-
Vascular cell proliferation is clearly a requisite com- rability and performance.
ponent of the neointimal hyperplasia that is known to com-
promise the long term success of autologous and prosthetic References
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Facilitation of Healing
Chemotaxis
CHAPTER 24
Signaling Mechanisms for Vascular Cell Migration
Ian Zachary
Introduction
I t is increasingly recognized that migration of vascular smooth muscle cells (VSMC) from
the media is a key event in progressive intimal thickening leading to atherosclerosis and
other occlusive vasculoproliferative complications.1,2 Transendothelial migration into the
tunica intima of other cell types, particularly monocytes and T lymphocytes, is thought to
be central both to the formation of the mature atherosclerotic lesion and to the pathogen-
esis of the disease.1,2 Arterial bypass graft surgery is one of the commonest treatments for
atherosclerotic cardiovascular disease, and a major cause of failure of bypass graft surgery is
stenosis of the graft, which occurs predominantly at, or contiguous to, the sites of anasto-
mosis.3-6 Stenosis of the grafted vessel is primarily due to the proliferation and migration of
VSMC.7 The use of prosthetic grafts in place of autogenous vessels has been an important
development in vascular surgery, but prosthetic materials have lower patency rates com-
pared to veins. The lower patency of prosthetic grafts is widely attributed to their inability to
form an endothelial lining, thus contributing to enhanced thrombogenicity and VSMC pro-
liferation/migration. Animal studies indicate that seeding prosthetic grafts with endothelial
cells (EC) prior to implantation can significantly reduce neointimal hyperplasia and
thrombogenicity.8,9 As these brief introductory remarks suggest, vascular cell migration may
be relevant for cardiovascular disease from more than one perspective. Thus, while VSMC
and monocyte migration contribute to progression of the primary disease and—in the case
of VSMC—complications consequent upon treatment, the promotion of EC migration and
proliferation may be of equal importance in clinical situations, either where prosthetic grafts
are used or where the endothelium is damaged (e.g., angioplasty).
The aim of this chapter is to provide an overview of the extracellular factors and intra-
cellular mechanisms implicated in the regulation of vascular cell migration. Emphasis will
be placed on the migration of VSMC and EC, though the role of the migration of other cell
types in the arterial wall and the atherosclerotic microenvironment (e.g., monocytes) will
also be considered. Emphasis will be placed on recent developments in our understanding
of the molecular basis of cell migration, such as the role of focal adhesion kinase (FAK) and
Rho proteins. Inevitably, many of the findings that will be discussed have come from studies
in other nonvascular cell types, but it is anticipated that these findings will have relevance
for the migration of vascular cells. The first section of the chapter (Migration Factors) will
describe the major factors known to modulate vascular cell locomotion in cell culture. The
second section (Chemotactic Signal Transduction) will go on to discuss intracellular mecha-
nisms implicated in cell migration and linked cellular processes.
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler
©1999 R.G. Landes Company.
252 Tissue Engineering of Prosthetic Vascular Grafts
Migration Factors PDGF in atherosclerotic lesions and following vascu-
Chemotaxis is the directed movement of cells along a lar injury may be derived from several sources. PDGF exists
gradient of a chemoattractant and should be distinguished as disulfide-linked dimers of two homologous isoforms, the
from the random movement or chemokinesis of cells in the A- and B chains, which give rise to three dimeric bioactive
absence of a gradient. In what follows, migration will gener- isoforms, AA, AB and BB. The BB form is the major species
ally be understood as synonymous with chemotaxis, i.e., the in porcine platelets15, while PDGF-AB is the predominant
directed movement of cells in response to a specific factor. form in human platelets.16,17 Expression of PDGF-A and -B
A diverse array of molecules have been shown to influence chains is induced in EC by interleukin-1 (IL-1), tumor ne-
the movement of vascular cells in vitro and are therefore crosis factor- # and by transformtin growth factor- !
potential regulators of vascular cell migration in vivo. The (TGF-!).18-21 Adult rat VSMC apparently produce only A
most important known chemoattractants and migration chains22,23 and PDGF-AA expression is upregulated in these
factors, together with their cellular and noncellular sources cells by TGF-! and IL-1.24-27 Macrophages also produce
and principal target cells are summarized in Table 24.1. PDGF-B and are an additional source of this factor in the
atherosclerotic plaque.1,28,29
PDGF and Other Growth Factors Two isotypes of the PDGF receptor have been identi-
Platelet-derived growth factor-BB (PDGF-BB) is the fied, the # and ! receptors, which exhibit different affinities
most potent known chemoattractant for cultured VSMC for PDGF ligand isoforms. The # receptor (PDGFR#) rec-
derived from a variety of species.1,2 The mRNA for PDGF ognizes all three possible isoforms of PDGF ligand while
ligand and receptors are upregulated both in human ath- the ! receptor (PDGFR!) exhibits high-affinity binding only
erosclerotic plaques10 and in rat and baboon neointimal tis- of the PDGF-B chain.30-33 Evidence is accumulating that the
sue induced by injury or vascular grafting.11,12 It has been different isoforms of PDGF ligand and receptor may medi-
reported that PDGF-stimulated neointima formation in the ate distinct cellular functions in VSMC. 34-36 The BB
rat model of balloon angioplasty predominantly involves homodimer is chemotactic for a variety of cell types includ-
migration of VSMC from the media. Thus, intimal thicken- ing every species of VSMC so far tested. 34-38 The AB
ing following infusion of PDGF-BB into rats subjected to heterodimer is also chemotactic where it has been exam-
injury of the carotid artery was primarily the result of mi- ined.35,36 In contrast, the chemotactic response to PDGF-AA
gration and was accompanied by only a small increase in is highly cell type dependent. Thus, PDGF-AA promotes the
VSMC proliferation.13 Furthermore, neointimal VSMC ac- migration of granulocytes but not of monocytes.35 Further-
cumulation in the rat carotid can be inhibited by an anti- more, in VSMC of several species and in monocytes and fi-
body to PDGF.14 These findings suggest that PDGF-induced broblasts this isoform has no chemotactic effects34-38 and
chemotaxis of medial VSMC may play a major role in the has been shown to antagonize migratory responses to
formation of a neointima both in atherosclerosis and in PDGF-BB.34-36 Our recent findings show that rabbit VSMC
restenosis following angioplasty. do not respond chemotactically to PDGF-AA and lack func-
Table 24.1. Factors regulating vascular cell migration
Factor Sources Target Cell(s) Biological Activities
PDGF-BB Platelets VSMC Stimulates chemotaxis and proliferation
EC Macrophages
PDGF-AA VSMC/EC VSMC Stimulates proliferation and inhibits chemotaxis
IGF-1 Circulation VSMC Stimulates chemotaxis and and is a co-mitogen
EC/VSMC
Platelets
HB-EGF Macrophages VSMC Stimulates chemotaxis and proliferation
EC/VSMC
VEGF VSMC macrophages EC Monocytes Stimulates EC proliferation and EC and monocyte chemotaxis
bFGF VSMC/EC VSMC/EC VSMC mitogen, mitogen and chemoattractant for EC
MCP-1 VSMC/EC Monocytes Specific monocyte chemoattractant
IL-1 Macrophages VSMC Possible chemotactic factor and mitogen; induces PDGF
EC/VSMC expression in EC and VSMC
CSFs T-lymphocytes Monocytes Chemoattractant
EC/VSMC
TGF-beta Platelets VSMC/EC Indues expression of ECM, beta 3 integrins and PDGF-A in VSMC;
EC/T-lymphocytes induces PDGF in EC. Bimodal effects on VSMC proliferation. Possible
direct bimodal effects on VSMC and EC migration.
This table lists polypeptide ligands for receptor PTKs and cytokines. For the effects of ECM components, the reader is referred to the text.
Abbreveiations: EC, endothelial cells. Other abbreviations are explained in the text.
Signaling Mechanisms for Vascular Cell Migration 253
tional # receptors.38 Low # receptor expression cannot en- is a distant relative of PDGF and is a member of a new fam-
tirely account for the lack of chemotactic responsiveness to ily of growth factors, which also includes VEGF-B, VEGF-C
PDGF-AA, however. For example, though human foreskin and placenta growth factor (PlGF).51-55 VEGF expression is
fibroblasts express 20,000-30,000 PDGF-AA binding sites per upregulated by hypoxia and by cytokines and growth fac-
cell, and rat and baboon VSMC possess PDGF # receptors tors in several cell types, including arterial VSMC56-58 and
albeit at relatively low levels, the AA isoform failed to stimu- EC.59,60 It has been established that VEGF plays a central
late migration of all these cells and inhibited their migra- role in angiogenesis in a variety of biological processes and
tory response to the BB isoform.34-36 In addition, TGF-!1 disease states, including embryogenesis, wound healing,
decreased migration of rat VSMC as a consequence of tumor growth, and collateral blood vessel formation in
PDGF-AA secretion.36 Koyama et al34 showed that the myocardial ischemia.61,62
PDGF-AA inhibition of baboon VSMC migration stimu- Most of the biological effects of VEGF are attributed
lated either by PDGF-BB or by fibronectin was abolished by to its ability to stimulate EC mitogenesis and recently this
specific antibodies to the # receptor and is therefore medi- has focused interest on the role of VEGF in accelerating re-
ated through this specific receptor isotype. These findings endothelialization following balloon angioplasty. Initial stud-
suggest that the stimulation of directed migration of VSMC ies in animal models of balloon injury have proved promis-
is mediated by the ! receptor, while the # receptor may play ing, and administration of VEGF protein and VEGF gene
a specific role in the negative regulation of chemotaxis. It is transfer have been shown to inhibit intimal thickening fol-
at present unclear, however, whether these conclusions can lowing balloon angioplasty and improve blood flow in is-
be extended either to human VSMC or to VSMC migration chemic limbs, effects mediated through stimulation of EC
in vivo. Human VSMC express # receptors37,38 and respond regrowth and angiogenesis respectively.63-66
mitogenically to PDGF-AA.39 Relatively few studies of the In addition to its mitogenic effects in EC, VEGF also
effects of PDGF-AA on the migration of these cells have been promotes the migration of EC and it is increasingly recog-
published, but it was reported that PDGF-AA stimulates nized that EC migration plays an essential role in angiogen-
chemotaxis in human VSMC and that this response esis and vascular modelling.61,62,67,68 VEGF also has a num-
correlated with the ability of human VSMC to respond ber of biological activities which may contribute to vessel
mitogenically to the AA isoform.39 It is unknown whether wall pathology. VEGF is a permeability-increasing factor,51,69
the # receptor has an inhibitory effect on migration of and also stimulates monocyte chemotaxis and tissue factor
human VSMC. production.70,71 It has also been proposed that VEGF may
Of the other polypeptide ligands for receptor protein play a role in neovascularization of the advanced ather-
tyrosine kinases (PTKs), heparin-binding epidermal growth osclerotic plaque.57,72-74 Induction of VEGF expression in
factor-like growth factor (HB-EGF) has been reported to the arterial wall may therefore be pro-atherogenic both
stimulate migration of bovine aortic smooth muscle cells in through its effects on the permeability of the endothelium,
a heparin-dependent manner.40 Insulin-like growth factor-I thereby facilitating the infiltration of monocytes, platelets
(IGF-1) is present at high levels in platelet #-granules, is se- and T cells, and by a direct effect on monocyte migration.
creted by cultured VSMC and EC41,42 and has increased ex- At later stages of atherogenesis, neo-vascularization induced
pression in rat aorta following balloon injury.43 IGF-1 has by hypoxia and mediated by VEGF may also contribute to
been reported to be a migration factor for human VSMC,37 disease progression.
and results from our laboratory show that IGF-1 promotes
chemotaxis in rabbit aortic VSMC (Abedi H, Zachary I, un- Angiotensin II
published observations). In both cell types, IGF-1 produces The vasoconstrictor angiotensin II is implicated in the
a weaker migratory response compared to PDGF-BB. regulation of VSMC proliferation, but it is unclear whether
Basic fibroblast growth factor (bFGF) is a mitogen for it contributes significantly to migration of these cells. Data
VSMC1,42 but there is little evidence that this factor has from in vitro studies are conflicting. Thus, it was reported
stimulatory effects on VSMC migration in vitro. This factor that angiotensin II stimulates migration of bovine aortic
may play a role in VSMC migration in vivo, however. Ad- VSMC,75 while MII and co-workers found no effect of an-
ministration of bFGF and neutralizing antibodies to bFGF giotensin II on migration of human VSMC.76 It is possible,
respectively stimulated and inhibited VSMC migration fol- however, that angiotensin II may play a role in migration of
lowing balloon injury of the rat carotid artery.44 There is VSMC in vivo. Consistent with this notion, infusion of an-
much stronger evidence that bFGF is a potent migration giotensin II enhanced neointima formation following bal-
factor for EC. Both basic and acidic FGF stimulate move- loon injury of the rat carotid artery77 and, using the same
ment of EC in vitro45-47and have been reported to promote model, it was shown that both inhibitors of angiotensin-
re-endothelialization following balloon injury.48,49 It is likely converting enzyme and antagonists of the angiotensin type 1
that the repertory of polypeptide migration factors for (AT1) receptor inhibited the accumulation of intimal
VSMC will be extended in the future. For example, a 58 kDa VSMC.78 It is unclear from these studies, however, whether
autocrine migration factor which appears to be different the effects of angiotensin II are direct or indirectly medi-
from PDGF was recently purified from the conditioned ated through increased expression of another migration
medium of rat aortic VSMC.50 factor(s) and/or receptor(s).
The secreted polypeptide vascular endothelial growth
factor (VEGF) (also known as vascular permeability factor)
254 Tissue Engineering of Prosthetic Vascular Grafts
Cytokines the case of these ECM components, it is the strength of at-
A variety of cytokines are released from macrophages, tachment as much as adhesion to the matrix itself that is
T lymphocytes, platelets and VSMC in the atherosclerotic one of the critical variables regulating cell motility.
microenvironment and stimulate monocyte chemotaxis and Recently, attention has focused on the role of matrix-
endothelial transmigration.1,27 They include interleukins, degrading metalloproteinases (MMPs) in VSMC migration.
colony-stimulating factors, monocyte chemotactic protein- MMP expression and secretion are upregulated in prolifer-
1 (MCP-1), oxidized low-density lipoprotein (ox LDL) and ating VSMC cultures and during neointimal thickening in-
TGF- ! (Table 24.1). TGF- ! is produced by activated duced by balloon injury of rat and pig carotid arteries.99-101
macrophages,79 VSMC80 and the #-granules of platelets81 and Components of the plasminogen activator system, and in
has been reported to have bimodal effects on VSMC particular the urokinase-type plasminogen activator have
migration.82 Interleukin-1 has also been shown to stimulate also been implicated in the regulation of VSMC migra-
migration of VSMC. 83 The specific monocyte tion.101-104 Receptors for the urokinase-type plasminogen
chemoattractant MCP-1 is secreted by arterial VSMC in activator are upregulated in atherosclerotic lesions. The
hypercholesterolemic primates.84 identification of endogenous tissue inhibitors of mmps
TGF-! regulation of vascular cell migration is as com- (TIMPs) has generated considerable interest in targeting
plex as its effects on VSMC proliferation, which can be both MMPs for the treatment of restenosis and stenosis.
positive and negative.25,85 TGF-! is a potent inducer of the Overexpression of TIMP-1 inhibited VSMC migration in
production of ECM components and also upregulates ex- vitro and attenuated neointimal hyperplasia in the balloon-
pression of integrins in VSMC,85 so it is unclear whether the injured rat carotid artery.105
modulation of VSMC chemotaxis by TGF-! is direct or in- The findings discussed above indicate that VSMC
directly mediated. In theory, TGF-! could also either migration in the arterial wall is modulated by a delicate bal-
stimulate or inhibit VSMC migration indirectly through its ance between degradation and deposition of matrix com-
respective abilities, mentioned above, to induce PDGF-B ponents as well as by the differential effects of distinct ma-
chain expression in EC and PDGF-A expression in VSMC. trix components. It is now increasingly recognized that
TGF-! may also regulate EC migration. In cocultures of bo- integrin-matrix interactions do not simply play a structural
vine EC and smooth muscle cells, TGF-! is secreted in a la- and biomechanical role in the regulation of cell movement.
tent form which is activated by plasmin and subsequently As discussed below, it has become well established that
inhibits EC movement.86 integrins transduce signals into cells which may be impor-
tant in the regulation of cell adhesion and motility.106
Extracellular Matrix
Dynamic adhesive interactions between cell surface Chemotactic Signal Transduction
integrins and components of the ECM play a central role in
cell migration by providing cell-substrate traction for the The Role of the Actin Cytoskeleton and Focal Adhesions
generation of intracellular mechanical stresses.87,88 The Three distinct processes occur during the movement
ECM-integrin network can regulate cell motility in at least of animal cells: protrusion, in which lamellipodia and
three ways: microspikes (or filopodia) extend from the leading edge of
1. Through specific ECM-integrin interactions; the cell; attachment, in which focal adhesion plaques form
2. By changes in the concentration of particular ECM adhesive interactions with the substratum; and traction,
substrates; and through which the intracellular mechanical forces generated
3. As a result of varying cell surface integrin density. by the locomotive cell machinery moves the cell body for-
The last of these possibilities will be considered in the ward. All of these processes are crucially dependent on the
next section. actin cytoskeleton and associated ultrastructural elements
Deposition of matrix is a major feature of the fibrous such as the focal adhesion.
atherosclerotic lesion.1,2 Several ECM components, includ- Migration is a highly dynamic process involving the
ing fibronectin34,89 and vitronectin,90 promote migration of rapid, organized and coordinated polymerization and
VSMC. The RGD-motif ECM molecule osteopontin is depoylmerization of actin filaments, crosslinking between
upregulated in proliferating rat VSMC, during rat neointima actin filaments, cell adhesion and detachment at focal con-
formation and in human atherosclerotic lesions.91,92 It was tacts, and the assembly and disassembly of macromolecular
recently reported that osteopontin promotes adhesion, complexes associated with both actin stress fibers and focal
spreading and chemotaxis of rat VSMC.93,94 TGF-!1 induces adhesions. Furthermore, directed cell movement implies that
expression of collagen types I, III and V and of fibronectin these dynamic changes in the configuration and organiza-
in VSMC.95-97 Other ECM components such as laminin, tion of components of the actin cytoskeleton is highly orga-
collagen and the glycosaminoglycan heparin may have in- nized in cells both spatially and temporally. While actin po-
hibitory effects on migration in vivo.1 However, VSMC do lymerization is essential for the rapid formation and pro-
migrate on collagen substrates in vitro.37,38,98 It was recently trusion of lammellipodia at the leading edge of a migrating
reported that maximal migration of human VSMC occurs cell, depolymerization of actin elsewhere is vital both to pro-
on fibronectin and collagen IV substrates at intermediate vide a source of monomeric actin for localized and rapid
attachment strengths.98 Since ECM concentration is one of stress fiber formation and to release or weaken adhesions
the key parameters determining the ‘stickiness’ of cell-sub- and thus allow cell movement. Similar considerations apply
strate interactions, this finding may suggest that, at least in to focal adhesions, whose rate of turnover and/or detach-
Signaling Mechanisms for Vascular Cell Migration 255
ment/attachment to the cellular substrate is also a crucial as yet showing unambiguous PDGF-BB stimulation of stress
parameter determining the rate and direction of cell loco- fiber or focal contact formation in vascular cells, suggesting
motion. In addition to dynamic adhesive interactions be- that regulation of actin cytoskeleton organization is complex.
tween the cell and its substrate, eukaryotic cell locomotion Given their pivotal cellular location at a point of close
requires the generation of force by the cytoskeleton and as- juxtaposition between the ECM and the actin cytoskeleton,
sociated components.87,88 attention has naturally focused on the role of components
Actin filaments organized in stress fibers and their sites associated with actin and with focal adhesions in regulating
of attachment at focal adhesions are both thought to play a signal transmission between the matrix and the
critical role in the regulation of cell adhesion and locomo- actin cytoskeleton.
tion.107,108 Focal adhesions are specialized juxtamembrane
regions which form at the termini of stress fibers and at sites Actin-Binding Proteins
of attachment of cells to the ECM. Integrins, the adhesive A large number of actin-binding proteins have been
receptors for ECM components, aggregate at focal contacts identified, some of which are thought to play an important
and many nonstructural cytoskeletal-associated components role in regulating the dynamics of actin polymerization. The
are localized to these subcellular structures (Table 24.2). scope of this review precludes a comprehensive discussion,
Interactions between integrins and the cytoskeleton and brief consideration will be directed to some of the find-
are themselves very dynamic. 109,110 For example, in ings most directly relevant to chemotactic signaling (see ref.
nonmotile cells, !1 integrins are localized in focal adhesions 108 for a recent review).
at the ends of stress fibers while in motile cells, integrins are Profilin is a 12-17 kDa protein which is associated with
found in organized, but dynamic substructures termed actin filaments in dynamic areas of the cell such as the lead-
macroaggregates that are in less intimate association with ing lamellae and at the termini of newly-formed stress fi-
focal contacts.109 bers. 108,119 In vitro profilin binds to actin, and
The formation of stress fibers and the assembly of fo- phosphatidylinositol 4,5-bisphosphate (PIP2). Earlier find-
cal contacts is stimulated in adherent cells including VSMC ings suggested that profilin had a strong inhibitory effect on
and EC by a variety of extracellular factors including PDGF, actin polymerization, but it is now believed that the effect
VEGF, bombesin, angiotensin II, the serum component of this protein on actin polymerization may be consider-
lysophosphatidic acid (LPA) and the ceramide sphin- ably more complex. Profilin binds and sequesters G-actin,
gosine.68,111-115 In serum-starved Swiss 3T3 cells and at rela- thus reducing the concentration of free actin monomers and
tively low concentrations, PDGF has been reported to in- lowering the rate of polymerization. On the other hand,
duce a slow increase in focal adhesion assembly and stress profilin also stimulates ATP/ADP exchange on G-actin,
fiber formation as well as intense membrane ruffling.115 The thereby promoting actin polymerization.120 Gelsolins are a
function of membrane ruffling is unclear but it appears to large family of proteins, the most dominant form of which
be closely associated with pinocytosis.115-117 In Swiss 3T3 is 80 kDa and contains multiple actin binding sites.108,121
fibroblasts, PDGF appears to have bimodal effects on actin Gelsolins are associated with focal adhesions and can both
cytoskeleton organization and, at higher concentrations, sever actin filaments and also cap the barbed (fast growing)
induces a marked disruption of the actin cytoskeleton.118 The ends of actin filaments.121 Like profilin, gelsolin binds to PIP2,
relevance of findings in Swiss 3T3 cells for the potent chemo- and a role for this protein in modulating agonist-induced
tactic effects of PDGF-BB in VSMC are unclear, however. actin filament organization through PIP2 hydrolysis has been
PDGF-BB causes no disruption of the actin cytoskeleton in proposed (see below).
cultured human and rabbit VSMC.38 There are no reports
Table 24.2. Cellular components implicated in cytoskeletal reorganization and cell migration
Focal Adhesion Components Small GTP-binding proteins SH2/SH3 Domain Proteins
!1, !3 Integrins 90-110 kDa p21 rho p85-a/PI3 kinase
Vinculin 116 kDa pp60src
Tensin 215 kDa p25 rac p130Cas
Talin 220 kDa GRB-2
Zyxin 82 kDa Cdc42Hs PLC-&
#-actinin 90 kDa Csk
FAK 125 kDa Crk
FRNK 41-43 kDa
Paxillin 68 kDa
Components implicated in the signaling mechanisms underlying cytoskeletal reorganization and cell migration. Alpha-actinin is a cross-
linking protein which binds to the cytoplasmic domain of beta-integrins, and to vinculin and zyxin. Talin binds to vinculin and beta-integrins
in vitro. Tensin is tyrosine phosphorylated in reponse to integrin activation and in cells over-expressing p125FAK. Other components listed
are discussed in the text. All non-standard terms and abbreviations are defined in the text.
256 Tissue Engineering of Prosthetic Vascular Grafts
Several lines of evidence suggest that the level of ex- Focal Adhesion Kinase
pression of actin-binding proteins may influence vascular FAK is a member of a growing family of protein ty-
cell motility. Gelsolin has been implicated in EGF-stimu- rosine kinases (PTKs) which also includes the recently iden-
lated cell motility and in histamine-induced changes in ac- tified FAKB and PYK-2.131-133,138,139 FAK was originally iden-
tin filament organization in HUVEC.122 Overexpression of tified as one of a group of proteins that are highly tyrosine
both gelsolin and CapG did not have significant effects on phosphorylated in chicken embryo fibroblasts transformed
actin filament content or organization, but did increase the with the v-src oncogene.140 The cDNA for FAK encodes a
rates of wound healing and chemotaxis. On the other hand, protein with a predicted molecular weight of 119-121 kDa
in rat aortic VSMC, it was reported that gelsolin protein and depending on the species, though on the basis of its migra-
mRNA are reduced during intimal thickening induced by tion in gels it is often designated p125FAK. FAK is widely ex-
endothelial injury of rat aortas.123 pressed in tissues, and is present in fibroblasts, VSMC, EC,
platelets, lymphocytes, monocytes and neuronal cells. A
Integrins 41-43 kDa protein corresponding to the noncatalytic car-
VSMC express !1 and !3 integrins as well as a variety boxyl-terminal FAK domain, termed FRNK (FAK-related
of # subunits including #v and #1.94,124-129 The !1 integrin is nonkinase) is autonomously expressed in some cells.139 A
constitutively expressed in most cells in the body106 and this salient feature of FAK is its subcellular localization to focal
also appears to be true for VSMC.127,128 In contrast, expres- adhesions and this has stimulated intense interest in the role
sion of !3 is upregulated in VSMC by TGF- ! and by of this kinase in the regulation of cell adhesion, cytoskeletal
PDGF-BB, and TGF-! was also reported to induce expres- organization and cell locomotion.131-133
sion of #1 subunits.127,128 Recent results suggest that !1 and The structure of FAK is unique, comprising a central
!3 integrins may have different functions in VSMC adhe- catalytic region flanked by two large noncatalytic domains
sion and migration. Thus, antibodies to !1 inhibit attach- with no homologies to other PTKs (Fig. 24.1). Tyrosine phos-
ment and spreading of rabbit arterial VSMC on collagen and phorylation of FAK is stimulated through the signal trans-
fibronectin substrates126 while osteopontin-stimulated mi- duction pathways initiated by v-src transformation,140,141
gration of VSMC was inhibited by antibodies to !3 but not activation of !1 integrins in fibroblasts142-144 and #IIb/!3
anti-!1 antibody.94 Interestingly, it has been reported that in integrins in platelets,145,146 by aggregation of high-affinity
the early stages of VSMC migration !1 integrins are orga- IgE receptors in mast cells,147 and in Swiss 3T3 fibroblasts
nized into focal adhesions which gradually cover the basal by regulatory peptides including bombesin and
surface of the cells, while !3 integrins remain at the leading endothelin148,149 and by LPA111 and sphingosine.112 It was
edges of cells.129 These findings suggest that !1 integrins are recently reported that tyrosine phosphorylation of FAK is
both necessary and probably sufficient for cell adhesion, also induced by hepatocyte growth factor in oral squamous
while !3 integrins may play a more specific role during cell carcinoma cells.150 The integrity of the actin cytoskeleton
chemotaxis or migration. is essential for FAK tyrosine phosphorylation.68,111,112,118,149
Most integrins have small cytoplasmic domains which Much evidence suggests that the noncatalytic amino-
lack any intrinsic enzymatic activity, and this suggests that and carboxyl-terminal domains mediate associations with
the mechanisms involved in integrin-mediated signaling are integrins, other focal adhesion components, and signaling
fundamentally distinct from those of receptor PTKs or molecules.132,133 Little is known about the function of the
G-protein linked receptors. In the past few years it has be- amino-terminal domain of FAK, but as mentioned above it
come clear that integrin-mediated cell adhesion stimulates may associate directly with the cytoplasmic domains of
several signaling pathways.106,130 In particular, it is now well integrins.151 The COOH-terminal noncatalytic domain is
established that activation of !1 integrins in adherent cells sufficient for targeting of FAK to focal adhesions152 and is
induces tyrosine phosphorylation of focal adhesion kinase also implicated in the association of FAK with paxillin, a
(FAK) and the focal adhesion-associated protein 68 kDa protein which colocalizes to focal contacts. 153
paxillin.107,131-133 Attachment of fibroblasts, EC and lympho- Bombesin, PDGF-BB, VEGF and cell adhesion to fibronectin
cytes to fibronectin also causes an increase in intracellular all induce paxillin tyrosine phosphorylation in fibroblasts
pH,106 and this response is implicated in cell proliferation.130 concomitantly with FAK phosphorylation38,68,143,154 and
Attachment of Swiss 3T3 fibroblasts to fibronectin and paxillin is a putative substrate for FAK.155,156 Paxillin has
laminin has also been reported to activate mitogen-activated recently been molecularly cloned and its sequence reveals a
protein (MAP) kinases,134-6 but, as will be discussed below, number of interesting features, including a proline-rich re-
the role of this pathway in cell migration is a matter of de- gion which is a potential binding site for SH3 domains, a
bate. Recent findings suggest that integrins may act syner- putative binding region for the focal adhesion proteins talin
gistically with receptors for extracellular growth factors.130 and vinculin and several consensus binding sites for v-Crk.157
In particular, attachment of some cells to fibronectin and Another component called p130Cas (Cas stands for Crk-as-
antibody-induced aggregation of the #5!1 integrin enhances sociated substrate) is also tyrosine phosphorylated in re-
the increase in cytoplasmic pH stimulated by PDGF.137 Such sponse to bombesin concomitantly with FAK in Swiss 3T3
cooperative interactions may underlie the well known but cells.148 P130Cas contains an SH3 domain and multiple bind-
poorly understood phenomenon of adhesion-dependent ing sites for v-Crk,158 and has been shown to associate with
proliferation. FAK.159,160
Several factors which potently stimulate stress fiber
and focal adhesion formation, including LPA, sphingosine
Signaling Mechanisms for Vascular Cell Migration 257
Fig. 24.1. The structure of FAK and its asso-
ciations with other cellular components. The
functions of the catalytic region and the
noncatalytic amino and carboxy-terminal do-
mains are indicated above the schematic.
Paxillin is known to interact with the carboxy-
terminal domain of FAK. Specific binding sites
for pp60Src, GRB-2 and p85# are indicated. A
proline-rich sequence has been shown to in-
teract with the SH3 domain of p85#. Other
SH2 domain proteins reported to associate
with FAK but for which the site of association
is unknown are boxed.
and bombesin, also increase tyrosine phosphorylation of receptor coupled to two G-protein pathways, one pertussis-
FAK. Recent findings have implicated FAK in the migration toxin sensitive which is implicated in activation of p21Ras,163
of other cell types.150,161 In oral squamous cell carcinoma the other pertussis toxin-insensitive leading to activation of
cells the protein tyrosine kinase inhibitor herbimycin blocked phospholipase C and tyrosine phosphorylation of FAK.164,165
the stimulation of FAK tyrosine phosphorylation and cell Consistent with the involvement of this second PLC-medi-
migration by hepatocyte growth factor.150 Intriguingly, the ated pathway, LPA-induced stress fiber formation was per-
ECM glycosaminoglycan hyaluronan has been shown to tussis toxin-insensitive.113 Though FAK lacks both src ho-
promote both the rapid assembly and disassembly of focal mology-2 (SH2) and SH3 domains,166 several recent reports
adhesions and a rapid phosphorylation-dephosphorylation indicate that FAK can associate with SH2-domain proteins.
of FAK in a c-H-ras-transformed fibroblast cell line.161 In FAK was originally identified as a prominently tyrosine phos-
VSMC and in EC, effects of several factors on FAK tyrosine phorylated protein in v-src-transformed fibroblasts,140 and
phosphorylation correlate with a chemotactic response.38,68 it has subsequently been shown that FAK associates with
Results from our laboratory show that LPA induces FAK ty- pp60c-src and pp59c-Fyn via an interaction between the SH2
rosine phosphorylation and directed migration in rabbit domain of Src-family PTKs and the major autophos-
aortic VSMC (Abedi H, Zachary I, unpublished observa- phorylation site of FAK, Y397.167 Several other SH2 domains
tions). Angiotensin II stimulates FAK and paxillin tyrosine (listed in Table 24.2) including those of the PDGF receptor
phosphorylation in rabbit VSMC (Abedi H, Zachary I, un- substrates p85#,168 GRB-2, NCK and phospholipase C-&169
published observations),89 but as already indicated it is un- have also been found to associate with FAK. Moreover, in
clear whether this factor can induce a direct migratory re- the case of the GRB-2 SH2 domain, association appears to
sponse in VSMC. PDGF-BB stimulates FAK and paxillin ty- result from the tyrosine phosphorylation of FAK at a spe-
rosine phosphorylation in VSMC through the ! receptor38,118 cific residue, Y925, induced by cell attachment to
and VEGF stimulates FAK and paxillin tyrosine phospho- fibronectin.170 Two other SH2 domain-containing proteins
rylation in human EC.68 IGF-1 has been reported to stimu- have recently been implicated in the regulation of focal ad-
late FAK and paxillin tyrosine phosphorylation concomi- hesion-associated components. The adapter protein p47Crk
tantly with lammellipodial advance in neuroblastoma associates with paxillin171 and the carboxy-terminal src ki-
cells,162 and our recent findings show that in VSMC, IGF-1 nase (Csk), which phosphorylates and negatively regulates
promotes tyrosine phosphorylation of FAK, directed migra- pp60 Src, has been shown to bind to both paxillin and
tion and an increase in immunolocalization of FAK to focal FAK.169,172 FAK also associates with SH3 domain-contain-
adhesions (Abedi H, Lobo M, Zachary I, unpublished ob- ing proteins. In platelets, FAK associates with the SH3 rather
servations). Taken together, these findings suggest that ty- than the SH2 domain of the p85 # subunit of
rosine phosphorylation of FAK might represent a conver- phosphatidylinositol 3' kinase (PI3 kinase) via a proline-rich
gent point in the signaling pathways stimulated by several sequence (residues 706-711) in the noncatalytic carboxy-
migration factors for vascular cells and other cell types. The terminal domain of FAK128.170 The same FAK motif also
role of FAK as a common target in the action of diverse fac- mediates association with p130Cas, and FAK-p130Cas com-
tors and cell surface receptors is highlighted in Figure 24.2. plexes are enriched in cytoskeleton-associated fractions in
FAK tyrosine phosphorylation may not be a common path- an adhesion-dependent manner.159,160 The associations of
way for all migration factors, however. There are, for example, FAK with other cellular components are summarized in Fig-
no reports to date that bFGF, a potent chemoattractant for ure 24.1.
EC, can regulate this pathway.
The signal transduction pathways which mediate ty- Rho-Related GTP-binding Proteins
rosine phosphorylation and presumably increased activity One of the most exciting developments in chemotac-
of FAK are unclear. LPA appears to act through a membrane tic signal transduction has been the discovery that
258 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 24.2. FAK as a convergent point in signaling
pathways for !1 and !3 integrins, receptor PTKs and
seven transmembrane domain, G-protein coupled
receptors. The diagram illustrates current evidence
concerning the signal transduction mechanisms
distal to FAK activation and downstream of FAK.
Small GTP-binding proteins of the Rho/Rac fam-
ily are implicated in mediating regulation of FAK
tyrosine phosphorylation through G-protein
coupled receptors and less certainly through the
PDGF receptor. Integrin-dependent activation of
FAK is thought to involve direct associations, pos-
sibly with the amino-terminal domain of FAK. The
substrates of FAK which are important in vivo are
still unknown, but FAK has been shown to associ-
ate in vivo and in vitro with several components in
the focal adhesion (paxillin, talin) and with pro-
teins containing either SH2 and/or SH3 domains
(p130Cas, Src, PI3 kinase). How signals are relayed
from FAK through FAK-associated proteins to the
focal adhesion remains unknown. Key: RPTK, re-
ceptor PTK; PI3K, PI3 kinase. Other nonstandard
terms and abbreviations are defined in the text.
Ras-related small GTP-binding proteins of the Rho and Rac mation and cdc42 induces peripheral actin microspikes and
family are implicated in regulation of cytoskeletal organiza- filipodia.175,176
tion.115,117,173 The first evidence for the role of Rho proteins Despite the interest in Rho and other members of the
in actin filament organization came from studies of the ex- Rho/Rac family in regulating actin cytoskeleton organiza-
oenzyme C3 transferase, which is produced by the bacte- tion, there are few studies of these proteins in vascular cells,
rium Clostridium botulinum and which ADP-ribosylates Rho and consequently their role in the migration (or other func-
at residue Asn 41 and thereby inactivates it. Introduction of tions) of vascular cells remains poorly understood. A few
C3 transferase into cells causes a loss of actin stress fibers.173 studies have, however, implicated Rho in some biological
Microinjection into fibroblasts of either wild type recombi- responses in smooth muscle, including contractility and
nant Rho proteins174 or constitutively activated Rho pro- myosin light chain phosphorylation.107
teins with a valine residue instead of glycine at residue 14115 Recent findings have implicated Rho in mediating
causes a rapid formation of stress fibers and an increase in phosphorylation of FAK (Fig. 24.2). C3 transferase inhibits
the localization of the focal adhesion associated proteins FAK tyrosine phosphorylation induced either by LPA,
vinculin and talin to focal adhesions. Importantly, introduc- bombesin or by the nonhydrolyzable GTP analog
tion of either C3 transferase or of ADP-ribosylated and in- GTP&S.165,177,178 Introduction of Rho into cells has also been
activated rhoA into cells blocked the stimulation of stress shown to stimulate tyrosine phosphorylation of FAK,
fiber formation and focal adhesion assembly caused by paxillin and p130Cas.179 These findings argue strongly that
bombesin, LPA or serum.115 Recent evidence suggests that Rho is involved in the activation of FAK through some
Rho-related proteins perform specialized roles in the regu- G-protein-coupled seven transmembrane domain receptors.
lation of actin filament organization. While Rho appears to Interestingly, it was reported that the SH2/SH3 domain pro-
be important for stress fiber and focal adhesion formation, tein crk, which as mentioned above binds to paxillin, asso-
Rac promotes membrane ruffling and lammellipodia for- ciates via its SH3 domains with the guanine nucleotide ex-
Signaling Mechanisms for Vascular Cell Migration 259
change factor C3G.180 Since C3G may regulate the activity with Rho, called Rho-kinase and PKN.107 Rho-kinase has
of Ras proteins, this finding suggests that other small GTP- been reported to phosphorylate MLC in vitro.182 The pri-
binding proteins may participate in the regulation of FAK mary phosphorylation site for Rho-kinase, Ser19, is identi-
and associated proteins in the focal adhesion complex. cal to the major phosphorylation site utilized by MLC ki-
Very little is known about the role of Rho family pro- nase,183 and phosphorylation of this residue is required to
teins in the activation of the FAK pathway through receptor enable actin activation of the myosin ATPase.184 Evidence
tyrosine kinases. The recent finding that PDGF-BB stimu- supporting a direct role for Rho-kinase in stress fiber and
lates an increase in the GTP-bound form of Rac, and that focal adhesion formation has come from microinjection
Rac activation is inhibited by the PI3 kinase inhibitor studies. 185 Rho has also been reported to stimulate
wortmannin,181 suggests that this small GTP-binding pro- phosphatidylinositol 4-phosphate 5-kinase (PI4P5K) which
tein may play a role in mediating PDGF stimulation of the catalyzes formation of PIP2 and promotes actin polymer-
FAK pathway (Fig. 24.2). Regardless of the precise sequence ization (see below). The central role of Rho in signaling path-
of interactions involved, it is likely that versatile proteins ways regulating actin cytoskeletal organization is illustrated
containing SH2 and SH3 modules will provide the crucial in Figure 24.3.
mediating links between the PDGF receptor, GTP-binding
proteins of the Rho family and FAK. Receptor Protein Tyrosine Kinase Signaling
How Rho is itself activated by these (or indeed any Given the importance of ligands for receptor PTKs in
other) receptors is unclear, but recent progress has been made the regulation of cell migration, it will be helpful to give a
towards the identification of candidate downstream effec- brief general discussion of some of the principal themes
tors which mediate the actions of Rho. Two serine/threo- which have emerged in studies of receptor PTK activation
nine protein kinases have been identified which associate and signal transmission. The PDGF receptor will serve as a
Fig. 24.3. The central role of Rho proteins in the
regulation of actin cytoskeleton. Regulation of Rho
activity is still poorly understood, partly because it
has been difficult to directly measure Rho-bound
GTP/GDP ratios. Studies with C3 exoenzyme and
dominant negative Rho mutants suggest that Rho
lies downstream of integrins and G-protein coupled
receptors. Little evidence is available for receptor
PTKs, though Rac may be activated by PDGF. Ef-
fectors for Rho include phosphatidylinositol
4-phosphate 5-kinase (PI5K) and Rho kinase. FAK
activation may also be mediated by Rho, though
the mechanism is unclear. Both PI5K and Rho-ki-
nase have been shown to stimulate actin filament
and focal adhesion formation. Effects of Rho-ki-
nase may in part be mediated through phosphory-
lation of myosin. Key: MLC, myosin light chain.
Other abbreviations are explained in the text.
260 Tissue Engineering of Prosthetic Vascular Grafts
paradigm. The receptor(s) for PDGF consists of an extra- through the tyrosine phosphorylation and activation of
cellular ligand-binding domain, a single membrane-span- PLC-&. PIP2 binds to the actin-binding proteins profilin and
ning region and a cytoplasmic domain with intrinsic PTK gelsolin. and it has been proposed that PIP2 can modulate
activity. Binding of PDGF in the form of homo- or actin filament formation through the sequestration of ac-
heterodimers of A and B chains induces dimerization of the tin-binding proteins like profilin and gelsolin. Anti-sense
receptor and subsequent receptor autophosphorylation. oligonucleotides to gelsolin and expression of a peptide
Autophosphorylation is thought to occur mainly by which competes for the PIP2-binding site on gelsolin both
transphosphorylation of each dimerized receptor molecule reduced epidermal growth factor-induced cell movement in
by its partner. Homodimers of # and ! receptors as well as NR6 cells.195 In platelets, gelsolin-derived PIP2-binding pep-
#/! heterodimers have all been identified, but as indicated tides also inhibited uncapping of F-actin induced by throm-
above the relative abundance of a given receptor dimer is bin receptor activation.196
highly dependent on cell-type and species. Dimerization and PIP2 has also been reported to bind to vinculin and
receptor autophosphorylation are essential for the activation unmask its talin and actin binding sites, thereby promoting
of the receptor.186 Subsequently, the receptor associates with focal adhesion and stress fiber formation.197 Profilin bind-
and tyrosine phosphorylates its intracellular targets via SH2 ing to PIP2, the substrate of PLC-&, inhibits PLC-& activity
domain interactions with specific tyrosine phosphorylated and this inhibition is overcome by tyrosine phosphoryla-
residues in the cytoplasmic domain of the receptor.186,187 tion. By effectively lowering the concentration of PIP2, acti-
Though an increasing number of the cellular compo- vation of PLC-& can potentially promote actin filament dis-
nents which are phosphorylated by the PDGF ! receptor have assembly both by raising the level of free profilin and gelsolin
been identified,186,187 it remains unclear which components and by reducing its vinculin unmasking activity (Fig. 24.4).
or intracellular signaling pathways specifically mediate the As indicated above, however, the role of profilin may be more
effects of PDGF-BB on cell migration and locomotion. In complex. Another consequence of PIP2 hydrolysis is the for-
addition, most studies of PDGF Receptor signal transduc- mation of inositol 1,4,5 trisphosphate, which in turn leads
tion have been performed on the ! receptor, which as dis- to the mobilization of intracellular Ca2+. Ca2+ also affects
cussed above is able to transduce mitogenic and chemotac- the equilibrium between actin polymerization and depoly-
tic signals in a wide variety of cells. In contrast, # receptor merization and also activates calcium-dependent enzymes
signaling has not been intensively examined. As a result, little such as Ca2+/calmodulin-dependent protein kinase II and
is known concerning either cell type-specific or receptor the Ca 2+ /calmodulin-activated protein phosphatase
isotype-specific differences in PDGF signal transduction.187 calcineurin, both of which have been implicated in the regu-
The main features of the activation mechanism that lation of VSMC migration (see Fig. 24.4).198,199
have been identified for the PDGF receptor have also been PIP2 levels can be increased in cells through the acti-
demonstrated for other receptor PTKs. In some cases, how- vation of phosphatidylinositol 4-phosphate 5-kinase
ever, the signal transduction mechanisms are not yet par- (PI4P5K). Overexpression of PI4P5K in COS-7 cells causes
ticularly well understood.188-190 The VEGF receptor will serve a striking increase in actin polymerization.200 This kinase
as an example. The diverse biological effects of VEGF are has been shown to be regulated by Rho,201 thus providing a
mediated through two specific PTK receptors, KDR/Flk-1 link with the pathway thought to be responsible for activa-
and Flt-1. Both receptor isotypes are expressed in ECs67 and tion of FAK by bombesin and LPA and potentially of other
Flt-1 is expressed in monocytes.71 Expression of VEGF re- factors which act through seven transmembrane domain,
ceptors has to date only been reported in these two cell types. G-protein coupled receptors. It is not known which extra-
KDR/Flk-1 is thought to be the receptor largely responsible cellular factors are responsible for activating PI4P5K, but it
for the angiogenic effects of VEGF and its mitogenic effects may be speculated that activation of this kinase and the con-
in EC.191,192 The signal transduction pathways which medi- sequent increase in cellular levels of PIP2 may contribute to
ate the diverse biological effects of VEGF either in EC or in the striking increase in actin filament and focal adhesion
monocytes remain unclear. Studies of VEGF signal trans- formation caused by bombesin, LPA and other factors which
duction have so far produced varying results.67,68,193,194 In activate FAK through a Rho-dependent pathway. Integrin
porcine aortic EC which have been transfected either with engagement has also been reported to activate PI4P5K.202
KDR or Flt-1, VEGF stimulated migration and proliferation
but had little effect on tyrosine phosphorylation of phos- PI3 Kinase
pholipase C-& (PLC-&), p120 GTPase-activating protein Activation of PI3 kinase is an early event in the action
(GAP) or on phosphatidylinositol 3 kinase (PI3 kinase) ac- of several receptor PTK ligands, including PDGF-BB and
tivity.67 In contrast, VEGF has been reported to stimulate IGF-1. Recent findings have implicated PI3 kinase in PDGF-
tyrosine phosphorylation of p120GAP, PLC-& and p85# sub- induced chemotaxis.203-205 PDGF-BB failed to stimulate
unit of PI3 kinase in bovine aortic EC and NIH 3T3.193,194 migration in Chinese hamster ovary cells expressing mu-
Our recent findings show, however, that in HUVEC, VEGF tant forms of the PDGFR! which are unable to associate with
activates MAP kinase and stimulates tyrosine phosphoryla- the p85# subunit of PI3 kinase.203 In addition, p85#/PI3 ki-
tion of PLC-&, FAK and paxillin.68 nase has also been implicated in membrane ruffling induced
by PDGF204 and IGF-1.205 PI3 kinase has also been impli-
Phosphatidylinositol Lipids and Calcium cated in mediating PDGF-stimulated FAK tyrosine phos-
Many chemoattractants for vascular cells, including phorylation. In Swiss 3T3 cells, inhibitors of PI3 kinase
PDGF, IGF-1, bFGF and VEGF, stimulate hydrolysis of PIP2 blocked PDGF-induced FAK and paxillin tyrosine phospho-
Signaling Mechanisms for Vascular Cell Migration 261
Fig. 24.4. The role of phosphoinositide metabolism in
the regulation of actin cytoskeleton organization. Many
migration factors, particularly ligands for receptor PTKs,
activate PLC-&, leading to hydrolysis of PIP2, and the gen-
eration of inositol 1,4,5-trisphosphate (IP3). Hydrolysis
of PIP2 may release actin-binding proteins (profilin and
gelsolin) which can promote depolymerization of actin.
Similarly, since PIP2 has been reported to allow vinculin
to bind to talin and actin, thereby promoting focal adhe-
sion formation, hydrolysis of PIP2 may also reduce focal
adhesion assembly. As explained in the text, at least in
the case of profilin, the role of PIP2-binding actin-asso-
ciated proteins may be more complex than suggested by
this model. Ca2+ mobilized from intracellular stores by
IP3 may also promote cell migration through its effects
on Ca2+/calmodulin-dependent protein kinase (CamKII)
and calcineurin.
rylation without affecting stimulation by bombesin. The sig- The mechanisms by which products of arachidonic acid
nificance of these findings for cell migration are unclear, metabolism stimulate chemotaxis in vascular cells are at
especially since PDGF causes a profound inhibition of FAK present not well understood.
tyrosine phosphorylation, migration and actin cytoskeleton
organization at concentrations higher than 10 ng/ml in the Nitric Oxide
same cells. Our own studies in rabbit aortic VSMC indicate Recent evidence suggests that NO may play a role in
that the PI3 kinase inhibitors wortmannin and LY294002 at angiogenesis and in the migration of both epithelial cells
concentrations which inhibit PDGF-BB-stimulation of PI3 and EC.211-213 In particular, NO production has been shown
kinase activity do not impair either the migratory response to be permissive for endothelin-induced migration of
to PDGF-BB or PDGF-BB-induced FAK tyrosine HUVECs.213 These findings indicate that NO production is
phosphorylation (Abedi H, Cospedal R, Zachary I, manu- at least permissive for EC migration in response to factors
script submitted). such as endothelin, but the mechanism through which the
NO pathway could act is unknown. Other EC migration fac-
Arachidonic Acid Metabolism tors may also act through the NO pathway. Our recent find-
Studies in several cell types implicate arachidonic acid ings show that VEGF is able to induce NO production in EC
and products of its metabolism in chemotactic signaling. (Zachary I, unpublished findings).
The best evidence so far is for a role of the products of the
lipoxygenase pathway in the migration of monocytes and The MAP Kinase Pathway
eosinophils. 5-oxo-15-HETE is a potent chemoattractant for Recent findings have established that the engagement
eosinophils,206,207 and other lipoxygenase products, lipoxin of integrins can activate the MAP kinase pathway.134-136 The
A4 and lipoxin B4, have been shown to stimulate chemot- role of integrin-mediated stimulation of this pathway in
axis of human monocytes.208 Other studies have implicated cytoskeletal changes is unclear, however. Thus, Clark and
the cytosolic phospholipase A2, thought to catalyze the ago- Hynes showed that while adhesion-dependent activation of
nist-driven release of arachidonic acid, in migration of Erk-2 is dependent upon Ras, inhibition of Ras signaling by
monocytes in response to the chemokines RANTES and expression of a dominant negative Ras mutant (N17Ras)
MCP-1,209 and in migration of EC stimulated by bFGF.210 blocked Erk-2 activation but had no effect either on FAK
262 Tissue Engineering of Prosthetic Vascular Grafts
tyrosine phosphorylation or on cell adhesion, spreading, of a common signaling network relaying information be-
focal adhesion formation or stress fiber assembly.136 Other tween the cell surface and the cytoskeleton. More precise
investigators have provided evidence that integrin activa- delineation of the interactions between the FAK and Rho
tion of Erks is mediated through Rho rather than Ras.202 pathways and of both with pathways involving proteins con-
Integrins failed to activate Erk-1/2 in the absence of Rho/ taining SH2 and SH3 domains, is likely to yield important
Rac activity, and integrin-dependent MAP kinase activity new insights into the mechanisms underlying the regula-
was blocked by C3 exoenzyme and was enhanced by expres- tion of vascular cell migration.
sion of a constitutively active Rho mutant (RhoQ63L).214 In the case of the most potent known VSMC migra-
Though some chemoattractants for VSMC and EC, includ- tion factor, PDGF-BB, the migratory response appears to be
ing PDGF-BB, activate the MAP kinase cascade, other fac- determined by a complex interplay between surface expres-
tors such as IGF-1 stimulate chemotaxis of human VSMC sion of receptor isotypes, the bioavailability of ligand
without activation of MAP kinase.37 These findings suggest isoforms and other as yet unidentified cell type specific de-
that the MAP kinase pathway is at least not obligatory for terminants. Identification of the differences in signaling in
VSMC migration, and it has been proposed that mitogenic different cells via # and ! receptor is likely to be a crucial
and chemotactic signaling pathways can be fairly sharply prerequisite for understanding the mechanisms underlying
dissociated.37,215 A degree of functional segregation of sig- the PDGF chemotactic response. It is plausible that alter-
nal transduction mechanisms underlying these processes is ations in the VSMC signaling pathways induced by PDGF
attractive, especially since the same factor (e.g., PDGF-BB) and associated changes in biological responsiveness, medi-
is often able to stimulate both mitogenesis and migration. It ated in part by changes either in autocrine/paracrine ligand
is possible, however, that MAP kinases may cooperate with production and/or receptor isotype expression, could also
other signaling pathways to augment a migratory response. have important implications for our understanding of the
Unpublished results from our laboratory show that a selec- mechanisms underlying the long term pathogenesis of ath-
tive inhibitor, PD98059, of MAP kinase kinase, the enzyme erosclerosis and other vascular pathologies. Whether a simi-
which specifically phosphorylates and activates Erk 1 and 2, lar complexity underlies the chemotactic effects of other fac-
caused partial inhibition of PDGF-BB-stimulated migration tors such as bFGF, IGF-1 and VEGF awaits further study.
without reducing FAK tyrosine phosphorylation. Stimula- A further challenge, with particular relevance to the
tion of rat VSMC migration by PDGF-BB has also been vessel wall, is posed by the diversity of extracellular factors
shown to be partially inhibitable by PD98059.216 Since IGF-1 which may regulate vascular cell migration. It is probable
is a less potent chemoattractant for VSMC than PDGF-BB, that this diversity will grow and that the repertory of VSMC
it might be speculated that activation of MAP kinase by the migration factors will be extended in the future both by the
latter factor could help to explain this difference. This pos- discovery of novel regulatory molecules and by identifying
sibility remains to be tested. new functions for existing components of the hemostatic
system. The dependence of VSMC migration on the com-
Future Perspectives plex interplay between diverse environmental factors has
Elucidation of the signal transduction mechanisms important ramifications for chemotactic signal transduction.
underlying the movement of cells remains a major unre- The finding that FAK is a point of convergence in signaling
solved problem in molecular cell biology. In the past few mediated both by integrins and by several receptor PTKs
years, however, our understanding of these mechanisms has raises the possibility that, in part at least, chemotactic sig-
been transformed by the identification of a variety of com- naling mechanisms through distinct receptors may share
ponents which are implicated in key cellular processes linked common features. It is equally likely that important differ-
to cell locomotion, including assembly of focal adhesions ences also exist creating scope for complementary, crosstalk
and the formation of stress fibers. Neither the precise role and synergistic interactions between different receptor-me-
played by these components in the molecular and cellular diated pathways. Establishing the relationships between the
sequelae leading to cell migration nor the relationships and pathways regulated by integrins, receptor PTKs, and other
interactions between them have been defined as yet. Future receptor types will be crucial for understanding the molecu-
effort will also need to address the relevance of putative lar basis of the migratory response in the vessel wall. The
chemotactic signaling pathways both for the action of spe- roles of metalloproteinases and the plasminogen activator
cific migration factors and for the migration of vascular cells system are likely to prove of particular importance for vas-
such as VSMC and EC. cular cell migration in vivo.
This chapter has highlighted recent work suggesting Paracrine interactions between VSMC, EC, monocytes
that FAK and other focal adhesion-associated molecules may and other cells in the atherosclerotic microenvironment adds
play a role in vascular cell migration in response to growth a further layer of complexity to the regulation of migration
factor ligands for receptor PTKs. The latter notion is espe- of vascular cells. The production of PDGF by EC in response
cially attractive in the light of recent findings that FAK has to monocyte-, platelet- and T lymphocyte-derived cytokines,
the ability to associate both with components of focal adhe- and the induction in VSMC of VEGF, an angiogenic factor
sions and with certain SH2 domain-containing substrates whose target cells are monocytes and EC, are two striking
of the PDGF receptor. The role of small GTP-binding pro- examples of such interactions. The production of VEGF by
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Facilitation of Healing
Chemotaxis
CHAPTER 25
Adhesion Molecules: Potent Inducers
of Endothelial Cell Chemotaxis
Zoltan Szekanecz, Alisa E. Koch
Introduction
A ngiogenesis, the formation of new blood vessels, plays an important role in a number of
physiological processes including development and tissue repair. Thus, it may also be
involved in the integration of newly implanted vascular grafts. Neovascularization is also
essential in a number of pathological situations, such as atherosclerosis, diabetic angiopa-
thy, rheumatoid arthritis, psoriasis and tumor growth.1-3
During angiogenesis, endothelial cells produce proteolytic enzymes, which leads to
the degradation of the basement membrane, followed by the emigration of endothelial cells.
Although there is another chapter on angiogenesis in this volume, here we will discuss the
role of cellular adhesion molecules and adhesive mechanisms in the induction of endothe-
lial cell migration and chemotaxis, which are essential steps in capillary formation. There is
evidence that certain adhesion molecules and extracellular matrix molecules may be chemo-
tactic for endothelial cells and thus may stimulate the migration of these cells into sites of
angiogenesis. It is possible that the induction of endothelial cell migration and
neovascularization around the vascular graft may facilitate its healing.
In this chapter, we will summarize our current understanding of the role of adhesion
factors in endothelial cell migration and chemotaxis. We will discuss the involvement of
extracellular matrix components and cellular adhesion molecules in these processes, also
highlighting our recently published data on recombinant, soluble adhesion molecule-in-
duced endothelial cell chemotaxis and neovascularization. We will also discuss the interac-
tions of other soluble mediators, such as growth factors and cytokines, with extracellular
matrix molecules and cellular adhesion molecules during endothelial cell migration and
angiogenesis. With regard to vascular surgery, we will present information on the role of
cellular adhesion molecules in the formation of human abdominal atherosclerotic aortic
aneurysms, as well as in wound healing. These data may support our theory that, after vascular
graft implantation, extracellular matrix macromolecules and cellular adhesion molecules
are involved in attracting endothelial cells and in the formation of new capillaries. Thus, the
induction of adhesion molecule expression may stimulate the revascularization of the sur-
rounding extracellular matrix, enhance blood supply and enable the survival of the graft.
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler
©1999 R.G. Landes Company.
272 Tissue Engineering of Prosthetic Vascular Grafts
The Role of Extracellular Matrix In contrast to other extracellular matrix components
Macromolecules in Endothelial Cell Motility described above, thrombospondin-1, which interacts with a
During Vessel Formation number of other extracellular matrix components such as
The extracellular matrix surrounding the emigrating heparin, fibronectin, fibrinogen and collagen, inhibits en-
endothelial cells consists of two major compartments: the dothelial cell chemotaxis to basic fibroblast growth factor as
basement membrane and the interstitial extracellular ma- reported by most investigators.16,17 The downregulation of
trix. The basement membrane contains various extracellu- the thrombospondin-1 gene expression by antisense oligo-
lar matrix macromolecules synthesized by the endothelium, nucleotides resulted in increased bovine aortic endothelial
including mainly type IV collagen and laminin, but also other cell chemotaxis to basic fibroblast growth factor on collagen
types of collagen, fibronectin, proteoglycans, entactin and gels. 17 Others, however, reported that soluble
thrombospondin.1,4-6 Many of these extracellular matrix thrombospondin-1 itself was chemotactic for bovine aortic
components play a role in the direction of endothelial cells and murine lung capillary endothelial cells. This chemotac-
during capillary formation.4-6 The interstitial, extravascular tic effect was highly !3 integrin-dependent, as determined
extracellular matrix contains mainly type I collagen, as well by blocking studies using monoclonal antibody to this
as other extracellular matrix components including integrin. 18 It is likely that, while recombinant
fibronectin, fibrinogen, vitronectin, tenascin and thrombospondin-1 promotes endothelial cell migration, it
thrombospondin.4-6 suppresses the chemotactic effect of angiogenic mediators,
It has been postulated that endothelial cell migration such as basic fibroblast growth factor.
and chemotaxis are stimulated by type I, and, to a lesser ex- The endothelial cell cytoskeleton is reorganized dur-
tent types II, III, IV and V collagen.1,7-9 In the in vitro col- ing endothelial cell migration and capillary formation on
lagen gel assay, capillary endothelial cells adhere to type I Matrigel. These processes can be blocked in vitro by colchi-
collagen, acquire elongated morphology, begin to produce cine, cytochalasin D and cycloheximide, indicating the in-
extracellular matrix components, and form tubular struc- volvement of microfilaments, microtubules and active pro-
tures when placed within, but not when layered onto, the tein synthesis, respectively.19
gel.6,10 These data suggest that collagen types in the intersti-
tial extracellular matrix stimulate endothelial cell chemo- Adhesion Molecules in Endothelial Cell
taxis. Fibronectin is also chemotactic for endothelial cells, Migration and Angiogenesis
promotes the elongation of microvessels in explant cultures Cellular adhesion molecules play an important role
in vitro and wound healing in vivo.1,8,11,12 Laminin is se- in endothelial cell adhesion to and migration into the
creted during endothelial cell proliferation1 and is chemo- extracellular matrix (Table 25.1). Endothelial cells also uti-
tactic for human endothelial cells.8 Matrigel, a laminin-rich lize a number of cellular adhesion molecules for their adhe-
basement membrane-like substrate, induces endothelial cell sion to each other, as well as to other cell types. Endothelial
migration and the formation of new vascular structures.9,13 cell-extracellular matrix interactions are mostly mediated
Tenascin is also an important stimulator of endothelial by integrins, while intercellular adhesion molecules include
sprouting.14 Fibrinogen also promotes endothelial cell mo- the #4!1 integrin, all !2 (leukocyte) integrins, members of
tility.15 Heparin and heparan sulfate proteoglycans are es- the immunoglobulin superfamily of cellular adhesion
sential for neovascularization, as a number of angiogenic molecules, selectins, cadherins and other cellular adhesion
growth factors bind to these proteoglycans in the extracel- molecules.20-24
lular matrix.1-3 Heparin itself is chemotactic for human en- Among integrins, the #1!1 and #2!1 heterodimers me-
dothelial cells and potentiates the chemotactic effect of diate endothelial cell adhesion to types I and IV collagen, as
growth factors.8 well as to laminin.20-22,24-26 The main endothelial cell laminin
Table 25.1. Endothelial cellular adhesion molecules involved in endothelial cell migration and neovascularization.
Cellular adhesion Adhesion molecule Reference(s)
molecule superfamily receptor*
1. Integrins !1 integrins (most) 10, 18, 24, 25, 29-31
!3 integrins (#v!3)
2. Immunoglobulin Vascular cell adhesion 23, 27, 35
superfamily molecule-1
CD31
3. Selectins E-selectin 24, 27, 35
4. Cadherins VE-cadherin 23
5. Others Sialyl Lewis-X 23, 24, 32-34
CD34
Endoglin
* See text for cellular adhesion molecule ligands and more information.
Adhesion Molecules: Potent Inducers of Endothelial Cell Chemotaxis 273
receptor, however, is #6!1. There are two important recep- We have recently shown the role of certain soluble,
tors for fibronectin, the RGD (arginine-glycine-aspartic recombinant endothelial cellular adhesion molecules in en-
acid)-dependent #5!1, as well as the RGD-independent dothelial cell chemotaxis, as well as in vivo
20-22,24,25
#4!1. The former is present on most endothelial neovascularization. Soluble vascular cell adhesion mol-
cells,25 while we and others have confirmed that #4!1 is also ecule-1 and soluble E-selectin have been tested in endothe-
expressed by some endothelial cells.27,28 Another fibronectin, lial cell chemotaxis assays in vitro and the rat corneal
laminin and collagen receptor, #3!1, is also present on en- neovascularization model in vivo.27 Recombinant E-selectin
dothelial cells.25,26 Integrins containing the !3 subunit are and vascular cell adhesion molecule-1 as chemotactic fac-
involved in endothelial cell adhesion to fibronectin, tors dose-dependently increased the chemotaxis of human
vitronectin, thrombospondin, von willebrand factor and fi- umbilical vein endothelial cells and dermal microvascular
brinogen. The #v integrin subunit can be associated with endothelial cells above the basal level. The chemotactic ac-
several ! chains (!1, !3, !5, !6, !8), and mediates endothelial tion of these soluble cellular adhesion molecules was com-
cell adhesion to a variety of extracellular matrix components, parable to that of basic fibroblast growth factor, a “classical”
depending on the ! subunit.20-22,25,26 angiogenic mediator. We have also shown in checkerboard
Extracellular matrix binding integrins can be classi- analyses that both soluble E-selectin and soluble vascular
fied into subgroups of “basement membrane” (collagen- cell adhesion molecule-1 were chemotactic rather than
laminin)-binding integrins (#1!1, #2!1, #3!1 and #6!1) and chemokinetic for endothelial cells. These soluble cellular
“inflammatory matrix” integrins (fibronectin-fibrinogen adhesion molecules seem to be mediating a direct effect, as
receptors: #4!1, #5!1, #v and !3). While microvessels express they did not stimulate umbilical endothelial cells to produce
the former, but not the latter type of integrins in situ, cellu- the angiogenic basic fibroblast growth factor, tumor necro-
lar adhesion molecules belonging to both subgroups are sis factor-# or interleukin-8 in vitro.27
present on capillary endothelial cells in vitro. These data In addition, monoclonal antibodies to the E-selectin
suggest that endothelial cells have a potential to alter their ligand sialyl Lewis-X and to the vascular cell adhesion mol-
cellular adhesion molecule profile during vascular morpho- ecule-1 counterreceptor #4!1 integrin on endothelial cells,
genesis.25 Additional studies have shown that the #2!1 mol- significantly reduced soluble cellular adhesion molecule-
ecule is involved in type I collagen-induced endothelial cell mediated human umbilical vein endothelial cell chemotaxis.
migration and neovascularization.10,24 The #v!3 molecule was We and others have detected both sialyl Lewis-X and #4!1
found to be involved in endothelial cell migration on on umbilical vein endothelial cells by immunohistochemis-
vitronectin.24 #v!3 is necessary for capillary formation in try.27,30 Although sialyl Lewis-X has been implicated in bo-
tumors and wound granulation tissue and promotes endot- vine capillary morphogenesis in vitro,34 our novel results
helial cell cord formation in vitro.29,30 It has recently been suggest a link between the soluble E-selectin-sialyl Lewis-X,
shown that the #v!3 integrin is required for the survival and as well as soluble vascular cell adhesion molecule-1-#4!1,
maturation of new blood vessels.30 As described above, !3 adhesion pathways, which mediate leukocyte-endothelial cell
integrins may also be important in thrombospondin-1-me- interactions, endothelial cell recruitment and angiogenesis.27
diated endothelial cell chemotaxis.18 The #6!1 integrin stimu- These data are supported by those of other investigators
lates angiogenesis on Matrigel.31 showing that sugars, which are also major constituents of
Adhesion molecules mediating endothelial cell adhe- selectins and sialyl Lewis-X, are chemotactic for bovine cor-
sion to other cells include vascular cell adhesion molecule-1, neal endothelial cells.36 In the rat corneal neovascularization
intercellular adhesion molecules-1 and -2, CD31, E- and assay both soluble cellular adhesion molecules induced in
P-selectin, CD34 and other carbohydrate ligands for vivo capillary formation at low concentrations in most ex-
L-selectin, and the transformtin growth factor-!1 receptor periments. We have also shown in neutralization studies that
endoglin.20-23,32,33 Antibodies to E-selectin, as well as to its soluble E-selectin and soluble vascular cell adhesion mol-
ligands sialyl Lewis-X and sialyl Lewis-A, inhibit the migra- ecule-1 account for a portion of rheumatoid arthritic syn-
tion and tube formation of capillary endothelial cells.24,34 ovial fluid-mediated chemotactic activity for human um-
CD31 has been implicated in tumor-associated bilical vein endothelial cells.27
neovascularization.35 The L-selectin ligand CD34 shows high In summary, we have shown that soluble E-selectin
endothelial cell expression in developing tissues and heal- and soluble vascular cell adhesion molecule-1 may act as
ing wounds.32 proangiogenic factors. They may directly trigger the migra-
Endothelial adherens junctions, which contain clus- tion and chemotaxis of endothelial cells by binding to their
tered cellular adhesion molecules and other membrane pro- respective endothelial cell ligands.27
teins, were also implicated in endothelial cell migration, ad-
hesion and angiogenesis. VE-cadherin, a major constituent Interactions of Soluble Mediators with Cellular
of these junctions, mediates homophilic binding between Adhesion Molecules and Extracellular Matrix
endothelial cells. Many other adhesive proteins, such as Components During Endothelial Cell
CD31, CD34, endoglin, occludin and connexins show Recruitment and Neovascularization
colocalization with VE-cadherin.23 As endothelial cells bind Cellular adhesion molecules and extracellular matrix
to each other through these cellular adhesion molecules, macromolecules may interact with other soluble mediators
these cells can migrate in organized clusters during capil- during endothelial cell migration and neovascularization.
lary formation. In fact, basic fibroblast growth factor treatment of
274 Tissue Engineering of Prosthetic Vascular Grafts
endothelial cells in vitro resulted in an increased expression RANTES, macrophage inflammatory protein-1# and mono-
of the integrin subunits #2, #3, #5, #6, !1, !3, !4 and !5, as well cyte chemoattractant protein-1 stimulate the expression and
as in better adherence to type I collagen, fibronectin, laminin avidity of both !1 and !2 integrins on T cells and mono-
and vitronectin compared to untreated cells.37,38 Interactions cytes.45,46 These chemokines also promote the adhesion of
between basic fibroblast growth factor, thrombospondin and mononuclear cells to extracellular matrix components, as
cellular adhesion molecules have also been demonstrated. well as recombinant human intercellular adhesion molecule-
Thrombospondin inhibits angiogenesis by blocking the ef- 1 and vascular cell adhesion molecule-1.45,46 However, most
fects of basic fibroblast growth factor on endothelial cell of these data are available on leukocytes, and not on endot-
chemotaxis.16,17,39 In addition, thrombospondin receptors, helial cells. Therefore, the possibility that either C-X-C or
such as !3 integrins, have been implicated in endothelial cell C-C chemokines may also influence endothelial cell migra-
migration and neovascularization.18,26,30 tion and chemotaxis via the modulation of endothelial cell
Transformtin growth factor-! and basic fibroblast adhesion molecule expression needs to be elucidated.
growth factor differentially modulate the integrin profile on
human microvascular endothelial cells, as transformtin The Relevance of Angiogenesis Studies
growth factor-! downregulates integrin expression on hu- in Vascular Surgery: Aortic Aneurysms
man dermal microvascular endothelial cells, both at the and Wound Healing
mRNA and protein level.38,40 This growth factor also inhib- We have performed a number of studies on the pos-
its fibronectin-mediated endothelial cell chemotaxis.40 One sible role of angiogenic mediators and adhesion molecules
receptor for transformtin growth factor-!1, endoglin, con- in the pathogenesis of human abdominal aortic aneurysms.
tains an RGD motif and thus serves as a cellular adhesion In this disease, atherosclerotic and inflammatory mecha-
molecule for endothelial cells.23,33 We found increased, well nisms occurring in the aneurysm wall are accompanied by
correlated expression of endoglin and transformtin growth the escalation of neovascularization. Small vessels of the vasa
factor-!1 in the highly vascularized synovium of rheuma- vasorum in the adventitial layer proliferate and the migra-
toid arthritis patients.33 tion of endothelial cells into the aortic wall plays an impor-
Treatment of endothelial cells with tumor necrosis tant role in the formation of new vessels and the growth of
factor-# stimulates #1!1 expression and adhesion to laminin, aneurysm.47-50
while the combination of tumor necrosis factor-# and in- Our immunohistochemical analysis carried out on
terferon-g downregulates #v!3 expression on these cells, sug- aortic aneurysm explants revealed that there was an increased
gesting the modulating effect of angiogenic and angiostatic expression of intercellular adhesion molecule-1 on endot-
mediators in endothelium-extracellular matrix interac- helial cells found in aortic aneurysm tissues compared to
tions.26 Tumor necrosis factor-# can also induce the expres- normal control aortas.50 Moreover, aortic aneurysm explants
sion of intercellular adhesion molecule-1, vascular cell ad- released significantly more soluble intercellular adhesion
hesion molecule-1, E-selectin and some integrins on syn- molecule-1 into their culture supernatants than did normal
ovial and other types of endothelial cells.3,20,21,25,26 The in- aortic tissues.47 We have used human aortic endothelial cell
creasing production of endothelial cellular adhesion mol- culture as an in vitro model for studying the interactions of
ecules may result in the perpetuated shedding of these cel- angiogenic cytokines and cellular adhesion molecules. The
lular adhesion molecules from the cell surface. The serum angiogenic cytokine tumor necrosis factor-# stimulated in-
and synovial fluid concentrations of soluble E-selectin and tercellular adhesion molecule-1 expression of, and soluble
soluble vascular cell adhesion molecule-1 are high in rheu- intercellular adhesion molecule-1 production by, these en-
matoid arthritis, a well-known “angiogenic disease”.41,42 As dothelial cells compared to control, unstimulated aortic en-
described above, these soluble cellular adhesion molecules, dothelial cells.47 We have also determined that aortic aneu-
in turn, may stimulate endothelial cell chemotaxis and an- rysm explants produce more tumor necrosis factor-#,
giogenesis.27 interleukin-6 and interleukin-8 into their conditioned me-
Chemokines, which are chemotactic for endothelial dia, than do normal aortic tissues.48,49,51,52 These cytokines
cells, as well as for leukocytes, may also be involved in this induced neovascularization in both in vitro and in vivo
regulatory network. Some C-X-C chemokines containing the models for angiogenesis.2,3,53-55 Moreover, we have shown,
ELR motif, such as interleukin-8, have high angiogenic ac- that tumor necrosis factor-# and interleukin-8 account for
tivity and they promote endothelial cell chemotaxis. In con- a portion of aortic aneurysm-associated endothelial cell
trast, C-X-C chemokines lacking the ELR sequence, such as chemotaxis.48
interferon-inducible protein-10 and platelet factor IV, in- Thus, soluble angiogenic and chemotactic mediators
hibit endothelial cell migration and angiogenesis.43 Although and certain cellular adhesion molecules, at least intercellu-
very little is known about the effects of C-X-C chemokines lar adhesion molecule-1, can be detected in aortic aneurysm
on cellular adhesion molecules, interleukin-8 stimulates !2 tissues. The increased production of these mediators results
integrin expression on neutrophils,44 while interferon-in- in increased endothelial intercellular adhesion molecule-1
ducible protein-10 induces integrin-dependent T cell adhe- expression. In conclusion, the interactions of angiogenic and
sion to fibronectin, laminin and collagen, as well as to re- chemotactic cytokines with cellular adhesion molecules may
combinant human intercellular adhesion molecule-1 and be important in the perpetuation of endothelial cell recruit-
vascular cell adhesion molecule-1.45 There are a number of ment and capillary formation underlying the growth of aor-
data available showing that C-C chemokines such as tic aneurysms.
Adhesion Molecules: Potent Inducers of Endothelial Cell Chemotaxis 275
Regarding wound healing, in the normal skin cellular diators of endothelial cell migration and angiogenesis. In
adhesion molecule expression is restricted to the basal layer addition, as one of the possible ligands for E-selectin is closely
of the epidermis. These cellular adhesion molecules medi- related to basic fibroblast growth factor, these cellular adhe-
ate the adhesion of the basal cells to the basement mem- sion molecules may also participate in neovascularization-
brane.56,57 During injury and wound healing, cellular adhe- associated signaling triggered by other mediators. Thus, the
sion molecules, mostly !1 and !3 integrins, appear in the complex interactions between endothelial cells, extracellu-
suprabasal layers of the skin, accompanied by the influx of lar matrix macromolecules, cellular adhesion molecules and
fibroblasts and endothelial cells, their adhesion to other angiogenic factors may lead to the perpetuation of
fibronectin, vitronectin and fibrinogen, as well as angiogen- angiogenesis. There are antagonistic factors, such as
esis and scarring.56,58 Similarly, we have found strong vas- transformtin growth factor-b and thrombospondin-1, which
cular cell adhesion molecule-1, P-selectin and CD44 expres- inhibit endothelial cell chemotaxis, as well as
sion in the injured, desquamating skin of scleroderma pa- neovascularization. The net balance of these factors result
tients.57 in the perpetuation or downregulation of endothelial cell
migration in the sites of angiogenesis. The detection of these
A Regulatory Network in Sites of Endothelial factors, as well as functional studies using in vitro chemot-
Cell Migration: Potential Target Promoting axis assays or in vivo animal models, can lead us to the bet-
Graft Healing ter understanding of the role of adhesive mechanisms around
In conclusion, in “angiogenic states”, such as rheuma- the healing vascular graft (Fig. 25.1).
toid arthritis, aortic aneurysms, wound healing or vascular In addition, targeting cellular adhesion molecules, ex-
graft implantation, cellular adhesion molecules such as !1 tracellular matrix components or other angiogenic media-
and ! 3 integrins, vascular cell adhesion molecule-1, tors by local injections of any mediators described above
E-selectin, sialyl Lewis-X and others, as well as extracellular may result in the local enhancement of endothelial cell
matrix molecules including collagen, fibronectin, laminin, chemotaxis and neovascularization, and thus may be useful
tenascin, fibrinogen and heparin may be involved in endo- for the healing and the prolonged survival of implanted vas-
thelial cell chemotaxis, migration and adhesion to extracel- cular grafts. Recently, putative progenitor endothelial cells
lular matrix components. A number of soluble mediators for angiogenesis were isolated from human peripheral blood.
secreted mainly by macrophages and endothelial cells in the These originally hematopoietic stem cells carry the CD34
extracellular matrix may interact with these cellular adhe- antigen, as well as Flk-1, a receptor for the angiogenic vas-
sion molecules and extracellular matrix components. Some cular endothelial growth factor. While mononuclear cells lose
of these factors, such as basic fibroblast growth factor, tu- these two antigens during in vivo differentiation, progeni-
mor necrosis factor-#, and possibly chemokines may induce tor cells plated onto fibronectin attach and become spindle-
cellular adhesion molecule expression on and shedding from shaped within days, and continue expressing CD34 and
the endothelial cell surface. Increased cellular adhesion mol- Flk-1, as well as endothelial cell marker cellular adhesion
ecule expression may result in stronger endothelial cell ad- molecules, such as E-selectin and CD31. In in vivo animal
hesion to extracellular matrix components. As we and oth- models of hindlimb ischemia, where one femoral artery is
ers have suggested, soluble, and possibly surface-bound en- excised, the progenitor endothelial cells isolated by magnetic
dothelial cellular adhesion molecules can act as direct me- bead separation using anti-CD34 or anti-Flk-1 monoclonal
Fig. 25.1. Interactions between cellular adhesion
molecules, extracellular matrix components and
angiogenic mediators in the regulation of
endothelial migration and angiogenesis. See text
for details.
276 Tissue Engineering of Prosthetic Vascular Grafts
antibodies migrated into sites of ischemia and angiogenesis. 17. DiPietro LA, Nebgen DR, Polverini PJ. Downregulation
Preliminary data suggest that these endothelial cells, possi- of endothelial cell thrombospondin 1 enhances in vitro
bly with the administration of angiogenic cytokines that angiogenesis. J Vasc Res 1994; 31:178-185.
enhance endothelial cell migration, could be used in autolo- 18. Taraboletti G, Roberts D, Liotta LA et al. Platelet
thrombospondin modulates endothelial cell adhesion,
gous endothelial cell transplantation in order to promote
motility, and growth: A potential angiogenesis regulatory
neovascularization.59 Similar techniques may be applied in factor. J Cell Biol 1990; 111:765-772.
vascular surgery as well. 19. Grant DS, Lelkes PI, Fukuda K. et al. Intracellular mecha-
nisms involved in basement membrane induced blood
Acknowledgments vessel differentiation in vitro. In Vitro Cell Dev Biol 1991;
This work was supported by NIH grants AR30692 and 27A: 327-336.
AR41492 (A.E.K.), funds from the Veterans’ Administration 20. Albelda SM, Buck CA. Integrins and other cell adhesion
Research Service (A.E.K.), Hungarian Scientific Foundation molecules. FASEB J 1990; 4:2868-2880.
grant No. T013239 (Z.S.) and Hungarian Medical Research 21. Springer TA. Adhesion receptors of the immune system.
Council grant No. 156/93 (Z.S.). Nature 1990; 346:425-433.
22. Szekanecz Z, Szegedi G. Cell surface adhesion molecules:
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Facilitation of Healing
Angiogenesis
CHAPTER 26
Angiogenesis in Tissues and Vascular Grafts
Paula K. Shireman, Howard P. Greisler
Overview of Angiogenesis
A ngiogenesis is a cellular process that starts during embryogenesis and continues through
out the life of the organism. It is defined as the formation of new blood vessels by a
process of sprouting from preexisting vessels, while vasculogenesis is the differentiation of
endothelial cells from mesodermal precursors and their subsequent organization into a pri-
mary capillary plexus. Unlike angiogenesis, vasculogenesis is limited to the embryonic pe-
riod of life.1
Angiogenesis is a highly regulated process that is predominantly inhibited except for
brief periods of time in which the normally quiescent vasculature is stimulated to form new
blood vessels. Many disease states such as arthritis, ocular neovascularization, and tumor
growth and metastasis, occur due to unregulated angiogenesis.2 Although the factors that
induce angiogenesis are poorly understood, hypoxia is thought to be a potent stimulus.
Hypoxia induces various cell lines to secrete angiogenic molecules. It is not understood how
cells sense hypoxia or how this triggers secretion of angiogenic molecules.3
Stimulating factors for angiogenesis are often categorized as direct or indirect. This
often leads to confusing, and sometimes conflicting, data in the literature due to the various
systems in which the molecules are tested. Target cell specificity for an angiogenic molecule
is usually determined using in vitro methods such as endothelial cell proliferation and mi-
gration assays. Angiogenic activity, however, is analyzed in vivo using bioassays such as the
chicken chorioallantoic membrane assay. When the two methods correlate, the angiogenic
factor is classified as being “direct” because it stimulates endothelial cell proliferation or
migration directly. When the angiogenic factor fails to stimulate activity in vitro, it is classified
as “indirect” because it is assumed that the endothelial cell activity observed in vivo must be
induced by some other factor or cell type that the indirect angiogenic molecule elicits.2
The formation of new vessels occurs by activation of endothelial cells from pre-exist-
ing capillaries or venules. Pericytes are cells that closely resemble smooth muscle cells. Pericytes
closely envelop endothelial cells in quiescent capillaries, and they become sparse or absent
in growing vessels. They reappear in mature, nongrowing capillaries where they downregulate
endothelial cell proliferation.3
Angiogenesis is thought to occur in four basic steps. Endothelial cells of a capillary
become activated by a variety of factors including hypoxia, acidosis and cytokines. This is
followed by local vasodilation and increased vascular permeability, both of which are caused,
at least in part, by vascular endothelial growth factor (VEGF). The initial step of angiogen-
esis involves degradation of the basement membrane of the parent vessel. This allows the
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
280 Tissue Engineering of Prosthetic Vascular Grafts
endothelial cells to migrate toward the angiogenic stimulus, of FGF-2 and TGF-! allow extracellular matrix degradation
which is the second step of angiogenesis. Migrating endo- to occur in a controlled fashion, thereby allowing endothe-
thelial cells elongate and align with one another to form a lial cell migration for angiogenesis. A feedback mechanism
capillary sprout. Thirdly, endothelial cells proliferate, which has been described for these two angiogenic molecules. In-
occurs proximal to the migrating tip, further increasing the creased plasminogen activation by FGF-2 results in high
length of the sprout. The solid sprout gradually develops a pericellular plasmin activity. Plasmin activates TGF-! by re-
lumen proximal to the region of proliferation. Contiguous leasing it from its latent complex. Activated TGF-! decreases
sprouts anastomose at their tips to form a functional capil- the overall proteolytic capacity by increasing synthesis of
lary loop in which blood flow can be established. The fourth PAI-1 and decreasing synthesis of uPA. The resulting de-
step is vessel maturation, which is accomplished by recon- crease in plasmin activity blocks the activation of further
stitution of the basement membrane and the reappearance TGF-!. When active TGF-! is no longer present, the effects
of surrounding pericytes (see Fig. 26.1).1,4 of FGF-2 again prevail, thereby increasing plasmin and uPA
New capillaries, as well as blood vessels that are stimu- levels. Plasmin also releases FGF-2 that is bound to the ex-
lated by VEGF, tend to be “leaky” and thus have an increased tracellular matrix by heparan sulfate proteoglycans. The
vascular permeability. This leads to the accumulation of ex- cleavage of the proteoglycan core releases FGF-2 in an ac-
travascular fibrin, which acts as a provisional matrix that is tive form that is protected from proteolytic degradation and
progressively removed and replaced by other matrix com- can diffuse to affect target cells.8
ponents.1 Fibrin itself is chemotactic for inflammatory cells Degradation of the extracellular matrix and the re-
and has been shown to regulate endothelial cell and fibro- lease of the growth factors stored there are also enhanced by
blast migration.5 macrophages. Macrophages contribute to angiogenesis in
Degradation of the extracellular matrix is a highly several ways. Macrophages release proteolytic enzymes, in-
regulated process that occurs secondary to the interplay of cluding plasminogen activators and matrix metallo-
proteolytic enzymes, their inhibitors, and angiogenic mol- proteinases, that assist in extracellular matrix breakdown.
ecules that induce the formation of these enzymes. Endo- Macrophages also degrade heparan sulfate, which binds
thelial cells produce both plasminogen activators, tissue-type many growth factors to the extracellular matrix, thus releas-
plasminogen activator (tPA) and urokinase-type plasmino- ing active cytokines to affect endothelial cells. Finally, mac-
gen activator (uPA), as well as the plasminogen activator rophages themselves secrete many angiogenic factors such
inhibitor (PAI-1).6 The plasminogen activators cleave the as FGF-2, TGF-!, VEGF, and angiotropin as well as factors
inactive plasminogen to form plasmin, a proteolytic enzyme that inhibit angiogenesis. Macrophages are important regu-
with broad specificity for fibrin, laminin, gelatin, and other lators of inflammation and tissue healing, and angiogenesis
extracellular components. Plasmin also activates the matrix is an integral part of both of these processes.9
metalloproteinases, a family of proteolytic enzymes that also Many different cytokines and growth factors have been
degrade extracellular matrix components. 7 Fibroblast implicated in either promoting or inhibiting angiogenesis,
growth factor-2 (FGF-2), also known as basic FGF, is an- and in some cases the same factor may have both stimulat-
other important angiogenic molecule that enhances endo- ing and inhibitory properties depending upon local condi-
thelial growth, proliferation, and migration. It also enhances tions. We will review the functions of several of these major
endothelial cell expression of uPA and collagenase. In con- cytokines: FGF, VEGF, TGF-!, and angiostatin.
trast, transformtin growth factor-! (TGF-!) dramatically The FGF family of growth factors plays an important
increases PAI-1 synthesis in endothelial cells. The interplay role in angiogenesis. FGF-2 stimulates endothelial cell pro-
Fig. 26.1. Four steps in angiogen-
esis. Parent mature vessel is on the
left. (1) Basement membrane and
ECM degradation. (2) Endothelial
migration. (3) Endothelial prolif-
eration (mitoses). (4) Organiza-
tion and maturation. (Used with
permission from ref. 4).
Angiogenesis in Tissues and Vascular Grafts 281
liferation, migration, and promotes formation of differen- meability occurs rapidly and is transient and reversible, per-
tiated capillary tubes in vitro.9 Endothelial cell production sisting for less than 30 minutes; it is not associated with any
of FGF-2 leads to its sequestration in the extracellular ma- detectable cell damage.12 This increased permeability leads
trix where it can be released to participate in angiogenesis to the accumulation of extravascular fibrin, which acts as a
during the degradation of the extracellular matrix. FGF-2 provisional matrix for migrating endothelial cells during
also induces the formation of plasminogen activators and angiogenesis.
collagenase, thereby promoting extracellular degradation VEGF induces endothelial cells to invade collagen gels
and its own activation.10 Both FGF-1, also known as acidic in vitro and also incites endothelial cell sprouting. Syner-
FGF, and FGF-2 are synthesized by a broad range of cell types gism between VEGF and FGF-2 was shown in a microvas-
including endothelial cells, fibroblasts, macrophages, and cular endothelial cell assay which demonstrated endothelial
smooth muscle cells. Both forms of FGF lack a signal se- invasion of a three dimensional collagen gel with formation
quence for secretion, and it is not entirely clear how FGF of capillary-like tubules. Both FGF-2 and VEGF were
release is regulated. As previously discussed, FGF binds to angiogenic in this model, with VEGF being about half as
heparan sulfate and can be released from the extracellular potent as FGF-2 in equimolar concentrations. However,
matrix. Furthermore, FGF is released from damaged and when both cytokines were added simultaneously, the angio-
dying cells and may therefore be important in inducing an- genic response was far greater than the additive effect of each
giogenesis in ischemic and injured tissues.3 For a more de- cytokine separately and occurred at a more rapid rate.13
tailed review of the functions of the FGF family of growth VEGF also promotes expression of tPA and matrix
factors in angiogenesis, see chapter 28. metalloproteinases in endothelial cells, which as already
VEGF, also known as vascular permeability factor, is discussed, induces degradation of the extracellular matrix
another important angiogenic molecule that is expressed in as well as the provisional fibrin matrix, releasing angiogenic
many different cell types including macrophages, fibroblasts, factors. PAI-1 is also increased by VEGF and may provide a
smooth muscle cells and endothelial cells. Hypoxia may, in negative feedback mechanism to balance the proteolytic
part, control the expression of VEGF.11 Unlike FGF, VEGF process.8,14
has a peptide leader sequence which probably controls cel- There are 4 major isoforms of VEGF and the relative
lular secretion of VEGF.10 Endothelial cells exhibit VEGF affinity to heparin binding may affect the bioavailability of
receptors, which explains the exquisite specificity of VEGF the various isoforms. VEGF-121 fails to bind heparin and is
for endothelial cells. Unlike FGF and other cytokines that therefore a freely diffusible protein that is secreted from cells.
induce endothelial proliferation as well as proliferation of VEGF-165, the major isoform, has an intermediate affinity
other cell lines, VEGF is generally considered to be a specific for heparin and is secreted from cells, although a significant
endothelial cell mitogen. Binding of VEGF to its receptor portion remains bound to the cell surface and the extracel-
induces a sequence of protein phosphorylation. VEGF in- lular matrix. Both VEGF-189 and VEGF-206 have a high af-
creases intracellular calcium levels and induces the release finity for heparin and these isoforms are almost completely
of von Willebrand factor from endothelial cells. sequestered in the extracellular matrix (see Fig. 26.2.).15,16
VEGF was originally discovered because of its ability The important role that VEGF plays in angiogenesis
to increase the permeability of blood vessels, primarily and vasculogenesis in embryo development is demonstrated
postcapillary venules and small veins. This increased per- by mouse VEGF knockout models. A heterozygous VEGF
Fig. 26.2. Schematic representation of
the actions of VEGF isoforms on the vas-
cular endothelium. Several stimuli may
result in the release of the diffusible al-
ternatively spliced VEGF isoforms
(VEGF-165 and VEGF-121) from a vari-
ety of cell types. These proteins may in-
duce a complex series of effects on the
vascular endothelium, including cell
sprouting, induction of interstitial col-
lagenase, plasminogen activators (PA),
and plasminogen activator inhibitor-1
(PAI-1), as well as extravasation of
plasma proteins. Plasminogen activation
results in generation of plasmin, which
may cleave extracellular matrix-bound
VEGF (VEGF-189 or VEGF-206) to release
a diffusible proteolytic fragment
(VEGF-110). Plasmin may also activate procollagenase. Activation of PAI-1 may constitute a negative regulatory step, by inhibiting
the action of PA. (Used with permission from ref. 15).
282 Tissue Engineering of Prosthetic Vascular Grafts
knockout resulted in embryonic lethality between days 11 came confluent at 4 weeks. Transmural capillaries were ob-
and 12. Homozygous knockouts of the VEGF receptor re- served to connect the graft lumen to extravascular granula-
sults in embryonic lethality. tion tissue. At 2 weeks, multiple small orifices 100-500 ∝m
Another growth factor that exhibits both angiogenic apart were located between the ridges of the graft. At 4 and
and antiangiogenic properties is TGF-!. TGF-! is angiogenic 12 weeks, the entire graft surface was covered with endothe-
in vivo but inhibits macrovascular proliferation of endot- lial cells and the number of small vessel orifices had dimin-
helial cells and the proliferation of endothelial cells not en- ished. Smooth muscle cells were seen underneath the en-
gaged in angiogenesis in vitro. TGF-! promotes prolifera- dothelial cells in the network of microvessels within the graft
tion of endothelial cells engaged in angiogenesis in vitro and as early as 2 weeks. A progressive thickening of the intima,
induces the formation of capillary-like tubes of microvas- which was the greatest at 12 weeks, was seen throughout the
cular endothelial cells. TGF-! potentiates FGF-2 and VEGF graft. Smooth muscle cells, which were thought to be de-
induced angiogenesis in vitro at low, but not high, doses.3 rived from pericytes, accompanied the capillary endothelial
As previously discussed, TGF-! modulates extracellular deg- cells into the graft lumen. In contrast, intimal thickening
radation by inducing PAI-1 synthesis and thus downregulates occurred only at the anastomotic ends in the 30 ∝m intern-
plasmin-mediated proteolysis. In view of its capacity to di- odal grafts (see Fig. 26.3.). These results indicate that capil-
rectly inhibit endothelial cell proliferation and migration, lary endothelium and smooth muscle cells can function in
reduce extracellular proteolysis, and promote matrix depo- the same manner as their large vessel counterparts and could
sition in vitro, TGF-! has been proposed to be a potential provide coverage of synthetic graft surfaces in contact with
mediator of the resolution phase of angiogenesis.1 the arterial circulation.24
Although many factors promote angiogenesis, uncon- The 60 ∝m internodal graft appeared to provide the
trolled angiogenesis is associated with many disease states, “optimal” porosity in the baboon model. In low porosity
including tumor formation and metastasis. It is therefore grafts, 10 and 30 ∝m internodal distance, endothelial cover-
the delicate balance of angiogenic and antiangiogenic mol- age was incomplete and was limited to the graft surface near
ecules that leads to controlled, appropriate angiogenesis. the anastomoses. In contrast, high porosity grafts, 60 and
Folkman and associates17 have recently isolated angiostatin, 90 ∝m internodal distance, revealed complete endothelial
an antiangiogenic molecule that inhibits the formation of coverage with a uniformly distributed intimal thickening
tumor metastases in the Lewis mouse lung model. throughout the graft. However, the 90 ∝m graft developed
Angiostatin is a 38 kDa fragment of plasminogen that in- areas of focal endothelial loss at 3 months, suggesting that
hibits endothelial cell proliferation in vitro. The primary the larger pore size resulted in intimal instability and en-
tumor secretes angiostatin, which inhibits the growth of dothelial cell loss in the ePTFE.25
metastases. Removing the primary tumor decreases Dacron, another porous graft material, was compared
angiostatin levels and this allows the metastases to reestab- to the 60 ∝m internodal ePTFE grafts in the baboon model.
lish their blood supply and grow. The Dacron grafts showed incomplete endothelialization at
12 weeks while all the ePTFE grafts revealed a confluent en-
Angiogenesis in Vascular Grafts dothelial layer. Although both types of grafts healed by pro-
Synthetic, small diameter vascular grafts fail prima- liferation of cells derived from invading capillaries, the au-
rily because of interactions at the blood-material and tis- thors hypothesized that the Dacron material itself may some-
sue-material interfaces.18 Currently available prostheses elicit how inhibit graft endothelialization.26
inflammatory and regenerative reactions, yielding graft Dacron may induce various cell types to secrete fac-
thrombosis and intimal hyperplasia.19 In humans, clinically tors that could inhibit angiogenesis and graft
utilized grafts of Dacron and 30 ∝m internodal distance ex- endothelialization. Evidence supporting this hypothesis is
panded polytetrafluorethylene (ePTFE) do not allow capil- provided by Greisler et al.27 Rabbit peritoneal macrophages,
lary infiltration despite the small wall thickness. Many re- grown in the presence of Dacron particles in vitro, released
searchers have attempted to improve synthetic grafts by us- more TGF-! than control macrophages. The effects of TGF-!
ing various techniques such as endothelial seeding20,21 and on angiogenesis and endothelial proliferation are complex.
bioresorbable grafts.19 Clowes and associates studied the ef- In vitro, TGF-! inhibits endothelial cell proliferation.28
fect of porosity on ePTFE grafts and demonstrated that these TGF-! instillation into newborn mice29 and into the chorio-
grafts could be lined by endothelial cells that were derived allantoic membrane assay30 induces angiogenesis, while
from graft transmural capillaries. This induction of “graft higher doses of TGF-! resulted in inhibition of angiogen-
angiogenesis” occurred in ePTFE grafts with 60 ∝m intern- esis in these models.31 The higher levels of TGF-! released
odal distance. Previous studies had shown that ePTFE grafts by macrophages in the presence of Dacron may contribute
with a 30 ∝m internodal distance were covered by an in- to the inhibition of endothelialization seen in Dacron grafts.
growth of endothelium from the adjacent artery as a con- In an attempt to apply the above research to the clini-
tinuous sheet across the anastomosis and along the graft.22 cal setting, 10 above-knee femoropopliteal composite grafts
This process was relatively slow with only 60% of grafts were placed into 8 patients. Each of these grafts consisted of
6-9 cm in length exhibiting complete coverage by 12 months equal lengths of 30 and 60 ∝m internodal distance ePTFE
in an aortoiliac baboon model.23 In contrast, when 60 ∝m with the 60 ∝m portion being randomly placed in either the
internodal grafts were used, endothelial cells began to ap- proximal or distal position. In contrast to the baboon ex-
pear on the graft surface at 1-2 weeks after surgery and be- periments which used unwrapped ePTFE, these grafts were
Angiogenesis in Tissues and Vascular Grafts 283
reinforced with a thin, nonexpanded wrap for human use. to the death of the host. On the other hand, tissue ischemia
Noninvasive assessment of endothelialization was performed caused by occlusive arteriosclerosis represents a relative de-
using 111Indium-labeled platelet imaging at 1 week and 3 ficiency of angiogenesis. Various cytokines have been used
months after surgery. There was no difference in indium to induce angiogenesis in animal models of tissue ischemia.
uptake between the 60 and 30 ∝m segments at either time Perhaps one of the most commonly used is the rabbit
point. Histologic sections were available from 60 ∝m seg- hindlimb ischemia model. The hindlimb is made ischemic
ments from 2 patients who underwent operations for graft by tying off the external iliac artery and removing the com-
thrombosis. Capillary ingrowth into the graft was observed, mon femoral and superficial femoral arteries. Growth fac-
but it rarely extended more than half the distance from the tors, such as FGF (reviewed in chapter 28) and VEGF, are
outside of the graft to the lumen. Smooth muscle cells were then introduced into the ischemic limb and their angiogenic
not seen on the luminal surface, indicating that a neointima effect is measured by counting the new vessels seen on an-
had not formed. The finding that capillary ingrowth can giography. VEGF has been extensively studied by Isner’s
occur in the 60 ∝m internodal graft, but does not lead to an group using this model. They found that a single bolus in-
endothelial lining in humans, could possibly be attributed jection of VEGF via the ipsilateral internal iliac artery caused
to an inadequacy of angiogenesis in adult humans or retar- an increase in flow at rest, in maximum flow velocity, and in
dation of capillary ingrowth by the reinforcing wrap.32 maximum blood flow at 30 days as compared to saline con-
Other attempts at inducing graft angiogenesis by coat- trols.35 VEGF treated rabbits also re-established an increased
ing the luminal surface of synthetic grafts with FGF-1 endothelium-dependent blood flow in response to seroto-
incorporated into a fibrin glue have been made by Greisler nin and acetylcholine injection as compared to saline treated
and associates. FGF-1 is a potent endothelial cell mitogen animals which did not respond to the serotonin or acetyl-
and the greater adhesivity of the FG surface for seeded choline injections, suggesting that VEGF facilitated endot-
endothelial cells plus the slow release of the suspended FGF-1 helial recovery after ischemic injury.36 VEGF also increased
suggest the tissue engineering applicability of this sub- endothelial cell proliferation 2.8-fold at day 5, followed by a
strate 33,34 For a more thorough review of this work, decrease to baseline by day 7 as compared to controls, while
see chapter 28. smooth muscle cell proliferation increased by 2.7-fold.37
Endogenous VEGF production in the ischemic hindlimb
Therapeutic Angiogenesis in Ischemic Tissues model was found to be decreased in older (4-5 years) as com-
Unregulated angiogenesis can be seen in many can- pared to younger (6-8 months) rabbits and the angiogenic
cers and is a biologically undesirable situation that often leads response was also decreased in the older rabbits. Exogenous
Fig. 26.3. Diagrammatic representation
of healing by endothelial and smooth
muscle cells of low-porosity (top) and
high-porosity (bottom) ePTFE grafts.
In low-porosity grafts endothelium is
derived from the cut edges of adjacent
artery and grows as a continuous
monolayer toward the center of the
graft. In high-porosity grafts, capillar-
ies derived from surrounding granula-
tion tissue penetrate the graft matrix
and provide multiple sources of endot-
helium at the luminal surface. (Used
with permission from ref. 24).
284 Tissue Engineering of Prosthetic Vascular Grafts
administration of VEGF induced a similar angiogenic re- Another human trial with transfection of phVEGF-165
sponse in both older and younger rabbits, suggesting that a by intramuscular injection or intraarterially revealed in-
relative VEGF deficiency may occur with aging and that re- creased serum levels of VEGF. Furthermore, 63% of the pa-
placement of VEGF may overcome this defect.38 This rela- tients had edema and the response seemed to correlate with
tive VEGF deficiency in response to ischemia was also seen disease severity, with no edema occurring in patients with
in diabetic mice. The diabetic mice exhibited less angiogen- claudication, variable amounts of edema occurring in pa-
esis and produced less VEGF than their normal mice coun- tients with rest pain, and profound edema occurring in pa-
terparts.39 Another model used by this group involved bal- tients with ischemic tissue loss.45 Isner’s group also reported
loon injury to the rat carotid artery with a 30 minute inocu- on a trial of 10 limbs in 9 patients with ulcers and rest pain
lation with either saline or VEGF. The VEGF treated ani- that received IM gene transfer two times at 4 week intervals.
mals re-endothelialized more quickly and exhibited less The clinical status was improved in 8, unchanged in 1, and
neointimal thickening.40 worse in 1 limb after a 3.7 month follow up. The patients
After establishing the angiogenic effect of the VEGF overall demonstrated an increased ankle-brachial index,
protein in ischemia, Isner’s group then proceeded to study improved blood flow by magnetic resonance angiography,
the effect of naked plasmid DNA encoding for human and angiographic evidence of new collaterals in 6/10 limbs.46
VEGF-165 (phVEGF-165). Using the rabbit ischemic hindlimb Further clinical trials are currently being performed using
model, they injected the phVEGF-165 intramuscularly; 30 this gene transfer technique.
days later, there were increased collateral vessels and histo- Yet another route for delivering VEGF to promote graft
logically identifiable capillaries were also seen. In addition, angiogenesis was used by Weatherford and associates.47 They
there was a superior calf blood pressure ratio in the incorporated VEGF and heparin into fibrin glue and found
phVEGF-165 treated group. They felt that ischemic skeletal that human aortic endothelial cell proliferation on this glue
muscle was a good target for gene therapy and that VEGF was increased as compared to fibrin glue alone. The addi-
was a good gene to use.41 Hypoxia upregulates VEGF recep- tion of heparin to the VEGF increased endothelial cell pro-
tor gene expression making ischemic tissues more likely to liferation while inhibiting smooth muscle cell proliferation.
respond to VEGF than normal tissues. VEGF also promotes The application of VEGF and heparin in fibrin glue to a pros-
physiological maturation of newly formed collateral chan- thetic vascular graft may be used in the future to promote
nels and restores appropriate endothelium-derived relaxing graft patency and endothelialization.
factor as well as the nitric oxide-dependent vasorelaxation
response initiated by the endothelium, thereby increasing Future Directions
the blood flow through the expanding vascular bed.42 Research into the mechanisms of angiogenesis is a rela-
An alternate method to deliver the phVEGF-165 in- tively new scientific endeavor and much progress has been
volved transfecting endothelial cells by balloon catheter made in a relatively short period of time. Control of angio-
angioplasty in the rabbit femoral artery. The phVEGF-165 genesis could provide enormous clinical benefits. Inhibition
treated animals showed enhanced endothelialization in the of angiogenesis could result in new treatment strategies for
transfected side as well as the nontransfected, control side, cancer, while promoting angiogenesis could relieve myocar-
decreased intimal thickening, decreased thrombotic occlu- dial and peripheral ischemia from atherosclerotic occlusive
sion, and accelerated recovery of endothelial-dependent va- disease. Other emerging technologies will probably utilize
somotor reactivity as compared to transfection with the LacZ our increasing knowledge of angiogenesis regulation to in-
gene in control animals.43 duce new vessel formation in ischemic tissues, perhaps some-
Recently, this work with the phVEGF-165 has been per- day making bypass grafting for chronic arterial occlusive
formed in humans. A patient with nonreconstructable oc- disease obsolete.
clusive disease received an angioplasty in a normal-appear-
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Facilitation of Healing
Angiogenesis
CHAPTER 27
Polypeptide Growth Factors with a Collagen Binding Domain:
Their Potential for Tissue Repair and Organ Regeneration
Bo Han, Lynn L.H. Huang, David Cheung, Fabiola Cordoba, Marcel Nimni
Introduction
T issue engineering is an emerging interdisciplinary field that applies the principles of en
gineering to the life sciences, with the aim of developing biological substitutes that re-
store, maintain or improve tissue function. In this process, extracellular matrix, cells and
regulatory signals are important in guiding, modulating and facilitating regenerative events.
Cellular activities are regulated by a large number of polypeptides which behave as
growth modulating factors. Such growth factors can either stimulate or inhibit cell division,
differentiation, migration or expression. The effects of such factors are cell type dependent
and can vary with the frequency and way of administration. As we increase our understand-
ing of growth factor functions and their clinical applications, the need for useful pharma-
ceutical forms becomes more apparent. Growth factor targeting to responsive cells and main-
tenance of adequate tissue levels becomes essential, particularly in view of their sometimes
opposite effects on various cells and the dose dependence of their response.
The extracellular matrix provides a scaffold for cell growth and differentiation, and
may eventually help to regenerate tissues. Since collagen is a major constituent of extracellu-
lar matrices and connective tissues, its use in designing a synthetic matrix becomes of spe-
cial interest. When growth factors contain collagen-binding domains, they can be targeted
to collagen matrices, their activities localized, and together with the collagen matrix, syner-
gistically affect the biological activities of cells. Therefore, collagen matrices impregnated
with growth factors become potentially useful for tissue repair and organ regeneration, stimu-
lating cell growth and extracellular restoration as well as remodeling.
In this chapter, currently developed collagen derived matrices, their interactions with
cells and related growth factors for tissue regeneration are reviewed. In addition, we will
discuss means of modulating growth factor release, including the use of recombinant pro-
tein strategies for targeting their delivery.
Development of the potential for addition of growth factors to bioprostheses in the
design of synthetic matrices is anticipated.
The Collagen Derived Matrix
The advantages of using collagen derived matrices as scaffoldings for tissue regenera-
tion include its excellent biocompatibility, its ability to resorb and to be obtained in a pure
form. In addition, collagen can be processed to generate minimal immune response and
crosslinked for attaining optimal degradation rates.1
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler
©1999 R.G. Landes Company.
288 Tissue Engineering of Prosthetic Vascular Grafts
Many investigators and manufacturers have estab-
lished the use of various collagen-derived matrices in der-
matology, cardiovascular surgery, neurosurgery, periodon-
tal and oral surgery, ophthalmology, orthopedic surgery,
drug delivery systems and other applications. Hyaluronan,
chondroitin sulfate, and other polysaccharides, as well as
growth factors and pharmacologically active compounds,
are frequently added to such collagen-derived matrices.
These matrices may be processed to attain different forms
such as sponges, films, sheets, tubes, membranes, felts, gels,
etc. Chemical modifications which include crosslinking with
glutaraldehyde, epoxy, or other chemicals may be carried
out to prolong the duration of such matrices.
Collagen Fibrillar Matrices / Hyaluronan Composites
In our laboratories, we have developed numerous col-
lagen derived matrices and the biological response to these
matrices has been studied.2-5 Various concentrations of bo-
vine skin type I collagen solutions were reconstituted to
fibrillar matrices under physiological conditions. The con-
tractility of these matrices by fibroblasts, a potentially un-
desirable side effect, has been investigated. To facilitate the
process of wound healing, hyaluronan has been added to
collagen fibrillar matrices. Through a contraction model
with a floating collagen fibrillar matrix, it has demonstrated Fig. 27.1. (A) Network of reconstituted type
that high concentrations (> 1 mg/ml) of hyaluronan can I collagen coating tissue culture well. (B)
significantly reduce matrix contraction by fibroblasts and Same as (A). but after seeding with human
the specificity documented. In addition, hyaluronan stimu- skin fibroblasts. Note contraction of the col-
lates cell outgrowth and migration.4 lagen network.
In order to prolong the beneficial effects of hyaluronan,
CNBr-activated hyaluronan was covalently linked to the
collagen fibrillar matrix to stabilize the interactions of designed for emergency treatment to prevent infection or
hyaluronan and collagen. It is found that covalently linked excessive fluid loss. The bilayer membrane consists of a
hyaluronan not only strengthens the collagen fibrils, but polydimethylsiloxane (silicone) top layer and a porous bio-
also blocks the direct communication between fibroblasts degradable collagen-glycosaminoglycan network bottom
and collagen fibrils. 5 Contractility of such developed layer. The glycosaminoglycan used in the network was mainly
hyaluronan-collagen fibrillar matrix by fibroblasts is chondroitin sulfate. Physicochemical and mechanical prop-
therefore abolished.3,4 erties such as moisture/air permeability, surface energy, flex-
In implantation studies, full-thickness wounds in the ural rigidity, and tear strength of the membrane are care-
dorsal skin of guinea pigs were filled with such fully controlled. About 14 days after covering the wounds
noncontractible hyaluronan-collagen fibrillar matrix, or with stage 1 membrane, the silicone layer must be removed
collagen fibrillar matrix, or hyaluronan, or nothing as a con- and the stage 2 membrane transplanted. The stage 2 mem-
trol. The wounds filled with hyaluronan-collagen fibrillar brane with basal cells seeded into the collagen-glycosami-
matrices healed faster than the open control group. In addi- noglycan layer can help the formation of neoepidermis. Sili-
tion, they manifest superior healing. The structure of the cone still covers the top of the membrane. The confluency
neomatrix can be hardly distinguished from the normal between new dermis and new epidermis is achieved about
counterpart evaluated by pathological examination and 15 days later. The result, after the silicone layer is peeled off
scanning electron micrographs.6 The results indicate that at zero strength, is a reconstructed skin.
the hyaluronan-collagen fibrillar matrix could provide ideal The choice of collagen-glycosaminoglycan network
tissue templates for big areas of wounds and may be very was claimed to be an important feature of their design
useful for the development of bioprostheses and implants (Fig. 27.2). The advantages of this crosslinked network are
(Fig. 27.1). as follows:
1. Both collagen and glycosaminoglycan are biodegrad-
Collagen / Glycosaminoglycan Networks able and relatively nonantigenic;
Yannas et al7-10 proposed a two stage artificial skin 2. Coprecipitation of collagen with one of several gly-
model for severe burn related healing. Clinical data show cosaminoglycans (including chondroitin 6-sulfate,
that stage 1 grafts are able to form a neodermis, but that the chondroitin 4-sulfate, dermatan sulfate, heparin and
epidermis layer has to be replaced by an autograft in the heparan sulfate) can control the biodegradation rate,
second stage. The membrane in stage 1 is a wound closure which is evaluated by collagenase susceptibility;
Polypeptide Growth Factors with a Collagen Binding Domain: Their Potential for Tissue Repair and Organ Regeneration 289
Fig. 27.2. Modified reconstituted or engineered biosynthetic extracellular matrices containing various portions of native components
and thiolated gelatin.
3. Glycosaminoglycan are also used to control the me- 2. Fibronectin molecules can assemble into fibrils as
chanical behavior, e.g., as a modulator of the ‘cables and connectors’ for linking cells to collagen
elasticity. and fibrin;11
It has been reported that certain collagen-glycosami- 3. Through binding to cell surface receptors, fibronectin
noglycan polymers have delayed or arrested the contraction can transduce signals internally to modulate cellu-
of full-thickness skin wounds. At least three conditions must lar function.14
be met to prevent wound contraction: Yaffe and Shoshan15 studied the behavior of gingival
1. The enzymatic degradation rate of the implant must epithelial cells cultured in dishes with gingival epithelial ex-
be below a certain threshold value; tracellular matrix. It was found that the movement of epi-
2. The average pore size must within the range of thelial cells stops at the contact point with extracellular ma-
20-500 ∝m in order to allow proper distribution of trix, and that this results in the piling of cells into several
mesenchymal cells; layers. This suggested that the movement of epithelial cells
3. Epidermal (basal) cells must be seeded into the po- may be influenced by collagen fibers in vivo. This also im-
rous implant before grafting. plies a role for collagen in preventing epithelial cells from
penetrating into the lower layer.
Interactions of Biosynthetic Matrices The studies of Donaldson et al16 failed to demonstrate
with Cells collagen type specificity for supporting the migration of
epidermal cells. Denatured collagen and glutaraldehyde
Cell Migration on Biosynthetic Matrices crosslinked collagen gels are also able to support the migra-
Cell migration requires a solid support such as an ex- tion of epidermal cells. It is found that the tertiary structure
tracellular matrix or a basement membrane. In the process of the collagen molecule is unimportant for the binding of
of wound healing, certain cells migrate extensively in a highly epidermal cells. It is also reported that the #1 chain of type I
organized manner.11 Evidence for the mechanisms of orga- collagen has at least three, and most likely more, epidermal
nized cell movement are: binding sites.
1. Cells are bound to the extracellular matrix through Epidermal growth factor (EGF) and transformtin
a variety of large glycoproteins;11-13 growth factor-! (TGF-!) have been shown to play a role in
290 Tissue Engineering of Prosthetic Vascular Grafts
stimulating the growth of epithelial cells.17,18 The structures mis-like appearance. It was suggested that both physical
of EGF and TGF-! are similar and both bind to the same forces and secreted factors from fibroblasts such as
cell surface receptor, tyrosine kinase.19 The interactions fibronectin or other proteoglycans contribute to the reor-
between the receptor and growth factors lead to intracellu- ganization of the collagen lattices.
lar changes in preparing cells for DNA synthesis and
proliferation. Barrandon and Green18 found that the effects Influence of Cells on the Contraction
of EGF and TGF-! in promoting cell proliferation depend of Collagen Matrices
on their abilities to increase the rate of cell migration, which Allen and Schor25 quantitatively measured the con-
is essential in the process of wound healing. Therefore, traction of collagen by fibroblasts. The spherical fibroblasts
appropriate application of EGF or TGF-! during tissue were initially incubated in the attached collagen lattice. Over
regeneration should be able to promote the re-epithelial- 90% of the cells change the morphology to stellate cells af-
ization process. ter incubation for two hours, but most of them subsequently
retract to a spherical morphology when the matrices are
Cell Infiltration into Biosynthetic Matrices carefully detached to produce a floating culture. Collagen
When cells are cultured on a hydrated collagen gel, matrices reduce to about one half of the original size within
the shape and motility of cells are very similar to cells in the first two hours in a floating culture. This initial con-
vivo.13 In addition to the biological effects of hyaluronan traction rate of collagen is proportional to the cell number
demonstrated by Huang et al5,6 Doillon et al20 have shown within the range of 105-106 cells/gel. The contraction of the
that the presence of either fibronectin or hyaluronic acid or collagen matrix appears to be the result of cell-collagen in-
both in a collagen sponge can enhance wound healing in teraction and collagen reorganization caused by fibroblasts
vivo. It has been found that fibroblasts can infiltrate and that exert a tension upon the surrounding collagen fibers. It
proliferate through the entire sponge, and subsequently in- is also suggested that cell-cell interaction does not contrib-
crease collagen biosynthesis. The effect of hyaluronic acid is ute significantly to the contraction process, since stellate fi-
to promote cellular infiltration and that of fibronectin is to broblasts and spherical fibroblasts are homogeneously dis-
mediate cell attachment to the collagen fibrils. Therefore, tributed within the collagen matrix in a floating culture.
incorporation of hyaluronic acid or fibronectin into collagen Bell et al also reported that human fibroblasts of dif-
sponges appears to improve the properties of the regener- ferent proliferative potential contract collagen lattices with
ated tissue.21 equal efficiency.26 They further found that the contraction
rate of collagen lattices can be regulated by varying the pro-
Influence of Cells on Matrix Reorganization tein content of the lattice, the cell number, or the concen-
Denefle et al22,23 used unstriated fibrils of purified tration of inhibitors such as colcemid. Inhibition of gel con-
type I collagen gels to study the anchoring process of traction by cytochalasin B and colchicine indicate that the
keratinocytes and their influences on collagen organization. integrity of the cytoskeletons inside the fibroblasts is im-
Two types of collagen striated fiber assemblies seem to ap- portant for the mechanism of gel contraction.
pear when isolated epithelium is superimposed on a col-
lagen gel. Numerous extended filopodia of cells arise at the Influence of Collagen Derived Matrices on Cells
contact sides with one type of collagen fiber assembling To improve the hematocompatibility of cardiovascu-
around and along them. In the inner side of the gel matrix, lar materials, Lee et al27,28 chemically crosslinked collagen
collagen fibers assume different shapes and widths depend- to polyurethane and investigated the growth of endothelial
ing on the concentration of acid soluble collagen.22 To fur- cells on the surface. A flossy-like collagen formed on the
ther distinguish the effect of preexisting fibronectin from polyurethane surface when the crosslinking reaction was
that of keratinocytes on the reorganization of collagen gels, carried out with (N-ethyl-N’-3-dimethyl aminopropyl)car-
frog skin fragments opposed to collagen coatings were stud- bodiimide at pH 5. A fibrillated collagen network was fur-
ied either in the presence or absence of fetal calf serum. The ther reconstituted on the polyurethane surface under physi-
results indicate that keratinocytes in the absence of serum ological conditions, using either the flossy-like collagen as a
appear to influence fibril organization of purified type I col- nucleus or an epoxy crosslinking reaction. When cultured
lagen gel. Pre-existing fibronectin appears to enhance the with endothelial cells, the collagen fibrillar networks better
capability of keratinocytes to modify collagen fibers, since supported the cell growth. The amounts as well as the den-
the presence of serum causes the collagen morphology to sity and diameter of collagen fibrils grafted on the polyure-
closely resemble that of the native wound healing process.23 thane surfaces correlated with the proliferation of endothe-
Grinnell and Lamke24 studied the reorganization of lial cells.
hydrated collagen lattices by human skin fibroblasts. They
found collagen reorganization to be similar whether the cells Endothelial cells in Tissue Engineering
were cultured on the top or at the bottom of the lattices. The main function of endothelial cells is to maintain
Initially, proximal collagen fibrils were aligned along the surface integrity, such as in the lining of heart, blood vessels
plane of cell spreading. Subsequently, the collagen fibrils and lymphatic vessels. They reduce the friction at the fluid
distal to the cells underwent reorganization. If the collagen to lumen interface, as well as control the flow of blood and
lattice is detached from the underlying substratum, the col- passage of cells in the circulation out of the vascular system
lagen fibrils roll up into a compact form which has a der- into the surrounding tissues.29 Resting endothelium ex-
Polypeptide Growth Factors with a Collagen Binding Domain: Their Potential for Tissue Repair and Organ Regeneration 291
presses low levels of adhesion molecules that prevent the branching tubular networks occurred. Based on these re-
binding of leukocytes and inhibit coagulation. Infection or sults they proposed that FGF-stimulated endothelial cells
injury stimulates the endothelium to express cell surface may be “switched” between growth and differentiation as a
molecules and adhesion proteins that promote cell interac- response to the mechanical integrity of their extracellular
tions.30 There has been a tremendous resurgence of interest matrices. The influence of the extracelluar matrix on en-
concerning the modulation of endothelial cell function by dothelial behavior is likely to be mediated through integrins,
factors, especially cytokines, and by the composition of the a family of heterodimeric cell surface receptors which trans-
extracellular matrix; this has encouraged the development mit signals from the extracellular matrix by organizing the
of endothelial cell culture techniques.31,32 Endothelial cells cytoskeleton, thus regulating cell shape, internal cellular ar-
cultured from the umbilical vein can be induced to switch chitecture and other cell functions.42 While Matrigel sup-
to a capillary-like morphology on interaction with base- ports the attachment, proliferation and differentiation of
ment.33-35 This morphologenesis results in formation of cel- endothelial cells, type I collagen containing alpha-elastin was
lular cords containing a central lumen surrounded by one found to inhibit the proliferation and migration of vascular
to three endothelial cells, and is thought to parallel capillary endothelial cells. While others found that type I collagen
formation in later stages of angiogenesis in vivo.33-35 Since inhibits the growth of endothelial cells, Lee et al found that
endothelial cells in vivo are organized in monolayers cover- endothelial cell growth is supported by large reconstituted
ing the basement membrane of the vessels, much work has type I collagen fibrils formed from collagen dialyzed in phos-
been done using in vitro models with endothelial cells seeded phate buffered saline.28 Chen et al studied human and bo-
on basement membrane equivalents such as Matrigel, re- vine capillary cells grown on surfaces coated with
constituted from solubilized basement membrane compo- micropatterned extracellular matrix substrates.43 As the ex-
nents such as type IV collagen, laminin and fibronectin ex- tracellular matrix-coated adhesive islands decreased in size,
tracted from Engelbreth-Holm-Swam mouse sarcoma.36-39 cell extension became more restricted and the endothelial
However, endothelial cells can also be successfully cultivated cells began to switch from growth to apoptosis. Spacing be-
on gelatin or poly-L-lysine-coated surfaces. The cultivation tween different adhesive islands also affected cell spreading.
of microvascular endothelial cells in Matrigel can result in Changes in the size and spacing of the micropattern of the
the formation of vessel-like cell assembly.40 Ingber and adhesive islands caused alteration in cell shape, which gov-
Folkman studied the effects of coating density of fibronectin, erned whether individual cells grow or die. The effect of the
type IV collagen or gelatin on culture dishes (adhesiveness) extracellular adhesiveness on cell growth and viability was
on the behavior of FGF-stimulated endothelial cells.41 They independent of the type of matrix protein used to mediate
observed extensive cell spreading and growth in dishes coated adhesion in the islands (Fig. 27.3).
with a high density of fibronectin or type IV collagen, while
cell rounding, detachment and loss of viability occurred in Modified Growth Factors: TGF-!
dishes with low density coating. Intermediate coating den- with a Collagen Binding Domain
sity promoted cell extension and partial retraction of multi- TGF-! appears to be an important regulator of the
cellular aggregates; cessation of growth and formation of cardiovascular system. The TGF- ! proteins are highly
Fig. 27.3. Endothelial cells grown on collagen matrix.
292 Tissue Engineering of Prosthetic Vascular Grafts
expressed during embryonic development of the heart, and control the biological activity of this growth factor during
can be elevated after hemodynamic stress. Hence, they pos- its use as regulatory signal for tissue engineering.
sess the potential to significantly influence the progression A collagen targeted TGF-! recombinant fusion pro-
of a number of vascular disorders, in particular those in- tein has been designed, expressed, and renatured into active
volving vessel remodeling and repair, such as hypertension forms.45,49 In these fusion proteins, a collagen targeting
following percutaneous balloon catheter angioplasty, ath- decapeptide derived from von willebrand factor50 was modi-
erosclerosis and under some circumstances, angiogenesis. fied49 and fused at the N-terminus of the active TGF-! frag-
These and many other effects of TGF-! proteins in cardio- ment by molecular manipulation. A protease sensitive site
vascular development, physiology and pathophysiology are was also inserted in the sequence between the collagen bind-
dependent upon processes that regulate their expression and ing domain and the active TGF-! fragment, in order to re-
release from latent complexes, the receptor types available lease the growth factor at appropriate times and sites. Bac-
for binding and the interactions between receptor-mediated terial expression systems provided a good source for this
intracellular signaling events, which ultimately lead to tis- recombinant protein and appear to guarantee sufficient
sue responses.44 amounts for tissue engineering applications. BL21 (DE3)
Major problems that hamper the use of growth fac- E. coli strain was transformed with a pET 28b expression
tors in tissue engineering are: vector which carried recombinant DNA. Under IPTG in-
1. The activity is quickly lost due to the protease duction, this fusion protein can be induced to express at high
digestion; yield. The induced fusion proteins were found in insoluble
2. They are not retained at the application site; inclusion bodies. After being solubilized by a denaturant and
3. They are carried away by the circulation. purified by Ni-chelating column, the purified TGF-! mono-
Therefore, targeting growth factors to the application mer was refolded in an in vitro optimized system, in which
site and controlling their release and activity becomes cru- reduced and oxidized forms of glutathione assist TGF-! in-
cial when applying growth factors as regulatory signals for ter- and intradisulfide bond exchange.45
tissue engineering. In addition to approaches that rely on
adsorption of ionically bound, physically entrapped or co- Binding Characteristics
valently bound growth factors to resorbable matrices, a novel Active TGF-!, a highly hydrophobic basic protein, is
approach developed in our laboratory relies on the construc- rapidly lost from the medium or extracellular fluid by pro-
tion of fusion proteins which include a matrix or cell bind- tease digestion. Many extracellular matrix components bind
ing domain for targeted delivery of such growth factors45 to TGF-! in a reversible manner and may serve as a reser-
(Fig. 27.4). voir for the growth factors.51,52 TGF-! binding proteins could
Transformtin growth factor-! (TGF-!) was used as an sequester TGF-! to the extracellular matrix and act as a buffer
example for studying a ECM targeted growth factor. TGF-! for its activities.53,54 Since collagen is a major component of
is a secreted multifunctional protein that regulates many the extracellular matrix, and the structural protein of most
aspects of cellular functions, including cell proliferation, tissues, it was chosen as functional carrier of soluble growth
differentiation, and extracellular matrix metabolism.46 Based factors in this study. In the fusion of TGF-! proteins, a col-
on the different physiological effects of TGF-!, a variety of lagen binding domain derived from von willebrand factor
potential clinical applications for this growth factor have was incorporated at the N-terminal of the active growth fac-
been suggested.47,48 Since TGF-! is a pleotropic agent which tor fragment by a bridging Gly. Takagi et al50 have reported
can stimulate, inhibit, and modulate cellular events in a time that the primary structure of the von willebrand factor con-
and concentration dependent manner, it is important to tains a high-affinity collagen binding domain. They further
Fig. 27.4. A schematic representation of the genetically engineered growth factor(s). The constructs contain: a collagen
binding sequence derived from von willebrand factor; a purification tag (e.g., (His)6 or GST); and a protease-sensitive
site for control release.
Polypeptide Growth Factors with a Collagen Binding Domain: Their Potential for Tissue Repair and Organ Regeneration 293
delimited the collagen binding site to a decapeptide with collagen binding domain in F2 afforded this high affinity
the amino acid sequence WREPSFCALS. The collagen tar- interaction with collagen.
geting characteristics were assayed as described below.
Collagen targeted TGF-! fusion proteins from bacte- Biological Activity
rial inclusion bodies were isolated, dehydrated in DMF, and
labeled by [3H]NaBH4 (100 mCi, Amersham) DMF solu- Released Collagen Targeted TGF-!
tion.55 Labeled recombinant proteins were purified and rena- Retains Biological Activity
tured as described and the bioactivity was also checked. Dif- TGF-! activity was assessed by transfected mink lung
ferent doses of labeled TGF-!1-F1 or -F2 fusion proteins were cell with PAI-1 promoter/luciferase reporter genes.45 The
then added to the collagen precoated wells. After incuba- specificity and sensitivity are the result of using a truncated
tion and extensive washes in PBS, the labeled TGF-! remain- PAI-1 promoter which retains the two regions responsible
ing bound to the collagen was counted after collagenase for maximal response to TGF-! (56). Collagen bound TGF-
digestion. The results are shown in Figure 27.5. TGF-!1-F2, !1-F1/F2 fusion proteins were released from the collagen gels
which has a collagen binding domain, binds to collagen in a with collagenase. The released solubilized TGF-!1-F1 and
dose dependent manner. TGF- ! 1-F1, which lacks the TGF-!1-F2 proteins were assayed for their biological activ-
collagen binding domain, binds to collagen with less ity. It was found that collagen bound TGF-!1-F2 was still
affinity through nonspecific interactions. The only dif- active when released with collagenase. These experiments
ference in F1 and F2 constructs is the auxiliary collagen demonstrate that the collagen binding domain enables TGF-
binding decapeptide; this suggests that with a collagen !1-F2 to be sequestered by the collagen matrix and that it
binding domain, TGF-! can be specifically targeted to can be released by collagenase digestion to display its bio-
collagen matrices. logical activity.
To determine the affinity of such binding, F2 fusion
protein was first immobilized on a Ni-NTA column and then Collagen Bound TGF-!1 Activity
exposed to biosynthetically labeled [3H]collagen, which was Even though we had proven that collagen-targeted
subsequently loaded onto the column. Under these condi- TGF-! can bind selectively to the collagen matrix, the bio-
tions a large portion of the radioactivity was found to bind logical activity of bound TGF-!2-F2 was unclear. When we
to the column. Washing the column with a linear gradient precoated culture wells with type I collagen, and then added
of NaCl from 0.15 to 2.0 did not release the [3H]collagen. TGF-!2-F1 and TGF-!2-F2 on the collagen gels, then washed
However, application of a urea gradient (0-5.0 M) was able extensively with PBS, only bound TGF-!2-F1/F2 remained
to quantitatively elute all bound radioactivity (Fig. 27.6). By on the gel. Subsequently, osteoblastic osteosarcoma cells were
contrast, when the F1 fusion protein containing only a (His)6 plated on top of the collagen gel. After 72 hrs, alkaline phos-
tag and the TGF-!1 active fragment was applied to the Ni- phatase activity was tested in the cell layer. Results (Fig. 27.7)
NTA column under the same conditions, most [3H]collagen showed that when TGF-!2-F1/F2 was added to the medium,
was eluted in the void volume, suggesting that the auxiliary the cellular responses to these two factors were similar; for
Fig. 27.5. Binding curve of renatured TGF-!2-F2 for
collagen matrices. [3H]labeled fusion proteins bind
to collagen in a dose-dependent manner. Radio-la-
beled TGF-!1-F1 or -F2 was incubated with collagen
gel. After extensive washing, the collagenase digest
solution was counted for radioactivity.
294 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 27.6. Binding of TGF-!1-F1 and
-F2 proteins with collagen. The tri-
partite fusion proteins F1 (dashed
line) and F2 (solid line) were first
bound to the Ni-NTA column (3
ml). [3H]collagen was then applied
to the column and bound protein
was eluted with a gradient of 0-5 M
urea in 0.05 M phosphate buffer at
pH 7.0 (3 ml/ fraction).
the collagen-bound TGF-!2, cells could still ‘recognize’ sistent with their playing a role during the rapid changes
bound TGF-!2 and display increased alkaline phosphatase seen in cardiovascular tissues in during development and
activity. On the other hand, TGF-!2-F1, washed away by PBS induced anomalies.
from the collagen gel because it lacks a collagen binding
domain, exhibited no effect on induction of alkaline phos- bFGF
phatase activity above control. These results not only con- bFGF is present in many tissues, intracellularly or
firmed that TGF-!2-F2 could specifically target collagen, but bound to heparin-like molecules of the extracellular ma-
also demonstrated that extracellular matrix-associated trix. It is thought to exert its physiological functions in tis-
TGF-! still performed its biological function. sue repair and neovascularizatoion after being released from
the extracellular matrix.58,59 bFGF causes mesenchyme for-
Other Growth Factors Which May Individually mation and stimulates skin wound healing by increasing cell
or Synergistically Contribute to Organogenesis recruitment, mitosis, and extracellular protein production.
Growth factor targeting to responsive cells and main- Though very potent, bFGF is rapidly degraded when injected
tenance of adequate pharmacological levels becomes essen- or ingested and applications of bFGF are hampered by the
tial, particularly in view of their different effects on various instability of the compound. Additionally, a bFGF dose-re-
cells and the dose dependence of their response. Using the sponse curve on bone growth study exhibits biphasic effects,
same recombinant strategy, other growth factors besides reflected by an exaggerated inhibited cell ingrowth.60 Con-
TGF-! have also been engineered with an ECM targeting trolling bFGF activity therefore becomes meaningful for
domain. These growth factors include basic fibroblast growth proper use and clinical application of this growth factor.
factor (bFGF), epidermal growth factor (EGF), vascular en- Preservation and stabilization of bFGF was previously ac-
dothelial growth factor (VEGF), #-ECGF and bone morpho- complished by binding the factor to heparin-Sepharose
genic protein-3 (BMP-3). The targeting sites of growth fac- beads. This permitted its prolonged storage, repeated hand-
tors are not limited to the collagen matrix, since fibronectin ing and encapsulation within a microspherical controlled-
and heparin targeting domains can also be incorporated into released device.61 Recently our laboratory has engineered
the growth factor molecules. The tissue distribution and collagen targeting bFGF recombinant fusion proteins to
cellular effects of platelet-derived growth factor (PDGF), ba- enable this fusion protein to be targeted to collagen matri-
sic fibroblast growth factor (bFGF), transformtin growth ces. Its in vitro activities have been evaluated and their ap-
factor-b1 (TGF-!1) and insulin-like growth factor 1 (IGF-1) plication is under investigation.
suggest a potential role for these factors in cardiovascular
matrix regeneration. Fibroblast replication is stimulated by VEGF
PDGF and by bFGF. IGF-1 and TGF-!1 have no effect on Recent studies show that exogenously administered
fibroblast replication. Collagen production is stimulated by angiogenic factors can induce the formation of new blood
all of the growth factors tested: in order of potency, TGF-!1. vessels in adult animals and enhance collateral blood flow
PDGF, IGF, bFGF. None of the growth factors affected the to ischemic tissues. It has been suggested that this approach
proportion of rapidly degraded newly synthesized collagen.57 may have a therapeutic potential.62 VEGF (vascular endo-
The sensitivity of cardiac fibroblasts to these factors is con- thelial growth factor) is a specific mitogen for endothelial
Polypeptide Growth Factors with a Collagen Binding Domain: Their Potential for Tissue Repair and Organ Regeneration 295
Fig. 27.7. Collagen bound TGF-!2-F2 increases alkaline
phosphatase activity of MG-63 cells. F1m and F2m: 1 ng
TGF-!2-F1 or -F2 fusion protein in the medium. F1b and
F2 b: 5 ng TGF-!2-F1 or -F2 incubated with precoated
collagen gel for 1 hour at 37°C followed by three exten-
sive washes with PBS. Cells were then plated on top of
gel. Alkaline phosphatase activity was evaluated after a
72 hour incubation.
cells in general and for vascular endothelial cells in particu- several BMP family members which belongs to the
lar. It has been shown to promote angiogensis, accelerate transformtin growth factor-beta superfamily. BMP-3 has
endothelial repaving, and to attenuate intimal hyperplasia been characterized by its ability to stimulate osteoblastic dif-
after arterial injury. The active forms of VEGF are disulfide- ferentiation and bone formation.64,65 It was first identified
linked, dimeric glycoproteins. The presence of two tyrosine and then purified from bovine bone by Reddi and col-
kinase receptors for VEGF have been identified in vascular leagues.65 Although in vivo tests support the role of BMP-3
endothelial cells. in bone formation, its mechanism of action is poorly un-
derstood. In our laboratory, we bioengineered human re-
EGF and ECGF combinant BMP-3 fusion proteins from E. coli and
Epidermal growth factor (EGF), a 53 amino acid mi- renaturated them into their native dimer conformation. The
togenic polypeptide present in many mammalian species, is large quantities of BMP-3 obtained enabled us to do exten-
one of a number of growth factors being investigated for sive studies on this growth factor in animal models. When
their potential to expedite the healing process. EGF has been combined with collagen sponge matrices or porous hy-
shown to stimulate keratinocyte division in vitro and epi- droxyapatite ceramics, it appears to generate a potential bone
dermal regeneration in vivo. It had been shown to have an substitute for fracture repair and regeneration.66
effect on mesenchymal cells by producing marked prolif-
eration of the dermis in partial thickness wounds and by Applications of the Growth Factor Matrix Technology
increasing the tensile strength of surgical incisions. In ex- to the Design of Bioprostheses
perimental animals a modified fibrin glue containing 1 ∝g One of the significant advances in medicine in this
of #-endothelial cell growth factor (ECGF) was implanted century is associated with the ability of the surgeon to re-
between the aorta and the myocardium of the left ventricle.63 place defective heart valves, veins and arteries with
After 9 weeks of implantation, angiography and histologic bioprosthetic devices. Nevertheless, these implants have as-
investigation showed a newly grown vascular structure be- sociated disadvantages since they may require anticoagula-
tween the aorta and the myocardium in all experimental tion, periodic monitoring for possible failure and in some
animals, but none in the controls. These studies proved the cases have a limited durability or only be usable in selected
feasibility of initiating site-directed formation of new blood cases or locations. It is obvious that if we can provide a scaf-
vessel structures to the heart by a modified fibrin glue im- folding which after being implanted would allow a tissue or
plant containing angiogenic growth factor #-ECGF. organ to regenerate, that we would be achieving a very meri-
torious objective. Our current understanding of cell biol-
BMP-3 ogy, that is, how cells differentiate from stem cells into dif-
One of the few tissues that can regenerate in mam- ferentiated cells, how cells interact with the extracellular
mals is bone. To a great extent this is due to the activity of matrix and the consequences of controlled cell-cell interac-
specific growth factors which stimulate its formation. Bone tions, should be able to lead to the ordered repair of tissues
morphogenetic protein-3 (BMP-3, osteogenin) is one of the and even organ regeneration in the near future.
296 Tissue Engineering of Prosthetic Vascular Grafts
Collagen is an ideal biomatrix, as it is the major com- lar interactions involving their cell membranes, as a result
ponent of the extracellular matrix. It can be rendered rela- of the movement of molecules such as peptides or steroids,
tively nonantigenic, even if obtained from another species, that can act locally or systematically to modulate cell func-
after removal of the nonhelical antigenic determinants and tions. Growth factors are included in the latter group.1
may soon be available in a recombinant form from human Growth factors can either stimulate or inhibit cell di-
DNA. Reconstituting soluble collagen into fibrous matrices, vision, differentiation, migration or gene expression, de-
alone or in combination with the other major component pending on the cells involved. Depending on the concentra-
of the extracellular space, namely the proteoglycans, is a rela- tion present in the cellular environment, growth factors can
tively simple procedure. Specific collagen types, including act in an opposing manner and up or downregulate the syn-
those which give rise to basement membranes, are also be- thesis of receptors. In general, both growth factors and
coming available in the design of biomatrices. mRNAs that code for them turn over very rapidly. They usu-
Growth factors are part of a large number of polypep- ally exist as inactive or partially active precursors that require
tides that transmit signals affecting cellular activities. Cells proteolytic activation, and may need to bind to matrix mol-
may communicate with each other through direct molecu- ecules for activity or stabilization. They may perform differ-
Fig. 27.8. Collagen-derived matrices
combined with collagen-targeted
growth factor as tissue substitutes.
(A) Modified, reconstituted or en-
gineered biosynthetic extracellular
matrices containing various propor-
tions of native components, rear-
ranged and crosslinked to each
other, provide sui carriers for the
local delivery of such factors. (B)
Recombinant collagen-targeted
growth factors are specifically tar-
geted to collagen matrices and
therefore their effects become local-
ized. Growth factor release can be
controlled by proteases due to the
insertion of protease sensitive sites
between the collagen targeting do-
main and the active growth factor
fragment or by the action of colla-
genase, which slowly degrades the
matrix. (C) Growth factor and
collagen composite materials serve
as scaffold for cell growth, migra-
tion, and differentiation. Under
growth factor modulation, cells
reorganize and synthesize new
extracellular matrix and tissue can
be regenerated.
Polypeptide Growth Factors with a Collagen Binding Domain: Their Potential for Tissue Repair and Organ Regeneration 297
Fig. 27.9. Schematic diagram of vascular graft substitute. A TGF-! impregnated crosslinked collagen sheet supports the growth of
fibroblast. VEGF impregnated modified collagen gel supports the growth of vascular endothelial cell growth.
ent functions on different cell types: For instance, TGF-! is ible with the surrounding tissue and should be able to anas-
stimulatory for fibroblasts and inhibitory for keratinocytes. tomose readily to the existing viable tissue.
With the increasing availability of growth factors de- Our increased understanding of the extracellular ma-
rived from cultured human cells and their expansion through trix, and of the role of agents such as cytokines and growth
recombinant technologies, coupled with an increasing un- factors in the various aspects of cellular activity associated
derstanding of their functions and clinical applications, the with tissue repair and remodeling, can now provide us with
need for useful pharmaceutical forms is becoming more and sufficient insight into the design of tissue compatible scaf-
more apparent. Growth factor targeting to responsive cells foldings. These scaffoldings can be populated with cells and
and maintenance of adequate pharmacological levels be- via processes of cell-matrix and cell-cell interactions
comes essential, particularly in view of their different effects modulated to acquire shapes and functions compatible with
on various cells and the dose dependence of their response. their endothelial and mesenchymal cells. There should have
An important issue is the short biological half life of a decreased tendency to attract platelets and be able to retain
growth factors. For example, platelet-derived growth factor growth modulating polypeptides which encourage
(PDGF), an important growth factor first isolated from plate- systematic repopulation by selected cells and yield functional
lets, cannot be detected in the circulation, and when injected vascular prostheses. We hope that the data present as well as
intravenously its half life is less than 2 min.67 the suggestions made during the course of this review
Our ability to target growth factors to the extracellu- continue to assist and encourage others to move in
lar matrix components and to couple their release to native this direction.
ability or inducible enzymatic activity, and our understand-
ing of stem cell biology, as well as our ability to cause such References
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factors, should allow us to generate functional tissues as re- systems. Biomaterials 1997; 18:1201-1225.
placement for damaged ones. In Figures 27.8 and 27.9 we 2. Huang-Lee LLH, Nimni ME. Preparation of type I col-
have provided models for the design of such a matrix, with lagen fibrillar matrices and the effects of collagen con-
centration on fibroblast contraction. Biomed Eng Appl
an emphasis on the construction of a vascular graft. Further
Basis Comm 1993; 5:664-675.
experimental work along these lines is very likely to gener- 3. Huang-Lee LLH, Nimni ME. Fibroblast contraction of
ate functional tissues which, starting with vascular grafts, collagen matrices with and without covalently bound
will continue to increase in complexity until we eventually hyaluronan. J Biomater Sci Polymer Edn 1993; 5:99-109.
are able to generate more complex functional organs. 4. Huang-Lee LLH, Nimni ME. Crosslinked CNBr-activated
hyaluronan-collagen matrices: Effects on fibroblast con-
Summary and Conclusion traction. Matrix Biology, 1994; 14:147-157.
The design of artificial vascular prostheses and recon- 5. Huang-Lee LLH, Wu JH, Nimni ME. Effects of
struction with collagen scaffoldings of damaged vessels rep- hyaluronan on collagen fibrillar matrix contraction by fi-
resents a very important field of endeavor. Vascular grafts broblasts. J Biomed Mater Res, 1994; 28:123-132.
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298 Tissue Engineering of Prosthetic Vascular Grafts
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replication by cardiacfibroblasts is enhanced in response
Facilitation of Healing
Angiogenesis
CHAPTER 28
Fibroblast Growth Factors in Angiogenesis
and Tissue Engineering
Karin A. Blumofe, Timothy J. Heilizer, Paula K. Shireman, Howard P. Greisler
Introduction
T he main treatment for arterial occlusive disease has become reconstruction. This involves
utilizing a graft, either in the form of a vein or synthetic material. These treatments have
been found to be limited in their long term therapeutic value due to stenosis formation, a
process in which there is luminal narrowing of the vessel due to smooth muscle cell
proliferation and connective tissue deposition in the intima of the vessel wall or at the vessel-
synthetic graft interface. The smooth muscle cells can continue to proliferate for up to one
year after the initial injury. This response is in part modulated by a variety of growth factors
such as fibroblast growth factor (FGF), platelet derived growth factor, angiotensin ii, insulin-
like growth factor I, and so forth. This course of events results in intimal hyperplasia,
ultimately leading to a decrease in blood flow, thrombosis and failure of the
interventional reconstruction.
A major focus of vascular research has been to investigate ways in which to decrease
the smooth muscle cell migration and proliferation, ultimately decreasing the intimal hy-
perplasia response. Fibroblast growth factor, as mentioned above, is one of the growth fac-
tors that stimulates smooth muscle cell migration and proliferation. Additionally, it serves
as a potent stimulator of endothelial cells, inducing angiogenesis, the formation of new blood
vessels. The process of neovascularization has also been studied extensively as a therapeutic
treatment for ischemic disease. With this in mind, the goal of this chapter is to discuss prop-
erties of FGF and its role in tissue engineering.
History
In the mid-1900s the brain was found to be a rich source of growth factors that stimu-
lated fibroblast proliferation. However, it was not until the 1970s that partially purified pitu-
itary extracts were shown to contain potent mitogens, particularly for 3T3 fibroblasts.1 In
1974 a mitogenic polypeptide from the pituitary was identified2-4 which was named fibro-
blast growth factor. Attempts to further characterize the growth factor were hampered by
high levels of contaminating fragments of myelin basic protein.5 Thomas in 1980 used iso-
electric points to isolate acidic FGF.6 The acidic form did not interact with antibodies against
myelin basic protein. It was also found that the acidic form possessed mitogenic activity for
endothelial cells.7 Bovine brain contained another type of FGF which had different proper-
ties from the acidic FGF, as it was a basic mitogen. Thus, FGF was separated into two forms:
FGF-1 and FGF-2. The structural characterization of acidic FGF 8 and basic FGF 9
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
302 Tissue Engineering of Prosthetic Vascular Grafts
demonstrated that these growth factors are related polypep- wards the perivascular space, forming parallel processes.
tides and established the basis for a larger family of polypep- With linear movement, a single endothelial cell projection
tide growth factors. Over the next 13 years eight more or pseudopod migrates from the parent capillary to the sur-
isoforms of FGF would be identified, most recently FGF-9 rounding connective tissue.11,17
in 1993.10 The next phase in this process is that of endothelial
cell proliferation. Mature endothelial cells normally have a
Angiogenesis slow turnover rate of approximately 2 months. However,
Angiogenesis is the formation of new blood vessels when necessary, as in response to angiogenic stimuli, the
from the microcirculation, by a process of cellular outgrowth. endothelial cell can quickly convert to a proliferative state.
In contrast, the development of new vessels from stem cells, The induction of endothelial cell proliferation is associated
as found in an embryo, is termed vasculogenesis. Angiogen- with disruption of cell-cell contacts18 and alterations in the
esis plays a major role in a variety of natural and disease cytoskeletal organization, such as microtubule destabiliza-
processes significant in the adult life, such as reproduction tion.19 Development of the new vessel lumen involves the
(placenta, corpus luteum formation, etc.), wound healing, endothelial cell forming tubular channels. It is thought that
bone repair, inflammation, neoplasia and collateralization the capillary lumen is generated by adjacent endothelial pro-
in response to ischemia (heart disease and peripheral vas- cesses, causing intercellular canalization of adjacent endo-
cular disease). thelial cells.20 A new basal lamina is generated consisting of
The phenomenon of angiogenesis begins with vascu- fibronectin, laminin and type IV collagen.13
lar sprouts that originate from the walls of preexisting cap- Another important cell in the angiogenic process is
illaries and small venules.11 It is possible that endothelial the pericyte. Initially the pericytes undergo intense prolif-
cells are recruited from venular segments, because these cells eration. During the stages of capillary sprouting, the exact
are less constrained by a well formed basal lamina. More role of the pericytes is unclear. These cells appear to bridge
recently, larger vessels have been shown to play a role in the the gaps between opposing endothelial sprouts by utilizing
formation of new blood vessels. For example, it has been the pericytic processes. Capillary sprouts are then able to
demonstrated that the rat femoral vein, with a discontinu- fuse, forming capillary loops and eventually a plexus net-
ous layer of internal elastic lamina and smooth muscle cells, work. The pericytes also play a role in the regulation of
can contribute to angiogenesis. When the vein is treated with angiogenesis. There is an absence of pericytes at the tips
prostaglandins E1 and E2, vascular sprouts arise from the of migrating endothelial cells while, in contrast, their
endothelial cells in the intima layer of the vessel.12 Angio- presence in the older regions of capillaries may inhibit
genesis does not seem to originate from the arterial side of endothelial cell proliferation and migration, leading to
the circulation.13 vessel maturation.21
Scanning electron microscopy has been used to study Many cells, including pericytes, macrophages, mast
the early changes in vasculature and sprouts appear rapidly, cells, lymphocytes, connective tissue cells, endothelial cells
as early as 27 hours after exposure to angiogenic stimuli.14 and tumor cells influence the formation of new vessels by
From three to five days these sprouts continue to flourish, secreting soluble angiogenic molecules. The balance of posi-
ultimately forming a rich anastomosing plexus. tive and negative regulators of angiogenesis control when a
On a cellular level, there are multiple processes in- vessel will proceed to neovascularization. A multitude of
volved in angiogenesis. Early on there is an increase in the molecules have been characterized as influencing this pro-
vascular permeability. There is an association of inflamma- cess. Initial studies were done with the chorioallantoic mem-
tory cells with intravascular accumulation of platelets and brane of the chick embryo (CAM) and the corneal pocket
polymorphonuclear leukocytes. The new vessels arise fol- of the rabbit. In the CAM system, a quantity of an angio-
lowing the margination and diapedesis of the leukocytes.13 genic substance was placed on exposed chick chorioallan-
Then the endothelial cells become activated. They display a toic membrane. Similarly with the rabbit experiments, an-
number of morphological changes, including hypertrophy giogenic substances were introduced into intracorneal pock-
with bulging into the vascular lumen, nuclear enlargement, ets. These two models were useful in examining the angio-
increase in the number of organelles and formation of pro- genic potential of a substance. However, it was not until the
jections from their surfaces.13 The fragmentation and disin- development of cultured endothelial cells and biological as-
tegration of the basal lamina is a necessary step for the mi- says that it became possible to directly examine the mecha-
gration of endothelial cells to occur.15 This degradation is nisms which regulate cell behavior in angiogenesis. The first
due to proteolytic enzymes synthesized and secreted by the purified angiogenic factor was FGF-2. This was followed by
activated endothelial cells.16 numerous other molecules such as vascular endothelial
Angiogenesis then proceeds with the migration of growth factor (VEGF), transformtin growth factor-beta,
endothelial cells, moving into the interstitial space. Two tumor necrosis factor, interleukin-8, platelet derived endot-
different types of endothelial cell migration have helial cell growth factor (PD-ECGF), angiogenin and
been described: angiotropin, to name a few.
1. Bicellular or telescoping formation; and FGF serves as a chemotactic stimulator for endothe-
2. Linear formation. lial cells. If a monolayer of cultured endothelial cells is dam-
In the bicellular type of migration, two or more en- aged, FGF stimulates cellular migration into the denuded
dothelial cells move from the wall of the parent vessel to- area.22 FGF-2 has been shown to increase the secretion of
Fibroblast Growth Factors in Angiogenesis and Tissue Engineering 303
plasminogen activator and collagenase from bovine aortic to the FGF family, with 30-40% homology. FGF-9, the most
endothelial cells. These substances are thought to digest the recently discovered, was purified from a human glial cell
basement membrane preceding migration of endothelial line.10
cells. Antibodies directed against FGF-2 cause impairment Of these nine FGF genes that have been identified,
of endothelial cell migration and inhibit the release of plas- different isoforms can be created through a variety of meth-
minogen activator.22 Additional experiments have demon- ods. For instance, varying isoforms are generated by using
strated that FGF-2 treatments can induce capillary endot- alternative initiation codons for translation.32,33 Novel FGF
helial cells to invade a collagen matrix. These cells then or- isoforms can also be generated by alternative splicing such
ganize themselves to form characteristic tubules that re- as with FGF-234 and FGF-8.30 Finally, posttranslational
semble blood capillaries.23 In animal studies, when FGF is modification techniques provide additional isoforms.
applied to the rabbit or mouse cornea, directed ingrowth of
new vessels is stimulated.24,25 Increased levels of FGF has Structure
not only been implicated in vessel growth but becomes lim- The gene for FGF-1 is located on chromosome 5 be-
ited when neovascularization ceases.23 tween bands 5q31.3 and 5q33.2, while the FGF-2 gene is on
chromosome 4.35 These two genes are similar in their orga-
FGF Characterization nization, as they both have three exons separated by two large
introns. The major difference in these two genes is the loca-
Isoforms tion of the amino-terminus of the two proteins. Nucleotide
Currently the FGF gene family comprises nine mem- sequence analysis of FGF-1 reveals that the open reading
bers (FGF-1 through FGF-9) in mammals. All are structur- frame is flanked by termination codons. The primary se-
ally related, with a homology from 35-55% and generally quence of FGF-2, however, has revealed an amino-terminal
encode proteins with a molecular mass of 20-30 kDa. Over sequence that extends 5' to a proposed initiator, methion-
the course of time and as research has developed, so has the ine.36 FGFs also possess a distinct structural feature known
nomenclature and characterization of FGF (Table 28.1). as a nuclear localization sequence, located near the NH2 end
In 1980, FGF-1 was discovered by detecting the pres- of the protein. This sequence in FGF-1 plays a role in mov-
ence of a polypeptide with a distinct acidic isoelectric point ing extracellular FGF towards the nucleus in a receptor-de-
from extracts of bovine brain.6 FGF-2 was purified by pendent manner. FGF-2 has similar features but has been
Abraham in 1986 by using cDNA clones from kidney, heart, found to be more complicated. FGF-2 mRNA contains mul-
liver, placenta and breast carcinoma.26 FGF-3 was identified tiple translational start sites and the resulting proteins are
as the site of insertion of the mouse mammary tumor virus. transferred to either a cytosolic or nuclear locale.37
Insertion of viral DNA within genomic int-2 led to gene ac- FGF-1 and FGF-2 are single chain polypeptides which
tivation and transformation of infected cells.27 FGF-4 and share 55% sequence homology. The complete structure of
FGF-5 were identified by screening neoplastic cells for the bovine FGF-1 was first described in 1985.38 The final struc-
presence of genes capable of transforming 3T3 fibro- ture is the result of multiple proteolytic cleavages of a more
blasts.28,29 Delli Bovi transfected DNA from Kaposi’s sar- primitive form. Sequence analysis of human FGF-1 demon-
coma into fibroblasts and two new mRNAs were produced. strates similar amino-terminal truncations, with a 92% se-
The protein product of one was termed FGF-4. It was 206 quence identity with the bovine protein.39 FGF-1 is an an-
amino acids in length had significant homology to both ionic mitogen with a molecular weight ranging from 15,000-
FGF-1 and FGF-2. Subsequently, Zahn discovered FGF-5 17,000 kDa. The X-ray crystal structure of human FGF-1
after transfecting fibroblasts with human tumor DNA. Us- has been identified.40 It has been shown to contain four in-
ing a human hst specific probe to screen a mouse cosmid dependent molecules arranged in an asymmetric unit. Each
library, Marics found FGF-6. Tanaka showed that a mouse molecule contains a sulfate ion which stabilizes the other
mammary carcinoma cell line was stimulated by a growth molecules through a hydrogen bond interaction, a potential
factor which was FGF-7.30 FGF-7 is a keratinocyte cell mi- heparin binding site.
togen31 which possesses a DNA sequence complementary FGF-2 is a cationic mitogen with an isoelectric point
of 9.6. Its molecular weight is 18,000 kDa. FGF-2 has been
found to contain 146 amino acids. Within this structure are
Table 28.1. Nomenclature of the FGF family two sequences similar to known heparin binding sites.9
FGF Secretion
Historical Name (23)
The FGF prototypes regulate biological activities as
FGF-1 acidic FGF, HBGF-1, ECGF
an extracellular protein; therefore it is important to exam-
FGF-2 basic FGF, HBGF-2 ine how these proteins are released (or secreted) from cells.
FGF-3 Int-3 FGF-1, FGF-2 and FGF-9 have been found to lack a “tradi-
FGF-4 Kaposi’s FGF, KS3, Hst-1 tional” leader sequence (similar to the interleukin family)
FGF-5 and consequently secretion via the endoplasmic reticulum
FGF-6 Hst-2 does not occur. When the endoplasmic reticulum-Golgi ap-
FGF-7 Keratinocyte growth factor (KGF) paratus path is disrupted by chemical agents, the release of
FGF-8 Androgen inducible growth factor
FGF-9
FGF-1 is not inhibited.41 It is thought that FGF is released in
304 Tissue Engineering of Prosthetic Vascular Grafts
the form of a dimer which is then separated by a reducing potentiates the biological activity of FGF-1 but does not have
agent. The monomer form of FGF is able to interact with a profound effect on the mitogenic activity of FGF-2. Both
the FGF receptors37,42 (Fig. 28.1). Various methods of FGF forms of FGF are protected from degradation when associ-
release have been proposed. For example, it has been dem- ated with heparin. It is suggested that this is due to the sta-
onstrated that the release of FGF-1 is regulated by tempera- bilization of FGF’s tertiary structure and prevention of pro-
ture. NIH 3T3 cells secrete FGF-1 in response to heat shock,43 teolytic modification. Heparin has additionally been found
and terminate release following treatment with either acti- to protect FGF from heat50,51 and acid51 inactivation. FGF-1
nomycin D or cyclohexamide. Recent studies of the kinetics is further protected from trypsin, plasmin50 and thrombin.52
of FGF-1 release suggest that the FGF-1 secretion pathway As previously mentioned, FGF is found in association with
may be limited to newly translated FGF-1.22 Inflammation heparin in the extracellular matrix and basement membranes
has also been proposed to serve as the initial stressor releas- of blood vessels. FGF is ultimately released from its interac-
ing FGF. FGF-2 has also been found to be secreted by an tions with heparin by an enzyme called heparinase, which is
independent pathway.44 FGF-9 also lacks a typical N-termi- expressed by platelets, neutrophils and lymphoma cells.46
nal signal sequence, but has been found to be secreted fol-
lowing transfection into COS cells.45 Biological Activity
In contrast, the remaining members of the FGF fam-
ily possess a functional signal sequence. Some of the FGF Tissue Expression
genes have been identified as oncogenes, reflecting an abil- High levels of FGF-1 are found in neural tissue.54 The
ity to function in an autocrine manner. For example, FGF-4 structural characterization of pituitary FGF was described
(hst-1) is a frequently identified oncogene in transforma- in 1985. In 1987 it was demonstrated that FGF is located in
tion assays of NIH 3T3 cells. the subendothelial extracellular matrix53 and basement
Storage of FGF has been localized to the extracellular membrane, particularly in arterial and capillary walls. FGF-1
matrix and basement membranes of medium and large is not as widely distributed as FGF-2, which is located in
blood vessels, as well as the anastomosis site of branching brain, kidney, adrenal, corpus luteum and the macrophage
capillaries. Heaprin sulfate, which is known to prevent pro- cell lines.23 Using radiolabeled immunoassays, circulating
teolytic degradation of FGF, is also found in high concen- FGF was established in bovine, rat and human serum. Since
trations in these areas. FGF is released from the extracellu- FGF is involved in cell growth, it stands to reason that its
lar matrix in a biologically active form.46 distribution would be widespread.
Interaction with Heparin In Vitro
In 1983 it was discovered that crude extracts of hep- FGF has been found to affect a variety of cell types in
arin bind to FGF.47 This was also demonstrated with pure vitro, including endothelial cells (as previously discussed),
forms of FGF-148 and FGF-2.49 The use of heparin affinity fibroblasts, smooth muscle cells, and neuroendocrine cells.
purification of FGFs allowed for a clearer understanding of
its biological activities. These initial studies showed that the Fibroblasts
binding of FGF-1 and FGF-2 to heparin were different, al- During tissue repair fibroblasts function as a source
lowing a means to differentiate between the two. Heparin of collagen and matrix that participate in the formation of
Fig. 28.1. Mechanism of FGF-1 secretion.
In response to a variety of stresses, FGF-1
is released as a homodimer. Reducing
agents (RSH) such as glutathione are able FGF-1 mRNA
to reduce the FGF-1 dimer, enabling it to FGF-1
interact with the heparan sulfate FGF-1 Signal
proteoglycan (HSPG). Once associated Stress
with HSPG at the cell surface, FGF-1 is
able to signal a biological response
FGF-1 dimer
through the FGF receptor (FGFR-1). (Re-
produced with permission from ref. 37)
RSH FGFR-1
HSPG
FGF-1 FGF-1
dimer monomer
Fibroblast Growth Factors in Angiogenesis and Tissue Engineering 305
granulation tissue. FGF is chemotactic for fibroblasts. It also Since angiogenesis plays such an important role in
acts as a negative regulator of collagen metabolism as dem- tumor biology, it has been postulated that the inhibition of
onstrated by a decrease in collagen production in cultured angiogenic growth factors may ultimately affect tumor
fibroblasts. growth and overall outcome. A number of different tech-
niques have been utilized to inhibit the effects of FGF. One
Smooth Muscle Cells particular model uses a monoclonal antibody against FGF-2
FGF-2 has been found to stimulate vascular smooth and successfully suppressed solid tumor growth.62 A differ-
muscle cell proliferation. Amplification of DNA synthesis ent approach to inhibit FGF is based on molecular targeting
occurs within the smooth muscle cell due to autocrine ef- of the gene transcripts. For instance, the use of antisense
fects of the FGF.55 Smooth muscle cell proliferation is also oligodeoxynucleotides will inhibit translation and reduce the
stimulated by FGF-1, but only in the presence of heparin (at stability of targeted RNA. For FGF-2 this method has been
physiologically relevant concentrations of FGF-1). shown to inhibit primary melanoma proliferation in tissue
culture.63
Neuroendocrine Cells
Both FGF-1 and FGF-2 are widely distributed Wound Healing
throughout the central nervous system. It has been found Wound healing consists of a series of complex pro-
that FGF has an effect on glial cells, playing a role in the cesses comprising inflammation, neovascularization, granu-
modulation of cell proliferation.56 lation tissue formation and matrix remodeling, which pro-
ceed in an overlapping manner. Exogenously applied FGF-2
In Vivo has been known to induce potent angiogenesis and granu-
The biological effects of FGF in vivo have been stud- lation tissue formation, and to result in stimulation of wound
ied in both natural and pathological processes such as an- healing in animal models. Localized immunostaining with
giogenesis (as previously described), intimal hyperplasia, antibodies shows increased FGF-2 levels in burn wounds.64
neoplasia, and wound healing. Using a variety of “healing-impaired” mice (such as obese,
diabetic, steroid treated, malnourished, etc.) the effects of
Intimal Hyperplasia FGF were observed. Each mouse sustained an injury (such
FGF has been found to be a smooth muscle cell mito- as a burn wound, decubitus ulcer, excisional wound, and so
gen and may contribute to the initial smooth muscle cell forth) and each wound was covered with an occlusive dress-
proliferation, playing a critical role in the development of ing, some with the application of FGF-2. The wound heal-
intimal hyperplasia. When rat arteries are injured to induce ing rate was monitored. It was found that the FGF-2 treat-
intimal hyperplasia, the response is limited when antibod- ment resulted in an acceleration of wound healing in every
ies to FGF-2 are applied, causing a significant reduction in condition.65 FGF has been found to have two major contri-
the smooth muscle cell response.57 By encoding porcine ar- butions to wound healing: It facilitates neovascularization
terial endothelial cells with a FGF-1 gene, increased intimal and causes a decrease in collagen production, ultimately re-
thickening was demonstrated after 3 weeks as compared to sulting in tissue with a reduced tensile strength.66
a control group.58 Finding ways to inhibit the smooth muscle
cell response to FGF while concomitantly stimulating en- Receptors
dothelial cell progression has been a major area of vascular Early studies suggested that FGF-1 and FGF-2 were
research and tissue engineering. bound by the same receptor.67 But later experiments revealed
that both high and low affinity binding sites exist.
Neoplasia
Tumor angiogenesis has properties somewhat differ- High Affinity Receptors
ent from normal angiogenesis. The fast growth of tumor The high affinity receptors for FGF-1 and FGF-2 have
cells requires that angiogenesis takes place at a high rate in been located on a wide range of cell types such as endothe-
order to keep up with the needs of an expanding tumor. lial cells, hepatoma cells, smooth muscle cells, fibroblasts,
FGF-1 and FGF-2 are found to be associated with the ma- smooth muscle cells, retinal cells, chondrocytes, hepatocytes,
jority of adult tumors (for instance, breast, lung, prostate myoblasts, and various tumor cells.68 The number of sites
cancer, renal cell and bladder carcinoma, brain tumors, hepa- per cell varies from 2,000 to over 100,000 and the molecular
tocellular carcinoma, melanoma, Kaposi’s sarcoma and oth- weight ranges from 105-165 kDa, depending on the type of
ers). A study of FGF localization in gastrointestinal tumors cell studied. The majority of FGF receptors are utilized by
found FGF-2 in the extracellular matrix. Cellular staining both FGF-1 and FGF-2. However, there is some heterogene-
also revealed FGF to be present in fibroblasts and endothe- ity among receptors, dependent on varying degrees of
lial cells of the host.59 There is a reported correlation be- glycosylation.
tween blood vessel density versus metastatic lesions in breast, The varying FGF receptor isoforms are produced by
lung, prostate, and head and neck cancers.60 This correla- the same gene. Since 1989 there have been four FGF recep-
tion was tested by injecting mice with anti-FGF antibodies, tor genes identified. The nomenclature is varied: FGFR1 (flg,
and a decrease in tumor blood vessel density was observed. bFGR, Cek1, N-bFGFR, h2, h3, h4, h5), FGFR2 (bek, Cek3,
FGF is even detectable at significant levels in the serum and K-sam, TK 14, TK 25, KGFR), FGFR3 (Cek2), and FGFR4.
urine of cancer patients.61 The FGF receptor genes share a large sequence homology
306 Tissue Engineering of Prosthetic Vascular Grafts
with each other. The gene structure reveals two mechanisms The low affinity receptor present on the cell surface
for the formation of the receptor isoforms: alternative mRNA shows many similarities to heparin. In fact, when cells are
splicing, resulting in deletions or alternate exon usage; and incubated with heparin, the interaction of FGF-2 and this
internal polyadenylation, resulting in truncated products.23 receptor is inhibited.69 Cloning of this receptor demonstrated
Thus the FGF receptor system is redundantly specific. That a cell-surface heparan proteoglycan. Heparan sulfates are
is, one receptor may bind with several different FGFs or, N-sulfated polysaccharide components of proteoglycans. It
alternatively, one FGF may interact with a number of is synthesized by most vertebrate cells, as compared to hep-
varying receptors. arin which is an exclusive product of connective tissue mast
The structure of the high affinity receptor, a trans- cells. Heparan sulfate and heparin synthesis occur within
membrane glycoprotein, is divided into an extracelluar and the Golgi complex, beginning with the nonsulfated precur-
intracellular component. The extracellular portion contains sor heparan. This precursor is transformed to heparan sul-
three Ig-like domains which vary in expression by alterna- fate or heparin by a series of polymer modifications. This
tive splicing; this area of the receptor is the least conserved results in heparin containing a significant higher concen-
region. The first Ig domain is not essential for the high af- tration of sulfate groups as compared to heparan sulfate.
finity binding of FGF-1 or FGF-2. There is also a signal pep- Heparan sulfates are mainly present on cell surfaces and in
tide and acid box region in the extracellular portion of the the extracellular matrix as proteoglycans. It is well estab-
receptor. The intracellular component has two tyrosine ki- lished that the heparan sulfates bind to a wide variety of
nase domains which are the most highly conserved regions cytokines and specifically to FGF-1 and FGF-2.70
(Fig. 28.2). The low affinity receptor is now known to be syndecan.
Syndecan is a proteoglycan initially cloned from mouse. It
Low Affinity Receptors is a polymorphic molecule with heparan and chondoitin
The low affinity receptors have been located on sulfate chains associated with a 31 kDa transmembrane
fibroblasts, endothelial cells, epithelial cells, neurons, tumor polypeptide. It is mainly found on the surface of epithelial
cells and in the basement membrane of a variety of tissues. cells from mature tissues such as skin, liver, and breast.68
These binding sites range from 0.5 x 106 to several million Syndecan is a family of proteoglycans (syndecan 1, 2, 3 or 4)
in number. based on four specific core proteins. They can interact with
Fig. 28.2. Structure of an FGF receptor (Reproduced with
permission from ref. 42)
lg loop I
acid box
lg loop II
lg loop III
transmembrane domain
tyrosine kinase domain
Fibroblast Growth Factors in Angiogenesis and Tissue Engineering 307
varying components of the matrix like fibronectin and col- Signal Transduction
lagen as well as FGF-2.72 FGF signaling immediately follows the binding of FGF
The amino acid sequence of the core protein contains to its receptor. Heparin is required for this interaction to
a transmembrane domain with an extracellular region con- occur. Although the exact mechanism is unclear, it is thought
taining six potential attachment sites for glycosaminogly- that heparin helps stabilize the FGF-FGF receptor complex.
can side chains.71 The receptor has been characterized to The binding leads to FGF receptor dimerization,
two major components: a proteoglycan of 250 kDa which autophosphorylation and an increase in the tyrosine kinase
appears in the plasma membrane and an 800 kDa which is activity. This initiates a responding cascade of events with
located in the extracellular matrix. It is the extracellular com- activation of a variety of signaling molecules, ultimately re-
ponent that can vary, allowing for diversity.72 sulting in transcription of genes necessary for the FGF re-
It is suggested that the low affinity receptor is an ac- sponse76 (Fig. 28.3).
cessory molecule required for the binding of FGF to the high
affinity receptor. Using cells deficient in heparan sulfates, it FGF and Vascular Grafts
was found that FGF-2 could not bind to its high affinity re- The major cause of failure of vascular grafts is 2-fold:
ceptor.73 Similarly, treatment of skeletal muscle or adrenal Early on it is due to thrombosis and later the primary cause
cells with heparinase demonstrated FGF’s inability to inter- is myointimal hyperplasia. The interface between the blood
act with its high affinity receptor, with a decrease in cell pro- and graft wall is where these pathological processes occur.
liferation.74,75 All of this data implicates the low affinity FGF In 1978 Herring developed the concept that endothe-
receptor as an accessory molecule, required for the lial cells transplanted onto a graft surface could prolong the
complexing of FGF in a biologically active form to its high graft’s survival.77 The presence of a confluent monolayer of
affinity receptor. endothelial cells could theoretically improve graft thrombo-
Fig. 28.3. Schematic presentation of intracellular signaling mediated by the FGF receptor (FGFR). The FGFR is activated upon
binding FGF and associated heparan sulfate proteoglycan (HSPG). The FGFR undergoes autophosphorylation on tyrosine resi-
dues. A signal transduction cascade results in the activation of various proteins, including the F-actin-binding protein cortactin.
Further propagation of the signaling cascade induces the activity of MAP kinase (MAPK), which phosphorylates transcription
factors (TF), activating the genes necessary for the FGF response. (Reproduced with permission from ref. 37)
308 Tissue Engineering of Prosthetic Vascular Grafts
resistance and prevent the development of intimal hyper- inhibit smooth muscle cells.89,90 Additional in vitro studies
plasia. Three sources of endothelial cells seen on a prosthetic have shown that at an optimized FGF-1 to heparin ratio,
vascular lumen have been observed. Endothelial cells may using a high dose of heparin, endothelial cell proliferation
infiltrate from a native artery over the anastomosis, are ob- could selectively be stimulated while smooth muscle cells
served as ingrowth through the interstices originating from are inhibited.91
host perigraft tissue, and may occasionally be circulating. In Others have studied the release of FGF from prosthetic
humans it is rare to see complete endothelialization on a grafts. For instance, Yamamura applied FGF-2 to 0.75 mm
prosthetic graft, even after it has been well incorporated into disks made of ePTFE. Biodegradable hydroxypropylchitosan
the host tissue. Direct seeding of endothelial cells onto pros- acetate (HPCHA) was applied to control the rate of release
thetic grafts has been extensively investigated, but in clinical of FGF from these disks. The disks were implanted into rab-
trials the benefits have been modest.78-81 One of the diffi- bit skin pockets and harvested over 24 hours. The disks
culties with this technique is the relatively low cell density treated with HPCHA and FGF released only 60% of the
initially applied to the graft and inadequate cell attachment. FGF-2, while disks treated with FGF-2 alone released all of
The use of specific endothelial cell chemoattractants and the cytokine in a 24 hour period. It was concluded that
mitogens, such as FGF, can theoretically stimulate HPCHA allowed the slow, reliable release of FGF from pros-
transinterstitial capillary ingrowth, resulting in enhanced thetic material.92 In another experiment, bone marrow cells
endothelialization of the graft surface. were applied to ePTFE vascular grafts and implanted into
Work by Greisler’s group has evaluated the applica- the abdominal aorta of dogs. At six months time the grafts
tion of FGF to synthetic surfaces as a means to either en- were still immunohistochemically reactive to FGF-2.93
hance spontaneous endothelialization of that surface or to The following experiments examined the FGF reten-
stimulate the proliferation of seeded endothelial cells. In early tion within injured vessels. Using a balloon injury model,
studies, FGF-1 was applied to both Dacron and [125I]FGF-1 in FG was applied to the carotid arteries of dogs.
polydioxanone (PDS) grafts by means of sequential appli- The result revealed a 41% and 37% retention of FGF-1 after
cation of fibronectin, heparin, FGF-1 and a second layer of 10 and 60 minutes of circulation respectively.94,95 In a simi-
heparin. Using [125I]FGF-1, the retention of the growth fac- lar fashion, the retention of 111Indium-labelled platelets was
tor in vivo was quantitated. One week after the application, quantitated in FG versus untreated identically injured
the retention was 44% in the Dacron grafts and 23% in the carotids. Platelet deposition was a significant 45% less in
PDS grafts.82,83 After the graft was explanted, the FGF-1 was the FG group.95 These studies seem to indicate that the ap-
eluted and shown to have retained its mitogenic activity on plication of FG to arterial surfaces following injury has po-
quiescent lung endothelial cells.83 A study by Tomizawa used tential clinical utility. The incorporation of FGF-1 plus hep-
minced canine adipose tissue that was applied to vascular arin within FG onto synthetic grafts may promote
grafts. These were implanted into the abdominal aorta of neovascularization and prevent pseudointimal hyperplasia,
dogs. When the grafts were removed, endothelial-like cells but this is still under investigation.
extending onto luminal thrombus was observed. These cells One technique that is now being utilized is the use of
were immunohistologically positive for FGF-2.84 “mutant” forms of FGF-1. In other words, by making spe-
Further studies have utilized fibrin glue (FG), a com- cific mutations in the structure of FGF, perhaps a growth
posite of thrombin and fibrinogen, as a controlled local de- factor with a more specific function can be developed. For
livery system for FGF-1 and heparin. Three dimensional example, an extensive characterization of FGF-1 mutants of
analysis of the FGF distribution has revealed that it was even lysine 132 to a glutamic acid or a glycine residue has been
throughout the graft.85 Studies comparing the fibronectin completed.96,97 This altered form of FGF-1 in bone assays
treatment to the FG illustrated greater endothelial cell re- has been shown to induce angiogenesis with a decease in
tention with the latter.86 Expanded polytetrafluoroethylene fibroblast proliferation. These functional characteristics, se-
(ePTFE—60 micron internodal distance) grafts were treated lective stimulation of endothelial cells and inhibiting fibro-
with the FG-FGF-1-heparin combination and implanted in blasts, are qualities that are likely to be beneficial when ap-
dogs as 5 cm aortoiliac grafts and 30 cm thoracoabdominal plied to models of vascular healing.
aortic grafts. When compared to control groups, the pre- To determine the relative percent coverage of seeded
treated grafts resulted in a significant increase in endothe- versus spontaneously ingrowing endothelial cells on FG
lial cell proliferation as assayed by en face autoradiography. treated surfaces, a method of fluorescent labeling with
There was also extensive transinterstitial capillary ingrowth PKH-26 has been developed. Proliferation assays in vitro
observed throughout the graft wall.87,88 Cross-sectional au- documented virtually identical growth rates for labeled ver-
toradiography showed a similar increase in subendothelial sus unlabeled canine jugular vein endothelial cells. There is
myofibroblast proliferation in the pretreated grafts at one nearly a linear relationship between the concentration of
month. Analysis at 140 days revealed the FG-FGF-1-hep- applied PKH-26 (from 0-10 ∝M of PKH-26) and the mean
arin grafts had developed a significantly thicker inner cap- intensity of fluorescence of labeled endothelial cells. Addi-
sule (139 microns versus <95 microns in the control groups) tionally, no PKH-26 induced toxicity or alterations in growth
consisting of myofibroblasts and collagen.88 kinetics were observed using concentrations of PKH-26 up
Although heparin and FGF-1 act synergistically to to 10 ∝M (although cellular toxicity was detected at 20 ∝M
stimulate smooth muscle cell and fibroblast proliferation, concentrations). Labeled endothelial cells are visualized on
in the absence of FGF-1, heparin alone has been shown to both Dacron and ePTFE surfaces under fluorescent illumi-
Fibroblast Growth Factors in Angiogenesis and Tissue Engineering 309
nation.98 This cell labeling technique is now being applied collateral vessels were found to be augmented.107 In a simi-
to study the effects on balloon injured carotid arteries treated lar fashion, FGF-1 was administered after the ischemia was
with FG plus FGF-1 and heparin and PKH-26 labeled au- allowed to subside for 10 days. Even after 30 days there was
tologous endothelial cells. evidence of angiogenesis and increased collateral blood flow
in the rabbit hindlimb.108 This method of investigation has
FGF and Ischemia been applied to other angiogenic growth factors such as
Compromised vascular circulation is an important VEGF109 (see chapter 26 for a full review).
component in many disease processes such as coronary is- The interest in FGF and ischemic disease has broad-
chemia, congestive heart failure, and peripheral vascular in- ened, and research is now being done on brain infarcts
sufficiency. In these conditions there is insufficient capillary (strokes)110,111 and trauma injuries.112
flow, which contributes to the extent of the damage to the The therapeutic potential of angiogenic cytokines re-
tissue. It is thought that increasing the number of capillar- mains to be fully defined, but clearly FGF plays an impor-
ies, and ultimately the oxygen supply, in these underperfused tant role in the development of collateral neovascularization
tissue beds may provide a palliative effect, thus limiting ne- in ischemic disease states.
crosis or loss of function. Angiogenesis has been investigated
in terms of its relation to the development of collateral blood Conclusions
flow. Studies examining canine and swine coronary circula- Over the past 15 years our knowledge and understand-
tions have established that cellular proliferation is a feature ing of angiogenic cytokines, FGF in particular, has made
of collateral vessel development that occurs as a result of great strides. Current vascular research is focused on more
arterial occlusion.99,100 By using angiogenic growth factors, bio-interactive grafts that optimize the microenvironment
such as FGF, collateral artery development has been of the tissue-graft-blood interfaces. The application of en-
augmented. dothelial cells and growth factors to synthetic grafts to in-
Since cardiac disease is a major cause of morbidity duce angiogenesis and limit the smooth muscle cell response
and mortality, the effects of FGF on myocardial are some of the most promising areas of research.
neovascularization have been studied extensively. When rab-
bits with chronic myocardial ischemia were subjected to References
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HP. Optimizing fluorescent labeling of endothelial cells
Facilitation of Healing
Matrix Degradation
CHAPTER 29
Role of Urokinase-Type Plasminogen Activator (uPA)
in In Vitro Angiogenesis in Fibrin Matrices
Pieter Koolwijk, Victor W.M. van Hinsbergh
Summary
T issue repair-associated angiogenesis, the formation of new blood vessels from existing
ones, usually involves cell invasion into a fibrin structure and the presence of inflamma-
tory cells. In this chapter the role of plasminogen activators and their receptors in the inva-
sion of endothelial cells into a fibrin matrix is described. At the basolateral side of the endo-
thelial cell, the urokinase-type plasminogen activator (uPA) bound to a specific cellular
receptor (uPA receptor) is involved in the proteolytic modulation of matrix proteins and
cell-matrix interaction. Interference in the activity of uPA and the binding of uPA to the
uPA receptor inhibits the in vitro invasion of human microvascular endothelial cells
(HMVEC) and the formation of capillary-like tubular structures of HMVEC in three di-
mensional fibrin matrices, induced by a combination of the cytokine tumor necrosis factor-#
(TNF-#) and the angiogenic factors basic fibroblast growth factor (bFGF) and vascular en-
dothelial growth factor (VEGF).
Introduction
Angiogenesis, the outgrowth of new blood vessels from existing ones, is an essential
process during development, but normally stops when the body becomes adult. In the ab-
sence of injury, overt angiogenesis in adults is limited to the reproductive system of females
(formation of corpus luteum and placenta).1 However, the formation of new blood vessels is
an essential factor in tissue repair (formation and regression of granulation tissue) which is
necessary to restore healthy tissue after wounding and/or inflammation. Furthermore, an-
giogenesis is associated with many pathological conditions, such as chronic inflammation
including rheumatoid arthritis,2 malignancies,3 and retinopathy caused by metabolic
dysregulation in particular diabetes.4 Common in these conditions is that angiogenesis is
accompanied by vascular leakage,5 the occurrence of inflammatory cells,6 and the presence
of fibrin.7,8 These latter factors are absent in angiogenesis during embryonic development.
Therefore, the possibility exists that “developmental angiogenesis” and adult “repair-associ-
ated or pathologic angiogenesis” are two processes with many identical features, but with
different properties with respect to their regulation.
Fibrin, a Temporary Repair Matrix
Fibrin is a temporary matrix which is formed after wounding of a blood vessel and
when plasma leaks from blood vessels forming a fibrous exudate. The fibrin matrix not only
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
314 Tissue Engineering of Prosthetic Vascular Grafts
acts as a barrier preventing further blood loss, but also pro- Inhibitors
vides a structure in which new microvessels can infiltrate The activities of the proteases of the fibrinolytic sys-
during wound healing. Proper timing of the outgrowth of tem are controlled by potent inhibitors which are members
microvessels as well as the subsequent (partial) disappear- of the serine protease inhibitor (serpin) superfamily. Plas-
ance of these vessels is essential to ensure adequate wound min, if not bound to fibrin, is instantaneously inhibited by
12
healing and to prevent the formation of scar tissue. An im- #2-antiplasmin. Because this interaction is facilitated by
portant role in the invasion of a fibrin matrix by endothelial the lysine binding domain of plasmin, it is attenuated when
cells is played by the plasminogen activator/plasmin system, plasmin is bound to fibrin. The predominant regulators of
which lyses fibrin at the basolateral side of the cell. How- tPA and uPA activities are PAI-1 and PAI-2.13,14 PAI-1 is a
ever, the endothelium must respond differently to fibrin 50 kDa glycoprotein present in blood platelets and synthe-
depending on whether the fibrin is present at its luminal or sized by endothelial cells, smooth muscle cells and many
abluminal side. If fibrin is generated at the luminal side, the other cell types in culture.15,16 PAI activity in human plasma
vessel may be occluded, which will cause serious damage of is normally exclusively PAI-1. PAI-1 binds to vitronectin,
the distal tissues. To prevent this the endothelium is able to which stabilizes its inhibitory activity. PAI-1 is the main if
instantaneously increase the fibrinolytic activity in the blood not the sole inhibitor of PAs synthesized by endothelial cells,
compartment. If, however, this would lead to strong systemic vascular smooth muscle cells and hepatocytes, whereas PAI-2
fibrinolytic activity, recurrent bleeding might occur. There- is produced by monocytes/macrophages, placental tropho-
fore, fibrinolysis must be restricted to a limited distance of blasts and certain tumor cell lines.14 It can be found as a
the endothelium. To fulfill the roles of fibrinolysis in both glycosylated secreted molecule and as an nonglycosylated
the prevention of local intravascular fibrin accumulation, molecule intracellularly.17
and a differently timed contribution in neovascularization
and tissue repair, endothelial cells are equipped with a com- Receptors
plex regulatory system which involves inhibitors, cell polar- Regulation of fibrinolytic activity also occurs by cel-
ity and cellular receptors. After discussing the components lular receptors. These receptors direct the action of PAs and
of the plasminogen activator/plasmin system, we will focus plasmin to focal areas on the cell surface, or are involved in
on the contribution of the urokinase-type plasminogen ac- the clearance of the PAs. High affinity binding sites for plas-
tivator (uPA) in pericellular proteolytic events associated minogen,18-21 tPA21,22 and uPA23-25 are found on various
with cell migration and the invasion of capillary-like tubu- types of cells, including endothelial cells.
lar structures into fibrin matrices. Endothelial cells in vitro bind plasminogen with a
moderate affinity (120-340 nM depending on whether the
Components of the Plasmin/Plasminogen Lys or Glu form of plasminogen is used), but with a high
Activator System capacity (3.9-14 x 105 molecules per cell).19,26 This binding,
which is also observed with many other cell types, is medi-
Proteases ated by the lysine binding sites of kringles 1-3 of the
Plasmin is formed from its zymogen plasminogen by plasmin(ogen) molecule. Because these lysine binding sites
proteolytic activation by plasminogen activators (PAs) are also involved in the interaction of plasmin with
(Fig. 29.1). Two types of mammalian PAs are presently #2-antiplasmin, occuPAtion of lysine binding sites protects
known: tissue-type plasminogen activator (tPA) and uroki- plasmin from instantaneous inhibition by #2-antiplasmin
nase-type plasminogen activator (uPA).9,10 The three serine not only when plasmin is bound to fibrin (see above), but
proteases, plasminogen, tPA and uPA, are synthesized as also when it is bound to the cellular receptors. The nature of
single polypeptide chains, and each of them is converted by the plasminogen receptors is not fully resolved. In addition
specific proteolytic cleavage to a molecule with two polypep- to gangliosides, which directly or indirectly contribute to
tide chains connected by a disulphide bond. The C-termi- the plasminogen binding,27 at least eight proteins have been
nal part of the molecule (the so-called B chain) contains the reported to be involved in plasminogen binding. Among
proteolytically active site, whereas the amino-terminal part them are members of the low density lipoprotein receptor
of the molecule (the A-chain) is built up of domains that family, such as gp330 and LRP; annexin II; a not yet identi-
determine the interaction of the proteases with matrix pro- fied 45 kDa protein; GbIIb/IIIa and #-enolase (see ref. 21
teins and cellular receptors. The proteolytic cleavage of plas- for review). In neural cells plasminogen binding to
minogen and single chain uPA to their respective two chain amphoterin was found. Lipoprotein LpA, which has strong
forms is necessary to disclose the proteolytically active site structural homology with a large part of the plasminogen
and to activate the molecule. The interaction of plasmino- molecule, can compete for plasminogen binding to endo-
gen with fibrin or the cell surface occurs predominantly via thelial cells.20,28 This competition also involves lysine bind-
binding sites in the kringle structures, which recognize lysine ing sites.
residues of proteins, in particular C-terminal lysines. Because Specific binding of tPA to human endothelial cells has
B-type carboxypeptidases remove C-terminal lysine residues been reported.29-31 tPA binds via its growth factor domain
from potential binding sites for plasminogen in fibrin or on with high affinity to annexin II on human endothelial cells
the cell surface, they can act as negative regulators of the in culture.32 Different epitopes of the annexin II molecule
fibrinolytic system.11 are involved in plasminogen and tPA binding. It is conceiv-
able that annexin II, like fibrin, forms a ternary complex
Role of Urokinase-Type Plasminogen Activator (uPA) in In Vitro Angiogenesis in Fibrin Matrices 315
Fig. 29.1. Scheme of the plasminogen activation system. +: activation; -: inhibition. Inhibitors are indicated in italics. PA: plasmi-
nogen activator; tPA: tissue-type PA; tcuPA: two chain urokinase-type PA; scuPA: single chain urokinase-type PA; PAI: PA inhibitor.
with tPA and plasminogen on the endothelial cell surface.21 binds to the uPA receptor and is subsequently converted to
In addition, tPA interacts with matrix-bound PAI-1.31 the proteolytically active two chain uPA. Since the endothe-
Binding of uPA with the cell surface limits plasmino- lial cell also contains plasmin(ogen) receptors, an interplay
gen activation to focal areas such as the focal attachment between receptor-bound uPA and receptor-bound
sites and cellular protrusions involved in cell migration and plasmin(ogen) and plasmin formation is likely to happen.
invasion. Furthermore, uPA interaction with the cell evokes The generated plasmin can degrade a number of matrix pro-
signal transduction and phosphorylation of several pro- teins. In addition, a direct plasmin-independent proteolytic
teins.33,34 A specific uPA receptor has been identified and action of uPA on matrix proteins may also occur.41 Like free
cloned. It is present on many cell types, including endothe- uPA activity, receptor-bound two chain uPA is subject to
lial cells.35 It is a glycosyl phosphatidyl inositol (GPI)-an- inhibition by PAI-1. As a consequence, uPA is only active
chored glycoprotein,25 which binds both single chain uPA over a short period of time. In contrast to receptor-bound
and two chain uPA via their growth factor domains.36 The single chain or noninhibited two chain uPA, the uPA-PAI-1
uPA receptor is heavily glycosylated and belongs to the cys- complex is rapidly internalized together with the uPA re-
teine-rich cell surface proteins. After synthesis it is proteolyti- ceptor,42 followed by the degradation of the uPA-PAI-1 com-
cally processed at its carboxyl-terminus and subsequently plex and the return of the empty uPA receptor to the plasma
anchored in the plasma membrane by a GPI group.37 It com- membrane. Internalization of the GPI-linked uPA receptor
prises three domains, which are structurally homologous to probably occurs after interaction with another receptor, such
snake venom #-toxins.25 The uPA receptor has been found as the #2-macroglobulin/low density lipoprotein receptor-
in focal attachment sites, where integrin-matrix interactions related protein (LRP)43 or the VLDL receptor.44 VLDL re-
occur, and in cell-cell contact areas.38,39 Human endothelial ceptors were demonstrated on capillary and arteriolar en-
cells in vitro contain about 140,000 uPA receptors per cell.40 dothelial cells in vivo.45 Recently it has been found that the
The uPA receptor both acts as a site for focal pericel- uPA receptor may have an additional role. The uPA receptor
lular proteolysis by uPA and is involved in the clearance of occupied by uPA interacts avidly with vitronectin.46 Hence,
the uPA-PAI-1 complex. Upon secretion, single chain uPA cell adhesion may represent an additional function of the
uPA receptor.
316 Tissue Engineering of Prosthetic Vascular Grafts
The LRP and LRP-like proteins on liver hepatocytes blast growth factor (bFGF, aFGF) and vascular endothelial
are involved in the clearance of plasmin-#2-antiplasmin, PA- growth factor (VEGF).65-67 The number of uPA receptors
PAI-1 complexes47-49 and probably free PAs from the circu- on human and bovine endothelial cells is also enhanced by
lation. In addition, tPA can also be cleared by mannose re- the activation of protein kinase c and by the elevation of the
ceptors present on macrophages and liver endothelial cells50 cellular cAMP concentration.68,69 Preliminary experiments
and by #-fucose receptors on hepatocytes.32 in our laboratory have demonstrated that the induction of
uPA receptor by VEGF in human endothelial cells is inhib-
The Regulation of Plasminogen Activation ited by protein kinase c inhibition. The effects of bFGF and
and Pericellular Proteolysis by Inflammatory VEGF on the induction of uPA receptor in human endothe-
and Angiogenic Mediators lial cells are regulated independently of their effects on cell
proliferation. Similarly, Presta et al70 have shown that the
Regulation of Proteases And Inhibitors induction of uPA by bFGF in bovine endothelial cells pro-
The primary cytokines interleukin-1 (IL-1) and tu- ceeds independently from the stimulation of mitogenesis by
mor necrosis factor-# (TNF-#) exert many effects on the this growth factor.
vascular endothelium. Their most prominent feature is the In addition to FGFs and VEGF, which induce mitoge-
induction or increase of the transcription of many genes, nesis, TNF-# also can induce angiogenesis, but this occurs
such as the leukocyte adhesion molecules E-selectin, without stimulation of cell proliferation.71,72 TNF-# increases
VCAM-1 and ICAM-1, cyclooxygenase-2, and a number of uPA receptor levels in human microvascular endothelial
proteases and protease inhibitors. The inflammatory me- cells65 and in monocytes,73 but not in endothelial cells from
diators TNF-#, IL-1 and the bacterial lipopolysaccharide human umbilical vein or aorta.65 However, simultaneous
(LPS) induce the synthesis of uPA in human endothelial cells exposure of the latter cells to TNF-# (which induces uPA
in vitro.51 Whereas uPA is normally not found in endothe- synthesis) to bFGF and VEGF (which enhance the expres-
lial cells in vivo, association of uPA with the endothelium sion of uPA receptors) potently increases cell-bound
was observed in acute appendicitis52 and in rheumatoid ar- uPA activity.
thritis.2 Induction of endothelial uPA by TNF-# in vitro is
associated by an increased degradation of matrix proteins.53 Interaction Between the uPA/Plasmin System
The enhanced secretion of uPA occurs entirely towards the and Matrix-Degrading Metalloproteases
basolateral side of the cell, whereas the secretion of tPA and The observation that TNF-# also increases the pro-
PAI-1 proceeds equally to the luminal and basolateral sides duction of matrix-degrading metalloproteinases (MMPs) by
of the cell.51 The polar secretion of uPA suggests that uPA endothelial cells is consistent with a putative role for TNF-#
may be involved in local remodeling of the basal membrane and IL-1 in inflammation-induced local pericellular pro-
of the cell. uPA activity is controlled in space by interaction teolysis. In human microvascular and vein endothelial cells,
of uPA with its cellular receptor and by the inhibitor PAI-1. TNF-# increases the mRNA levels and the synthesis of in-
TNF-# and IL-1, as well as LPS, also elicit another ef- terstitial collagenase (MMP-1), stromelysin-1 (MMP-3)
fect on the regulation of plasminogen activator production and—if protein kinase c is also activated—gelatinase-B
in endothelial cells. Simultaneous with the increase in uPA, (MMP-9), whereas the mRNAs levels and synthesis of their
these inflammatory mediators markedly increase the pro- physiological inhibitors TIMP-1 and TIMP-2 are not
duction of PAI-1 in endothelial cells in vitro.54-57 This in- changed. 74,75 Furthermore, activation of gelatinase a
duction was also demonstrated at the transcriptional level, (MMP-2) was observed after exposure of the cells to
and was largely inhibited by the isoflavone compound TNF-#.74 Recently, it has been shown that activation of
genistein.58 In vivo, administration of TNF-#, IL-1 or LPS gelatinase a depends on the activity of membrane-type MMP
causes an increase in PAI-1 concentration in the circulation. (MT-MMP), 76,77 and that MMP-2 interacts with #v!3
After infusion of LPS in animals, PAI-1 mRNA increased in integrin.78 Interestingly, secretion of gelatinases by bovine
vascularized tissues and PAI-1 mRNA was elevated in the endothelial cells occurs predominantly towards the
endothelium of various organs.59,60 The increase of PAI-1 basolateral side of the cells,79 similarly to the TNF-#-induced
induced by inflammatory mediators may represent a pro- production of uPA.51 A role of MMPs in endothelial cell-
tective mechanism of the cell against uncontrolled uPA matrix remodeling has indeed been shown in a three dimen-
activity. sional collagen matrix in vitro.80 Furthermore, MMPs have
The effect of the angiogenic growth factors basic and been detected in vivo in proliferating endothelial cells dur-
acidic fibroblast growth factor (bFGF, aFGF) and vascular ing development,81 and in endothelial cells present in ath-
endothelial growth factor (VEGF) on the production of uPA erosclerotic plaques and growing tumors.82-84
by endothelial cells is species dependent. Whereas bFGF and The plasmin-plasminogen activator system and the
VEGF are potent inducers of both uPA in bovine cells,61,62,63 matrix metalloproteinases cooperate in the degradation of
bFGF and VEGF do not enhance uPA production in human extracellular matrix proteins.85 Figure 29.2 depicts the in-
endothelial cells.64,65 PAI-1 production by human endothelial teraction between the two systems. It should be noted, how-
cells is slightly decreased by the addition of bFGF or VEGF.65 ever, that this schematic picture is based on in vitro data,
and that it is still uncertain whether all the depicted steps
Regulation of the uPA Receptor also act in vivo. Nevertheless, uPA and MMP expression
The expression of the uPA receptor is enhanced by frequently coincide in time and location in pathological
angiogenic growth factors including basic and acidic fibro- tissues. If these data are taken together, it will be clear that
Role of Urokinase-Type Plasminogen Activator (uPA) in In Vitro Angiogenesis in Fibrin Matrices 317
activation of endothelial cells by TNF-# affects multiple sites tPA.94,95 These data suggest that uPA and plasminogen have
in the proteolytic cascades involved in the degradation of a role in cell recruitment.
matrix proteins to such an extent that it markedly enhances An involvement of uPA and the uPA receptor in the
the breakdown and remodeling of the endothelial cell migration of bovine endothelial cells has been demonstrated
basal membrane. by several investigators.63,96,97 After the wounding of a mono-
layer of these endothelial cells, the cells that migrate into the
Involvement of uPA and uPA Receptor wounded area express uPA activity96 bound to the uPA re-
in Cell Migration and Realignment ceptor.63 The migration and expression of uPA depends on
of Endothelial Cells the release of bFGF from the wounded area.97 bFGF is a
Concentration of uPA activity at the cellular protru- potent inducer of plasminogen activator activity, in particu-
sions of migrating or invading cells has been frequently ob- lar uPA, in bovine endothelial cells,61,98 but not in human
served.86,87 Blasi88 suggested that a continuous activation and endothelial cells.65 In both species bFGF increases the num-
removal of uPA bound to the receptor could contribute to ber of uPA receptors on endothelial cells.63,65,66
the formation and detachment of focal attachment sites and Receptor-bound uPA was demonstrated in focal ad-
hence to locomotion of the cell. Indeed, migrating and in- hesion sites of fibroblasts89 and endothelial cells.39 It may
vading cells, such as monocytes and tumor cells, express uPA act proteolytically on these structures and hence influence
activity bound to uPA receptors on their cellular protru- cell-matrix interactions and cell migration. After exposure
sions87 and on focal attachment sites.38,89 Receptor-bound of endothelial cells to shear forces induced by fluid flow, the
uPA activity is also thought to be involved in smooth muscle cells realign according to the direction of flow.99 This pro-
and endothelial cell migration and in the formation of new cess is paralleled by a movement of the cellular focal contact
blood vessels (angiogenesis). Inhibition of plasminogen ac- sites of endothelial cells.100 Ponfoort et al101 observed that
tivation interferes with smooth muscle cell migration in the production of uPA by human iliac vein endothelial cells
vitro90,91 and affects smooth muscle migration and prolif- was elevated several fold after exposure of the cells to fluid
eration in vivo.92 In mice lacking uPA, intimal hyperplasia flow. Interestingly, the realignment of iliac vein endothelial
of injured arteries is less pronounced than in wild type or cells according to the direction of flow appeared to be re-
tPA-deficient mice.93 Moreover, PAI-1-deficient mice show lated to cell-bound uPA activity. Anti-uPA antibodies, which
an exacerbated intimal proliferation.93 Animals made defi- inhibited uPA activity, reduced the cellular realignment in-
cient for plasminogen show comparable pathological fea- duced by fluid shear forces.101
tures to those with a combined deficiency of uPA and
Fig. 29.2. Schematic representa-
tion of the interaction between
the uPA/plasmin system and the
presumed activation of matrix
degrading metalloproteinases
(MMPs). Abbreviations: PA:
plasminogen activator; uPA:
urokinase-type PA; sc-uPA:
single chain uPA; tc-uPA: two
chain uPA; uPAR: uPA receptor;
Plg: plasminogen; Plg-R: Plg
receptor; PAI-1: PA inhibitor-1;
MT-MMP: membrane-bound
MMP; TIMP: tissue inhibitor of
MMP. +: stimulation; -:
inhibition.
318 Tissue Engineering of Prosthetic Vascular Grafts
Role of the Plasmin/Plasminogen Activator cells, and thus inhibits PA activity.61 In addition to bFGF,
System in the Formation of Endothelial Tubes VEGF can also stimulate bovine endothelial cells to form
in a Fibrin Matrix tubular structures. It acts cooperatively with bFGF in
The outgrowth of new blood vessels from existing this induction.113
ones, angiogenesis, is an essential process during develop- Studies in human endothelial cells showed that no
ment, but normally stops when the body becomes adult. The tubular structures are formed when a quiescent monolayer
half life of endothelial cells in the adult body varies between of human microvascular endothelial cells grown on a fibrin
100-10,000 days in normal tissues, whereas it is reduced to matrix is exposed to bFGF or VEGF. However, when bFGF
several days in placenta and tumors.102 With the exception or VEGF are added simultaneously with TNF-#, a large num-
of the female reproductive system, angiogenesis in the adult ber of capillary-like tubular structures are formed
is associated with tissue repair after injury by wounding or (Fig. 29.3).65 The outgrowth of tubular structures requires
inflammation. Repair-associated angiogenesis in the adult uPA activity and is completely reduced by anti-uPA immu-
is usually accompanied by the presence of fibrin and inflam- noglobulins but not by anti-tPA antibodies (Fig. 29.4). It is
matory cells or mediators, in contrast to developmental an- also reduced by inhibiting the interaction of uPA with its
giogenesis in the embryo. Fibrin is a temporary matrix, which receptor. Furthermore, proteolytic activation of plasmino-
is formed after the wounding of a blood vessel and when gen appears to be involved, because the plasmin inhibitor
plasma leaks from blood vessels forming a fibrous exudate, aprotinin largely inhibits the formation of tubular struc-
often seen in areas of inflammation and in tumors.7,103 The tures.65 Recent immunohistochemical studies performed on
fibrin matrix acts as a barrier preventing further blood loss, cross sections of these invading capillary-like tubular struc-
and provides a structure in which new microvessels can in- tures showed an enhanced expression of both uPA and the
filtrate during wound healing. A proper timing of the out- uPAR antigen by invading endothelial cells compared with
growth of microvessels as well as the subsequent (partial) noninvading endothelial cells (Kroon et al; manuscript sub-
disappearance of these vessels is essential to ensure adequate mitted for publication). These data agree with the data on
wound healing and to prevent the formation of scar tissue. bovine endothelial cells, except that in human endothelial
Although essential for the formation of granulation tissue cells a second mediator is required to induce uPA synthesis.
and tissue repair, angiogenesis, once under the control of In animals bFGF and aFGF have been shown to induce
pathological stimuli, can contribute to a number of patho- neovascularization.114,115 Because it is difficult to rule out
logical conditions, such as tumor neovascularization, pan- the involvement of a limited number of leukocytes in these
nus formation in rheumatoid arthritis, and diabetic retin- experiments in vivo, it remains to be elucidated whether these
opathy. Understanding the mechanisms involved in angio- growth factors act in vivo in conjunction with inflamma-
genesis may provide clues to preventing pathological angio- tory mediators or independently of the latter mediators. Min
genesis without seriously impairing tissue repair. A number et al116 have recently shown that prevention of the binding
of studies and reviews have focused on angiogenic factors of uPA to the uPAR by a fusion product of the epidermal
and the formation of capillary-like structures.4,5,108-109 growth factor-like domain of murine uPA and the Fc por-
Among them, Dvorak7 and Colvin8 have pointed to the im- tion of human IgG reduced bFGF-induced angiogenesis in
portance of fibrin in angiogenesis. Furthermore, the work vivo, and the growth of a B16 melanoma in syngeneic mice,
of Polverini and colleagues have demonstrated the involve- indicating that uPA-mediated angiogenesis also occurs in
ment of monocytes and their products in the induction of these in vivo systems.
angiogenesis.6,71,72,110 Because of the specific roles of fibrin Proteolysis of the basement membrane of endothelial
and inflammatory cells and mediators in pathological an- cells and invasion of endothelial cells into the underlying
giogenesis in the adult, we have focused our studies on the matrix are prerequisites for angiogenesis.117 However, not
invasion of human endothelial cells into three dimensional only proteolysis, but also the formation of new cell attach-
fibrin matrices and the role of endothelial plasminogen ac- ment sites, is important for the formation of tubular struc-
tivators in this process. This model resembles recanalization tures. It should be noted that fibrin contains cell binding
of a fibrin clot by invading endothelial cells. This invasion is domains for endothelial cells: a RGD sequence in its #-chains
usually preceded by the infiltration of inflammatory cells, which binds to the vitronectin receptor, i.e., the #v!3
which interact with the vascular structures from which en- integrin;118,119 and another site in the !-chain (residues
dothelial cells subsequently migrate into the fibrin clot.111 5-42),120 which binds to an 130 kDa receptor.121 Brooks et
A three dimensional fibrin matrix model was used by al122,123 and Hammes et al124 have shown that inhibition of
Pepper and Montesano to demonstrate a direct correlation the #v!3 integrin reduces angiogenesis in several in vivo
between the expression of PA activity and the formation of models. Friedlander et al125 recently reported that both #v!3
capillary sprouts by bovine microvascular endothelial cells and #v!5 integrin are involved in growth factor-stimulated
in vitro.106,112 The outgrowth of tubular structures was in- angiogenesis in the rabbit cornea, but via distinct mecha-
creased by bFGF, which increases both uPA activity and uPA nisms. It is of interest to note that bFGF- and TNF-#-in-
receptor in bovine endothelial cells. Interestingly, the extent duced angiogenesis is inhibited by an antibody against the
of tube formation and the diameter of the formed tubes were #v!3 integrin, whereas angiogenesis induced by VEGF or by
reduced by the simultaneous presence of TGF-!.112 The latter a protein kinase c activating phorbol ester required #v!5
is a growth factor which, amongst others, exerts a strong integrin. It remains to be established whether both #v!3- and
enhancement of PAI-1 synthesis in cultured endothelial #v!5-dependent mechanisms are active in the invasion of
endothelial cells into a fibrin matrix. The involvement of
Role of Urokinase-Type Plasminogen Activator (uPA) in In Vitro Angiogenesis in Fibrin Matrices 319
Fig. 29.3. Formation of capillary-like tubular structures in a three
dimensional fibrin matrix by human endothelial cells. (A) Phase
contrast microscopy of human microvascular endothelial cells grown
under control conditions on top of a three-dimensional fibrin ma-
trix. The bar represents 300 ∝m. (B) Formation of tubular struc-
tures is induced by the simultaneous addition of the growth factor
bFGF (20 ng/mL) and the cytokine TNF-a (20 ng/mL). (C) Light
microscopy of haemotoxylin/ploxin-stained cross-secion perpen-
dicular to the fibrin matrix surface of a capillary-like tubular struc-
ture of microvascular endothelial cells.
320 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 29.4. Effect of various inhibitors of the uPA/plasmin system on the bFGF/VEGF/TNF-#-induced formation of tubular struc-
tures of human microvascular endothelial cells in a three dimensional fibrin matrix in vitro.
#v!3 integrin interaction with the fibrin matrix is likely,126 as fibronectin, vitronectin, hyaluronic acid and
while the role of #v!5 integrin, which more selectively inter- thrombospondin, and by the environmental conditions like
acts with vitronectin, has to be evaluated. Irrespective of the the pH and the ionic strength, determines whether new cap-
exact role of fibrin in stimulating angiogenesis, the fibrin illaries are formed or not. The invasion of endothelial cells
structure has important consequences for wound healing, into a fibrin matrix and the formation of capillary-like struc-
and proteolytic modification of fibrin, e.g., by leukocyte tures in the fibrin matrix in vitro is an attractive model to
elastase, or interaction of fibrin with other matrix proteins, study this essence of the angiogenesis process associated with
such as vitronectin and fibronectin, may affect cell invasion tissue repair. A recent paper of Nehls and Herrmann128
and angiogenesis and the success of wound healing. Fur- points to the importance of the fibrin structure in endothe-
thermore, the recent observation that MMP-2 (gelatinase-A) lial cell migration and the formation of tubular structures.
binds to #v!378 further points to the complexity of interac- These authors show that the rigidity of the fibrin gel has a
tions that the #v!3 integrin may have in fibrinous exudates, strong impact on tube formation by bovine endothelial cells
in which the matrix consists of a mixture of fibrin, collagens in response to bFGF and VEGF. In addition, we have found
and other extracellular matrix components. that this was also the case when human microvascular en-
dothelial cells were used (Collen et al, manuscript submit-
Perspective ted for publication). It remains to be determined whether
Delineation of the various cellular pathways that are this effect of the fibrin structure reflects a mechanical bar-
involved in angiogenesis is essential to selective interference rier to movement of cells by a dense fibrin network, or is
with unwanted angiogenesis, such as in rheumatoid arthri- due to an inadequate spacing of cell-binding epitopes in the
tis and tumors, and to stimulation of neovascularization at fibrin network.
sites where it is needed, such as in normal wound healing Nevertheless, these data obtained using in vitro ex-
and collateral formation. periments may be of great value when fibrin gels are used in
The structure of fibrin clot, determined not only by vivo as “temporary” matrix with regard to the initiation of
the concentration of fibrinogen and thrombin present in site-directed formation of new blood vessel structures or
the tissues but also by the presence of matrix molecules such collaterals, as shown by Dvorak et al129 and Fasol et al.130
Role of Urokinase-Type Plasminogen Activator (uPA) in In Vitro Angiogenesis in Fibrin Matrices 321
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Facilitation of Healing
Matrix Modulation
CHAPTER 30
How Does Extracellular Matrix Control
Capillary Morphogenesis?
Robert B. Vernon, E. Helene Sage
Angiogenesis: An Introduction
T he blood vascular system has evolved a significant capacity for change. During embry-
onic and fetal life, the vasculature increases in quantity and complexity to serve develop-
ing tissues and organs. Vasculature of the adult is, in general, quiescent, yet it retains a capac-
ity for growth, remodeling, and regression in response to the menstrual cycle, placentation,
changes in adiposity, wound repair, and inflammation. During these processes, growth of
new blood vessels is regulated and eventually ceases. In contrast, certain conditions such as
tumor growth, diabetic retinopathies, arthritis, and psoriasis involve excessive proliferation
of blood vessels that contributes directly to the pathological state.1 Therefore, an under-
standing of the principles and mechanisms that direct the assembly new blood vessels, and
the processes that start and stop vascular growth, are central to the development of agents
and strategies to control vascularization in disease.2
Tissue engineering involves the fabrication of tissue or organ-like implants comprised
of living cells in contact with a supportive matrix. Like cells of natural tissues and organs,
cells associated with an implant must interface with the circulatory system for reception of
oxygen and nutrients and removal of waste products. When implants are membranous, the
metabolic needs of the resident cells can be met by simple diffusive exchanges with sur-
rounding tissue fluids. However, a direct connection between the implant and the vascular
system may be necessary when diffusion distances are long as a consequence of thickness or
bulk of the implant. Moreover, in certain cases, resident cell types might require access to
circulating blood so that they can sense and respond rapidly to changes in body chemistry
and distribute their biosynthetic products (e.g., hormones) rapidly throughout the body.
Exchanges between cells and blood in vivo are most efficient across blood vessels with small
diameters and thin walls (e.g., capillaries); therefore, it would be desirable for implants to be
invested by a microvasculature that is connected to the blood supply of the host via large-
caliber arteries and veins. Artificial capillaries made from nonliving, semipermeable materi-
als are likely to be occluded by thrombi and rendered impermeable by precipitated plasma
proteins within a short period of time after exposure to flowing blood. Thus, it is likely that
long term function of implants will require a microvasculature derived from the host. This
microvasculature would be derived from the growth of extant blood vessels into the im-
plant, a process termed neovascularization, that is typical of the vascularization of tumors.
However, unlike the vasculature of a tumor, which grows and remodels continuously as the
tumor grows, the vascular supply of an implant of finite mass must stop growing once a
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
326 Tissue Engineering of Prosthetic Vascular Grafts
level of neovascularization sufficient to sustain the resident 1. Migration—ECs must penetrate and move through
cell population has been achieved. Excessive and unceasing interstitial ECM;
vascularization of the implant must be prevented, since an 2. Cellular alignment—Unlike a migrating epithelium
overexpression of some of the processes that mediate vas- in which the cells move as a sheet, migrating ECs
cular growth (e.g., proteolysis or forces of cellular traction, must follow one another in a “head-to-tail” orienta-
which are discussed below) could compromise the function tion for nascent vessels to have a tubular form;
of the implant or destroy it altogether. Thus, the develop- 3. Cell division—ECs must increase in number to
ment of methods to control vascular growth will be an im- lengthen the nascent vessel;
portant goal for tissue engineers. 4. Cell-cell adhesion—Migratory ECs must at some
The sprouting of new blood vessels from extant vas- point make firm connections with one another to
culature, termed angiogenesis, is the principal form of vas- form a mechanically-integrated structure;
cular growth in the adult. Sprouts arise from endothelial cells 5. Formation of a lumen—Individual ECs must assume
(ECs) that line the lumens of capillaries and postcapillary a curved shape to enclose a tubular space. Moreover,
venules—the smallest, most delicate branches of the vascu- adjacent cells must join their individual tubular
lar system where ECs are not confined by sheaths of smooth spaces to form a continuous lumen;
muscle or fibrous adventitia. The smallest capillaries are 6. Branching and anastomosis—ECs must send off
comprised of ECs alone and are capable of generating new branches from the main sprout. These branches must
capillary networks in the absence of other cell types. Al- find branches from other sprouts and fuse with them
though ECs contain all of the genetic information required to form anastomoses (also referred to as vascular
for morphogenesis, their potential to organize into capillar- loops or arcades).
ies is realized only by interaction with a noncellular entity— It is generally appreciated that genes regulate the ac-
the extracellular matrix (ECM). This review will discuss ways tivities of cells. Therefore, it has been proposed that the be-
in which ECM mediates and modulates the behaviors of ECs haviors listed above are mediated and regulated by a spe-
that lead to the development of a capillary bed. Much of cific “angiogenic program” or cascade that orchestrates the
what we will relate is speculative, because mechanistic stud- expression of EC gene products at specific times and in spe-
ies of the effects of ECM on vascular morphogenesis are in cific places.5 This program is, in turn, controlled by a vari-
their infancy. ety of soluble extracellular molecules (“growth factors”) se-
creted by target tissues, organs, and tumors.6,7 The role of
The Process of Angiogenesis growth factors in angiogenesis has been intensively studied:
Angiogenesis is a complex, multistep process initiated Much is known about the sources and structure of growth
in response to specific signals (e.g., diffusible, angiogenic factors, receptors for growth factors on the surfaces of ECs,
proteins) that emanate from sites of potential vasculariza- and signaling pathways within EC cytoplasm that are acti-
tion. The stages of angiogenesis induced by solid tumors have vated by growth factors. Moreover, the chemotaxis of EC
been described3,4 and can be summarized as follows: sprouts in response to gradients of growth factors has been
1. Upon receipt of an angiogenic stimulus, ECs of the analyzed in detail.8 What is not understood, however, is how
parent vessel degrade and penetrate the basement regulation of expression of EC genes by growth factors speci-
membrane that invests them, and subsequently mi- fies the actual construction of a capillary. Somehow, geneti-
grate into the surrounding interstitial ECM as a cally programmed, quantitative changes in the synthesis of
knoblike or cone-shaped vascular sprout; specific proteins by individual ECs are translated into spa-
2. The invading ECs assume bipolar shapes and align tial and vectorial information that directs the cooperative
behind one another in a follow-the-leader fashion; assembly of ECs into complex, three dimensional structures
3. ECs at the base or the middle of the sprout begin to (Fig. 30.1). Studies of vascular development indicate that
divide and add to the population of migrating cells; ECM plays a critical role in this translation. We will illus-
4. ECs organize into solid cords, which subsequently trate this concept by reviewing the development and char-
develop lumens; acteristics of the most important models of angiogenesis—
5. Neighboring sprouts move toward one another and an exercise that will reveal the special relationship between
fuse to form vascular anastomoses; ECs and ECM.
6. Circulatory flow begins in the vascular anastomoses;
7. Pericytes emerge along the length of capillary Models of Angiogenesis:
sprouts, and a new basement membrane is The Essential Role of ECM
synthesized.
It is likely that the above mentioned steps are typical Windows In vivo
of many forms of angiogenesis; however, solid cords of ECs Angiogenesis is difficult to observe and manipulate in
may be absent from advancing sprouts in wounds and cer- vivo because of the thickness and opacity of most tissues.
tain developing systems, such as the retina. Instead, nascent Tissue sections offer only a static view and provide little in-
lumens form immediately behind the tips, which are com- formation about the three dimensional structure of sprout-
prised of one or two spindle-shaped ECs. Although specific ing vasculature. Indeed, it is nearly impossible to identify
elements of angiogenesis might vary, the molecular processes the advancing fronts of sprouts (the critical areas of inva-
of capillary morphogenesis must mediate one or more of sion and morphogenesis) in sectioned tissue. To circumvent
the following six individual or cooperative behaviors of ECs: these problems, investigators have attempted to find or make
How Does Extracellular Matrix Control Capillary Morphogenesis? 327
Fig. 30.1. ECs that initiate angio-
genesis exhibit genetically derived,
quantitative changes in the synthesis
of specific proteins of the nucleus,
cytoplasm, cell surface, and extra-
cellular milieu. These changes,
which constitute an “angiogenic
program”, are translated by poorly
understood mechanisms into
spatial and vectorial information
that directs the assembly of ECs into
a three dimensional capillary
network.
windows into the living animal. In the late nineteenth and details of vascular morphogenesis toward clinical investiga-
early twentieth centuries, angiogenesis had been observed tions of the role of angiogenesis in the growth of tumors.
directly in the thin, transparent tails of amphibian larvae From these largely descriptive studies came validation of the
(for references and description of early work, see ref. 10). In central paradigm of tumor-induced vascular chemotaxis and
the 1930s, Clark and Clark10 viewed the revascularization of the corresponding discovery of diffusible, tumor-derived
wounds in rabbit ears through transparent chambers and molecules that promoted vascular growth. An increasing
made elegant drawings of the time course of angiogenic in- interest in an understanding of angiogenesis at the molecu-
vasion, anastomosis, formation of lumens, and interaction lar level led to the development of models in vitro, in which
of ECs with the ECM of the fibrin clot. Subsequent studies angiogenesis-related behaviors of ECs were studied under
with the rabbit ear chamber and other window-type mod- highly simplified, defined conditions. It was from such mod-
els (e.g., transparent chambers in mouse skin11 and hamster els that ECM was identified as a key element of vascular
cheek pouches,12 rabbit corneal pockets,13 and chick chorio- morphogenesis.
allantoic membranes)14 shifted the focus away from specific
328 Tissue Engineering of Prosthetic Vascular Grafts
EC Monolayers In Vitro the formation of endothelial networks. In 1988, Kubota et
In the 1970s, methods were developed to isolate ECs al30 reported that subconfluent ECs seeded on top of a mal-
from large vessels and culture them in flat dishes for extended leable, hydrated gel of basement membrane ECM
periods of time.15,16 ECs grown under these conditions in (Matrigel™) aligned in network-like patterns within one
vitro proliferated until they formed a confluent, pavement- hour and formed a planar, capillary-like meshwork of cellu-
like monolayer that simulated the endothelial lining of a lar cords within eighteen hours. In later studies of this cul-
blood vessel. It was appreciated that denuding injuries to ture system,30 we discovered that ECs formed networks as a
the endothelium of large vessels induced ECs at the periph- consequence of the mechanical response of Matrigel to cel-
ery of the wound to proliferate and migrate on the exposed lular tensile forces. The process (summarized in Fig. 30.2)
ECM of the basement membrane until an unbroken endo- begins as each EC continually pulls Matrigel toward itself
thelium was reestablished.17,18 A similar progression of re- by the motility-related phenomenon referred to as traction
sponses—proliferation, migration, re-establishment of in- (described below). The centripetal movement of Matrigel
tegrity, quiescence—could be elicited in vitro from confluent toward ECs generates radiating fields of strain in the sur-
monolayers of ECs “wounded” by removal of adherent cells rounding Matrigel (traction fields) that emanate from the
with a scraper.19,20 Thus, early studies of the relationship ECs (which act as traction centers). Where adjacent fields of
between ECs and ECM were designed in the context of re- traction overlap, tension within the Matrigel is enhanced
endothelialization as comparisons between confluent mono- (the “two center effect”32) and causes fibers of ECM that
layers and wounded (or simply subconfluent) ones. By this comprise the Matrigel to align into narrow matrical tracks
experimental approach, it was discovered that cultured bo- that connect the traction centers. The connection of neigh-
vine aortic ECs secreted fibronectin, laminin, heparan sul- boring traction centers soon establishes a tessellated (net-
fate proteoglycan, and type I through V collagens; 20-22 more- work-like) pattern on top of the flat field of Matrigel. ECs
over, they migrated better on a monomolecular coating of migrate along the network of matrical tracks by contact guid-
mixed type I and type III collagens than on a coating of ance and, with time, colonize the tracks to form a corre-
fibronectin.23 Studies of ECs (including ECs from capillar- sponding network of cellular cords. In situations where
ies) cultured as monolayers on molecular coatings of ECM Matrigel is rendered highly malleable by a cycle of freezing
have yielded important information; however, the migra- and thawing, the centripetal movement of Matrigel to trac-
tory, chemotactic, proliferative, and synthetic responses of tion centers actually perforates the sheet of matrix and re-
ECs that are elicited in these systems are not integrated in organizes it into a web of cables that subsequently become
situ such that vessel-like structures are formed. For such an surrounded by cords of cells.31 Matrical webs will form un-
integration to occur in vitro, a significant quantity of mal- der variable circumstances in vitro, e.g., they can be gener-
leable ECM is required. ated from malleable layers of gelled, fibrillar type I collagen.33
Moreover, the network of tension that tessellates a sheet of
Spontaneous Angiogenesis In Vitro ECM can be developed by a confluent monolayer of ECs:33
In 1980, following the successful, long term culture of By this process, endogenously-synthesized ECM is organized
capillary ECs,24 Folkman and Haudenschild reported that into a web during spontaneous angiogenesis in vitro.33
20-40 day cultures of bovine or human capillary ECs devel-
oped a two dimensional (planar) cellular network on top of Limitations of Planar Angiogenesis In vitro
the confluent cellular monolayer—a process they termed The matrical scaffolds that arise in cultures that com-
“angiogenesis in vitro”.25 By transmission electron micros- bine monolayers of ECs with sheets of ECM offer an attrac-
copy, the ECs of the networks appeared to form tubes with tive mechanism to explain how ECs orient themselves into
lumens that were filled with variable amounts of fibrillar, networks in vivo. However, these planar models33 lack cer-
membranous, and amorphous substances. This “string-like” tain defining characteristics of true angiogenesis:
extracellular material traversed the anastomoses between 1. Invasion—ECs in planar models form networks on
adjacent ECs and was proposed to act like a “mandril for the top of ECM and show little propensity to burrow
alignment of tubes from cell to cell”.25 Later studies showed into the ECM;
that ECs from large vessels (e.g., human umbilical vein26 and 2. Directionality—planar networks of ECs form in vitro
calf aorta27) could also form networks spontaneously in vitro. by tessellation that is more or less simultaneous
Here also, the lumens of the tubular ECs were filled with throughout a field of prepositioned cells, whereas
ECM that included fibronectin,26,27 laminin, and type IV and angiogenesis in vivo involves the vectorial invasion
V collagens.28 Direct evidence of a morphogenetic role for of ECM by filamentous sprouts that arborize by
ECM in what was now called “spontaneous angiogenesis in multiple levels of branching;
vitro” was provided by Ingber and Folkman,29 who showed 3. Polarity—Although the ECs in planar models make
by time-lapse cinematography that the luminal ECM formed unicellular tubes that markedly resemble capillar-
a “web” or “scaffold” on which the ECs organized themselves. ies,26,27,34 they deposit basement membrane mate-
rial on their luminal surfaces and have their throm-
Traction, Tension, and Tessellation Mediate bogenic surfaces facing outward to the surrounding
Spontaneous Angiogenesis In Vitro culture media,26,27 opposite to the situation in vivo.
A serious drawback of spontaneous angiogenesis in Despite these deficiencies, the central characteristic of
vitro was the length of time (up to six weeks) required for planar models—alignment of ECM in response to tension
How Does Extracellular Matrix Control Capillary Morphogenesis? 329
Fig. 30.2. Cellular traction generates patterns of order in pla-
nar ECM in vitro. (A) Viewed from above, a cellular traction
center (shaded circle) in contact with malleable, planar ECM
(rectangle) generates radial lines of tension (arrows) in the ECM
that constitute a traction field. (B) Tension in ECM between
adjacent traction centers is enhanced (large arrows) as a conse-
A quence of the two-center effect. (C) Fibers of ECM (black lines)
align along the direction of principal stress. Fibers influenced
by the two-center effect align to form a matrical track that con-
nects the traction centers. (D) Traction centers (small black dots)
arranged in a field on planar ECM become connected by mat-
rical tracks (e.g., arrow) that form a network. (E) left panel:
Where planar ECM (shaded) is highly malleable, centripetal
movement (arrows) of ECM to traction centers (black dots)
B results in clearance of ECM from central areas (X) that are bor-
dered by two-center effects (dotted lines). (E) right panel: Clear-
ance process diagrammed in left panel is manifested as perfo-
rations (white areas) in the sheet of ECM (shaded). Perfora-
tions are small (S) initially but enlarge with time (L). ECM
aligned by two-center effects (dotted lines) between traction
centers (black dots) is resistant to cell-generated stresses. Re-
printed from Vernon et al.33 Copyright © 1995 by the Society
C for In Vitro Biology. Reproduced with permission of the copy-
right owner.
entation of ECs could be influenced significantly by the to-
pology of their interaction with ECM. Although this par-
ticular three dimensional model lacks the characteristics of
invasion (the ECs are pre-embedded in the ECM) and di-
rectionality (the ECs are evenly dispersed throughout the
D culture), it has proven useful in studies of lumen forma-
tion37 and responses of ECs to growth factors.38-41
Three Dimensional Models
with Invasive Characteristics
In 1982, Nicosia et al described the sprouting of ECs
from sections of rat aorta (“aortic rings” one millimeter in
thickness) embedded in a clot of chicken plasma.42 Within
E 36 h, solid sprouts arose from the incised ends of the aorta
and invaded the fibrin-rich ECM of the clot. Over a period
of two weeks, ECs within sprouts migrated and proliferated
generated by ECs—is likely to regulate the migration of ECs to form branching, anastomosing cords that subsequently
in vivo (discussed below). Fortunately, the problems of in- developed large, patent lumens. As in the three dimensional
vasion, directionality, and polarity have been addressed by model with dispersed ECs, sprouts from aortic rings exhib-
three dimensional models of angiogenesis, which are de- ited polarity like that in vivo. In addition, however, sprouts
scribed below. from aortic rings were truly invasive and exhibited a direc-
tional, branching morphogenesis. Aortic explants have been
Angiogenesis in Three Dimensions used to study the influence of tumors,43 growth factors,44,45
The recognition that capillary ECs are surrounded by and various forms of ECM46-50 on angiogenesis. Deficien-
ECM in vivo led to the development, in 1983,35 of a three cies in this model are typical of explant cultures, i.e., results
dimensional model of angiogenesis in which subconfluent can be influenced by non-EC types51 and by growth factors
bovine adrenal capillary ECs were embedded in a lattice of that diffuse from the explant.44 These problems are addressed
native type I collagen fibrils, commonly referred to as a col- by yet another model of vascular invasion in vitro. In 1985,
lagen “gel”.36 Within two days, the ECs formed a network of Montesano and Orci52 cultured confluent monolayers of
cellular cords and tube-like structures with a polarity simi- bovine microvascular ECs on top of a layer of gelled type I
lar to that of capillaries in vivo, i.e., basal lamina-like mate- collagen, and thereby generated an endothelium-like sheet
rial was deposited between the abluminal surfaces of the ECs of cells with a highly polarized morphology, i.e., the ECs
and the collagen matrix. This result indicated that the ori- possessed “luminal” surfaces that faced the overlying culture
330 Tissue Engineering of Prosthetic Vascular Grafts
medium, lateral surfaces that contacted adjacent ECs, and however, relatively little is known about the means by which
“abluminal” surfaces that faced the collagen substrate. This ECs move through ECM. Indeed, the basic mechanism of
topology resulted in an angiogenesis-like response to the ameboid movement by animal cells remains unclear, despite
tumor-promoting ester phorbol myristate acetate: ECs pen- intensive study of selected cell types such as fibroblasts.56
etrated the collagen as invaginations of the cellular sheet Moreover, almost all detailed mechanistic studies of cell
(much like the outpocketing that occurs during early stages movement have been conducted in vitro with cells on top of
of sprout formation in vivo) and formed branched, multi- planar substrates that are rigid (i.e., glass or plastic), or
cellular tubes with abluminal basal lamina-like material and semideformable (e.g., silicone rubber), and hence unlike
patent, fluid-filled lumens similar to those of capillary malleable ECM in vivo. On such artificial surfaces, fibro-
sprouts in vivo10 (Fig. 30.3). The induction of invasive blasts, ECs, and many other cell types exhibit a “crawling”
sprouting by both micro- and macrovascular ECs53-55 rein- form of motility characterized by a repeated cycle of:
forces the notion that the structure of the parent vessel (i.e., 1. Protrusion of lamellipodia or filopodia at the lead-
a tube of mutually adherent cells with fluid plasma inside ing edge of the cell;
and ECM outside) provides vital cues and constraints that 2. Adhesion of the protrusive structures to the support-
position ECs for correct orientative behaviors once their ive substrate;
angiogenic programs are initiated. 3. Movement of the cell body and nucleus forward by
the process of traction, during which the cell pulls
Interactions Between ECs and ECM strongly on the supportive substrate;
That Mediate Angiogenesis 4. De-adhesion of the rear, pointed “tail” of the cell from
As discussed above, differently-conceived models of the substrate; and
angiogenesis all agree on the central role played by ECM in 5. Retraction of the tail toward the cell center 56
vascular morphogenesis. In the following section, we will (Fig. 30.4).
move beyond descriptive studies to explore ways in which Classic time-lapse observations by Bard and Hay show
the behaviors of ECs and the structure of ECM interact chick corneal fibroblasts exhibiting this cycle of motility as
mechanistically to promote the morphogenesis of capillaries. they migrate through corneal stroma and through a gel of
fibrillar type I collagen in vitro.57 It is likely that ECs move
Characteristics of EC Motility through malleable three dimensional ECM in a simil-
Locomotion of ECs is a critical element of angiogen- ar manner.
esis. A significant number of studies have examined the mi-
gration of ECs in response to various angiogenic molecules;
Fig. 30.3. Vectorial invasion of type I collagen gels
by confluent monolayers of ECs in vitro. In re-
sponse to stimulatory molecules, such as phorbol
esters, ECs of the monolayer penetrate the un-
derlying collagen gel as invaginations of the cel-
lular sheet. The invaginations elongate to form
multicellular tubes with patent, fluid-filled lu-
mens (L) that are continuous with the overlying
culture medium. ECs that comprise the tubes
deposit basal lamina-like material (BL) on their
abluminal faces. It is likely that similar material
is deposited underneath noninvasive ECs of the
confluent monolayer (not shown).
How Does Extracellular Matrix Control Capillary Morphogenesis? 331
Fig. 30.4. Cycle of movement of an EC
through three dimensional ECM. (1) The
cycle begins with actin-mediated protru-
sion at the cell front. Focal adhesions
(small gray ovals) anchor the cell body
to the ECM (shaded background). (2)
New focal adhesions anchor the protru-
sion to the ECM. (3) Cytoplasmic actin
filaments and associated proteins gener-
ate traction forces (double-headed ar-
rows) that pull on the ECM at the cell
front and move the cell body forward past
the focal adhesions, which remain fixed
in position relative to the ECM (dotted
lines). The rear of the cell remains at-
tached to the ECM and is drawn out to
form a pointed “tail”. (4) Focal adhesions
of the tail become altered (white ovals)
and detach from the ECM. (5) The tail
retracts into the cell body. At the cell front,
a new protrusion begins the next cycle of
forward progression.
332 Tissue Engineering of Prosthetic Vascular Grafts
Malleability of ECM as a Function of Its Composition trusions of ECs in ECM usually consist of one or more very
The migration of ECs and other cell types can be af- thin, elongate filopodia that sometimes exhibit filamentous
fected by the physical properties of the supportive ECM, branches, i.e., structures with minimal frontal area that are
notably malleability. In the context of angiogenesis, mallea- thermodynamically favored in a viscous environment. The
bility is defined as the degree to which ECM deforms in re- possibility that protrusive activity governs the rate of cell
sponse to forces generated by sprouting ECs. ECM is com- locomotion65 might explain why movement of ECs into
prised of a hydrated gel of proteoglycans, glycoproteins, and gelled collagen66 or fibrin67 is inhibited as the density of the
hyaluronan in which fibrillar proteins (e.g., collagens and ECM fibrils increases.
fibronectin) are embedded. The malleability of this mixture
of macromolecules is significantly affected by the concen- Traction of ECs as a Spatial Organizer of ECM
tration and relative proportion of the major fibrillar pro- In crawling cells, actin filaments in the cytoplasmic
teins, such as type I and type III collagens that predominate cortex are driven rearward by association with other motive
in interstitial ECM. An additional element of physical het- proteins, such as myosin.56,68,69 Movement of actin is trans-
erogeneity involves the stiffness of individual protein fibrils, duced into propulsive traction force by focal adhesions
which depends in part on interactions with other compo- (Fig. 30.4), which are spot or bar-shaped specializations of
nents of ECM. For example, the polymerization of type I the plasma membrane that adhere to the substrate (i.e.,
collagen in vitro yields thinner, more pliable fibrils in the ECM) via their extracellular faces and connect to the actin
presence of type III collagen,58 the ECM protein TRAMP,59 via their cytoplasmic faces.68-70 Traction is applied vectorially,
and the ECM proteoglycan decorin.60 The malleability of and generates tension in malleable substrates in vitro (e.g.,
ECM is decreased by increases in the physical entanglement silicone rubber) that is directed parallel to the cellular axis
of fibers (shear-drag) and by the presence of covalent of motion.64,71 Remarkably, fibroblasts exert traction that is
crosslinks, such as the lysyl oxidase-catalyzed crosslinking at least six orders of magnitude greater than is needed to
of type I collagen fibers,61 and the transglutaminase-cata- propel the cell at a normal speed of 1 ∝m/sec against the
lyzed generation of fibronectin multimers.62 The rigidify- viscous drag of aqueous medium.64 It is important to real-
ing effects of shear-drag and crosslinks are counteracted by ize that this magnitude of force and its vectorial application
specific molecular interactions (e.g., shear-drag between type to the substrate are specialized features of crawling cells that
I collagen fibers is reduced by the binding of type XII and are not intrinsic to ameboid locomotion. Neuronal growth
type XIV collagens)63 and by more general influences such cones and fish keratocytes that exhibit an alternative “glid-
as the reduction in fiber length and number by cell-medi- ing” form of ameboid motility64 move 10-60 times faster
ated proteolysis. ECs within a vascular sprout influence the than fibroblasts in vitro,56 generate only 3-12% of the trac-
structural properties of ECM in their immediate vicinity by tion force,64 and apply tension to the substrate at right angles
their synthesis of new forms of ECM and by chemical modi- to the direction of motion.64,72
fication and/or degradation of extant ECM. These processes It is thought that the “excessive” levels of traction de-
can be modulated to facilitate or inhibit vascular invasion, veloped by fibroblasts allow these cells to compress and align
contingent upon the type of signals received by the ECs. ECM in healing wounds73 and within developing tendons,
Additional control over the rate, extent, and locale of vascu- ligaments, and organ capsules.74 The traction of ECs in vitro
lar invasion resides with non-EC types (e.g., fibroblasts) that is similar in magnitude to that of dermal fibroblasts75 and
can control the composition of surrounding ECM via spe- therefore is likely to serve a morphogenetic function. Trac-
cific synthetic and degradative activities. tion at the advancing front of sprouts would tend to align
ECM in the direction of invasion and thus would facilitate
Malleability of ECM Influences the Motility of ECs protrusion of filopodia from leading ECs. Moreover, elastic
It is likely that the malleability of ECM directly influ- interactions among ECM fibrils allow them to be aligned
ences the generation of protrusive structures by cells. Pro- over great distances (as much as one centimeter in vitro),32,74
trusion of filopodia and lamellipodia by crawling cells in- and to form pathways on which invading ECs would move
volves the polymerization of cytoplasmic actin that either by contact guidance.76 Such matrical pathways mediate mi-
pushes the plasma membrane forward or stabilizes bulges gration of ECs in planar angiogenesis in vitro and have been
in the plasma membrane made by other means (e.g., via ther- associated with the movement of endocardial cells into the
mal movements or the local influx of water).56 Counterforces endocardial cushions of the developing chicken heart.77
exerted by ECM on the front of a cell (generated, for ex- Moreover, the existence of matrical pathways is consistent
ample, by the resistance of stiff, fibrillar ECM to bending or with the follow-the-leader behavior of ECs that migrate
the resistance of charged, hydrated glycans to compression) through three dimensional ECM in vitro78 and in vivo.3
would slow the rate or extent of protrusion by shifting the Matrical pathways could also mediate the development of
thermodynamic equilibrium in favor of actin depolymer- anastomoses between vascular sprouts. ECs at the tips of
ization. Another consequence of ECM counterforces would adjacent sprouts would align ECM between them by a trac-
be their effect on the shape of protrusions. For example, tion-mediated two-center effect, approach one another via
during progressive movement over rigid, planar substrates the matrical pathway, and fuse to establish a common lu-
in vitro, during which viscous drag from the culture me- men (Fig. 30.5).
dium is negligible,64 fibroblasts and ECs frequently project Beyond the role of traction to facilitate vascular inva-
blunt or fan-shaped lamellipodia. In contrast, forward pro- sion and anastomosis of sprouts is the potential for traction
How Does Extracellular Matrix Control Capillary Morphogenesis? 333
Fig. 30.5. Hypothetical role of matrical pathways in the anasto-
mosis of angiogenic sprouts in vivo. (A) Migratory ECs (small
arrows) at tips of adjacent sprouts create a connecting pathway
of aligned ECM (matrical pathway) as a consequence of a trac-
tion-mediated two-center effect. Lumens (L) of sprouts are in-
dicated. (B) Migratory ECs approach one another (arrows) via
the matrical pathway. (C) The vascular loop is completed as
ECs meet, adhere, and interact to form a patent lumen. Adapted
from Vernon RB, Sage EH. Between molecules and morphol-
ogy: Extracellular matrix and the creation of vascular form. Am
J Pathol 1995; 147:873-883. Copyright © 1995 by the American
Society for Investigative Pathology. Reproduced with permis-
sion of the copyright owner.
embryo,33 capillary plexuses of acinar exocrine glands and
pulmonary alveoli, and the microvasculature of the retina.
For a given system of planar vasculature, the architecture of
the cellular network and, indeed, whether such a network
forms, are direct functions of the malleability of the ECM31,79
and the levels of traction force generated by the component
ECs79—parameters that are controlled by a multiplicity of
regulatory signals.
Adhesion Between ECs and ECM as a Regulator
of EC Migration
A crawling cell generates new focal adhesions at the
front that persist and remain fixed to the substrate as the
cell pulls itself over them.80,81 Upon reaching the rear of the
cell, the adhesions are released.80 This dynamic circumstance
suggests that an asymmetry of adhesive strength exists be-
tween the front and rear of the cell: Strong adhesion at the
front enables the cell to pull itself forward and weaker adhe-
sion at the rear allows the cell to free itself to begin the next
cycle of movement. Mathematical models82 predict that for
a given level of cytoplasmic contractile force, the opposing
force of substratum adhesion influences the rate of loco-
motion in a biphasic manner: Weak adhesion inhibits the
conversion of cytoskeletal contractility into propulsive trac-
tion force, whereas excessive adhesion prevents the cell from
freeing its rear from the substrate. Speed of migration is pre-
dicted to be maximal at an intermediate level of adhesion
that allows dynamic making and breaking of cell contacts.
This hypothesis is supported by studies in vitro that show
biphasic relationships between the rate of cellular motility
and the concentration of laminin,83 laminin fragment E8,83
fibronectin,84 and type IV collagen84 adsorbed to plastic.
ECs exhibit variable adhesion to different components
of ECM in vitro: Bovine aortic ECs attach more avidly to
type I collagen and fibronectin than to laminin and type III
to organize ECs into large-scale networks in vivo. This type collagen.85 Differential adhesion is correlated with varied
of network organization has been modeled by planar an- rates of motility; for example, adhesion and migration of
giogenesis in vitro and by a recent mathematical simula- ECs on coatings of type IV collagen are higher than on coat-
tion.79 A variety of network-like microvascular systems ex- ings of laminin.86 Thus, the composition of extant ECM
hibit planar characteristics in vivo that render them ame- could influence the migration of sprouting ECs, not only
nable to morphogenesis via traction. Examples include the through mechanical resistance to cell protrusion, but also
para-aortic and vitelline vascular plexuses of the early avian through adhesive control of traction. Moreover, migratory
334 Tissue Engineering of Prosthetic Vascular Grafts
ECs could control their adhesion directly by secretion of hesive receptors (e.g., integrins) to their cognate ECM
specific types of ECM in their immediate vicinity (referred ligands. Alternatively, the cell-binding domains could inter-
to as pericellular matrix). For example, when stationary bo- act with specific cell surface receptors to trigger
vine aortic ECs are induced to migrate in vitro, they switch counteradhesive responses through second messenger sig-
from synthesis of an ECM rich in heparan sulfate naling pathways. In addition to matricellular proteins, in-
proteoglycan (which stabilizes the binding of focal adhesions vestigators have identified other important classes and fami-
to fibronectin and inhibits cell migration) to an ECM en- lies of counteradhesive molecules, including some novel
riched in chondroitin/dermatan sulfate proteoglycans which members.119 It is likely that further study of counteradhesion
destabilize adhesion sites and promote cell motility.87,88 will improve our understanding of how angiogenesis and
other types of morphogenesis are regulated.
Matricellular Proteins and Counteradhesion
In the context of EC adhesion, of particular relevance Integrins Mediate Adhesion to ECM
are matricellular proteins which reside (often transiently) and Signaling in Focal Adhesions
in the ECM and influence adhesion between cells and their Integrins are a major class of transmembrane recep-
ECM ligands.89,90 Unlike proteoglycans and other adhesion- tors that mediate the attachment of cells to ECM proteins
modulating components of ECM, matricellular proteins do via focal adhesions. They comprise a family of at least 16 #
not contribute significantly to ECM structure. Matricellular and 8 ! subunits that associate noncovalently in over 20 dif-
proteins are secreted by many cell types and consist of three ferent #/! combinations on the cell surface. Both # and !
families exemplified by SPARC (secreted protein, acidic and subunits are required for high-affinity binding to an ECM
rich in cysteine), 91 thrombospondin-1 (TSP),90,92 and ligand: The specific ligand is determined by the combina-
tenascin-C (TN).93 Although SPARC, TSP, and TN are struc- tion of distinct # and ! subunits. Some integrins recognize a
turally unrelated, a commonality is their enhanced expres- single ECM protein (e.g., integrin #5!1 recognizes only
sion by migrating, dividing cells of subconfluent cultures fibronectin); however, it is more common for an integrin to
and in tissues undergoing embryogenesis and wound re- bind to two or more ligands. An ECM protein may be rec-
pair.94 Moreover, these proteins have similar effects on cells ognized by more than one integrin; for example, fibronectin
in culture: They inhibit attachment and spreading, and cause and laminin are bound by the !1 subunit in combination
partial detachment and rounding of spread cells. In accor- with one of four and five different # subunits, respectively.
dance with these counteradhesive properties, SPARC, TSP, Different integrins that share an ECM ligand may bind to
and TN reduce the number of focal adhesions made by at- the ligand at the same site, as do #5!1 and #3!1, or they can
tached, subconfluent ECs.94-96 Although these proteins are recognize different regions of the ligand, as do the #1!1 and
associated with capillaries in vivo,97-99 their influence on #6!1 laminin receptors, or the #5!1 and #4!1 fibronectin
angiogenesis remains unclear. Whereas SPARC itself has receptors.120 ECs and other cell types typically express sev-
minimal effects on angiogenesis in vivo, specific peptide frag- eral different integrins on their surfaces concurrently and
ments of SPARC have angiogenic properties.100 TSP is de- can alter the expression of one integrin or more in response
posited in a trail behind migrating ECs101 and promotes their to stimuli.
chemotaxis in vitro.102 It is also reported to inhibit the mi- Unbound integrins are freely diffusive in the plane of
gration and sprouting of ECs in vitro.103,104 TSP is generally the plasma membrane; however, binding to ECM or other
believed to be anti-angiogenic in vivo;98,103,105 however, it is ligands causes adjacent integrins to cluster, an event medi-
proposed that TSP stimulates angiogenesis by indirect ef- ated by the cytoplasmic domains of the ! subunits.121 Clus-
fects on non-EC types.106 The association of TN with pro- tering of unligated #/! heterodimers is prevented by the #
liferating vessels in the vicinity of tumors99,107,108 could in- subunit; however, this inhibition is removed following en-
dicate that it promotes angiogenesis. In accordance with this gagement of the ligand. Integrin clusters increase in size by
hypothesis, planar angiogenesis in vitro is inhibited by anti- further aggregation and eventually become large enough to
bodies against TN.104 be recognized as focal adhesions. As a focal adhesion grows,
Matricellular proteins bind both to cells and to ECM. the clustering of the short cytoplasmic domains of the # and
SPARC binds TSP109 and type I through V collagens,110,111 ! integrin subunits nucleates the formation of large molecu-
and also interacts with ECs via as yet uncharacterized bind- lar complexes comprised of cytoskeletal linking proteins and
ing proteins or receptors.112 TSP binds collagen, fibronectin, a variety of catalytic signaling molecules. The cytoskeletal
laminin, fibrinogen,89 and heparan sulfate proteoglycans of linking proteins, which include #-actinin, talin, vinculin,
ECM or the cell surface.92 Moreover, TSP binds to the cell paxillin, and tensin, connect the clustered integrins to the
surface integrin #v!3,92 an ECM receptor that is expressed actin cytoskeleton and thereby strengthen the focal adhe-
on ECs and plays an important role in angiogenesis. TN has sion mechanically and couple it to the motility apparatus.
a low affinity for ECM proteins such as collagens, fibronectin, The catalytic signaling proteins (notably tyrosine kinases and
and laminin113 but binds to several cell surface receptors, their substrates) initiate cascades of molecular association
including integrins #2!1 and #v!3 on ECs,114,115 integrin and enzymatic activity in both the cytoplasm and the nucleus
116
#9!1, proteoglycans,117 and annexin II.118 The cell and that regulate the cell cycle, responses to growth factors, and
ECM-binding domains of matricellular proteins, either sepa- expression of specific gene products via several convergent
rately or in combination, could mediate counteradhesion pathways (see refs. 122-125 for general reviews of integrin-
by physical interference with the binding of cell surface ad- mediated signal transduction). The sequestration of signal-
How Does Extracellular Matrix Control Capillary Morphogenesis? 335
ing proteins in focal adhesions of ECs is rapid enough to vitronectin, fibronectin, fibrinogen, laminin, von willebrand
play a role in integrin-mediated responses to cytokines. Fif- factor, and denatured type I collagen. Thus, expression of
teen to thirty minutes after bovine capillary ECs bind to this integrin enables a cell to adhere to, migrate on, or re-
fibronectin or the immobilized peptide Arg-Gly-Asp (the spond to almost any ECM protein it might encounter.138
recognition site in fibronectin for integrin #5!1) in vitro, their Integrin #v!3 is expressed preferentially in angiogenic ves-
nascent focal adhesions are enriched in Flg (a high-affinity sels135,139 and is necessary for angiogenesis, since a mono-
receptor for the angiogenic protein basic fibroblast growth clonal antibody (LM609) against this integrin blocked
factor [bFGF]), the tyrosine kinases pp60c-Src and pp125FAK, cytokine-induced angiogenesis in chick chorioallantoic
and other signaling molecules including phospha- membranes,135 tumor-induced angiogenesis in human skin
tidylinositol-3-kinase, phospholipase C-&, and the Na+/H+ transplanted on severe combined immunodeficiency (SCID)
antiporter.126 mice,140 and bFGF-induced angiogenesis in the rabbit cor-
nea.141 LM609 also disrupted vascular morphogenesis and
Functional Characteristics of Integrins Expressed by ECs lumen formation within vasculature of early quail em-
Human umbilical vein ECs in vitro express integrin # bryos.142 In the chick chorioallantoic membrane, antago-
subunits 1-6127,128 and ! subunits 1 and 3,127 with #2!1 as the nists of #v!3 induced programmed cell death (apoptosis) of
predominant combination. Integrin #2!1 is present on cap- ECs in sprouting blood vessels. Engagement of #v!3 with an
illaries in vivo,129 and this integrin is responsible for most of ECM ligand might therefore provide a “survival signal” to
the binding of ECs to type I and IV collagens in vitro.128 ECs during angiogenesis.143
Integrin #2!1 promotes the migration of ECs on type I col-
lagen130 and mediates the reorganization of type I collagen Integrins, Cell Shape, and Gene Expression
by EC traction in vitro.131 Although #2!1 contributes signifi- The genetic programs that mediate proliferation and
cantly to the binding of human umbilical vein ECs to differentiation of ECs and other cell types tend to be mutu-
laminin-coated plastic,127,128 it plays little or no role in the ally exclusive in vitro; e.g., synthesis of cell-specific gene
traction-mediated organization of ECs into networks on products diminishes when cells are stimulated to divide.144
Matrigel in vitro132—a process dependent on the laminin- Since both programs are triggered by the binding of cell sur-
binding integrin #6!1.132,133 Despite the involvement of !1 face integrins to ECM, additional elements of control must
integrins in adhesion and traction-related processes in vitro, exist to determine which program is selected. A variety of
the role played by this integrin subfamily in vascular mor- studies point to cell shape as an important element in the
phogenesis is unclear. A monoclonal antibody (CSAT) switch between proliferation and differentiation. In general,
against the avian !1 integrin subunit induced abnormalities cells that assume rounded shapes exhibit a differentiated
of aortic vasculogenesis in the quail embryo that included phenotype. For example, levels of synthesis of the milk pro-
inhibition of luminal development.134 CSAT, however, does tein !-casein by mouse mammary cells (induced by the bind-
not inhibit angiogenesis in the chick chorioallantoic mem- ing of laminin to cell surface !1 integrins) are high in cells
brane despite the presence of !1 integrins in nascent blood made round by culture on nonadhesive substrates, but are
vessels.135 In accordance with the effects of CSAT in the quail low in flattened, spread cells grown on surfaces that permit
embryo, antibodies against the #2 and !1 integrin subunits cell adhesion.145 Spreading of cells facilitates proliferation:
inhibited development of lumen-like intracellular vacuoles Ligand-induced clustering of integrins, in the presence of
within human ECs cultured in three dimensional type I col- mitogenic factors, is sufficient to induce ECs and other cell
lagen gels.37 It is not known how the engagement of type I types to exit from G0 and progress into the late G1-phase of
collagen by #2!1 promotes the formation of lumens. Pos- the cell cycle in vitro;146-149 however, the cells will not enter
sible mechanisms include: S-phase unless they are allowed to spread.148,149 The path-
1. Induction of a “program for lumen formation” via ways by which cell shape is coupled to proliferation and gene
intracellular signaling pathways; expression are not understood, although evidence points to
2. Application of traction force to the collagen that involvement of the cytoskeleton. It is known that the net-
causes distortion of cell structure (cavitation) rather work of actin filaments generates tension in the cytoplasm
than cell migration or reorganization of collagen; and that promotes cell retraction and rounding. This tension is
3. Clustering of #2!1 at selected sites to provide cell- opposed both intracellularly by stiff, compression-resistant
matrix anchor points for the development of pinocy- microtubules and extracellularly by the counteracting ten-
totic membrane invaginations.37 sion of ECM, which is coupled directly to the actin network
Exposure of capillary ECs to bFGF augments their through integrins within focal adhesions. It is hypothesized
expression of integrin subunits #2, #3, #5, #6, !1, !3, !4, and !5 that the equilibrium between these forces places the cytosk-
and results in a corresponding increase in adhesion of ECs eleton at a “setpoint” of stress which defines a specific pat-
to collagen, fibronectin, laminin, and vitronectin.136,137 The tern of gene expression via mechanically sensitive biochemi-
anti-angiogenic transformtin growth factor-! (TGF-!) cal processes in the plasma membrane, cytoplasm, and
stimulates #2, #5, and !1 in these cells;137 the pattern of nucleus.150,151 It follows that a change in the setpoint of stress
integrin expression by ECs is thus altered selectively by dif- caused by alterations in:
ferent cytokines. 1. Actin-generated forces;
Integrin #v!3 binds to a variety of ECM proteins with 2. Cytoskeletal structure;
an exposed tripeptide Arg-Gly-Asp moiety, such as
336 Tissue Engineering of Prosthetic Vascular Grafts
3. Strength, number, or spatial arrangement of adhe- by ECs include the interstitial collagenase MMP-1, which
sive contacts to ECM; or cleaves native type I, II, and III collagens at a single site;158,159
4. Malleability of ECM gelatinases A (MMP-2) and B (MMP-9), which degrade de-
would alter the biochemical equilibrium to favor ex- natured collagens (gelatins), type IV and V collagens, and
pression of a different set of genes. Electron microscopic elastin,160,161 and stromelysin (MMP-3), which has a broad
studies of resinless sections152 reveal that tension applied to range of substrates that include laminin, fibronectin, the pro-
the cell surface could be transmitted first to the nuclear en- tein core of proteoglycans, and gelatin.161,162 Recently, it has
velope, via bridges of intermediate filaments, and then into been demonstrated that MMP-2 is an interstitial collagenase
the nuclear interior, via nuclear fibrils that insert into the with a specificity similar if not identical to those of MMPs-1
inner side of the nuclear envelope. In support of these ob- and -8.163 Type I and type III collagens are highly resistant
servations, Maniotis et al have shown recently that a me- to proteolysis by trypsin, plasmin, and other members of
chanical pull applied to integrins on the EC surface causes a the serine and sulfhydryl proteinase families. These collagens,
physical distortion of the nucleus and a reorganization of however, are degraded by MMPs via a two-stage process in-
nucleoli along the axis of applied tension.153 The functional volving initial cleavage by interstitial collagenases and sub-
consequence of this chain of mechanical events might in- sequent digestion of the larger fragments by gelatinases.
clude control of DNA synthesis, since the degree to which Studies in vitro indicate a role for MMPs in angiogen-
ECs spread on coatings of ECM in vitro, the physical expan- esis. Exposure of ECs to phorbol esters and angiogenic
sion of the nucleus, and the rate of DNA synthesis are closely growth factors in vitro induces production of MMPs-1, -2,
correlated.149 -3, and -9.164-167 MMPs are regulated in vivo by tissue in-
During angiogenesis in vitro, ECs are likely to hibitors of metalloproteinases—a family of low molecular
encounter a shifting pattern of external forces that are modu- weight proteins (TIMPs-1, -2, and -3) that specifically block
lated by: the catalytic activity of MMPs.161 Inhibition of endogenous
1. Cell-cell adhesion; MMP activity of ECs in vitro, either by metal ion chelators
2. Enzymatic modification of extant vicinal ECM; or by TIMPs, suppresses traction-mediated reorganization
3. Synthesis of new ECM; of type I collagen gels75 and the corresponding angiogen-
4. Changes in patterns of integrins or other cell-sur- esis-like invasion of these gels.52,55,168
face receptors that mediate adhesion to ECM; and The inherent complexity of proteolysis and its regula-
5. Restructuring of ECM by the traction of ECs and tion are well illustrated by the plasmin/PA system. PAs are
non-EC types. serine proteinases that cleave the zymogen plasminogen (a
Asymmetrical or focal application of forces to specific plasma protein) to plasmin. Plasmin is itself a proteinase
areas of a sprout could cause specific ECs to proliferate and/ that degrades several components of ECM, such as
or express distinct gene products. Such localized behaviors fibronectin, laminin, the protein core of proteoglycans, and
could alter the morphology of the sprout—for example, the gelatins.169 The tissue-type (tPA) form of PA is expressed by
sprout could bend or branch. Clearly, much remains to be ECs of quiescent microvasculature and is neither modulated
discovered regarding the complex role of biomechanical during angiogenesis nor expressed by sprouting ECs in vivo.
processes in vascular morphogenesis. In contrast, the urokinase (uPA) form of PA is expressed by
angiogenic ECs associated with tumors, inflammation, he-
Angiogenesis Requires Proteolysis of ECM mangiomas, ovarian follicles, corpora lutea, and maternal
At the site of a new vascular sprout, ECs of the parent decidua. Little or no uPA is expressed in quiescent vessels.168
vessel secrete proteases that effect a focal degradation of the Plasminogen is present in most tissues at high levels;
basement membrane.154 Proteolysis continues as the sprout therefore, its activation by PAs results in high local concen-
invades the interstitial ECM: Clark and Clark observed that trations of plasmin. Moreover, plasmin-mediated, limited
invasive capillaries within rabbit ear wounds dissolved fi- proteolysis activates latent elastase (a serine protease) and
brin in their immediate vicinity and thus created a “clear MMPs-1, -2, -3, and -9. Thus, small quantities of PA can
space” around each sprout.10 Experimental studies have generate significant levels of proteolytic activity with differ-
shown proteolysis is necessary for angiogenesis within the ent substrate specificities.169
developing chorioallantoic membrane of the chicken155 and The position of uPA as the initiator of a proteolytic
for invasion of ECs into three dimensional ECM comprised cascade dictates that its activity be strictly controlled during
of fibrin,156 type I collagen,55 and the amniotic basement angiogenesis. Expression of uPA by ECs is modulated at the
membrane.157 The basis of the permissive effect of proteoly- transcriptional level by a number of angiogenic growth fac-
sis on angiogenesis is not fully understood. It is known that tors. Moreover, angiogenic ECs in vitro and in vivo express
various angiogenic factors (e.g., bFGF) bound to the ECM high levels of PA inhibitor-1 (PAI-1),170,171 a protein which
are made available to ECs after the matrix is degraded. It is blocks the formation of plasmin by PA and thereby inhibits
likely, however, that the most important effect of proteoly- the activation of elastase and MMPs. An additional element
sis is to alter the malleability and adhesivity of the ECM to of control is the confinement of active uPA to the surface of
facilitate the movement of ECs through it. ECs. uPA is secreted as a proenzyme (pro-uPA) that binds
Two major groups of proteinases that regulate angio- to uPA receptor (uPAR), a glycoprotein which is anchored
genesis are the matrix metalloproteinases (MMPs) and the to the surface of ECs at the site of focal adhesions.172 The
plasmin/plasminogen activator (PA) system. MMPs secreted bound pro-uPA is subsequently activated by limited pro-
How Does Extracellular Matrix Control Capillary Morphogenesis? 337
teolysis.173 The proteolytic function of uPA at the cell sur- of neovascularization for efficient healing of wounds, bio-
face is facilitated by the binding of its substrate, plasmino- integration of prosthetic devices, functionality of the repro-
gen, to the plasma membrane by #-enolase-related proteins ductive system, and regulation of tumor growth and me-
and cell-surface chondroitin-sulfate proteoglycans.174,175 tastasis, we could not overemphasize the need for a more
After a period of time (possibly several hours), active uPA is complete and mechanistic understanding of angiogenesis
inactivated by binding to PAI-1, internalized, and rapidly as a therapeutic means for the prevention and treatment of
degraded.176 The importance of PAI-1 to the control of uPA human disease. In the field of tumor biology, for example,
activity is indicated by the effects of angiogenic growth fac- few would disagree that some form of anti-angiogenic
tors on the expression of the two proteins. bFGF and vascu- therapy might be beneficial for the treatment of solid tu-
lar endothelial growth factor (VEGF) increase the ratio of mors. However, there is considerable debate regarding the
uPA mRNA to PAI-1 mRNA in ECs, for a net increase in type of intervention (i.e., which stage of blood vessel growth
proteolysis. In contrast, TGF-!1 reduces the uPA:PAI-1 should be targeted), as well as the duration and route of
mRNA ratio for a net reduction in proteolysis—an effect administration of angiostatic drugs or compounds.
consonant with its inhibition of angiogenesis and invasion We have chosen to focus on questions and models that
of ECM by ECs in vitro.168 address the angiogenic process itself and have recapitulated
Migratory ECs exhibit increased expression of uPA and the acknowledged stages of vascular sprout formation and
uPAR relative to nonmigratory ECs in confluent monolay- progression—EC migration, alignment, proliferation, cohe-
ers. 168 Nanomolar concentrations of uPA or of its sion, lumen formation, and branching/anastomosis—in the
noncatalytic A-chain stimulate motility of ECs.177 Blockage context of cell adhesion, movement, and shape, molecular
of uPAR with monoclonal antibodies or a specific antago- biology, receptor-ligand interactions, and extracellular pro-
nist inhibits the migration of ECs on serum-coated substrates teolysis. To date, there are no “perfect” models in which to
in vitro to a greater degree than has been observed after the test angiogenic regulation, although much can be learned
inhibition of endogenous plasmin activity.178 Moreover, from culture systems in which ECs form capillary-like struc-
uPAR binds to the ECM component vitronectin and inhib- tures. At best, each of the models in vitro that were covered
its !1 integrin-dependent adhesion to fibronectin.179 Col- in this chapter appears optimal for one or more of the stages
lectively, these observations point to a direct involvement of requisite to blood vessel formation. In contrast, tissues of
the uPA/uPAR complex in the modulation of cell adhesion living animals provide excellent examples of angiogenesis,
during motility by a mechanism independent from proteoly- but their disadvantages include the complex interactions
sis. Since PAI-1 is also increased in migratory ECs,168 it is among several types of cells and the limitations in visualiza-
possible that this inhibitor could influence adhesive inter- tion and quantification of blood vessel growth.
actions between uPA/uPAR and ECM ligands. A synergistic We have also stressed the importance of the ECM and
role for PAI-1 in counteradhesion would explain the find- its capacity for the regulation of EC behavior and vascular
ing that synthesis of PAI-1 by subconfluent, migratory ECs morphogenesis. Clearly, alteration of the properties of ECM
is increased in the presence of the counteradhesive, through its molecular composition and/or proteolysis has
matricellular protein SPARC.180 profound effects on EC migration, proliferation, and bio-
The degree to which ECM must be degraded to facili- synthetic capacity, all of which can be affected, in part, by
tate angiogenesis in vivo is unclear; however, it appears that changes in cell shape and adhesion. Both adhesive (e.g., col-
the range of optimal proteolysis has an upper limit. For ex- lagen, fibronectin, laminin) and counteradhesive proteins
ample, the increased production of endogenous proteases (e.g., SPARC, TSP), as well as the integrins and other cell
by murine ECs that express the polyoma virus middle T surface receptors for ECM macromolecules, are important
oncogene is correlated with their organization into large, in the regulation of EC shape and function.
hollow cysts within fibrin clots in vitro.181 In the presence There remain several unanswered questions that are
of inhibitors of serine proteases, however, the cyst currently receiving considerable attention in the field of vas-
morphotype is replaced by capillary-like tubes that are gen- cular biology:
erated by the invasion of ECs into the fibrin matrix. The 1. Which are the macromolecules that are truly critical
fact that proteolysis of ECM inhibits the formation of en- for angiogenesis?
dothelial tubes indicates that angiogenic sprouts are not elon- 2. Is all angiogenesis the same (e.g., in a normal tissue
gated by forces derived solely from the parent vessel (e.g., by versus a tumor, and in the endometrium versus the
a hydraulic push). Alternatively, the sprouts pull themselves brain)?
outward by a process (e.g., traction) that requires mechani- 3. What is the initiating stimulus for sprouting?
cal support from the ECM. Accordingly, excessive proteoly- 4. How is selective or focal proteolysis controlled?
sis of the ECM would disrupt its mechanical continuity to 5. What stage of angiogenesis provides the most rea-
an extent that ECs would lack a fixed substrate against which sonable site for intervention to effect either stimula-
they could pull. tion or stasis?
Reagents and methodology are largely in place to ad-
Concluding Remarks dress these problems, although there is a continuing need
In this chapter we have introduced the fundamental for better quantitative models in vivo and in vitro. Within
characteristics of angiogenesis and have discussed the cell the next decade we should expect major and significant
biology of this highly complex process. Given the necessity progress in basic research that will allow clinicians,
338 Tissue Engineering of Prosthetic Vascular Grafts
bioengineers, and molecular/cellular biologists to develop tochemical localization and role in cell attachment. Lab
and test novel strategies for the control of vascular growth. Invest 1983; 49:362-370.
22. Sage H. Collagen synthesis by endothelial cells in culture.
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Facilitation of Healing
Matrix Modulation
CHAPTER 31
Collagen Matrices Attenuate Fibroblast Response to TGF-!
Richard R. Clark, John M. McPherson
Modified with permission from J Cell Sci 1995; 108:1251-1261.
Introduction
F ollowing loss of soft tissue, fibroblasts proliferate and produce an initially loose-weave
provisional matrix which is heavily vascularized and which contains fibronectin, hyalur-
onate, and relatively little collagen.1 Gradually this cell-rich granulation tissue becomes a
predominantly collagen-rich tissue. The ultimate outcome of this biological process is an
imperfect, yet expedient repair called scar formation. The rate and extent of repair depends,
in part, on the net balance of negative and positive modulatory signals that impinge on
fibroblasts at such sites.2
Fibroblasts’ responses to various soluble mediators, e.g., growth factors and cytokines,
have for the most part been studied with cells grown on tissue culture plastic.2-5 However, in
vivo fibroblasts are surrounded by an extracellular matrix (ECM), and ECM can have pro-
found effects on cell function.6-12 Thus the responsiveness of fibroblasts to growth factors
and cytokines has been evaluated when the cells were cultured on or in various ECM com-
ponents, especially collagen.13 Such studies provide important insights into the integration
of mediator and ECM signals on fibroblasts.11-13
Transformtin growth factor-! (TGF-!), a potent multifunctional cell regulatory pro-
tein factor,5 induces increased fibroblast synthesis of collagen and fibronectin when the fi-
broblasts are grown on tissue culture plastic.14-16 Since fibroblasts are usually surrounded by
a collagen matrix in vivo, determination of the response of fibroblasts cultured in collagen
gels to TGF-! would help elucidate the action of this factor in vivo.
It has become evident that the fibroblast phenotype that develops as a consequence of
collagen matrix differs dramatically depending on whether the matrix is relaxed, as occurs
in a floating collagen gel, or under tension, as occurs in an anchored collagen gel.13 Cells in
a relaxed collagen gel become stellate, mitogenically quiescent, and produce little col-
lagen.9,17-20 These are the morphological, proliferative and synthetic characteristics of nor-
mal dermal fibroblasts. In contrast, fibroblasts in an anchored collagen gel develop an elon-
gated fusiform morphology as they attempt to contract the attached gel; furthermore, they
actively proliferate and synthesize collagen.19-21 These are the morphological, proliferative
and synthetic characteristics of wound fibroblasts which have deposited substantial amounts
of collagen in the granulation tissue, and which have begun to compact the collagen matrix
and contract the wound.1
In this paper, we review evidence that type I procollagen and TGF-! are coexpressed in
fibroblasts of early, collagen-poor granulation tissue, while fibroblasts in collagen-rich granu-
lation tissue lack type I procollagen despite the persistence of TGF-!. Furthermore, human
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
344 Tissue Engineering of Prosthetic Vascular Grafts
dermal fibroblasts cultured in stressed collagen matrices, an B, respectively); however, type 1 procollagen was not appar-
environment that simulates late collagen-rich granulation ent in day 10 fibroblasts despite the persistence of TGF-!
tissue, synthesize little collagen in response to TGF- ! (panels C and D, respectively). Collagen matrix density in-
compared to fibroblasts cultured on tissue culture plastic, creased rapidly from day 5 to day 10 as judged by trichrome-
an environment that simulates early collagen-poor granu- stained histologic sections1 and photometric analysis of
lation tissue. Sirius red stained sections (Fig. 31.4). This led to the hy-
pothesis that the rapidly accumulating collagen matrix mark-
Results edly diminishes the ability of fibroblasts to produce addi-
tional collagen despite the presence of TGF-!.
Synchronous Expression of Type I Procollagen To test this hypothesis, human dermal fibroblasts were
and TGF-! in Wound Fibroblasts cultured in conditions to simulate early, collagen-poor
of Early Collagen-Poor Granulation Tissue granulation tissue or late, collagen-rich granulation tissue.
Fibroblasts in 5 day full thickness porcine wounds were Fibroblasts cultured in plastic wells are closely juxtaposed
examined as noted in Figure 31.1 for expression of type I to one another and produce abundant amounts of
procollagen and TGF-! using a murine monoclonal anti- fibronectin, similarly to fibroblasts in early granulation tis-
type I procollagen22 or a polyclonal rabbit anti-TGF-!,23 re- sue.24 Fibroblasts cultured in a stressed collagen gel, i.e., the
spectively. The two proteins demonstrated coordinate in- gel remains attached to a plastic well, simulate 10 day, col-
creasing expression from the wound edge to the wound cen- lagen-rich granulation tissue, since in both situations fibro-
ter (Fig. 31.2). No type I procollagen or TGF-! appeared in blasts form tension across a dense collagen matrix.1,20
cells near the wound edge (panels A and D, respectively),
expression occurred in some fibroblasts between the wound Collagen Matrix Attenuates the Ability of TGF-!
edge and wound center (panels B and E, respectively), and Stimulated Fibroblasts to Make Collagen
in many fibroblasts at the wound center (panels C and F, TGF-! stimulation of collagen synthesis was attenu-
respectively). ated when neonatal human dermal fibroblasts were cultured
in attached collagen gels compared to tissue culture plastic
Discordant Expression of Type I Procollagen and TGF-! (Fig. 31.5A). In contrast, TGF-! stimulation of noncollagen
in Wound Fibroblasts of Late Collagen-Rich synthesis was not substantially affected by the collagen ma-
Granulation tissue trix environment (Fig. 31.5B). The apparent TGF-! stimu-
When day 7 and day 10 wounds were probed for type I lation of noncollagen protein synthesis was largely attribut-
procollagen and TGF-! (Fig. 31.3), both proteins appeared able to a 2-fold increase in the specific activity of intracellu-
in most, if not all, wound fibroblasts at day 7 (panels A and lar proline pools (control 19,900 dpm/nmole; 100 pM TGF-!,
Fig. 31.1. A schematic of a 5 day old cutaneous wound in the flank of a Yorkshire pig. The boxes with
capital letters represent the areas of the wound shown in the photomicrographs illustrated in Fig. 31.2.
e = epidermis, d = dermis, g = granulation tissue, ft = fat, f = fibrous septa.
Collagen Matrices Attenuate Fibroblast Response to TGF-! 345
Fig. 31.2. Detection of procollagen and TGF-! in granu-
lation tissue fibroblasts 5 days after excisional wound-
ing of swine corium. (A)-(C): Photomicrographs taken
from the edge (A), intermediate zone (B), and center
(C) of wound granulation tissue stained with a mono-
clonal antibody specific for type I procollagen. Fibro-
blasts in the middle of granulation tissue stain strongly
for procollagen (C), but this molecule was not detected
in fibroblasts at the edge of the granulation tissue (A).
Some but not all fibroblasts in the intermediate zone
stained weakly positive (B). (D)-(F): Photomicrographs
from the edge (D), intermediate zone (E), and center
(F) of granulation tissue of adjacent sections of the same
wound, stained with rabbit antibody to a synthetic
polypeptide identical to the N-terminal 30 amino ac-
ids of TGF-!. Fibroblasts in the center of the granula-
tion tissue stained for TGF-! (F). Some but not all fi-
broblasts in the intermediate zone stained weakly posi-
tive for TGF-! (E) while none of the fibroblasts at the
periphery of the wound stained positive (D). Thus, the
distribution of staining for TGF-! and procollagen in
fibroblasts in granulation tissue in excisional swine skin
wounds 5 days after wounding appeared similar. Bar =
20 ∝m.
Fig. 31.3. Detection of procollagen (A,C) and TGF-!
(B,D) in granulation tissue fibroblasts at 7 days (A,B)
and 10 days (C,D) after wounding. All photomicro-
graphs were taken at the center of the wound. Anti-
type I procollagen antibodies reveal intense perinuclear
fluorescence in day 7 fibroblasts (A) but not in day 10
fibroblasts (C). Anti-TGF-! antibodies reveal intense
perinuclear fluorescence in day 7 fibroblasts (B) which
persists in day 10 fibroblasts (D). Bar = 20 ∝m.
38,900 dpm/nmole) as previously reported.15 Parenthetically, Five lines of evidence suggest that attenuation was not
TGF-! did not increase the total intracellular proline pool a trivial event. First, [14C]-labeled mixed amino acids and
(control, 1.62 nmole/∝g DNA; 100 pM TGF-!, 1.68 nmole/∝g albumin freely diffused out of collagen gels, obtaining an
DNA). Attached collagen matrix culture environment also equilibrium between the gels and the medium within 30
consistently attenuated the collagen synthetic response of minutes. Second, total DNA of cells cultured for 3 days in
adult human dermal fibroblasts to TGF- !, while the collagen gels was not appreciably different than the DNA of
noncollagen synthetic response was not affected (Table 31.1). cells cultured on plastic (Table 31.3). Third, LDH release
Four strains of fibroblasts were examined, two in duplicate from cells cultured 3 days on plastic or in collagen was not
experiments. Although marked differences were noted in appreciably different and was always less than 5% of the to-
TGF-! response among these strains, all strains demon- tal LDH. Fourth, basal collagen synthesis of fibroblasts cul-
strated attenuation of their TGF-! response when grown in tured in collagen gels was usually similar to basal collagen
collagen gels. In contrast, attached fibrin gels did not alter synthesis of fibroblasts cultured on tissue culture plastic
the collagen synthetic response to TGF-! (Table 31.2). (Table 31.4). Fifth, TGF-! equally stimulated noncollagen
synthesis of fibroblasts cultured in collagen gels or on tissue
346 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 31.4. Increase of collagenous matrix
in granulation tissue over time as deter-
mined by Sirius red birefringence. Acid-
alcohol fixed specimens from 5, 7, 10, and
14 day wounds were stained with Sirius red
and the birefringence of the central granu-
lation tissue photometrically measured.
Accumulation of wound collagen was then
determined as the quotient of granulation
tissue birefringence intensity divided by
normal dermal collagen birefringence in-
tensity x 100. The data points and brack-
ets represent the means ± SEM, respec-
tively, of three wound replicates at each
time point.
Table 31.1. Collagen matrices attenuate the collagen Table 31.2. Fibrin matrices do not attenuate the
synthetic response of adult human dermal fibroblasts to collagen synthetic response of adult human dermal
TGF-!1. fibroblasts to 100pM TGF-!.
Strain # Condition Collagen Noncollagen Collagen Fold Increase Noncollagen Fold Increase
!
synthesis with TGF-! synthesis with TGF-!!
1 TC Plastic 2.1a 1.2 dpm/ug dpm/ug
Collagen gel 1.5 1.4 DNAx10-3 DNAx10-3
2 TC Plastic 4.6 3.0
Collagen gel 2.7 2.5 TC Plastic 16.24±3.03a 87.60±27.13
3 TC Plastic 4.5 3.1 TC Plastic 69.07±30.09 4.3x 314.37±76.75 3.6x
Collagen gel 3.0 1.9 +TGF-!
3 TC Plastic 5.0 3.0 TC Plastic 15.78±9.78 157.14±11.35
Collagen gel 3.8 2.1 Fibrin gel 69.63±15.95 4.4x 539.30±96.25 3.4x
4 Tc Plastic 9.1 3.4 +TGF-!
Collage Gel 1.8 3.6 aMean of 3 replicates ± standard deviations
4 TC Plastic 9.9 3.3
Collagen gel 2.8 2.9
p < 0.004b NS
aFold increase of fibroblasts stimulated with 100pM TGF-b over Beta-aminoproprionitrile did not alter the collagen gel ef-
control
fect (data not shown).
bStatistics were performed on the ranks by the Wilcoxon test
Autoradiographic Analysis of TGF-!1-Stimulated
Proteins
When equal aliquots of medium from each experi-
mental condition shown in Figure 31.5 were analyzed on
culture plastic (Table 31.1). In addition, nonspecific adsorp- SDS-PAGE autoradiographs (Fig. 31.7A), most protein
tion of TGF-! to the collagen matrix was not observed. TGF-! bands were notably increased by 100, 300, or 1000 pM TGF-!
preincubated for 24 hours in collagen gels or in TC dishes (lanes 2-4, respectively and lanes 6-8, respectively) compared
stimulated collagen synthesis to the same extent when added to no TGF-!1 (lanes 1 and 5). Type I procollagen #1-and
subsequently to fibroblasts cultured on plastic. #2-chains and fibronectin (arrows) were increased to a
TGF-! had no consistent effect on the proportion of greater degree than most other proteins. When fibroblasts
collagen retained in the cell layers of neonatal or adult der- were cultured in collagen gels, the TGF-!-induced increase
mal fibroblasts whether the cells were cultured on plastic or of some protein bands was clearly attenuated (lanes 5-8)
in collagen gels (Fig. 31.6). For reasons that may be biologi- compared to cells cultured in tissue culture plastic (lanes
cally important but are presently unclear, more collagen was 1-4). To resolve whether the attenuated TGF-! response in-
retained in the cell layers of adult fibroblasts cultured in col- volved mostly collagenous or noncollagen proteins, the newly
lagen gels regardless of whether TGF-! was added (Fig. 31.6). synthesized proteins were digested with either pepsin or col-
lagenase, respectively, prior to SDS-PAGE.
Collagen Matrices Attenuate Fibroblast Response to TGF-! 347
Fig. 31.5. Effect of collagen matrix
environment on protein synthetic re-
sponse of neonatal human dermal fi-
broblasts to TGF-! 1. (A) Collagen
synthesis. (B) Noncollagen synthesis.
Fibroblasts were plated at 5 x 105 cells/
35 mm dish on TC plastic or sus-
pended in 4 mg/ml collagen gels,
treated with 0, 100, 300 or 1000 pM
TGF-!1, and labeled with [14C] pro-
line. Total collagen synthesis of fibro-
blasts on TC plastic (•) and in collagen
gels (▲) was the sum of collagen in
the medium and in the cell layer. Like-
wise, total noncollagen synthesis of fi-
broblasts on TC plastic (o) and in col-
lagen gels ()) was the sum of values
obtained for the medium and cell
layer. The total [14C]proline incorpo-
ration was normalized per ∝g DNA.
Basal collagen synthesis in (A) was
30,130 dpm/∝g DNA on TC plastic
and 20,480 dpm/∝g DNA in collagen
gels. Basal noncollagen synthesis in
(B) was 170,750 dpm/∝g DNA on TC
plastic and 95,420 dpm/∝g DNA in
collagen gels. Data shown is represen-
tative of three experiments with neo-
natal fibroblasts.
Increased collagen synthesis induced by TGF-!1 was 300, 1000 pM TGF-!1 (lanes 2-4 and lanes 6-8) compared
best demonstrated by SDS autoradiography of medium to no TGF-!1 (lanes 1 and 5); however, the TGF-! response
samples that had been pepsin digested to remove was markedly attenuated when fibroblasts were cultured in
noncollagen protein.25 The major bands appearing on SDS- collagen gels (lanes 6-8) compared to fibroblasts in tissue
PAGE autoradiography after pepsin digestion were #1(I) and culture plastic wells (lanes 2-4).
#1(III) chains (Fig. 31.7B, upper arrow), and #2(I) chains Using interrupted gel electrophoresis26 and autorad-
(Fig. 31.7B, lower arrow). These bands of collagen proteins iography on pepsin-digested samples, the TGF-! stimula-
were increased when fibroblasts were incubated with 100, tion of #1(I) and #1(III) collagen chains was equally attenu-
348 Tissue Engineering of Prosthetic Vascular Grafts
(Fig. 31.8, panels B and D) compared to unstimulated fi-
Table 31.3. Total DNA of fibroblasts cultured on plastic broblasts (Fig. 31.8, panels A and C), whether the fibroblasts
or in collagen gels. were cultured on tissue culture plastic (Fig. 31.8, panels A
and B) or in collagen gels (Fig. 31.8, panels C and D). How-
DNA (ug)* ever, whereas most fibroblasts on tissue culture plastic stained
TGF-B (pM) TC Plastic Collagen gel for type I procollagen after TGF-!1 treatment (> 90%) only
a rare cell in collagen gels did so (< 10%). To maximize the
0 4.8±1.2(9) 5.2±2.4(6) observable response to TGF-!, strain #4 fibroblasts were uti-
100 5.4±1.0(9) 5.3±1.2(6) lized (see Table 31.1). These immunofluorescence data cor-
300 4.5±0.7(2) 4.1±1.0(2)
roborated the biochemical data that TGF-! 1 stimulates fi-
1000 4.4±1.1(2) 4.0±1.2(2)
broblasts to produce more collagen in tissue culture plastic
*Mean ± standard deviation of (n) replicates. conditions and that collagen gel matrices attenuate this
response, apparently by inhibiting synthesis by most but not
all cells.
Table 31.4. Effects of attached collagen gels on basal Discussion
collagen synthesis (DPM/∝g DNA x 10-3) of human Fibroblasts undergo striking phenotypic modulation
dermal fibroblasts. during tissue repair, embryogenesis and morphogenesis. Elu-
cidating the controls of such phenotype switching will fa-
Exp.# Condition Neonatal Adult cilitate our ability to intercede when tissue development or
repair goes awry. As a first step we designed a series of ex-
1 TC Plastic 15.6 14.1 periments to identify the temporal relationships of certain
Collagen gel 15.5 13.0 fibroblast phenotypic markers with ECM maturation and
2 TC Plastic 30.1 8.3 growth factor appearance. We observed that F-actin bundle
Collagen gel 20.5 10.6 formation and fibronectin receptor expression are coordi-
3 TC Plastic 61.6 5.6 nately regulated with fibronectin matrix assembly in
Collagen gel 55.5 7.8
4 TC Plastic 8.4 13.1
wounds1 as had been previously observed in cultured fibro-
Collagen gel 12.9 11.9 blasts.27-29 Here we review data demonstrating that type I
5 TC Plastic 7.1 8.4 procollagen expression and TGF-! are coordinately expressed
Collagen gel 6.2 14.3 in early stages of a healing wound (Figs. 31.2 and 31.3 A,B),
NS NS however, by 10 days type I procollagen is no longer appar-
Statistics were performed on the ranks by the Wilcoxon test ent while TGF-! expression persists (Fig. 31.3 C,D).
The coordinate expression of TGF-! in early wounds
was not surprising since TGF-! is known to induce fibro-
blast production of types I and III collagen in vitro14-16 and
ated by collagen gels (data not shown). Thus, types I and III to induce a fibrotic reaction in vivo.15,30-32 However, the dis-
collagen are regulated synchronously under the conditions appearance of type I procollagen in fibroblasts that still ex-
of these experiments. pressed TGF-! was unanticipated. Although the antibody
Fibronectin was the major noncollagen band in auto- used for immunoprobing is specific for mature TGF-!,33 the
radiographs of collagenase digested medium samples mature TGF-! may be latent or inactivated.34-36 Alternatively,
(Fig. 31.7C, arrow). Western blot analysis and immunopre- the fibroblasts may have become relatively refractory to
cipitation with anti-fibronectin antibodies confirmed that TGF-!. Since the density of the collagen-rich ECM rapidly
the band was fibronectin (not shown). TGF-! markedly in- accumulated (Fig. 31.4) as type I procollagen expression dis-
creased fibronectin synthesis as previously shown,14 and appeared (Fig. 31.3C), we hypothesized that collagen ma-
collagen matrices appeared to have little effect on the TGF-! trix might attenuate the fibroblasts’ response to TGF-!.
stimulation of fibronectin in either the medium (Fig. 31.7C, Initially we were disturbed that there was a 40% in-
arrow) or cell layer (Fig. 31.7D, arrow). Unfortunately, syn- crease in collagen density between days 10 and 14 (Fig. 31.4)
thesized proteins in the cell layers of fibroblasts grown in despite the apparent absence of further collagen production
collagen gels could not be visualized by SDS-PAGE (Fig. 31.3C); however, the wound area decreased by approxi-
autoradiography except after collagenase treatment, since the mately 33% from day 10 to day 14,1 presumably secondary
large amount of collagen present caused distortion of the to fibroblast-driven reorganization and compaction of
protein bands. wound matrix collagen bundles. Thus the increase in wound
collagen density between days 10 and 14 is probably attrib-
TGF-! Stimulation of Type I Procollagen utable to collagen compaction.
as Visualized by Immunofluorescence Microscopy To test the hypothesis that collagen matrix attenuates
Using a monoclonal antibody specific for the carboxy- fibroblasts’ response to TGF-!, we assayed the ability of cul-
terminal globular domain of type I procollagen,22 we ob- tured human fibroblasts to respond to TGF-! in the pres-
served increased intensity and numbers of fluorescent, peri- ence of stressed collagen gels, a culture system in which fi-
nuclear granules in fibroblasts after stimulation with TGF-! broblasts are under tension similar to that of collagen-rich,
Collagen Matrices Attenuate Fibroblast Response to TGF-! 349
Fig. 31.6. Effect of TGF-! and collagen matrix on compartmentalization of newly synthesized collagen
between the media and cell layers of neonatal (A) and adult (B) human dermal fibroblasts. Fibroblasts
were cultured on TC plastic or in collagen gels and metabolically labeled with [14C]proline as described
in Fig. 31.5. Collagen production in the culture medium and cell layer was determined using the modi-
fied Peterkofsky method. The amount of [14C]proline incorporated into newly synthesized collagen
was normalized per ∝g DNA. The percent shown in each bar represents the percent collagen retained in
the cell layer.
350 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 31.7. SDS-PAGE autoradiography confirms that collagen matrices attenuate the collagen synthetic response of neonatal
human dermal fibroblasts to TGF-! 1. (A) Total newly synthesized protein in the medium. (B) Newly synthesized pepsin-
resistant protein in the medium. (C,D) Newly synthesized collagenase-resistant protein in the medium and in the cell layer,
respectively. Medium samples from equal cell numbers based on DNA were analyzed by SDS-PAGE autoradiography. Samples
were taken from fibroblasts cultured in the absence of TGF-!1 (lanes 1 and 5), and in the presence of TGF-!1 (100 pM, lanes
2 and 6; 300 pM, lanes 3 and 7; 1000 pM, lanes 4 and 8). Arrows indicate fibronectin (FN), type I procollagen #1 (pro #1) and
#2 (pro #2) and type I collagen #1 and #2.
Fig. 31.8. Immunofluorescence staining for
procollagen in cultured neonatal human der-
mal fibroblasts. Cultures containing 5 x 105
fibroblasts per 35 mm dish were prepared on
tissue culture plastic (A and B) or suspended
in 4 mg/ml hydrated collagen gels (C and D)
and treated with 0 or 300 pM TGF-! 1 (A, C
and B, D, respectively) for 22 hours. All cells
were fixed with acid-alcohol. Cells in collagen
gels were embedded in paraffin and sectioned.
Cells on tissue culture plastic and in the sec-
tioned collagen gels were immunostained for
type I procollagen using an indirect avidin-
biotin technique. Arrows indicate the areas of
procollagen staining. Nuclei of cells in col-
lagen gels (arrowheads) were counterstained
by the 1% paraphenylenediamine used in the
mounting medium. Bar = 20 ∝m.
late (e.g., day 10), granulation tissue in which fibroblasts are environment did not appear to be a trivial effect of the col-
under tension as they reorganize and compact the collagen lagen gels on TGF-! or fibroblasts, as TGF-! was not bound
matrix.1,20 As shown in Figs. 31.4-31.7 and Table 31.1, such by the collagen gels, and collagen gels did not cause nutrient
collagen gels consistently and significantly attenuated the deprivation or cell injury. Furthermore, TGF-! stimulation
collagen synthetic response of fibroblasts to TGF-! compared of noncollagen synthesis was usually not altered by the at-
to fibroblasts cultured on plastic. When cultured on plastic, tached collagen gels used in these experiments (Table 31.1,
fibroblasts grow in close juxtaposition and deposit an abun- Figs. 31.4 and 31.6). Specifically TGF-! stimulation of
dance of fibronectin in the pericellular matrix, a situation fibronectin synthesis appeared unaffected by stressed col-
that simulates early, fibronectin-rich and collagen-poor, lagen gels. Biochemical results were confirmed by immun-
granulation tissue. The attenuation by a collagen matrix ofluorescence studies of fibroblasts (Fig. 31.8). Most if not
Collagen Matrices Attenuate Fibroblast Response to TGF-! 351
all fibroblasts cultured on plastic appeared to respond to if any, when fibroblasts are cultured in attached collagen gels
TGF-! by expressing perinuclear granular staining for type confirm the studies from the Grinnell laboratory.19-21 Al-
I procollagen (Fig. 31.8B), while most cells in collagen were though the attached collagen gel paradigm predicts that fi-
negative even after TGF-! stimulation (Fig. 31.8D). Never- broblasts under tension as observed in 10 day, collagen-rich
theless, an occasional cell in collagen stained at least as in- wounds would not show a decrease in basal collagen syn-
tense for type I procollagen as the TGF-! stimulated fibro- thesis1 ( Fig. 31.1), our studies clearly demonstrate that fi-
blasts cultured on plastic (Fig. 31.8D). Thus, there appeared broblasts in these conditions do lose their responsiveness to
to be a marked heterogeneity of cultured fibroblasts in their TGF-!. Recently we have found that while PKC-3 and Nf-∀b
ability to respond to collagen matrix. are activated by both stressed and relaxed collagen matrices,
The mechanism by which collagen matrix alters the other signal transduction pathways are, however, affected
responsiveness of fibroblasts to TGF-! is not known. One differentially by these two physical forms of collagen matrix
possibility is that the collagenous environment (unpublished data).
downregulates one or more of the three known cell surface The concept of collagen matrix attenuation of fibro-
receptors for TGF-!.37,38 In fact, given that we have observed blast collagen synthesis to TFG-! is appealing and is consis-
a differential effect of collagen gels on the biosynthesis of tent with previous results which showed that collagen ma-
collagen and fibronectin in response to TGF-! (Fig. 31.6), trices downregulated the responsiveness of fibroblasts to
collagen matrix may differentially affect these receptors. As PDGF and other growth factors.42,45 These data provide in
a first step toward investigating these possibilities, we probed some measure an explanation for the orderly progression of
RNA extracts of human adult dermal fibroblasts for type II the wound repair process by a means of positive signals that
and type III TGF-! receptor expression when fibroblasts were stimulate cells to generate an environment that both pro-
grown in plastic tissue culture dishes verses collagen matri- vides for repair and establishes feed-back regulation of the
ces. Using cDNA probes for the types II and III receptors process.
obtained from Drs. Herbert Lin, Harvey Lodish, Robert In contrast to the suppression effects of collagen gels
Weinberg, and Xiao-Fan Wang,39,40 we found no detectable on collagen synthesis, the permissive effects of fibrin gels
differences in mRNA expression among these conditions. are consistent with the role of this molecule in providing a
Nevertheless, at least one study has shown that the extracel- provisional matrix during the early phases of the wound
lular matrix environment can modulate TGF-! receptor ex- repair to support cell invasion, proliferation and matrix
pression with remarkable effects on cell responses to TGF-!.41 deposition. Thus the normal process of wound repair ap-
Alternatively, collagen matrix may influence signal pears to be under the control of both soluble factors that
transduction42 and this may influence, perhaps differentially, stimulate certain events and the extracellular matrix, which
the biosynthesis of ECM molecules. The pathway(s) through can modulate the responsiveness of cells to these signals. It
which collagen matrix transmits its effect on fibroblasts are is likely that certain pathological fibrotic conditions develop
currently a focus of study in our laboratory. It is clear that when these feedback regulation systems become ineffective.
collagen matrix activates protein kinase-3 (PKC-3) and
Nf-∀b, which leads to upregulation of #2!1 collagen recep- Acknowledgments
tors;43,44 however, the relationship of these pathways to col- Funding for this work was provided by NIH grants
lagen matrix attenuation of TGF-!-stimulated collagen syn- HL27353 and AM31514 to Richard A. F. Clark.
thesis is not known.
An interesting byproduct of this study was the failure References
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Facilitation of Healing
Matrix Modulation
CHAPTER 32
Extracellular Matrix Proteins Are Potent Agonists
of Human Smooth Muscle Cell Migration
Terry L. Kaiura, K. Craig Kent
Introduction
T he current treatments of atherosclerotic occlusive disease are multiple and include by-
pass, endarterectomy, and angioplasty. Unfortunately, the long term success of these
interventions is significantly jeopardized by a process known as intimal hyperplasia.1,2 Tis-
sue engineering and subsequent creation of a prosthetic graft that is resistant to the forma-
tion of an intimal plaque would be instrumental in improving the long term outcome of
vascular reconstructions. In order to synthesize the “ideal” vascular graft, however, it is im-
perative that the critical cellular processes that precede the formation of intimal hyperplasia
be understood.
The three essential components of intimal hyperplasia are smooth muscle cell (SMC)
migration, proliferation, and extracellular matrix (ECM) production.3 Although all three of
these events are necessary for restenosis, their relative importance is not well understood. In
normal human vasculature, the intima is almost completely devoid of SMCs. Thus, migration
of SMC from the vessel media into the subintimal space is a critical initial step in the initiation
of the restenotic process. Confirming the importance of SMC migration are studies that
demonstrate that over 50% of the neointimal plaque is composed of nonproliferating SMC.4
Following vascular injury, SMC are exposed to a variety of growth factors, cytokines,
and ECM proteins, all of which can potentially affect SMC migration. The role of growth
factors in SMC chemotaxis has been studied in great detail and the platelet-derived growth
factor BB homodimer (PDGF-BB) has been identified as an extremely potent agonist of
SMC migration.5 Moreover, a plethora of other growth factors including fibroblast growth
factor (FGF), epidermal growth factor (EGF), and transformtin growth factor-! (TGF-!)
have been found to affect SMC chemotaxis.6,7 ECM proteins are also abundant in the arterial
wall, and SMC are exposed to these proteins following vascular injury as well.8 The role of
ECM proteins in SMC migration, however, is less well understood, and is the focus of
this chapter.
Cell Migration
Cell migration is a dynamic, complex process, the molecular biologic basis of which
has not been fully described. The central component of the cellular apparatus that is neces-
sary for migration is the actin cytoskeleton.9 Actin is organized into stress fibers that create
the framework of a cell. These filaments are anchored by actin binding proteins such as
#-actinin10 to the cell membrane via a series of proteins termed the focal adhesion complex.11
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
354 Tissue Engineering of Prosthetic Vascular Grafts
The focal adhesion complex itself is composed of clusters of Therefore, cellular secretion of proteolytic agents such as
proteins such as tensin, p125 focal adhesion kinase (FAK), matrix metalloproteinase (MMP)-2, MMP-9, tissue type
paxillin, talin, and vinculin, that bind to transmembrane plasminogen activator, and urokinase plasminogen activa-
receptors called integrins (Fig. 32.1).12 Integrin receptors in tor appear to be necessary for cell locomotion.18,19 It has
turn bind via their extracellular domains to surrounding been demonstrated that PDGF increases the expression of
matrix.13 several proteases found in the ECM;20 therefore PDGF may
Cellular migration is a cyclic process that initially re- contribute to cellular migration by acting directly on the
quires detachment of the leading edge of a cell, reorganiza- cytoskeleton or also by modifying the environment that sur-
tion of the actin filaments to allow forward movement, fol- rounds a cell. Heparin, for example, which is a known de-
lowed by reattachment of this “pseudopod” in a new loca- terrent of intimal hyperplasia inhibits expression of tissue
tion.14 The body of the cell is then pulled forward to “catch type plasminogen activator and collagenase.21
up” with the leading edge, resulting in forward movement The migratory response of SMC to an agonist can as-
of the entire cell. This complex process requires that both sume three different patterns, depending upon the state and
focal adhesions and actin filaments be cyclically dissolved solubility of the stimulatory protein. Chemokinesis describes
and then reformed. random cellular migration in response to a soluble factor,
The integrin receptor is central to the interaction be- chemotaxis refers to directed migration toward a positive
tween the cytoskeleton and the external cellular environ- gradient of soluble attractant, and haptotaxis defines directed
ment.15 Integrin receptors have large extracellular domains, cellular migration toward a positive gradient of an insoluble
a single membrane-spanning domain and a short cytoplas- agonist such as a substrate bound matrix protein.21 Whether
mic domain. The extracellular domain attaches to matrix or not matrix proteins are in a soluble or insoluble form at
proteins that are specific for each integrin receptor and the the time of vascular injury is not clear. This may be an im-
cytoplasmic domain interacts with focal adhesions and ac- portant distinction, since the magnitude of the migratory
tin filaments. It is increasingly recognized that integrin- response may vary depending upon whether the stimulus is
matrix interactions are not purely structural or biomechani- that of haptotaxis or chemotaxis. Moreover, it has been
cal, but that integrins transduce signals into cells which may shown that the signaling pathways that mediate these two
be important in the regulation of multiple processes includ- responses are distinct.22 Possibly all three stimuli have the
ing cell motility.16 Thus, integrins are similar to growth fac- potential to influence vascular SMCs during the repair and
tor receptors in that ECM proteins can act as ligands that remodeling process that follows vascular injury.3
stimulate signaling events that ultimately influence cellular
behavior. In the case of migration, integrins not only pro- Extracellular Matrix Proteins
vide the structural mechanism that allows a cell to attach ECM proteins are ubiquitous in the normal vessel wall.
and migrate across ECM, but these same integrin receptors Endothelial cells manufacture an underlying basement mem-
may transduce messages via ECM proteins into the cell that brane that is superficial to the internal elastic lamina. This
influence the propensity of a cell to migrate.17 layer is composed primarily of type IV collagen, laminin,
Lysis of ECM may also be an important component and heparan sulfate proteoglycan.23 The media is composed
of the process of migration. Lysis of these proteins allows a of smooth muscle cells that are surrounded by a plethora of
pathway to be created through which the cell can migrate. ECM proteins that include fibronectin,24 types I, III, and V
Fig. 32.1. The focal adhesion complex,
illustrating clusters of proteins in-
cluding paxillin, tensin, vinculin,
p125FAK, and talin which interact with
both actin filaments and integrins.
Extracellular Matrix Proteins Are Potent Agonists of Human Smooth Muscle Cell Migration 355
collagen,25 as well as elastin,26 and proteoglycans.27 The ad- following vascular injury, its expression rapidly increases.45
ventitia, which consists mainly of collagen, is bound on its Yabkowitz et al have found that TSP is a potent mediator of
outer aspect by loose connective tissue that includes collagen calf pulmonary artery SMC migration.47 Either inhibition
type I and proteoglycans.23 of TSP synthesis or TSP-SMC interactions decreased in re-
ECM comprises as much as 60-80% of a mature inti- sponse to SMC migration. Interestingly, SMC migration to
mal plaque.28,29 The predominant proteins are collagen types the combination of TSP and PDGF was greater than the
I and III,30 elastin,31 fibronectin,32,33 and proteoglycans such maximal response observed with PDGF alone.47
as versican-like chondroitin sulfate.34 Two weeks following The glycosaminoglycan hyaluronan (HA) is
vessel injury and after the period of maximal SMC prolif- synthesized and deposited in the neointima soon after vas-
eration and migration, cell numbers in the intima reach a cular injury.48 Riessen et al found through histological analy-
maximum. The continued expansion of intimal plaque is ses of both rat and human vascular tissues that HA is a
then due to the synthesis of the ECM proteins previously predominant component of the early vascular lesion.48
mentioned.28 A variety of less predominant proteins have Savani et al demonstrated that HA stimulated the migra-
also been identified in intimal plaque including osteo- tion of arterial bovine SMCs, and that an antibody to the
pontin,35 vitronectin,36 thrombospondin,37 and tenascin.38 HA-binding RHAMM receptor blocked migration.49 CD44
These proteins are synthesized and secreted primarily by is another HA binding receptor that is upregulated in the
SMC. The prevalence of ECM in the normal and pathologi- neointima and may also play a role in SMC migration.20
cal vessel has spurred our investigation into the effect of these Moreover, the ability of HA to bind to large amounts of water
proteins on SMC migration. and to create a gel-like environment may also potentially
facilitate SMC migration.48
Extracellular Matrix Protein and Migration Vitronectin, otherwise known as the serum-spread-
In recent years, the effect of a number of ECM pro- ing factor or S-protein is present as an ECM protein in the
teins on SMC migration has been investigated. In the fol- circulation, as well as in the vessel wall.36 This ECM protein
lowing paragraphs, the results of these investigations will be is also increased in atherosclerotic tissues.50 Vitronectin is
reviewed. involved in the adhesion of cells to the ECM and in the regu-
Osteopontin has been widely studied because of its lation of complement and coagulation pathways.51 Naito et
expression in tissues that undergo remodeling, such as the al found that vitronectin invoked SMC haptotaxis and pos-
smooth muscle layer of the intestinal tract and the normal tulated that vitronectin, which is found in the subendothe-
intima of the ductus arteriosus.20 Liaw et al have demon- lial matrix, may stimulate migration of SMCs from the me-
strated that osteopontin is dramatically elevated in the rat dia to the intima.52 Moreover, Brown et al have shown that
carotid artery and aorta following balloon injury.39 Studies vitronectin induces human aortic SMC migration via the
of human vascular tissue have revealed focal expression of #v!3 integrin to an extent that is comparable to that of the
osteopontin in atherosclerotic plaques but not in normal growth factor PDGF-BB.53
human arteries.40 Osteopontin, in vitro, has a strong stimu- Although all of the above described proteins are ex-
latory effect on SMC chemotaxis.41 Moreover, antibodies pressed following arterial injury and promote SMC migra-
directed against osteopontin inhibit SMC migration and also tion, these proteins constitute a relatively small portion of
neointima thickening in the injured rat carotid artery.42 It is the total ECM within both normal and diseased vessels. More
likely that osteopontin works in conjunction with other ECM predominant components of normal artery and vein and
proteins, integrins, and matrix proteases to promote adhe- intimal plaque include proteins such as collagen types I and
sion and migration of SMCs. IV, fibronectin, and laminin.24 The effect of these ECM pro-
Chen et al has shown that the large ECM glycopro- teins on SMC migration has been less well investigated. As a
tein, tenascin, is distributed throughout the neointima of result, our group has recently studied the effect of these four
hyperplastic lesions derived from human arteriovenous proteins on all three mechanisms of migration (namely
polytetrafluoroethylene grafts that have been removed for chemokinesis, chemotaxis, and haptotaxis) using human
revision.43 Tenascin has also been found in the intimal layer smooth muscle cells derived from saphenous veins.
of injured rat aorta and carotid arteries.38 This glycopro- We found that collagen types I and IV, fibronectin,
tein, which has been reported to have both adhesive and anti- and laminin were all very potent agonists of SMC migra-
adhesive domains, is a mediator of focal adhesion disassem- tion.54 In our initial studies, we determined the concentra-
bly as well.44 As such, Majesky has speculated that this dis- tion of each matrix protein that produced maximal cell
bursement of focal adhesions, or sites of cellular attachment movement. Maximal migration was achieved with interme-
to the ECM, may facilitate the transition of a cell from a diate concentrations of fibronectin and laminin. Higher con-
nonmotile to a migrating state.45 Accordingly, there is centrations of these two proteins did not produce a further
evidence that tenascin supports the migration of rat aortic increase in the migratory response. Type I and IV collagen
smooth muscle cells, although the effect of this ECM on similarly produced maximal migration at intermediate con-
human SMC migration has not been investigated.46 centrations of protein; however, higher concentrations of
Similarly to tenascin, thrombospondin (TSP) pos- both collagens led to a decrease in SMC migration. Corrobo-
sesses both adhesive and counteradhesive properties and is rating our findings, DiMilla et al found that high concen-
a prominent facilitator of focal adhesion disassembly.44 TSP trations of type IV collagen increased SMC attachment, but
is present in the ECM of the normal arterial wall; however, decreased SMC migration.55 These authors concluded that
356 Tissue Engineering of Prosthetic Vascular Grafts
cell migration on a matrix protein may be inversely related We found that simultaneous stimulation of SMC with
to the ability of a cell to attach this protein. PDGF-AB and collagen type I or IV, fibronectin, or laminin
To determine the relative potency of these proteins as synergistically enhanced SMC migration to a level that was
stimulants of migration, we studied SMC chemotaxis, greater than the addition of their individual effects.60 At both
haptotaxis, and chemokinesis in response to optimal con- low and high concentrations of the collagens, the chemo-
centrations of each matrix protein.54 We also compared the tactic response was enhanced 20-60% by the simultaneous
relative effect of these matrix proteins to the prototypical stimulation of SMC with PDGF and collagen. Alternatively,
migratory stimulus, PDGF. Collagen types I and IV produced with low concentrations of laminin or fibronectin the re-
the most significant increase in chemotaxis, with a respec- sponse with PDGF was additive, whereas, at higher concen-
tive increase of approximately 17- and 21-fold in the rate of trations, laminin and fibronectin produced a 40-70% en-
migration compared to control. Providing less of a stimula- hancement of PDGF-induced migration compared to what
tory effect was fibronectin (5-fold increase), followed by would be anticipated by the addition of these two proteins.
PDGF-AB (3-fold increase) and finally laminin (2-fold in- Surprisingly, we did not find synergy between the growth
crease). This same hierarchy was demonstrated for both factors EGF or bFGF and any of the ECM proteins.60
chemokinesis and haptotaxis as well. Synergism between growth factors and ECM proteins
Thus, despite the previous focus on growth factors as has been previously observed in other cell types. As previ-
stimulants of SMC migration, our findings suggest that ously mentioned, Yabkowitz et al, who studied the effect of
matrix proteins, which are plentiful in the vessel wall after TSP on PDGF-dependent migration of calf pulmonary
arterial injury, are as a group more potent stimuli of SMC artery SMCs, noted a 60% increase in PDGF-induced mi-
migration.54 Although PDGF-BB is a stronger stimulant for gration with concentrations of TSP that alone had no effect
migration than PDGF-AB, neither of these growth factors on SMC migration.47 Vitronectin and type IV collagen have
could produce a migratory response that equaled that of also been noted to synergistically increase chemotaxis
collagen type I or type IV.54 We and others have also previ- induced by insulin-like growth factor I in a human breast
ously demonstrated that soluble growth factors such as EGF cancer cell line.61
and bFGF are less potent agonists of SMC migration than
PDGF-AB.56 These findings imply that matrix proteins may Integrins
be the predominant “in vivo” stimulus for SMC migration The above outlined effect of matrix proteins on SMC
following arterial injury. locomotion are mediated largely through activation of
In these studies, we consistently found that haptotaxis integrin receptors.62 It is through these receptors that ma-
(SMC migration toward a gradient of insoluble matrix pro- trix proteins are able to influence the intracellular machin-
tein) was the most profound stimulus of human SMC mi- ery that is necessary to initiate the migratory response. Iden-
gration.54 These findings were reproducible for all four ma- tification of the integrin receptors that are required for ma-
trix proteins; the SMC response to haptotaxis was on aver- trix protein-driven SMC migration is critical to the under-
age 33% greater than the response of SMC to chemotaxis standing of this complex process.
for the same agonist. Haptotaxis of neoplastic cells has been Integrins are a family of heterodimeric cell surface
previously studied in some detail and is thought to play a receptors composed of # and ! subunits.63 Approximately
significant role in the invasion and spread of metastatic tu- 17 different # subunits and 8 ! subunits and over 20 differ-
mor cells.57 It appears that haptotaxis and chemotaxis are in ent #! pairings have been identified. Three families of
fact very distinct processes rather than an extension of the integrins have been designated according to their ! subunits,
same process.58 In studies with thrombospondin, haptotaxis which include !1, !2, or !3. Beta 2 is localized largely in plate-
and chemotaxis were found to be mediated by completely lets; however, !1 and !3 have been detected in vascular
separate peptide domains of the thrombospondin molecule SMCs.15 A single ECM protein is able to bind multiple
and the signaling pathways activated by these two processes integrin receptors and a single integrin receptor is able to
were also very different.59 bind multiple ligands.64 In this way, the same ECM protein
After investigating the influence of individual growth can mediate different cellular responses via a variety of
factors and ECM proteins on SMC migration, we next stud- integrin receptors. Therefore, past associations of a single
ied the chemotactic response of SMC to combinations of integrin and receptor such as the designation of #5!1 as the
these proteins.60 At the time of arterial injury, SMCs are ex- “fibronectin” receptor and #v!3 as the “vitronectin” receptor
posed to a plethora of growth factors and ECM proteins. are too restrictive, since both #5!1 and #v!3 are able to be
Presumably these proteins can act in concert to influence affected by multiple ECM proteins. Thus, the control over
SMC migration. Possible interactions include: cellular function exerted via integrin receptors is complex
1. One factor may mask the other, with the combined and critically important to normal and abnormal cellular
response being equivalent to that of the more po- function. Although there has been extensive investigation
tent agonist; of integrins and their importance in vascular biology, here
2. The two factors may have an additive response; we will discuss the different integrins involved in matrix
3. The two factors may act in a synergistic manner in protein driven SMC migration.
which the sum of both factors is greater than the sum Choi et al explored the role of the #v!3 integrin in hu-
of their individual effects.60 man and rabbit PDGF-AB stimulated SMC migration.65
These investigators chose to study #v!3 because of its ability
Extracellular Matrix Proteins Are Potent Agonists of Human Smooth Muscle Cell Migration 357
to bind to several ECM proteins by its RGD-containing bind- haptotaxis to type I and IV collagen, fibronectin, and
ing site, making it a possible candidate as a mediator of cell laminin.68 This effect was consistently greater for type I col-
motility. Antibodies to #v!3, as well as an RGD peptide an- lagen and laminin versus type IV collagen and fibronectin.
tagonist of this same integrin, both inhibited PDGF-AB Moreover, a blocking antibody to the #2 integrin subunit
stimulated SMC migration in vitro, albeit incompletely, inhibited chemotaxis and haptotaxis to type I and IV col-
implicating involvement of other integrins in this process. lagen and laminin, though these effects were less than those
They did not assess the in vitro role of #v!3 in matrix pro- observed with the !1 antibody.68 In similar experiments,
tein driven SMC migration. In a rabbit carotid injury model, Skinner et al found that a blocking antibody to the #2!1
these same investigators found that a monoclonal antibody integrin also inhibited migration of cultured human SMCs
to #v!3 did not inhibit intimal hyperplasia, although a spe- on type I collagen.64
cific RGD peptide antagonist did. Further supporting the There is some evidence that the #1!1 integrin might
role of #v!3 in SMC migration, Clyman et al studied migra- also be involved in SMC migration. Clyman et al detected
69
tion in fetal lamb ductus arteriosus SMC and found that #1!1 in cultured rat vascular SMC. Moreover, Gotwals et
SMC migration on type I and IV collagen, fibronectin, and al, again using rat SMC, found that #1!1 is the primary fa-
laminin was mediated predominantly by the #v!3 integrin.66 cilitator of SMC migration on collagen.70 It is possible that
The importance of the #v!3 integrin in SMC migration, how- the findings of the latter two studies were related to the use
ever, remains controversial. Recent immunocytochemical of rat SMC, and the critical integrin necessary for SMC mi-
studies by Hosiga et al revealed that the #v!3 was localized gration may differ between human and rat.
only in the media and not the neointima of rat aortic tissue In our immunohistochemical studies, the #5 subunit
following injury.67 Skinner et al were not able to identify the was found homogeneously distributed throughout normal
64
#v!3 integrin in an arterial SMC line. In immunohis- human saphenous vein vascular SMCs and in hyperplastic
tochemical studies from this laboratory, cultured human plaques from saphenous vein grafts; however, a blocking
saphenous vein SMC stained only weakly positive for the antibody to the #5 integrin subunit had no effect on chemo-
68
#v!3 integrin and only after permeabilization. We were not taxis or haptotaxis of saphenous vein SMC following their
able to identify the #v!3 integrin in the intima or media of stimulation with any of the ECM proteins.68 Though the
hyperplastic lesions excised from patients following saphe- #5!1 has been reported by other investigators to be the pro-
nous vein bypass grafting (despite positive staining for this totypical integrin for fibronectin,21 our results suggest that
integrin in the microvessels of the adventitia of these same this integrin does not mediate fibronectin migration of hu-
lesions), nor was #v!3 present in atherosclerotic plaque re- man saphenous vein SMC.
moved at the time of carotid endarterectomy. Using SMC It is important to note that there is tremendous
derived from human saphenous vein and blocking antibod- variation in the distribution and function of integrins from
ies, we found that #v!3 was not required for ECM protein one tissue to another (animal versus human, primary versus
(collagen type I or IV, laminin, fibronectin, or vitronectin) cultured cells, artery versus vein). Therefore, it is necessary
stimulated SMC migration; however #v!3 did play a role in to critically analyze data from the appropriate cell type before
PDGF mediated chemotaxis.68 making conclusions about the importance of integrins in
Our studies and those of others suggest that the !1 remodeling after vascular injury. In studies of SMC derived
integrin subunit has a more dominant role in SMC migra- from human saphenous vein, we and Skinner et al have found
tion.68 Skinner et al studied several !1 integrin combinations the #2 and !1 subunits to be the most critical receptors
and demonstrated that #1!1, but not #2!1, was expressed in required for ECM protein stimulated chemotaxis and
vivo in normal intact human arterial tissue; however in cell haptotaxis.64,68 Thus, modulating the #2!1 integrin may be a
culture the expression of #1 was downregulated and #2!1 was potentially effetive method for controlling intimal hyper-
upregulated.64 These authors postulated that #1!1 may be plasia.
present in normal venous tissue, but that hyperplastic plaque
might then preferentially express #2!1, and that this conver- Cell Signaling
sion might contribute to the pathophysiology of intimal We and others have extensively evaluated the intrac-
hyperplasia. In immunohistochemical studies of normal ellular signaling pathways associated with PDGF-induced
human saphenous vein and specimens of intimal hyperpla- cell migration. A variety of signaling proteins and ions ap-
sia derived from excised vein grafts, we found the !1 subunit pear to be involved, including phosphatidylinositol-3-OH
to be homogeneously distributed throughout both normal kinase (PI3K),71 phospholipase C (PLC),72 MAPK,73 Src,74
and abnormal tissues.68 Similarly, the #1 integrin was found calcium, 75 calmodulin-dependent protein kinase II
in normal human saphenous vein as well as hyperplastic tis- (CamKII),76 and the small G-protein rho.77 The intracellu-
sue from saphenous vein grafts. Interestingly, the #2 integrin lar signaling events that mediate matrix protein-induced
was only identified in specimens of intimal hyperplasia, cor- SMC migration have been less well studied. Our early inves-
roborating the hypothesis proposed by Skinner et al that the tigations suggest that at least some of these pathways may
#2!1 integrin is expressed only following arterial injury. be distinct from those used by growth factor receptors.
We conducted further studies which verified the physi- Matrix protein-driven signaling events are initiated
ologic importance of the #2!1 integrin in human SMC mi- through activation of integrin receptors.62 As previously
gration. We found that a blocking antibody to the !1 integrin mentioned, matrix proteins attach to the extracellular do-
subunit produced an inhibitory effect on chemotaxis and main of integrin receptors, and the short cytoplasmic
358 Tissue Engineering of Prosthetic Vascular Grafts
domains of both the # and ! integrin subunits allow trans- We have also studied the role of large G-proteins (Gs)
fer of messages to the intracellular environment.15 These in the intracellular signaling pathways involved in matrix
cytoplasmic domains have direct access to the signaling ma- protein-driven SMC migration.54 Cholera toxin, which leads
chinery in the cell, and studies have demonstrated that mu- to constitutive activation of Gs,21 profoundly inhibited SMC
tations or truncations of the distal end of both the # and the migration in response to collagen types I and IV, fibronectin,
! cytoplasmic tail can interfere with integrin signaling and and laminin and even eliminated baseline migration in
cytoskeletal organization.78 Further evidence of the impor- unstimulated cells.54 Activation of Gs increases levels of
tance of the ! subunit in intracellular signaling can be de- cAMP through stimulation of adenylate cyclase, and ago-
rived from studies where overexpression of a single ! cyto- nists of cAMP such as forskolin and 8-bromo-cAMP have
plasmic tail leads to inhibition of tyrosine phosphorylation been found to inhibit SMC migration.86 Thus, the inhibi-
of intracellular proteins such as FAK, 79 whereas tory effect of Gs activation with cholera toxin may be due to
overexpression of multiple ! subunits configured in clusters elevated levels of cAMP.87 Surprisingly, pertussis toxin, which
results in an increase in tyrosine phosphorylation.80 Through inhibits the Gi subtypes, including Gi1, Gi2, and Gi3, and
these and similar studies, the concept has evolved that thereby releases the inhibitory effect that Gi has on adeny-
integrin clustering is a prerequisite for the initiation of in- late cyclase,21 had no effect on chemotaxis or haptotaxis to
tracellular signalling.81 In in vitro studies, the # and ! cyto- fibronectin, type I or type IV collagen.54 However, pertussis
plasmic domains have been found to couple with a variety toxin did partially inhibit haptotaxis and completely elimi-
of cytoplasmic proteins such as p125FAK, #-actinin, talin, nated chemotaxis to laminin.54 Similarly, Aznavoorian et al
vinculin and paxillin.17 It is activation of these proteins that found that pertussis toxin decreased chemotactic migration
allows for the induction of intracellular signaling pathways of A2058 human melanoma cells when stimulated with
that lead to migration. laminin; however, with these same cells there was no inhibi-
Focal adhesion kinase (FAK) is a nonreceptor tyrosine tion of fibronectin-induced chemotaxis.88 Although Gi pro-
kinase that has been found to be activated by both ECM teins can influence adenylate cyclase, the multiple Gi sub-
and soluble signaling factors.81 Ligand attachment leads to types can also interact with enzymes such as phospholipase
direct phosphorylation of FAK by the ! subunit of the A2, C, and D, and ion transporters such as potassium and
integrin receptor.16 FAK can then interact with Src, another calcium channels.89 Thus, the varying role that pertussis
nonreceptor tyrosine kinase, which is localized to focal ad- toxin has on SMC migration may be due to either inhibi-
hesions by its SH2 domain.82 Src in turn can activate the tion of the inhibitory influence of Gi on cAMP or perhaps
Ras/MAPK signaling pathways,83 both which have been pre- the lack of interaction of Gi with one or more of these other
viously implicated in SMC migration.73,84 MAPK may pro- signaling substrates.
mote migration either through the phosphorylation and ECM proteins are known to play a role in the turn-
activation of transcription factors which regulate integrin over of phospholipids. In fibroblasts, fibronectin increased
gene expression, or by phosphorylating and activating cyto- the production of phosphatidylinositol 4,5 bisphosphate
plasmic phospholipase A2 (cPLA2), which hydrolyzes (PIP2) and dissociation of these same cells from fibronectin
glycerophospholipids to produce arachidonic acid.16 It has resulted in a rapid decrease in cellular PIP2.90 PIP2, in turn,
been previously demonstrated that inhibition of cPLA2 pre- has been implicated in cellular migration91 and this effect
vents cell migration, an effect that can be reversed by the may be mediated by several pathways. It has been specu-
readdition of arachidonic acid.16 lated that the upregulation of the production of PIP2 may
We and others have shown a relationship between lev- facilitate the polymerization of actin because of a regula-
els of intracellular calcium and PDGF-induced SMC migra- tory effect of PIP 2 on actin binding proteins such as
tion.75,76 We postulated that a rise in intracellular calcium profilin.16 PIP2 is also an important substrate for the en-
might also be required for SMC migration in response to zyme PLC.63 PLC hydrolyzes PIP2 into the active second
matrix proteins.60 Despite the potent stimulatory effect of messengers diacylglycerol (DAG) and inositol trisphosphate
collagen types I and IV, fibronectin, and laminin on SMC (IP3), which then respectively activate protein kinase C
migration, none of the matrix proteins produced a sustained (PKC) and the release of intracellular calcium. Both PKC
or prolonged rise in intracellular calcium.60 This was true and calcium have been implicated in the signaling pathways
even if calcium measurements were carried out for more for cell migration.75,92
than 30 minutes after stimulation. This finding was surpris- We have previously demonstrated that growth factors
ing, since activation of the #v!1 integrin by fibronectin has and matrix proteins act synergistically to promote SMC
been shown to produce a persistent rise in intracellular cal- migration.60 The mechanism through which these two ago-
cium in endothelial cells.85 These findings imply that cal- nists might interact is not clear; however, the point of inter-
cium plays little if any role in matrix protein stimulated SMC section may be either at the level of the receptor or at a mu-
locomotion. We did find that fibronectin, laminin, and type tual downstream signaling pathway. There is some evidence
I and IV collagen augmented the early peak in intracellular that ECM activation of integrins can up or downregulate
calcium produced by PDGF.60 Although it is possible that growth factor receptors. The term “crosstalk” has been used
this augmentation provides at least a partial explanation for to describe such interactions between growth factor recep-
the synergistic enhancement by matrix proteins of PDGF- tors and integrins.93 We have recently reported that type I
induced SMC migration, the physiologic relevance of this collagen enhances clustering and activation of the PDGF-!
finding is still unclear. receptor.94 Tyrosine phosphorylation by growth factor re-
Extracellular Matrix Proteins Are Potent Agonists of Human Smooth Muscle Cell Migration 359
ceptors of the cytoplasmic domains of integrins, or of pro- 7. Chen P, Gupta K, Wells A. Cell movement elicted by epi-
teins associated with the focal adhesion complex is an addi- dermal growth factor receptor requires kinase and
tional mechanism by which these two ligands/receptors autophosphorylation but is separable from mitogenesis. J
might interact.60 Other potential sites of interaction are at Cell Biol 1994; 124:547-555.
8. Madri JA, Bell L, Marx M et al. Effects of soluble factors
the level of the proteins, MAPK, PI3K and/or PIP2. It is im-
and extracellular matrix components on vascular behav-
portant that we investigate the synergistic interactions be- ior in vitro and in vivo: Models of de-endothelialization
tween matrix proteins and growth factors, since these points and repair. J Cell Biochem 1991; 45(2):123-30.
of interaction may represent exceedingly effective targets for 9. Stossel TP. From signal to pseudopod. J Biol Chem 1989;
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10. Vandekerckhove J. Actin-binding proteins. Curr Opin in
Conclusion Cell Biol 1990; 2:41-50.
In this chapter, we have reviewed the effect of the nu- 11. Burridge K, Fath K, Kelly T et al. Focal adhesions: Trans-
merous matrix components of the vessel wall on SMC mi- membrane junctions between the extracellular matrix and
gration. It is clear that many of these proteins produce a the cytoskeleton. Ann Rev Cell Biol 1988; 4:487-525.
strong stimulus for migration. Moreover, the effect of these 12. Luna EJ, Hitt AL. Cytoskeleton-plasma membrane inter-
actions. Science 1992; 258:955-964.
matrix proteins on SMC migration may be more profound
13. Huttenlocher A, Ginsberg MH, Horwitz AF. Modulation
than that of many of the growth factors that have been tra- of cell migration by integrin-mediated cytoskeletal link-
ditionally studied. We have also shown that these matrix ages and ligand-binding affinity. J Cell Biol 1996;
proteins can interact with growth factors to provide a syn- 134(6);1551-62.
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tors of which, at least for human venous SMC, #2!1 appears 15. Hynes RO. Intergrins: Versatility, modulation and signal-
to be the most critical. The signaling pathways that mediate ling in cell adhesion. Cell 1992; 68:11-25.
matrix-induced migration have not been fully elucidated but 16. Clark EA, Brugge JS. Integrins and signal transduction
appear to be distinct from those that mediate migration of pathways: The road taken. Science 1995; 268:233-238.
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Vascular disease remains a major cause of morbidity
18. Bendeck MP, Irvin C, Reidy MA. Inhibition of matrix
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21. Zetter BR, Brightman SE. Cell motility and the extracel-
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Facilitation of Healing
Matrix Modulation
CHAPTER 33
Extracellular Matrix Effect on Endothelial Control
of Smooth Muscle Cell Migration and Matrix Synthesis
Richard J. Powell
Introduction
I ntimal hyperplasia remains the most common cause of early failure following angioplasty
and bypass surgery.1,2 Intimal hyperplasia is a particularly prevalent problem in small di-
ameter synthetic grafts used for extremity bypasses performed to tibial and peroneal tar-
gets.3 The development of intimal hyperplasia following smooth muscle cell (SMC) injury
is characterized by SMC dedifferentiation from a contractile to synthetic phenotype.4,5 Fol-
lowing phenotypic modulation, SMCs migrate into the subintimal space, proliferate and
secrete extracellular matrix. For the first several weeks following injury the intimal hyper-
plastic lesion is predominantly cellular. However, as the lesion continues to enlarge, extra-
cellular matrix gradually becomes the predominant component.1,4-7 Several months follow-
ing injury, extracellular matrix comprises 80% of the mature intimal hyperplastic lesion.8
In several recent studies, endothelial cell (EC) seeding of injured arterial wall seg-
ments and synthetic grafts has been shown to limit the development of intimal hyperplasia.
Bush and co-workers have shown that EC seeding of endarterectomized canine arteries de-
creased the intimal hyperplastic response.8,9 Conte has shown that EC seeding of injured
hypercholesterolemic rabbit femoral arteries also limits the intimal hyperplastic response.10
In addition, several in vivo human studies have shown improved patency of endothelial
sodded synthetic grafts when compared to unsodded grafts.11-14 The improved patency has
been attributed to decreased thrombogenicity and increased resistance to intimal hyperpla-
sia in the sodded grafts. Zilla and co-workers have recently shown that EC lined synthetic
grafts have a 73%, 5 year primary patency rate in the below knee position compared to 31%
for unlined grafts.12 They now have follow up data out to 6.5 years with a patency rate of
71% in the EC lined grafts and 0% in the controls.11 However, not all studies of EC sodded
grafts have shown improved patency rates. A report by Herring and co-workers failed to
show any improvement in EC sodded graft patency when compared to control grafts.3 One
explanation for the lack of effect of EC sodding was thought to be the low EC seeding den-
sity used to line the grafts, which resulted in an incomplete coverage of the graft lumen.
Extracellular matrix profoundly affects both endothelial and smooth muscle cell func-
tion. Following arterial wall injury, not only is extracellular matrix quantity increased but
alterations in matrix composition occurs as well. Matrix composition can affect SMC func-
tions such as migration and proliferation, and may also affect matrix synthesis.15 For in-
stance, following vascular wall injury there is an increase in type I collagen present within
the restenotic lesion. Type I collagen has been shown to stimulate smooth muscle cell
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
364 Tissue Engineering of Prosthetic Vascular Grafts
dedifferentiation and increase SMC proliferation.16 In ad- are established by plating ECs on the outer side of the mem-
dition, type I collagen has been shown to upregulate the ex- brane and grown to confluence over 3-5 days. Once the ECs
pression of the growth factor TGF-!1.17 TGF-!1 is a potent are confluent (2-3 days, as determined by phase contrast
stimulant of extracellular matrix synthesis and inhibits ma- microscopy), SMCs are plated on the opposite side of the
trix degradation.18 TGF-!1 has been shown to potentiate the PET membrane (see Fig. 33.1). After 24 h both cell types are
development of intimal hyperplasia in animal models fol- placed in DMEM/2.5% CS for the duration of each
lowing arterial injury.19 In similar studies, neutralizing an- experiment.
tibodies to TGF-!1 have been shown to inhibit the develop- This coculture model allows for the diffusion of soluble
ment of intimal hyperplasia following arterial injury. Tran- factors across the membrane, yet the two cell types remain
scripts to TGF-!1 are upregulated in intimal hyperplastic separated for examination. Previous work has shown that
human coronary lesions retrieved by coronary ECs maintain SMCs in a more differentiated contractile
atherectomy.20 TGF-!1 appears to be an important media- phenotype when compared to SMCs cultured alone, which
tor of the increased extracellular matrix deposition which exhibit a synthetic phenotype.21,22 There is limited physical
occurs during vascular wall remodeling. contact across the membrane during the first 4-6 days
The effects of various extracellular matrix proteins on following coculture; after which, however, SMC cytoplas-
endothelial cell control of smooth muscle cell function is mic projections begin to cross the membrane and contact
unknown. This information is important in the study of tis- the ECs.23
sue engineering of prosthetic grafts. To which particular
matrix protein endothelial cells and smooth muscle cells are Effect of Matrix on Endothelial Cell Control
exposed will determine what effect ECs have on such SMC of Smooth Muscle Cell Migration
functions as phenotype expression, proliferation, migration Migration of SMCs into the anastomotic site follow-
and matrix synthesis. This could obviously affect the patency ing bypass grafting is an early and important event in the
of EC sodded prosthetic grafts. The purpose of the work development of intimal hyperplasia.24 SMC migration oc-
performed in our laboratory is to define the effect of matrix curs following phenotypic modulation to a synthetic phe-
composition on the ability of ECs to regulate smooth muscle notype and prior to SMC proliferation and extracellular ma-
cell functions. In particular, we are interested in the matrix trix synthesis. The process of migration is distinct from cel-
effect on EC control of SMC migration and extracellular lular proliferation. Soluble factors such as bFGF, PDGF and
matrix synthesis. To study this we have developed a coculture extracellular matrix are involved in the control of SMC mi-
model in which ECs are cultured opposite SMCs on a semi- gration and appear to play a role in the intimal hyperplastic
permeable membrane. response.24,25
The role of ECs in modulating the SMC response to
Model injury is unclear. Previous work evaluating the effect of ECs
Bovine aortic ECs and SMCs are harvested using the on SMC proliferation have yielded controversial results and
collagenase method for ECs and the explant method for have not directly addressed the effect of ECs on SMC mi-
SMCs. Cells are grown for 3-5 passages from primary cul- gration. In vivo studies by Fingerle and co-workers have
tures in Dulbecco’s Modified Eagles Media/10% calf serum demonstrated that the loss of ECs from the arterial intima
(DMEM/10% CS). ECs are identified by their cobblestone results in the development of an intimal hyperplastic lesion
morphology and positive uptake of di-I-acetylated ldl. SMCs and that this lesion formation can be limited by rapid re-
are identified using an anti-#-actin antibody (Sigma A-2547, endothelialization.26 Although this is suggestive of the im-
Sigma Chemical, St. Louis, MO). For each component of portant role of ECs in limiting this process, these authors
these studies, SMC cultures are established by plating cells have not identified an EC released substance which might
on a 13 ∝m thick polyethylene terapthalate (PET) membrane account for this phenomenon. Conte and co-workers failed
with 0.45 ∝m pores configured at a density of 1.6 million to show any attenuation of the intimal hyperplastic response
pores/cm2 (Cyclopore membrane, Falcon cell culture insert, following early luminal EC seeding of balloon catheter in-
Becton Dickinson, Lincoln Park, NJ). EC/SMC cocultures jured arterial wall segments in rabbits with normal choles-
Fig. 33.1. Diagram of the
coculture model consist-
ing of a Falcon coculture
insert shown in plate
(A). This cell insert is
placed in a six well tissue
culture plate as shown in
plate (B). Endothelial
cells (ECs) are plated on
the under surface of the membrane and smooth muscle cells (SMCs) are plated on the upper surface. The membrane contains 0.45
∝m pores configured at a density of 1.6 million pores/cm2.
Extracellular Matrix Effect on Endothelial Control of Smooth Muscle Cell Migration and Matrix Synthesis 365
terol levels, but did show a reduction in intimal hyperplasia Previous investigators have shown that SMC migration
in a hypercholesterolemic model.10 In addition, Reidy and can be modulated by the extracellular matrix substrate
Silver failed to show any increase in SMC replication fol- composition. Our laboratory has shown that shown that
lowing gentle denudation of ECs from the intimal surface fibronectin decreases the migration of SMCs cultured alone
as long as there was no injury to the underlying SMCs.27 when compared to cells cultured on plastic (see Fig. 33.2).15
Several reports describing the increased resistance of EC The presence of ECs stimulated SMC migration on both
sodded grafts to the development of intimal hyperplasia have plastic and fibronectin coated membranes. This finding was
been previously mentioned. More recently, synthetic graft different when SMCs were cultured on type I collagen which
seeding studies performed by Lewis and co-workers have stimulated migration in SMCs cultured alone compared to
shown that EC sodded grafts produce EC-derived vasoac- SMCs cultured on plastic or fibronectin. In addition, the
tive factors which could limit SMC proliferation and mi- presence of type I collagen significantly inhibited the ability
gration.28 Thus it is unclear how important the role of the of ECs to stimulate SMC migration such as that observed
EC is in controlling SMC, migration and proliferation fol- on plastic and fibronectin (see Fig. 33.2). Thus, matrix
lowing vascular wall injury in-vivo. composition affected the EC ability to modulate
In vitro cell culture studies by Castellot and co-work- SMC migration.
ers have shown that ECs can inhibit SMC proliferation as a Cell matrix interactions which allow the cell to attach
result of secreted heaprin sulfates from the abluminal en- and detach from the extracellular matrix are also important
dothelial cell surface.29 These results conflict with studies in the migration process.31 It has been shown that there is
performed by Fillinger and co-workers as well as from our an intermediate cell attachment strength at which cell mi-
own laboratory which have shown that ECs stimulate SMC gration rate is optimized. Altering either the expression or
proliferation when these two cell types were cocultured on organization of cell surface integrin and nonintegrin attach-
opposite sides of a semipermeable membrane.22,30 Since pro- ment molecules will subsequently affect cell migration, as
liferation and migration are unassociated processes, these will altering extracellular matrix composition. In support
above studies cannot be used to address what effect ECs have of this hypothesis are the recent reports which demonstrate
on SMC migration. Using a steel fence assay our laboratory that following dedifferentiation SMCs alter their expression
has previously demonstrated that ECs stimulate SMC mi- of cell surface integrins, upregulating #2!1 integrin and de-
gration in vitro and that this process can be mediated by a creasing #1!1 integrin expression.32
secreted soluble factor.15 Possible factors involved include Basson and co-workers have shown that the growth
PDGF and endothelin-1, growth factors which have been factor TGF-!1 alters integrin expression, whereas matrix
shown to be secreted by cultured ECs. In addition, we have composition can change the cell surface integrin organiza-
previously shown that ECs may regulate TGF-!1 activation tion.33 Our laboratory has shown that adhesion molecule
in coculture.22 Changes in TGF-!1 levels could as a result expression and/or organization as determined by cell adhe-
alter SMC migration. Finally, ECs may secrete extracellular sion is altered by the presence of ECs.15 This is true regard-
matrix proteins which could deposit on the opposite side of less of whether SMCs are cultured on fibronectin, type I
the membrane and subsequently affect SMC migration. collagen or plastic. The mechanism by which this occurs is
Fig. 33.2. Migration of SMCs cultured
alone (Black bars) compared to SMCs
cocultured with ECs (gray bars) on dif-
ferent extracellular matrices. ECs in-
creased SMC migration on plastic and
fibronectin (p < 0.01, SMC vs.
EC/SMC). Type I collagen stimulated
migration in SMCs cultured alone
(p < 0.01, SMC type I collagen vs. SMC
plastic and fibronectin). In addition, the
presence of ECs resulted in only a mini-
mal increase in migration on type I col-
lagen. Results are expressed as the mean
+ standard error of the mean (SEM).
366 Tissue Engineering of Prosthetic Vascular Grafts
unclear, but is likely related to the EC release of a soluble EC stimulation of SMC migration is counterintuitive
factor. At present antibodies specific for various bovine to what one would expect. However migration is just one
integrins are not available; however, future studies need to process in the restenotic process. As we will see in the
be performed to identify the specific integrin compositional upcoming section of this monograph, ECs may limit the
and organizational changes in integrin expression that oc- development of intimal hyperplasia by affecting other
cur in SMCs as a result of ECs. mechanisms in the process, namely by inhibiting SMC
Cell spreading has been shown to correlate more matrix production.
closely with migration rate than cell attachment.34 Whereas
cell attachment is likely to be primarily integrin mediated, Matrix Effect on Endothelial Cell Control
cell spreading appears to involve nonintegrin matrix bind- of Smooth Muscle Cell Matrix synthesis
ing proteins as well.34 Differences in cell spreading on vari- The continued accumulation of extracellular matrix
ous matrix types were similar to those seen in SMC migra- forms the bulk of the mature intimal hyperplastic lesion.1,7
tion (plastic<fibronectin<type I collagen).15 We have shown For the most part, the origin of extracellular matrix is dif-
that ECs increase SMC cell spreading. SMC cell spreading ferentiated SMCs which have migrated into the subintimal
was increased in SMCs cocultured as a bilayer with ECs com- space following vessel wall injury. In addition to increased
pared to SMCs cultured alone. Thus, ECs may alter SMC matrix deposition, the matrix composition of the
matrix adhesion molecule expression or organization to ac- fibroproliferative vascular lesion is altered, containing in-
count for the increased migration in cocultured SMCs. How- creased amounts of the fibrillar collagens such as type I col-
ever, this is indirect evidence and does not exclude the pos- lagen (8). We have shown that ECs can inhibit both SMC
sibility that ECs may stimulate SMC migration by other collagen protein synthesis and type I collagen gene expres-
mechanisms as well. For example, cultured ECs have been sion in bilayer coculture (see Fig. 33.3). It is interesting to
shown to secrete PDGF, a factor which stimulates SMC mi- note that in these studies, ECs inhibited SMC type I col-
gration. This may be an alternative or additional mecha- lagen RNA levels by 50%, which is similar to the 40% EC
nism by which ECs stimulate SMC migration in vitro. inhibition of SMC collagen protein synthesis. The ability of
Fig. 33.3. (A) Representative North-
ern blot which shows a decrease in
transcripts for type I collagen in
cocultured SMCs compared to SMCs
cultured alone. Densitometry for
three experiments shows a 60% re-
duction in type I collagen transcripts
in SMCs cultured opposite ECs
(*p < 0.01). (B) Compared to SMCs
cultured alone (black bars), ECs
cocultured with SMCs(hatched bars)
inhibited SMC collagen synthesis
measured as trichloroacetic acid
precipitatable tritiated proline counts
per ug of DNA in SMC conditioned
media by 50% (*p < 0.01).
Extracellular Matrix Effect on Endothelial Control of Smooth Muscle Cell Migration and Matrix Synthesis 367
ECs to inhibit SMC collagen synthesis in vitro suggests that organization and cytoskeletal structure.36 In order to deter-
this may be one mechanism by which ECs may alter vascu- mine the effect of extracellular matrix composition on EC
lar wall remodeling and smooth muscle cell function fol- inhibition of SMC collagen synthesis coculture, we repeated
lowing vascular wall injury in vivo. experiments on various matrix proteins. The extracellular
The mechanism by which ECs inhibit SMC collagen matrix proteins studied were types I and IV collagen,
synthesis in bilayer coculture is unclear. TGF- ! 1 is Matrigel and fibronectin. Type I collagen is a fibrillar col-
upregulated in the injured arterial wall.19,20 It is a potent lagen which is increased in intimal hyperplasia and has been
stimulant for extracellular matrix synthesis, and antibodies shown to increase SMC proliferation. Fibronectin is a high
against TGF- ! 1 have been shown to limit the fibro- molecular weight glycoprotein which is also present in the
proliferative response following balloon angioplasty in ani- extracellular matrix of restenotic lesions and affects SMC
mal models.19 However, in our coculture studies the addi- proliferation and migration. Type IV collagen is a constitu-
tion of TGF-!1 antibody and aprotinin (a plasmin inhibitor ent of the normal EC basement membrane and has been
which prevents the activation of TGF-!1) did not inhibit shown to maintain ECs in a more differentiated state.
SMC extracellular matrix in SMCs cultured alone. This sug- Matrigel is a reconstituted composition of basement mem-
gests that, at least in this in vitro coculture model, EC effects brane proteins laminin and type IV collagen. Laminin is a
on TGF-!1 activation do not play a major role in this pro- component of the EC basement membrane and has been
cess (see Fig. 33.4). Though we did not perform a dose re- shown to maintain ECs in a differentiated state. We have
sponse curve using the TGF-!1 antibody, our studies indi- found that type IV collagen and Matrigel decreased collagen
cate that the antibody did at the least partially inhibit TGF-!1 synthesis in SMCs cultured alone (see Fig. 33.5). These data
based on the diminished SMC hill and valley growth seen in demonstrate that matrix proteins which normally reside in
these cultures. Hill and valley growth has been shown to the EC basement membrane can inhibit SMC matrix syn-
occur as a result of active TGF-!1.35 The plasmin inhibitor thesis. In addition, the data indirectly support the concept
aprotinin completely suppressed hill and valley growth, sug- that EC synthesized matrix proteins could be a possible
gesting complete inhibition of TGF-!1 activation. mechanism by which ECs inhibit SMC matrix synthesis.
Endothelial cells have been shown to secrete various In contrast to the effects of the EC basement mem-
extracellular matrix proteins which could cross the semi- brane matrix proteins, type I collagen, a fibrillar collagen,
permeable membrane and as a result affect SMC function. actually increased collagen synthesis of SMCs cultured alone
Endothelial cells are typically seeded on the membrane for to the greatest degree. In other studies, type I collagen has
4-6 days ( to allow the ECs to grow to confluence), prior to been shown to potentiate SMC dedifferentiation to a syn-
adding SMCs. This allows ample opportunity for ECs to coat thetic phenotype and is abundant in fibroproliferative vas-
the opposite surface of the membrane with matrix proteins. cular lesions.16
Previous investigators have shown that extracellular matrix To determine if differences in cell proliferation and
composition strongly influences SMC integrin expression, thus cell density could account for the differences in collagen
Fig. 33.4. Inhibition of TGF-!1 activity
either with TGF-!1 neutralizing antibody
(TGF-!1 Ab, 25 ∝g/ml) or with aprotinin
(plasmin inhibitor which prevents the
plasmin-mediated activation of TGF-!1,
SMC aprotinin, 200 ∝g/ml) did not in-
hibit SMC collagen synthesis when com-
pared to SMCs cultured alone. ECs in-
hibited collagen synthesis in cocultured
SMCs (EC/SMC)(*p < 0.01 vs. all SMC
groups). The addition of 5 ng/ml TGF-!1
had a modest stimulatory effect on SMC
collagen synthesis (EC/SMC TGF-!1,
p=NS).
368 Tissue Engineering of Prosthetic Vascular Grafts
synthesis observed in this study, we examined cell density at Matrix substrate composition can also affect the abil-
the completion of each experiment. As shown in Figure 33.4, ity of ECs to inhibit SMC collagen synthesis.37 We have
the cell density (cell number after three days in culture) of shown that ECs decrease smooth muscle cell collagen syn-
SMCs cultured alone was greatest in cells cultured on thesis when these cells are cocultured together on type I and
Matrigel. There was no difference in cell density when IV collagen and Matrigel (see Fig. 33.6). Fibronectin pre-
fibronectin, type I collagen, and type IV collagen were com- vented the EC inhibition of SMC collagen synthesis, and in
pared; however, collagen synthesis was significantly less in fact actually increased collagen synthesis. Thus, the ability
the type IV collagen group. This suggests that the matrix for grafts sodded with ECs to resist the development of inti-
protein effect on SMC collagen synthesis is due not only to mal hyperplasia may depend upon the matrix substrate the
changes in cell proliferation and cell density, but is extracel- ECs are cultured on.
lular matrix specific. We have not as yet explored the mecha- In conclusion, using an in vitro coculture model, we
nisms by which matrix proteins effect SMC collagen syn- have shown that ECs stimulate SMC migration but inhibit
thesis. As is the case with the effect of matrix proteins on SMC collagen synthesis and transcripts for type I collagen
proliferation and migration, it is likely that specific extra- gene expression. The effect of ECs does not appear to be
cellular matrix proteins interact with cell membrane adhe- mediated through changes in active TGF-!1 levels but may
sion molecules and as a result activate various intracellular be due to secreted matrix proteins. Extracellular matrix pro-
signaling pathways. teins have a profound effect on SMC collagen synthesis and
on the ability of ECs to modulate SMC functions such as
migration and matrix synthesis.
Fig. 33.5. Collagen synthesis (tritiated proline counts per ∝g DNA in conditioned media;
black bars) of SMCs cultured alone on Matrigel (MG) and type IV collagen (CIV) was
decreased when compared to plastic (Control). Fibronectin (FN) and type I collagen (CI)
both significantly increased SMC collagen synthesis when compared to SMCs cultured
on plastic (*p < 0.01 vs. other groups). SMC density is shown in the hatched bars. Cell
density (cell number after three days in culture) was greatest in the SMCs cultured on
MG. There was no difference in cell density when FN, CI and CIV were compared; how-
ever, collagen synthesis was significantly less in the CIV group. This suggests that matrix
effects on SMC collagen synthesis are due not only to changes in cell proliferation and cell
density but are extracellular matrix specific.
Extracellular Matrix Effect on Endothelial Control of Smooth Muscle Cell Migration and Matrix Synthesis 369
Fig. 33.6. (A) ECs inhibited soluble
tritiated proline levels in the condi-
tioned media of cocultured SMCs
(hatched bars) on type I collagen
(EC/SMC CI) and Matrigel (EC/
SMC MG) when compared to SMCs
cultured alone (black bars).
Fibronectin prevented the EC effect
on SMCs (EC/SMC FN vs. SMC FN).
(B) ECs inhibited cell-associated tri-
tiated proline levels in cocultured
SMCs (hatched bars) on type I col-
lagen (EC/SMC CI) and Matrigel
(EC/SMC MG) when compared to
SMCs cultured alone (black bars).
ECs stimulated collagen synthesis in
cocultured SMCs on fibronectin(EC/
SMC FN) when compared to SMCs
cultured alone.
References cultured from human coronary atherosclerotic and
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2. Liu MW, Roubin GS, King SB. Restenosis after coronary muscle cells from injured rat aortas display elevated ma-
angioplasty: Potential biologic determinants and role of trix production associated with transformtin growth fac-
intimal hyperplasia. Circulation 1989; 79:1374-1387. tor-b activity. Am J Path 1995; 147:1041-1048.
3. Herring M, Smith J, Dalsing M et al. Endothelial cell seed- 8. Mesh CL, Majors A, Mistele D et al. Graft smooth muscle
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The failure of low-density seeding to improve patency. J 1995; 22:142-149.
Vasc Surg 1994; 20:650-655. 9. Bush HJ, Jakubowski JA, Sentissi JM et al. Neointimal
4. Kocher O, Gabbiani F, Gabbiani G et al. Phenotypic fea- hyperplasia occurring after carotid endarterectomy in a
tures of smooth muscle cells during the evolution of ex- canine model: Effect of endothelial cell seeding vs
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1991; 65(4):459-470. 10. Conte MS. Endothelial cell resurfacing improves remod-
5. Mosse PRL, Campbell GR, Wang ZL et al. Smooth muscle eling of balloon-injured arteries in the hyper-
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53(5):556-562. 11. Zilla P. Long term effects of clinical in-vitro
6. MacLeod DC, Strauss BH, DeLong M et al. Proliferation endothelialization on grafts. Research Initiatives in Vas-
and extracellular matrix synthesis of smooth muscle cells cular Disease 1997:82-85.
370 Tissue Engineering of Prosthetic Vascular Grafts
12. Zilla P, Deutsch M, Meinhart J et al. Clinical in vitro 26. Fingerle J, Au YP, Clowes AW et al. Intimal lesion for-
endothelialization of femoropopliteal bypass grafts: An mation in rat carotid arteries after endothelial denuda-
actuarial follow-up over three years. J Vasc Surg 1994; tion in absence of medial injury. Arteriosclerosis 1990;
19:540-548. 10(6):1082-87.
13. Zilla P, Fasol R, Callow A. Endothelialization of vascular 27. Reidy MA, Silver M. Endothelial regeneration: Lack of
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1992;:78-131. 1985; 118(2):173-177.
14. Magometschnigg H, Kadletz M, Vodrazka M et al. Pro- 28. Lewis DA, Lowell RC, Cambria RA et al.. Production of
spective clinical study with in vitro endothelial cell lining endothelium-derived factors from sodded expanded
of expanded polytetratfluoroethylene grafts in crural re- polytetrafluoroethylene grafts. J Vasc Surg 1997;
peat reconstruction. J Vasc Surg 1992; 15:527-535. 25:187-197.
15. Powell R, Carruth J, Basson M et al. Matrix specific ef- 29. Castellot J, Addonizio ML, Rosenberg R et al. Cultured
fect of endothelial cell control of smooth muscle cell mi- endothelial cells produce a heparin like inhibitor of
gration. J Vasc Surg 1996. smooth muscle cell growth. J Cell Biol 1981; 90:372-379.
16. Yamamoto M, Yamamoto K, Noumura T. Type I collagen 30. Fillinger MF, O’Conner S, Wagner RJ et al. The effect of
promotes modulation of cultured rabbit arterial smooth endothelial cell coculture on smooth muscle cell prolif-
muscle cells from a contractile to a synthetic phenotype. eration. J Vasc Surg 1993; 17(6):1058-1067.
Exp Cell Res 1993; 204:121-129. 31. DiMilla P, Stone J, Quinn J et al.. Maximal migration of
17. Streuli CH, Schmidhauser C, Kobrin M et al. Extracellu- human smooth muscle cells on fibronectin and type IV
lar matrix regulates expression of the TGF-!1 gene. J Cell collagen occurs at an intermediate attachment strength. J
Biol 1993; 120(1):253-260. Cell Biol 1993; 122:729-737.
18. Penttinen RP, Kobayashi S, Bornstein P. Transformtin 32. Skinnere M, Raines E, Ross R. Dynamic expression of al-
growth factor-! increases mRNA for matrix proteins both pha 1 beta 1 and alpha 2 beta 2 integrin receptors by
in the presence and in the absence of changes in mRNA human vascular smooth muscle cells. Am J Path 1994;
stability. Proc Natl Acad Sci 1988; 85:1105-1108. 145:1070-1081.
19. Majesky MW, Lindner V, Twardzik DR et al. Production 33. Basson C, Kocher O, Basson M et al.. Differential modu-
of transforming growth factor !1 during repair of arterial lation of vascular cell integrin and extracellular matrix
injury. J Clin Invest 1991; 88:904-10. expression in vitro by TGF-!1 correlates with reciprocal
20. Nikol S, Isner JM, Pickering JG et al. Expression of effects on cell migration. J Cell Phys 1992; 153:118-128.
transformtin growth factor-b1 is increased in human vas- 34. Basson C, Knowles W, Bell L et al.. Spatiotemporal seg-
cular restenosis lesions. J Clin Invest 1992; 90:1582-92. regation of endothelial cell integrin and nonintegrin ex-
21. Powell RJ, Cronenwett JL, Wagner RJ et al. Endothelial tracellular matrix-binding proteins during adhesion events.
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22. Powell RJ, Cronenwett JL, Fillinger MF et al. Effect of fies organizational behavior in vascular smooth muscle cell
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23. Fillinger MF, Sampson LN, Cronenwett JL, Powell RJ, and extracellular matrix components on vascular cell be-
Wagner RJ. Coculture of endothelial cells and smooth havior in-vitro and in-vivo: Models of de-
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Surg Res 1997; 67:169-178. 45:123-130.
24. Casscells W. Migration of smooth muscle and endothe- 37. Powell RJ, Hydowski J, Frank O et al.. Endothelial cell
lial cells. Circulation 1992; 89:723-729. effect on smooth muscle cell collagen synthesis. Accepted
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tured bovine aortic endothelial and smooth muscle cells.
Circ Res 1989; 65:1057-1065.
Facilitation of Healing
Cell Entrapment from the Circulation
CHAPTER 34
Circulating Stem Cells: A Fourth Source for the
Endothelialization of Cardiovascular Implants
Willie R. Koen
Introduction
A n endothelialized blood-contacting surface remains the key to long term cardiovascular
implants. The source for this endothelium could be either the intima of the adjacent
artery or perigraft capillaries. Accordingly, the mode of endothelialization would either be
through transanastomotic outgrowth or transmural ingrowth.1-4 However, certain devices
such as artificial hearts cannot rely on either form of ingrowth, due to device isolation from
adjacent tissue. Therefore, the only means of obtaining an endothelium on these surfaces is
either by seeding,5-10 or by capturing from the blood. The latter possibility of a blood borne
source of endothelial cells has been postulated and debated for the past three decades.11-15
However, in the last few years enough data was produced to confirm that this fourth source
of endothelium is no longer a myth but a fact. As a logical consequence, the question arose
as to whether circulating cells could be harnessed in the development of a long lasting tissue
engineered cardiovascular implant.
The History of Spontaneously Healing Surfaces
In 1963 Stump et al noticed that a 4.5 cm Dacron vascular implant was fully
endothelialized after 7 days of implantation into a pig. Since this short period of implanta-
tion could not be explained with transanastomotic pannus ingrowth, it was speculated that
a different cell source, such as the circulating blood, could have been responsible for this
spontaneous endothelialization. To further elucidate this possibility, a cell trapping device
was constructed which included a small square Dacron hub suspended in the center of the
lumen of a Dacron prosthesis. This hub was anchored with stitches, which prevented it from
coming into contact with the graft wall. After a 4 week period of canine implantation, silver
nitrate stain demonstrated an endothelial coverage of the entire hub surface. Although the
conclusion of a circulating cell source was drawn from these experiments, explanations for
this phenomenon remained vague.11
To shed some light onto the chronology of events taking place in a spontaneous
endothelialization of grafts, Mackenzie (1968) implanted into dogs 20 crimped terylene vas-
cular grafts which were isolated with a surrounding Nylon sheath. After two and four weeks,
patches of silver-stained round cells were found on the luminal side, lining the underlying
amorphous protein mass. By four weeks these patches were composed of cells that looked
like endothelial cells.12
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
372 Tissue Engineering of Prosthetic Vascular Grafts
Later on, spontaneous endothelialization was also Properties of Hematopoietic Stem Cells
observed on other cardiovascular implants such as ventricu- and Progenitor Cells
lar assist devices. In 1982 Bossart13 described a pseudo- A hematopoietic stem cell can be defined as a cell with
neointoma (PNI) consisting of mainly myofibroblasts and self-renewal proliferative potential, coupled with the poten-
endothelial cells on a textured surface of an assist device af- tial to differentiate into progenitor cells of all blood lineages.
ter two years of implantation. This PNI consisted of a ma- Furthermore, a stem cell is typically not terminally differ-
trix layer of collagen bundles embedded in proteinaceous entiated and can divide without limit. However, when it di-
material and interspersed with myofibroblasts. Furthermore, vides, each daughter cell has a choice either to remain a stem
this layer was covered by a monolayer of cells with electron cell, or embark on a course leading irreversibly to terminal
microscopic characteristics of endothelial cells. Based on differentiation. Therefore, the pluripotent stem cell normally
these findings as well as literature evidence that similar fi- generates either more pluripotent stem cells (with self-re-
brous plaques can arise from cultured guinea pig bone mar- newal potential), or committed progenitor cells (also referred
row, Bossart postulated that the source of PNI cells could be to as colony forming cells—CFC). Committed progenitor
multipotential bone marrow stem cells.13 This hypothesis cells are irreversibly determined to produce only one or a
of circulating cells as a source for spontaneously endo- few types of blood cells.20 Moreover, committed progenitor
thelializing grafts was once again brought up by Feigel in cells divide rapidly (amplification divisions) for only a lim-
198514 after a series of experiments investigating the pro- ited number of times, and at the end of this series of divi-
cess of intravascular thrombus organization. He summarized sions they develop into terminally differentiated cells. These
clot organization as a chronological sequence of events which terminally differentiated cells usually have a limited num-
starts off with activation of the mononuclear/macrophage ber of divisions and eventually die after several days. Other
system. Large numbers of early monocytic cells are noted in properties frequently ascribed to stem cells include the abil-
the thrombus, which he subscribed to chemotactic mecha- ity to undergo asymmetric cell divisions as well as the abil-
nisms as well as the ability of monocytic cells to adhere to ity to exist in a mitotically quiescent form.21 During asym-
surfaces. This event of macrophage activation is then fol- metrical division, the stem cells divide into two daughter
lowed by the elongation of monocyte nuclei, the appear- cells, of which one remains a stem cell and the other be-
ance of myofibroblastic cells and eventually endothelial cell comes a differentiated (progenitor) daughter cell. However,
formation, suggesting a transformation of mononuclear cells during symmetrical division of stem cells, the stem cell di-
into endothelial cells.14,16 In 1994 distinct evidence in favor vides into two daughter cells, both of which either remain
of a circulating endothelial cell source was brought forward stem cells or become differentiated progenitor cells. There-
by Shi et al, 15 which he referred to as fallout endo- fore, symmetrical divisions allow the size of the stem cell
thelialization. By using impervious Dacron grafts implanted pool to be regulated.22 Yet, stem cells can also enter a mi-
into different anatomic positions in dogs, a healed surface totic quiescent stage where the divisions take place very
could be demonstrated as early as four weeks after implan- slowly or rarely. This feature of mitotic quiescence is thought
tation. Furthermore, endothelial cell presence was for the to be true for stem cells in the skin and bone marrow. In
first time confirmed by using immunohistochemistry, which contrast, stem cells in the mammalian intestinal crypts have
included von Willebrandt factor staining. been estimated to divide every 12 h.22
With enough evidence at hand that a synthetic sur-
face does have the ability to spontaneously endothelialize The Location of Hematopoietic
simply by being exposed to the circulating blood, the ques- Progenitor Cells
tion remains concerning the origin of these cells. Over the The main source of pluripotential hematopoietic stem
years two basic theories evolved: The first theory is that the cells is the bone marrow microenvironment. This complex
source of fallout endothelialization is cells that detached from space is occupied by hematopoietic cells at all different stages
the vascular wall in areas of high shear stress. This explana- of differentiation. In addition to the hematopoietic cells,
tion has also been supported by the observation that in dy- stroma cells account for the rest of the bone marrow con-
namic tissue culture studies endothelial cells are most prone tent. Stroma cells consist of fibroblasts, endothelial cells,
to detach at the time of mitotic division.17 The second theory adipocytes, monocytes and osteoclasts and can be regarded
is that the fallout endothelial cells arise from circulating pre- as the backbone of the marrow microenvironment. The func-
cursor cells which eventually differentiate into mature en- tion of the stroma is 3-fold namely:
dothelial cells.13-15,18 Although this source has been specu- 1. The production of extracellular matrix;
lated on for many years, it was only recently that published 2. The secretion of cytokines, and
data confirmed that isolated putative progenitor endothe- 3. The mediation of direct cellular contacts regulating
lial cells from the human circulation were able to differenti- hematopoiesis.23
ate in vitro into endothelial cells.19 If this differentiation From the bone marrow, hematopoietic cells enter the
phenomenon is to be the link to a nonthrombogenic tissue blood stream via the bone marrow endothelium (BMEC),
engineered synthetic cardiovascular implant, one needs to which also acts as a gatekeeper in regulating this passage.24
understand stem cell physiology to identify aspects which Furthermore, it has been hypothesized that BMEC is a
could be exploited for facilitated surface endothelialization. unique endothelium that may regulate hematopoiesis by
direct cellular contact and/or expression and secretion of
specific cytokines.25
Circulating Stem Cells: A Fourth Source for the Endothelialization of Cardiovascular Implants 373
Apart from the bone marrow, a second source of he- increase in CD34 cell concentration, suggesting surface colo-
matopoietic stem cells is the peripheral blood. Mature pe- nization with hematopoietic precursor cells.16 Therefore,
ripheral blood cells are derived from a small pool (1-3%) of should these CD34 hematopoietic precursor cells possess the
primitive precursor cells in the bone marrow that bear a ability to mature and differentiate into endothelial cells, then
unique surface glycoprotein, CD34 (Fig. 34.1). Furthermore, the answer to the origin of fall-out endothelialization be-
CD34+ cells are also found at lower concentrations in pe- comes more unclouded. The speculation that circulating
ripheral blood, where they are capable of reconstituting he- hematopoietic precursor (CD34+) cells possess the ability
matopoiesis.26 As a result of this feature, transplantation of to differentiate into endothelial cells was eventually answered
allogeneic peripheral blood stem cells has become a success- by Asahara et al (1997).19 In his study, mononuclear cells
ful and common modality of treatment in clinical practice.27 were isolated from human peripheral blood by magnetic
bead selection on the basis of cell surface antigen expres-
Recognition of Stem Cells sion. In vitro, cells isolated with anti-CD34 or anti-Flk-1
The direct microscopic recognition of stem cells is very antibodies differentiated into endothelial cells. This was con-
intricate and unreliable for various reasons, namely: firmed by the expression of ecNOS, Flk-1/KDR and CD31
1. In bone marrow samples, it is basically impossible mRNA by using the reverse transcription-polymerase chain
to distinguish between distinct precursor cells in their reaction (RT-PCR).
different stages of differentiation; and also
2. In peripheral blood, the concentration of stem cells Control of Stem Cells
is simply too low. The ligand-receptor mechanism remains the most
However, this dilemma has been overcome by the de- commonly researched topic in the control of stem cell dif-
tection of specific surface markers on hematopoietic stem ferentiation. In general, a receptor can be defined as a struc-
cells that can be harnessed for cell recognition. Probably the ture (generally a protein) located on or in a cell, which spe-
most prevalent surface antigen in stem cell isolation at cifically recognizes a binding molecule, a ligand, and thereby
present is cluster derivative 34 (CD34). This glycosylated initiates either a specific biological response (the wide sense
protein has a molecular weight of 115 kDa and is expressed of receptor) or the transduction of a signal (the narrow sense
on human hematopoietic stem and progenitor cells as well of receptor). Furthermore, receptors can be situated in the
as on vascular endothelial cells.26 During vasculogenesis, plasma membrane (receptors for neurotransmitters, growth
hematopoietic stem cells form clusters with angioblasts and factors, sensory stimulants and most circulating hormones),
share common antigenic determinants such as CD34 and in an organelle membrane (e.g., Ca2+ release transduction)
fetal liver kinase-1 receptor (Flk-1).28 Furthermore, the CD34 or in the cytosolic solution (as for the receptors for steroid
surface antigen is also detected in peripheral blood cells, but and thyroid hormones). A receptor of particular interest in
only on 0.03-0.09% of circulating hematopoietic cells.16 the field of tissue engineering as well as stem cell biology
Compared to peripheral blood, the fallout cells noticed on involves the growth factor receptor. By definition, a growth
explanted ventricular assist device surfaces express a 100-fold factor receptor is a cell surface protein whose purpose is to
Fig. 34.1. The main source
of pluripotent hemato-
poetic stem cells is the
bone marrow microenvi-
ronment, which forms
97-99% of the total stem
cell pool. Ther peripheral
blood contributes to the
remaining 1-3%. Of this
peripheral pool, opnly
0.03-0.09% are CD34
positive cells.
374 Tissue Engineering of Prosthetic Vascular Grafts
receive information from outside cells and convey it across nies in soft agar culture. Currently, four major colony-stimu-
the cell membrane. It binds to a receptor and converts the lating molecules are known, namely:
receptor to an active state which then interacts with pro- 1. Granulocyte/macrophage colony-stimulating factor
teins on the inner surface of the membrane. This interac- (GM-CSF);
tion consequently stimulates a program of events leading, 2. Granulocyte colony-stimulatin factor (G-CSF);
amongst other effects, to cell division.29-31 3. Colony-stimulating factor-1 (CSF-1) or macrophage
Growth factor receptors can be classified into three colony stimulating factor (M-CSF); and
groups, namely: 4. Multi-colony stimulating factor (multi-CSF) or
1. The tyrosine kinase receptors. interleukin-3 (IL-3).
2. Seven transmembrane domain receptors; and Interleukin-3 (IL-3) is primarily an activated T cell-
3. The nontyrosine kinase receptors. derived pleiotropic factor (Fig. 34.2) that can stimulate the
The tyrosine kinase receptors have been grouped into proliferation and differentiation of pleuripotent hematopoi-
families on the basis of kinase domains, general structure etic stem cells as well as various lineage-committed progeni-
and ligand structure similarities. Among tyrosine kinase re- tors. Following the purification and cloning of IL-3, it be-
ceptors, the type I receptor family includes the receptors for came apparent that this same protein had been studied un-
epidermal growth factor as well as transformtin growth fac- der different names, including mast cell growth factor, P cell
tor-! (TGF-!). The type II receptors include insulin-like re- stimulating factor, burst promoting activity, multi-colony
ceptors and the type III receptors consist of the platelet-de- stimulating factor, Thy-1 inducing factor and WEHI-3
rived growth factor (PDGF) receptors A and B, c-Kit as well growth factor.32-34
as c-Fms (also known as colony-stimulating factor). Finally, However, more specific growth factor receptors are
the type IV family is a group of receptors whose ligands are now known to be present on stem cells. For example, the
not yet known (Eph, Elk, Eck, Eek and Erk).29-31 fetal liver kinase-1 receptors (Flk-1 in mouse, also known as
The most commonly used ligands in stem cell research KDR in human) are expressed by both the vasculogenic
as well as in clinical practice are the colony stimulating fac- angioblasts and the hematopoietic stem cells as well as vas-
tors (CSF). In bone marrow transplantation, CSF have been cular endothelial cells. Yet, although this receptor is specific
used to mobilize hematopoietic stem cells to optimize the for vascular endothelial growth factor (VEGF) (Fig. 34.3), it
harvest of peripheral blood stem cells. CSF were first de- ceases to be expressed during hematopoietic differentia-
fined by the stimulation of granulocyte/macrophage colo- tion. 28,35 Furthermore, it has been demonstrated in
Fig. 34.2. A summary of the
control and regulation of
hemotopoietic stem cells in
their differentiation towards
the mature endothelial cell.
Circulating Stem Cells: A Fourth Source for the Endothelialization of Cardiovascular Implants 375
Flk-1/KDR knockout mice that few, if any, vascular endot- Do Precursor Endothelial Cells Have Self-Renewal
helial cells are present.36 Moreover, Asahara et al (1997) dem- Potential?
onstrated the differentiation of Flk-1 positive precursor cells Per definition, a stem cell has the potential to renew
into VEGF producing vascular endothelial cells, thereby itself. From experiments carried out in baboons it could be
underlining once again the theory of a circulating endothe- demonstrated that highly enriched populations of autolo-
lial cell source. In contrast to the stimulation of stem cells, gous CD34+ marrow cells can restore hematopoiesis and lead
there are also known factors that inhibit the differentiation to a long term recovery of bone marrow function in lethally
and proliferation of stem cells. So far, such inhibiting fac- irradiated baboons.37 Furthermore, it has been known for
tors include nitric oxide (NO) and transformtin growth fac- more than 20 years that, after chemotherapy, circulating
tor-! (TGF-!). myeloid progenitor cells are recruited from the bone mar-
row to the periphery. This recruitment can be further en-
Speculative Application hanced by using colony stimulating factors to produce a more
In summary, our knowledge of specific surface mark- profound mobilization of CD34+ cells. Today, peripheral
ers for circulating progenitor endothelial cells is at this stage blood stem cell (CD34+) transplantation is regarded as
limited to the CD34 antigen as well as the Flk-1/KDR recep- equivalently successful, even superior in certain cases, to bone
tor. Furthermore, VEGF is a specific ligand for the Flk-1/KDR marrow transplantation.38-42 Yet, can we assume that the self-
receptor, as opposed to the less specific ligands for the CD34 renewal of CD34+ stem cells also applies to the precursor
antigen (including !-selectins) (Fig. 34.3). Therefore, circu- endothelial cell? This question was addressed by Hammond
lating progenitor endothelial cells could be addressed by tar- et al: Pigs were lethally irradiated and subsequently trans-
geting the Flk-1 receptor. planted with sibling bone marrow cells. After 6 weeks of
In summary, it seems as if in our striving toward a implantation, hematopoiesis was restored and the explanted
spontaneously healing, long lasting cardiovascular implant, vascular grafts showed the presence of donor related endo-
we could rely on circulating endothelial progenitor cells. Our thelial cells staining positive for factor VIII. Therefore, this
optimism is based on two key facts, namely: study suggests that peripheral blood stem cells exert self-
1. The proof of the presence of circulating progenitor renewal potential to eventually restore hematopoiesis as well
endothelial cells which have the potential to differ- as graft endothelialization.43
entiate into functioning endothelial cells19; and
2. Available receptors on these precursor cells making To What Extent Are Circulating Endothelial Precursor
manipulation with specific ligands possible. Cells Committed?
However, a number of questions still remain open Using fluorescence-activated cell sorting (FACS) the
before one can exploit circulating stem cells for the sponta- CD45 leukocyte common antigen could be demonstrated
neous endothelialization of synthetic impermeable implants. on 94% of freshly isolated CD34+ mononuclear blood cells.
Fig. 34.3. The hematopoietic
stem cell has two well known sur-
face antigens, namely CD34 and
Flk-1. The Flk-1 receptor has a
specific ligand, namely VEGF.
The ligands for the Cd34 surface
antigen are the !-selecings, which
have a less specific binding.
376 Tissue Engineering of Prosthetic Vascular Grafts
However, attached CD34+ cells, after being in culture for a the further differentiation into CD34+CD45– cells and even-
week on a fibronectin-coated surface, expressed the CD45 tually endothelial cells could be more specifically addressed,
antigen on only 27% of cells.19 Therefore, it seems that en- for instance with VEGF.
dothelial precursor cells circulate as CD34 and CD45 posi-
tive without being finally committed. After attachment, these At What Stage of Differentiation Do Endothelial
noncomitted cells become CD45 negative (after a week) and Precursor Cells Attach to a Surface?
are then committed to further differentiate into endothelial Freshly isolated CD34+ cells attach to a fibronectin
cells. This CD34+CD45+ circulating cell stage reminds us of coated culture flask as early as on day 3 and express at that
the burst forming cell unit (BFU) in erythropoiesis that dif- stage the CD45 antigen in 94% of cells (Fig. 34.4). After a
ferentiates into colony forming cell units (CFU) or, in our week in culture, the CD45 antigen practically disapears.19
case, CD34+CD45– cells. The colony forming cells in turn Therefore, these cells attach at the CD34+CD45+ stage, the
differentiate into erythrocytes, or in our scenario, endothe- burst-forming stage. From here, they could then further dif-
lial cells. Furthermore, it is well described that erythropoi- ferentiate into CD34+CD45– cells, the colony-forming stage,
etin does not act on burst forming unit-erythroid (BFU-E) and eventually endothelial cells, or they can remain
alone, but in concert with other growth factors. The forma- CD34+CD45+ cells and further differentiate into myeloid
tion of BFU-E necessitates the presence of other bioactive leukocytes. This decision could well be influenced by
molecules, originally termed burst-promoting activities, of fibronectin and/or VEGF.
which IL-3 was the first to be well characterized.
Interleukin-3 acts synergistically with erythropoietin to in- A Practical Vision for Future
duce proliferation of BFU-E,44 but does not appear to aug- Cardiovascular Implants
ment the effect of erythropoietin on CFU-E.45 Therefore, Tissue engineering can be defined as the application
with IL-3 also stimulating the formation of circulating he- of engineering disciplines to either maintain existing tissue
matopoietic stem cells (CD34+CD45+), it might well be that structures or to facilitate tissue growth. From the materials
Fig. 34.4. A summary of the proposed endothelial cell pathway. Hematopoietic stem cell in the bone marrow enters the blood
stream and changes its surface status after a) surface attachment as well as b) the presence of IL3, to form a committed endothelial
precursor cell. This precursor differentiates into an endothelial cell in the presence of VEGF.
Circulating Stem Cells: A Fourth Source for the Endothelialization of Cardiovascular Implants 377
engineering point of view, tissues are considered to be cel- 10. Zilla P, Grimm M, Fasol R, Eberl T, Fischlein T, Preiss P,
lular composites representing multiphase systems.46 In or- Krupicka O, Deutsch M, vonOppell U. Growth proper-
der to support tissue engineered tissue, researchers used scaf- ties of cultured human endothelial cells on differently
folds made of various absorbable as well as materials in- coated artificial heart materials. J Thorac Cardiovasc Surg
1991; 101:671-680.
cluding polidioxanone, polyglycolic acid, polyesther
11. Stump MM, Jordan GJ, De Bakey, Halpert B. Endothe-
(Dacron), ePTFE, polyurethanes and collagen.47-49 Today, lium grown from circulating blood on isolated intravas-
scaffold research plays a major role in the overall drive to- cular dacron hub. Am J Path 1963; 43:361-367.
wards tissue engineered vascular implants. Furthermore, the 12. Mackenzie JR, Hackett M, Topuzlu C, Tibbs DJ. Origin
technology of covalent bonding of certain peptides, includ- of arterial prosthesis lining from circulating blood cells.
ing cytokines, to polymeric surfaces holds the promise that Arch Surg 1968; 97:879-886.
scaffolds may play a much more bioactive role in tissue en- 13. Bossart MI, Turner SA, Milam JD, Connor DJ, Urrutia
gineering in the future. It may well be possible to produce a CO, Frazier OH. Multipotential cells in the circulating
synthetic polymeric scaffold for a cardiovascular implant blood: Ultrastructural evidence in the calf. Trans Am Sac
with specific ligands within or on the surface of the struc- Artif Intern Organs 1982; 28:185-189.
ture which elicits a specific biological response. Such a re- 14. Feigl W, Susani M, Ulrich W, Matejka M, Losert U,
Sinzinger H. Organization of experimental thrombosis by
sponse, in our vision, will be the spontaneous endotheli-
blood cells. Virchows Arch (Path Anat) 1985; 406:133-148.
alization of the blood-contacting surface from recruited cir- 15. Shi Q, Wu M, Hayashida N, Werchezac AR, Clowes
culating precursor cells. Therefore, one could hypothesize AW,Sauvage LR. Proof of fallout endothelialization of
that the immobilization of VEGF onto a synthetic surface impervious Dacron grafts in the aorta and inferior vena
could be one possible step towards spontaneous cava of the dog. J Vasc Surg 1994; 20:546-547.
endothelialization, as it may promote stem cell attachment 16. Rafii S, Oz MC, Seldomridge JA, Ferris B, Asch A,
and differentiation, as well as maintenance. Nachman RL, Shapiro F, Rose EA, Levin HR. Character-
However, since surface endothelialization will even- ization of haematopoietic cells arising on the textured
tually need a mature underlying connective tissue matrix, surface of ventricular assist devices. Ann Thorac Surg
the wide open question regarding the existence of circulat- 1995; 60:1627-32.
17. Werchezac AR, Viggers RF, Coan DE, Savauge LR. Mito-
ing cells promises the next exciting chapter in this field.
sis and cytokinesis in subconfluent endothelial cells ex-
posed to increasing levels of shear stress. J Cell Physiol
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effective hematopoietic reconstitution of breast cancer
Facilitation of Healing
Cell Entrapment from the Circulation
CHAPTER 35
Surface Population with Blood-Borne Cells
William P. Hammond
Background
T he developmental biology of blood vessels has long been believed to consist of two phases.
The first, vasculogenesis, is the development during embryologic life of the original pre-
cursors to all vessels.1 Our understanding of this phenomenon has been built around detailed
studies of the development of chick and quail embryos, and the elegant tracing of cellular
movement during early development. Angiogenesis, on the other hand, is the term applied
to development of new blood vessels during adult life, which largely serves a reparative func-
tion.2 In addition to wound healing, this form of new blood vessel formation by sprouting
from existing blood vessels is believed to explain the development of vessels to feed growing
tumor cell masses. The understanding of regulatory factors for this form of new vessel growth
in the adult has been a principal theoretical model for the last few decades.
The introduction of vascular prostheses prompted study of the basic biology through
which these grafts maintain patency and integrity as blood conduits. As early as 1955, Sauvage
and Wesolowski had noted the apparent importance of tissue ingrowth to prosthesis perfor-
mance.3 Wesolowski’s experimental studies convinced him of the importance of porosity to
tissue ingrowth and prompted him to develop a high porosity knitted prosthesis.4,5 In 1962,
Florey et al observed islands of endothelium around mouths of vascular channels seen on
the flow surface of knitted Dacron grafts implanted in the baboon.6 Although they could
not determine the origin of these channels, they noted that they appeared to connect with
perigraft vessels. In 1985 Greisler et al demonstrated arterial regenerative activity in
polyglycolic acid absorbable aorta replacements in the rabbit,7 and in 1986, Clowes and
colleagues demonstrated in the baboon the capacity of a special porous form of PTFE, with
an internodal distance of 90 ∝m, to admit the development of transmural microvessels from
the perigraft tissue;8 this suggested that the usual tissue response to the placement of vascu-
lar prostheses involves a local variant of angiogenesis, which subsequently leads to endothe-
lial resurfacing of the graft, thereby contributing to its patency. In 1989 Kogel et al studied
transprosthetic vascularization in the dog, using a polyester-based resin to make three di-
mensional casts, and concluded that midgraft endothelialization in PTFE grafts with 60 ∝m
internodal distances was attributable to ingrowth of microvessels from the perigraft tissue
through the graft and into the lumen; this phenomenon was not observed in microporous
polyurethane grafts.9 In 1990 Golden and Clowes et al reported the importance of graft
porosity to healing of PTFE prostheses in the baboon; they found that a 60 ∝m internodal
distance produced optimal endothelial coverage.10 In 1995, Bull et al also reported midgraft
endothelialization of 60 ∝m internodal distance PTFE grafts implanted in the dog carotid,
and attributed it to microvessel ingrowth to the flow surface; they also noted that an external
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
380 Tissue Engineering of Prosthetic Vascular Grafts
jugular vein wrap around the grafts produced endo- dothelial precursor cell and suggest several implications for
thelialization as early as 7 days.11 In 1996 our center reported the engineering of vascular prostheses in the future.
histological demonstration of the continuity of a microvessel
between the perigraft vessels and flow surface microostia in Impervious Grafts
a porous Dacron prosthesis; however, we were unable to In 1994, Shi and Sauvage et al reported the results of a
determine whether its origin was in the outer wall or on the study designed to demonstrate unequivocally that perigraft
inner flow surface of the graft.12 tissue ingrowth is the sole source of healing beyond the pan-
In 1963, Stump et al described the presence of endot- nus areas.19 Dacron grafts were made impervious by a coat-
helium on Dacron hubs suspended in the descending tho- ing of silicone rubber and shielded from pannus ingrowth
racic aorta of young pigs, and cited this as evidence for for- (which is very limited in humans and dogs)20 by 2.5-6 cm
mation of endothelium from circulating cells in the blood.13 lengths of microporous (30 ∝m internodal distance) PTFE
More recently, Scott et al found endothelium on the sur- at each end. Suitable lengths of this composite graft were
faces of pledgets of vascular graft material implanted in dog implanted in the descending thoracic aorta, abdominal aorta
aortas.14 A direct demonstration of endothelium in the blood and inferior vena cava of the same dog. At the end of the
stream was developed by Sbarbati et al using an endothe- implant periods, light microscopy study confirmed that there
lial-specific monoclonal antibody to analyze patients’ arte- was no perigraft tissue ingrowth (Fig. 35.1). Instead of the
rial and venous blood before and after heart catheteriza- expected unhealed flow surface, by 4 weeks all these imper-
tion,15 and in the following year George et al identified vious grafts had scattered islands of endothelialization
postangioplasty circulating human endothelial cells with (Fig. 35.2). We named this phenomenon “fall-out” healing,
S-Endo1 coupled to immunomagnetic beads.16 Further evi- because the only possible source appeared to be the blood
dence of circulating endothelium has been supplied by flowing through the graft.
Frazier’s17 and Rafii’s18 studies of human endothelial cells The experimental study was extended to include 70 cm
found on the lining of ventricular assist devices. long impervious grafts implanted in the carotid-femoral
position in dogs.21 At 3 months, each of the 5 extra-ana-
Previous Studies tomic grafts that remained patent had results similar to the
The following studies have produced data that, taken aorta and inferior vena cava grafts, with scattered endothe-
together, strongly support the concept of a circulating en- lial islands in the midgraft area despite a lack of ingrowth
from the outer wall.
Fig. 35.1. NOTE: See color insert for color representation. Blockage of pannus and transmural ingrowth for study of
fallout endothelialization of isolated central Dacron limb of a prosthesis made impervious by a coating of silicone rubber
and implanted for 8 weeks in the descending thoracic aorta of a dog. (A) No transmural ingrowth from perigraft tissues
and limited pannus from native aorta onto PTFE (LM, x50). (B) PTFE graft, beyond the pannus zone, showing no perigraft
tissue growth into the graft (LM, x25). (C) No perigraft tissue ingrowth into the impervious Dacron graft, but presence of
endothelial-like cells on flow surface, with #-actin positive smooth muscle cells beneath (H&E; LM, x50). Reprinted with
permission from Shi et al. Proof of fallout endothelialization of impervious Dacron grafts in the aorta and inferior vena
cava of the dog. J Vasc Surg 1994; 20:549.
Surface Population with Blood-Borne Cells 381
Fig. 35.2. NOTE: See color insert for color representation. Findings for specimens taken from areas beyond the pannus zone of
Dacron grafts made impervious to perigraft tissue ingrowth by a silicone rubber coating and implanted in the dog. (A)-(L)
Endothelial-like cells on flow surfaces of Dacron prostheses implanted in the descending thoracic aorta, abdominal aorta and
inferior vena cava of the dog: (A)-(C) LM, AgNO3, x70; (D)-(F) LM, x500; (G) SEM, x1000; (H) SEM, x500; (I) SEM, x3000;
(J)-(L) TEM, x4000. (M)-(O) Proof of endothelium by FVIII/vWF positivity on flow surface of isolated Dacron grafts: (M)-
(N) x500; (O) x1000. Reprinted with permission from Shi et al. Proof of fallout endothelialization of impervious Dacron
grafts in the aorta and inferior vena cava of the dog. J Vasc Surg 1994; 20:550.
382 Tissue Engineering of Prosthetic Vascular Grafts
Porous Grafts, Using Early Implantation Periods 1. A fibrin matrix layer;
My colleagues have also demonstrated the phenom- 2. A fibrin matrix layer topped with a thin layer of mac-
enon we refer to as fall-out healing in porous grafts by using rophages and leukocytes;
early implant periods.22-24 In 2 studies, they accelerated 3. Multiple layers of #-actin positive cells; in a few in-
endothelialization by wrapping a resected segment of the stances, microvessels were associated with the latter
autogenous inferior vena cava around knitted Dacron grafts (Fig. 35.1C).19
implanted in the abdominal aorta of dogs. In the first study, It is possible that this manifestation was time depen-
at 2 weeks they found active angiogenesis between the wrap dent: It was seen in descending thoracic aorta grafts at 8 and
and the outer graft wall, regardless of whether the intima or 12 weeks, but not in 4 week descending thoracic aorta, ab-
adventitia of the inferior vena cava was placed against the dominal aorta and inferior vena cava grafts (all carotid-femo-
graft (Fig. 35.3).22 Endothelialization and ostia formation ral grafts were implanted for 3 months).
occurred on some flow surface areas at 2 weeks, either be-
fore the tissue ingrowth had reached into the graft wall, or Human Grafts
before it had grown close to the lumen (Fig. 35.4). In a sec- In 1975 Sauvage et al reported endothelium on the
ond study of this accelerated endothelialization model, BrdU flow surface of a human axillofemoral graft; unfortunately,
staining revealed endothelial cells and increased cellular pro- their analysis was limited because the only technique avail-
liferation near the flow surface as early as 7 days, with no able to them was silver nitrate staining for morphology.25
associated tissue ingrowth from the outer wall.23 Porous However, in 1995 we observed a patch of endothelium far
grafts without inferior vena cava wraps were then studied from areas of pannus ingrowth on a woven Dacron human
for short periods ranging from 7-14 days in the dog’s de- axillofemoral bypass graft, and endothelial factor VIII/von
scending thoracic aorta: At 7 and 10 days, endothelial cells Willebrand factor and Ulex europaeus agglutinin were avail-
were found on the midgraft area of the flow surface, with no able to confirm this finding (Fig. 35.5).26 This graft had no
connection to pannus or perigraft tissue ingrowth.24 perigraft tissue attachment due to serous fluid that had sur-
rounded it for two years,26 a factor that suggested fall-out
Substrate Observations healing was the endothelial source, as did microscopic evi-
We have seen three basic types of substrates beneath dence that there was no tissue in the interstices.
single-layer endothelium in impervious grafts:
Fig. 35.3. NOTE: See color insert for color rep-
resentation. Ostia and microvessels in knit-
ted Dacron grafts wrapped in segments of the
inferior vena cava and implanted in the ab-
dominal aorta of dogs. (A) At 2 weeks, flow
surface microostia identified by AgNO3 stain
(LM, x74). (B) At 2 weeks, flow surface
microostium (SEM, x444). (C) Microvessels
in the flow surface lining at 2 weeks (H&E;
LM, x486). (D) A 4 week serial section show-
ing a large microvessel between the flow sur-
face and the perigraft tissue (H&E; LM, x108).
Reprinted with permission from Onuki et al.
Accelerated endothelialization model for the
study of Dacron graft healing. Ann Vasc Surg
1997; 11:147.
Surface Population with Blood-Borne Cells 383
Fig. 35.4. NOTE: See color insert for color
representation. Two week specimens from
knitted Dacron grafts wrapped in autog-
enous inferior vena cava and implanted in
dogs. (A) A monolayer of endothelial-like
cells on the flow surface (H&E; LM, x108).
(B) Proof of endothelium (FVIII/vWF;
x460). (C,D) SEM and TEM, respectively,
showing morphology typical of endothe-
lium (SEM, x1918; TEM, x4640). (E) #-ac-
tin stained smooth muscle cells in the
neointima (LM, x108). (F) Control graft,
which was not wrapped in inferior vena
cava, showing incomplete healing (H&E
stain; LM, x42). Reprinted with permission
from Onuki et al. Accelerated endo-
thelialization model for the study of Dacron
graft healing. Ann Vasc Surg 1997; 1:146.
Present Studies developed as a result of fall-out endothelialization, a con-
cept that is supported by the 1983 report of tube formation
Ontogeny of Endothelial Cells Observed on the Flow from a monolayer of endothelium in a collagen matrix.27
Surface of Impervious Grafts The presence of smooth muscle cells in the neointima of
Sbarbati’s and George’s direct demonstrations of cir- the graft surface beneath the fall-out endothelium may also
culating endothelial cells dislodged from the vascular tree be attributed to cells from the blood stream, because there
by heart catheterization and angioplasty indicate one pos- seems to be no other source; however, unlike endothelial cells,
sible source for fall-out endothelialization.15,16 smooth muscle cells have not been observed in circulating
We are currently testing the hypothesis that the cell of blood, although Stump reported elongated cells resembling
origin is a primitive mesenchymal cell, possibly related to smooth muscle cells on isolated Dacron hubs,13 and in Scott’s
hematopoietic cell populations, and is not derived from ad- study of suspended PTFE and Dacron pledgets smooth
jacent existing blood vessels. One possibility is that hemato- muscle cells also were observed.14 Recently, a clinical case
poietic progenitor, or “stem”, cells can differentiate into report from Deutsch et al reported a matrix containing cells
endothelial cells. Our experimental in vivo studies are con- between a PTFE graft and an endothelial monolayer, which
tinuing, with implantation of impervious Dacron grafts in resulted from preoperative lining with autologous cultures
the descending thoracic aorta of beagles that have received of cephalic vein endothelial cells.28 Many of the cells in this
bone marrow transplants, and in which the DNA of donor matrix were actin and desmin positive, resembling smooth
marrow cells can be distinguished from the DNA of host muscle cells; this group also mentions the blood as a pos-
tissue cells. sible source, in addition to coseeding or pannus migration.
Character of Other Cells Lining Impervious Grafts Related Findings
In our studies we have demonstrated the presence of Until our report in 1995,26 it was believed that endo-
smooth muscle cells and microvessels beneath endothelium thelium did not appear on the flow surfaces of artificial vas-
on the flow surface of completely impervious Dacron grafts, cular grafts implanted in humans. However, we have now
or porous grafts retrieved before transmural angiogenic in- demonstrated endothelium on the flow surface of three
growth could traverse the wall.19,21,23 At 14 days we observed additional Dacron arterial prostheses explanted from hu-
unconnected microvessels in the intima and perigraft areas mans.29 As discussed earlier, one of these grafts was sur-
of the graft, suggesting that microvessel ingrowth also oc- rounded by seroma, which prevented any perigraft tissue
curs from the flow surface.24 The microvessels may have ingrowth into the graft.26 Of the remaining 3, 2 bifurcated
384 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 35.5. NOTE: See color insert for color repre-
sentation. Sections taken from the midgraft area
of a woven Dacron axillofemoral bypass graft ex-
planted from a patient during redo surgery after
an implant period of 26 months. (A) TEM show-
ing typical endothelial-cell morphology (x3050).
(B) Positive factor VIII/vWF staining confirms en-
dothelium (x485). (C) Confirmation of endo-
thelium by positive Ulex europaeus agglutinin
staining (x1210). (D) HAM 56 staining confirms
presence of macrophages (x485). Reprinted with
permission from Wu et al. Definitive proof of
endothelialization of a Dacron arterial prosthesis
in a human being. J Vasc Surg 1995; 21:865.
woven grafts had endothelium in areas with no tissue in- area. Similar results were obtained in rabbits. Although they
growth.29 The endothelium in all 4 instances was located in refer to this process as “therapeutic angiogenesis”, it suggests
the midgraft area, far from any possible extension of pan- a form of vasculogenesis, as do our experimental and clinical
nus ingrowth from the native vessel. findings of endothelialization of synthetic flow surfaces in
The occurrence rate for endothelialization of human the absence of blood vessel ingrowth. If hematopoietic
vascular grafts is, of course, impossible to estimate. How- progenitor cells can be shown to differentiate into endothelial
ever, the usual failure to find it in explanted specimens may cells, the case for an adult form of vasculogenesis will
be due more to the amount of elapsed time after death be- be strengthened.
fore removal and preservation than to the actual frequency:
The 3 specimens we have reported were all fixed either im- Implications
mediately or within 5 hours after death. The implications of these findings are potentially far
The possibility of clinical graft endothelialization raises reaching. In basic science our understanding of the devel-
the question of whether it would be possible to encourage opment of new blood vessels, and in particular their endo-
this phenomenon, either through treatment of the graft or thelial cell linings, would be transformed by a new under-
the patient. standing of the origins of these cells. The existence of a cir-
Recently, Asahara et al reported in vitro magnetic bead culating “angioblast” would significantly alter how one ap-
studies in which they isolated putative endothelial cell proaches disorders of new blood vessel formation, includ-
progenitors from human peripheral blood.30 They then in- ing the management of tumor metastases. Should we dis-
jected DiI-labeled CD34 positive mononuclear blood cells cover the existence of a true multipotential “hemangioblast”,
into the tail veins of mice with unilateral ischemia of a hind it would be necessary to dramatically reshape our concepts
limb; later examination demonstrated proliferative DiI- of angiogenesis and vasculogenesis. In fact, definitive proof
labeled cells in the reparative vascularization of the ischemic of the presence of a mesenchymal precursor for both he-
Surface Population with Blood-Borne Cells 385
matopoietic cells and vascular cells would suggest an adult grafts is influenced by graft porosity. J Vasc Surg 1990;
version of vasculogenesis which would profoundly alter the 11:838-845.
view of both normal physiology of the vasculature as well as 11. Bull DA, Hunter GC, Holubec H, Aguirre ML, Rappaport
pathologic changes in blood vessels. WD, Putnam CW. Cellular origin and rate of endothelial
cell coverage of PTFE grafts. J Surg Res 1995; 58:58-68.
In the clinical realm the application of these concepts
12. Wu MH-D, Shi Q, Onuki Y et al. Histologic observation
could be very important. Many efforts over the past decade of continuity of transmural microvessels between the
or so employing techniques for “seeding” of vascular grafts perigraft vessels and flow surface microostia in a porous
would have to be rethought in light of new concepts in stem vascular prosthesis. Ann Vasc Surg 1996; 10:11-15.
cell biology applied to endothelium, and possibly smooth 13. Stump MM, Jordan GL Jr, De Bakey ME, Halpert B. En-
muscle cells as well. The potential for specific identification dothelium grown from circulating blood on isolated in-
of cells giving rise to new endothelial surfaces could signifi- travascular Dacron hub. Am J Pathol 1963; 43:361-367.
cantly alter the basic approaches to design of experiments 14. Scott SM, Barth MG, Gaddy LR, Ahl ET Jr. The role of
using seeding. A second clinical implication is that the evo- circulating cells in the healing of vascular prostheses. J
lution of design concepts in vascular prostheses has moved Vasc Surg 1994; 19:585-593.
from “bioinert” to “biocompatible” materials,31 and will al- 15. Sbarbati R, deBoer M, Marzilli M, Scarlattini M, Rossi G,
van Mourik JA. Immunologic detection of endothelial cells
most surely need to evolve further to a concept of
in human whole blood. Blood 1991; 4:764-769.
“biospecific” materials. For example, where a goal in the past 16. George F, Brisson C, Poncelet P et al. Rapid isolation of
may have been the synthesis of biopolymers that failed to human endothelial cells from whole blood using S-Endo1
activate coagulation processes of blood and subsequently monoclonal antibody coupled to immuno-magnetic beads:
moved toward materials that are capable of passive, Demonstration of endothelial injury after angioplasty.
nonactivating interactions with their surroundings, the Thromb Haemost 1992; 67:147-53.
present studies suggest a need to develop materials with spe- 17. Frazier OH, Baldwin RT, Eskin SG, Duncan JM. Immu-
cific affinities for the circulating cells, which would also pro- nochemical identification of human endothelial cells on
mote both their landing and growth in situ on the appro- the lining of a ventricular assist device. Texas Heart Inst
priate graft materials. If circulating endothelial progenitor J 1993; 2:78-82.
18. Rafii S, Oz MC, Seldomridge JA, Ferris B, Asch AS,
cells truly exist, the clinical approach to accelerated healing
Nachman RL, Shapiro F, Rose EA, Levin HR. Character-
of prostheses will be totally transformed, and the tissue en- ization of hematopoietic cells arising on the textured sur-
gineering required for vascular graft prostheses will be en- face of left ventricular assist devices. Ann Thorac Surg
tirely different in the future. 1995; 60:1627-1632.
19. Shi Q, Wu MH-D, Hayashida N et al. Proof of fallout
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267:10931-10934. ing of porous arterial prostheses. Observations, 1960 to
3. Sauvage LR, Wesolowski SA. The healing and fate of ar- 1974. Arch Surg 1974; 109:698-705.
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Rationale for the development of the gossamer small ar- endothelialization model for the study of Dacron graft
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lining fabric grafts of the aorta. Br J Exp Pathol 1962; erated graft healing, with BrdU identification of cellular
43:655-60. proliferation. Ann Vasc Surg, 1998; 12:207-215.
7. Greisler HP, Kim DU, Price JB, Voorhees AB Jr. Arterial 24. Onuki Y, Kouchi Y, Yoshida H et al. Early presence of
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Surg 1985; 120:315-323. rial prostheses implanted in the descending thoracic aorta
8. Clowes AW, Kirkman TR, Reidy MA. Mechanisms of ar- of the dog. Ann Vasc Surg, 1997; 11:604-11.
terial graft healing. Rapid transmural capillary ingrowth 25. Sauvage LR, Berger K, Beilin LB, Smith JC, Wood SJ,
provides a source of intimal endothelium and smooth Mansfield PB. Presence of endothelium in an axillary-
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123:220-230. surface. Ann Surg 1975; 182:749-753.
9. Kogel H, Amselgruber W, Frosch D, Mohr W, Cyba- 26. Wu MH-D, Shi Q, Wechezak AR et al. Definitive proof
Altunbay S. New techniques of analyzing the healing pro- of endothelialization of a Dacron arterial prosthesis in a
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10. Golden M, Hanson SR, Kirkman TR, Schneider PA, tion of endothelial cells into capillary-like networks is
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386 Tissue Engineering of Prosthetic Vascular Grafts
28. Deutsch M, Meinhart J, Vesely M et al. In vitro 30. Asahara T, Murohara T, Sullivan et al. Isolation of puta-
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29. Shi Q, Wu MH-D, Onuki Y et al. Endothelium on the interface. How to build a blood vessel; Lifeline Founda-
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Facilitation of Healing
Cell Entrapment from the Circulation
CHAPTER 36
Cellular Population of the Textured-Surface Left Ventricular
Assist Devices Leads to Sustained Activation of a
Procoagulant and Proinflammatory Systemic Response
Talia B. Spanier, Ann Marie Schmidt, Mehmet C. Oz
Overview
T he use of LVAD technology has a potentially critical role in the management of patients
with end-stage cardiac failure.1-6 The ability of this device to enhance left ventricular
function as a bridge to transplantation or for longer periods is well established. However, as
with any foreign material implanted into a host, the issue of host-graft interaction becomes
an integral part of LVAD biology, beyond its mechanical function as a pump. Compared
with early design LVADs, whose surface was mainly of smooth contour, the
Thermocardiosystems HeartMate LVADTM was designed with a textured surface polyure-
thane diaphragm, intended to minimize the potential for thromboembolic complications
by facilitating the formation of a tightly adherent “pseudoneointima” on the blood-contact-
ing surface7-12 (Fig.36.1). The goal of this strategy was to reduce the risk of development of
thromboemboli by eliminating direct contact between the device and circulating blood.11
The apparent success of this modification in LVAD design was suggested by studies which
reported a thromboembolic rate of < 2% associated with use of this modified surface.12
Furthermore, patients receiving this device were apparently safely maintained with no or
minimal forms of systemic anticoagulation.
The implantation of this device with its subsequent cellular population and creation
of a biological lining, however, has significant impact on its host, with both procoagulant
and proinflammatory consequences. Of considerable interest, we believe that so few in-
stances of clinically apparent thromboembolic phenomena are seen because of the creation
of a delicate balance between the generation of thrombin and activation of fibrinolytic path-
ways which occurs as a result of device implantation. Furthermore, it is tempting to specu-
late that perturbations of vascular homeostasis associated with infection, inflammation or
surgery, for example, might easily tip the balance of hemostatic mechanisms to formation of
thrombi or consumptive coagulopathy. Thus, dissecting the mechanisms by which
prothrombotic tendencies, anticoagulant defenses and fibrinolysis are balanced in this LVAD
environment become essential for understanding clinically relevant prothrombotic disor-
ders in these patients.
The development of clinically important immune alterations in patients related to the
implantation of the textured surface LVADs also is of significant interest. It has been ob-
served that LVAD patients develop increased levels of anti HLA antibodies13-15 in a time-
dependent manner over the course of LVAD implantation.15 In our own experience, the
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
388 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 36.1. LVAD. Left Panel. Schematic illustration of the LVAD implanted in a patient. The pump is positioned intraperitoneally in
the upper left quadrant of the abdomen. Dacron conduits are used to connect the pump between the apex of the left ventricle and
the ascending thoracic aorta. The LVAD is either pneumatically or electrically driven via a percutaneous drive line powered by an
external console or battery pack, respectively. Porcine valves placed in the inflow and outflow conduits ensure the unidirectional
flow of blood. Right Panel. The blood-contacting surfaces of both the titanium housing component and the flexible polyurethane
diaphragm are textured to encourage the formation and adherence of a biological lining. The flexible diaphragm consists of a
fibrillar surface integral with the base material to eliminate detaching of fibrils. The individual fibrils measure approximately
18 ∝m in diameter to 300 ∝m in length. The nonflexible housing surface is fabricated from titanium microspheres sintered together
to form a porous nonpermeable topography. The diameter of the spheres ranges from 50-75 ∝m, with a resultant pore size ranging
from 25-50 ∝m.
clinical consequences of these antibodies are significant and Thrombin Generation and Fibrinolysis
unique to each class of antibody produced. Anti-MHC class In order to better understand the hematologic profile
I antibodies, measured in the panel reactive antibody assay which underlies the low thromboembolic risk associated
(PRA), are associated with a prolonged waiting time to trans- with the textured surface LVAD, and to more fully under-
plantation because of difficulty in finding a negative stand the long term physiologic implications of its use, we
crossmatched donor organ. Anti-MHC class II antibodies analyzed measurements of thrombin generation and fibrin-
are associated with increased severity and frequency of cel- olysis in patients supported by the TCI LVAD. Our data in-
lular rejection episodes after transplantation. Of note, our dicate that despite both apparent clinical stability and “nor-
data do not suggest that these findings are related to such mal” screening values of routine hemostatic parameters such
factors as number of blood transfusions, use of filtered blood, as platelet count, prothrombin and activated partial throm-
age or gender. However, we propose that one potential boplastin times, patients with textured surface LVADs nev-
perturbant in this setting may be the presence of CD34 posi- ertheless demonstrate significant activation of coagulation
tive cells and other activated inflammatory cells populating with secondary fibrinolysis.16 The etiology (ies) underlying
the LVAD. We believe, in fact, that interactions between the these hemostatic and immune abnormalities are of consid-
LVAD surface and host mononuclear cells leads to a Th2 erable interest. Such LVAD-specific etiologies as the textured
pattern of cytokine expression by activated T cells, which polyurethane surface, the titanium housing, and the dacron
contributes to B cell hyperreactivity in vivo (Fig.36.2). These inflow / outflow lines need to be considered. Other etiolo-
interactions ultimately culminate in immune alterations in gies, such as altered flow characteristics and the presence of
the host, further emphasizing the necessity of understand- preexisting clots in the failed ventricles, must also be inves-
ing the identity and functional significance of the cellular tigated (Fig. 36.3). Our data do not suggest that these find-
populations of the LVAD surface. ings are a direct result of the surgical implantation proce-
Sustained Activation of a Procoagulant and Proinflammatory Systemic Response 389
Fig. 36.2. B cell antibody pro-
duction results from antigen
driven CD4 positive Th2 T cell
activation: B cell antibody pro-
duction results from the follow-
ing complex pathway. First: Mac-
rophages on the LVAD surface
activate CD4 positive T cells by
presenting antigen in the context
of MHC class II molecules
together with IL-1 production.
The subsequent activation of Th2
T cells leads to production of
IL-10 and CD40-CD40L interac-
tions, which act on B cells to in-
duce antibody production.
dure of the LVAD, as hemostatic findings remain abnormal macrophage procoagulant activity (Fig. 36.5B), is consistent
at least up to the 335th day of LVAD therapy. Jeevanandam with the hypothesis that the microenvironment created by
and colleagues have shown that in the perioperative period, the LVAD was indeed reflective of a systemic pro-
compared with coronary artery bypass graft (CABG) pa- thrombotic effect.
tients, LVAD patients consistently demonstrate significantly
higher markers of thrombin generation (TAT and F1+2) and LVAD Surface Cellularization
fibrinolysis (D-dimers and FDPs).17 Our studies examining Indeed, these data suggest that a specific characteris-
LVAD patients in this regard do not, however, demonstrate tic of the LVAD textured surface itself is, at least in part, re-
that these perioperative findings are sustained, as the overt sponsible for these observations. The blood contacting sur-
indices of thrombin generation and fibrinolysis are acute faces of both the titanium housing component and the flex-
and procedure-related. In fact, prospective analysis of 20 ible polyurethane diaphragm are textured to encourage the
LVAD patients from the time of LVAD insertion for up to formation and adherence of a biological lining (Fig. 36.1,
335 days revealed an initial rise in markers of thrombin gen- right panel). The flexible diaphragm consists of a fibrillar
eration as measured by elevated levels of prothrombin frag- surface integral with the base material to eliminate detach-
ment 1+2 (F1+2) and thrombin-antithrombin III complex ing of fibrils. The individual fibrils measure approximately
(TAT). The data demonstrated an increase in each of these 18 ∝m in diameter to 300 ∝m in length. The nonflexible hous-
parameters in the immediate perioperative period which ing surface is fabricated from titanium microspheres sin-
declined progressively by days 5-7. Subsequently, indices of tered together to form a porous nonpermeable topography.
thrombin generation and fibrinolysis rose in a time-depen- The diameter of the spheres ranges from 50-75 ∝m, with a
dent manner, reaching an apparent maximum by day 35 resultant pore size ranging from 25-50 ∝m.9
which was sustained through at least day 335 (Fig 36.4A,B). Although either the sintered titanium surface or the
Similar results were observed for levels of D-dimers polyurethane diaphragm should in theory be capable of cel-
(Fig. 36.4C). lular entrapment, we and others have demonstrated in stud-
These data suggest that activation of coagulation in ies of explanted LVADs considerable cellular entrapment
LVADs is biphasic. Initial / immediate activation of coagu- specifically by the polyurethane diap-Bposits on the sintered
lation and fibrinolysis in the perioperative period is likely titanium housing.18-22 Flow cytometry studies have demon-
secondary to acute contact activation of the blood with the strated that the majority of these diaphragm cells are my-
extensive foreign surfaces of the cardiopulmonary bypass eloid/monocytic in origin. In addition, a smaller percentage
and with the LVAD itself, as well as perioperative surgical of the cells are pluripotential hematopoietic cells which can
response. Later, a second, sustained phase of activation of be induced to differentiate in culture to mature hematopoi-
coagulation/fibrinolysis is created in these LVAD patients, etic cells.22 Menconi and colleagues21 found that the surface
which is caused by the progressive cellular population of the of explanted LVADs contained adherent mononuclear cells,
LVAD surface with cellular entrapment, activation and en- platelets, and myofibroblasts intermingled with areas of com-
hanced procoagulant activity. Furthermore, although we pact fibrinous material, as well as areas of collagenous tis-
found no evidence of increased IL-1 ! or TNF-# in the pe- sue. Similarly, Salih and colleagues18 found the surface of
ripheral plasma of LVAD patients over the time course of the explanted LVAD polyurethane membrane to consist of
LVAD implantation, sustained elevations in levels of tissue fibrinous and cellular layers. The immediate layer atop the
factor antigen were present in LVAD patients compared to membrane was composed of a compact fibrin coating as well
normal controls (Fig 36.5A). This, together with the finding as numerous mononuclear cells and spindle-shaped cells.
of increased tissue factor activity as reflected by enhanced This was followed by an intermediate middle layer consisting
390 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 36.3. Activation of coagulation
and fibrinolysis. As demonstrated by
our studies, evidence exists for signifi-
cant thrombin generation (as reflected
by increased levels of thrombin-AN-
TITHROMBIN COMPLEX (TAT)
and prothrombin activation peptide
(F1+2)) and fibrinolysis (increased lev-
els of D-dimers and fibrinogen (fi-
brin) degradation products (FDPs) )
in patients with TCI LVAD compared
with normal control population val-
ues and patients with end-stage heart
failure not supported by LVAD or an-
ticoagulant therapy. The mechanism
(s) underlying the generation of a
procoagulant state are not yet clear
since, for example, it is not known if
activation of the intrinsic and/or ex-
trinsic pathways of coagulation under-
lie these findings. Closed arrows are
indicative of the procoagulant pathway resulting in the formation of a fibrin clot. Open arrows follow the fibrinolytic pathway.
Dotted arrows and bold font signify the markers of these pathways reported in this study.
of cells resembling fibroblasts and fewer mononuclear cells of a stimulus releasing increased numbers of dendritic cells
than in the immediate inner layer, and the outermost layer, into the blood of patients with LVAD and/or the presence of
at the biomaterial/tissue interface, contained a foreign body highly efficient mechanisms capable of trapping them on
type reaction with numerous multinucleated giant cells. Sec- the LVAD surface. In the first context, the presence of sig-
tions of tissue islands removed from the titanium surface nificant hemolysis in LVAD patients (as defined by elevated
revealed organized fibrous and collagenous tissue with few plasma free hemoglobin and absent haptoglobin) suggests a
cellular areas, except for occasional mononuclear cells. While possible mechanism underlying bone marrow stress. The
Salih and colleagues found no evidence of endothelial cells presence of activated macrophages on the LVAD surface pro-
on the surface of the LVAD, other studies have disputed this. vides an environment potentially enhancing recruitment of
Specifically, Frazier and colleagues19 found evidence for the CD34 positive and other cells, such as lymphocytes. In this
presence of endothelial cells on the pseudointimal lining of context, the work of Browning, Gallo and colleagues24 con-
the textured LVAD surface. Using antibodies to von cerning Kaposi’s sarcoma-like spindle cells (derived from the
Willebrand factor, they demonstrated positive immunore- peripheral blood of HIV-1-infected individuals) is especially
activity in certain cells on the lining, suggestive of the pres- of interest. They found that such cells expressed markers
ence of endothelial cells. (Of course, platelet material may found on both activated cells of monocyte/macrophage and
also be responsible for this positive immunostaining for von endothelial lineage, similar to what we have observed in cells
Willebrand factor). The significance of defining the pres- populating the LVAD. The authors speculated that a likely
ence of endothelial cells in this setting cannot be overstated, origin of Kaposi’s spindle cells would be a circulating bone
since their presence may suggest the potential for restora- marrow progenitor cell. The finding that culturing of these
tion of normal control of the vascular pro- and anticoagu- spindle cells required the presence of conditioned medium
lant pathways. from activated lymphocytes suggested the requirement for
In an attempt to more clearly delineate the cellular an additional stimulus to promote differentiation and pro-
content of the LVAD surface, we began to try to more de- liferation of Kaposi’s cells. By analogy with the situation in
finitively define the phenotypes of cells which are adsorbed LVAD, we propose that the initial recruitment of immune/
to the LVAD surface. inflammatory cells to the LVAD surface establishes a local
Our demonstration of CD34 positive cells on the milieu facilitating recruitment of CD34 positive cells from
LVAD surface (Fig. 36.6) confirms the results of Rafii and the blood. This further promotes the establishment of the
colleagues.22 Such cells, likely to be derived from the bone LVAD as an organ with impact on diverse host response
marrow, are progenitor cells which under normal conditions mechanisms.
differentiate into red blood cells, myeloid cells and platelets. Until future studies identify whether there are elevated
Under homeostatic conditions, their numbers in the periph- levels of CD34 positive cells in the periphery and whether
ery are quite low (0.01-0.1%).23 This suggests the presence there are specific stimuli recruiting them to the LVAD in
these patients, an alternate explanation must be considered.
Sustained Activation of a Procoagulant and Proinflammatory Systemic Response 391
Fig. 36.4. Prospective analysis of hemo-
static parameters obtained from patients
with textured surface LVAD. Twenty pa-
tients with LVAD as described above were
examined at various time points as indi-
cated for levels of: (A) prothrombin frag-
ment 1+2 [F1+2] (nmol/ml); (B) throm-
bin-antithrombin III complex [TAT]
(∝g/ml); and (C) D-dimers (ng/ml). Each
point represents the mean of duplicate
values + standard error. Normal values for
each parameter are indicated in the inset
of each figure. * denotes p < 0.01 com-
pared with normal values by analysis of
variance (ANOVA)
392 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 36.5. (A) Analysis of systemic
inflammatory/procoagulant me-
diators in the peripheral blood of
LVAD patients. Peripheral blood
was obtained from 11 LVAD pa-
tients at random times during
LVAD implantation (but at least
at or beyond 35 days). Plasma was
prepared by centrifugation and
assayed by ELISA for IL-1 ! ,
TNF- # or tissue factor as de-
scribed above. Data from LVAD
patients are reported as % above
normal population values as re-
ported by the manufacturer. Each
point represents the mean of du-
plicate determinations per pa-
tient (reported as mean ± SE). *
denotes p < 0.001 compared to
normal values by ANOVA. Open
bars represent normal values, and
black bars represent values ob-
tained from LVAD patients. (B)
Enhanced tissue factor activity in
peripheral blood monocytes of
patients with LVADs. Peripheral
blood mononuclear cells were
isolated from LVAD patients
(n = 5) or normal controls
(n = 3) and tissue factor activity
assays performed. TF activity was
then performed as described in
triplicate and reported as the
mean ± SE. * indicates p < 0.05
by ANOVA.
Fig. 36.6. Immunohistochemical analy-
sis of cells derived from the LVAD sur-
face: CD34. A distinct cellular popula-
tion was found to be positive for CD34,
a marker of pluripotent hematopoietic
stem cells.
Sustained Activation of a Procoagulant and Proinflammatory Systemic Response 393
Previous studies have suggested that bone marrow trans- left panel). Suggestive of enhanced macrophage procoagulant
planted onto vascular prostheses (composed of activity of these cells was the presence of increased
polytetrafluoroethylene, PTFE), results in complete immunostaining for tissue factor (Fig. 36.8). And, the cells
endothelialization by three weeks. Such graft areas popu- which were phenotypically similar to the population of CD34
lated with a confluent layer of cells were completely patent positive cells appeared to bear markers suggestive of endo-
and without evidence of thrombotic occlusion. In contrast, thelium, such as thrombomodulin (Fig. 36.9, left panel) and
control grafts (without bone marrow transplantation) were von Willebrand factor (Fig. 36.9, right panel).
covered by thrombi, as expected. Despite the fact that these Since CD34 positive cells are known to express adhe-
authors did not immunotype the cells transplanted onto the sion molecules, we postulated that such cells on the LVAD
prostheses, it is likely that the complex mixture contained surface might express vascular cell adhesion molecule-1
multiple hematopoietic precursor CD34 positive-like cells. (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1),
While it is possible that the LVAD surface-derived cells have thereby favoring the recruitment and adherence of further
the capacity to form a confluent monolayer, potentially ex- mononuclear/inflammatory cells. Immunocytochemistry
plaining why frank thromboses are not often observed, there revealed immunoreactivity for both of these adhesion mol-
is a critical distinction between the experimental previous ecules (Fig. 36.10, right and left panels, respectively). In the
results on PTFE and our data on the LVAD surface. Over- context of the LVAD surface, this is likely to be significant,
whelming evidence of angiogenesis was evident on the vas- since the presence of adhesion molecules provides a mecha-
cular PTFE prostheses, manifested by new capillary forma- nism for triggering the further recruitment of inflamma-
tion.25 In contrast, our study of multiple explanted LVAD tory/immune effector cells, thus propagating cellular acti-
surfaces has not shown angiogenesis, evidence of crude cap- vation on the LVAD. In contrast to the presence of cells bear-
illary formation or continuous cell monolayers covering the ing dendritic and monocytic markers, there was no staining
LVAD surface. Rather, we consistently observe that there are with antibody to smooth muscle actin or nonimmune IgG
patches of discrete cells (data not shown). Therefore, at this (Fig. 36.11 right and left panels, respectively).
time, we conclude that it is less likely that the cells on this Finally, the presence of proinflammatory leukocytes
surface are directed toward the formation of discrete vascu- was confirmed in the abundance of T lymphocytes which
lar, confluent, nonthrombogenic structures or monolayers. expressed strong immunoreactivity for CD3/CD4 and CD25
Further immunophenotypic characterization of cel- (Fig. 36.12), consistent with activated helper T cells.
lular populations on the LVAD surface suggested that inter- There was also a distinct B cell population immunore-
actions among different lineages of cells was likely occur- active for CD20 (Fig. 36.13).
ring. Quiescent round monocyte lineage cells expressing The presence of dendritic type CD34 positive cells with
CD14 were present (Fig. 36.7, right panel). In addition, nu- a procoagulant phenotype, as well as activated macrophages
merous larger cells with prominent cytoplasmic processes surrounded by proinflammatory leukocytes, certainly set the
and multinucleated morphology were present which express stage to explain the proinflammatory/procoagulant pheno-
the macrophage marker CD68, not CD14, and are therefore type created as a result of device implantation.
most likely activated macrophage lineage cells. (Fig. 36.7,
Fig. 36.7. CD14/CD68. Immuno-
phenotypic characterization of the
cellular populations on the LVAD
surface demonstrated that the most
prominent cell types were a popu-
lation of quiescent monocytic cells
bearing the marker CD14 (right
panel) as well as activated macroph-
ages bearing the marker CD68 (left
panel).
394 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 36.8. Procoagulant TF: Tissue
factor on the surface of these cells
confirms their procoagulant
phenotype.
Fig. 36.9. Endothelial markers: TM
and vWB. These cells express typi-
cal endothelial type markers like
thrombomodulin (sTM, left panel)
and von Willebrand factor (vWB,
right panel).
Cellular Activation demonstrated amplicons of the appropriate size for the
Consistent with the hypothesis that the LVAD surface proinflammatory cytokines IL-1, IL-2 and TNF as well as
itself contributes to the multiple biologic phenomena ob- the proinflammatory adhesion molecules VCAM-1 and
served in the course of its use, studies have revealed that this ICAM-1 (Fig. 36.14). RT-PCR for !-actin was utilized as the
lining is metabolically active. Menconi et al20 showed by RNA positive control.
hybridization analysis that the colonizing cells actively ex- To specifically demonstrate interactions between mac-
pressed genes encoding proteins for cell proliferation mark- rophages and T cells, our results were consistent with mac-
ers, cell adhesion molecules, cytoskeletal structures and ex- rophage driven T cell activation as evidenced by the pres-
tracellular matrix components. ence of mRNA for macrophage derived IL-1 and T cell de-
Evidence for immunological cellular activation on the rived IL-2R and IL-2 (Fig. 36.15). To determine whether there
LVAD surface was confirmed by evaluating the presence of was a predominance of Th1 or Th2 T cell activation and to
transcripts for inflammatory mediators. RT-PCR of RNA evaluate potential T cell/B cell interactions, we looked at
prepared from the cells harvested from the LVAD surface cytokine gene expression for IFN-&, IL-4 or IL-5 which was
Sustained Activation of a Procoagulant and Proinflammatory Systemic Response 395
Fig. 36.10. Inflammatory cell re-
cruitment: Cell adhesion molecules.
These cells strongly express intercel-
lular adhesion molecule (ICAM-1,
left panel) and vascular cell adhe-
sion molecule (VCAM-1, right
panel) consistent with their capac-
ity to recruit other circulating cells
thereby sustaining the inflamma-
tory phenotype.
Fig. 36.11. Smooth muscle actin and
nonimmune IgG. There was no
staining with antibodies to smooth
muscle actin (left panel) or
nonimmune IgG(right panel).
found to be absent. In contrast, prominent expression of vated macrophages on the LVAD surface activate CD4 posi-
IL-10 mRNA was detected. Importantly, the production of tive T cells by presenting antigen in the context of MHC
IL-10 is known to have a stimulatory effect on B cells and class II molecules together with IL-1 production. The sub-
has been reported to be increased in SLE and implicated in sequent activation of a Th2 subset of T cells leads to pro-
the pathogenic mechanism of autoantibody production in duction of IL-10 and CD40-CD40L interactions, which act
this disease. Evidence for T cell / B cell interactions involved on B cells to induce antibody production (Fig. 36.2).
in B cell hyperreactivity was shown by the presence of mRNA
for CD40 ligand. (CD40 on B cells and CD40 ligand on T LVAD as Inflammatory/Immune Organ
cells are the principal molecules involved in T cell contact- Taken together, these data suggest that considerable
mediated B cell stimulation.)(Fig. 36.16) Together these find- interactions between the LVAD surface cells and the host
ings led us to our supposition, as described earlier, that acti- are occurring throughout the time course of LVAD
396 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 36.12. Activated T cells. There
was an abundance of T lymphocytes
which expressed strong immunore-
activity for CD3/CD4 and CD25,
consistent with activated helper
T cells.
Fig. 36.13. Antibody-producing B
cells. There was also a distinct B cell
population which was immunore-
active for CD20.
Fig. 36.14. Activated cells
are present on the LVAD
surface: Examination by
RT-PCR. RT-PCR was
performed from material
directly removed from the
LVAD surface at the time
of explantation as de-
scribed above using spe-
cific primers. Positive
amplicons for the follow-
ing were identified: (A)
interleukin-1 ! , (B)
interleukin-2, (C) TNF-#,
(D) Vascular cell adhesion
molecule-1 and (E) inter-
cellular adhesion mol-
ecule-1. Markers are indi-
cated on the right hand
side of each panel.
Sustained Activation of a Procoagulant and Proinflammatory Systemic Response 397
implantation. We postulate that the mononuclear cells, plate- a cell-associated and released form, suggests a direct mecha-
lets and pluripotential stem cells initially trapped by the poly- nism for activation of coagulation. This is consistent with
urethane surface, not the titanium housing or Dacron grafts, the presence of tissue factor antigen in the plasma of pa-
become activated, thus leading to the generation of a local tients with LVADs,16 and suggests a mechanism for dissemi-
proinflammatory/procoagulant state, and that this mecha- nating the locally intense inflammatory and procoagulant
nism is, at least in part, responsible for triggering and sub- stimuli in the LVAD milieu. Furthermore, when monocytes
sequently sustaining the systemic activation of the coagula- were placed onto the LVAD surface in tissue culture, their
tion and fibrinolytic cascades. Furthermore, inflammatory capacity to respond to exogenously added LPS was enhanced
cells present on the LVAD surface appear to demonstrate (unpublished observation). This supports the concept that
production of proinflammatory cytokines and expression the LVAD primes adherent cells for heightened responsive-
of adherence molecules which promote cell activation, as ness to superimposed inflammatory stimuli.
well as facilitate recruitment of other cell types from the cir- We believe, therefore, that the biology of cells in the
culating blood, thereby sustaining the immune alterations LVAD resembles a two-hit model in which the initial place-
(Fig. 36.17). Our observation that monocyte-derived mac- ment of the device within the circulation results in recruit-
rophages seeded on the LVAD surface generate tissue factor, ment of adherent cells, initially likely to be CD34 positive
Fig. 36.15. Macrophage-driven T cell ac-
tivation. To demonstrate interactions
between macrophages and T cells we
evaluated cytokine gene expression by
mononuclear cells harvested from the
LVAD surface. Our results were consis-
tent with macrophage-driven T cell ac-
tivation as evidenced by the presence of
mRNA for macrophage derived IL-1 and
T cell derived IL-2R and IL-2. Positive
controls for each gene product were sup-
plied by the manufacturer. (As shown by
the lanes marked ‘control’).
Fig. 36.16. Th2 pattern of T cell activa-
tion. To determine whether there was a
predominance of Th1 or Th2 T cell acti-
vation and to evaluate potential T cell/B
cell interactions, we looked at cytokine
gene expression by mononuclear cells de-
rived from the LVAD surface. As shown
in this representative individual, no mes-
senger RNA for IFN-&, IL-4 or IL-5 was
present. In contrast, prominent expres-
sion of IL-10 mRNA was detected. Im-
portantly, the production of IL-10 is
known to have a stimulatory effect on B
cells and has been reported to be in-
creased in SLE and implicated in the
pathogenic mechanism of autoantibody
production in this disease. Evidence for
T cell/B cell interactions was shown by
the presence of mRNA for CD40 ligand.
CD40 on B cells and CD40 ligand on T cells are the principal molecules involved in T cell contact-mediated B cell stimulation.
398 Tissue Engineering of Prosthetic Vascular Grafts
pluripotent hematopoietic cells and monocytes, which then NF-∀B is involved in the transcriptional activation of in-
undergo subsequent differentiation and activation with the flammatory cytokines such as IL-1, IL-2, IL-6 and IL-8 in
capacity to recruit other cells, such as dendritic-type cells response to LPS or TNF, thereby facilitating the further
and lymphocytes, consequently sustaining and expanding propagation of the inflammatory response. Electrophoretic
the local host response. From such a view, the LVAD emerges mobility shift assays using nuclear extracts prepared from
as an immune/inflammatory organ which redirects the host cells removed from the LVAD surface26-27 as well as a
response, modulating multiple effector systems such as co- [32P]-radiolabeled consensus probe for NF-∀B demonstrated
agulation and immune mechanisms. a marked gel band shift, confirming NF-∀B activation in this
setting (Fig. 36.18). This finding, in addition to lending
further support to the concept that the cells colonizing the
NF-∀∀B Is a Marker of Cellular Activation in the surface of the LVAD are activated with respect to the
LVAD Surface Milieu and a Target for Anti- generation of a proinflammatory/procoagulant environ-
Inflammatory Intervention ment, also provided a potential target for therapeutic anti-
An important goal of our studies in attempting to inflammatory intervention in LVAD patients. In this context,
understand the intricate interactions between the populated a number of studies have suggested that NF-∀B’s role as a
LVAD surface and the host was to determine if therapeutic central regulator of the inflammatory response can be
strategies might be designed to limit the generation of downregulated by anti-inflammatory agents such as high
proinflammatory and prothrombotic mediators that appear dose salicylates (ASA).28-33 The specific cellular mechanism
to be central to the activation of coagulation/immune path- of ASA seems to be dependent on its ability to act as an an-
ways observed in the setting of the textured surface LVAD. tioxidant and free radical scavenger, thereby preventing the
We therefore sought evidence that the generation of inducible decay of I-∀B and therefore the subsequent
cytokines and tissue factor reflected ongoing cellular stimu- activation of NF-∀B. This effect has been shown to be
lation and activation of cell signaling pathways in the cells independent of the cycloxygenase pathway, as agents such
populating the diaphragm. A potential culprit in this set- as indomethacin are ineffective in blocking
ting is the nuclear transcription factor NF-∀B, an inducible I-∀B-NF-∀B signaling.
transcription factor of the Rel family which is known to have To test this hypothesis, we began a prospective evalu-
a central role in the inflammatory response. Specifically, in ation of ASA (325 mg PO/PR QD) started in LVADs within
endothelial cells, NF-∀B is required for the transcriptional three days of implantation. Markers of thrombin genera-
activation of VCAM-1, ICAM-1 and E-selectin in response tion (TAT and F1+2) and fibrinolysis (D-dimers) were sig-
to TNF-#, thus contributing to the expression of cell adhe- nificantly reduced compared to untreated LVADs (n = 20),
sion molecules involved in the recruitment of leukocytes to as were peripheral TF antigen and macrophage procoagulant
an inflammatory nidus. And, in proinflammatory leukocytes activity (p < 0.05). Inflammatory cell activation was also
Fig. 36.17. LVAD surface
cellularization by circulating
blood elements promotes
proinflammator y/pro-
coagulant environment. To
explain our findings, we pro-
pose that the textured sur-
face LVAD selectively
adsorbs and activates den-
dritic type cells and mac-
rophages from the circulat-
ing blood, allowing them to
create a proinflammatory/
procoagulant microenviron-
ment with the expression of
cell adhesion molecules and
cytokines which favor the
further recruitment and ac-
tivation of T cells and B cells,
as well as the expression of
procoagulant tissue factor.
Together, these forces con-
tribute to the activation of
both the coagulation and immune pathways associated with the implantation of this device.
Sustained Activation of a Procoagulant and Proinflammatory Systemic Response 399
abated, as none of the treated LVADs developed elevated all consistent with a primary decrease in inflammation at
PRAs up to 90 days post implantation. Analysis of cells on the LVAD surface as well as diminished ability to further
the LVAD surface at explant (n = 3) compared to untreated recruit inflammatory cells. Taken together, these data sug-
LVADs (n = 6) confirmed marked downregulation of the gest that targeted anti-inflammatory intervention with ASA
inflammatory response: may be an effective means to diminish cellular adsorption/
1.Markers of macrophage activation (CD68) were distinctly activation by the textured surface LVAD, thereby attenuat-
absent; ing the associated inflammatory response and improving
2.Cells did not express procoagulant TF or vascular cell ad- long term tolerance and eventual clinical outcome
hesion molecule-1; and (Fig. 36.19).
3.There was a marked absence of B cells (CD20) and T cells
(CD3/4/25)—
Fig. 36.18. Binding of nuclear proteins derived from LVAD cells
to NF-∀B consensus probe: Analysis by electrophoretic mobil-
ity shift assay. Cells were harvested from the LVAD surface and
immediately frozen in liquid nitrogen. Nuclear extracts were
subsequently prepared as described. Nuclear protein (10 ∝g)
was incubated with [32P]-labeled consensus probe for NF-∀B
alone (lane 1 and 4) or in the presence of an 100-fold molar
excess of unlabeled NF-∀B (lane 2) or unleveled Sp1 (lane 3).
In certain experiments, nuclear protein was preincubated with
anti-p65 IgG (lane 5), anti-p50 IgG (lane 6), anti-p50 and anti-
p65 IgG together (lane 8) or anti-c-Rel IgG (lane 7). In all
supershift assays, IgG (7.5 ∝g/ml) was utilized. This experiment
was performed from cells obtained from two different LVAD
surfaces, with identical findings. A representative assay is dem-
onstrated.
400 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 36.19. Anti-inflamma-
tory intervention with ASA
attenuates the proinflam-
matory/procoagulant re-
sponse created by LVAD im-
plantation. These encourag-
ing results support our hy-
pothesis that indeed the criti-
cal phenotype defining the
textured surface LVAD is its
ability to selectively trap and
adsorb dendritic and mono-
cytic cells from the circulat-
ing blood elements. Activa-
tion of these cells, however, is
clearly dependent on the in-
teractions between the host
and the cellular population of
the LVAD surface and is criti-
cally linked to activation of
the NF-∀B signal transaction
pathway. Inhibition of this
mechanism, therefore, does
appear to quiet these
adsorbed cells, inhibiting the production of cytokines and adhesion molecules necessary for the further recruitment and activation
of circulating B cell and T cells. NF-∀B inhibitors, therefore, do appear to be effective in downregulating the proinflammatory/
procoagulant systemic milieu associated with LVAD implantation.
Conclusion 3. Burton NA, Lefrac E, Macmanus Q et al. A reliable bridge
We propose that the textured surface LVAD microen- to cardiac transplantation: The TCI left ventricular assist
vironment presents a prototypic model for the study of device. Ann Thorac Surg 1995; 55:1425-30.
cellular mechanisms that underlie the pathogenesis of 4. Frazier OH, Macris MP, Myers TJ et al. Improved sur-
vival after extended bridge to cardiac transplantation. Ann
prothrombotic/proinflammatory disorders caused by
Thorac Surg 1994; 57:1416-1422.
implantation of foreign devices. These data suggest that 5. McCarthy PM, James KB, Savage RM et al. Implantable
modification of the present LVAD surface might be left ventricular assist device. Approaching an alternative
warranted to minimize cellular entrapment and activation. for end-satge heart failure. LVAD study group. Circula-
The activation of NF-∀B in this setting provides insight into tion 1994; 90(5 pt 2):II83-6.
a possible target of anti-inflammatory intervention in pa- 6. Pennington DG, McBride LR, Peigh PS et al. Eight years
tients with LVADs (especially as the success of this device experience with bridging to cardiac transplantation. J
has prompted moves toward its use as a permanent form of Thorac Cardiovasc Surg 1994; 107:472-80.
cardiac replacement), and also an excellent setting in which 7. Bernhard WF, Lafarge CG, Robinson et al An improved
to delineate the intricate molecular mechanisms that un- blood pump interface for left ventricular bypass. Ann Surg
derlie the potent proinflammatory/ prothrombotic environ- 1968; 168:750.
8. Clark R, Boyd J, Moran J. New principles governing the
ment created by this device. Future studies designed to ad-
tissue reactivity of prosthetic materials. J Surg Research
dress the role of both anti-inflammatory intervention in 1974; 16:510-522.
LVAD patients, as well as modifications in LVAD surface 9. Dasse KA, Chipman S, Sherman C et al. Clinical experi-
design and composition as a means of maximizing host- ence with textured blood contacting surfaces in ventricu-
device compatibility and long term tolerance, are under ac- lar assist devices (VADs). Trans Am Soc Artif Intern Or-
tive investigation. gans 1987; 33:418-425.
10. Rose EA, Levin HR, Oz MC et al. Artificial circulatory
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Facilitation of Healing
Transdifferentiation
CHAPTER 37
Transdifferentiation and the Vascular Wall
William A. Beresford
T he solidity of the idea of a continent hindered accepting that continents move and change.
Likewise, the long-accumulated robust bases for defining tissue and cell identities obliged
one to doubt that the occasional clearcut switches in character by mature, differentiated
cells—transdifferentiations1-3—were typical of cells in general and had far-reaching signifi-
cance. But today, such ‘plasticity’, although a vogue word, demands the attention of surgeons
and bioengineers if they are to understand their live working materials. For instance, has
what one grows in culture for grafting the phenotype one wants? Will this introduced tissue
be stable, and how will it interact with other tissues to maintain or alter their characters?
Will reactions to foreign materials or viral vectors for gene transfer4 encourage cells to take
on undesirable phenotypes? Can new and beneficial phenotypes be created in vitro or in
animals, for example, cells resistant to rejection or lacking dangerous cytokines? The de
novo generation of cardiac myocytes by transdifferentiation is a recent proposal.5 Certain
smooth muscle cells and fibroblasts can become epithelial and glandular. Hence, can one
graft to a vascular site cells transfected for long term prolific liberation of a therapeutic
substance, but which still have a sufficiently ‘vascular’ nature to be properly integrated? Or
which might also be regulated or transfected to express adhesion molecules to hold the cells
in place? Answers lie in seeing how each cell type of the many vascular walls can change its
phenotype (Figs. 37.1 and 37.2), uncovering the stimuli and their sources, and tracking the
regulatory pathways and interactions.
Endothelial Cells
Endothelial cells (ECs) have multiple personalities, recognized as their many respon-
sibilities at any particular site,6 and their different properties and responses at various loca-
tions7,8 and in the regions of a vascular bed.9 Examples are urea transport in the renal me-
dulla,10 and directing T lymphocyte homing to superficial rather than deep vessels of the
dermis.11 ECs’ biochemical profiles must reflect this diversity, but the transdifferentiations
detected thus far are mainly morphological.
To further aid lymphocyte homing and emigration, ECs in venules of nodes, tonsils,
and mucosal lymphoid organs become cuboidal, quite rich in organelles, and express
discernible antigens—high endothelial venules (HEVs).12 Coagulating and severing the
afferent lymphatic vessels to rat popliteal nodes causes the high endothelium to flatten and
lose its secretory ultrastructure by three weeks. Injecting sheep erythrocyte antigen into
such a node rapidly restores some of the high ECs and the lymphocyte traffic; thus the
conversion is reversible.13
HEVs develop from normal endothelium in chronically inflamed synovium, both
human and experimental animal.14,15 Other experiments demonstrate the role of an
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
404 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 37.1. Transdifferentiations
of vascular grafts.
Fig. 37.2. Less gene-flexing
transdifferentiations.
immunoreactive environment and the generality of the microfilaments with focal densities as to lead some authors
transdifferentiation. An immune mediator—gamma-inter- to term them ‘myoendothelial’.23
feron—induces expression of an HEV antigen in cultured The contrary phenomenon—an induction of fenes-
nonlymphoid ECs from mouse lung and marrow.16 Inter- trae in continuous ECs—also occurs in vivo:
feron-!, IL-2, and mitogens for ECs did not result in HEV- 1. During regrowth of capillaries in wounded guinea-
marker expression. The HEV marker and plumper ECs ap- pig cremaster muscle;24
peared in dermal venules where sheep red blood cells in 2. In vessels growing into the rat retinal pigment epi-
Freund’s adjuvant had been injected. Subcutaneous implants thelium after urethane-induced loss of the photore-
of sponges loaded with splenic lymphocytes acquire vessels ceptors;25 and
which display a high endothelium, if the lymphocytes are 3. In human dermal papillary vessels affected by pso-
allogeneic.17 riasis.26
The second and also reversible EC transdifferentiation Experimental instances are in:
is between fenestrated and continuous endothelium. The 1. The new capillaries of hypertrophied guinea pig gut
change may come about rapidly, within 10 minutes,18 on a muscle;27
physiological time scale (Fig. 37.3), or be slow and endur- 2. Rabbit lingual microvessels a few hours after destruc-
ing. Cirrhosis of the liver in man19 and in rats injected with tion of the animal’s platelets by anti-serum;28 and
CCl4 20 is accompanied by the fenestrated sinusoids acquir- 3. Rat cremasteric and dermal capillaries treated locally
ing a basement membrane and a continuous endothelium: with vascular endothelial growth factor (VEGF).29
a capillarization. If the cirrhotic liver progresses to hepato- A month of continuous infusion of retinoic acid or
cellular carcinoma, the tumor cells appear to cause the de- phorbol acetate into the rat cortex30 results in fenestrated
velopment of a basement membrane and endothelial CD34 vessels in cerebral cysts. After cessation of the infusion and
expression in the sinusoids of the tumor.21,22 ECs of these removal of the cannula, the vessels in the cystic cavity slowly
capillary-like tumor vessels are peculiar in having so many become continuous, despite the absence of astrocytes. There
Transdifferentiation and the Vascular Wall 405
Fig. 37.3. Electron micrographs de-
picting acute effects of hu rVEGF on
the microvasculature. After topical
application of VEGF-165 for 10 min-
utes, the rat cremaster muscle was
processed for EM (inset: topically ap-
plied VEGF-121). The cremaster
muscle is supplied with a continuous
(nonfenestrated) endothelium, but
within 10 minutes of exposure to
VEGF, fenestrations (arrowheads) are
formed in the microvascular endo-
thelium. Another effect of VEGF is
the loosened interendothelial junc-
tion (j). Both fenestrations and open/
loose junctions contribute to the per-
meability-enhancing properties of
VEGF. Bar: 500 nm; inset bar: 200 nm.
Figure kindly provided by Dr. W Gre-
gory Roberts, University of Califor-
nia, San Diego.
is other evidence in vivo31 that coculture work may have A differentiative role for basement membrane and
been misleading as to the importance of astrocytes in regu- neighboring tissue39 is clear in the conversion of embryonic
lating how brain ECs create the blood-brain barrier. How- cardiac endothelium of the atrioventricular canal to the mes-
ever, factors in the neural environment are significant, since enchymal cells that will form the cardiac cushion, from which
local chick vessels growing into intracoelomically grafted the AV valves and septa develop (Fig. 37.4).40 This trans-
embryonic quail brain acquire morphological and his- differentiation, in keeping with certain others of embryonic,
tochemical features of brain capillaries, with their special but partly differentiated, cells is termed an ‘epithelial-mes-
variety of continuous endothelium.32 enchymal transition/transformation’. It appears to require
For an analysis of factors controlling trans- specific matrix glycoproteins41,42 and, in the chick, TGF-!3,43
differentiations, we note that ECs are a form of epithelium, but in mouse the TGF isoform responsible and whether it
and epithelial-mesenchymal interactions are well investi- comes from the myocardium are uncertain.44 Preliminary
gated, and slowly making sense, in a variety of organs. Simi- evidence suggests that, in the earliest development of the
lar work in vitro on ECs indicates that fenestrated adrenal avian dorsal aorta, the first SMCs may be derived from the
capillaries lose their pores unless cultured on basement endothelium in chick45 and quail,46 from the sharing of en-
membrane, newly formed by MDCK cells,33 or reconstituted dothelial and smooth muscle markers. The endothelial-mes-
as Matrigel. However, the same MDCK-derived matrix was enchymal conversion may not be restricted to embryonic
unable to switch continuous capillary ECs to fenestrated.34 times. Human foreskin microvessel ECs grown in cyclic
Two specific differentiative agents, having opposite effects AMP-deficient medium lose their epithelial and EC charac-
on fenestrae of adrenal ECs, were retinoic acid, increasing teristics and become mesenchyme-like,47 as do dermal ECs
their number, and TGF-!, causing a marked reduction.35 exposed to interleukin-1!.48 That this was more than an in
A different and more comprehensive approach in vivo vitro dedifferentiation needs evidence that the resulting cells
is to examine markers of continuous endothelium and of have true mesenchymal potential, but there are other in-
sinusoids, along with changes in the molecular composition stances of possible EC to SMC conversions.49
of the adjacent matrix, as human liver sinusoids develop.36 The localized cardiac loss of endothelial character early
After an early phase of matrix and cellular structural change in development raises the issue of homeobox phenotype
(and the loss of capillary markers), the sinusoidal EC markers determining genes and their products. Receptors for the vas-
(CD4, ICAM-1, CD32, and CD14) appeared. Unexpectedly, cular endothelial growth factor (VEGF) family are the ty-
tenascin, although present, seemed not to regulate EC rosine kinases Flk-1 and Flt-1. Two related receptors, also
development, but laminin probably does.37 The influence almost restricted to endothelial cells, are Tie and Tek, but
of adjacent tissue stands out in the rabbit’s nose, where the their ligand is unknown. Working upstream of the genes for
endothelium of the capillaries and venous sinuses is these receptors is the product of a gene altered in the cloche
fenestrated only on the side of the vessel facing the mutation in zebrafish.50 This mutation causes the develop-
nasal epithelium.38 ing heart to lack endothelial cells. Here one can ask a question
406 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 37.4. Diagrammatic summary of endocardial cytodifferentiation. In (A) the endocardium of an 8 somite embryo is shown as
consisting of uniformly similar cell types in all areas of the simple tubular heart. In (B) the two pathways of endocardial develop-
ment are shown as they first occur in 16-18 somite embryos: In the atrium and ventricle, the endocardium becomes attenuated
with reduced secretory potential and acquires an “endothelial look”. Trabeculation brings the myocardium in close proximity to
the endocardium (B) and (C) and appears to be temporally related to “endothelization” of atrial and ventricular endocardium.
Conversely, in sites where prospective valvular and septal primordia (cushion tissue) will form, the endocardium hypertrophies
and acquires and amplifies secretory potential. The initial formation of cushion tissue cells (C) beneath the endocardium, and
their morphologic similarity to the latter, suggest that valvular and septal primordia are endocardial derivatives. Reprinted from
Markwald G et al. Devel Biol 1975; 42:160-180, with kind permission from Academic Press Inc., Orlando, Florida.
basic to any transdifferentiation, of whether in the endothe- can develop in vascular walls.61 Phagocytic cells removing
lial to mesenchymal switch the cell type specific cloche, Flk-1, surplus collagen from the tadpole’s degenerating tail are
flk-2, tie and tek genes stop expression. If so, one asks, ‘When?’ adapted local fibroblasts.62
since there is a sequence to gene activation during embry- When the bladder outlet is partially obstructed,
onic endothelial differentiation. Secondly, are the Hox and submesothelial fibroblasts of the rabbit’s urinary bladder
other genes needed to generate a cell type in normal devel- proliferate and thicken the serosa. The proliferating cells
opment (better understood in blood cell lineages51,52) again progress from expressing keratin-18 to become myo-
required when that type derives later from a fibroblasts, and finally resemble fetal smooth muscle cells,63
transdifferentiation? If so, this would offer an opportunity based on their immunoreactive cytoskeletal profiles. The
for experimental influences on mature cells. authors suggest that the keratin positivity indicates that some
of the proliferating fibroblasts become mesothelial cells to
Fibroblasts replace lost mesothelium. A dual potency of submesothelial
The tantalizing variety of site-related fibroblastic phe- fibroblasts is further indicated by the mixture of smooth
notypes53 extends to several kinds of transdifferentiation— muscle cells (SMCs) and decidua-like cells in the guinea pig
glandular, skeletal, and muscular, for example. Stromal fi- peritoneum, resulting from giving progesterone after three
broblasts of the rodent endometrium become glandular months of estrogen priming.64
epithelial decidual cells in normal physiology, and in re- In vitro studies mirror most of the above
sponse to various experimental provocations.54 Stromal cells transdifferentiations in vivo, and add more. Notable is the
of the human uterus, tube, and ovary behave similarly. Re- conversion of the fibroblast-like 3T3 cell line to adipocytes.65
nal medullary interstitial fibroblasts are laden with lipid Blocking mitosis with relaxin does not prevent the
droplets and may be endocrine,55 but in anemia the cortical adipogenic conversion, 66 giving further evidence that
fibroblasts also acquire significant lipid56 and are recruited transdifferentiation can be direct,67 i.e., not dependent on
to produce erythropoietin (Fig. 37.5).57,58 Fibroblasts from cell division. A mesenchymal to epithelial switch is demon-
somatic rather more than visceral sources become strated by 3T3 cells transfected with hepatocyte growth fac-
chondroblasts and/or osteoblasts, depending on the stimu- tor (HGF/SF) and the human Met receptor.68 The cells be-
lus and the animal species,59,60 but ectopic cartilage and bone come epithelial-like in vitro and tumorigenic in nude mice.
Transdifferentiation and the Vascular Wall 407
Fig. 37.5. Distribution of interstitial
ecto-5'-nucleotidase activity (ena)
(A,B) and of mRNA of erythropoi-
etin (C) in cortex of rat kidney. (A)
In normemic rats interstitial staining
for ena (arrows) is strong only in deep
levels of the cortical labyrinth (above
broken line); extent and intensity of
staining decease toward superficial
labyrinth (bar = 200 ∝m). (B) In an
anemic rat the extent of superficial
staining is increased, and staining in-
tensity is strong; (C) In situ hybrid-
ization of mRNA for erythropoietin
in same animal as in (B), showing
same distribution pattern as for ena.
Reprinted from Hir ML, Kaissling B.
Am J Physiol 1993; 264:F377-F387,
with kind permission of the Ameri-
can Physiological Society, Bethesda,
Maryland.
Taxol treatment of mouse embryonic fibroblasts makes them 4. Myofibroblasts occur as subtypes based on how far
adopt an epithelial morphology.69 they share other cytoskeletal components, such as
Hypoxic pulmonary vasoconstriction and hyperten- desmin with smooth muscle,79 and there are species
sion result from keeping newborn calves at simulated high differences;80
altitude. Among the structural remodelings is the switch of 5. Some myofibroblasts may be caught part way though
adventitial fibroblasts of the pulmonary arteries to making a fibroblast to smooth muscle conversion;81 many
elastin.70 Medium from culturing SMCs from hypoxic ar- others seem to be a stable endpoint of a
teries brings about an elastogenic response in cultured ad- transdifferentiation, although still able to revert to
ventitial fibroblasts from normal pulmonary arteries. In gen- fibroblasts;82
eral, the role of adventitial fibroblasts in vessel-wall remod- 6. Study of the production of extracellular matrix by
eling may have been underestimated.71 myofibroblasts has focused on collagen until re-
The ‘myofibroblast’72—the cell combining features of cently;83
fibroblast and SMC—is a practical and theoretical challenge. 7. The organ diversity of fibroblastic forms brings va-
Uncovering and understanding transdifferentiations of fi- riety to the starting cell: How fibroblastic are hepatic
broblasts to or towards muscle cells stumble on six issues: stellate cells,84 renal mesangial cells,85 and renal in-
1. How does one know that the starting cell was a fi- terstitial fibroblasts?86
broblast, since many organs normally contain The bulky literature on myofibroblasts concentrates
myofibroblasts, including aortic valves?73 on visceral fibrosis and dermal wound healing, but some of
2. Somatic connective tissues by and large lack the experiments are exciting, with new stimuli acting on the
myofibroblasts, unless disturbed or diseased,74,75 and usual cells, and old stimuli provoking newly examined cells.
help with this problem; The general phenotype-altering agent 5-azacytidine confers
3. How easily are SMCs, fibroblasts, and myofibroblasts an SMC-like phenotype on mouse mesangial cells.87 Expos-
to tell apart? They are distinguished by certain de- ing mouse dermal fibroblasts continuously to culture me-
finitive ultrastructural features, such as microfila- dium from growing C2C12 skeletal myoblasts causes the fi-
ment bundles and dense bodies which got the broblasts to express a skeletal muscle-specific transcription
myofibroblast idea started, but mostly by quantita- factor—MyoD—and desmin.88 And the differentiation-per-
tively different profiles76 rather than by specific turbing factor TGF-!1 similarly has cardiac fibroblasts in
markers, although #-smooth muscle actin has been culture becoming like cardiac myocytes.89 Intriguingly, stri-
the main standby, signifying a shift towards muscle. ated muscle, smooth muscle, and fibroblasts are already
(Specific cell type markers now exist only as linked in that all three, and endothelium, synthesize a spe-
smoothelin for smooth muscle77 and certain mAbs cial collagen—type XV.90
for fibroblast surface epitopes.78)
408 Tissue Engineering of Prosthetic Vascular Grafts
The cells involved in colonizing and incorporating a cytokines in myofibroblast activity.84 Lastly, as rat cardiac
Dacron graft to the canine aorta imply a similar connec- fibroblasts convert to myofibroblasts in response to injury,
tion. The graft becomes lined with collagen-synthesizing they express angiotensin-converting enzyme (ACE) on their
myofibroblasts, with a gradation of phenotype from fibro- surface. The resulting angiotensin ii may stimulate the re-
blasts in the pseudointima. Filaments in the early central lease of TGF-!l by autocrine and paracrine modes from the
endothelioid cells indicated that some of the endothelial cells myofibrolasts themselves and macrophages, respectively
might derive from the adjacent myofibroblasts,91 a counter- (Fig. 37.6).95
point to embryonic aortic endothelial cells being a source
of VSMCs. However, fibroblasts and endothelial cells are not Smooth Muscle Cells
directly tied to muscular phenotypes in that it is only the The diversity of smooth muscle phenotypes expresses
three muscle varieties that share use of the Gax different embryological germ layer origins, locations,
homeodomain protein during mouse embryogenesis.92 changed physiological demands, pathological effects, and
Lastly, angioplastic balloon injury elicits proliferation and a species. Ectodermal neural crest gives rise to smooth muscle
myofibroblastic reaction from adventitial fibroblasts in por- of great arteries, but, if ablated, is replaced inadequately by
cine coronary arteries.93 mesodermal SMCs.96 Lineage maintains its influence on the
Analyzing how cytokines influence the phenotypic thoracic versus abdominal aortic SMCs in their adult be-
conversions is of general interest. Four experiments point havior. Vascular smooth muscle cells (VSMCs) are distinct
the way. Fibrosis is an end result requiring adequate num- from gastrointestinal, uterine and tracheal smooth muscles,
bers of myofibroblasts, so which cytokines promote prolif- and experience most of the known transdifferentiations. In
eration? And which cause transdifferentiation? When the kidney, the afferent arterioles become glandular and se-
TGF-!1 induces quiescent normal human breast fibroblasts crete renin in response to a variety of stimuli,67 including
to express #-smooth muscle actin in culture without divid- an inhibitor of angiotensin I converting enzyme.97 SMCs of
ing, this shows the differentiative role of TGF-!, and that some muscular arteries become fibroblastic in situ, produc-
the phenotype does not arise by selection of a subpopula- ing excessive collagen and other ECM materials in medial
tion.74 This is, incidentally, another example of a direct fibromuscular dysplasia,98,99 a condition reproduced in dogs
transdifferentiation. Overexpression of GM-CSF in the rat after several months by blocking the vasa vasorum with a
lung in vivo expands and activates the macrophage popula- mixture of thrombin and gelatin.100 A more physiological
tion, which then provides the TGF-! causing the myo- phenomenon is the remodeling and increase in medial col-
fibroblasts to develop and make collagen.94 Detection of lev- lagen in mesenteric arteries of spontaneously hypertensive
els of, and relations between, cytosolic signal-transduction rats,101 although the changes precede the increase in blood
molecules, such as Ras and MAPK, in cells responding to pressure,102 so that what prompts the response is unclear.
TGF- ! can rule out the involvement of certain other
Fig. 37.6. A theoretical
model depicting inflamma-
tory and postinflammatory
phases of tissue repair.
Reprinted from Weber KT et
al. Int J Biochem Cell Biol
1997; 29:31-42, with kind
permission from Elsevier
Science Ltd., Kidlington
OX5 1GB, UK.
Transdifferentiation and the Vascular Wall 409
Knowing the ontogeny of the cells, migratory ties, qualifies as a transdifferentiation. An eclectic lumping
possibilities, and the actual state of the tissue believed to of all such phenomena is justified, since so little is known of
undergo conversion is essential for accurate interpretation. what aspects of phenotype are significant, and any rules of
For example, veins grafted in place of arteries are thought to regulation learned in a suspect transdifferentiation illumi-
undergo ‘arterialization’. But the smooth muscle of the nate differentiation mechanisms in general. The contractile
neointima may have migrated from host arterial wall,103 to synthetic shift is reflected in abundantly altered gene ex-
or come in as myofibroblasts from the remodeling venous pressions, with activation or reactivation for cytokeratins 8
perivascular tissue, 104 or even have been present and 18,121 tenascin,122 collagen type VIII,123 #3 integrin,124
before grafting. 105 nonmuscle myosin heavy chain,93 platelet-derived growth
Intense clinical interest focuses on atherosclerosis- factor A chain, 125 and the related transmembrane
mimicking fibroblastic behaviors of VSMCs, which include proteoglycans, syndecans 1 and 4,126 with changed balances
proliferation and migration—striking changes in the nor- of alternative RNA-splicing,127 a marked downregulation of
mally quiescent contractile cells. Circumstances evoking a ‘contractile’ genes, and upregulation for collagen,128 c-myc,129
contractile to synthetic arterial VSMC transition (Fig. 37.7) sulfated proteoglycans, 130 and matrix
include: culturing in serum; 106 luminal injury; 107 metalloproteinases.131,132 Caveolin is not reduced, but is re-
intraperitoneal injections of benzo[a]pyrene;108 uremia from distributed internally.133
subtotal nephrectomy;109 adjacent mesenteric fibrosis;110 Analysis of phenotypic regulation has penetrated to
giant-cell arteritis; 111 immune injury from allogeneic the coordination of gene transcriptions and the search for
transplantation;112 and, perhaps closer to etiological mecha- shared type-specific transcription factors.134 The control of
nisms, topical application of cytokines.113 The examples key cytokines occurs both by the sequestration by binding
show that VSMCs from both muscular and elastic arteries in the ECM135 and by localization to cell surface proteoglycan
execute the switch. modulators.136 Signals come via cell-matrix binding from
How much of a change is it for VSMCs to become the ECM made by the cells themselves137 and by adjacent
fibroblastically ‘synthetic’?106 First, the cells in development cells.124 The confirmation and stabilization of phenotype also
are substantial synthesizers of ECM, while acquiring con- derive from the linking of cells by cell-cell junctions, includ-
tractility. Secondly, normal human aortic VSMCs contain ing the innervation.138 For the earliest establishment of vas-
the synthetic organelles—granular endoplasmic reticulum cular smooth muscle, tissue factor (used later to help initiate
and a Golgi complex, in addition to the contractile machin- clotting) is needed, as shown by a gene inactivation experi-
ery,114 whereas those of the usual experimental animals are ment in mice,139 but homeotic gene expression patterns spe-
far fewer. In some species the media includes ‘nonmuscle’ cific for VSMC precursors at this stage are not yet known.
cells.115-117 Thirdly, VSMCs becoming synthetic in culture Other smooth muscles than vascular resemble VSMCs
can be returned to a contractile state.118-120 Some would in their heterogeneity140 and plasticity: Tracheal SMCs
question whether the acquisition of a phenotype by a rever- become ‘synthetic’ in culture;141 gut SMCs make more ECM
sion to embryonic behavior, or by boosting existing activi- material, if the ileum is partially obstructed in guinea-pigs;27
Fig. 37.7. (A) shows a contractile smooth muscle cell from the media of
the rat carotid artery, and (B) a synthetic smooth muscle cell from the
neointima two weeks after balloon injury. E, elastic fibers; ER, endoplas-
mic reticulum; F, myofilaments; M, mitochondria; N, nucleus. Magnifi-
cation x15,000; bar = 1 ∝m. Reprinted from Thyberg J et al. Cell Tissue
Res 1995; 281:421-433, with kind permission from Springer-Verlag
GmbH, Heidelberg, Germany.
410 Tissue Engineering of Prosthetic Vascular Grafts
and TGF-!1 stimulates collagen production by human gut Macrophages
SMCs in vitro.142 However, the different layers of smooth The working macrophage has already experienced two
muscle in the gut require independent developmental profound changes of phenotype, which are, in a sense,
control, including neural and epithelial influences, and the transdifferentiations: from circulating monocyte to tissue
major lability is back and forth between an #-SM actin-ex- macrophage;162,163 and from quiescent macrophage to acti-
pressing smooth muscle myoblast and a &-SM actin-endowed vated macrophage.164 An activation step, under the prompt-
SMC.143 ing of cytokines, happens to endothelial165 and other cells,
and is not peculiar to leukocytes. These conversions are rel-
Pericytes evant to transdifferentiation,166 but will be set aside for more
Pericytes resemble smooth muscle cells in their con- extreme changes of macrophage phenotype.
tractile proteins, membrane receptors, and membrane prop- Surface and other markers indicated that most of the
erties,144,145 but junctions between pericytes are lacking.146 foam cells of atherosclerotic lesions are modified macro-
Pericytes attach to endothelial cells, but whether there are phages.167 Providing modified cholesterol to macrophages
gap junctions between endothelial cells and SMCs is con- in culture turns them into foam cells,168 a process accompa-
troversial.147 Pericytes bind a specific antiganglioside anti- nied by changes in the expression of many genes.169 Circu-
body.144 A transdifferentiation related to their probable func- lating monocytes might convert to myofibroblasts organiz-
tion of contraction is from pulmonary arteriolar pericytes ing thromboses170 and intraperitoneal blood clots,171 but an
to SMCs in rats kept for several days in hypobaric cham- origin from fibroblasts has not totally been excluded. Mac-
bers.148 Also, cells already intermediate in ultrastructure rophages may become producers of collagen, while creating
between SMCs and pericytes became SMCs. The idea of a a capsule around implanted mammary tumor cells.172 In the
stable, normal, intermediate pericyte phenotype149 is met developing chick optic nerve, some of the macrophages dis-
elsewhere as the myofibroblast of normal tissues, renal cells posing of dying redundant neurons elongate and become
expressing both myoid and renin-secreting properties, and like microglia.173 Embryonic macrophages give rise to the
normal VSMCs with dual synthetic and contractile roles. Hofbauer cells of the placental villi,174 yet another example
Other pericyte transdifferentiations include cultured where organ-specific circumstances fine tune and stabilize
bovine retinal pericytes becoming bone-forming osteo- cell phenotypes.
blasts,150,151 and a fibroblastic conversion in excessive hu- Two proposed additional origins for macrophages also
man dermal scarring.152 Based on double immunofluores- rest on transdifferentiations: Coculturing mouse B lympho-
cent reactivities for indicators of collagen synthesis and a cytes with splenic fibroblasts confers some macrophage
pericyte marker, a switch of pericytes to perivascular fibro- properties on the B cells;175 and renal glomerular podocytes
blasts takes place in the scarring. become macrophage-like in rat cell culture, and perhaps in
human crescentic glomerulonephritis.176 A fair conclusion
Mast Cells from this miscellany of phenomena is that the macrophage
Mast cells of rats and mice express distinct phenotypes bears close watching and is more experimentally challeng-
related to where they are located—the gastrointestinal mu- ing because of its undoubted heterogeneity,177 functional
cosae versus connective tissue and the peritoneum (MMCs plasticity, and metaplastic potential. Quickly aroused at the
vs. CTMCs). The cell types are well delineated by granule scene of injury and a potent source of cytokines, the mac-
and mediator chemistry and responses.153 By contrast, the rophage and its characteristics must be known for its con-
two main types of human mast cell (MC) usually occur to- duct to be steered.
gether, but may work with different ends in mind,154 which
locally influences the balance of numbers. Mucosae and Conclusions
immune defense require the MCT, whereas the MCTC is ac- The lessons apply to transdifferentiation generally, and
tive in connective tissues, fibrosis, and atherosclerosis.155 to any organ, because it will have vessels with cells exquis-
The phenotypic distinction is based on the MCTCs itely adapted to serve that organ’s tasks. For cell phenotype,
having chymase, carboxypeptidase, and cathepsin G, in ad- location is almost everything. Knowing very few of the ele-
dition to mast cell tryptase. Furthermore, the granule ultra- ments of phenotype, one can still experiment fruitfully us-
structure also does not completely distinguish the two.156 ing the shorthand of organ and site, e.g., renal medullary
The clearer cut phenotypes of murine mast cells, mast cell- interstitial fibroblasts. The picture one thereby builds of the
deficient mutant mice, and the ability of mouse precursor human body is not like the four entities (tissues) of the
cells to differentiate in culture render convincing the evi- United Kingdom, but rather the astounding patchwork of
dence for transdifferentiations in rodents. MMCs become 18th century Germany, with autonomous parts ranging from
CTMCs when injected intraperitoneally 157 or into the the Margraviate of Braunschweig-Luneburg to the imperial
skin,158 and a CTMC introduced into gastric mucosa gave cities of Schweinfurt and Wangen.
rise to MMCs.159 More elaborate experiments indicate that Cell lineage and current local microenvironment
the switches are reversible.160 Helping to pinpoint how their specify what cells are and do within a normal physiological
environment switches mast cell types was the conversion of range. For convenience, one speaks of ‘modulations’ of
mucosal-like MCs to CTMCs by coculture with 3T3 fibro- activity and characteristics within this range, but excursions
blasts,161 and a variety of studies on how cytokines affect beyond enter into the territory of transdifferentiation, but
mast cell differentiation.153 not necessarily immediately, in the sense that there is
Transdifferentiation and the Vascular Wall 411
implicitly, and often overtly, an intermediate zone—reflected provocation. To be useful a new phenotype must integrate
in the myofibroblast, epithelioid SMC, and such others. harmoniously over the longer term. The invasive growth
However, as the investigation probes phenotype more deeply, within the brain of fibroblasts expressing a transgene for
the extent of molecular change involved in, say, a contractile tyrosine hydrolase184 illustrates the danger of modifying only
SMC becoming a synthetic SMC may amount to a one aspect of phenotype. Fortunately, cell proliferation is
transdifferentiation. Differential gene and protein analysis not a prerequisite for a switch of phenotype, making it easier
and display will help define exactly how related cell to aim for a new cell type while taking steps to prevent run-
types differ.178 away growth in vivo.
Progress requires that the differentiated state and its Another route to sufficient cells of a desired type is to
conversions be understood in terms of cell structure, be- steer stem cells to differentiate while they proliferate, but
havior (including polarity, proliferation, migration, adhe- again the problem arises of matching in vitro cell type to
sion,179 and inclination to apoptosis180) the distinctive work- host organ locale. Can one achieve sufficient refinement in
ing molecules, the transcriptional and translational regula- this before grafting? Detection of the critical environmental
tors producing those molecules, the receptors and transduc- factors, along with coculture or transfer of medium, may be
tion mechanisms controlling the production and deploy- an avenue.
ment of the principal molecules, the external relations of The paucity of known controlling factors constitutes
the cell with signals, cells, and matrix, and what the cell ‘re- another obstacle—TGF-! and retinoic acid influence the
members’ of its lineage, i.e., its precursor states. Relative to differentiation of several different, but in vivo adjacent, cell
what should be known, transdifferentiations are testified to types. For selectivity, much more needs to be known of how
by a very few structural and chemical markers, expressing such factors are released, bind, are activated, work in se-
what available technique, curiosity, current concerns, and quence and combination, and affect the transduction sys-
often chance happened upon in regeneration and disease. tems. Beyond the signals are the transcriptional controls,
Routes to a more systematic study include such questions where the complexity multiplies almost by the month for
as: Can cells return along lineage pathways that they have any given cell type specific gene, including sometimes the
traversed? (Osteoblasts and mast cells may be more termi- discovery that the gene is expressed in cell types not con-
nal than fibroblasts and pericytes.) And, if reversion occurs, ventionally thought to be related, such as prostate-specific
what is the story of homeodomain genes and ‘master’ tran- antigen in the female breast. Nevertheless, cures can be ef-
scriptional factors, and of signaling mechanisms? Answers fective without one knowing how, and induced bone for
learned from the classic transdifferentiations in amphibia, skeletal repair60,185 shows that transdifferentiations186 can
worms, hydra, plants, and other mammalian systems2,3 be applied to healing before they are satisfactorily under-
remain pertinent. stood at cellular and molecular levels.
The question of what restrains adult cell phenotype
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Facilitation of Healing
Transdifferentiation
CHAPTER 38
Endothelial Cells Transformed
from Fibroblasts During Angiogenesis
Takashi Fujiwara, Kazunori Kon
Introduction
A ngiogenesis, the formation of new blood vessels, is of fundamental importance for sev
eral physiological and pathological processes. It occurs, as is well known, during organ
development, wound healing and tumor growth.1-6 Endothelial cells of newly formed blood
vessels are widely believed to derive from preexisting blood vessels. According to Furcht,7
and Rhodin and Fujita,8 the process of angiogenesis can be broken down into a number of
discrete yet overlapping steps:
1. Increase of vessel permeability;
2. Dissolution of basal lamina caused by a variety of enzymes and penetration of basal
lamina by endothelial projections;
3. Matrix ‘channel’ formation in the interstitial connective tissue and migration of en-
dothelial cells from the preexisting vessel;
4. Proliferation of endothelial cells;
5. Sprout and loop formation;
6. Canalization of the loops; and
7. Association of pericytes and fibroblasts with the newly formed blood vessels.
In such an angiogenic process, endothelial cell migration from preexisting vessels and
its proliferation is requisite. However, questions have been addressed to this current angio-
genic mechanism9 because of several puzzling phenomena outlined as follows:
1. Nonendothelial cells are integrated into the linings of newly formed blood vessels;10-12
2. Angiogenesis takes place without apparent endothelial cell proliferation13 and with-
out endothelial cell migration from preexisting blood vessels;10 and
3. Factors inhibiting in vitro proliferation of endothelial cells, tumor necrosis factor-#
(TNF-#)14 and transformtin growth factor-! (TGF-!),15 stimulate in vivo angiogen-
esis.16
Therefore, we aimed to examine whether endothelial cells of preexisting blood vessels
are the only origin of endothelial cells of newly formed blood vessels.
Angiogenesis in Rabbit Ear Chamber
Ultrastructure of the Tips of Newly Formed Blood Vessels
A transparent rabbit ear chamber (REC), 6 mm in diameter and 100 ∝m in depth, was
implanted into an ear of an adult rabbit under anesthetization with pentobarbital sodium
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
418 Tissue Engineering of Prosthetic Vascular Grafts
(40 mg/kg body weight).17 Newly formed blood vessels ap- was confirmed by their spindle shape and by the inhibitory
peared in the chamber space about one week after the cham- effect of cis-hydroxyl proline on fibroblast proliferation.20
ber implantation. The newly formed vessels invaded the When 100 mg/ml of cis-hydroxyl proline was added to the
space which extravasated fibrin and red blood cells filled. cultures, the cellular number decreased as shown previ-
For mapping the network of newly formed blood vessels, ously.20 Furthermore, no contamination of endothelial cells
invaginated red cells prepared by treating with trifluopera- in culture was ascertained by the negative uptake to fluores-
zine were perfused through the ear artery, enabling us to cence labeled acetyl low density lipoprotein by the cultured
detect tips of blood vessels growing among numerous ex- cells.21 During the fifth passage, fibroblasts were cultured in
travasated red cells in the REC. After fixation, newly formed MEM containing India ink particles for 10 h to mark fibro-
blood vessels were processed for electron microscopy in usual blasts by their ingestion of India ink particles.22 Fibroblasts
way and observed with an electron microscope. contain India ink particles in the cytoplasm for as long as 9
Some newly formed blood vessels containing invagi- months, enabling us to keep fibroblasts marked with India
nated red cells were lined with partially discontinuous en- ink particles for a long time. The marked fibroblasts, about
dothelium (Fig. 38.1). Endothelial cells of the blood vessel 1 x 104 cells, were washed to remove free carbon particles
were provided with well developed rough ER and many mi- from the suspension, with MEM solution two times and with
tochondria, morphologically resembling fibroblasts in the Hanks’ solution three times, and then transplanted
interstitium. This phenomenon suggests that endothelial autologously into the space between protective covers of the
cells may derive from fibroblasts. Then we paid particular chamber with a fine needle at 2 days after the chamber im-
attention to the possible role of fibroblasts as progenitors of plantation. On 8 days after the autologous transplantation
endothelial cells, since mesenchymal cells are known to be of the marked fibroblasts into REC, growing vessels in the
the origin of vascular endothelial cells in fetal tissue, and REC were fixed and cut into sections for light microscopy.
fibroblasts function as embryonic mesenchymal cells in some Newly formed blood vessels of various diameters were
circumstances in adult tissue.18 found in the connective tissue, identified as venules, capil-
To give an insight into the transformation of fibro- laries and arterioles by their morphological characteris-
blasts into endothelial cells, transplantation of cultured fi- tics.23-25 Endothelial cells containing carbon particles in the
broblasts into the space of REC was designed. For this pur- cytoplasm were, though only occasionally, found to line
pose, the fibroblasts were marked so as to trace them. We newly formed blood vessels (Fig. 38.2), indicating that the
used two methods for marking fibroblasts, carbon particle transplanted fibroblasts transformed into the endothelial
ingestion and [3H]thymidine uptake of fibroblasts. cells. Extravasation of red cells from the openings of newly
formed blood vessels was sometimes observed in the speci-
Transformation of Fibroblasts Marked with Carbon mens prepared after autologous transplantation of carbon-
Particles into Endothelial Cells marked fibroblasts (Fig. 38.2). This is consistent with the
Fibroblasts were separated from rabbit ear skin as re- well known high permeability and fragility of newly formed
ported previously19 and were cultured in Eagle’s minimal blood vessels.26
essential medium (MEM). The identification of fibroblasts
Fig. 38.1. Ultrastructure of newly formed
blood vessel in the rabbit ear chamber. The
presence of invaginated red cells which
were infused through the ear artery show
that the structure is a vessel. The endot-
helial cells have well developed rough ER
in the thick cytoplasm, suggesting a close
resemblance to fibroblasts in the intersti-
tial space. Note a large intercellular gap be-
tween adjacent endothelial cells. x5400:
Bar = 2 ∝m.
Endothelial Cells Transformed from Fibroblasts During Angiogenesis 419
Transformation of Fibroblasts Labeled with 66.8 ± 8.1 for endothelial cells and 55.9 ± 4.5 for fibroblasts
[3H]Thymidine into Endothelial Cells in 10 preparations examined, indicating that the radioactive
We labeled fibroblasts with [ 3 H]thymidine to labels are not transmitted to the endothelial cells by uncon-
confirm the result obtained by fibroblasts marked with trolled uptake from degenerating transplanted fibroblasts.
carbon particles. In electron microscope autoradiography, a few silver
Fibroblasts were separated from rabbit ear skin and grains were found over the nuclei of the endothelial cells of
cultured as mentioned above. During the fifth passage, capillaries, arterioles (Fig. 38.4A) and venules (Fig. 38.4B).
the fibroblasts were cultured in MEM containing The labeled cells were joined together by intercellular junc-
[3H]thymidine (185 Kbq/ml) for 6 days for labeling. To re- tions and were surrounded by a basal lamina on their
move free [3H]thymidine, the fibroblasts were washed twice abluminal surface, provided with some of the morphologi-
with MEM solution, then with Hanks’ solution three times. cal characteristics of endothelial cell. These data also indi-
Two days after the chamber implantation, the labeled fibro- cate that fibroblasts transform into endothelial cells of newly
blasts, about 1 x 104 cells, were autologously transplanted formed blood vessels.
into the space between protective covers of the chamber with Silver grains were observed over the nuclei of the
a fine needle. Tissues of the proximal part of the vascular pericytes of capillaries (Fig.38.3A) and venules (Fig. 38.3F),
bed were prepared for light microscopic autoradiography and the nuclei of arteriolar smooth muscle cells (Figs.
on days 7, 9, 12 and 23 and for electron microscopic autora- 38.3C,D, 38.4A), suggesting that both pericytes and vascu-
diography on day 12 after the chamber implantation. lar smooth muscle cells are derived from fibroblasts.
Capillaries (Figs. 38.3A,B), arterioles (Figs. 38.3C,D)
and venules (Figs. 38.3E,F) were found in autoradiographic Discussion
preparations. Numerous autoradiographic silver grains were
frequently observed over the endothelial cell nuclei of these Endothelial Cell Origin and Formation
newly formed blood vessels.27 There were also many fibro- of New Blood Vessels
blasts labeled with silver grains in the connective tissue Endothelial cells of newly formed blood vessels are
(Figs. 38.3A,D). In our experiments, it could not be excluded generally believed to derive from preexisting blood vessels.
that endothelial cells took up radioactive compounds which Pioneering studies on transformation of nonendothelial
might be released from degenerated fibroblasts and diffused cells, such as mesenchymal cells10 and tumor cells,11 into
toward the endothelial cells. If so, however, the concentra- endothelial cells have been carried out. These studies, how-
tion of [3H]thymidine in the interstitium surrounding en- ever, have not attracted general notice, probably because their
dothelial cells of newly formed blood vessels must be lower evidence is indirect. Here, we clearly showed by tracing cul-
than that in our culture medium, predicting that silver grain tured fibroblasts marked with carbon particles that fibro-
density on endothelial cell nucleus must be lower than that blasts transform into endothelial cells of newly formed blood
of fibroblasts. Furthermore, if diffusion of [3H]thymidine vessels, challenging the general agreement on angiogenic
from degenerated fibroblasts occurred in the interstitium, mechanism. The transformation of fibroblasts into endot-
almost all of the endothelial cells and fibroblasts should be helial cells has been supported by an experiment with quail
more or less labeled. In our experiment, the silver grain chick chimeras. Stein et al28 transferred limb buds of three
density on each nucleus of endothelial cells was very similar day old quail embryos to the chorioallantoic membrane of
to that of fibroblasts, and not all of the endothelial cells and 10 to 14 day old chick embryos. Six days after grafting, sev-
fibroblasts were labeled: The percentage of labeled cells was eral quail-derived capillaries were found in each of the
Fig. 38.2. A microphotograph of newly formed blood
vessels in the rabbit ear chamber. Cultured fibroblasts
marked with India ink carbon particles were trans-
planted into the ear chamber two days after the im-
plantation of the chamber. Eight days after the trans-
plantation of the fibroblasts, the tissue was fixed and
processed for light microscopy. A carbon particle
(arrow) is observed in the cytoplasm of an endothe-
lial cell. Red blood cells extravasate from the gap of
endothelium. x1700: Bar = 5 ∝m.
420 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 38.3. Light microscopic autoradiography. Autoradiographic silver grains are found over the nuclei of the endothelial cells of
capillaries (A, B), arterioles (C,D) and venules (E,F). Silver grains are also observed over the nuclei of the pericytes of capillaries (A)
and venules (F) and over the nuclei of the smooth muscle cells of arterioles (C, D). Fibroblasts labeled with silver grains are
distributed in the connective tissue (A, D). Tissues were prepared on days 7 (E), 9 (A), 12 (C,D and F) and 23 (B), after chamber
implantation. e: endothelial cell; p: pericyte; s: smooth muscle cell. x930: Bar = 10 ∝m.
Endothelial Cells Transformed from Fibroblasts During Angiogenesis 421
Fig. 38.4. Electron microscopic autoradiography. The endothelial cells of an arteriole (A) and a venule
(B) and the smooth muscle cell of an arteriole are labeled with silver grains over their nuclei. The
endothelial cells are joined together with intercellular junctions and surrounded by a basal lamina,
exhibiting typical features of endothelial cells. The specimens were prepared on day 12 after cham-
ber implantation. e: endothelial cell; s: smooth muscle cell (A) x7600: Bar = 1 ∝m; (B) x5400: Bar =
1 ∝m.
transplanted limb buds, suggesting that quail fibroblasts, ing a new blood vessel. Such an incorporation of cells from
better to say mesenchymal cells, participate in new blood the abluminal side into newly formed blood vessels has been
vessel formation. Similar findings that some mesenchymal drawn schematically.29
cells have an ability to transform into endothelial cells have
been reported by several studies on quail chick transplanta- 0rigin of Pericytes and Vascular Smooth Muscle Cells
tion in the embryonic stage,29-31 and on mammalian em- from Fibroblasts
bryos.32 These studies indicate that the endothelial cells from Fibroblasts have also been clearly demonstrated here
preexisting blood vessels are not the only origin of endothe- to be the origin of both pericytes and smooth muscle cells
lial cells of newly formed blood vessels. Recently Asahara et of blood vessels, as shown previously.8,28,34,35 Accordingly,
al12 have clearly demonstrated that putative endothelial cell these results indicate that fibroblasts can be the common
progenitors in circulating blood differentiated into endot- origin for all cellular elements of blood vessels, i.e., endo-
helial cells in vitro and incorporated into sites of active an- thelial cells, pericytes and smooth muscle cells. A similar phe-
giogenesis in vivo. nomenon of cell origin is known in fetal vasculogenesis, in
We observed that endothelium of newly formed ves- which mesenchymal cells are the common origin for these
sels was occasionally discontinuous and that blood cells cir- three cellular elements.36 This is consistent with the view
culating in the vessels extravasated into interstitial space that fibroblasts function in adult tissue as embryonic mes-
through the gaps of endothelium. This extravasated blood enchymal cells in some aspects.18
may flow back and forth in the interstitium and show only
oscillatory movements as described by Hadlicka,33 forming Possible Contribution of Fibroblasts to Angiogenesis
a matrix channel in the interstitial space.8 Fibrin deposits in From our experiments, an increase in fibroblast den-
the intersititial space are supposed to play a critical role in sity can be expected to promote angiogenesis. This view is
providing a matrix channel, from our experimental finding supported by the experimental evidences that activated fi-
that fibrinosis of accumulated fibrin in REC by plasmino- broblasts are associated with angiogenesis in electrically
gen activator causes complete inhibition of new vessel for- stimulated skeletal muscle37 and that inhibition of fibroblast
mation. Fibroblasts in the interstitium may find an oppor- proliferation by proline analogs,20 on the contrary, inhibits
tunity to face and directly contact flowing blood in the ma- angiogenesis.38 The puzzling phenomenon of TNF-# and
trix channel. Then they may come to line the luminal sur- TGF-! promoting in vivo angiogenesis, in spite of their in-
face of the channel in place of normal endothelial cells, form- hibitory effect on endothelial cell proliferation, has
422 Tissue Engineering of Prosthetic Vascular Grafts
previously been rationalized by the suggestion that stimu- ever, we need further studies to reveal whether transforma-
lated secondary cells produce angiogenic factors.4,16 Based tion of fibroblasts into endothelial cells generally takes place
on our data, however, we suggest that TNF-# and TGF-! in angiogenesis.
contribute to promoting angiogenesis by the increasing
population of fibroblasts.39,40 Both TNF-#39 and TGF-!41 are References
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Facilitation of Healing
Transdifferentiation
CHAPTER 39
Mechanical Forces and Cell Differentiation
Ira Mills, Bauer E. Sumpio
Introduction
O ur laboratory and others have been rigorously studying the influence of mechanical forces
on vascular cell biology. It has been our contention that static conditions commonly
utilized to study vascular cells in culture may not reflect their in vivo milieu. Vascular cells in
vivo are subjected to a variety of flow-related forces including shear stress, hydrostatic pressure,
and cyclic strain1,2 (see Fig. 39.1, ref. 3). Shear stress is the tangential stress applied across the
endothelial cell surface due to the bulk flow of blood. Hydrostatic pressure is the normal
stress acting radially on the vessel wall due to the propagation of the pressure wave. Cyclic
strain represents the stress acting along the vessel wall due to circumferential deformation.
Endothelial cells are exposed to all three forces, whereas the underlying smooth muscle cells
are exposed to hydrostatic pressure and cyclic strain. There are a number of recent reviews
of the effects of shear stress on endothelial cells.3-5 Therefore, we will mention but not pro-
vide details for shear studies. We will not consider the effect of shear stress on smooth muscle
cells, since this force is considered insignificant as compared to cyclic strain.3
Cyclic strain, along with the other forces listed above, plays an important role in the
regulation of vascular tone, remodeling, and the genesis of atherosclerosis in vivo.6,7 The
mechanism by which cultured vascular endothelial cells and smooth muscle cells perceive
cyclic strain, utilizing in vitro devices to model this force, is the subject of this chapter.1,2 The
strains described for the in vitro studies herein (~10% strain) are functionally significant
based on in vivo models that replicate the major geometric features of blood vessels. These
studies report a 5-6% wall excursion at peak systole under normal conditions, which can
increase to 10% in the hypertensive state.8-10
Several recent studies in the literature suggest that the study of mechanical forces on
vascular cell biology may have fruitful applications in the field of tissue engineering of
prosthetic vascular grafts.11 In one such study, Ott and Ballerman12 found that shear stress
preconditioning of endothelial cells led to an improved tolerance (i.e., cell adherence) of
endothelialized grafts to acute shear stress. Assessment of endothelial cell tolerance was
determined by counting the number of dislodged cells, examining the grafts by light and
scanning electron microscopy and by measurement of whole blood clotting time. Shear
stress preconditioned grafts, as compared to control grafts, showed fewer numbers of
dislodged cells, the presence of an intact monolayer, and a prolonged clotting time. Thus,
this study provides evidence to suggest that preconditioning by shear stress leads to
amelioriation of the poor retention of endothelial cells, as well as the thrombogenicity of
prosthetic vascular grafts.
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
426 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 39.1. Schematic showing mechanical forces of shear stress, cyclic strain, and hydrostatic pressure and associated signalling
pathways that occur in vascular cells. Adapted from ref. 3.
The mechanism responsible for the improved status In a recent study, Kraiss et al15 have provided mecha-
of the shear stress-preconditioned prosthetic grafts remains nistic data to explain the smooth muscle cell proliferation
unclear, but Ballermann and Ott11 suggest that a shift of associated with abrupt reductions in fluid shear stress. They
endothelial cells towards a more differentiated state may be found that PDGF-A gene expression in both subluminal
involved. Endothelial cells cultured under normal conditions endothelium and subadjacent smooth muscle cells is in-
tend to become dedifferentiated. In the presence of shear creased in low flow grafts. This was convincingly shown by
stress, they observed the appearance of more differentiated Northern blot analysis, in situ hybridization studies and
features, including a tighter adherence to the substratum that immunohistochemical studies. Increases in the PDGF-A
resembled in vivo endothelial cells displaying an organized transcript of 2.9-fold were noted in neointimal areas in the
cytoskeleton, Weibel-palade bodies and basal stress fibers presence of reduced shear stress. Even greater changes were
with focal adhesion plaques. indicated by in situ studies, presumably due to the predomi-
Clowes and colleagues13-15 have characterized the ef- nance of upregulated message in the luminal surface of the
fects of blood flow and shear stress on subendothelial smooth neointima. This study emphasizes the need to delineate the
muscle cell proliferation in baboon prosthetic grafts. They responsiveness of vascular cells to mechanical perturbation
have found that neointimal hyperplasia in endothelialized to better understand their behavior in situations such as tis-
grafts is inhibited by increased blood flow. In the presence sue engineering of vascular grafts.
of a distal arteriovenous fistula that caused nearly 3-fold The complexity of the nature of the forces perceived
elevations in flow and shear stress, smooth muscle cell by grafts was illustrated by Dobrin et al,16 who subjected
number was reduced with a consequent reduction in the autogenous vein grafts to a variety of conditions of pressure
cross-sectional area of the neointima at 3 months. More- and flow including three static deformations, three static
over, ligation of the fistula after 2 months caused a rapid stresses, increased pulsatile deformations, pulsatile stresses,
increase in neointimal thickness,13 showing a strong asso- and altered shear stress at the blood-intima interface. Three
ciation between blood flow and the smooth muscle cell re- sequential experiments were performed to sequentially
sponse in vascular grafts. determine the role of nine factors. In agreement with Clowes’
work,13-15 low flow velocity was associated with intimal hy-
Mechanical Forces and Cell Differentiation 427
perplasia. Moreover, medial thickening of autogenous vein tive of a dedifferentiated state occur, such as elevated prolif-
grafts was observed with increased deformation of the vein eration.38 For example, recent work by Reusch et al39 sug-
wall in the circumferential direction, which mimics gests that smooth muscle cells become more differentiated
clinical findings. upon cyclic strain as evidenced by the upregulation of myo-
Over the years, we have acquired a large body of data sin heavy chain expression. As described in greater detail
to suggest that the study of vascular cells challenged with below, other responses of smooth muscle cells to cyclic strain
mechanical stimuli is indeed a more appropriate method portend a dedifferentiated state such as enhanced prolifera-
for studying their biology.17-32 This chapter represents an tion.24,40 It is clear that much work needs to be done in char-
update of an earlier review of the literature describing the acterizing the modulation of cyclic strain on vascular cell
effect of cyclic strain on vascular cell biology.22 Data obtained biology before any mechanistic significance can be ascribed
from our laboratory and that of others have begun to delin- to its role in advancing tissue engineering of prosthetic grafts.
eate the signaling pathways that may be involving in affect- As recently reviewed by Zilla et al,41 “manipulation of
ing vascular cell phenotype and is commented on in great the endothelium might provide the next major advancement
detail in this review. We also discuss the recent discovery of for therapeutic and preventive measures for cardiovascular
strain-sensitive genes that appear to be finely tuned in their disease”. It is with this objective that the following studies
ability to distinguish mechanical perturbations based on are outlined that characterize the mechanical manipulation
unique strain sensitive or shear sensitive cis-elements in their of both endothelial cells and smooth muscle cells. It is hoped
promoter regions that respond to unique strain-activated that a greater delineation of the mechanisms involved in the
or shear-activated transcriptional factors that either induce strain-induced modulation of vascular cell phenotype will
or repress gene induction. lead to better diagnosis and treatment of vascular disease
Recent studies suggest that the state of differentiation and the implementation of improvements in tissue engineer-
of vascular cells in culture is fluid,33-35 and a novel stimulus ing of prosthetic grafts.
for controlling the phenotypic state of these cells is mechani-
cal strain.36 As described below, recent studies suggest that Effect of Cyclic Strain
cyclic strain may direct endothelial cells to a more differen- on Endothelial Cell Biology
tiated state as evidenced by phosphorylation of focal adhe-
sion proteins.37 However, the state of differentiation of en- General Phenotype
dothelial cells as well as smooth muscle cells in response to Previous studies in our and others’ laboratories have
cyclic strain is complex, since many other responses indica- shown that cyclic strain can alter the general phenotype of
Mechanical
Stress
Ion Channels
Adenylyl/Guanylyl
Cyclase
ECM & Surface Proteins
Phospholipids Cell
Response
Transmembrane Proteins Integrin
Superfamily
Gene
Cell-cell Expression
Proteins
Linker Proteins
alpha-actinin, talin,
vinculin, etc.
Cytoskeleton
Fig. 39.2. Schematic showing mechanical force transmission and transduction pathways in vascular cells. Adapted from ref. 47.
428 Tissue Engineering of Prosthetic Vascular Grafts
Table 39.1. Effect of cyclic strain and shear stress on endothelial cell phenotype. (Adapted from ref. 3)
Response Cyclic Strain Ref. # Shear Stress Ref. #
Proliferation Increase 27,38 Increase; 52
Turbulent flow
Alignment Perpendicular 18,42, Parallel 53,54
43
F-Actin Yes 18 Yes 48,55
Redistribution
Focal Adhesion Site Yes 37,50, Yes 49,55
Modification 56
Macromol: PG12 Increased 29,57 Increased 58
NO Increased 44,45 Increased 59
tPA Increased 20,32 Increased 60
Endothelin-1 Decreased (Koo, Increased/ 61,62
unpublished data) Decreased
endothelial cells, including their proliferative rate,27,38 mor- analysis, they found a remodeling of focal adhesion sites in
phology18,42,43 and the secretion of macromolecules such as the direction of flow. In addition to this response, shear stress
prostacyclin,29 endothelin,30 nitric oxide,44,45 tissue plasmi- caused a redistribution of intracellular stress fibers, align-
nogen activator20 and plasminogen activator inhibitor-1 ment of individual focal adhesion sites, and the coalescing
(PAI-1)46 (see Fig. 39.2, see ref. 47). of smaller sites to larger, and fewer, focal adhesions per cell.
Changes in endothelial cell phenotype are also modu- Yano et al50 recently studied the involvement of dif-
lated by shear stress (see Table 39.1). Changes include alter- ferent integrins in signaling induced by cyclic strain, since
ations in proliferation, morphology and the synthesis and integrins have been localized to focal adhesion sites. Fur-
secretion of many of the same proteins influenced by cyclic thermore, these focal adhesion sites are known to exhibit
strain.3-5 However, the nature of the changed phenotype can pp125FAK phosphorylation by cyclic strain as well as by
be markedly different, whether the perturbation is either integrins. Cyclic strain of 4 h led to a redistribution of # and
50
shear stress or cyclic strain. For example, the orientation of ! integrins in human umbilical vein endothelial cells. In
endothelial cells in response to shear stress is parallel to the addition, !1 integrin reorganized in a linear pattern parallel
direction of flow.48 In contrast, cyclic strain causes a per- with the long axis of the elongated cells, creating a fusion of
pendicular alignment of endothelial cells in relationship to focal adhesion plaques in cells plated on fibronectin (a ligand
the strain vector.18 for #5!1) or collagen (a ligand for #2!1) coated plates. In con-
More recent studies performed in our laboratory sup- trast, the vitronectin receptor, !3 integrin, failed to redis-
port the involvement of focal adhesion plaques in the trans- tribute in endothelial cells subjected to cyclic strain. Cyclic
mission of cyclic strain to the cell. Yano et al37 demonstrated strain also caused a reorganization of #5 and #2 integrins in
phosphorylation of cytoskeletal proteins that reside at the a linear pattern on cells seeded on fibronectin and collagen-
cytoplasmic face of the focal adhesion plaques, namely coated plates, respectively. However, the expression of #5,
pp125FAK and paxillin. The strain-induced phosphorylation #2, and !1 was not influenced by 24 h of cyclic strain as as-
of these proteins occurs on tyrosine residues and can be in- sessed by immunoprecipitation of these integrins. Strain-
hibited by specific tyrosine kinase inhibitors. The tyrosine induced phosphorylation of pp125FAK occurred concomi-
phosphorylation of pp125FAK occurs earlier and is more ro- tantly with the reorganization of !1 integrin. Thus, #5!1 and
bust than that observed with paxillin. However, after a four #2!1 integrins may play an important role in transducing
hour exposure to strain, both cytoskeletal proteins were mechanical stimuli into intracellular signals.50
found to align as shown by confocal microscopy.37 This ob- Thoumine et al51 demonstrated effects on the organi-
servation was in sharp contrast to their random distribu- zation and composition of the extracellular matrix upon
tion found in stationary controls. Tyrphostin A25, a tyrosine subjecting endothelial cells to shear stress. They examined
kinase inhibitor, is blocked by the strain-induced phospho- the pattern and levels of various extracellular matrix pro-
rylation of pp125FAK and paxillin as well as by their strain- teins including fibronectin, laminin, type IV collagen, and
induced alignment. Moreover, the importance of focal ad- vitronectin. Of these, fibronectin was found to be the most
hesion proteins on the mechanically induced gross morpho- affected by shear stress, with a thickening and alignment of
logical changes of endothelial cells was implied by the abil- its fibril tracts. Moreover, the level of fibronectin increased
ity of tyrphostin A25 to reverse both cellular alignment and after 1 or 2 days of shear stress after an earlier reduction at
migration caused by strain. 12 h. The extracellular matrix response to shear was found
Davies et al49 have studied the effect of shear stress on to be specific for fibronectin, since both laminin and type
focal adhesion sites in bovine aortic endothelial cells. By tan- IV collagen showed thickening of fibrils, but no alignment.
dem scanning confocal microscopy and digitized image Vitronectin was unaffected in any respect.
Mechanical Forces and Cell Differentiation 429
Table 39.2A. Effect of cyclic strain on second messenger pathways in endothelial cells. (Adapted from ref. 3)
Effect Strain Cell Type Response Time Significance Reference Number
/ [Ca2+] 10% avg., BAEC 12 s, initial peak: 2nd Activation of Ca2+ 63
60 cpm phase sustained, 15- sensitive pathways
35 s
/ IP3 10% avg., BAEC 10 s peak Phosphoinositide 74
60 cpm sensitive pathways
/ Diacylglycerol 10% avg., BAEC d10 s, initial peak, 2nd Activation of the 65,74
60 cpm phase sustained to PKC pathway
500 s
/ PKC 10% avg., BAEC 10 s initial peak, 2nd Phosphoinositide 74
60 cpm phase from 100 s sensitive pathways
sustained to 8 min
/ Adenylyl cyclase 10% avg., BAEC 5-60 min Activation of the 68
60 cpm cAMP pathway
/ cAMP 10% avg., BAEC 15-30 min Activation of the 68
60 cpm cAMP pathway
/ PKA 10% avg., BAEC 10-60 min Activation of the 68
60 cpm PKA pathway
Table 39.2B. (Adapted from ref. 3)
Effect Shear Cell Type Response Time Significance Reference Number
/ [Ca2+] 0.2-4.0 BAEC 15-40 sec Activation of Ca2+ 64
dynes/cm2 sensitive pathways
/ IP3 30 and 60 BAEC, 10 sec peak Phosphoinositide 66, 75
dynes/cm2 HUVEC sensitive pathways
/ cGMP 0-40 BAEC 60 sec Vasoregulation 76
dynes/cm2 Mechanisms
/ MAP kinase 3.5-117 BAEC 5 min peak, Involvement of 77
dynes/cm2 20-30 min membrane mitogen
receptor-like pathway
Cell Signaling Shear stress has been demonstrated to induce calcium
transients in single endothelial cells as measured by fura-2.64
Second Messenger Pathways (See Table 39.2A, B) Geiger et al64 studied the effect of shear stress at 30 dynes/cm2
in endothelial cells subjected to flow in parallel plate flow
[Ca2+]i, Inositol Phosphates and Diacylglycerols chambers and glass capillary tubes. They found a rapid in-
Previous studies have demonstrated that cyclic strain crease in [Ca2+] that peaked by 30 seconds and decreased
causes a rapid but transient generation of 1,4,5 inositol slowly to a plateau that persisted for greater than 5 minutes.
trisphosphate (IP3), which peaked by 10 seconds of cyclic Recent studies performed in our laboratory show that
strain. To address the effect of cyclic strain on intracellular cyclic strain causes a biphasic increase in diacylglycerol.65
[Ca2+] and its temporal relationship to IP3 generation, bo- These data support a mechanism of phosphatidylcholine
vine aortic endothelial cells were grown on flexible mem- hydrolysis and diacylglycerol generation at both an early and
branes, loaded with aequorin and the membranes placed in late phase. Cyclic strain was shown to stimulate phospholi-
a custom designed flow through chamber.63 The chamber pase C acutely and phospholipase D activity in an immedi-
was housed inside a photo multiplier tube and vacuum was ate and sustained manner.65 Bhagyalakshmi et al66 also
utilized to deform the membranes. The initiation of strain showed stimulation of DAG by shear stress. In another study
caused a rapid increase in intracellular calcium of two com- of shear stress-activated platelets, pathological levels of shear
ponents. The first component was a large initial peak 12 sec- (i.e., 90 dynes/cm2) failed to elevate DAG despite increased
onds after the initiation of stretch that closely followed the PKC activity.67
IP3 peak. The second component was a subsequent lower,
but sustained, phase. Pretreatment with 5 ∝M gadolinium Adenylyl Cyclase
for 10 minutes or nominally calcium-free medium for 3 min- Cyclic strain causes activation of the adenylyl cyclase/
utes reduced the magnitude of the initial rise and abolished cyclic AMP/protein kinase a pathway in bovine aortic en-
the sustained phase. dothelial cells.68 In membranes deformed with 150 mm Hg
430 Tissue Engineering of Prosthetic Vascular Grafts
(average 10% strain) vacuum at 60 cycles per minute (0.5 sec lished a role of G proteins in the responsiveness of endothe-
strain; 0.5 sec relaxation) for 15 minutes, there was a 1.5- to lial cells to shear stress. Both shear stress mediated
2.2-fold increase in adenylyl cyclase, cAMP and PKA activ- prostacyclin and nitric oxide production were found to be
ity as compared to unstretched controls. The strain-induced mediated by pertussis-toxin sensitive and insensitive G pro-
activation of this pathway appears to occur by exceeding a teins, respectively.70,71
strain threshold, since no change in adenylyl cyclase, cyclic
AMP or PKA was observed in cells subjected to 37.5 mm Hg Protein Phosphatase Activity
(average 6% strain). Further studies demonstrated an in- As described above, we have reported previously that
crease in cAMP response element activity as shown by gel PKC in the membrane fraction of endothelial cell lysates is
shift analysis. Thus, cyclic strain may stimulate the expres- activated in response to cyclic strain.72 To better understand
sion of genes containing cAMP-responsive elements. cellular responses that are dependent on the phosphorylated
We next tested the hypothesis that cyclic strain modu- state of proteins, it is also important to study the role of
lates G protein function, since G proteins are intimately in- protein phosphatases. In a recent study,73 we examined the
volved in the activation of adenylyl cyclase (Mills, unpub- effect of cyclic strain on protein phosphatase-1 and -2A ac-
lished observations). To test this hypothesis, we examined tivity in bovine aortic endothelial cells. Protein phosphatase-
the effect of acute cyclic strain on the immunoreactivity of 2A activity in the cytosol was decreased by 36.1% in response
the alpha subunits of the heterotrimeric G proteins, Gs and to 60 minutes of cyclic strain, whereas activity in the mem-
Gi, that promote stimulation and inhibition of AC activity, brane was unchanged. Furthermore, treatment with a low
respectively. We observed a transient decrease in the immu- concentration of okadaic acid (0.1 nM) enhanced prolifera-
noreactivity of the inhibitory G protein alpha subunits tion of both static and stretched endothelial cells in 10%
(Gi#1,2).In contrast, immunoreactivity of Gs in bovine aor- fetal bovine serum. These data suggest that protein phos-
tic endothelial cells and Gi#1,2 and Gs in bovine aortic phatase-2A acts as a growth suppresser, and cyclic strain may
smooth muscle cells were unaffected by cyclic strain. enhance cellular proliferation by inhibiting protein phos-
Frangos and colleagues have shown shear stress acti- phatase-2A.73
vation of G proteins, particularly G#q/#11 and G#i3/#o.69
This was determined by labelling of flow-stimulated G pro- Transcriptional Factor Activation (See Table 39.3B)
teins with a nonhydrolyzable GTP photoreactive analog. The In a recent study, Cheng et al46 demonstrated strain-
rapidity by which this response is obtained (1 sec) suggests induced activation of PAI-1 secretion. Although this effect
a key role of G proteins in the mechanotransduction of shear could be accomplished directly on the secretory process, the
stimuli. Previous studies by the same group further estab- authors postulate the upregulation of the PAI-1 gene via a
Table 39.3A. Effect of cyclic strain on transcription factor activation in endothelial cells. (Adapted from ref. 3)
Effect Strain Cell Type Response Time Significance Reference Number
/ AP-1 10% avg., HUVEC 4h Activation of genes 78
60 cpm w/AP-1 binding sites
/ AP-1 10% avg., HAEC 2h Activation of genes 78
60 cpm w/AP-1 binding sites
/ CRE 10% avg., HUVEC Biphasic; Activation of genes 78
60 cpm 15 min, 24 h w/CRE binding sites
/ CRE 10% avg., HAEC Biphasic; 2-4 h Activation of genes 78
60 cpm 24 h w/CRE binding sites
/ Nf-∀b 10% avg., HUVEC 4h Activation of genes 78
60 cpm w/Nf-∀b binding sites
/ Nf-∀b 10% avg., HAEC By 2 h, Activation of genes 78
60 cpm peak at 4 h w/Nf-∀b binding sites
Table 39.3B. Effect of shear stress on transcription factor activation in endothelial cells. (Adapted from ref. 3)
Effect Shear Cell Type Response Time Significance Reference Number
/ AP-1 12 dyn/cm2 BAEC Biphasic, 20 Activation of genes 79
min and 2 h w/AP-1 binding sites
/ Nf-∀b 10% avg., HUVEC 30 min-2 h Activation of genes 79
60 cpm w/Nf-∀b binding sites
/ Nf-∀b 10% avg., BAEC 1h Activation of genes 5, 81
60 cpm w/Nf-∀b binding sites
Mechanical Forces and Cell Differentiation 431
functional AP-1 binding site in its promoter regions. Inter- Selective seeding of the endothelial cells to the high strain
estingly, the strain-induced secretion of PAI-1 was shown region (7-24% strain) led to a greater response than found
to involve reactive oxygen species, a known activator of both in those cells grown in the central, low strain region (0-7%).
AP-1 and NF-∀B transcription factors. Further studies will An increase in CRE binding activity was not observed in
be required to test this hypothesis. However, studies per- cells subjected to an average 6% strain. The reasons for ob-
formed in our laboratory support the hypothesis of increased servation of a strain response in stimulation of CRE bind-
activity of transcriptional activators in endothelial cells sub- ing activity of bovine aortic endothelial cells in this study,68
jected to cyclic strain.78 but not the earlier one,78 is unclear and may suggest other
Since previous studies demonstrated that cyclic strain variables aside from species and vascular bed diversity that
stimulates protein kinase c activity in endothelial cells,72 we remain to be uncovered. The nature of the mechanical force
tested the hypothesis that downstream induction of the fos may also be critical in activating a particular transcription
and jun genes and the transcription factor activator protein-1 factor. For example, Lan et al79 and Resnick et al80 have both
(AP-1) were also activated by strain. Sumpio et al31 showed shown NF-∀B activation in bovine aortic endothelial cells
that human umbilical vein endothelial cells subjected to cy- subjected to shear stress; a finding we have not been able to
clic strain for as short a duration as 30 minutes leads to the replicate in response to cyclic strain.
induction of c-fos, fosB and c-jun genes. In contrast, junB
and junD were not altered by cyclic strain under identical Gene Expression
conditions. We did detect an increase in AP-1 binding activ-
ity as measured by EMSA conducted after 2 h of cyclic strain. Endothelin
It may be concluded that cyclic strain may be coupled to the Cyclic strain stimulates endothelin-1 secretion and
endothelial cell response via activation of protein kinase c mRNA levels in human umbilical vein endothelial cells.82
and elevated steady state levels of different Fos and Jun prod- Elevated expression of endothelin-1 mRNA was detected in
ucts, which may enhance the activity of the transcriptional cells subjected to 2 h or longer duration of cyclic strain. This
activator AP-1.31 was abolished by treatment with actinomycin D. The strain-
In more rigorous studies performed to characterize induced elevation of ET-1 mRNA synthesis was found to be
the induction of transcriptional factor activation, AP-1, CRE mediated by the activation of the PKC pathway and was
binding protein and NF-∀B were examined in endothelial shown to require extracellular Ca2+. Involvement of the PKC
cells obtained from a variety of beds and species.78 These pathway was determined since the PKC inhibitor calphostin
include endothelial cells obtained from human umbilical C prevented strain-induced entothelin-1 expression. In con-
vein, human aorta and bovine aorta. In human umbilical trast, the cAMP-dependent protein kinase inhibitors KT5720
vein endothelial cells subjected to cyclic strain, AP-1 activity or KT5823 only partially blocked endothelin-1 expression
was elevated as compared to unstretched controls.31 The stimulation by cyclic strain. A requirement of extracellular
onset of strain-induced AP-1 activity was at 2 h, peaked by Ca 2+ was determined by blockade of strain-induced
4 h, and returned to baseline levels by 24 h. Similar findings endothelin-1 mRNA by EGTA as well as a lesser effect by
were obtained in human aortic endothelial cells but not in BAPTA/AM, an intracellular calcium chelator.82 In our
bovine aortic endothelial cells.78 A more pronounced retar- hands, we have observed strain-dependent reduction in ET-1
dation indicative of a supershift was noted in the presence (Koo J et al, unpublished data).
of antibodies to c-Jun in HUVECs. Similar contradictory effects of shear stress have also
CRE binding activity was also influenced by cyclic been detected in measurements of ET-1.61,62,83 Malek et al83
strain in a species and vascular bed-dependent manner.78 have shown shear stress-mediated downregulation of ET-1
CRE levels were significantly stimulated in human umbili- expression in bovine aortic endothelial cells conferred by a
cal vein endothelial cells in a biphasic manner with a 2-fold cis-element between -2.5 kb and -2.9 kb of the 5'-upstream
increase at 15 minutes and a nearly 5-fold increase at 24 h. promoter region that does not involve the AP-1 or GATA-2
Similar trends and kinetics were found in endothelial cells factor binding site. In contrast, Morita et al61,84 have shown
derived from bovine aorta. Similar to what is described above shear stress-induced ET-1 expression in porcine aortic en-
for AP-1, we failed to detect a change in CRE binding activ- dothelial cells that appears to be related to cytoskeletal
ity in this study.78 disruption.
NF-∀B binding activity in response to cyclic strain was
also dependent on the vascular bed and species under study. Prostacyclin
NF-∀B binding activity was stimulated in a monophasic Previous studies indicate that cyclic strain29 and shear
58,85
manner in endothelial cells obtained from umbilical vein stress can increase PGI2 secretion by endothelial cells,
with a 4.6-fold increase at 4 h. As observed for AP-1, NF-∀B but the effect of these forces on prostacyclin synthase (PGIS)
was stimulated earlier by 2 h in human aortic endothelial gene expression remains unclear. In a recent study, we ex-
cells, with a peak observed at 4 h. NF-∀B binding activity, as amined this question by studying PGIS gene expression by
shown above for AP-1 and CRE, was not altered by cyclic Northern blot analysis and protein level by Western blot
strain in bovine aortic endothelial cells in this study.78 analysis.57 In addition, the effect of cyclic strain on the PGIS
However, in a later study we did show evidence for promoter was determined by the transfection of a 1 kb hu-
strain-induced stimulation of CRE binding activity in bo- man PGIS gene promoter construct coupled to a luciferase
vine aortic endothelial cells.68 In this study, CRE binding reporter gene into EC, followed by determination of lu-
activity was found by 30 minutes and diminished by 4 h. ciferase activity.
432 Tissue Engineering of Prosthetic Vascular Grafts
PGIS gene expression increased 1.7-fold in EC sub- Awolesi et al 44 showed that the strain-induced
jected to cyclic strain for 24 h.57 Likewise, EC transfected upregulation of eNOS expression in bovine aortic endothe-
with a pGL3B-PGIS(-1070/-10) construct showed an ap- lial cells translates into an increase in functional activity. In
proximate 1.3-fold elevation in luciferase activity in EC sub- this study, 10% cyclic strain for 24 h led to an increase in
jected to cyclic strain for 2, 4, 8 and 12 h. The weak stimula- both citrulline production and accumulated nitrite (Greiss
tion of PGIS gene expression by cyclic strain was reflected reaction) as indices of eNOS activity. Specificity of this re-
in an inability to detect alterations in PGIS protein levels in sponse was confirmed by blockade with EDTA and L-NAME.
EC subjected to cyclic strain for as long as 5 days. These data As is the case for strain-induced expression, strain-induced
suggest that strain-induced stimulation of PGIS gene expres- eNOS functional activity was more pronounced at 10% av-
sion plays only a minor role in the ability of cyclic strain to erage strain as compared to 6% strain. The effect of 10%
stimulate PGI2 release in EC. These findings, coupled with strain was as potent as that observed with the calcium iono-
our earlier demonstration of a requisite addition of exog- phore, A23187.44
enous arachidonate in order to observe strain-induced PGI2 Shear stress has also been shown to increase nitric
release,29 implicates a mechanism that more likely involves oxide gene expression in endothelial cells.59,86,87 In a recent
strain-induced stimulation of PGIS activity. study, Uematsu et al59 reported 2 to 3-fold elevations in eNOS
by shear stress of 15 dynes/cm2 for up to 1 day. The involve-
Tissue Plasminogen Activator (tPA) ment of K+ channels was suggested, since shear stress in-
Previous studies showed an increase in tPA mRNA, duction of nitric oxide mRNA was blocked by tetraethylam-
immunoreactive tPA protein and tPA activity in the medium monium chloride, a K+ channel antagonist.
of cultured bovine aortic endothelial cells exposed to 10%
average strain.19,20 Previous studies documented that tPA MCP-1
expression is upregulated by shear stress.60 We have more Wang et al88 studied the effect of cyclic strain on mono-
recently examined the regulation of tPA gene expression in cyte chemotactic protein-1 (MCP-1) expression in cultured
endothelial cells by cyclic strain.32 A functional analysis of human umbilical vein endothelial cells (HUVECs). They
the tPA promoter was performed by transfecting bovine found a two-fold enhancement in MCP-1 expression as early
aortic endothelial cells with a 1.4 kb construct of the hu- as one hour after the initiation of strain. This response was
man tPA promoter coupled to CAT. After 4 h of cyclic strain, maintained for at least 24 h in the presence of strain, but
a nearly 3-fold increase in the activity of the 1.4 kb tPA pro- was restored to baseline levels 3 h after its release. Calphostin
moter was detected.32 A 60% drop off in activity was found C was able to abolish strain-induced MCP-1 expression,
between position -145 and -105 by analysis of deletion mu- implicating a role of PKC as a mediator of this response. In
tants. DNase I protection analysis of the segment down- addition, a strong Ca2+ requirement for strain-induced, as
stream of position -196 suggested involvement of AP-2 or well as basal, expression of MCP-1 was determined by block-
CRE-like regions, which was confirmed by EMSA analysis. ade with either EGTA pretreatment, BAPTA/AM chelation,
Site directed mutants of either the AP-2 or CRE-like regions or verapamil addition to HUVECs.88
resulted in a 65% decrease in activity compared to the wild Shear stress-induced expression of MCP-1 has been
type. In addition, double mutations abolished basal tran- shown to involve a cis-acting TRE-responsive element in its
scription and any strain-induced activity. A SSRE binding promotor.89 A construct of multiple copies of the TRE-re-
site was found to be present at -945, but site directed mu- sponsive element coupled to a prolactin minimal reporter
tants failed to show any drop in strain-induced activity as and luciferase gene was found to be sufficient to confer shear
compared to the wild type. Overall, our studies demonstrate sensitivity in transfected bovine aortic endothelial cells.
that cyclic strain regulates tPA gene transcription in bovine
aortic endothelial cells by a mechanism similar to that shown ICAM/VCAM
previously for phorbol ester. In recent studies conducted in our laboratory, we have
obtained evidence to suggest that ICAM and VCAM expres-
Nitric Oxide sion are downregulated by cyclic strain in human umbilical
Awolesi et al45 demonstrated that cyclic strain can vein endothelial cells (Lee et al, unpublished observations).
upregulate the expression of endothelial nitric oxide syn- In contrast ICAM-1 expression is upregulated in the same
thase (eNOS) in bovine aortic endothelial cells. At an aver- cell type under conditions of shear stress.87,90
age strain of 10%, eNOS expression in these cells was shown
to be increased as compared to unstretched controls as de- Effect of Cyclic Strain on Vascular Smooth
termined by Northern blot analysis. A milder elevation in Muscle Cell Biology
eNOS expression was measured in cells exposed to a lesser
degree of strain of 6%. The strain-induced activation of General Phenotype
eNOS expression was attributed to stimulation of transcrip- Smooth muscle cells are known to exist in two classic
tional activity as shown by nuclear runoff assays. Western phenotypic states.26 In vivo, under normal conditions,
blot analysis and immunochemistry confirmed that eNOS smooth muscle cell proliferation is slow and the contractile
protein levels were similarly increased in response to strain; or differentiated phenotype predominates. The differenti-
again 10% strain led to a heightened response as compared ated phenotype is characterized by a heterochromatic
to 6% strain. nucleus, abundant actin and myosin filaments. The contrac-
Mechanical Forces and Cell Differentiation 433
tile smooth muscle cells contract in response to both chemi- is mediating opposing activities; PDGF secreted in response
cal and mechanical stimulation. In vivo counterparts of the to cyclic strain opposes the effect of strain to upregulate
contractile phenotype are smooth muscle cells from media smooth muscle heavy chain isoforms.
of vessels of normal adults. The findings of Reusch et al39 also confirm the earlier
However, the smooth muscle cell is not terminally dif- work of Birukov et al95 who showed a dual effect of cyclic
ferentiated and is able to undergo phenotypic modulation strain in modulating rabbit aortic smooth muscle cell phe-
under a variety of circumstances. This phenotypic modula- notype. Cyclic strain was found to both increase prolifera-
tion occurs upon vessel injury and is characterized by a re- tion and also increase expression of markers of smooth
duction in smooth muscle contractile proteins such as #- muscle cell differentiation, in this case h-caldesmon. Com-
actin and smooth muscle myosin heavy chain, and by a shift mon attributes of these studies included a dependence on
toward a synthetic state accompanied by the laying down of quiescent conditions to observe many of their effects (i.e.,
extracellular matrix molecules.35 Such changes can be mim- strain-induced myosin heavy chain expression and prolif-
icked in vitro by growing smooth muscle cells in a serum- eration ), as well as modulation by extracellular matrix pro-
fed preconfluent condition. Morphological characteristics teins that served as a substratum for the smooth muscle cells.
of the synthetic smooth muscle cell phenotype include a In the study by Birukov et al,95 smooth muscle myosin heavy
euchromatic nucleus as well as a prominent endoplasmic chain was increased in the presence of 0.5% serum, but not
reticulum and Golgi complex. These smooth muscle cells observed at concentrations of 2, 5 and 10%. However, strain-
resemble those found in diffuse intimal thickenings of ath- induced h-caldesmon expression was less dependent on se-
erosclerotic lesions.91 rum levels. Laminin was a preferred substrate for detection
However, the identification of smooth muscle cells as of strain-induced h-caldesmon levels95 and was also a pref-
belonging to either a contractile or synthetic phenotype may erable substrate, along with type I collagen, in promoting
be a gross simplification.92,93 Rather, the behavior of smooth differentiation of smooth muscle cells in the study by Reusch
muscle cells is not locked into a particular phenotype such et al.39
that those classified as belonging to the “synthetic” pheno- The authors also studied the effect of extracellular
type are not able to contract and vice versa. For example, matrix molecules on strain-induced SM-1 expression,39 since
Chamley and Campbell94 found that single smooth muscle earlier studies indicated its modulation of strain responsive-
cells from newborn guinea pig vas deferens underwent mi- ness.96 In this study, matrix was again found to be an im-
tosis (as observed by time-lapse photography) despite the portant factor in how SMCs transduce the strain stimuli.
fact that the cells were in a morphologically-defined differ- The ability of cyclic strain to increase SM-1 was 2-fold in
entiated state. This finding emphasized the need to examine SMCs grown on type I collagen, 3-fold on laminin, and ab-
biochemical parameters, in addition to morphology, in or- sent in fibronectin-coated wells. It was concluded that me-
der to better define the phenotypic state of smooth muscle chanical strain can alter myosin isoform expression to a
cells in culture.93 more differentiated state in a matrix-dependent manner.96
As described recently by Owens,36 the factors that nor- The findings of Reusch et al39 confirmed earlier ob-
mally control the differentiation of the smooth muscle cell servations of Kanda and Matsuda97 that indicated stress-in-
are not understood. However, a recent paper by Reusch et duced differentiation of bovine aortic smooth muscle cells.
al39 is lauded as providing the most definitive study to date In their study, Kanda examined smooth muscle cells cul-
identifying a positive differentiation influence/factor in tured in a three dimensional type I collagen gel. Smooth
smooth muscle cells. The work by Reusch et al39 describes muscle cells were exposed to 3 conditions including isotonic
cyclic strain as the factor. Notably, the story as described control, isometric stress or static stress and dynamic stress
below is most complex, as strain has been shown to not only consisting of periodic stretching and recoiling of 5% above
upregulate markers of differentiation (i.e., SM-1, SM-2) but and below the resting tissue length at 60 cycles per minute.
also to stimulate smooth muscle cell growth. Whether this After 4 weeks of this regimen, smooth muscle cell morphol-
represents an artifact of the system due to its heterogeneous ogy was examined by light microscopy and transmission
strain gradient, is due to distinct subpopulations of smooth electron microscopy. Smooth muscle cells subjected to the
muscle cells, or reflects the fact that growth and differentia- dynamic regimen were unique in exhibiting a more differ-
tion are not necessarily exclusive properties remains to be entiated state as shown by increased fractions of contractile
established.36 apparatus. Over time on stress, the content of myofilaments,
Since previous studies had shown that PDGF secre- dense bodies and extracellular filamentous basement mem-
tion is elevated by cyclic strain,40 the authors examined brane-like materials increased. This was an indication that
whether secreted PDGF affects the strain-induced changes dynamic stress in cells laid down in a 3-dimensional collagen
in contractile protein expression. Interestingly, PDGF-AB matrix inclines them toward a more differentiated state.97
was shown to cause a qualitatively opposite change in myo- The effect of cyclic strain on smooth muscle cell pro-
sin isoform expression, decreasing smooth muscle myosin liferation is predominantly stimulatory40,95,98,99 Davis99 ob-
by greater than half. Moreover, neutralization of PDGF-AB served strain-induced cell proliferation of 33% in serum-
with neutralizing antibodies led to a strain-induced increase starved rat aortic smooth muscle cells supplemented with
in SM-1 that was enhanced 10-fold. Thus, PDGF fails to insulin and transferrin. Although significant, this response
mediate the effect of cyclic strain on smooth muscle myosin was only one fifth that observed in the presence of repleted
isoform distribution. Instead, it is concluded that cyclic strain calf serum of 10%. Likewise, Yang et al98 showed a significant
434 Tissue Engineering of Prosthetic Vascular Grafts
increase, albeit slight (13%), by 6 days of cyclic strain expo- muscle cell proliferation, depending on the exact source of
sure in cultured smooth muscle cells obtained from the tissue and the regimen under study. For example, Sumpio
media of human left descending coronary artery. In neona- and Banes28 showed that the proliferative response of por-
tal rat smooth muscle cells grown to quiescence, Wilson et cine aortic smooth muscle cells is inhibited by cyclic strain.
al40 observed a similar 40% increase in the proliferative re- In another study using early passaged smooth muscle cells
sponse to cyclic strain as compared to static controls. In rab- from lamb pulmonary artery, Kulik and Alvarado100 failed
bit aortic smooth muscle cells, cyclic strain also caused an to detect any increase in cell number after 24 h of 20% stretch.
increase in cell number.95 Interestingly, the ability to detect Similarly, DNA synthesis as measured by [3H]thymidine
an increase in cell proliferation induced by cyclic strain was incorporation was unaffected by stretch. In the same study,
dependent on the cells being serum activated. In quiescent a passaged clonal line of rat cells (PAC1) did show a modest
cultures, smooth muscle cells failed to respond to cyclic elevation (17% increase) in the percentage of
strain. In this study, Birukov et al95 also showed that the [3H]thymidine-labeled cells. However, TCA-precipitated
number of detached cells (i.e., an index of cell attachment [3H]thymidine was unaffected by cyclic strain.
to membranes) was unaffected by cyclic strain. In another In a follow up study to the earlier work of Kulik and
control experiment, motion effects of circulating medium Alvarado100 failing to demonstrate an effect of mechanical
in response to cyclic strain was ruled out as a contributory forces on the growth of smooth muscle cells from pulmo-
factor to the stretch-induced increase in smooth muscle cell nary artery, Kolpakov et al101 reexamined this finding in a
proliferation, since no difference was noted in control ver- whole vessel model. Interestingly, they found that subject-
sus nutational conditions. ing rabbit pulmonary arteries to varying magnitudes of
The effect of cyclic strain on DNA synthesis in smooth stretch led to an increased rate of protein synthesis (as mea-
muscle cells is more pronounced than for proliferation. In sured by quantitative autoradiography) and replication of
coronary artery smooth muscle cells obtained from human smooth muscle cells. Moreover, synthesis of extracellular
left descending coronary arteries, Yang et al98 found that matrix proteins, elastin and collagen, were elevated in 4 day
cyclic strain stimulates DNA synthesis greater than 2-fold stretched segments as compared to static controls. The au-
after 24 h. In rat aortic smooth muscle cells, Davis et al99 thors also showed that these effects were independent of
showed a 2-fold increase in [3H]thymidine incorporation endothelium interaction, as its removal did not prevent the
by cyclic strain. In smooth muscle cells obtained from new- aforementioned changes.
born rat, Wilson et al40 demonstrated a nearly 3-fold increase This study illustrates the caution that should be exer-
in [3H]thymidine incorporation by cyclic strain of -20 kPa cised in interpretation of in vitro experiments that may not
at 60 cycles/min for 48 h. This elevation afforded by strain be analogous to the condition in vivo or even in an ex vivo
was less potent as compared to that observed with 1 U/ml model. The authors rightly assert that their earlier negative
thrombin or 5 ng/ml platelet derived growth factor (PDGF). findings of in vitro studies could be attributed to cell shape,
Further study showed that combinatorial addition of strain its orientation relative to the applied strain, matrix substra-
and thrombin led to even greater stimulation of DNA syn- tum, and of course, the cell culture milieu.101
thesis. However, strain and PDGF did not exhibit signifi-
cantly greater stimulation of [3H]thymidine as compared Cell Signaling
to that observed with PDGF alone. The authors suggest that
this observation may be explained by strain itself acting via Second Messenger Pathways
autocrine production of PDGF.40
Wilson et al96 further studied the matrix dependence Adenylyl Cyclase
of the strain-induced mitogenic response in rat vascular In bovine aortic smooth muscle cells, we have dem-
smooth muscle cells. They found that cyclic strain increased onstrated strain-induced activation of the adenylyl cyclase/
DNA synthesis in SMCs grown not only on type I collagen, cyclic AMP/protein kinase a pathway.24 Basal adenylyl cy-
but also fibronectin or vitronectin. However, on plates coated clase was elevated nearly 2-fold in SMC subjected to
with elastin or laminin, strain induction of DNA synthesis 150 mm Hg vacuum deformation for 30 min and returned
was not observed. The integrin binding peptide GRGDTP to basal levels by 60 min. Likewise, cyclic AMP accumula-
was shown to block the mitogenic response of SMCs to strain tion was also stimulated by cyclic strain at 30 min and re-
in cells seeded on type I collagen. Specificity of this response turned to basal values by 60 min. Protein kinase a activity
was noted by the inability of the inactive peptide GRGESP was also stimulated by cyclic strain. The activation of this
to also block strain-induced mitogenesis. In addition, pathway was strain specific, such that cells subjected to low
strain-induced expression of a PDGF-A chain promoter- strain (0-7%) in the center of the well were found to signifi-
CAT construct transiently transfected into SMCs was blocked cantly stimulate PKA activity, as compared to cells in the
by the integrin binding protein GRGDTP. A role for specific peripheral, high strain (7-24%) region of the well. Similar
integrins involved in sensing mechanical strain in SMCs was findings of an elevated response from cells obtained from
shown by studies with antibodies to both !3 and #v!5 the center region were observed for CRE binding protein
integrins, which also blocked the mitogenic response levels as measured by gel shift analysis.
to strain. The relationship between strain-activated signaling
However, there are conflicting reports in the litera- events and downstream responses is complex. This is exem-
ture regarding the effect of mechanical strain on smooth plified by our inability to demonstrate any effect of
Mechanical Forces and Cell Differentiation 435
Rp-cAMP, a PKA inhibitor, on strain-induced proliferation Our data suggested that cyclic strain causes dephosphoryla-
or alignment.24 Thus, although strain activated the adenylyl tion of MLC20 in SMCs, which may be partially due to the
cyclase signaling pathway, the lack of an effect of PKA inhi- activation of MLC20 phosphatase and/or inhibition of
bition suggests either the lack of involvement of this path- MLC20 phosphorylation.
way or the multifactorial nature of these responses.
Gene Expression
Tyrosine Kinase
Davis et al99 showed that cyclic strain stimulates the PDGF
phosphotyrosine accumulation in smooth muscle cells ob- PDGF-AA and -BB secretion and PDGF-A expression
tained from rat aorta. Proteins with upregulated were stimulated in smooth muscle cells subjected to cyclic
phosphotyrosine content in response to cyclic strain included strain.40 Moreover, neutralizing antibodies to both PDGF-
species of 110-130 kDa and 70-80 kDa as determined by AA and -AB were found to block strain-induced stimula-
Western blot analysis. The identity of these proteins was not tion of DNA synthesis. In contrast, bFGF antibody failed to
ascertained, but ascribed to proteins of corresponding mo- prevent the mitogenic response to cyclic strain.40
lecular weight, including GTPase and p74Raf, respectively.
Specificity of tyrosine kinase activity induced by cyclic strain Myosin Heavy and Light Chain
was shown by blockade with the phosphotyrosine kinase Reusch et al39 recently examined the effect of mechani-
inhibitor herbimycin A. Moreover, both herbimycin A and cal strain on the expression of myosin heavy chain isoform
genistein, another tyrosine kinase inhibitor, were found to expression in neonatal rat vascular smooth muscle cells. As
have cyclic strain-induced DNA synthesis as measured by a marker of differentiation, they studied expression of the
incorporation of [3H]thymidine. Thus, it was concluded that smooth muscle myosin heavy chain isoforms, SM-1 and
cyclic strain stimulates vascular smooth muscle cell prolif- SM-2 (see Fig. 39.7 in ref. 39). These isoforms are well char-
eration through activation of tyrosine kinases.99 acterized markers of differentiation expressed at high levels
in postconfluent, quiescent SMC.102 In quiescent smooth
Phosphatases muscle cells grown in 0.5% fetal bovine serum, SM-1 and
Phosphorylation of the 20 kDa regulatory myosin light SM-2 protein content was elevated by 4.5- and 3.5-fold,
chain (MLC20) is the seminal event in the initiation of vas- respectively, after 72 h of cyclic strain. In addition, nonmuscle
cular smooth muscle cell contraction. We sought to deter- myosin A (NM-A) protein content decreased to 30% of con-
mine whether cyclic strain affects MLC20 phosphorylation trol levels. Cyclic strain was also found to increase gene ex-
in bovine aortic smooth muscle cells. We studied the effect pression of SM-1 and decrease that of NM-A, with peak
of cyclic strain on serum-fed SMCs already displaying a high changes occurring at 12 h and 3 h, respectively.
level of MLC20 phosphorylation. Confluent SMCs were sub-
jected to 10% average strain at 60 cycles per minute for 30 Summary
and 60 minutes. Basal MLC20 phosphorylation (N=non, In summary, recent data support the hypothesis that
M=mono, D=di) of serum-fed SMC was as follows: N=34%: vascular endothelial and smooth muscle cells are keenly sen-
M=27%: D=39%. After 60 minutes of cyclic strain, both the sitive to mechanical perturbation such as cyclic strain. Our
mono and diphosphorylated species of MLC20 were de- group and others have characterized changes in the pheno-
creased by 21% and 15%, respectively. The strain-induced type of both cell types and have begun to delineate the rel-
dephosphorylation of MLC20 was partially inhibited by the evant signaling pathways and strain-sensitive genes. Con-
protein phosphatase 1/2A inhibitor calyculin A. However, tinued advances in this field may prove beneficial in devel-
phosphorylase A phosphatase activities in Triton-soluble and oping novel approaches to improve tissue engineering of
insoluble fractions of SMCs were unaffected by cyclic strain. prosthetic grafts.
Table 39.4. Effect of cyclic strain on smooth muscle cell signaling pathways
Effect Strain Cell Type Response Time Significance Reference Number
/ PKC 10% avg., BASMC 10 sec Activation of the 24
60 cpm PKC pathway
/ Adenylyl 10% avg., BASMC 30 min Activation of the 24
cyclase 60 cpm cAMP pathway
/ cAMP 10% avg., BASMC 30 min Activation of the 24
60 cpm cAMP pathway
/ PKA 10% avg., BASMC 30 min Activation of the 24
60 cpm PKA pathway
/ Tyrosine 10% avg., RASMC 10 min Role in SMC 99
Phosphorylation 15 cpm growth
436 Tissue Engineering of Prosthetic Vascular Grafts
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PART III
Biointeractive Prostheses: Complete Healing
Engineering Components
Scaffold Engineering
Structural Designs
CHAPTER 40
An Integral Mathematical Approach to Tissue Engineering
of Vascular Grafts
Greg R. Starke, A.S. Douglas, D.J. Conway
Introduction
T he development of neointimal hyperplasia near the anastomosis of small diameter grafts
has been positively linked to changes in the arterial fluid dynamics. Graft materials such
as woven Dacron or polytetrafluoroethylene (PTFE) are relatively rigid when compared to
the native artery. The implantation of these less compliant grafts causes a considerable alter-
ation in the fluid flow patterns of the arterial system. Altered flow patterns result in high
shear stresses on the vessel wall1,2 and turbulence downstream of the anastomosis,3 possibly
causing endothelial cell damage.4-6 Several studies7-10 have shown a positive correlation be-
tween compliance matching and graft patency.
Other mechanical issues, apart from altered blood flow, have also been implicated in
the formation of intimal hyperplasia. Various research groups11,12 have suggested that the
development of hyperplasia is a consequence of the normal remodelling response, as a re-
sult of the stress concentrations that will occur around the individual sutures. They argued
that the proliferation of smooth muscle cells, and the deposition of extracellular matrix
around the suture line, is a process which increases the amount of load bearing material in
an attempt to reduce the strain to more physiological levels. Finite element models of a
bypass graft have been used to investigate the stress distribution in the region of the anasto-
mosis.11 This study showed that the presence of the graft resulted in stress magnitude of
approximately 1.5 times the normal value. Increasing the amount of tissue in the region, to
reproduce the experimentally observed geometry, resulted in a reduction in stress to 1.2
times the normal value. In a subsequent study12 the same research group examined the de-
velopment of cellular hyperplasia around individual sutures and showed that thickening
was more pronounced around the sutures. In a corresponding finite element model they
showed that, in the early post-operative period, stresses were concentrated around the su-
tures—which were subsequently dispersed once healing had progressed. However, it may be
argued that the observed remodelling was a response to the surgical injury—although it is
likely that both mechanisms play a role.
Minimising the stress gradient at the anastomosis requires the correct sizing of the
graft in relation to the host artery. To predict the size of the vessels during diastole and
systole requires the accurate characterisation of the materials and the loading. Paasche et
al13 investigated the choice graft sizes in an effort to minimise suture line stresses. In this
investigation they assumed that the graft could be regarded as rigid when compared to the
host. They found that the suture line stresses were at a minimum when the ratio between the
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
442 Tissue Engineering of Prosthetic Vascular Grafts
graft and the host artery sizes was approximately 1.45. How- ing the design process by allowing the developer to test de-
ever, in a polymeric tissue engineered graft the implant sign concepts and parameters without the time consuming,
should not be assumed to be rigid. Therefore the optimal and costly, process of prototyping. Furthermore, it is often
size of the graft in relation to the host needs to be investi- difficult—if not impossible—to measure quantities such as
gated in the light of graft compliance. In addition, the pres- stress, strain or displacement. These quantities can, how-
ence of soft tissue within the graft structure will alter the ever, be readily calculated from mathematical models of the
compliance, and will also alter the suture line stresses at the loaded implant. Accurate predictions require sound consti-
anastomosis. Therefore the compliance matching and graft tutive (material) laws describing the behavior of the intact
size selection needs to consider the evolutionary nature of artery as well as the proposed graft material. Analytical so-
the implant compliance. Reproducing the stress/strain be- lutions of arterial systems are rarely possible because of the
havior of the native artery also means that diameter com- complexity of the material formulations, structures, bound-
patibility can be examined. If diameter matching is achieved ary conditions and initial loading conditions. However,
in the long term, then stress concentrations at the anasto- making use of sophisticated numerical techniques such as
mosis will be kept to a minimum and thus progressive re- the finite element method can approximate solutions.
modelling will not be necessary to reach a homeostatic state. Many of the mechanical design requirements can,
A tissue engineered vascular graft is intended to pro- however, be derived from the mechanics of the intact ar-
vide a scaffold for the development of tissue; therefore, the tery—as the success of the graft will depend in part on rec-
structure has to be sufficiently porous, with interconnectivity, reating the mechanics of the natural vessel. However, the
so as to allow for vascularization and the subsequent infil- mechanics of the native vessel needs to be reproduced in a
tration of cells. However, in the early post-operative period, synthetic material with a highly porous structure. In addi-
before tissue ingrowth has taken place, the scaffold needs to tion, the process of tissue infiltration and development will
have sufficient structural integrity to withstand the applied alter the mechanical properties of the graft. The remainder
loads, without leading to excessive deformation or rupture. of this chapter is therefore concerned with the development
Greer et al14 investigated the material properties of a col- of a numerical formulation for mechanics of the native ar-
lagen/smooth muscle cell gel and found that the stress/strain tery as well as a polymer graft.
characteristics, as well as the stress relaxation, were both
strongly dependent on the extent of cell seeding. This pre- The Mechanics of Arteries
liminary study shows how the biological features of a tis- The flow of blood can be related to the elasticity of an
sue-engineered graft can alter the mechanics of the graft. artery by considering the feedback loop shown in Figure 40.1.
Furthermore, the development of tissue may depend on the The bottom block shows the artery as a rigid conduit allow-
loading environment. Therefore, the graft structure needs ing blood flow. A pulsating pressure results from this flow.
to create optimal strain conditions, during both diastole and If the artery is treated as an elastic body, as shown in the top
systole, that will facilitate the development and maintenance block, the pressure will then act to deform the conduit, which
of smooth muscle cells within the porous structure. in turn will affect the nature of the flow. The flow character-
Mathematical modelling provides a means for char- istics can then be analyzed assuming that the vessel is rigid,
acterizing the mechanical response to loading of the native and has a diameter that has been altered by the flow pres-
artery, as well as the structured polymer from which a tissue sure. The deformation of the artery can then be determined
engineered graft might be constructed. Such models allow using the laws of elasticity assuming a constant pressure and
for the investigation of compliance, remodelling, sizing and determining the nature of the deformation. In this way the
failure prediction. This approach offers the potential for aid- flow of blood is coupled to the elasticity of the vessels.
Fig. 40.1. Interaction between the
flow of blood and the mechani-
cal deformation of an artery,
adapted from Fung, 1996.
An Integral Mathematical Approach to Tissue Engineering of Vascular Grafts 443
Structural Morphology ure 40.2, which shows experimentally determined and nu-
The structural characteristics of arteries arise from the merically predicted stress/strain relationships for the arte-
tissue composition of the media and the adventitia. The rial walls of four normal rabbit arteries.17 The effective
media is made up of smooth muscle cells and bundles of Young’s modulus of the material can be viewed as the slope
collagen fibrils which are layered between a number of elas- of the stress/strain curve. Since the modulus increases with
tic sheets. The structure of the adventitia results from col- strain, arterial stiffness is a non-linear function of strain.
lagen fibres, fibroblasts and ground substance. A relatively The stiffening of the arterial wall, which occurs when the
thick layer of elastin separates the media and adventitia. The collagen bundles become straightened and go into tension,
endothelial cells of the intima do not have any significant is observed in the region of 150 mm Hg pressure. This be-
influence on the artery mechanics, and therefore the intima havior protects the arteries from excessive dilation and there-
and media can essentially be grouped together in so far as fore offers protection against the development of aneurysms.
the elastic response is concerned. In the low strain region The complex composition and structure of arteries
(near zero stress) the stiffness of the intima-media layer is means that the material properties of the wall are not the
almost an order of magnitude greater than that of the ad- same in all directions (the material is therefore anisotropic).
ventitia. However, at larger strains the collagen bundles be- Several research studies have shown that the stiffness of the
gin to straighten and then stretch, which increases the stiff- arterial wall in the circumferential direction is greater than
ness contribution from the adventitia. The relative thick- in the axial direction.19,20 However, this result is by no means
ness of the intima-media/adventitia varies as a function of conclusive, as previously Tanaka and Fung21 found the op-
the position in the body and therefore so too does the elas- posite result. The are many factors, including the specimen,
tic response.15 The walls of the artery are structurally aniso- species and testing methodology, to which the difference in
tropic, incompressible and highly deformable, exhibiting results may be attributed.
non-linear stress/strain characteristics. As a result of the high fluid content in the arterial wall
tissue, the material is often assumed to be incompressible—
Non-Linear Elastic Response hence volume is preserved during deformation. Such an as-
The most striking aspect of the mechanical response sumption considerably simplifies the mechanics of the walls,
of arteries is the non-linear relation between the pressure as the deformation in the radial direction can be directly
acting within an artery and the resulting change in diam- related to the deformation in the longitudinal and circum-
eter. Experimental evidence16-18 has shown that as the pres- ferential directions. The assumption of incompressibility has
sure increases, and hence the stress within the arterial walls, been validated by Choung and Fung23 who found that the
the change in diameter is initially rapid, but as the stress percentage of fluid extrusion of the undeformed tissue was
increases the change in strain becomes smaller until increases between 0.5 and 1.26% as a result of radial compression of
in stress are accompanied by only very small increases in rabbit aortic tissue. This result showed that, while the arterial
strain. This high degree of non-linearity is illustrated in Fig- tissue is slightly compressible, it would suffice to assume
incompressibility.
Fig. 40.2. True stress plotted
against true strain for arteries from
the canine coroid, left iliac as well
as the lower and upper aorta,
adapted from Fung et al 1979.
444 Tissue Engineering of Prosthetic Vascular Grafts
Viscoelasticity pears to deform indefinitely under a constant load, and the
Thus far the non-linear large strain elastic behavior recovery is so slow that the material appears to have crept.
typically exhibited by arteries has been discussed. However, There have been a number of important studies over
no mention has been made of a further (often very typical) the last 40 years that have contributed greatly to our under-
material characteristic of soft tissues, namely their time-de- standing of the specific viscoelastic behavior of arterial wall
pendent nature. In such materials, the current stress is de- tissue and biological soft tissues in general. Fung24 found
pendent not only on the current strain but also on the strain that while arterial tissue does indeed exhibit a delayed re-
history. Since no materials respond truly instantaneously to sponse to loading and increased stiffness with increased load-
a load (indeed, neither can loading be applied instanta- ing rate, the amount of lag is fairly insensitive to strain rate.
neously without inducing large inertia effects), the response This result allowed Fung to postulate a fairly simple con-
is always delayed to some degree, and in so-called viscoelas- tinuous modulus function from which he found model pa-
tic materials this delay is significant enough to necessitate rameters using canine aortas. Some detail on this approach
quantification and modelling. Typically the elastic response is given in the viscoelastic properties subsection of the sec-
to a sudden stress is characterized by an equally sudden strain tion “Materials Testing”. Fung’s models assume linear vis-
(the glassy response) followed by a gradual increase in the coelastic behavior—an assumption that was consistent with
strain (the rate of which decays exponentially). The sim- the results of some later animal studies by Hutchinson,25
plest model of such a material is one which is described by Newman26 and Greenwald.27 In contrast, however, work
one time scale, 4, and one elastic modulus, G. Typically, 4 is done by Langewouters et al28,29 has shown that human arte-
defined as the time it takes for a material to reach a set fac- rial wall tissue has a non-linear viscoelastic response (i.e.,
tor (approximately 0.37) of the asymptotic, long term elas- the viscoelastic parameters vary significantly with varying
tic response to a step load (force or displacement). Real vis- strain). There now appears to be consensus in the literature
coelastic materials may have infinitely many time scales that the true viscoelastic behavior of biological soft tissues
(ranging from milliseconds to days and longer), each with (including arterial tissue) is indeed non-linear,16,30-35 and
its own associated elastic modulus. These parameters are the reason for this phenomenon has been explained with
defined by way of a continuum modulus function that must reference to the ultrastructure of the tissue. The two major
be determined experimentally. Details of these continuum stress-bearing constituents in arterial tissue are elastin, which
functions and the salient aspects of viscoelastic constitutive maintains a fairly constant Young’s modulus over the range
modelling are presented later. of physiological strains, and collagen, whose stiffness in-
Depending on the type of loading conditions experi- creases considerably with increased stretch. The exponen-
enced by a viscoelastic material, it will display one of the tial stiffening with increased strain is attributed to the
following responses: straightening out and tensioning of the wavy collagen strands
1. Delayed elastic (deformation, strain) response to a as they appear in an unstretched state.36,37 This process is
constant, suddenly applied load referred to as collagen recruitment. Thus the purely elastic
2. Stress relaxation under a constant strain; behavior of arterial tissue is almost always modelled non-
3. Delayed recovery on removal of all loads. linearly. It has been shown38 that collagen exhibits double
These responses are all illustrated in Figure 40.3. If, in the amount of stress relaxation as that of elastin, but at a
addition to all these fully reversible responses, a material fifth of the rate of elastin relaxation at room temperature
exhibits gradually increasing permanent deformation un- (assuming simple rheological models with only one relax-
der a constant load, the material is said to creep. This is not ation time constant per material). Collagen recruitment
a property of purely viscoelastic materials since creep is not means that collagen takes up proportionally more of the load
recoverable, and hence not elastic. However, some viscoelas- as the artery wall dilates and so its viscoelastic properties
tic materials do have very large time constants with associ- also become increasingly predominant—which explains the
ated moduli that are large enough so that the material ap- non-linear viscoelastic behavior.
Fig. 40.3. Schematic illustration of
delayed elastic response, stress re-
laxation, and delayed recovery of
viscoelastic materials.
An Integral Mathematical Approach to Tissue Engineering of Vascular Grafts 445
In spite of this evidence, most research in this field radially along the longitudinal axis. Pre-stressing has been
still assumes quasi-linear viscoelasticity (i.e., non-linear in investigated by several researchers,41,42 resulting in the “uni-
the elastic deformation response but linear in the history of form strain” hypothesis.
deformation). This approach avoids difficulties associated Fung et al42 examined the no load and no stress state
with implementing non-linear viscoelasticity numerically, of the pulmonary and ileac arteries of rats. Figure 40.4a
and the more complex materials testing that would be re- shows the cross section of the pulmonary artery at a blood
quired for the constitutive characterisation. Huyghe et al39 pressure of 15 mm Hg. In the no load state (Fig. 40.4b) the
implemented a quasi-linear viscoelastic formulation for pas- artery has reduced considerably in diameter and has there-
sive heart muscle tissue. They found that all the mechanical fore increased in wall thickness. Figure 40.4c shows the sec-
characteristics of tissue—which are essentially the same for tion of the artery following the radial cut that has removed
arterial wall tissue (including the weak frequency depen- the pre-stress. In order to quantify the extent of pre-stress-
dency of the dissipation energy during cyclic loading (i.e., ing within the wall, the angle to which the artery opens is
lag time))—were accommodated by their model. However, measured. This angle determines the amount of displace-
when they set the relaxation parameters in order to correctly ment caused by the pre-stress and can therefore be related
predict the dissipation energy, they found that the model to the magnitude of pre-stress.41
overpredicted the amount of stress relaxation by over 100%. In a further study, Greenwald et al43 measured the
This discrepancy they attributed to the non-linear viscoelas- opening angle of rat aortic arteries following the removal of
ticity of true cardiac tissue. various of the structural components. They found that the
Young et al,16 Decraemer et al,32 Wu and Lee33 and removal of smooth muscle and collagen had no effect on
Johnson et al35 have all incorporated non-linear viscoelas- the opening angle, while the removal of elastin had a sig-
ticity in their constitutive models for soft biological tissues. nificant effect, resulting in a decrease in the opening angle.
These laws were reported to predict results that correlate This study concluded that the residual stresses are stored in
fairly well with existing experimental test data for tissues the elastic component of the media.
such as human patellar tendon, canine collateral ligament The opening of the artery shows that the pre-stress-
and canine arterial wall tissue. Such models do have poten- ing puts the internal surface of the wall into a state of com-
tial to make more accurate predictions in the design process pression and the external surface into tension. Subsequently,
than the simpler quasi-linear laws. However, the experimen- the tensile stresses that are experienced during normal load-
tal determination of the constitutive parameters is neces- ing are reduced by the initial compressive component, re-
sarily a far more complex task with these more realistic sulting in a more uniform distribution of stress, as is shown
models. Until accurate and accessible experimental meth- schematically in Figure 40.5.
ods are developed which enable such parameterization, it
can be argued that the simpler, numerically more robust Material Laws for the Native Artery
quasi-linear laws are sufficient. Myers et al40 discuss the limi- and Graft Materials
tations of quasi-linear theory and suggest methods for re- The extent of the porosity of a polymeric graft, as well
ducing the effects of this simplifying assumption. as the nature of the structure, requires careful engineering
design which will consider not only the mechanical aspects,
Pre-Stress but also the coupling with the biological design. In design-
In a thick walled cylinder under internal pressure the ing an artery graft, many of the design objectives can be
distribution of the circumferential strain through the wall learned from studying the mechanics of the intact vessel.
varies considerably, with a peak at the inner surface which Also, failures of grafts typically occur in the region of the
decays exponentially towards the outer surface. It has been anastomosis, and a mechanical model of the interaction
shown that several tissue types, including bone, will remodel between the graft and the host is therefore needed for graft
so as to reach a homeostatic strain condition. In order to design. Since any mechanical model will require the consti-
moderate the peak strain, and produce a more uniform dis- tutive response of both the graft and the natural artery, the
tribution through the thickness, the artery walls are pre- fundamentals of the mechanical response of compliant
stressed. This pre-stressing is evident by the way an artery materials are reviewed.
will spring open if a transverse section is removed and cut
Fig. 40.4. Sectional geometry
of the rat pulmonary artery at
(a) in vivo loading of
15mmHg, (b) no-load and (c)
no stress. Reproduced with
permission from Fung et al
1992.
446 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 40.5. Schematic representation of
the influence of artery pre-stressing on
the resultant stress distribution through
the artery wall.
Compliant engineering materials (such as polymers) 5(X), to the displacements of each material point, u(X), if
and soft tissues deform appreciably under load and care must we note that the deformed position is merely the sum of the
be taken to describe their behavior. Most soft tissues sus- reference position and the displacement of that point, viz.
tain large strains, are generally non-linear in their con- x = X + u(X). To characterize the deformation of this mate-
stitutive response (see Fig. 40.2), exhibit either transverse rial we need to examine how the deformed position or the
isotropy or anisotropy and are either incompressible or displacements change spatially. Both the deformation gra-
nearly so. Compliant materials, or rubber-elastic materials, dient tensor, F, the components of which are given by
also sustain large strains, may be anisotropic and are often
incompressible. (1)
To describe the mechanical behavior of both soft
tissues and compliant engineering materials, researchers where i, j=1,2,3, and the displacement gradient ten-
usually use a hyperelastic (Green elastic) strain energy sor, H, with components given by
function of the local deformation or strain, W. The existence
of a strain energy function for a material implies that it is (2)
hyperelastic and that, regardless of deformation path, equal
work is done in going from one deformation (or strain) state characterize the deformation at the point X. The deforma-
to another. In order to characterize compliant graft materials tion gradient tensor F also contains information about the
and the natural artery, we need to formulate appropriate local rotation. However, since we would like to relate the
strain energy functions W. To do this we must first develop strain energy function, W, to deformation alone, we define
a framework with which to describe the deformations, strains the right and left Cauchy-Green deformation tensors, C=FTF
and stresses required. Note that, since soft tissue is not strictly and B=FFT, respectively, which depend only on the defor-
hyperelastic, it is characterized by a pseudostrain mation (not the rotation) at any point in the material. Strain
energy function. is also a measure of deformation, independent of rotation,
and used to characterize the strain energy function. The
Characterizing Finite Deformation Lagrangian strain, E, is defined by
The motion of a compliant material can be described
as a mapping,5, which defines the deformed position, x, of E = 12 F T F % I =
[ ] [H + H
1
2
T
+ H TH ] (3)
a particle which occupied the position, X, in the reference
configuration; thus x = 5(X). We can also relate the motion, where I is the identity tensor (Note that F = H + I).
An Integral Mathematical Approach to Tissue Engineering of Vascular Grafts 447
In soft tissue mechanics, since deformations and ro-
tations are finite, we need to be precise about the definitions ( = % p I + 2 W1 B % 2 W2 B %1 + 1# W# F (N < N )FT
of stress. The true, or Cauchy, stress is the symmetric sec-
ond-rank tensor, (, (with components (ij) which relates the (9)
actual loads to the current deformed configuration. Since
where W1, W2 and W# are the derivatives of the strain
the Second Piola-Kirchhoff stress, S, considers the reference
energy function with respect to its arguments and N < N is
configuration, it is more easily measured in experiments and
the tensor product of the two unit vectors.
is related to the Cauchy stress by
We can also cast the strain energy functions in terms
6∋9
( ij = 8 ; Fik Skl Fjl ,
of the Lagrangian strain components, Eij, since these are easy
(4) to measure in experiments. Using the notation of Fung,22 if
7 ∋0 :
W is the strain energy per unit mass of the tissue, then ∋0 is
where ∋ and ∋0 are the current and reference tissue
the strain energy per unit volume in the zero stress (refer-
densities, respectively, and repeated indices imply a sum-
ence) state (∋0 is the density in the reference state). The Sec-
mation from 1 to 3.
ond Piola-Kirchhoff stress is then given by
= ∋ 0 W(E )
[ ]
S ij =
Hyperelasticity
(10)
= Eij
The mechanical constitutive description of both soft
tissues and compliant engineering materials assumes that
for compressible materials, and by
these materials are hyperelastic. Tests therefore need to be
conducted to determine the functional form and parameters = ∋ 0 W (E)
[ ]
S ij = % p Fik%1 Fjk%1 + (11)
which define the strain energy (or pseudo-strain energy)
= E ij
function, W. The simplest strain energy functions describe
for incompressible materials.
isotropic materials. Since the behavior of these materials is
For the special case in which the strain energy func-
independent of orientation, the strain energy is a function
of the three invariants of the Cauchy-Green deformation tion is quadratic in strain, or ∋ 0 W = 1
2
Dijkl Eij E kl , there
tensor, viz.,
is a linear relationship between the Piola-Kirchhoff stress,
W = W ( I1 , I 2 , I3 ) ,
Sij, and the Lagrangian strain, Eij, given by
(5)
where S ij = Dijkl E kl . (12)
For any non-linear material, the increment in stress,
(6) dsij, is related to the increment in strain, dEij through
= 2W
dSij = dE kl = Dijkl dE kl ,
If the material is incompressible, then I3=1 and
= E ij = E kl
W=W(I1,I2) only. (13)
For transversely isotropic (these have a single preferred
where Dijkl is the incremental material modulus.
orientation defined by the unit vector N in the reference
configuration) and incompressible materials, the strain en- Viscoelasticity
ergy is dependent on four invariants,
Thus far we have outlined constitutive modelling of
W = W ( I1 , I 2 , I4 , I5 ) ,
the time independent elastic response of soft tissues materi-
(7) als and described how a postulated strain energy function is
used to calculate the stress for any given strain state. As dis-
where
cussed earlier, both arterial wall tissue and many elastomeric
materials exhibit significant viscous behavior at strain rates
I 4 = N − C N = # 2 and I 5 = N − C2 N . (8) typical of heart rates. Since the concern in the design of ar-
tery grafts is to construct the graft so that it closely matches
Note that, in Equation (8), # is the stretch in the di-
the instantaneous compliance of the native artery, we need
rection N, so that if N is the muscle fibre orientation, # is
to include the viscoelastic nature of both the arterial tissue
the muscle fibre stretch.
and the graft material in our constitutive law.
If the material is incompressible, the hydrostatic stress
Fortunately, the addition of viscoelasticity in the ma-
is not determined by the material deformation, but is set by
terial model does not mean that we have to discard our stati-
the boundary conditions and the equations of equilibrium.
cally determined hyperelastic strain energy function. A fi-
The constitutive response must therefore introduce a
nite strain viscoelastic material law still requires the strain
Lagrange multiplier to accommodate this hydrostatic stress.
energy function, W, for the long-term modulus.
The deviatory stress is determined by the strain energy, so
The description of viscoelasticity that follows is re-
that the Cauchy stress, for a material with a strain energy
stricted to linear (or quasi-linear) viscoelasticity—where the
function dependent on three invariants, W=W(I1,I2,#), is
rate of the delayed response to a given load is only a func-
given by Humphrey et al,44,45
tion of time (not of the magnitude of load). Recall that quasi-
linear viscoelasticity does allow for non-linear long term
hyperelastic behavior.
448 Tissue Engineering of Prosthetic Vascular Grafts
Spring and dashpot models are a good analogy for s(t) = ket=0e–t/t for the
modelling material viscoelastic behavior, since they allow ( Maxwell Model.
the characterisation of the material both qualitatively and The relaxation time,
quantitatively. The simplest model for the elastic behavior t, is the time taken for
of a material is the linear spring where stress, (, is simply the stress to
proportional to the strain, >, relax by a factor
e -1Χ0.37 of the initial
( ( = k>) spring , (14) t value.
where k is the spring constant. The spring element A true viscoelastic material will exhibit a delayed elas-
stores elastic energy. The lagging component of a material tic response, stress relaxation and strain recovery (i.e., a
response to an applied load can be modelled with a dashpot gradual return to the original undeformed configuration on
(which dissipates energy), where the stress is proportional the removal of all applied loads). Neither the Kelvin-Voigt
to the strain rate >: nor the Maxwell model adequately represents all these char-
acteristics. However, a combination of the two models, giv-
( ( = ?>& ) dashpot, (15) ing the 3-element or standard linear models shown in Fig-
ure 40.7, will accommodate all the required characteristics.
where ? is the viscosity constant. The two simplest vis-
In order to capture the full range of time scales (i.e., a
coelastic models are the Kelvin-Voigt and Maxwell models,
continuous spectrum) that typically exist in a true viscoelas-
illustrated in Figure 40.6.
tic material, one would need to assemble an infinite num-
In the Kelvin-Voigt model, the strain is the same in
ber of these 3-element units in series or parallel. However,
both the spring and the dashpot, and the stresses in each
the major viscoelastic trends can usually be satisfactorily
element add to balance the applied load. Thus the equilib-
characterized with only a few such units. Langewouters et
rium equation is:
al29 found good correlation to human aortic creep curves
( = k > + ?>&
for their 5-element model (i.e., with only two time scales).
(16) van Dijk46 also characterized smooth muscle cell stress re-
laxation with two time constants. These models are param-
In the Maxwell model, the strains are additive, while
eterized by curve fitting to delayed elastic or stress relax-
the stress is constant throughout, thus:
ation experimental data.
1 1 For a multi-element Maxwell model (with N units),
>& = (
& + ( (17)
k ? the stress relaxation under a constant strain is:
N
((t ) = > 0 G(t ) where G ( t ) = G≅ + Α Gn e %t /4
(The Maxwell equilibrium is written in terms of strain
rate since the dashpot equilibrium is defined in terms of
strain rate). Solving these two differential equations yields: n=1
1. The retarded strain at time, t, for a constant stress (18)
applied at time, t = 0
where G≅ is the long term modulus—calculated from
> the hyperelastic strain energy function, and G? is the in-
stantaneous modulus of element n. For a continuous spec-
trum of relaxation times, the relaxation modulus can be
written:
> ( t ) = ( t = 0 1 % e % t /4
( ) ≅
k G( t ) = S (4 )e %t 4 d4
t for the Kelvin-Voigt
Β0
(19)
2. The relaxation stress at time, t, for a constant strain where S (4 ) is the relaxation spectrum. Experimen-
applied at time, t = 0 model where t = h/k;
tally, the determination of this S(4) is perhaps easier than
finding discrete time scales and moduli from creep test data.
The methodology for finding such a relaxation spectrum is
described in the section “Materials Testing”.
Fig. 40.6. Simple Kelvin-Voigt and
Maxwell vscoelastic models.
An Integral Mathematical Approach to Tissue Engineering of Vascular Grafts 449
Thus far we have only considered the time-varying fore, in this case it would be advantageous to include infor-
response to a constant load applied at some time in the past. mation about the microstructure in the description of the
Assuming linear viscoelastic behavior, we can use the prin- material behavior. The porous microstructure is then mod-
ciple of linear superposition to calculate the response to a eled as a continuum (solid) region that has the apparent (or
time-varying load. Casting the problem into an incremen- effective) properties of the porous material. This idea can
tal form, an increment in strain for the function, >(t), over a be taken one step further by including structural properties,
small time increment )t is written as such as porosity, as variables in the material formulation.
d> 4 Obviously the relation between the material behavior and
)> (4 ) = ) t at time, t = 4 . (20) the structural property needs to be determined. But once
dt this has been achieved, it becomes a simple matter to exam-
Thus from Equation (18) the stress response at time t ine the influence of changes in material microstructure on
(where t > 4) to this strain increment (held constant after its the structural response of the component without having
application) is to undertake complex geometrical modeling.
d> 4 In most porous materials the single most important
((t ))> (4) = G (t % 4 ) )t . (21) parameter in determining the mechanical properties is the
dt relative density ∋/∋s, where ∋ is the apparent density while
Now, the principle of linear superposition allows us ∋s is the density of the structural members. The porosity is
to sum the elastic responses to all the past increments in therefore defined as 1-∋/∋s. It turns out that cell size is gen-
load from time -≅ to t. By taking infinitesimal increments in erally far less important in the determination of material
time we can write Equation (21) in integral form as properties; however, cell shape will have a notable influence.
t
d> The distinction needs to be made between open and closed
(( t) = G ( t % 4) d4 .
Β %≅
Integrating by part yields
dt
(22) cells: In an open configuration, the cells are interconnected,
whereas in a closed cell arrangement each cell is isolated from
its neighbor. As interconnectivity is vital in a tissue-engi-
t
dG(t % 4) neered graft, only the mechanics of open celled structures
(( t) = G(0 ) > (t ) % > (4 )d4
Β -≅ dt
where G(0) is the so called glassy or instantaneous
(23) will be considered. Most porous structures are anisotropic
to a greater or lesser extent. Anisotropy arises from the shape
of the cells, and is characterized by the ratio of the largest to
modulus. the smallest cell dimension (R). Typically in a foam that is
Similarly, the delayed strain response to a smooth stress considered to be isotropic, R Χ 1.3; however, values in the
function of time is range from one to ten are not uncommon.
t
d J (t % 4) For most linear-elastic, isotropic, cellular structures
> (t) = J ( 0) ((t ) % (( 4) d4
Β %≅ d4
≅
(24) the apparent material modulus E can be related to the ap-
parent density by
J (t ) = T(4)(1 % e %t 4 )d4 ,
where
Β 0
and E E s ∆ (∋ ∋ s )
M
(25)
where Es is the elastic modulus of the solid material,
T(4) is the retardation spectrum.
and M is a constant which is generally greater than one. How-
Equations (23) and (24) are constitutive laws for a lin- ever, if the material structure is anisotropic, and therefore
ear viscoelastic material. They can be implemented in a nu- the stiffness cannot be represented by a scalar quantity, a
merical algorithm, such as the finite element method, to more complex description is required. Common porous
predict the history of any geometry with a continuum vis- materials have distinct directionality and can be regarded as
coelastic material behavior subjected to a time-varying stress either transversely isotropic or orthotropic. A transversely
or strain. isotropic material has one preferred direction and any trans-
verse plane will be isotropic. An orthotropic material will
Modeling Microstructure have three orthogonal planes of symmetry. For these mate-
It was noted earlier that the complete geometry of the rials the moduli in the preferred directions will be a function
structure must be fully characterized. However, in the case of the density as well as the anisotropy ratio. For most porous
of a porous material, it would neither be possible, nor desir- materials the elastic modulus is approximately proportional
able, to model precisely the structure of each pore. There- to R2 while the strength is typically proportional to R.47
Fig. 40.7. Standard linear visco-
elastic models.
450 Tissue Engineering of Prosthetic Vascular Grafts
Based on analytical and experimental studies,47 pa- amount of work, and therefore a strong dependence on strain
rameters such as the yield strength and the compressive col- rate exists. In the early postoperative stages of a tissue-engi-
lapse load of a porous material can also be determined as a neered graft, the graft material will contain fluid. In addi-
function of relative density and material structure. How- tion, the normal loading rate of approximately one cycle per
ever, these investigations are limited to the case where the second will likely be high enough to make the influence of
material comprising the structure of a foam is operating in loading rate noticeable. For these reasons the fluid phase of
the linear elastic regime. In a highly porous material sub- the porous structure needs to be given consideration. The
jected to large forces this would not be the case, and there- foam can be treated as a porous material with an absolute
fore other means of characterizing the material response are permeability K. The velocity of the fluid v within the porous
required. One approach for determining the apparent prop- material can then be determined from Darcy’s law
K dp
erties of a material is to develop numerical models of the
microstructure under investigation. If the behavior of the v= % , (26)
material from which the structure is made has been com- ∝ dx
pletely characterized, and if the material has a uniform struc- where ∝ is the dynamic viscosity of the fluid and dp/dx
ture, then a single structural unit can be tested in order to is the pressure gradient within the porous structure. The per-
obtain the apparent properties of the material. meability can be determined from the relative density and
An example of this approach is shown in Figure 40.8. the pore structure, while the pressure gradient can be re-
The polyurethane microstructure (Fig. 40.8a) has been ide- lated to the applied stress. After some mathematical manipu-
alized and modeled using the finite element method lation the strength of the porous material can be related to
(Fig. 40.8b). This model is then subjected to a range of nu- the viscosity of the fluid, the strain rate and some geometri-
merical tests, from which the apparent material behavior can cal information.
be determined. This approach has great flexibility, as it makes
possible the investigation of the influence of relative den- Material Testing
sity, cell shape and size. It also becomes a relatively simple Material testing forms the basis of the characteriza-
matter to distort the cells so as to create an anisotropic struc- tion of the mechanical properties of the native artery and
ture and then quantify the structural response. Once the the graft material. The material morphology and the extent
nature of the apparent material has been determined this to which the material needs to be characterized determine
information can be used to model a complete implant the complexity of the testing. For isotropic materials, simple
(Fig. 40.8c) without having to consider the geometry of the uniaxial extension tests may be sufficient to obtain the nec-
internal microstructure. essary information for characterizing the material. However,
The presence of a viscous fluid within a porous mate- for transversely isotropic and orthotropic materials, biaxial
rial will have a considerable influence on the strength. When testing is required.
the material is compressed the fluid will be squeezed out, Hayashi et al9 developed a highly elastic, blood com-
while it will be drawn in when the material is extended. As patible, small diameter vascular graft fabricated from seg-
the fluid has viscosity, work is done during material defor- mented polyurethane. Introducing porosity into the mate-
mation. The higher the loading rate the greater will be the rial, which also allowed for possible tissue ingrowth, created
Fig. 40.8. (a) Micrograph of a poly-
urethane microstructure and (b) and
idealized model of the micro-
structure which is then used (c) for
the determination of an equivalent
material for the properties of a po-
rous structure.
An Integral Mathematical Approach to Tissue Engineering of Vascular Grafts 451
a physiologically realistic compliance for the graft. Electron more, the time dependent behavior (viscoelasticity) of the
microscopy revealed the graft material to be approximately material will influence the sizing of the graft, and therefore
isotropic, and therefore the mechanical properties were de- needs to be considered in the design of the implant.
termined using simple uniaxial tests. Their strength tests
revealed the importance of pretreatment of the material prior Hyperelastic Properties
to mechanical testing. They found that the material became In order to develop the constitutive models for soft
stable, in terms of mechanical properties, after having been tissues and compliant engineering materials, the general
immersed in a saline solution for 30 minutes. form of the strain energy function must be postulated. This
They compared the stress/strain characteristics of their should be as simple and convenient as possible, as labora-
graft material to those of traditional grafts as well as the tory tests will be required to set the relevant parameters in
natural artery (Fig. 40.9). In order to present excessive dila- the strain energy function. These energy functions are based
tion, the graft was coated with a woolly polyester net. How- on qualitative observations of the material behavior and on
ever, such a net will reduce the compliance of the graft. The prior testing of similar materials. The functional form of
mechanical test results showed that the porous polyurethane the strain energy function is subject to much debate and
exhibited an almost linear stress/strain response (KP), with both polynomial and exponential forms are in current use.
a slight tendency to strain more at higher stresses. This re- The development of sound constitutive laws to de-
sult is in contrast to the highly non-linear behavior of the scribe the mechanical behavior of arterial vessels has been
calf ’s thoracic aorta (Ao) which shows a very strong decline the focus of numerous research efforts.17,48-50 Since these
in the amount of strain at elevated stresses. However, in the constitutive laws are set out in terms of large strain
low strain region (up to approximately 30%) the stiffness of hyperelasticity, experiments are required to determine both
the polyurethane graft is very similar to the thoracic aorta. the functional form and the coefficients defining the strain
This result further shows the considerable difference in stiff- energy function, W.
ness between the compliant polyurethane graft and the more Essentially three forms of strain energy functions are
stiff traditional Dacron grafts (CW, GT, CV and DB). used to describe arterial walls: polynomial, exponential and
Although the material developed for this graft is very logarithmic. The polynomial function of Vaishnav et al,51
compliant and exhibits strain of over 200% before failure, it which was used to describe a cylindrical vessel subject to
does not show the stiffening behavior normally associated internal pressure, is the simplest; in general it has seven co-
with polymeric materials. The slight softening at high strains efficients and is given as follows:
may be a result of the failure of the woolly coating. Further-
(27)
Fig. 40.9. Comparison of stress—
strain characteristics for a porous poly-
urethane graft (KP) with other tradi-
tional graft materials (CW—Cooley
low porosity woven Dacron, GT—
Gore-Tex thin walled EPTFE, CV—
Cooley double velour knitted Dacron)
as well as the thoracic aorta (Ao).
Adapted from Hayashi et al 1989.
452 Tissue Engineering of Prosthetic Vascular Grafts
where a, b, c, d, e, f, g are material constants, and E∗∗ Dynamic testing with sinusoidal loading over a large
and Ezz are the longitudinal strain components in the cir- range of frequencies will provide data for the determina-
cumferential and axial directions, respectively. tion of the continuous spectrum functions S(4) and T(4).
Fung et al17 proposed an exponential function of the The viscoelastic strain response to a sinusoidal stress,
form
( ( t ) = ( 0 sin Ε t , (30)
W = C exp a1 E∗∗
2
(+ a 2 E 2zz + a4 E∗∗ E zz ) (28)
at frequency Ε, and amplitude (0 is
where c, #1, #2 and #4 are the material constants.
Experimental data was determined from the cardiac, left iliac, >( t ) = > 0 sin(Ε t - Φ ) (31)
lower aorta and upper aortic arteries of rabbits, which was
used to determine the coefficients defining the strain energy where the peak strain >0 lags the peak stress by Φ—the
functions. The comparison of the experimental and theo- loss angle. These relations can be written in complex form
retical results, using Equation (10), are shown in Figure 40.2. as
Lastly, Takamizawa and Hayashi18 proposed a loga-
( ( t) = ( *0 e iΕ t and >(t ) = >*0e
i (Ε t % Φ )
rithmic function that again has four coefficients (32)
where ∋*o and >*0 are the complex amplitudes.
W = % C ln 1 % 12 a ∗∗ E∗∗
[ 2
% 21 a zz E zz2 % a ∗z E∗∗ E zz (29)
] The complex modulus, made up of a real (stiffness)
where C, aqq, azz and aqz are material constants. and complex (damping) part, is
The different layers and components making up the
( 0 iΦ
arterial wall result in an inhomogenous structure—that is GΓ = e = G1 + iG 2 (33)
to say, the properties of the material change through the >0
thickness of the wall.52 The models that have been previ- with an amplitude >0 lagging the stress. The loss tan-
ously discussed have not included the different layers when gent is the ratio of the complex to real parts of G*, viz.
G2
analyzing the mechanical response. To address this limita-
tion, von Maltzahn et al53 modeled the artery using two lay- tanΦ = . (34)
ers, each having different properties. The arterial wall is gen- G1
erally considered to be anisotropic; however, this does not Thus, measuring ∋o, >0, and Φ for a range of frequen-
necessarily mean that each layer within the wall is anisotro- cies and using Equations (33) and (34), we can plot G1 and
pic. Their model consisted of a transversely isotropic media G2 as functions of frequency. An appropriate continuous re-
and an anisotropic adventitia. This two layer model was com- laxation spectrum S(4) is chosen based on the form of these
pared to the single layer model of Vaishnav et al,51 and it curves. Fung22 describes this procedure in detail. See also
was found that it more accurately predicted the experimen- Ferry54 for a comprehensive treatise on this topic.
tal data. This was the case even when the fourth order terms Fung24 found that for a wide range of frequencies, the
were included into the polynomial function. The model also damping modulus is constant for biological soft tissues. This
had the advantage that the material parameters could be result allows for a very simple relaxation spectrum, S(4)=c/4.
related to specific mechanical properties within the artery. Lockett 55 reported the same result for polymethyl-
methacrylate (PMMA). However, he found a considerable
Viscoelastic Properties frequency dependency above 1 Hz for a lightly crosslinked
The appropriate choice of experiment for the deter- amorphous polymer.
mination of viscoelastic properties depends on a number of One other important consideration when undertaking
factors. If the modulus or compliance function is to be ap- materials testing is the fact that viscoelastic properties tend
proximated with only a few relaxation times, then either a to be highly temperature dependent. There are established,
stress relaxation or a retarded strain test would suffice. The proven laws which allow one to simply shift the compliance/
time constants and associated moduli or compliance coeffi- time curve for a particular material up or down the time
cients would be found by curve fitting to the assumed form line if the temperature is decreased or increased respectively.
of the rheological model. To characterize the full constitu- In the case of tissue engineered products this is not an issue
tive behavior, tensile, shear and volumetric (if the material for developing a constitutive model. However, it is vital that
is compressible) testing must be done. Such time-history tests all materials tests are done at normal core body temperature.
are problematic in practice because they require the appli- A detailed treatment on the issues involved in vis-
cation of sudden large stresses or strains without inducing coelastic materials testing is given by Lockett.55 Other use-
significant inertial effects. This means that the loading can- ful literature includes Christensen56 and Sternstein.57
not be applied too quickly and so very small relaxation time
scales cannot be measured with such tests. The determina- Finite Element Implementation
tion of very large time constants—longer than a few days—
is also impractical. The long term behavior of materials General Outline
which do creep (such as non-crosslinked polymers) can thus The finite element method is a numerical procedure
not realistically be fully characterized. frequently used for analyzing complex deformable bodies
An Integral Mathematical Approach to Tissue Engineering of Vascular Grafts 453
subjected to mechanical loading. The finite element proce- tor {u}represents these displacements. The finite element
dure essentially involves discretizing a component into a method seeks to minimize the potential energy by selecting
number of small (finite size) regions called the elements, the values of {u} which minimize
U({u}) = W (E ({u}))dV % {f } {u}
(which are defined by nodes—or nodal points), and approxi- T
mating the mechanical response in each element by assum-
ing that the local deformation can be represented by low-
order polynomials.
Β V
for all kinematically admissible {u}, where V is the vol-
(35)
For time independent linear problems, the potential ume of the body and {f} is the vector of nodal loads.
energy of the entire system can be minimized in one step
that produces a large number of algebraic simultaneous Element Behavior
equations in the nodal displacements, which are solved us- To calculate elastic strain energy in the body, the ma-
ing a computer. The results obtained using this method are terial domain is discretized into small regions called elements
not exact but with increased model refinement, and subse- (see, for example, Fig. 40.10). The geometry of each element
quently more computational effort, solutions to the approxi- is described by the location of a set of nodes (different ele-
mated displacement field of the desired resolution may ment types have different numbers of nodes) which also
be obtained. serve as inter-element connections.
For the non-linear and time-dependent problems pre- Within each element the displacement field is approxi-
sented in the design of artery grafts, the problem is broken mated by low-order piecewise continuous polynomials, spe-
down into a number of linearized increments. The method cially chosen such that their coefficients are the nodal dis-
therefore requires repeated solution of simultaneous equa- placement components for that element, {#e}, which is a sub-
tions to solve for increments in nodal displacements. How- set of the global displacements, {u}. In each element, the el-
ever, to illustrate the finite element method here, we limit ement strains, {E}, are obtained from the displacements of
the discussion to the linear, time independent problem. the nodes in that element {#e}, by writing Equation (3) as
The finite element method requires: the compatibility requirement
1. That the behavior of the component material be pre-
cisely defined; {E} = B({a e }) {a e }.
[ ] (36)
2. That the geometry of the component in the unloaded
state be known; and The Piola-Kirchhoff stress components in each ele-
3. That the boundary conditions, which include the ment are written as a vector {S}, so that the constitutive re-
mechanical loading, the supports or restraints hold- lation, Equation (10), which accounts for all of the interac-
ing the structure and, for time dependent problems, tions between the stress and strain components in a linear
the initial velocities, are fully characterized. elastic body, can now be written as
From this problem definition the finite element pro-
cedure will be able to analyze the structure and calculate the {S }= [D]{E} (37)
design parameters, such as the deformations and stresses over
the entire component domain. These results will allow deci- where [D] is the square matrix of coefficients
sions on the appropriate material choice and component representing the tensor Dijkl in Equation (12). Essentially,
geometry to be made. The flexibility and predictive power the matrix [D] describes the stresses required to produce
of the finite element method allows engineers the ability to the given strains in the material, and is determined by the
test various design parameters. In the case of a porous graft, strain energy function, W, using Equation (12). The strain
for example, the finite element method is an efficient way to energy W is determined by laboratory testing on the mate-
examine the effect on overall component compliance of rial of interest.
changes in the graft diameter, thickness, material stiffness, Materials such as soft tissue exhibit non-linear time
porosity, pre-stress, etc. dependent behavior and Equation (13) must be written in
an incremental form
Minimizing the Potential Energy
In the continuum description of hyperelastic mate- {dS }= [D({E}, {E& })]{dE} . (38)
rial behavior, it was most convenient to use tensor notation
to describe the field quantities (stresses, strains and displace- Therefore the components of [D] depend on the strain
ments) in a deformable body. However, since the finite ele- {E}and the strain rate {E}. Materials of this type can cause
ment method describes these fields with their values at dis- significant computational difficulties, but the general pro-
crete points (for example, at the nodes for the displacements) cedures are the same as in the linear case.
it is most effective to use matrix notation for the finite ele- For time-independent hyperelastic materials, the elas-
ment equations. tic strain energy in each element, We, is computed by inte-
Since the finite element method computes the dis- grating the hyperelastic strain energy potential W over the
placement field that minimizes the potential energy, the pri- volume of the element, Ve, viz.,
mary variables are the displacements of the nodes. The vec-
454 Tissue Engineering of Prosthetic Vascular Grafts
displacement field, can be used to compute the nodal dis-
We = W dV e
Β Ve
placements, {#e}, from which the strains, {E}, and the stresses,
{S}, can be computed within each element. Hence the dis-
T tribution of stress and strain can be found over the region
=
Β1
2 {S } {E}dV e of interest.
Ve
Examples of Artery Graft Modeling
T
T
{a} [B] [D][B]dV e {a}
1 (39) Once the mechanics of a material has been fully char-
= 2
Β V e
acterized, the geometry of the graft can be modeled using
the finite element method. To complete the modeling pro-
cess, the loading and boundary conditions need to be de-
where we have used both the material constitutive scribed in terms of the finite element modeling. In the case
of the natural artery, or a vascular graft, the loading magni-
equation (37) and the compatibility relationship (36) for
that element (note that the superscript ( )T denotes the trans- tudes are easily quantified from blood pressures. In addi-
pose of the vector or matrix). It is convenient to define the tion, the time dependence of the loading pulse can also be
determined and used as input to the numerical model.
element stiffness matrix [ke], by
In order to illustrate some aspects of the mechanical
T
[k ]= Β [B] [D][B]dV
e
Ve
e
(40)
design process, a few examples of artery graft modeling are
presented. Figure 40.10 shows the displaced shape of a simple
so that the elastic potential energy (or strain energy) finite element model of the anastomosis region of the natu-
for a single element is given by ral artery and a porous polyurethane graft. The artery
(shaded) is modeled as a single layer, using the simple
e T
W e ae({ })= {a } [k ]{a } 1 e e hyperelastic material model proposed by Fung et al,17 and
2 (41)
does not include any pre-stress. The graft is modeled as a
hyperelastic and isotropic material, using the experimental
Element Assembly data from the uniaxial tensile test data of the porous poly-
To compute the strain-energy for the entire structure, urethane. The graft is analyzed for a range of relative po-
WΗ({u}), the element strain energies, We({#e}), are summed. rosities. The different porosities were created by altering the
This is done by summing the element stiffness matrices, [ke], ratio of the salt to polymer concentration in the manufac-
for each element. Since each set of element displacements, turing process (values of 1:1, 3:1, 5:1 and 7:1 have been in-
{#e}, is a different subset of the global displacements, {u}, vestigated). Both the native artery and the graft are assumed
we must relate the local to the global displacements, which to have an internal diameter (D) of 6 mm and a wall thick-
can be done by defining a locator matrix for each element, ness of 1 mm. The model was loaded with an internal pres-
from which {#e}=[Pe]{u}. This allows us to compute a glo- sure, which is initially set to 70 mm Hg, and then increased
bal stiffness matrix, which represents the structural stiffness linearly to 120 mm Hg—thus )P is 50 mm Hg.
of the entire component. Mathematically, The resultant compliance of the artery and the graft,
as a function of the axial distance, is shown in Figure 40.11.
A simple measure of vascular compliance58
6 2 96 ) D 9
Cv = 8 ; 8 ;, (45)
7 D :7 ) P :
(42)
which relates the change in graft diameter )D to the
Problem Solution change in pressure, is used to compare the compliance of
To compute the total energy of the system, U({u}), we the native artery to the porous graft. This relation is chosen,
require the work done by the loads on the component, which as it does not contain any information about the properties
is merely the inner product of the loads at each node, {f}, of the two materials, which are non-linear and therefore
and the nodal displacements, {u}. Thus, the total energy of cannot be characterized with a single constant parameter.
the system The compliance of the native artery was predicted to be
0.273%/mm Hg (or 13.65% for a pulse pressure of
U ({u}) = 1
2 {u}T [K ]{u}% {u}T {f }. (43) 50 mm Hg), which was greater than any of the four grafts
analyzed. In the region of the anastomosis the compliance
Minimizing the total energy requires the solution of of the artery is affected by the presence of the graft and there-
the (usually large) system of simultaneous linear equations fore reduces towards the junction, which is halfway (5 mm)
along the length. The lowest porosity graft (1:1) results in
[K ]{u}% {f } = {0}, (44) the smallest compliance, which is 0.05%/mm Hg. As the
porosity increases to 7:1, the graft compliance increases to
for which modern computers are highly effective. The approximately 80% that of the host. It is interesting to see
displacements, {u}, which are an approximation to the true that with this more compliant graft material a hyper-com-
An Integral Mathematical Approach to Tissue Engineering of Vascular Grafts 455
pliant zone of about 2 mm, on the arterial side of the anas- The results of the proceeding example are based on a
tomosis, is predicted. In this region the compliance of the static analysis and do not show the influence of the time
artery is increased by approximately 0.4%. A similar zone of dependent behavior of the material. The viscoelasticity of
hyper-compliance was predicted by Chandan et al59 and the polyurethane will result in a gradual increase in the graft
observed in vivo by Hasson et al.60 However, in the in vivo diameter, as a result of strain accumulation during cyclic
study the extent of hyper-compliance was more dramatic, loading. The increase in diameter will reach a steady state
with a localized increase in compliance of up to 50%. after some time. Therefore the inclusion of viscoelasticity is
The graft compliance as a function of the porosity is important in determining the final size of the graft after
shown in Figure 40.12. The result shows that the percentage implantation.
compliance increases exponentially as the porosity increases. The flexibility of the modeling approach means that
However, increase in porosity will also be accompanied by a complex geometrical features, with a host of different
decrease in yield strength of the material. interacting materials, can be simulated. Figure 40.13 shows
Fig. 40.10. Simple axisymmetric fi-
nite element model of anastomosis
region of the native artery and the
polyurethane graft.
Fig. 40.11. Compliance
(%/mmHg) of the native artery
and the polyurethane graft for
various porosity’s of the graft
material shown as a function of the
axial distance.
456 Tissue Engineering of Prosthetic Vascular Grafts
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An Integral Mathematical Approach to Tissue Engineering of Vascular Grafts 457
Fig. 40.13. Finite element model of a possible graft
structure showing an underlying porous graft
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24:841-849. 59. Chanran KB, Gao D, Han G, Baraniewski H, Corson JD.
40. Myers BS, McElhaney JH, Doherty BJ. The viscoelastic Finite element analysis of arterial anastomoses with vein,
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Biomech Eng 1986; 108:189-192. 2:419-423.
Scaffold Engineering
Material Aspects
CHAPTER 41
Bioinertness: An Outdated Principle
David F. Williams
The Convention of Inertness
B iomaterials have been with us for the majority of the twentieth century. Their nature has
evolved during this time, and the applications for which they have been used have in-
creased in complexity and diversity. However, for much of this time, the functions required
of these materials and the performance parameters of the medical devices in which they
have been used have been relatively straightforward and largely confined to mechanical and
physical characteristics. The selection and design of biomaterials have therefore been simi-
larly constrained by the conventional engineering concepts underlying these applications.
During the last few years the situation has changed quite radically as new concepts and new
treatment modalities have required a fundamental reappraisal of the scientific principles
upon which biomaterials science are based. Nowhere is this seen more vividly than in the
use of biomaterials for implantable devices within the cardiovascular system, and specifi-
cally in relation to the application of tissue engineering concepts in reconstructive vascular
surgery. In this chapter we attempt to provide a rationale for this fundamental change and
to demonstrate the pivotal role of surface reactivity in the products and devices of the future.
If we consider the historical role of biomaterials in implantable devices we can iden-
tify several clinical conditions that underlie their use, including congenital and develop-
mental defects and trauma, but must recognize that it is in the treatment of diseases which
have caused irreversible changes in tissues and organs that biomaterials play their biggest
part. Thus, we see the widespread use of total joint replacements associated with the very
widespread incidence of osteoarthritis, of intraocular lenses necessitated by cataracts, of
prosthetic heart valves in the treatment of valvular disease, of dental implants following
tooth and periodontal destruction, and of vascular prostheses and devices used for the pros-
thetic reconstruction of arteries.
In all of these situations the functional requirements of the materials are simple and
essentially nonbiological. The major implantable devices of the twentieth century either
transmit loads, facilitate sliding motion, passively direct fluid flow, control fluid flow or
simply fill a space. The materials that permit the devices to perform these functions need to
have the appropriate mechanical properties and little else. Indeed, in the majority of these
cases, the mechanical and physical requirements are not themselves very onerous, and a
wide range of materials is theoretically available for the construction of these devices.
In reality, of course, the situation is not quite so straightforward, since the devices
usually have to perform their function for a long period of time within the confines of the
human body. This implies that the materials have to supply these mechanical or physical
characteristics without interference from the hostile physiological environment and without
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
460 Tissue Engineering of Prosthetic Vascular Grafts
causing any untoward or adverse effects in the patient. The There are three fundamental reasons why bioinertness
latter two aspects are subsumed within the phenomenon of may not necessarily be the sensible choice. The first is that
biocompatibility, and it has been a combination of the me- complete inertness is unachievable, so that any strategy
chanical and physical properties together with predicated on this quality is ultimately doomed to fail. Sec-
biocompatibility considerations that have determined the ondly, if a device is made from materials which are inert
selection of materials available for implantable medical de- and which do not interact with the body in any way, then it
vices at the present time. is unlikely that it can be truly incorporated into the body.
The term biocompatibility sounds simple to interpret For effective, long term performance in the dynamic tissue
since, as alluded to above, it implies compatibility or har- environment, it is usually preferable for there to be func-
mony with living systems. This concept, however, is too tional incorporation, which implies that the device should
simple to be useful and the meaning of compatibility has to be stimulating the tissues to react to it positively rather than
be explored further, both to understand the real logic of permitting them to ignore it. Thirdly, the emergence of tis-
today’s material selection and the opportunities for the fu- sue engineering principles, in which biomaterials are usu-
ture. It is intuitively obvious that a biomaterial or an im- ally seen to act in a supporting role to biological compo-
planted medical device should cause no harm to the recipi- nents, has placed a very considerable emphasis on the utili-
ent. This is the underlying principle of biological safety, and zation of intentionally degradable materials. These three fea-
it is this rather than the totality of biocompatibility which tures, which are determining the changing emphasis away
has shaped the historical evolution of biomaterials. Most from bioinertness, are explained below.
crucially, the requirements of biomaterials have been domi-
nated by the perceived necessity to be safe, which has usu- The Impossibility of Inertness
ally been interpreted as a requirement that a biomaterial The convention described above is based on the as-
should be totally inert in the physiological environment and sumption that a prosthetic device replaces, augments or
should itself exert no effect on that environment. In other modifies tissues or organs and is intended to remain within
words, there should be no interaction between biomaterials the patient for the length of time that the function is re-
and their host, implying that the material should be non- quired, typically for the natural lifetime of that patient. The
toxic, nonirritant, nonallergenic, noncarcinogenic, non- inertness was considered to be of crucial importance, both
thrombogenic and so on. to allow the device to retain its structural integrity and
This concept of biocompatibility which equates “bio- thereby maintain its performance, and to prevent or at least
logical performance” to inertness and biological indifference limit the release of any substance or component from the
has resulted in the selection of a portfolio of acceptable or material which would have some toxicological or other ad-
standard biomaterials that have widespread usage in appli- verse biological consequence. In the vast majority of circum-
cations such as those mentioned above. These range from stances, sufficient control has been exercised over the chemi-
the passive alloys such as stainless steel and titanium alloys, cal stability of biomaterials to ensure that no catastrophic
the noble metals of gold and platinum, some oxide ceram- loss of that integrity occurs as a result of compromised
ics such as alumina and zirconia, various forms of carbon bioinertness. There have been some exceptions and there
and a range of putatively stable polymeric materials includ- are no grounds for undue complacency in this respect, but
ing silicone elastomers, polyolefins, fluorocarbon polymers this is generally not the most important of considerations.
and some acrylics and polyesters. The precise material cho- On the other hand, the potential to initiate harmful biologi-
sen for an application from this type of list would depend cal processes in the host will always be finite as long as there
on the precise mechanical or physical property specifications. are structures capable of degradation or components free to
It cannot be denied that this approach has led to the be released.
successful deployment of many types of prostheses in wide- Several features of the inherent structure of materials
ranging clinical areas, and, apart from a few notable and and of the complex nature of the tissue environment aggra-
controversial situations, such implantable devices rarely vate this situation. In particular, inertness in the physiologi-
impact adversely on the health of the patient. The concept cal sense requires a great deal more than resisting degrada-
of bioinertness has therefore served a useful purpose. Even tion at the atomic or molecular level and, furthermore, even
when we look at these generally successful areas, however, it if it were that straightforward it would be extremely diffi-
can be seen that there are significant limitations to their per- cult to achieve. Indeed, it is now recognized that no material
formance and to the type of patient in which they can be is totally inert in the body. Even those very stable materials
used with confidence. Total joint replacements are generally mentioned earlier will interact to some extent with tissues.
contra-indicated in the younger, more active patient; the Titanium, although one of the most corrosion-resistant en-
performance of vascular prostheses decreases as the devices gineering alloys, corrodes in the body, judging by the pres-
are used more distally; bioprosthetic heart valves give greater ence of the metal in surrounding tissues. Gold and plati-
cause for concern in very young patients; and intraocular num will interact electrochemically with the saline-based
lenses may not give satisfactory results in patients with un- extracellular fluid. For reasons not entirely understood, the
derlying inflammatory conditions. Questions may be raised, most inert of the oxide ceramics suffer some long term
therefore, as to whether bioinertness is the most acceptable changes within the body, and almost all known polymers
principle underpinning material selection or whether alter- will undergo oxidation, hydrolysis or other changes upon
native concepts would be preferable. implantation.
Bioinertness: An Outdated Principle 461
With many materials, while the main component it- that there is no such thing as an inert biomaterial. It is never
self may be exceptionally inert, there are often minor com- a question of whether a biomaterial will interact with the
ponents, perhaps impurities or additives, which can be re- body but rather when and how. Under these circumstances,
leased under some circumstances. The leaching of plasticiz- the preferred alternative strategy is to accept that such in-
ers and other additives from plastics provide good examples teractions take place and to attempt to control these inter-
of interactions which are not necessarily related to the mo- actions proactively and to incorporate these interactions into
lecular breakdown of the material, but which nevertheless the design specifications.
confer a degree of instability to the product.
It should also be noted that descriptions of material The Requirement for Controlled Reactivity
degradation mechanisms have to take into account the spe- The above arguments indicate that complete inertness
cial and indeed, unique, features of the tissue environment. may not be possible, but still do not indicate that it is unde-
Whatever its location, a biomaterial will continuously en- sirable. The possibility that the products of any interaction
counter an aqueous environment during its use. This is not between a biomaterial and its physiological environment
simply a saline solution, however, but a complex solution could be released into the host has generally been consid-
containing a variety of anions and cations, a variety of large ered a sufficient deterrent to utilizing any material that was
molecules, some of which are very reactive chemically, and significantly reactive in that environment. This logic is clearly
a variety of cells. There are occasions when a degradation only valid if those products were going to initiate an unde-
process can be explained, mechanistically and qualitatively, sirable response from the host, either locally or systemically.
by the presence of electrolyte. This is the situation with most If interactions took place whereby the products were totally
metals when they suffer from corrosion in a physiological harmless, then there would be less cause for concern over
environment. Even here, however, it is known that the ki- the inability to achieve inertness. More importantly, if the
netics of corrosion may be influenced by the organic species nature of the interaction were one which produced a more
present, especially the proteins, and it is indeed possible for appropriate response from a tissue, then a positive virtue
the corrosion mechanism to be somewhat different from that could be made of this lack of inertness.
found in nonbiological situations. This quite different thinking is now enshrined in the
This phenomenon is even more pronounced with current concepts and definition of biocompatibility. Instead
other groups of materials and it is clear that with polymers of biocompatibility being equated with inertness, it is now
the kinetics and mechanism of degradation are fundamen- recognized that it should encompass a wide range of reac-
tally related to the precise details of the environment. Al- tivity, with the caveat that any reactivity is beneficial rather
though hydrolysis remains the substantive mechanism for than harmful. On the basis of these ideas biocompatibility
degradation of most heterochain polymers, including polya- was redefined a few years ago as “the ability to perform with
mides and polyesters, this hydrolysis may be profoundly in- an appropriate host response in a specific application”. It is
fluenced by the active species present in the tissue. In par- apparent that this definition still encompasses the situation
ticular, the lysosomal enzymes synthesized and released from where inertness is required, since the most appropriate re-
cells of the inflammatory response to biomaterials may in- sponse in some situations is indeed no response. A tradi-
fluence the degradation process. Moreover, the hydrolysis tional bone fracture plate is most effective when it is attached
may be supplemented by oxidative degradation, again oc- mechanically to the bone and does not corrode. No response
curring not only by virtue of passively dissolved oxygen in of the tissue to the material is required under these
body fluids, but by active oxidative species such as superox- circumstances.
ides, peroxides and free radicals generated by activated in- In the type of device mentioned earlier in this chap-
flammatory cells such as macrophages. It is thus possible ter, in which the performance is dependent upon its physi-
for homochain polymers not particularly susceptible to hy- cal replacement of diseased tissues and its incorporation into
drolysis and not normally oxidized at room temperature to the structure of the body, inertness of all of the components
undergo oxidative degradation upon implantation. may prevent optimal performance from being achieved. In
Polyolefins such as polyethylene and polypropylene come particular, if a material is inert and unreactive within tis-
into this category. sues, the long term host response will be associated with a
On the basis of a vast amount of experimental work lack of recognition and a lack of functional incorporation.
and clinical experience, it is now clear that all biomaterials An inert polymer such as polyethelyne or PTFE will induce
are inherently susceptible to some degradation process the formation around it of a thin layer of collagenous fi-
within the physiological environment. It is equally clear that brous tissue which can neither facilitate incorporation of
although a variety of surface treatment methodologies are the device into the tissue nor assist the device in achieving
available to reduce or ameliorate the degradation, none of any of its functions. Moreover, this fibrous layer is unlikely
these are entirely effective and their availability does not to be stable and may alter its characteristics over time, this
negate the now accepted principle that complete inertness often being the ultimate cause of the device failure. In such
cannot be achieved. Moreover, in the context of interactions circumstances, the biocompatibility characteristics of the
which affect the overall performance of the material in the materials in contact with their host tissues should be those
physiological environment, it is important to note that an which favor a positive interaction between the molecules of
interfacial reaction involving a physicochemical process such the material and the relevant molecules of that tissue, such
as protein adsorption will inevitably take place, further em- that there is a functional attachment between the two. A to-
phasizing the fact that inertness is a very relative term and tal joint replacement which has the nonarticulating surfaces
462 Tissue Engineering of Prosthetic Vascular Grafts
composed of materials or substances that are able to inter- with the delivery of active tissue repair promoting molecules
act with the cellular components that promote bone regen- and with the physical support necessary for the developing
eration at the interface should yield a more appropriate host tissue structure. It goes without saying of course that such a
response in the context of the long term mechanical stabil- material should not degrade to produce harmful side effects,
ity of the joint. Similar arguments could be put forward for which could be associated with either the chemical charac-
many of today’s implantable devices that are used in recon- teristics of any degradation products or their physical mor-
structive surgery. phology. Although this is not a trivial matter, such that in-
This concept may be extended to many other types of flammatory reactions associated with the degradation can-
implantable device, including those which function through not be ruled out, there are several biodegradable polymers
an intervention in healing processes. For example, whilst it which may now be used with reasonable confidence in these
was noted before that conventional fracture plates need to situations.
be inert, on the basis that degradation or corrosion prod- Tissue engineering products should, however, involve
ucts could be harmful to the local tissue, it is not impossible more than a degradable structural polymer. The real essence
for such devices to be made with surfaces that interact with of such a product is the biological activity which it imparts
osteoblasts, thus actively promoting or accelerating the heal- to the host site. A simple, empty, porous scaffold of a de-
ing process. Similarly, while many intravascular stents are gradable polymer implanted within connective tissue may
fabricated from alloys chosen on the basis of their corrosion become infiltrated with repair tissue and may ultimately
resistance and general inertness, the application of substances degrade to leave an area of reconstituted tissue derived from
to the surfaces of these alloys which potentially have an ability that infiltration. This, however, is not tissue engineering,
to interact at the molecular level with those agents that are since the tissue will not have been directed or controlled in
responsible for restenosis provides a new concept of terms of its structure and nature. In order to be really use-
bioreactivity rather than bioinertness for these devices. ful, this product would have to incorporate the appropriate
Almost every type of implantable device which has cellular components, normally previously derived from the
hitherto been associated with materials chosen for their ap- hosts, or certain molecules that are able to signal to the host
parent inertness may now be reconsidered in this light. This cells the appropriate instructions to produce the desired tis-
applies equally to devices where active surface molecules can sue morphology and function. Under these circumstances,
control thrombogenicity, to devices which are able to en- the biodegradable polymer has to be chosen very carefully,
courage tissue repair and to devices which are able to mini- such that at the very least it will be compatible with the de-
mize the undesirable consequences of inflammation. sired cell function in its vicinity or, preferably, such that it is
capable of delivering these cell signaling molecules with the
The Essential Requirement of Reactivity desired activity and in the desired manner. Inherent inert-
in Tissue Engineering ness is therefore unacceptable in these tissue-engineered
The desirability of some degree of reactivity in con- products. The challenge is to introduce the desired level of
ventional implantable devices can be seen as a precursor to reactivity without compromising the biological safety of the
the essentiality of reactivity associated with the concepts and material.
products of tissue engineering. It is important to remember
here that the underlying principle of tissue engineering in- Conclusions
volves the combination of biological principles and sub- For all the reasons outlined above, bioinertness can
stances with medical engineering principles and devices in be considered as an outdated principle. Although there are
order to provide products that are able to persuade the body some types of implantable device which are still better served
to heal itself. In this rapidly emerging field, the major prod- by inert materials, the majority should preferably incorpo-
ucts utilize some material structure as a support, matrix or rate intentionally interactive materials, whilst others, espe-
vehicle for the delivery of active biological molecules or cel- cially in this new area of tissue engineering, positively de-
lular species to a target site in the host where repair or re- mand this characteristic.
construction is to be effected. Important as these material It has to be said, however, that academic arguments in
structures are, they do not normally constitute a compo- favor of replacing a concept that is perceived to be out of
nent that is intended to remain in the body for very long, date with a new principle cannot be sustained unless this
and certainly their function is incompatible with the char- new principle can be verified and reduced to practice. In
acteristic of total inertness. view of the fact that bioinertness has never produced any
There are several different levels at which reactivity is harm, it is indeed a brave new world which is predicated on
desirable or essential. As alluded to above, the most obvious the availability of biomaterials that are both functionally
of these is the desirability for intentional biodegradation in interactive with their host and intrinsically safe. Many might
the polymeric support structures that form the basis of tis- argue that these in fact are contradictory requirements.
sue-engineered reconstruction devices. Whether this is con- Clearly that need not be the case, but the whole future of
cerned with the repair of cartilage defects, of nerve tissue, of tissue engineering products that are able to demonstrate ef-
damaged skin or diseased tissue within the vascular system, ficacy and safety is dependent on the development of the
the template of a device usually involves a polymer that can appropriate materials. However outdated bioinertness may
degrade over a period of time ranging from a few weeks to a be, it should not be replaced by materials based upon hope
few years and where the degradation profile is consistent rather than scientific reality.
Scaffold Engineering
Material Aspects
CHAPTER 42
Bioinert Biomaterials: Are Their Properties Irreplaceable?
Patrick T. Cahalan
n 1955 Sewell and colleagues1 performed a study comparing ovine and bovine sources of
I catgut sutures in three animal models. The objective of the study was to quantitatively
compare the tissue response to the implanted material, and this event is often cited as the
beginning of biocompatibility testing. Coincident to this was the rapidly expanding postwar
technology in high performance plastics, with mounting evidence that biological grafts, even
homografts, were not going to survive the challenges in vascular graft applications.2 Voorhees’
observations in 1952 of a neointima free of thrombi on a silk thread, and his later experi-
ments with Vinyon “N” cloth in dog aortas, clearly showed that a synthetic material could
serve as a conduit, and be accepted by the tissue.3 Studies would follow showing the merits
of other materials, particularly Teflon and Dacron.4 The paradigm was set, and materials
were classified as very reactive to inert.
What followed was once described as “20 years of frustration”.5 During this period
commercial efforts were driven by the promise of a very large market, estimated between 0.6
to 1 billion dollars annually. Scientific efforts were focused on comparing properties of ma-
terials with limited physiological markers, primarily the occurrence of thrombi on the ma-
terial surface. An example of surface property concepts that were hypothesized to be benefi-
cial are seen in Figure 42.1.6 The majority of the surfaces listed are in essence attempts to
create surfaces with minimal effect, whether it be termed low protein or platelet adhesion.
In the same 1972 article, Baier refers to much of the individual surface property character-
ization towards blood compatibility as people working in a maze within a maze.
Hydrophilic surfaces, for example, while showing low platelet adhesion and therefore
thought to be blood compatible, when tried in a vascular graft position did not prove to
provide patency. Thus, researchers thinking they had found a way out of the blood compat-
ibility maze found themselves in another maze with new barriers to solve. Baier’s represen-
tation of the surface properties compared to the living blood vessel intima serves to high-
light the focus of blood compatibility to the vascular application. This narrow focus, plus
the lack of clearer understanding of cellular and molecular biology, helped continue the
quest for the “holy grail” of inert materials.
Not until it was shown in the late 1970s that endothelial cells were more active in
maintaining blood compatibility7 did the paradigm begin to change. Until this time it was
even possible to hypothesize that endothelial cells were the perfect inert surface. The emer-
gence of RIA and ELISA in the mid-70s opened up the possibilities of looking at the mo-
lecular and cellular aspects involved at the surface. The common use of these tools did not
make its way into blood compatibility research until well into the 1980s, and today a signifi-
cant number of papers are still offered that rely largely on platelet adhesion alone to deter-
mine blood compatibility of surfaces. The increase in understanding of reactions between
coagulation factors and formed elements of the blood has led to a change in thinking that
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
464 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 42.1. General approaches to improve blood compatibility: early years (70s).
previously focused on the bulk reactions in blood to the re- explanation for this failure may be the nondiscriminatory
alization that these critical reactions “occur almost exclu- nature of some of the cell adhesion receptors to adhesion
sively on surfaces”.8 The formerly thought to be inert endot- proteins. Some new approaches to deal with the nondiscrimi-
helial cell has been shown to have as many procoagulant natory cell receptor binding to adhesion proteins attempt
properties as anticoagulant.9 The pat answer for years to the to attach peptide adhesion ligands to surfaces.12 A critical
question, “What is the perfect surface?” was: “An requirement stated by Hubbell in such attachment is to ren-
endothelialized surface.” The fact that an endothelialized der the surfaces resistant to protein adsorption by first graft-
surface can be thrombogenic opens up a new maze. It also ing with a hydrophilic nonionic polyethylene oxide (PEO)
begs the question, “Bioinert biomaterials: Are their proper- chain to which the peptide ligand could be tethered.
ties irreplaceable?” The latter stated requirement could serve to finally
“The long sought after biologically inert material has propose a more pragmatic application of the “inert” con-
not as yet been developed, and the realization of the inher- cept. Inertness with respect to a nonionic hydrophilic spacer
ent ‘biointeractivity’ of all implanted polymers has led many that is resistant to protein adhesion permits a selectivity or
investigators to pursue strategies aimed at optimizing the control of molecular and cellular events at surfaces. The chal-
biological interactions with the synthetic polymer rather lenges left with this approach are to achieve the proper den-
than to minimize all biological interaction.”10 This statement sity of ligands on the surface, their orientation, and the abil-
would seem to put an end to the question, “Is there a need ity to accomplish this on synthetic substrates in the geomet-
for bioinert materials?” Unfortunately, vascular grafts and ric configuration of a biomedical device such as a vascular
other biomedical devices still use synthetic materials of con- graft, artificial heart, heart valve, or ventricular assist de-
struction, and their replacement with other biological ma- vice. While this elegant concept represents a major advance
terials or repair therapies is not on the very near horizon. in the effort to make an endothelialized and hopefully long
The great promise of biodegradable materials has not been term blood-compatible surface on a vascular graft, it is not
realized, and attempts at endothelialization of synthetic certain whether another maze will not open up, such as con-
materials, while showing promise, have not consistently ex- tinued hyperplasia. Due to noncompliance of synthetic
ceeded the performance of autologous tissue. Preadsorption structures, some might argue that a truly new totally func-
of synthetic vascular grafts with cellular adhesive proteins tioning vascular tissue might not be possible, and that even
tends to enhance the short term attachment of endothelial though there is an endothelial layer on the lumen, the struc-
cells, but platelet adhesion, and therefore increased ture will succumb to other mechanisms of failure. More
thrombogenicity, typically increases with time.11 A possible optimistically, it could be hoped that compliant structures
Bioinert Biomaterials: Are Their Properties Irreplaceable? 465
could be engineered that also could be modified to promote as optimal pretreatments followed by confirming XPS and
spatially correct cellularization of smooth muscle cells, and TofSIMS data is required to assure optimal presentation of
even include neovascularization. The progress made by per- PC moieties on the surface before testing. There is support-
sistent researchers10,12 in the vascular graft field suggests that ing evidence for the inertness of a pure PC surface.19 Stud-
the latter possibility is not out of the question. ies performed at the University of Maastricht have demon-
As mentioned earlier, the vascular graft focus may have strated that a phospholipid bilayer of pure dioleoyl-phospha-
actually been in part responsible for a lack of more rapid tidylcholine (DOPC) on an ellipsometry plate is indeed an
progress in understanding the cellular and molecular events inert surface, and assembly of the prothrombinase complex
at the surface, particularly in development of more blood- requires the addition of dioleoyl-phosphatidylserine (DOPS)
compatible surfaces. Other application areas such as dialy- in significant amounts.28 DOPS is present on the internal
sis and cardiopulmonary bypass (CPB) have created a strong surface of the platelet membrane, and platelet activation
research effort in providing surfaces with enhanced blood causes a flip-flop of the DOPS to the external surface of the
compatibility, and in this effort industry, academia, and cli- membrane. When DOPS is on the surface, the
nicians have invested large amounts of time and capital in prothrombinase complex can be assembled and thrombin
investigating improved biocompatible surfaces on devices generation can take place. Unfortunately, the stability of a
not necessarily designed for permanent implant. From a DOPC bilayer is very poor, and at this time has not been
strictly clinical perspective, the ability to monitor coagula- attained with consistency on a polymeric surface. The MPC
tion and inflammatory responses to foreign surfaces during polymer approach would be to assure uniformity and den-
CPB, and evaluate devices after use, has created a plethora sity of the PC moiety on the surface, and thus impart through
of scientific publications involving measurement of molecu- mimicry an inertness to the surface.
lar and cellular events on blood-contacting surfaces. This While the DOPC surface appears to be the closest thing
has also created a commercial stimulus for funding research to an inert surface, the actual surface of the plasma mem-
on blood materials interactions and research in surface brane is in fact made of more than just DOPC. Transmem-
modification of biomedical devices. brane proteins assure that platelets in the end are very reac-
In our laboratory, surfaces deemed “inert” have been tive with their environment. It is perhaps appropriate to re-
studied using multiple ELISA markers such as TAT, F1.2, flect back to the comments of Spaet8 with respect to all im-
elastase, and SC5b-9 (terminal complement complex) in portant reactions in the body taking place on surfaces, and
addition to measuring platelet adhesion and activation. The that these surfaces we have learned to be anything but inert.
most often cited inert molecule is poly(ethylene glycol) or In the end, one of the roles or properties of inert
(PEG). It has been suggested by some that a PEG surface has biomaterials that is needed is their ability to enhance the
little or no protein adsorption or interaction.13,14 This expression of another molecule, such as a peptide as sug-
premise has also been challenged by some researchers.15,16 gested by Hubbell. This may be accomplished by minimiz-
It has further been suggested that PEG surfaces may serve to ing competing phenomena such as protein adsorption, as
delay rather than reduce protein adsorption and activation.17 well as presenting a molecule in a conformation more ac-
With respect to blood compatibility, we found that while a cessible to its target receptor. This property is indeed possi-
PEG surface does in fact show lower protein adsorption and bly irreplaceable. Our studies specific to the attachment of
platelet adhesion, it does not show any improvement in TAT, heparin to surfaces clearly indicates that the bioactivity of
complement (SC5b-9), or elastase. These studies used a an attached molecule is dependent on its ability to interact
blood loop system that contained a valve to effect physiologi- with target molecules, and the ability to control the loading
cal flow, and human blood containing 1 U/ml of heparin. and the reactivity of the attached molecules is critical to
While PEG surfaces have been studied and widely reported achieve optimal functioning on the surface.20 Heparin on
to approach inertness, it is obvious that there is yet much to these surfaces was tethered to a nonionic hydrogel that was
be learned before this technology is optimized. Neverthe- derivatized to achieve different loadings of heparin. Here
less, the suggested use of this molecule to tether a peptide, the surface is certainly not inert, but it is designed to opti-
or possibly a specific adhesion molecule, may in the end mally compete with the undesirable physiological mecha-
prove to be the best application of PEG. nisms of coagulation and platelet adhesion. These surfaces
Another molecule that is receiving considerable atten- do not prevent protein adhesion, but the proteins that have
tion for its low protein and platelet adhesion is phosphatidyl- adhered show different relative concentrations to control
choline (PC). Methacrylate copolymers with pendant PC surfaces, and the performance of the surfaces correlates with
groups (MPC) have been coated with a facile process to poly- these adhesion profiles.21 In nature, a denuded endothelial
mers, and the PC moiety is expressed to a high degree on lining exposes a procoagulant surface, yet this surface can
the uppermost surface.18 Conceptually, the MPC coating is rapidly passivate without excessive thrombin formation. Fi-
said to mimic the natural phosphatidylcholine present on brinolysis, controlled inflammation and, finally, remodel-
the surface of nonactivated platelets. In testing in our labo- ing give a normal healing response that restores the tissue to
ratory, this surface does show reduced platelet adhesion and a functional state, barring the introduction of complications
less TAT formation. Additional testing needs to be completed of a biomaterial, ongoing disease state or flow restrictions.
to evaluate the complement and white cell response to this A normal healing response replaces inertness with spatially
surface. In our hands this polymer has shown material-de- correct signals via the subendothelial extracellular matrix
pendent coating characteristics, and evaluation is tedious, and thus controls the first step of repair, which is platelet
466 Tissue Engineering of Prosthetic Vascular Grafts
adhesion and coagulation. A naturally formed and controlled of these materials can easily be controlled by coupling the
hemostatic response (clot) presumably is resolved without release of anti-inflammatory drugs; this may possibly open
the excessive inflammatory reactions associated with the up yet another maze.
presence of foreign materials. If those foreign materials pos- In summary, the 20 years of frustration27 to which
sess an inert surface, the resultant histology should resemble Andrade referred have been replaced by numerous
that of a injury without the presence of a foreign material. multidisciplined efforts, aided by a much better understand-
Since injury exposes the blood to nonendothelialized sur- ing of molecular and cellular events at surfaces. The knowl-
faces, and these surfaces do bind plasma proteins, it might edge gained has opened up many approaches to finding the
be a better approach to attempt to modify a surface to have holy grail, and also created a number of positive spin-offs in
a similar protein adsorption profile. Implants of biomate- therapies apart from vascular grafts. The concept of bioinert
rial surfaces modified with collagen (data to be published) materials still persists, not as an end in itself, but rather as a
after 10 weeks show minimal capsule formation with mi- tool to enhance positive mechanisms or to retard undesir-
crovascular structures within two fibroblast layers of the bio- able competing mechanisms. In the quest for new
material surface. The control biomaterial surface (polyure- biomaterials, and for tissue engineering to replace diseased
thane) has macrophage and foreign body giant cells (two tissue with new functioning tissue, we will surely progress
layers) followed by 12-15 layers of densely packed fibroblasts in an iterative manner. Mundane issues such as sterilization
adjacent to the material surface (a typical fibrotic response). may render new concepts nonfunctional, but may stimulate
The attachment of the collagen to the above mentioned sur- new sterilization technology in order to bring them to mar-
face was not simple adsorption, but rather a covalent at- ket. Mechanical property limitations may require creative
tachment with isolation of the collagen layer to the upper- processing or fabrication advances. Promising surface modi-
most layer (70 Å).22 XPS data showed a pure surface of col- fications may first produce composite hybrid structures with
lagen, and this was attainable based on the inherent proper- traditional materials that can later be replaced by resorbable
ties of the grafted surface beneath. This should be a target materials. What in the end is probably irreplaceable is the
for modification of surfaces whether inert or interactive, and learning process involved in trying to find inert biomaterials.
that being complete and uniform coverage without influ-
ence from intermediate chemistries used to couple the mol- References
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In addition to the property of inertness being used to paring suture of ovine catgut with sutures of bovine cat-
enhance the presentation of molecular species to the physi- gut in three species. Surg Gynecol Obstet 1955; 100:483.
ological environment, there is another possibly irreplace- 2. Szilagyi DD, McDonald RT, Smith RF et al. Biological fate
of human arterial homografts. Arch Surg 1957; 75:506.
able property of inertness. That is the relative chemical in-
3. Voorhees AB, Jaretski A, Blakemore AH. The use of tubes
ertness of biomaterials. In addition to finding the optimal constructed from Vinyon “N” cloth in bridging arterial
protein adhesion mosaic on a surface, the biomaterial of defects. Ann Surg 1952; 135:332.
construction of a device must withstand the degradative 4. Deterling RA, Bhonslay SB. An evaluation of synthetic
environment of the body. Numerous attempts have been materials and fabrics suitable for blood vessel replacement.
made, for example, to fabricate vascular prostheses using Surgery 1955; 38:71.
polyurethane, when several instances of urethane degrada- 5. Andrade JD, Nagaoka S, Cooper S, Okano T, Kim SW.
tion in vivo have been documented.23,24 Attempts to mimic Surfaces and blood compatibility: Current hypotheses. Vol
the in vivo response in an in vitro system have shown that XXXIII Trans Am Soc Artif Organs 1987.
adsorption of the plasma protein #2-macroglobulin could 6. Baier RE. The role of surface energy in thrombogenesis,
Bulletin of The NY Academy of Medicine 1972;
play a role in biodegradation of the urethane.25 It might be
48(2):257-272.
hypothesized that a surface modification that could isolate 7. Weskler BB, Marcus AJ, Jaffe EA. Synthesis of prostag-
the biomaterial surface from protein and cell attachment landin 12 (prostacyclin) by cultured human and bovine
could serve to protect its stability. Those who would choose endothelial cells. Proc Natl Acad Sci USA 1977; 74:
to continue the use of polyurethanes for vascular grafts will S.3922-3926.
need to deal with the potential degradation mechanisms 8. Spaet TH. Blood in contact with artificial surfaces: Where
demonstrated. An alternative approach to elastomeric ma- have we been and where are we going. Annals of The New
terials may be fabricated grafts using very stable materials York Academy of Sciences, 1987; 516.
such as PTFE and PET that have mechanical properties cre- 9. Gimbrone Jr MA. Vascular endothelium: Nature’s blood
ated by the bulk design of the device. container. Vascular Endothelium in Hemostasis and
Biodegradable materials have boasted the promise of Thrombosis. Gimbrone Jr MA, Ed. Edinburgh: Churchill
Livingstone, 1986; 1-13.
ultimate inertness in that they are not left behind for reac-
10. Greisler HP, Gosselin C, Ren D, Kang SS, Kim DU.
tion with the body. While very impressive advances are be- Biointeractive polymers and tissue engineered blood ves-
ing made in resorbable polymers,26 their application at this sels. Biomaterials 1996; 17:329-336.
time may still be limited by the need for mechanical proper- 11. Seeger JM, Klingman N. Improved endothelial cell seed-
ties that are best met with traditional biomaterials. An addi- ing with cultured cells in fribronection-coated grafts. J
tional issue with resorbable materials is the control of the Surg Res 1985; 38:641-647.
rate and type of resorption, ablative or eroding. It is also 12. Massia SP, Hubbell JA. Tissue engineering in the vascu-
somewhat taken for granted that the inflammatory responses lar graft. Cytotechnology 1992; 10:189-204.
Bioinert Biomaterials: Are Their Properties Irreplaceable? 467
13. Merrill EW, Salzman EW. Polyethylene oxide as a bio- with enhanced infection resistance. University of
material. Am Soc Artif Intern Organs 1993; 6:60. Eindhoven, The Netherlands. 1996:127-148.
14. Harris JM. Poly(ethylene glycol) Chemistry: Biotechnical 23. Stokes K. Environmental stress cracking in implanted
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1992:15. Engineering. Plank H, Egbeers G, Syre I, eds. Amsterdam:
15. Llanos G, Sefton MV. J Biomater Sci Polymer Edn, 1993; Elsevier, 1984:243.
4:381-400. 24. Takahara A, Hergenrother RW, Coury AJ, Cooper SL.
16. Sheth SR, Leckband D. Measurements of attractive forces Effect of soft segment chemistry on the biostability of seg-
between proteins and end-grafted poly(ethylene glycol) mented polyurethanes. II. In vitro hydrolytic degradation
chains. and lipid adsorption. J Biomed Mater Res 1992;
17. Kulik E et al. Poly(ethylene glycurface: Reduced vs de- 26:801-818.
layed protein adsorption and activation. Abstract from 25. Schubert MA, Wiggins MJ, Schaefer MP, Hiltner A,
Firth World Biomaterials Congress, Toronto, Canada. Anderson J. Oxidative biodegradation mechanisms of bi-
1996; 29 May-2 June. axially strained poly(etherurethane urea) elastomers. J
18. Ishihar K, Nakabayashi N. Part A: Polymer Chemistry: Biomed Mater Res 1995; 29:337-347.
Specific interaction between water-soluble phospholipid 26. Sawhney AS, Pathak CP, Hubbell JA. Bioerodible
polymer and liposome. J Poly Sci 1991; 29:831-835. hydrogels based on photopolymerized poly(thylene gly-
19. Andree HAM. Phospholipid binding and anticoagulant col)-co-poly(#-hydroxy acid) diacrylate macromers. Mac-
action of annexin V. Ph.D. thesis at the University of romolecules 1993; 26:581-587.
Maastricht, The Netherlands 1992. 27. Andrade JD, Coleman DL, Didisheim P, Hanson SR,
20. Lindhout T et al. Antithrombin activity of surface-bound Mason R, Merrill E. Blood-materials interactions: 20 years
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21. Sapatnekar et al. Blood-biomaterial interactin in a flow 28. Smeets E. Scrambling of Membrane Phospholipids in
system in the presence of bacteria: Effect of protein ad- Platelets and Erythrocytes. Ph.D. Thesis, University of
sorption. J Biomed Mater Res, 1995; 29:247-256. Maastricht, The Netherlands, 1996.
22. HendriksM. A study on the covalent surface-immobiliza-
tion of collagen. Ph.D. thesis, Development of biomaterials
Scaffold Engineering
Material Aspects
CHAPTER 43
Biostable Polymers as Durable Scaffolds
for Tissue Engineered Vascular Prostheses
Arthur J. Coury
Introduction
T he successful implementation of any medical device requires a systematic development
process from concept through use in humans. Rigorous quality systems and design con-
trols are now mandated by law,1 and the framework they provide2 is especially relevant to
the development of systems as complex as tissue engineered vascular prostheses.
The selection of polymeric materials to serve as structural scaffolds for the vascular
prostheses should only be made in the context of the design intent of the device. In the spirit
of design control, this chapter begins with a concept statement—the first stage of the design
process. The intent is not to completely design the device but to provide a minimum set of
characteristics which will set rather loose boundaries, to allow a range of prosthetic materi-
als to be considered in this chapter.
Design Concept
The product is a vascular prosthesis intended to address the unmet medical need for
functional replacement of small (, 6 mm) and medium (6-12 mm) diameter arterial seg-
ments.3-4 The device consists of a structural matrix (scaffold) of available synthetic
biomaterials and possibly other components assembled prior to implantation. The scaffold
promotes and retains attachment and/or ingrowth of biological tissues. The synthetic scaf-
fold is biostable, that is, resistant to excessive structural deterioration for the projected life-
time of the device, which is considered to be a “permanent” implant. The synthetic compo-
nents of the device elicit an acceptable host response alone or through pharmacologic inter-
vention. The device can be produced by available techniques into configurations which pro-
vide adequate hemodynamic flow. It may be packaged, sterilized, stored and implanted by
accepted processes. It is amenable to any special techniques (e.g., in vitro cell seeding or
surface modification) which are required for its proper function.
Material Requirements and Limitations
Given the design intent of the device, specific material requirements for the structural
matrix are discussed as subtopics of this section. It must be stated at the outset that no
material is optimal in all of the requirements. The final selection of a material will involve
compromises from the ideal, but demonstration of acceptable characteristics by appropri-
ate testing is required.
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
470 Tissue Engineering of Prosthetic Vascular Grafts
Availability by Purchase or Synthesis Modes of Polymer Degradation
Particular attention should be paid to long term avail- The requirement of biostability does not imply that
ability of the polymer. In recent years, major suppliers of the biomaterial is inert in the medium under consideration.
polymers have withdrawn their products from consideration It must be appreciated that a polymer, and all materials, re-
for implantable devices, 5-6 chiefly for liability reasons. In spond continuously to changes in environment before and
the United States, several legislative initiatives have led to during the implantation period.8 All polymers are permeable
the passage of the Biomaterials Access Assurance Act on July to gases and liquids. They absorb, transmit and desorb com-
30, 1998 (Public Law 105-230) which offers protection to ponents. Their surface and bulk properties change with time
biomaterials suppliers. The effect of this legislation will likely and the imposition of external forces, sometimes reversibly,
take several years to be fully realized. The supply problem is other times irreversibly. The changes can be favorable or
especially serious for stable biomaterials which often require unfavorable.8 Degradation leading to compromised perfor-
capital-intensive processes available only to large chemical mance of a scaffold in vivo can be physical, chemical, or a
companies—the sources of highest liability exposure. This combination of both. Both types of degradation can also
problem has led device manufacturers to search internation- necessitate rejection of a component or device prior to use.9
ally for biomaterial equivalents or to work with smaller com- Physical changes may include swelling, plasticization,
panies with limited manufacturing capability, but lower po- crystallization, decrystallization, fatigue fracture, creep,
tential liability consequences. simple stress cracking and kinking, among others.10 Chemi-
cal degradation generally consists of covalent bond cleav-
Fabricable into Intended Configuration age, but, in some cases, covalent crosslinking or ionic bond
Reasonable mechanical characteristics of the tubular transformations are involved. Variables such as heat, mois-
device include flexural compliance, kink resistance and ture, oxygen, light, radiation, even mechanical stress, induce
suturability. While materials with a wide range of physical chemical degradation.10 Many or all of these factors may be
properties can achieve these goals, there are property- encountered by a biomaterial prior to its implantation. Each
dependent limitations on the configurations of these mate- of these destructive agents is generally manageable to ac-
rials that can be used. Fiber-based or expanded wall con- ceptable levels by process and exposure control and the use
figurations are probably required if the material has a rela- of additives10 if the material is inherently stable in the body.
tively high to medium modulus (e.g., poly(ethylene tereph- The longest intended phase in the lifetime of a “per-
thalate), polypropylene, polytetrafluoroethylene). Low manent” implant is residence in the body. The polymeric
modulus compositions such as elastomers (e.g., polyure- scaffold for a tissue engineered vascular prosthesis is subject
thanes) can be considered as solid-wall, porous-wall or fi- to all of the forces listed above which may induce physical
ber-based tubes. Extrusion, molding or casting processes degradation. Appropriate device design and processing (e.g.,
must be available to produce the base configuration. If sur- to provide burst strength and minimize residual stress) and
face modification is required, acceptable processes must be proper implantation techniques (e.g., to minimize kinking
applicable to the material. and applied stress) must be implemented to minimize physi-
cal degradation.10
Stable to Processing, Storage and Use Conditions Chemical degradation forces are also encountered at
Maintenance of structural integrity by the biomate- every phase of the biomaterial scaffold’s lifetime. The ob-
rial scaffold is the main requirement of this chapter and is jective is to understand these forces and the material’s sus-
addressed in the sections on degradation mechanisms and ceptibility to them and to protect against their effects. For
discussions of individual materials. The major point to make example, antioxidants can protect polymers such as polypro-
here is that there are stability considerations at each of the pylene and polyurethanes from thermooxidative, photo-
many stages of processing and storage of the biomaterial, oxidative and autooxidative (oxygen induced) degradation.10
not just during residence in vivo.8 The material, at the time In the body environment, modes of chemical degra-
of implant, is the product of its prior history and attention dation consist of three major types: hydrolysis, oxidation
to preserving its integrity throughout can assure stability of and mineralization (calcification). The latter mode may or
the implant. may not induce significant covalent bond cleavage, but does
involve chemical transformations that cause structural dam-
Elicits Acceptable Host Response for Life of Device age to the polymer and will be treated as one of the three
Inherent nonthrombogenicity of the biomaterial is not subtopics discussed next.
likely and is not assumed to be required. A biological re-
sponse will be induced, and control of that response is the Hydrolysis
function of the tissue engineering component of the vascu- Hydrolysis is the cleavage of susceptible chemical
lar product. It is assumed that the matrix material has passed bonds with water. Simple hydrolysis can be catalyzed by acid
a standard biocompatibility protocol2 and that control of or base and occurs as a consequence of the number and re-
other responses can be achieved by mechanical design, local activity of the cleavable bonds. Enzyme-catalyzed hydroly-
or systemic drug delivery, surface or bulk modification. Any sis (e.g., by esterases, proteases) has been reported9,11-14 but,
additives used for processing, stabilizing or effecting bio- for most synthetic polymers, enzymatic effects are superfi-
logical responses are likewise assumed to be nontoxic. cial and minor and will not be further discussed here.
Biostable Polymers as Durable Scaffolds for Tissue Engineered Vascular Prostheses 471
Hydrolytically susceptible bonds generally consist of Inside the body, biomaterials are subject to oxidative
carbonyl groups with a heteroatom (O,N,S) on one or both insult as a consequence of inflammation due to the foreign
sides of it. These structures and several other hydrolyzable body reaction (phagocytic attack).9,17 Powerful oxidative
groups are listed in Figure 43.1. products result from phagocyte activation (e.g., hydroxyl
Hydrolysis rates differ among the susceptible groups. radical, peroxynitrite, hypochlorite), which can attack oxi-
For example, carbonyl group reactivity decreases in the order: dizable bonds. Susceptible sites generally are those that can
anhydride > ester > urethane > amide. Ethers, ketals and stabilize a free radical (Figure 43.2).9
acetals are labile to acidic pH but stable to basic conditions. Oxidatively stable structures include straight-chain
Backbone structure and intermolecular interactions hydrocarbons, halocarbons and fully-oxidized groups (e.g.,
of hydrolyzable polymers also affect degradation rate. Fac- ketones, sulfones, carbonates, esters, urethanes). We have,
tors such as crystallinity, hydrophobicity and crosslink den- however, produced some evidence that the carbon atom next
sity tend to decrease hydrolysis rate9 and render polymers to the heteroatom of the latter carbonyl groups may be ame-
based on hydrolytically labile groups potentially viable can- nable to oxidation.17
didates for long term implant. Evidence for direct (receptor-ligand) enzymatic
Polymer structures that resist hydrolysis include hy- catalysis of oxidation is limited and inidcative of minor con-
drocarbons, silicones, sulfones, halocarbons and isolated sequences.12,18
carbonyl-containing molecules (i.e., ketones). In vivo oxidation of polymers has been most exten-
sively studied for polyurethanes and polyethylenes.9,15-24
Oxidation Structural degradation due to oxidation is usually manifest
Oxidative degradation of polymers involves destruc- as surface fissuring, deep crack formation, fragmentation
tion or modification of molecular structure via electron or wear, often in zones of applied stress. Radiation steriliza-
transfer reactions.9 The polymers to be considered have vary- tion (e.g., of polyethylene) can produce long-lived radicals
ing degrees of susceptibility to oxidation. Prior to implanta- with autooxidation and embrittlement leading to increased
tion, specific operations such as melt processing, radiation wear and fragmentation in use.
sterilization or exposure to light containing ultraviolet wave- Mitigation of structural degradation due to oxidation
lengths can promote oxidative degradation. Autooxidation can be accomplished by minimizing residual and applied
involves reaction with molecular oxygen and is a likely stress, controlling exposure to destructive radiation, use of
mechanism of many oxidative events.15 Other oxidants (e.g., antioxidants, isolating susceptible polymers in a device from
metal ions, hypochlorite, hydroperoxides)16 are also effec- direct attack by phagocytes or soluble oxidants and, finally,
tive in causing degradation. by the use of oxidation resistant materials.9,10,15,16
Fig. 43.1. Hydrolytically susceptible
O O groups.
O
X C Y C Z C
X=O,N,S,H
Y=O,N,S,H Z=O,N,S
X or YΙH
Hydrolyzable carbonyl structures
O CH2 (ether)
C N (nitrile) O CH2 O (ketal or acetal)
O
CH CH
(cyanoacrylate)
O P O (phosphate)
C N C OR
O O
O
S NH (sulfonamide)
R=alkyl
O
Other hydrolyzable groups
472 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 43.2. Oxidatively susceptible groups.
* * *
CH X CH
R
hydrocarbon
chain branch X=O,NH1-2 ,S
(R=alkyl, aryl, allyl) (eg, ether, amine, alcohol, sulfide)
*
OH
*
C O
H
phenol aldehyde
* = susceptible site
Calcification The emphasis of this section is primarily on
Calcification or mineralization involves the deposition biostability, although product design and biocompatibility
of calcium phosphate salts on or within implanted struc- are not ignored. The general assumption is that the latter
tures. These phenomena are termed extrinsic and intrinsic two requirements can be met by approaches described in
calcification respectively. Extent of calcification is related to this book if the scaffold can perform its function for the
the level of soluble calcium and the device function, more intended period.
than the chemical composition of the material.25,26 Calcifi- O O
cation has been reported to occur with synthetic vascular Poly(Ethylene Terephthalate)(PET) OC COCH2CH2 n
prostheses, causing stiffening and obstruction.25 The most PET vascular prostheses have dominated the large di-
prominent effects of calcification occur, however, in devices ameter field since their introduction in 1957.27 PET displays
that undergo extensive cyclical deformations in vivo (e.g., several advantages in its “standard” or “micro-denier” fiber
prosthetic heart valves, heart assist devices).25 Generation form. It is a strong material having a tensile strength in ori-
of microscopic defects in the polymer may produce nucle- ented form of 170-180 MPa and a tensile modulus of about
ation sites for the initiation of crystal formation, which can 14,000 MPa.28 It is readily fabricable into woven or knitted
lead to extensive mineralization and, ultimately, mechani- textile or mesh form. It can be crimped to enhance kink re-
cal failure of the device. Chemical degradation of the sistance. It is relatively stable. Although it is based on the
biomaterials has not yet been implicated in initiating or pro- hydrolytically susceptible ester group, it is highly crystalline
moting calcification. Anticalcification additives have been (MP ~ 265°C) because of its ordered chain structure and
applied to synthetic biomaterials with some success.26 Vas- deep drawing during fiber formation.9 Implant durability
cular implants that undergo moderate deformation should studies have shown or estimated progressive deterioration
present a relatively low risk for calcification. of physical properties over decades as a consequence of hy-
drolysis, and, possibly, some oxidation induced by activated
Polymers with Potential to Serve phagocytes.29-31 In vivo studies have projected approximately
as Biostable Scaffolds 30 years to full resorption in humans.29,30
This section describes polymers with potential for use PET is relatively stable to sterilization by ethylene ox-
as scaffolds for permanent tissue engineered vascular pros- ide and ionizing radiation;32,33 however, techniques involv-
theses. Commercial polymers used in existing prostheses will ing steam, dry heat or chemically reactive sterilants32-34 may
be described first. Polymers that may not have been com- cause deformation or degradation.
mercialized but have been described in the literature for use Mitigating against the use of PET for small diameter
in medical devices will be briefly described. Finally, com- prostheses is the high reactivity of blood and vascular tissue
mercial polymers that have not been reported in vascular toward the implant with consequent high inflammation,
prosthesis literature, but may satisfy the design requirements neointimal proliferation and inhibition of cellular regenera-
for scaffolds listed above, will be mentioned. tion. This response to PET has been demonstrated with tex-
All of the polymers to be described are assumed to tile vascular prostheses35-39 and vascular stents in open mesh
contain additives such as processing aids and stabilizers. configuration.40,41 The blood and vascular tissue reactivity
Different grades, lots or brands of polymers with the same of PET can vary with the proprietary additives and treat-
generic structure may be more or less stable as a result of ments applied to the individual manufactured devices.42
additives, processing conditions, molecular weight differ- PET has successfully served as the structural matrix
ences, crystallinity differences and other variables. for composite vascular devices. Albumin, collagen or gela-
Biostable Polymers as Durable Scaffolds for Tissue Engineered Vascular Prostheses 473
CH3
tin impregnation of PET textile prostheses is performed to
seal them against blood leakage.38-43 Healing responses of Polypropylene(PP) CH CH2 n
the composite are comparable to the unsealed PET.44 The PP has considerable appeal as a biostable scaffold. It is
Omniflow Vascular Prosthesis (Bio Nova, International) is a relatively strong (tensile strength = 400 MPa), crystalline,
formed from PET mesh placed on a silicone mandrel and high modulus (tensile modulus = 2.6 GPa) thermoplastic in
implanted in sheep to produce a collagen-PET composite its isotactic form.56 Its hydrocarbon structure renders PP
device. It has been used successfully for peripheral vascular insensitive to hydrolytic attack. However, since every other
replacement.45 Other modifications, such as incorporation atom on the polymeric chain is a tertiary carbon, it is sus-
of bioactive molecules46,47 and surface modification for ceptible to oxidation during processing, storage and implan-
blood compatibility48 have shown promise. tation.57 This type of degradation can be effectively coun-
In sum, PET should be considered a highly credible tered by the use of antioxidants, which are universally used
candidate for biostable vascular scaffolds. in PP products.57,58 Consequently, stabilized PP is consid-
ered a “permanent” implant material.
Polytetrafluoroethylene(PTFE) CF2CF2 n Biostability, strength, and durability combined with a
PTFE is a member of the fluorocarbon class of poly- relatively low inflammatory tendency59-61 make stabilized PP
mers. It is, by far, the most commonly used fluorocarbon49 the material of choice for vascular anastomotic sutures,59-61
in implants because of its demonstration of long term certain ligament augmentation devices58,62 and mesh for
biostability and biocompatibility in vivo. PTFE vascular pros- surgical repair.63
theses are prominent in the medium diameter (~7-9 mm) PP yarns have been woven into multifilament tubes
and dialysis access markets.27 for investigation as single component vascular prostheses59
The polymer is prepared in powdered form from or as the reinforcing matrix for partially absorbable com-
tetrafluoroethylene monomer. The product is a highly crys- posite devices.64 Chronic animal implant studies indicated
talline material (> 90% crystallinity) which is not completely that PP offered potential advantages in efficacy over ex-
fusible, but is formed into shapes by sintering.49 A densely panded PTFE and PET in small diameter vascular applica-
sintered configuration approaches the intrinsic properties tions.59 Biomechanical behavior, which could be readily
of the material and gives a moderate stiffness (tensile modu- modulated by varying fiber diameter and weaving condi-
lus of elasticity = 0.5 GPa) and tensile strength (14 MPa).49 tions, was shown to have a significant effect on tissue in-
Implantable forms of the material of interest for vas- growth and the resulting hybrid bio-artificial composite.64,65
cular prostheses include the “expanded” and textile prod- PP is one of the least resistant structural polymers to
ucts.50 Expanded PTFE (ePTFE) is made by an extrusion, sterilization by ionizing radiation.32,33 However, it is safely
drawing and sintering process50 to produce a tube with a sterilized by chemical agents and autoclaving.32-34
porous wall consisting of fibrils and nodules which is con- Recent innovations in the technology of PP include a
trollable to different pore sizes (e.g., 30 and 60 ∝m). The syndiotactic product and an ionomer (ion-containing poly-
porosity can be used advantageously to promote ingrowth ethylene)-modified isotactic PP which produce lower moduli
of tissue and formation and retention of an endothelial layer (0.5 GPa) than isotactic PP (2.6 GPa).56 Finally, a formula-
in vascular prostheses.51,52 tion of polypropylene rendered radiation resistant by the
Knitted PTFE cloth is effectively used as sewing rings incorporation of hindered amine light stabilizers and a
for heart valve prostheses,53,54 and should be considered plastomeric ethylene polymer has been described.66
when a textile configuration for a biostable scaffold is pre- PP is normally considered a low surface energy, hy-
ferred. It should be noted here that the preparation of PTFE drophobic material to which surface bonding is difficult.
fibers requires the use of sizing agents which must be re- However, certain highly hydrophobic cellular strains adhere
moved by vigorous (and proprietary) cleaning processes. strongly to PP.67 This suggests that the adherence of tissue
PTFE has a notable shortcoming—its susceptibility to generated on PP scaffolds should be studied on a case by
degradation by ionizing radiation as experienced during case basis with consideration of the possibility surface modi-
gamma sterilization.32,33 It is completely stable to other forms fication to control delamination.
of sterilization, however. Polypropylene should be considered in the top tier of
Another potential issue with PTFE concerns the ad- candidates for biostable vascular matrices.
herence of other materials to its surface. The polymer is noted O
for its release properties, and this is an advantage for
declotting vascular access prostheses.27 However, if a hybrid Polyurethanes(PUR) NHCO
bio-artificial device requires the bonding of a substantial PURs comprise a large family of polymers which is
amount of tissue to the PTFE surface (i.e., without com- notable for its diversity. The only required attribute that in-
plete mechanical interlock), a potential for delamination may dividual members have in common is the presence of the
exist. This factor has been recognized and promising ap- urethane [-NH(CO)O-] group in some repeating sequence
proaches to enhance the binding of, for example, endothe- on the main chain, from the reaction of an isocyanate group
lial cells are being developed. These include denucleation of with an alcohol group. Most likely, however, there are other
ePTFE by saturating its pores with water, use of surfactants functional groups which make up the soft segment of the
and treatment with adhesion molecules (e.g., fibronectin) PUR, which is generally a copolymer consisting of hard and
or adhesion peptides.52,55 soft segments. The hard segment generally consists of the
474 Tissue Engineering of Prosthetic Vascular Grafts
Diisocyanates Soft Segment Macroglycols
OCN CH 2 NCO (MDI) HO CH 2 O H (PTMEG)
4 n
OCN CH 2 NCO (HMDI)
HO CH CH 2 O n H (PPG)
OCN CH 2 NCO (HDI) CH 3
6
OCN D NCO (DDI) O
HO R OCR' O H (polyester diol)
n
Short-chain Diols/Diamines
O
HO CH 2 OH (BDO) HO ROCO H (polycarbonate diol)
4 n
HO CH 2 OH (EG)
2 HO D OH (Dimerol)
HOCH 2 CH 2OH (CHDM)
*H2NCH 2CH2NH2 (EDA)
*
NH2 (CHDA)
NH2
O Where: R, R' = aliphatic hydrocarbon
*Amines form urea NHCNH D = C36 Hydrocarbon from
linkages by reaction with isocyanates. dimerized fatty acid
n = repeating units to produce MWs of ~500-3000
Fig. 43.3 Monomers for polyurethanes.
reaction product of a diisocyanate and a diol or diamine. biodegradation is the soft segment (ester, ether, carbonate).
The soft segment is derived from a macromonomer rang- While hydrolysis of the urethane (or urea) hard segment link-
ing from several hundred to several thousand in molecular ages is possible, those groups are relatively stable and do not
weight.10 Typical monomers for polyurethanes which have comprise the primary mode of biodegradation.
been used as “permanent” implants are listed in Figure 43.3.10 Polyester soft segments have generally degraded
Most of the polyurethanes used in medical devices are based quickly and severely by hydrolysis.16 Polether urethanes are
on difunctional monomers. susceptible to oxidative degradation as described in the
Commercial PURs usually contain additives such as “Modes of Polymer Degradation” section, above.71,72 Since
catalyst residues, processing aids and stabilizers. These ad- oxidative susceptibility is generally proportional to ether
ditives often migrate to the surface of the PUR part and serve content,9,10,15,16 relatively hard (low ether content) PURs
as the material in contact with blood or soft tissue.10 Cer- have performed with minimal degradation for periods as
tain PURs have demonstrated relatively low long as the lifetime of pacemakers (8-10 yr). Even soft
thrombogenicity.10 polether urethanes are capable of performing as intended
PURs range in physical properties from soft, tough for years in the absence of high stress or strong oxidative
elastomers to strong, rigid structural polymers (e.g., tensile attack.15,16
strength = 20-50 MPa, tensile modulus = 5-1150 MPa).10 As Over the past few years, several industrial concerns
a result, PURs have been considered for soft, flexible im- have reported on the development of polyurethanes based
plantable device components such as pacemaker lead insu- on polycarbonate soft segments.69,73-75 These have shown
lation15 tubing and compliant vascular prostheses68-70 as well very high resistance to degradation in short term studies,
as hard, rigid components such as pacemaker lead connec- although minor hydrolytic effects have been noted on the
tors.10 The polyurethanes used in medical devices are usu- implant surfaces. 69,76 However, in vascular implants
ally formed into shapes by solution or melt processes10 and approaching one year, structural degradation of the
can be solution or melt spun into fibers, cast into porous or poly(carbonate urethane) fibers was detectable.69 This is
solid-wall structures, or extruded into solid-wall tubing. predictive, in my opinion, of a progressive hydrolytic degra-
The record for PURs in “permanent” implants is dation that should make a device designer very wary of
mixed, because of hydrolytic and oxidative degradative choosing poly(carbonate urethanes) as “permanent”
mechanisms that may come into play. Generally, the site of
Biostable Polymers as Durable Scaffolds for Tissue Engineered Vascular Prostheses 475
scaffolds until long term (> 5 years) studies have confirmed ecules” in the zones between crystalline regions in these semi-
their stability. crystalline polymers.84 ESC is reduced in PE with lower crys-
An approach to polyurethane design that uses soft seg- tallinity (LLDPE) or higher molecular weight (UHMWPE)
ments composed entirely of hydrocarbon molecules (i.e., a because of higher concentrations of tie molecules relative
36 carbon structure derived from dimerized fatty acids) with to crystalline regions.84 Therefore, two forms of PE, LLDPE85
no ether, ester or carbonate groups to degrade77,78 has theo- and UHMWPE, have theoretical appeal for ESC resistant
retical appeal. However, this concept must also pass the test fiber-based prostheses. The latter has been used as fibers for
of long term implant stability before it may be used with sutures.86 The LLDPE is reported to be resistant to crack-
confidence. ing.85 A previously unmentioned form of PE deserves men-
Polyurethanes are generally resistant to sterilization tion—gel-spun UHMWPE. These fibers achieve levels of
of all types except steam and, possibly, dry heat.10 They are tensile strength (2-4 GPa) and tensile modulus (125-175
susceptible to calcification, especially in highly dynamic ap- GPa) that are multiples of the PE levels reported above.87
plications such as leaflet heart valves.25 Their high stability and strength at very low fiber diameters
Although polyurethanes display some of the most fa- are potentially useful design features for scaffolds.
vorable mechanical properties for compliant grafts, no com- PE is resistant to chemical sterilization and suscep-
position has yet demonstrated dependable long term stabil- tible to degradation by ionizing radiation.22 Most forms of
ity in vivo, especially as a fibrous vascular implant. thermal sterilization would warp the relatively low melting
PE (MP 104-135°C).83
The PE family, in my opinion, has not received
Polyethylene(PE) CH2 CH2
n adequate consideration for use in filament-based
PE is produced in several forms including very low vascular prostheses.
density (VLDPE), low density (LDPE), medium density CH3
(MDPE), high density (HDPE), linear low density (LLDPE) O Si
and ultra high molecular weight (UHMWPE), each with n
Polydimethylsiloxane (PDMS)
different thermal and mechanical characteristics.49 The CH3
variations in density and physical properties result from dif- The PDMSs comprise a family of thermoset elastomers
ferences in polymerization conditions or the use of other with a long history of successful use in implantable de-
olefinic hydrocarbon comonomers.49 All of these molecular vices. 79,88 Structural devices are usually produced by
structures are amenable to fabrication into fibers or cellular crosslinking 2-part systems at room temperature or higher.
structures, which are the forms most likely to meet the re- The crosslinked products are highly elastic and extensible.
quirements of the “design concept.” The tensile modulus (2-9 MPa) and ultimate tensile strength
Although PE has been reported as being used in vas- (2-10 MPa) are relatively low compared to other polymers
cular prostheses40 and is regularly used in several forms of considered in this chapter.10 However, excellent flex fatigue
permanent implants,79 recent literature describing its use in resistance and tear resistance of some compositions,10 com-
vascular prostheses is sparse. More commonly reported is bined with high biostability,88,89 make PDMS a worthy con-
the use of solid-wall polyethylene tubing with or without tender for compliant vascular prosthesis scaffolds.
coatings for thrombogenicity studies.80-82 The studies show PDMS has been studied in vascular prostheses. Be-
that PE can be modified to improve surface wetability for cause of its low modulus, porous-wall structures have been
binding of coatings (e.g., heparin)81 and, potentially, other favored over filamentous configurations. The
components for tissue engineered prostheses. Structurally, “Replamineform” process uses sea urchin spine machined
PE compositions range from relatively low to medium into tubular shape as template for fabrication of porous
modulus when thermally processed (Table 43.1).83 PDMS vascular devices. 90,91 The spine, consisting of
PE in its various structural forms is stable to hydro- microporous calcite, is dissolved in acid, leaving behind an
lytic media and resistant to oxidation in proportion to its open-cell PDMS structure with adequate strength and the
ratio of linear to branched chain structure and crystallinity. potential for tissue ingrowth.90
PE is susceptible to the phenomenon of environmental stress PDMS derives much of its strength from silica filler.
cracking (ESC) produced by exposure of stressed specimens The filler is actively thrombogenic; however, PDMS with-
to aggressive media such as detergents (and, possibly, blood). out filler can be used as a less thrombogenic coating.82 PDMS
Cracking occurs because of stress relaxation of “tie mol- has been used as a surface for endothelial cell growth;82 how-
ever, it is a relatively low energy surface and the authors cau-
tion that resistance to cell detachment under conditions of
blood flow must be verified before a hybrid device based on
PDMS is deemed suitable for long term function.82
Table 43.1. Properties of PE compositions83
PDMS can be sterilized under relatively mild ionizing
radiation conditions and by heat or chemical sterilants.32
2 Tensile Strength (MPa) Tensile modulus (MPa) A final note on PDMS is in order. The PDMS class of
elastomers has come under severe scrutiny in recent years
LDPE 15-80 55-170
because of its use in mammary prostheses. Reports of
MDPE 10-20 170-380
HDPE 15-35 415-1035 autoimmune responses to implanted devices have focused
476 Tissue Engineering of Prosthetic Vascular Grafts
on PDMS oil-filled PDMS shell designs. Leakage of the oil mers (LCPs). They are so named because of their high de-
is alleged to cause systemic responses to PDMS.92 However, gree of order in the liquid phase. Upon cooling, these poly-
decades of successful use of PDMS in solid form79,93 provide mers solidify to structures with anisotropic skin-core mor-
a massive body of evidence that these materials are phologies and tensile properties among the highest of known
chemically stable and safe as implants. The list of suppliers polymers (i.e., tensile strengths to 240 MPa, tensile moduli
of PDMS for “permanent” implants is currently short, but to 32 GPa).98 Chemical degradation resistance is also excep-
PDMS raw materials are available93 and these elastomers tionally high. The materials can be readily extruded into
merit serious consideration as scaffolds for “permanent” tubing and fibers.99 If the fibers are not too stiff, LCPs may
hybrids. be worth considering as structural matrices for hybrid
vascular prostheses.
Other Polymer Scaffold Materials A group of rigid thermoplastics having amorphous
This subsection speculates briefly on polymers that or semicrystalline morphologies and superior resistance to
have not received much reported consideration for vascular chemical degradation includes the polysulfones (polysulfone,
prostheses, but are judged to have the potential to meet the polyethersulfone, polyarylsulfone) the polyketones (PEK,
design criteria for a tissue engineered device. The selection PEEK, PAEK, PEKK, where P=poly; A=aryl; E=ether;
is based on my experience in working with or considering K=ketone), poly(ether imide), polyimide, poly(methyl meth-
the materials for use in “permanent” implants. The sparse- acrylate) and others.98,100-104 The stated polymers normally
ness of available information offers the opportunity for would comprise the nonfibrous component of a fiber-rein-
technical advancement associated with several risks. At best, forced composite used, for example, in orthopedic de-
there will be substantial development expense to assure vices.100-104 Investigation of fibrous forms of these materials
successful implementation. There will likely be increased is a worthwhile undertaking, in my opinion. This is being
regulatory complexity for new vascular materials. A new done with poly(methyl methacrylate) currently, and the re-
vascular material is riskier, because an established clinical sults are impressive.103 For example, the ultimate tensile
history would have revealed characteristics of a material that strength of PMMA can be increased from 25-50 MPa to as
might not be anticipated in the development plan. Finally, high as 220 MPa while enhancing ultimate elongation from
there is the risk that some of these materials have been stud- 5-35% with fiber drawdown ratios of ~20.103,104 Tensile
ied by others and rejected for good cause without the results moduli increase from ~2 GPa in castings to ~8 GPa in the
being published. fibers, which would suggest the use of very thin fiber diam-
The subsection is now separated into two parts, the eters in vascular scaffolds.
first listing high-modulus linear polymers which are best Poly(acrylonitrile-co-vinyl chloride) has been fabri-
utilized in fiber form, the second listing low-modulus elas- cated into hollow fiber form as a cell encapsulation mem-
tomers which may be fabricated into fiber-based, solid-wall brane for hybrid bio-artificial organs and hemo-filtration
or porous devices. membranes. It has been shown to be stable in long term
implants and can likely be extruded or solution spun into
High Modulus Polymers fibers suitable for consideration for vascular scaffolds.105
PTFE is just one of several polymers which belong to
the fluoropolymer category. It also includes: poly(ethylene Low Modulus Elastomers
tetrafluoroethylene), poly(ethylene chlorotrifluoroethylene), Elastomers meriting consideration in this subsection,
poly(vinylidine fluoride), fluorinated ethylene propylene, in addition to the ones already considered, may be either
perfluoroalkoxy resin, polychlorotrifluoroethylene and oth- thermoplastic or thermoset. Both types may be fabricated
ers.49,94 They all offer exceptional resistance to hydrolysis and into fibrous, porous or solid wall form. Thermoset rubbers
oxidation. Just as with PTFE, they are sterilizable by all com- are often prepared from prepolymers (“millable gums”) by
mon means except, possibly, ionizing radiation.32 The “other” hot molding processes. In such cases, residues from the “vul-
fluoropolymers are worth considering as an alternative to canization” (crosslinking) process may need to be removed
PTFE because most are more readily proccessable by melt by extraction to make the scaffold nontoxic. The elastomers
or solution techniques. They also offer a broad range of are often compounded with fillers, stabilizers, processing aids
modulus characteristics to choose from.94 and other ingredients to control physical and chemical prop-
Several rigid polyamides should have the chemical erties of the final product.
resistance to provide long term service in vivo. These in- Certain of the compositions to be described may not
clude Nylon 11, Nylon 1295 and aromatic polyamides (Ara- be available in prepolymer form for fabrication into desired
mids).96,97 Their stability arises either from hydrophobicity shapes, but are sold in finished form by the materials manu-
of the backbone (Nylon 11, 12) or high crystallinity (Ara- facturer. Therefore, any evaluation of a scaffold configura-
mid). Braided Aramid fiber prostheses have been studied tion would require the approval and fabrication by the ma-
for artificial ligaments.96,97 While mechanical degradation terial supplier. In these times of high liability risk, some elas-
was observed under dynamic conditions,96 long term chemi- tomer suppliers may not approve the use of their product as
cal stability in human plasma was confirmed. “permanent” implants.
Certain aromatic polyesters and poly(ester amides) Nonetheless, elastomers offer the potential advantage
belong to a class of polymers called liquid crystalline poly- of radial compliance and deserve consideration. This sec-
Biostable Polymers as Durable Scaffolds for Tissue Engineered Vascular Prostheses 477
tion provides a very brief overview, but recent reviews of indeed, a worthy one which I believe will ultimately be
elastomers are more detailed and comprehensive.106,107 successful.
Polyolefins include rubbers synthesized from olefinic
hydrocarbons, generally crosslinked with dienes. Rubbers of Acknowledgments
ethylene, propylene, isobutylene and higher olefins are in- The literature provided by Drs. Giovani Galletti, Rob-
cluded here. They may be compounded to various ranges of ert Guidoin, Howard Greisler, Vince Medenhall, Colin Pitt
strength, hardness and elongation. They are highly resistant and J. Paul Santerre is gratefully acknowledged. I sincerely
to hydrolysis, and have low residual olefin content for oxi- thank Mrs. Sandra Brigham for her expert preparation of
dation stability (in contrast to rubbers from dienes such as this manuscript.
butadiene). However, they have branched-chain sites which
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Scaffold Engineering
Material Aspects
CHAPTER 44
Biophilic Polymers: What’s on the Horizon?
Patrick T. Cahalan
T his chapter was outlined for a section of this book entitled ‘Bio-Interactive’ Prostheses,
and was further subdivided to a section including biostable polymers/materials. The other
chapter in this subsection, titled “Biostable Polymers as Durable Scaffolds for Tissue Engi-
neered Vascular Prostheses” is being written by a dear colleague, Art Coury, who shares this
author’s sense of trepidation as to achieving substance in the context of the remaining chap-
ters of the overall section. It is hoped that, although this chapter will be written from an
industrial perspective, it will have some practical value for the reader.
The word biophilic cannot be found in the dictionary, but then neither can the word
biocompatible. In the early 1980s materials were placed in four basic groups:
1. Bioinert;
2. Biocompatible;
3. Bioactive; and
4. Biointeractive.
Bioinert surfaces were hypothesized to be “invisible” to the body, and to have little or
no interaction. Proposed early examples were negative surface charge to repel cell adhe-
sion,1 high surface energy materials2 such as pyrolytic carbon and low critical surface ten-
sion materials3 such as fluoropolymers, which also claimed low protein and platelet adhe-
sion. Also in the early 1980s, hydrophilic materials were suggested to be low protein adsorbing
and platelet adhering,4 and in particular PEG-like surfaces were claimed to have increased
surface motion (flagella-like activity) that served to decrease protein adsorption and dena-
turation.5 The term biocompatible could be applied to any material that when implanted
showed an equal or better tissue response compared to a control material, such as polypro-
pylene, that was accepted as biocompatible. Bioactive surfaces were surfaces designed to
promote an advantageous effect such as preferential adsorption of albumin by C-18 alky-
lated surfaces.6 Finally, biointeractive surfaces were those designed to interact with a specific
physiological mechanism such as coagulation; an early example is ionically bound heparin
for local release to decrease coagulation.7,8 Heparin was finally covalently immobilized and
shown to have bioactivity without releasing by Larm;9 this surface has been shown in vitro
to bind ATIII to produce a catalytic rate of thrombin deactivation. These categories were
arrived at largely from the perspective of the empirical surface response, and arguably need
some revaluation with more instrument-intensive analytical methods now available to prop-
erly order them by rank in a new era of tissue engineered materials.
The editors have chosen new labels, and have done so with the healing response in
mind. Thus they have set the stage for the conceptual advantages of tissue engineering. In
labeling bio-inert materials as those exhibiting insufficient healing, biolized (endothelialized
surfaces) as surface healing, and bio-interactive as complete healing, they have set the goal
to incorporate all that has been learned to date towards designing materials and structures
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
482 Tissue Engineering of Prosthetic Vascular Grafts
that should more closely approximate naturally function- plete loss of heparin activity, it is highly optimistic to hope
ing tissue. In the early years of vascular graft implantation, for a synthetic anionic polymer with heparin properties.22
Voorhees’ observations with Vinyon “N” cloth in dog aortas Studies in our laboratory with compounds reported as
clearly showed that a synthetic material could serve as a con- heparinoid or anticoagulant fail to produce heparin-like
duit, and be accepted by the tissue.10 Tissue engineering, or activity when measured in solution with ATIII and throm-
the integration of biology and biomaterials, holds out the bin. Surfaces with immobilized cationic or anionic charac-
promise of producing more than an accepted conduit, and ter can adsorb ATIII and thrombin respectively, and give false
obviating all the problems associated with the current com- positives for heparin activity. An amine functional surface
mercial products such as thrombotic occlusion, anastomotic can show binding of ATIII that is resistant to extensive 0.15
hyperplasia, aneurysmal dilatation, and infection. Presum- ionic strength rinsings, and if given time will deactivate
ably, bio-interactive prostheses are the desired approach to thrombin, though not in a catalytic fashion. An anionic sur-
achieving this goal. face can bind thrombin that does not rinse off in the nor-
During the period between 1982 and 1988, this au- mal rinse steps used before difference measurement using
thor was involved with research to find a small diameter chromogenic substrate, giving a false measure of thrombin
(4 mm or less) vascular graft that could exhibit patency at deactivated. Active thrombin can be witnessed by adding
least equivalent to that of autologous saphenous vein. In re- substrate back to the surface and obtaining a color change
ality the effort was more a development, or an evaluation in the substrate. Since it is well known that anionic surfaces
exercise, that included animal implantation of numerous are contact activating, it is of concern to have an anionic
technology platforms to include: surface present that does not have high heparin activity. Since
1. Biodegradable polyurethane-PLLA grafts; coagulation is so dependent on platelets, it is hypothesized
2. Biostable polyurethane grafts made by phase inversion, that by using a polymer that mimics the nonactivated plate-
electrostatic spinning, and spray techniques; let membrane, namely phosphatidylcholine, it would be
3. Alternatively (non glutaraldehyde) fixed heterografts; possible to prevent platelet interaction with the polymer
4. Plasma TFE coated Dacron prostheses; surface (Fig. 44.1).
5. Silicone replamarinaform porous conduits; and Phosphatidylcholine in the form of commercially
6. Dacron and PTFE control grafts from commercial sources. available lecithin can coat surfaces of hydrophobic polymers,
Predominant modes of failure were: aneurysmal dila- but is easily removed with flowing plasma. In attempt to
tation (1 and 2); in vivo degradation across species (3); re- remedy this problem, commercial efforts have given rise to
jection by surgeons at the need to cut the prostheses with an a network polymer with strong adhesive properties to poly-
electrical device (the flaming graft), and undesirable suture mers, and containing pendant phosphorylcholine moieties
characteristics (4); poor crush resistance leading to throm- as seen in Figure 44.2.23
botic occlusions, and poor suture pull out properties (5). The most common network polymer is an acrylate
The Dacron and PTFE grafts had patency rates not com- backbone that has good adhesive properties for PVC and
petitive with autologous vein, and in general showed a fi- polyurethanes. The polymer gives excellent wetting proper-
brotic nonintegrated histology. Fabricating prostheses for ties to the surfaces it coats, and has very low platelet adhe-
implantation gives one an appreciation for necessary me- sion. Human blood testing in our laboratories show this
chanical properties such as suture strength, ease of suture, polymer to be promising, but material-dependent.
burst strength, porosity, and wall thickness for matching Perhaps the most innovative biophilic polymer syn-
vessel anastomosis. Since these properties have been achieved thesis to date is the work of David Tirrell in recombinant
for the most part in commercially available materials, it was artificial structural proteins. These polymers hold out the
felt that applying surface modification technologies to ex- potential of engineering proteins with controlled crystallin-
isting materials might be able to control problems such as ity and expression of specific peptide sequences at surfaces.
thrombosis and anastomotic hyperplasia, and possibly If desirable mechanical properties can be engineered into
achieve a healing response that would result in the protein polymers, and if processing methods are devised
neovascularization, endothelialization, and a tissue response to make fibers, extrusion, or coating possible, then these
that was not primarily a fibrotic or a chronic inflammatory polymers can make their way into devices.
response. The latter could help a graft towards long term There has been somewhat of a rebirth in surface modi-
patency and infection resistance. fying additives (SMAs). If a commercial group could blend
If one defines biophilic polymers as those specifically in additives to polymers that would express themselves on
synthesized to interact in an advantageous manner with the surfaces of devices that their customers manufacture, and
body, there are many claims, but few real candidates are on demonstrate enhanced biological response, then a premium
the near horizon for application. There are numerous claims price could be charged for such materials. To date, because
for heparinoid polymers.21 These polymers often have car- of the lack of tonnage of polymers used in biomaterials, it
boxyl and sulfate functionality approximating the ratio con- has been less than attractive for larger chemical companies
tained in heparin. Studies showing less adhered platelets, or to specifically manufacture biomaterials. That, together with
changing clotting times, are generally used as proof of hep- the liability issues surrounding medical devices, makes it
arin-like activity. In light of studies demonstrating the unique difficult to see new formulations coming from the larger
pentasaccharide sequence required for activation of ATIII, chemical companies. SMA technology is particularly being
and slight changes in this structure resulting in 90% to com-
Biophilic Polymers: What’s on the Horizon? 483
Fig. 44.1. Membrane lipid—phosphatidyl-
choline.
Fig. 44.2. Phosphorylcholine surface.
applied for blood-contacting surfaces for short term use, attachment, then, with some license in nomenclature, we
such as cardiopulmonary bypass circuits. have created new polymers. In light of later chapters on sur-
For the most part, biophilic polymers on the near ho- face modification for specific surfaces, we hope that it is
rizon are going to be polymers that have been surface modi- appropriate in this chapter to discuss the more general meth-
fied. Simple adsorbed coatings will be problematic from the ods of surface modification that can be used to get to the
standpoint of assuring stability for permanent implants, and biophilic surface, and point to critical concerns and oppor-
there continues to be a quest for the universal biophilic poly- tunities that may be afforded from our experiences.
mer that can coat all materials. It was the choice of our group In 1989 we created a biomaterials laboratory in
to attempt to comprehensively evaluate methods to co- Maastricht, The Netherlands. The main objective was to fo-
valently couple molecules to the surfaces of common poly- cus on surface modification technologies to improve the
meric biomaterials in hopes of creating new modified biocompatibility of materials used to construct biomedical
biophilic polymers that are stable and have controlled load- devices. The industrial laboratory was ideally situated
ing of bioactive molecules. If we achieve covalent and stable 100 meters from the Biomedical Research Institute of The
484 Tissue Engineering of Prosthetic Vascular Grafts
University of Maastricht, and the group of Professor Coen boxyl groups. This can also be accompanied by crosslinking
Hemker, an experienced and respected expert in thrombo- or chain scission, the former leading to increase in tensile
sis and hemostasis. A partnership was established between properties and the latter to polymer degradation. The first
the University of Maastricht (RL), The Bakken Research consideration on the use of irradiation should be if the
Center (BRC) and The Center for Surface and Materials polymer to be modified is predominantly a crosslinking or
Analysis (CSMA), in Manchester, England. The BRC was to chain scission polymer. Some common chain scission poly-
provide modified biomaterials for characterization by mers are polymethylmethacrylate (PMMA), polytetra-
CSMA, using predominantly XPS and TofSIMS, and fluoroethylene (PTFE), poly(vinylidene chloride), polyiso-
biocompatibility testing by RL. The partnership received a butylene (and copolymers) and polypropylene. Attempting
three year research subsidy from the EC in the form of a to modify these materials with irradiation may give a sur-
Brite EuRam grant (BE 5972). In this project a broad survey face modification, but on a degraded material. Recent high
of physical and chemical methods to modify biomaterials voltage accelerators with high penetration potential have
was performed (Table 44.1), and these methods were evalu- been suggested as an alternative, because relatively low total
ated on seven common biomaterials for feasibility. 11 dosage is required. It has also been suggested that total de-
Equipped in our laboratory with a plasma reactor, corona vices can be modified.12 The listed materials that are claimed
treater, ozone generator, UV photopolymerization equip- to have been successfully modified include in particular the
ment, and a nearby gamma-beta commercial irradiation scission polymer PMMA. These irradiation techniques, par-
source, we were prepared to generate many of the surfaces ticularly when grafting is performed in situ, require near
proposed to be more biocompatible for head to head com- oxygen free environments and thus create some problems
parisons with our partners’ assistance. What will be presented for ease of manufacture. Nevertheless, irradiation is a very
in this chapter are our somewhat narrow perspectives on effective method for covalently grafting molecules to bio-
making polymers biophilic; our experience with synthesis material surfaces.
is limited to modifying the surface of biomaterials, and does
not include creating new bulk polymers. With respect to the Plasma
latter, we maintain surveillance of the literature and sample Plasma polymerization has been touted as the most
new materials through our testing schemes when available exact method to modify biomaterial surfaces; this is in a large
from the developers. part due to the control over the plasma gases, and the thick-
The processes mentioned in Table 44.1, although not ness and uniformity of the modified surface. Some draw-
complete, represent treatments that were performed on at backs to plasma are:
least 3-7 common materials for each method. Some brief 1. Requirement for reactor specific design based on ge-
comments will be given here that are felt to be important ometry and materials of construction;
concerning these surface modification methods and their 2. In almost all cases the process will be a batch opera-
applicability to biomaterials and biomedical devices. tion; and
3. Contamination of the reactor and thus the require-
Physical Methods ment for cleaning cycles and for single process dedi-
cation of reactors. Several investigators have pro-
Irradiation posed simple plasma treatment to make materials
Early work by Ratner and Hoffman4 showed the po- more hydrophilic and thus more blood compatible.13
tential of grafting biomaterials with irradiation sources. Our Plasma treatments may show initial lowering of con-
efforts included irradiation using electron beam sources to tact angle, and will follow with an inversion within
successfully graft to polyolefins. E-beam or gamma radia- hours back to a hydrophobic surface. The latter is
tion can introduce several functionalities to polymeric ma- particularly true of elastomers.
terials, such as free radicals, vinyl bonds and, in the presence Plasma polymerized surfaces can be directly depos-
of oxygen, hydroperoxides, carbonyls, aldehydes and car- ited on a surface, or plasma can be used to activate a surface
for subsequent grafting of molecules. The latter is difficult,
as the surface generally requires a short plasma discharge
time, or pulsed plasma discharge. It has been hypothesized
Table 44.1. Methods to functionalize/activate surfaces that free radicals are short lived on the surface of plasma
(Brite EuRam) treated materials, due largely to the rapid termination by
vicinal radicals.14 A final observation on plasma-modified
Physical Chemical surfaces is that the reactivity in terms of coupling further
biomolecules has been less than expected. Evaluation of cou-
Irradiation Oxidation pling to plasma functionalized surfaces based on the results
Plasma Reduction of XPS data suggests that a significant portion of the func-
Corona Hydrolysis tional groups do not react. One possible explanation is that
CVD and PVD Ozonization organic chemistry, and particularly biomolecules, react in
“Simple” Coating Silanization three dimensions, and the plasma surface is highly ordered
Entanglement Grafting
and rather two dimensional in its ability to enter into reac-
Textruing Photocuping
Ion Beam Implant Add’n/Subst’n tions.15 If one sees new biophilic surfaces as containing high
Biophilic Polymers: What’s on the Horizon? 485
density biomolecules such as heparin, proteins and/or cations where the device was simply coated with the poly-
growth factors, they will have to be coupled postplasma treat- urethane polymer. This was best effected by finding a sol-
ment and the ability to control the quantity and density of vent that could attack the surface of the material to be coated
coupling may prove to be a challenge. and result in a mixing of polymer chains that would give a
mechanical bond upon solvent removal. The same method
Corona has been used to imbrue drugs into polymers using solvents
Corona treatment has been used extensively in indus- that could swell the polymer, taking in the drug and leaving
try for improvement of adhesion. It is also the subject of it upon solvent evaporation. The latter method has been used
several papers on surface activation to effect grafting to sur- for introducing antimicrobial agents to polymers. The same
faces.16-18 Several affinity schemes can be used to attach principle applies for SMAs or blooming agents. Addition of
biomolecules to the grafted surfaces. Corona treatment is a amphipathic molecules to polymers can result in the more
rough process compared to plasma discharge, and takes place hydrophilic portion being expressed on the polymer sur-
in an air atmosphere. This results in a highly oxidized sur- face, anchored by the more hydrophobic end entangled in
face, to include sufficient free radicals to effect grafting of the polymer surface. In the case of polyurethanes, most con-
vinyl monomers. It can be used to graft to most polymers, tain processing waxes that migrate to the surface and pre-
but does not work for most fluoropolymers. Corona and vent the polymer from being tacky during extrusion. One
plasma can be used to make surfaces more wettable for such additive, ethylene bis-stearamide wax, has been reported
simple adsorptive coating approaches. There are several sug- to be in part responsible for improved blood compatibility.
gested universal adsorptive coating schemes that often re- Studies in our laboratory of such additives reveal that per-
quire a pretreatment such as corona to improve the unifor- formance in blood is optimal immediately after thermal pro-
mity of coating as well as the adhesive strength of the coat- cessing, but changes with time. XPS shows considerable in-
ing to the base material. crease over time in amide functionality, which may be less
effective than the long fatty chain in improving blood com-
CVD and PVD patibility. It appears that perhaps entanglement or SMAs may
Chemical vapor deposition (CVD) and physical va- be difficult to control at the surface, and thus present some
por deposition (PVD) have long been used in industry as challenges to be solved.
barrier coatings. In the European food industry, PVD has
been used to apply a layer of SiO to packaging material to Texturing
prevent moisture transmission through wrapping. CVD has Professor Andreas von Recum has been one of the
been used in electronics to provide moisture barriers and leading researchers in the field of tissue response to textured
insulative coatings. It was hoped that these methods could surfaces. He has demonstrated that textured implants with
be used as a base for engineering biologically active surfaces. internodal distances of approximately 1-3 microns appear
The ability to deposit a metallic surface such as gold would to have much thinner capsules, indicating less fibrotic re-
give a base for self assembled surfaces.19 It has also been sug- sponse.38,39 One of the hypotheses offered in explanation is
gested that a ceramic based PVD surface could be activated that the textured surface allows for more stable anchorage
to produce functionality (carboxyl groups) to which of fibroblasts, and less destruction of these cells due to
biomolecules could be coupled.20 microvibration that can lead to further inflammatory re-
sponse. A possible further explanation is that the
Simple Coatings microtextrue allows for an improved production and attach-
We define simple coatings as those that are designed ment of extracellular matrix on the material surface. Re-
to be applied by simple dipping, spraying, or by pumping search on immunoisolation membranes by Becton
solutions through devices. There is a plethora of coatings of Dickinson presented at an ACS workshop indicated that
this nature offered for short term blood compatibility. All optimal surface texture could result in minimal capsule for-
of these coatings have performance that is material depen- mation and vascular structures present very close to the
dent; this problem is often overcome by a pretreatment such material surface. The latter was independent of the material
as corona. Special attention should be paid to devices made of construction for the membrane. Applying microtextrue
from materials that undergo bending and flexing, as these to devices is a developing technology, and one promising
coatings may have a tendency to crack. Dislodgement of the approach is the use of photopolymerization. This technique
coating may cause a problem, depending on the application. holds out the promise of introducing texture and chemistry
Most manufacturing engineers of biomedical devices will to surfaces, plus the ability to use photo resists or screening
require a battery of tests to assure integrity of the coating, techniques to pattern the sights for cellular attachment to
especially if the device is to be a permanent implant such as surfaces. Application of this technology to vascular grafts
a vascular graft. While the list of short term coatings is long, will be more complicated than simply applying this tech-
the list for permanent implants is very limited. nique to the inside of a polymeric tube that has laser drilled
holes for porosity if one hopes to maintain other important
Entanglement features for grafts such as porosity for ingrowth, kink resis-
In the late 70s and early 80s the wide acceptance of tance, compliance, and suture characteristics.
Biomer as a blood compatible material led to several appli-
486 Tissue Engineering of Prosthetic Vascular Grafts
Ion Beam Implantation can be done fairly easy in aqueous solutions. The ozone pen-
Ion beam has been used to impart surface texture in etrates materials at different rates and this can be measured
addition to surface chemistry. In the orthopedic area it is using colorimetric methods.29 Materials modified by ozone
useful in improving fixation of prostheses to tissue. The Spire may need final reduction to assure that radicals are not
Corporation offers ion beam treated surfaces for improved present, and should undergo rigorous mechanical testing to
slip properties and antimicrobial surfaces, and has made assure no loss in durability of the native polymer substrate.
claims to improved blood compatibility.24 As with PVD, a
metallic surface could provide advantages for further sur- Silanization
face modification using self assembling techniques. Ion beam Silanization is most frequently used for coupling to
implantation can result in crosslinking the surface, and im- metal surfaces. The use of organofunctional silanes allows
part mechanical properties to the material. The tendency the addition of reactive organofunctional groups to the metal
for cracking of the surface on elastomers and materials that oxide surface. These groups can the be used to directly couple
will see chronic flexing should be considered. For elastomeric biomolecules. The most common mistake made with
materials, and in particular silicone, the resultant surface may silanization treatments is improper application and cure
still present the elastomer chemistry primarily. methods. Excessive coating with silanes results in weak sur-
faces that will delaminate, and failure to properly hydrolyze
Chemical Methods the silane before drying will fail to create enough reactive
silanol groups to couple during the dehydration and final
Oxidation, Reduction, Hydrolysis formation of the polysiloxane network. Covalently attached
Most synthetic polymers do not possess functionality heparin via silane activation has been released for commer-
to perform coupling reactions. Several of the physical meth- cial application to intravascular stents in Europe.30
ods mentioned can oxidize the surface and produce radi-
cals, hydroxyl, carboxyl, and carbonyl groups. This oxida- Photocoupling
tion can also be induced by chemical means such as acid In 1969 Knowles demonstrated the effectiveness of
etching or treatment with peroxides.25 For fluoropolymers 2-nitro-4-azidobenzene in forming a nitrene radical upon
that are oxidation resistant, reduction by exposure to so- exposure to UV that could extract a proton directly from a
dium metal in napthalene can produce unsaturation in the polymeric backbone and substitute the organo group to
polymer, and subsequent direct grafting of hydrogels can be which it was attached directly to the polymer.31 Application
effected.26 Hydrolysis, particularly of esters, can introduce of this method to the biological field was pioneered by
functional groups such as carboxyls to surfaces. All these Patrick Guire,32 leading eventually to commercialization by
methods involve degradation of the polymer, and it is nec- the BSI (Biometric Systems Inc.) Corporation.33 Numerous
essary to control or limit the degradation to have stable sur- applications are envisioned by BSI to include enhancement
faces, as well as to prevent degradation of the mechanical of endothelial cell attachment, blood compatible surfaces,
properties of the base material. and infection resistant surfaces. While photocoupling ap-
pears to be a facile process of direct coupling to polymers, it
Addition/Substitution is also material dependent, and often requires a pretreat-
Some polymers such as polyurethane have sites that ment of the surface to remove contaminants or promote
can be attacked by proton extracting bases such as sodium better wetting of the coupling reagents. It also often requires
hydride. The reduced urethane group can then undergo alkyl repeated coatings and light exposures to effect a uniform
substitution using alkyl halides such as octadecly iodide.27 and dense coupling. This latter process can result in
An impressive body of work has been documented by the crosslinking of photobiomolecules within a matrix and
group of Cooper, showing improvement in blood compat- somewhat diminish their activity. Careful cleaning of these
ibility and the ability to control the loading of alkyl groups surfaces is required to remove any unreacted or leachable
on the surface. This method of introducing alkyl groups to species, as is the case with almost all surface modification
the surface may provide better and more stable performance approaches. Nevertheless, this method is receiving very fa-
than previously mentioned methods using SMAs. vorable attention as a commercially feasible method to
modify biomaterials, especially for short term use.
Ozonization
Treatment of materials by exposure to ozone has Grafting
proven effective in activating surfaces for subsequent graft- Direct grafting of hydrogels to surfaces is only pos-
ing reactions.28 Ozone can be applied as a gas or dissolved sible on a limited number of materials. The best know ma-
in water, which gives very attractive manufacturing features terial is polyurethane, where the carbamate group can be
for devices with complex geometry and multiple materials, activated using ceric ion initiation.34 Another material also
as ozone is effective on almost all polymers save shown many years ago to undergo direct grafting is dialysis
fluoropolymers. Ozone is primarily an oxidative approach, membrane.35 Early studies on hydrogel grafted surfaces
and can produce peroxy radicals, but also can introduce func- showed promises of being cell friendly.36 Ratner and
tionality from oxidative degradation. Because it can result Hoffman, using a baboon shunt model, later showed that
in degradation, care needs to be taken to control the expo- hydrogel surfaces may produce emboli and, while not being
sure time, concentration of ozone, and temperature. These thromboadherent, were in fact capable of being thrombo-
Biophilic Polymers: What’s on the Horizon? 487
genic. Attempts to utilize the positive aspects of grafted 4. Ratner BD, Weathersby P, Hoffman AS, Kelly MA,
hydrogels by coupling bioactive agents is now commonplace. Scharpen LH. Radiation-grafted hydrogels for biomaterial
A possible advantage of this approach is lower protein ad- applications as studied by the ESCA technique. J Appl
sorption and nonspecific cell adhesion. Grafted hydrogels Polym Sci 1978; 22:643-664.
5. Merrill EW, Salzman EW. Polyethylene oxide as a bio-
do not require crosslinking and when coupled with
material. ASAIO Journal 1983; 6:60-64.
biomolecules can express more bioactivity of the attached 6. Munro MS, Eberhart RC, Make NJ, Brink BE, Fry WJ.
molecule. For most grafted hydrogels some sort of activa- Thromboresistant alkyl-derivatized polyurethanes. J Am
tion of the base polymer is required, such as plasma, co- Soc Artif Int Organs 1982; 6:65-75.
rona, irradiation, or ozonization. Depending on the grafted 7. Leininger RI. Polymers as surgical implants. CRC Critical
species, additional functionalization may be required to pro- Reviews in Bioengineering 1972; 1:333-381.
vide optimal control of coupling with respect to loading and 8. Gott VL, Koepke DE, Daggett RL, Zarnstorff W, Young
spacing chemistry. A good stable grafted hydrogel allows for WP. The coating of intravascular plastic prostheses with
numerous coupling strategies available from affinity chro- colloidal graphite. Surgery 1961; 50:382.
matography techniques. Some affinity schemes have unstable 9. Larm O, Larsson R, Olsonn P. A new nonthrombogenic
bonds that are allowable for chromatographic application, surface prepared by selective covalent binding of heparin
via a modified reducing terminal residue. Biomat Med Dev
but not desirable for biological application. The coupling
Art Org 1983; 11:161-173.
scheme should be tested rigorously to assure that there is no 10. Voorhees AB, Jaretski A, Blakemore AH. The use of tubes
leaching. In our laboratory we have found that grafting fol- constructed from Vinyon “N” cloth in bridging arterial
lowed by intermediate functionalization allows for easier defects. Ann Surg 1952; 135:332.
control and clean up of the grafted surface, as well as opti- 11. Cahalan PT. Brite EuRam Final Technical Report, Con-
mal loading of biomolecules, even to the point of present- tract Number: BREu-336, Project Number BE-5972-
ing the biomolecule in high purity at the uppermost layers 92.Title: Surface modification of Biomaterials for Biomedi-
of the hydrogel.37 cal Devices. European research subsidy granted in Nove.
1992 running until Nov. 1995. Report available through
Summary European Commission, Director General XII, Science, Re-
The horizon for biophilic polymers was discussed in search, and Development, Rue de la Loi 200, B-1049 Brus-
sels, Wetstraat 200, Brussels, Belgium Office: M075 1/5.
this chapter with an obvious bias toward surface modifica-
12. Goldberg et al. Surface modified surgical instruments, de-
tion of polymers to make them biointeractive. Our efforts vices, implants, contact lenses and the like. U.S. Patent
have led to covalent grafted surfaces on almost all common 5,100,689, Assignee: University of Florida, Gainesville
biomaterials including metals, and stable covalent March 31, 1992.
biomolecules coupled to these surfaces. In our opinion, the 13. Andrade JD, Triolo PM. Surface modification and evalu-
closest thing to a biophilic commercially available polymer ation of some commonly used catheter materials. I. J
in the near future would be one that maintains high me- Biomed Mater Res 1983; 17:129-247.
chanical performance characteristics of existing polymers 14. Suzuke M, Kishida A, Iwata H, Ikada Y. Graft copoly-
and contains pendant reactive groups that can have merization of acrylamide onto a polyethylene surface pre-
biomolecules coupled to these functionalities in an economic treated with a glow discharge. Macromolecules 1986;
19:1804-1808.
process. It is doubtful that a biomolecule could be reacted
15. Dias AJ, McCarthy TJ. Synthesis of a two-dimensional ar-
into a material and still maintain its activity through pro- ray of organic functional groups: Surface-selective modi-
cessing. It would be also difficult to expect optimal presen- fications of poly(vinylidene). Macromolecules 1984;
tation of the biomolecule to the blood or tissue. It may be 17:2529-2531.
possible for certain molecules or drugs that are targeted for 16. Okada T, Ikada Y. Tissue reactions to subcutaneously im-
release, but probably not for scaffolding applications. planted, surface modified silicones. J Biomed Mater Res
It is hoped that this chapter gives some useful infor- 1993; 27:1509-1518.
mation regarding several of the methods employed to make 17. Okada T, Ikada Y. In vitro and in vivo digestion of col-
surfaces more biocompatible, and some appreciation for lagen covalently immobilized onto the silicone surface. J
problems and opportunities that are attendant upon these Biomed Mater Res 1992; 26:1569-1581.
methods. Knowledge of several techniques can allow for a 18. Okada T, Tamada Y, Ikada Y. Surface modification of sili-
cone for tissue adhesion. Biomaterials and Clinical Ap-
number of combined processes that can bring optimal per-
plications, 1987.
formance as well as economic reality to creating biocom- 19. Whiteside et al. Langmuir, 1988; 4:365.
patible surfaces. 20. Gauckler L. Personal communication at The Monte Verita
Conference on Biocompatible Materials Systems. Ascona,
References Switzerland, 1993: October 11.
1. Sawyer PN. Surface charge and thrombosis. Ann NY Acad 21. Csomor K, Karpati E, Nagy M, Gyorgyi-Edeleny J,
Sci 1984; 416: 561-584. Machovich R. Blood coagulation is inhibited by sulphated
2. Chin TH, Nyilas E, Turcotte LR. Microcalorimetric and copolymers of vinyl alcohol and acrylic acid under in vitro
electrophoretic studies of protein sorption. Trans Am as well as in vivo conditions. Thrombosis Research, 1994;
SocArtif Intern Organs 1978; 24:389-402. 74(4):389-398.
3. Andrade JD. Interfacial phenomena and biomaterials. Med
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488 Tissue Engineering of Prosthetic Vascular Grafts
22. van Boeckel CAA. From heparin to a synthetic drug: A 30. Cahalan L et al. Biocompatible medical article and
multi-disciplinary approach. Trends in Receptor Research. method. U.S. Patent 5,607,475. Assignee: Medtronic Inc.
Elsevier Publishers B.V. 1993. 1997:March 4.
23. Ishihara K, Nakabayashi N, Nishida K, Sakakida M, 31. Knowles JR. Accounts of Chemical Research 1972;
Shchiri M. Designing biocompatible materials. Chemtech, 5:90-119.
1993. 32. Guire P, Fliger D, Hodgson J. Photochemical coupling of
24. Sioshansi P. Ion beam modification of materials for bio- enzymes to mammalian cells. Pharmacological Research
medical application. Seminar: Biomaterials: Medical and Communications, 1977; 9(2).
Pharmaceutical Applications, sponsored by Technomic 33. Guire PE. Binding reagents and methods. U.S. Patent
Publishing Company, Inc, 851 New Holland Ave., 4,722,906, Assignee: Bio-Metric Systems Inc. 1988:Feb. 2.
Lancaster, PA: 1990. 34. Annual Report (July 1, 1971-June 30, 1972), Medical De-
25. Larsson N, Senius P, Eriksson JC, Maripuu R, Lindberg vices Applications Program of the National Heart & Lung
B. J Colloid Interface Sci, 1982; 90:127-136. Institute, Bethesda, MD.
26. Yun JK, DeFife K, Colton E, Stack S, Azeez A, Cahalan L, 35. Luttinger M, Cooper CW. J Biomed Mater Res 1967; 1:67.
Verhoeven M, Cahalan PL, Anderson JM. Human mono- 36. Ratner BD, Horbett T, Hoffman AS. Cell adhesion to
cyte/macrophage adhesion and cytokine production on polymeric materials: Implications with respect to
surface-modified poly(tetrafluoroethylene/ hexafluoro- biocompatibility. J Biomed Mater Res, 1975; 9:407-422.
propylene) polymers with and without protein 37. Hendriks M. A study on the covalent surface-immobili-
preadsorption. J Biomed Mater Res, 1995; 29:257-268. zation of collagen. Development of biomaterials with en-
27. Pitt WG, Cooper SL. Albumin adsorption on alkyl chain hanced infection resistance; a surface modification ap-
derivatized polyurethanes: I. The effect of C-18 alkylation. proach, Ph.D. Thesis from University of Eindhoven, 1966.
J Biomed Mater Res 1988; 22:359-382. 38. von Recum AF, Park JDB. Permanent percutaneous de-
28. Yamauchi J, Yamaoka A, Ikemoto K, Matsui T. J Appl vices, CRC Crit Rev Bioeng 1981; 5:37-77.
Polym Sci 1991; 43:1197-1203. 39. von Recum AF, van Kooten TG. The influence of micro-
29. Kulik E et al. Poly(ethylene glycol) enriched surface: Re- topography on cellular response and the implications for
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Canada: 1996:May 29-June 2.
Scaffold Engineering
Material Axpects
CHAPTER 45
Bioresorbable Grafts: A Counterintuitive Approach
David Fox, David A. Vorp, Howard P. Greisler
Overview
T his chapter reviews the use of bioresorbable materials in vascular grafting. First, the theo
retical basis for the use of bioresorbable materials is presented. Next, the various materi-
als and the results of experimental work with them are discussed. Bioresorbable materials
have been incorporated into vascular prostheses in a number of novel ways. Accordingly, the
chapter is organized primarily by the manner in which bioresorbable materials have been
incorporated. These include the bioresorbable material as a stand-alone graft, in combina-
tion with nonresorbable materials as a partially resorbable bi-component, or compound
graft, and finally as a supportive scaffold over a biological graft. The chapter also discusses
the use of biological grafts that have been chemically modified so as to be at least partially
resorbable.
Definitions
The terms biodegradable, bioresorbable and bioabsorbable have been used relatively
interchangeably in the literature reporting this heterogeneous class of biomaterials. An at-
tempt was made to reach a consensus on precise definitions at The First International Scien-
tific Consensus Workshop on Degradable Materials held in Toronto in 1989. Biodegradation
was defined as “loss of a property (of a biomaterial) caused by a biological agent.” The Work-
shop was unable, however, to agree on definitions of degradation, bioabsorption and
bioresorption.1 Therefore, in this chapter the terms biodegradable, bioresorbable and
bioabsorbable are in general applied in accordance with the original authors, without impli-
cation of specific mechanisms of degradation.
Introduction
Despite the successful application of prosthetic grafts in the replacement of large ar-
teries, the performance of prosthetics as medium and small caliber replacements has been
less than satisfactory, resulting in both early and late graft failures, increasing the need for
reoperation and limb loss. Factors contributing to these failures include thrombogenicity,
inadequate tissue ingrowth and hyperproliferation of tissue with deposition of extracellular
matrix resulting in myointimal hyperplasia.2
Biodegradable grafts have been investigated as a potential alternative to conventional
biostable prosthetic grafts. The concept of a biodegradable graft emerged from the work of
Arthur Voorhees’ group at Columbia University. In 1954 Voorhees reported the first truly
successful clinical application of prosthetic arterial prostheses, replacing 17 abdominal aor-
tas and 1 popliteal aneurysm with grafts composed of Vinyon-N cloth tubes.3 The success of
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
490 Tissue Engineering of Prosthetic Vascular Grafts
the Vinyon-N fabric prostheses was in large part a function Single Component Resorbable Grafts
of its porosity, which allowed the ingress of capillaries and The materials most commonly used in the fabrication
fibroblasts. This granulation tissue served as the nidus for of resorbable grafts have been synthetic polymers based upon
the formation of an organized inner surface of flattened naturally occurring hydroxy acids such as lactic and glycolic
cells.4 Thus, it became apparent that the graft material itself acid.11 The rate of resorption of these copolymers is deter-
may be needed only transiently, to function as a scaffolding mined to a degree by the ratio of lactide to glycolide rings
for regeneration of the vascular wall rather than as a perma- and decreases as the percentage of lactide component in-
nent conduit. creases.12 The primary mechanism of resorption is by the
After Voorhees’ success, many other materials were hydrolytic cleavage of ester bonds. Inflammatory cells such
proposed. Sigmund Wesolowski and his co-workers, work- as macrophages, lymphocytes and neutrophils perform the
ing at Walter Reed, evaluated 45 prospective materials and final degradation and resorption.11
confirmed the concept that the porosity of a graft material
and not its inertness determined the success of a prosthetic Polyglactin 910 (Vicryl)
vascular graft.5 Prior to this time, the prevailing concept was Bowald’s group from Sweden is credited with the first
that the biological inertness of a prosthetic material was the report of the use of a totally biodegradable prosthesis as a
critical determinant of its clinical success as a vascular graft.6 stand-alone replacement for large caliber vessels.7 Knitted
Wesolowski determined that grafts constructed of materials mesh composed of Polyglactin 910 (PG910), “Vicryl,” manu-
of relatively high porosity were resistant to late calcification factured by Ethicon Inc., was preclotted and interposed as
and occlusion. The problem of hemorrhage during implan- tube and patch grafts into the thoracic aorta of growing pigs.
tation, however, remained an important obstacle. To over- PG910 is a polymer composed of glycolic and lactic acids
come this problem, the concept of constructing a compos- and thus is absorbed by hydrolysis within 70 days.13 The
ite graft emerged. The ideal composite graft would exhibit mesh had a pore size of 400 x 400 microns.14
the property of low porosity at implantation, by virtue of a The grafts were explanted at intervals up to 6 weeks.
degradable component combined with a permanent com- All grafts remained patent and no instances of graft rupture
ponent of high porosity. Over time, the degradable compo- or aneurysmal dilatation occurred. At 40 days only small
nent would be depleted, leaving a high porosity graft ca- fragments of the PG910 remained and the grafted areas dis-
pable of being incorporated by tissue.7 played some similarities to normal aortic walls. The lumi-
The feasibility of incorporating absorbable collagen nal surface was covered with a monolayer of mature endo-
and gelatin components into Dacron grafts had been re- thelial-like cells in a mosaic pattern. Beneath this was a layer
ported by Humphries and Bascon in 1961.8,9 Weselowski of longitudinally directed smooth muscle-like cells. No in-
took a different approach, suggesting the use of a tempo- ternal elastic lamina was seen, although scattered fibrillar
rary scaffold fabricated from a slowly absorbable polymeric elastic material was found interspersed amongst the smooth
material as a vascular graft.5 This scaffold would enable the muscle-like cells. An outer “adventitial” layer was reconsti-
arterial wall to reorganize by virtue of natural repair pro- tuted as well.13 With respect to the source of the repopulat-
cesses.7 The American physiologist Claude Guthrie has been ing cells, Bowald observed that the endothelium and smooth
credited with the origin of this concept. In 1919 he wrote, muscle-like cells seemed to be derived from the cut edges of
“To restore and maintain mechanical function an implanted the native vessel. Occasional inflammatory and giant cells
segment only temporarily restores mechanical continuity were noted around residual PG910 filaments in the 20 day
and serves as a scaffolding or bridge for the laying down of explants.14
an ingrowth of tissue derived from the host.”10 Late results after 2 years were reported for one pig that
Since Wesolowski’s report, many additional biode- received a 10 mm by 4 cm tube graft in the thoracic aorta.
gradable materials have been proposed for use as compo- The graft was patent angiographically. On gross examina-
nents in the fabrication of vascular prostheses. These mate- tion it was free of thrombus, calcification or fatty infiltra-
rials have been incorporated into grafts in a number of in- tion. Of note, the diameter of the grafted area had increased
teresting and novel ways that will be described in this chapter. proportionally in size with the nongrafted aorta and was
The majority of investigators have used the biodegradable not aneurysmal. They also reported reconstitution of a coarse
material as a stand-alone graft. Others have constructed com- internal elastic lamina.15
pound grafts wherein the degradable material is combined Based on these observations, Bowald sought to deter-
with a nonabsorbable prosthetic or biological graft. The mine the maximal regenerative capacity of arterial tissue.
degradable materials have been applied as a lining or wrap To this end, the descending thoracic aorta of 20 pigs was
and in some cases have been interweaved with the prosthetic. bypassed with 8-10 mm diameter double layer Vicryl seg-
The biomaterials used in the fabrication of ments ranging from 7-20 cm in length. All animals receiv-
bioresorbable vascular grafts have a unique dual function. ing a graft greater than 15 cm in length died from graft rup-
They serve as temporary vascular conduits while simulta- ture. There were no ruptures in the animals receiving grafts
neously inducing the regeneration of the arterial wall. As less than 15 cm and the histologic appearance of these grafts
will be elaborated, complex interactions between the was equivalent to those in the previous reports.16
bioresorbable material and host macrophages are a critical Extensive implant and in vitro work with lactic and
step in the process of arterial regeneration. glycolide copolymers have also been done by our group in
the research laboratories of Loyola University. We have in-
Bioresorbable Grafts: A Counterintuitive Approach 491
vestigated PG910 and other copolymers including turally primitive mesenchymal cells that differentiated into
polyglycolic acid (PGA) and polydioxonone (PDS), as well smooth muscle-like myofibroblasts and the establishment
as combinations of these polymers. PG910 grafts were im- of a confluent endothelial-like luminal lining cells was dem-
planted into the abdominal aorta of rabbits. Likewise, the onstrated, thus confirming Bowald’s earlier observations
transinterstitial migration and proliferation of ultrastruc- (Figs. 45.1A-C).14 This process was found to parallel the time
Fig. 45.1. Hematoxylin-eosin histologic sections
(x25) from 1 month post implantation. (A, top)
Dacron prosthesis demonstrating a confluent
endothelial surface with a small subendothelial
layer. (B, middle) Polyglactin 910 prosthesis dem-
onstrating a thick subendothelial cellular inner
capsule with an endothelial luminal surface.
(C, bottom) Polydioxanone prosthesis demon-
strating an endothelial lined luminal surface with
an intermediate subendothelial layer.
492 Tissue Engineering of Prosthetic Vascular Grafts
course of macrophage-mediated prosthetic dissolution. Peak ated aorta was unknown, as was the atherogenicity of the
inner capsule (IC) thickness was achieved at 2 months. Again, vessels.19
there was no graft related morbidity or mortality. Twenty Stimulated by these early results, our group compared
percent of the PG910 grafts become aneurysmal.17 Dacron aortic prostheses to PGA. The Dacron grafts were
In order to further assess the potential contribution fabricated to specifications that closely matched the physi-
of transanastomotic pannus ingrowth to the arterial regen- cal characteristics of woven PGA, most notably in terms of
eration seen, prostheses were constructed of three 10 mm pore diameter, pore area and wall thickness. Over the 12
segments with a PG910 segment interposed between two month observation period both the PGA and Dacron grafts
Dacron segments. Grafts were again implanted in rabbit remained free of thrombosis, infection or hemorrhagic com-
aortas and harvested at intervals up to 4 months. All grafts plications. Again, 10% of PGA grafts showed moderate an-
remained patent with no aneurysms or stenoses. Inner cap- eurysmal dilatation and 15% demonstrated significant inti-
sule thickness in the PG910 segments increased only during mal hyperplasia.20
the interval from 2 weeks to 2 months and was statistically After three months the PGA, as before, was found to
greater than either Dacron segment at 1 and 2 months. In- have been resorbed and in its place a thick, dense neointima
ner capsules of PG910 segments at 1 month were composed lined with a confluent layer of endothelial cells was seen.
predominantly of myofibroblasts. Pannus ingrowth was lim- Beneath this layer were numerous smooth muscle like
ited to the first 2 mm of the inner capsules of the Dacron myofibroblasts in a circumferential and longitudinal orien-
segments, the remainder consisting of a fibrin coagulum. tation. The inner capsules of the Dacron grafts, however,
These findings essentially ruled out transanastomotic pan- exhibited only matrix and small numbers of myofibroblasts
nus ingrowth as the primary source of cells replacing ab- in a radial orientation. The flow surface was thin, fibrinous
sorbable vascular prostheses.18 and acellular.
The Dacron grafts explanted at 1 year contained ex-
Polyglycolic Acid (Dexon) tensive calcium deposits and some advanced atherosclerotic
In 1982, we reported the regeneration of major com- plaques. The PGA explants at 1 year remained remarkably
ponents of the rabbit aorta over a scaffold of absorbable stable with the exception of an occasional lipid-laden his-
porous polyglycolic acid (PGA). PGA has the simplest struc- tiocyte, as had been previously observed.
ture of the linear aliphatic polyesters. It is hydrophilic and Despite their similarities in mechanical porosity, these
loses its strength rapidly over 2-4 weeks.11 Woven PGA, two graft materials achieved remarkably different histologic
manufactured by Davis & Geck, was fabricated into 3.5 mm outcomes, suggesting that porosity was not the only factor
diameter grafts and implanted into the abdominal aorta of active in graft healing. Insight into the pathophysiology of
rabbits and explanted over a seven and a half month period. these differences was gained by studying the grafts explanted
Seventy-six percent (76%) of the animals were found to have after two weeks.
maintained a lumen of 3-4 mm in the region of implanta- As in the previous experiment, an inflammatory re-
tion. Eleven percent (11%) of the animals demonstrated an- action containing macrophages, polymorphonuclear leuko-
eurysmal dilatation in this region; however, no animal died cytes and giant cells was observed in the tissues surround-
of graft rupture. In addition, there was no evidence of ing the PGA grafts. Additionally, numerous macrophages
perigraft hematomas or of false aneurysm formation. There were found within the mesh of the PGA grafts. The Dacron
were no instances of graft thrombosis or infection. Experi- grafts as well were surrounded by an inflammatory reac-
mental regenerated aortas were subjected to bursting tion; however, cellular infiltration was limited to the outer
strength determinations and withstood hemodynamic forces capsule with little or no permeation of the woven Dacron. It
at three to five times systolic pressure. was hypothesized that phagocytosis of PGA by macrophages
Histologic analysis revealed some marked similarities inducing their phenotypic alteration and synthesis of growth
to native aorta. The regenerated aortas were composed of a factors chemotactic and mitogenic for endothelial cells and
luminal surface containing factor VIII positive endothelial myofibroblasts, as well as subtle differences in the surface
cells, a subendothelial zone containing smooth muscle-like electronegativity of the materials, could have accounted for
myofibroblasts amidst dense fibrous tissue and an outer layer differences in transinterstitial migration of inflammatory
of vascularized connective tissue. Elastin regeneration, how- cells.20
ever, was minimal. Some lipid-laden macrophages and his- Additional speculations were made regarding the role
tiocytes were observed in the six month explants, suggest- of growth factors in inducing myofibroblast and endothe-
ing possible early atherogenesis. Intimal hyperplasia result- lial cell ingrowth and deposition. Macrophages were known
ing in severe graft stenosis was observed in 13% of animals. to stimulate the proliferation of endothelial cells, smooth
The PGA graft material underwent progressive disso- muscle and fibroblasts through the elucidation of “macro-
lution. At two weeks the PGA was densely invaded by mes- phage-derived growth factors (MDGFs).”21-23 The degree of
enchymal cells, histiocytes and giant cells. Complete resorp- macrophage infiltration corresponded temporally with
tion occurred between three weeks and three months. myofibroblast proliferation as quantitated by autoradiogra-
Important questions emerged from this study. Prin- phy with tritiated thymidine and suggested the possibility
cipally, it remained to be defined what were the factors re- that a macrophage derived factor played an important role
sponsible for the initiation and modulation of the repair in the modulation of myofibroblast response. The possibil-
process. Additionally, the origin of the cells in the regener- ity that the degradable materials had directly resulted in
Bioresorbable Grafts: A Counterintuitive Approach 493
Fig. 45.3. Midportion of PDS specimen at 2 months show-
Fig. 45.2. Top: Midportion of 1 month Dacron specimen ing capillary invading inner capsule and communicating
showing thin inner capsule (IC) composed of fibrin co- with luminal surface. Endothelial-like cellular luminal
agulum and minimal cellular repopulation. (Hematoxy- surface of specimen appears to be continuous with capil-
lin-eosin; x50). Bottom: Midportion of 1 month lary wall.
polyglycolic acid specimen showing greatly thickened IC
of circumferentially and longitudinally oriented smooth
muscle-like myofibroblasts beneath endothelial-like flow
surface. More peripheral are prosthesis and well vascular-
ized outer capsule. (Hematoxylin-eosin; x50).
Fig. 45.4. Midportion of PDS specimen
at 12 months showing maintenance of
smooth confluent layer of endothelial-like
cells on luminal surface. (Hematoxylin-
eosin; x390).
494 Tissue Engineering of Prosthetic Vascular Grafts
macrophage stimulation was suggested by the demonstra- to have real potential for improved efficacy over prosthetics
tion of PGA remnants within the cytoplasm of macrophages in current use.
by TEM. Additional speculations surrounded the potential
roles of PDGF, ECGF (now FGF-1), and FGF-2 and of me- Characteristics of the Healing Response
chanical forces on the tissue bed, which were found to in- In addition to rate of resorption and kinetics of IC
crease as the PGA degrades and the integrity and mechani- formation, additional studies were performed in order to
cal strength of the graft material was lost.20 further characterize differences between the healing re-
sponses induced by the various materials. The prostaglan-
Polydioxanone (Pds) din metabolite contents of inner capsules formed in the pres-
The ability of lactide/glycolide polymers to induce ence of PDS, PG910 and Dacron containing grafts were com-
arterial regeneration was also demonstrated with grafts fab- pared. PDS inner capsules were found to have greater ratios
ricated from PDS, a more slowly resorbed polymer.24 Grafts, of 6-keto-PGF1#:TxB2 as compared to PG910 and Dacron
24 x 4 mm in size and composed of woven PDS were again implants. Notably, the ratio of 6-keto-PGF1#:TxB2 for PDS
interposed into the abdominal aorta of rabbits. The animals was nearly identical to its normal aortic control. This favor-
were studied at intervals up to one year. Again, no aortic- able ratio suggested that grafts containing PDS may be less
related deaths or hemorrhages occurred. Aneurysmal dila- thrombogenic and again suggests the potential for clinical
tation developed in 10% of specimens, whereas stenosis from efficacy of resorbable grafts.31
neointimal hyperplasia occurred in 15%. As with PGA The kinetics of collagen deposition induced by PDS,
(Fig. 45.2), a vessel wall composed of myofibroblasts and PG910 and Dacron containing grafts was determined by
matrix covered by a confluent endothelial-like flow surface examining the inner capsules of grafts explanted at 1, 3 and
resulted. The PDS implants (Figs. 45.3 and 45.4) yielded an 12 months. In the Dacron grafts, collagen formation peaked
inner capsule (IC) thickness as well as compliance and at 1 month at approximately 12 ∝g/mg. In contrast, the col-
strength characteristics that were comparable to the PGA lagen content of the inner capsules of the PDS and PG910
grafts. Not unexpectedly, tissue formation with the PDS grafts at 1 month was around 27 ∝g/mg. This amount of
grafts occurred more gradually. These grafts reached maxi- collagen is essentially equivalent to that found in normal
mal IC thickness between 3 and 6 months as compared to 2 infrarenal rabbit aorta. The level of collagen in the Dacron
months for PGA. The difference in the rates paralleled the grafts at one year remained the same as at 1 month. The
differing dissolution rates of the two materials.25 These ob- level of collagen in the PDS and PG910 grafts increased to
servations reinforced the concept that the process of arte- approximately 38 ∝g/mg at 3 months, after which it stabi-
rial regeneration was regulated by some factor or factors re- lized.32
lated to the absorption of the material by macrophages. The kinetics of myofibroblast proliferation in the in-
ner capsules of PDS, PG910 and Dacron implants was also
Mechanical Stimuli of Arterial Regeneration analyzed, again in the rabbit infrarenal aorta model. Grafts
A study by Vorp et al to evaluate the stress history were explanted at intervals up to 52 weeks. Mitotic indices
within the inner capsule demonstrated that, early in the re- were determined by autoradiography using tritiated thymi-
sorption phase, the neointima is under a state of circumfer- dine. Of interest was the finding that the mitotic index asso-
ential compression.26 Later in the resorption phase, as the ciated with each graft material was parallel to that material’s
graft material weakens upon dissolution and as the rate of resorption. The peak mitotic index for PG910 was
neointimal tissue strengthens upon reorganization, the in- 28.34 in a 24 h period and occurred at 3 weeks postimplant,
ner capsule stress becomes tensile. The occurrence of com- whereas the peak for PDS was 7.50; this occurred at 4 weeks
pressive stresses parallels the time point and location of and was prolonged. There was little evidence of inner cap-
maximal mitotic index for the neointimal tissue. We showed sule formation in the Dacron grafts until 12 weeks
using the well established rabbit model that the mitotic in- postimplant and the mitotic indices for Dacron grafts never
dex of inner capsule myofibroblasts in PG910 grafts was sig- exceeded 1.22.27
nificantly higher during the early period of resorption than
in the later period.27 Macrophage/Biomaterial Interactions
Using a pulse duplicator apparatus developed at the In order to further characterize the contribution of
University of Pittsburgh,28 changes in the dynamic compli- the macrophage/biomaterial interaction in the elaboration
ance of PG910 grafts occurring over 0 to 36 weeks were as- of factors regulating the process of arterial regeneration, a
sessed.29 Using the rabbit model, explanted PG910 grafts series of in vitro experiments were performed.33-36 In each
were exposed to realistic pulsatile hemodynamics in vitro. A series of experiments, rabbit peritoneal macrophages were
rapid increase in compliance was noted after 3 weeks, level- harvested and cultured in Minimum Essential Medium
ing off at 13 weeks. In a companion study, compliance in- (MEM) with platelet-poor serum and containing particles
creased over time in the proximal and distal graft segments of Dacron, polyglactin 910 or no biomaterial (Fig. 45.5A,B).
compared to baseline. Loss of compliance has been cited as MEM conditioned in this way was collected and mitogenic-
an important mechanism in the failure of small and me- ity assays were performed with quiescent fibroblasts
dium sized Dacron and ePTFE implants.30 Thus, by achiev- (BALB/c3T3), rabbit aortic smooth muscle cells, and mu-
ing greater compliance over time, resorbable grafts appear rine capillary lung (LE-II) endothelial cells. Mitogenic ac-
Bioresorbable Grafts: A Counterintuitive Approach 495
Fig. 45.5. (A, top) Phase contrast photomicrograph of perito-
neal macrophages following six weeks in culture in the pres-
ence of the bioresorbable polyglactin 910. Intracytoplasmic
polyglactin 910 inclusions can be seen (original magnification
x400). (B, bottom) Phase contrast photomicrograph of perito-
neal macrophages following five weeks in culture in the pres-
ence of Dacron. Macrophages can be seen adherent to Dacron
particles but no intracytoplasmic inclusions are observed (origi-
nal magnification x100).
tivity was assayed by scintillation counting of tritiated thy- rophages exposed either to Dacron or to no biomaterial. In
midine incorporation into deoxyribonucleic acid (DNA). In fact, it could be demonstrated that macrophages harvested
order to examine these processes in a model more accurately from rabbits fed a cholesterol rich atherogenic diet exposed
reflecting the clinical setting, macrophages were also har- in culture to Dacron produced conditioned media that ex-
vested from rabbits that had been rendered hyperlipidemic erted an inhibitory effect on endothelial cell thymidine in-
through an atherosclerotic diet.33-35 corporation. In order to isolate the factor or factors respon-
All cell types grown in conditioned media from mac- sible for these alterations in mitogenic activity, Western blot
rophages exposed to PG910 demonstrated significantly analysis was performed using antibodies raised against
greater increases in tritiated thymidine incorporation as PDGF-B chain, acidic fibroblast growth factor (FGF-1) and
compared to those grown in media conditioned from mac- basic fibroblast growth factor (FGF-2). Only anti-bFGF
496 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 45.6. (A) Rabbit aortic smooth
muscle cell bioassay results. Condi-
tioned media from macrophages har-
vested from New Zealand White rab-
bits fed a normal diet and cultured
from 1 through 7 weeks in the presence
of either no biomaterial (first bar),
polyglactin 910 (second bar), or
Dacron (third bar). Results are ex-
pressed as counts per minute of the
sample divided by counts per minute
of the fetal bovine serum positive con-
trol x 100 (mean ± SD). (B) Rabbit aor-
tic smooth muscle cell bioassay results.
Conditioned media from macrophages
harvested from New Zealand White
rabbits fed a 2% cholesterol/6% pea-
nut oil diet and cultured from 1
through 7 weeks in the presence of ei-
ther no biomaterial (first bar),
polyglactin 910 (second bar), or
Dacron (third bar). Results are ex-
pressed as counts per minute of the
sample divided by counts per minute
of the fetal bovine serum positive con-
trol x 100 (mean ± SD).
antibody immunoreacted with samples of the conditioned smooth muscle cells grown in conditioned media from mac-
media.33 rophages exposed to Dacron (Fig. 45.6A). When macro-
In order to further define the contribution of bFGF phages were obtained from atherosclerotic rabbits a gener-
to the mitogenic response, conditioned media was alized decrease in the incorporation of tritiated thymidine
preincubated with neutralizing anti-basic-FGF antibody. In by smooth muscle cells occurred in both the Dacron and
rabbits fed normal diets, Dacron induced mitogen release PG910 groups. The macrophages from atherosclerotic rab-
was completely inhibited by anti-basic-FGF antibody. PG910 bits cultured with Dacron, however, showed a particular in-
mitogen release was diminished, but to a significantly less crease in the mitogenic factors most active on the smooth
degree (36%). Thus it appeared that much of the mitogenic muscle cells (Fig. 45.6B). To further characterize this effect,
effect on smooth muscle cell was mediated via basic-FGF.35 Western blot and immunoprecipitation studies were per-
In another series of experiments, the effect of condi- formed, and as before, the presence of FGF-2 was demon-
tioned media on rabbit aortic smooth muscle cells was as- strated, whereas PDGF and FGF-1 were not. TGF-! inhibi-
sessed. Smooth muscle cells grown in conditioned media tory activity was demonstrated to be increased for the ath-
from macrophages exposed to PG910 again resulted in sig- erosclerotic macrophage containing media. These findings
nificantly higher tritiated thymidine incorporation than. support the hypothesis that interactions between Dacron,
Bioresorbable Grafts: A Counterintuitive Approach 497
macrophage and smooth muscle cells play a role in the patho- all groups; however, only the PU/PLLA grafts reconstituted
genesis of graft failure.34 an elastic lamina. They concluded that both biodegradation
and compliance were important stimuli of arterial
Polyurethane/Poly-L-Lactide Copolymers (Pu-Plla) regeneration.40
A different approach has been taken by the polymer To characterize the functional characteristics of the
chemists at the University of Groningen in The Netherlands. regenerated arteries, endothelialization and prostacyclin syn-
They have worked extensively with copolymers of polyure- thesis were determined using a bioassay and RIA for the
thane and poly-L-lactide (PU-PLLA) with the goal of com- prostacyclin metabolite 6-oxo-PGF1#. Comparisons were
bining the properties of the two materials to produce an anti- made between PU/PLLA and PTFE grafts and normal en-
thrombogenic, flexible as well as resorbable material. PU- dothelium. Looking at prostacyclin production per unit graft
PLLA was described by Gogolewski in 1982.37 area covered with neoendothelium, there were no signifi-
Lommen et al interposed 1.5 x 10 mm PU-PLLA grafts cant differences between the two graft types and native ar-
into the abdominal aorta of rats.2 These grafts were of tery. Endothelialization and healing, however, as determined
40-50 ∝m porosity and were explanted at 3, 6 and 12 weeks. by light and electron microscopy were incomplete in the
These grafts were compared to a control group that received PTFE grafts, even after 12 weeks.41 Additional ultrastruc-
PTFE grafts of the same dimensions and of 30 ∝m porosity. tural analyses were performed and, as was observed by our
At explant all grafts were patent and there was no evi- group, epithelioid cells and multinucleated giant cells were
dence of aneurysm formation, dilatation or other compli- seen to engulf polymer particles of the disintegrating grafts.42
cations in either group. By 6 weeks the PTFE grafts had de- The long term fate of PU/PLLA grafts was assessed in
veloped a complete “endothelial” lining that was continu- a group of 8 implants. After 1 year all grafts were found to
ous with that of the adjacent vessel. However, this lining be patent. Three of the grafts were of normal shape and were
detached easily during processing, suggesting that there was visibly pulsatile. Two grafts were mildly dilated, 2 were an-
poor attachment with the prosthesis. The internodal spaces eurysmal and 1 had sustained a dissection. These grafts were
of the graft appeared to contain fibrin. Similar findings were not visibly pulsatile. Histologic analysis of the “neomedia”
noted in the 12 week PTFE explants. revealed circularly arranged smooth muscle cells (as in nor-
The PU-PLLA grafts demonstrated arterial regenera- mal artery) in the normally shaped implants. However, in
tion. A complete “endothelial” lining was observed at three the nonpulsatile implants, the neomedia had a predomi-
weeks. Likewise, the outer surface of the grafts was covered nantly longitudinal arrangement. They concluded that the
with vascularized connective tissue that was morphologi- pattern of arrangement of smooth muscle cells in the
cally indistinct from native adventitia. At 6 weeks giant cell neomedia affects the fate of the regenerated arteries.43
activity was observed within the graft walls, most promi- There was uncertainty as to the origin of the cells in
nently at the periphery. By 6-12 weeks a subintimal layer the regenerated arteries. Hypothesized origins have included
containing “smooth muscle cells,” collagen and elastin (dem- migration from the cut ends of the adjacent vessel and the
onstrated by Orcein staining) had developed. Multiple fe- granulation tissue surrounding the graft, as well as deposi-
nestrated elastic laminae were observed as well. The body of tion of circulating cells onto the luminal surface.44 To gain
the PU-PLLA graft gradually became infiltrated with cells, insight into the source origin of the cells in the arteries re-
and this process was associated with progressive degrada- generated from PU/PLLA, sequential explants at intervals
tion of the graft substance.2 of 1 h to twelve weeks were performed. They observed that
Having demonstrated that biodegradable PU-PLLA the endothelium originated from adjacent intima. The
stimulates the regeneration of an arterial wall they next smooth muscle cells seemed to originate from the media of
sought to optimize some of the properties of the grafts re- the adjacent aorta; however, they could not exclude the other
sponsible for this process. They postulated that the compli- postulated mechanisms which would be required for clini-
ance of the grafts was an important property and they per- cal applicability, and proposed experiments with longer
formed in vitro stress/strain measurements of grafts com- grafts to decrease the likelihood of trans-anastomotic cell
posed of several combinations of PU and PLLA and com- migration.44
pared them to native artery. They determined that grafts Other work with lactide polymers has been done by
composed of 5-20% PLLA possessed compliance before Hanson’s group at the University of Texas. They fabricated
implantation comparable to rat aorta. 38 They subsequently grafts with copolymers of L-lactide, DL-lactide, and
investigated the effect of varying the pore size of grafts com- >-caprolactone in various compositions. Lactide polymers
posed of 95%/5% PU/PLLA in the rat model. They deter- were chosen for their strength, >-caprolactone for their flex-
mined that there was no distinct advantage of a pore size of ibility. The grafts were 3 mm by 8 cm, nonwoven, nonpo-
40 ∝m as compared to pores in a gradient of 10-100 ∝m from rous and completely resorbable. The mechanical properties
the inner to the outer regions of the graft in terms of arterial and degradation rates of the grafts were evaluated in vitro.
regeneration. The advocated the use of a pore gradient, how- Circumferential tension, compliance, and kink angle were
ever, as the smaller pores on the luminal surface were thought determined. Tensile strength and modulus of elasticity were
to decrease thrombogenicity.39 found to be in the range of normal artery; however, the grafts
They compared these compliant grafts to less compli- were significantly less compliant than artery. It was antici-
ant grafts composed of biostable PU and of PU/PLLA sur- pated that the addition of porosity, a necessity for in vivo
rounded by PTFE. Partial arterial regeneration was seen in
498 Tissue Engineering of Prosthetic Vascular Grafts
studies, would improve compliance. Further investigations which more closely approximate the situation of clinical
were planned.45 vascular grafting, resorbable grafts have had an increased
incidence of graft dilation, aneurysm formation and rup-
Polyurethane Elastomers (PU) ture.47-49 Investigators have taken several approaches to
Extensive work has been done in the evaluation of counteracting this problem, including incorporating
small diameter vascular grafts composed of polyurethane resorbables with enhanced resistance to degradation,7,50-56
elastomers. Polyurethanes hold promise by virtue of their increasing the rapidity of tissue regeneration with growth
superior compliance as compared to conventional prosthet- factors57,58 and cell seeding.59,60 Others have taken an inter-
ics. By and large, these polyurethanes are nonresorbable mediate approach and constructed compound or partially
(biostable).46 Gogolewski fabricated vascular grafts from a resorbable vascular prostheses combining resorbable and
bioresorbable segmented aliphatic polyurethane (PU). Like permanent materials.
the PU/PLLA grafts, these were degradable, microporous and
compliant. Grafts of 1.5 mm inner diameter and 0.7 mm Enhancing the Resistance to Degradation
wall thickness were interposed into 13 mm defects in rat of Resorbable Materials
abdominal aorta and explanted at intervals between 3 and Our group evaluated woven bicomponent totally
16 weeks. The luminal surface of the graft consisted of in- bioresorbable grafts fabricated from compound yarns con-
terconnected pores of 5-10 ∝m. The outer surface of the graft taining 74% PG910 with 26% polydioxanone (PDS). The
had a porosity of 20-50 ∝m. The tissue response was reported resorption kinetics of PDS are slower than PG910. Thirty-
as being comparable to that previously observed with seven grafts were implanted into the infrarenal aorta of rab-
polylactide and polyglycolide. Again, prosthetic debris were bits and explanted at intervals between 2 weeks to 12 months.
surrounded by giant cells and macrophages. A “glistening Complete resorbtion of the PG910 occurred by 2 months
neointima” was observed at 3 weeks, with “no tendency” to- and the PDS by 6 months. All grafts were patent at explant.
wards intimal thickening. Smooth muscle cells and elastin One graft developed a 50% stenosis that appeared to be
fibers were observed in the intima. By four months almost thrombotic in origin. No aneurysmal dilatation was seen in
complete degradation of the PU had occurred. Stress/strain any graft. Inner capsule thickness was intermediate to that
analysis revealed mechanical characteristics similar to na- seen with PDS and PG910.51
tive aorta.46 The Groningen group hypothesized that the dilata-
Encouraged by these results, 8 mm by 6 cm grafts were tion observed with their PU/PLLA grafts was related to tech-
implanted into the infrarenal aorta of 50 young pigs. The nical problems with the dip-coating process of graft prepa-
grafts were explanted at intervals of 1 month to 1 year. Eigh- ration.52 They produced a modified 2-ply graft with a more
teen pigs died as a result of prosthetic rupture, infection or biostable inner layer. The inner layer was made highly anti-
thrombosis. Ten pigs received no aspirin and a regular diet. thrombogenic by crosslinking of a mixture of linoleic acid
These grafts were all thrombosed by 90 days. Twenty-five and an aliphatic polyetherurethane with dicumyl peroxide.
pigs received aspirin; 55% of these grafts remained patent The outer ply was a (95/5) mixture of polyesterurethane and
at 90 days. Only 3 were patent at 1 year. Of 15 pigs receiving poly(L-lactide).
a diet high in lipid, 7 remained patent at 1 year. Three of Twenty-two two-ply grafts were implanted in the ab-
these animals were noted to have dilation of the prosthesis dominal aorta of rats. All grafts remained patent at least up
similar to that observed by our group in PG910 grafts placed to 1 year and did not exhibit any aneurysm formation. As in
in the abdominal aorta of rabbits fed an atherogenic diet. 47 the earlier formulations, the inner layer was covered with
It was postulated that the enhanced patency of these grafts endothelial cells and several layers of smooth muscle cells.53
was related to an anti-thrombotic effect of the lipid-rich Galletti’s group at Brown University approached the
diet.48 problem of aneurysmal degeneration of Vicryl grafts by coat-
Histologic analysis again revealed macrophage and ing the yarns used in the fabrication process with slowly re-
giant cell infiltration of the prosthesis and phagocytosis of sorbed polyesters, thus extending their functional life. They
prosthetic debris. A neointima and neomedia containing produced tubular conduits, 1 mm by 3 mm, composed of
longitudinal and transverse smooth muscle cells developed triple ply Vicryl coated with a 1:1 composite of poly-DL-
by 180 days. A fibrous neoadventitia was observed at early lactic acid and poly-2,3-butylene malate or poly-2,3-buty-
time points; however, at 1 year this was comprised of fatty lene fumarate. Coated or uncoated Vicryl grafts were im-
tissue. Elastic fibers dispersed throughout the muscle layer planted subcutaneously in mice. Samples were retrieved at
were observed. The prostheses were completely resorbed by intervals between 2 and 12 weeks and the remaining mass
1 year. Prostacycline synthesis was found to be comparable of polymer implant was analyzed for the presence of Vicryl
to normal aorta with no apparent differences between using gel permeation chromatography.
groups.48,49 After 8 weeks Vicryl was undetectable in the uncoated
implants. Grafts coated with poly-2,3-butylene malate or
Preventing Aneurysmal Dilatation poly-2,3-butylene fumarate, however, retained 48 and 88%
The chief obstacle in the development of a clinically of their Vicryl content respectively.54
useful resorbable graft has been the potential for loss of Galletti’s group next constructed 8 mm by 13 cm grafts
mechanical and structural integrity in the graft prior to the of triple ply Vicryl coated with a 1:1 composite of poly-DL-
regeneration of an arterial wall. Adding to this concern is lactic acid and poly-2,3-butylene fumarate. To minimize
the finding that when evaluated in hyperlipidemic models, oozing through the graft interstices, they added a fourth
Bioresorbable Grafts: A Counterintuitive Approach 499
outer layer of a 1 cm wide ribbon of Vicryl mesh wrapped Cell Seeding
in a spiral around the graft. The grafts were inserted in the The Groningen group sought to accelerate the regen-
infrarenal aorta of dogs. Grafts were retrieved at intervals eration of the neomedia through the application of smooth
up to 12 weeks. They found that at 8 weeks the polymer was muscle cell seeding.59,60 Cultured smooth muscle was seeded
substantially reduced and by 12 weeks had nearly disap- into PU/PLLA biodegradable vascular grafts and implanted
peared. Stress/strain analysis of the resulting inner capsules into the abdominal aorta of rats. Arterial wall regeneration
demonstrated properties similar to natural vessels.55 was evaluated at intervals between 2 h and 1 week. The seeded
They next investigated prostheses composed of Vicryl grafts showed a more rapid and uniform development of
coated with either poly-DL-lactic acid and poly-2,3-buty- the neomedia (clearly discernible at 2 days) as compared to
lene fumarate (PDLA-PBF) or poly-DL-lactic acid and poly- nonseeded control grafts. No grafts demonstrated aneurysm
2,3-butylene succinate (PDLA-PBS). Grafts (8-9 mm in in- formation or dilatation in these short term implants.60
ternal diameter, 8-10 cm long) were implanted in the
infrarenal aortic position of dogs and explanted at intervals Compound Partially Bioresorbable Vascular Grafts
up to 24 weeks. All animals receiving coated prostheses sur- Others have taken an intermediate approach and de-
vived, whereas one animal receiving an uncoated Vicryl pros- veloped compound, or partially resorbable, vascular pros-
thesis died from graft rupture. Grafts were patent in 14 of theses combining resorbable and permanent materials. Early
18 animals at the time of retrieval. studies were performed by Wesolowski’s group in the late
Histologic analysis revealed no cellular invasion of the 1950s and 1960s. They evaluated 24 different combinations
Vicryl mesh in the PDLA-PBF coated grafts. The PDLA-PBS of graft materials in dogs and pigs. They found, in general,
coated grafts, however, demonstrated cellular elements, and that coated grafts performed less favorably than compound
fusion of inner and outer tissue layers could be demon- grafts wherein the resorbable component was an integral
strated. Isolated patches of endothelium-like cells were noted part of the fabrication. Deleterious changes (calcification)
in the mid-region of the PDLA-PBS coated grafts; other- did occur in the compound grafts; however, this occurred
wise, composition of the inner and outer capsules did not in a delayed fashion thought to be a result of the resorbable
differ significantly between groups.56 component. The most satisfactory results were achieved with
grafts composed of a compound yarn of resorbable catgut
Increasing the Rapidity of Tissue Regeneration that had been wrapped with multi-filament polyester yarn.5
with Growth Factors More recent work on compound vascular grafts has
Our group sought to minimize the likelihood of graft focused largely on the of incorporation of lactide polymers.
rupture by accelerating the regeneration of the arterial wall An early report came from Ruderman’s group at Walter Reed.
through the local application of growth factors. We They evaluated woven grafts composed of 24% PLA and 76%
documented our ability to attach FGF-1, a potent endothelial Dacron implanted into the abdominal aorta of dogs. After
cell mitogen, and FGF-1-heparin complexes to PDS 100 days they found all grafts to be patent and to show ex-
prosthesis.58 Dacron or PDS grafts were then interposed into tensive tissue ingrowth.63
the infrarenal aorta of rabbits and explanted at intervals up
to 30 days. We confirmed the method of attachment of FGF-1 Compound Grafts: PG910
to biomaterial surfaces and documented the retention of Bowald’s group investigated compound grafts as well.
FGF-1 to the graft while in circulation. At 1 month all grafts They compared double layer Vicryl grafts with Vicryl grafts
were patent and without stenoses or aneurysm formation. anchored inside PTFE or Dacron mesh tube grafts. The grafts
No significant differences were demonstrated between FGF-1 were implanted into the descending aorta of pigs. Nearly
and control groups in terms of cellularity of luminal sur- half of the pigs receiving Vicryl grafts experienced graft rup-
faces, inner capsule thickness or capillarization of the ture. The pigs receiving grafts with external support did not
inner capsule.61 experience graft rupture. Histologically, the supported Vicryl
After explantation, residual [125I]FGF-1 was eluted grafts were found to have the microscopic picture of arterial
from the prostheses. Intact FGF-1 was identified by SDS gel regeneration, although areas of degenerative change and
electrophoresis. Similarly prepared prostheses were ex- calcification were noted. Increased collagen formation was
planted after 7 days and their FGF-1 eluted off for in vitro noted in the Vicryl + Dacron group.16
documentation of activity. Eluted FGF-1 was found to have Our group as well investigated Vicryl (PG910) in com-
retained its mitogenic properties, causing a 1000-1200% bination with Dacron and ePTFE.17,31 In addition, we in-
increase in [3H]thymidine incorporation into newly synthe- vestigated combinations of Vicryl and polypropylene. 64-66
sized DNA in test murine LE-II cells.61 We hypothesize that Vicryl grafts surrounded by Dacron or PTFE showed no in-
the lack of difference in healing seen between control and cidence of aneurysmal dilatation as compared to a 20% di-
growth factor treated grafts relates to the inactivity of bound latation rate for unwrapped Vicryl grafts. Arterial regenera-
FGF-1. It is also possible that differences may have occurred tion occurred in the wrapped grafts, but in an incomplete
earlier than the 30 day explant time used in this study. We manner. Grafts were also fabricated from compound yarns
have subsequently altered our approach and are applying composed of blends of Vicryl and Dacron. The rate of an-
growth factors to grafts in polymerized suspensions of fi- eurysmal dilatation increased proportionally with the per-
brin glue.62 We have not yet applied this technique to centage of Dacron in the blended yarn. A maximum dila-
resorbable grafts. tion rate of 83% was seen with the 56% Dacron grafts. No
500 Tissue Engineering of Prosthetic Vascular Grafts
dilatation was seen when 20% Dacron was used. Cellularity finding to the decreased compliance associated with the
of the regenerated tissue in general was decreased in the pres- PTFE wrap.40
ence of Dacron, suggesting an inhibitory effect of the bio-
material.17 Subsequent work demonstrated decreased pros- Compound Grafts: Polyethylene Oxide
taglandin content of the inner capsules of Dacron/Vicryl and Polylactic Acid Copolymers (PELA)
composite grafts as compared to those of Vicryl grafts.31 The Biomaterials Research Laboratory at The Hebrew
In order to avoid the deleterious effects of Dacron, University of Jerusalem has developed and evaluated com-
compound grafts were constructed using polypropylene, pound vascular prostheses fabricated from resorbable block
which possesses greater biocompatibility and a incites only copolymers of polyethylene oxide and polylactic acid (PELA)
a relatively low-grade inflammatory response.65 Grafts fab- applied as a coating to knitted Dacron and polether ure-
ricated from yarns containing 69% Vicryl (PG910) and 31% thane urea (Lycra) grafts. PELA is an elastomer that fully
polypropylene were implanted into rabbit infrarenal aortas. degrades in 3 weeks after implantation.68-71 The in vivo per-
Over 2 weeks to 12 months all grafts remained patent with- formance of the Dacron grafts was evaluated in dogs as right
out aneurysm formation. Only residual polypropylene re- ventricle to pulmonary artery conduits. Unlike control wo-
mained in the prostheses after 2 months. Production of ven grafts, the PELA coated grafts showed complete trans-
6-keto-PGF1# was in the normal range.64 mural tissue ingrowth after 14 months.68,69
PG910/polypropylene prostheses were also implanted In a separate study, the Hebrew University group com-
into the aorto-iliac positions of dogs. Again, all grafts were pared ePTFE grafts to grafts comprised of Lycra fibers coated
without aneurysm formation over one year. Patency was with PELA. 6 mm by 6 cm grafts were implanted in the ca-
enhanced as compared to Dacron and ePTFE control grafts. nine carotid artery for 90 days. Unlike the ePTFE grafts, the
Arterial regeneration was demonstrated.65 PELA coated grafts remained compliant and demonstrated
successful healing and incorporation. Further work with
Compound Grafts: PGA small caliber prostheses was anticipated.70
Our group also evaluated grafts made of polyglycolic
acid (PGA) fabric reinforced by an outer wrap of Dacron. Resorbable Outer Wraps
In this series, the incidence of aneurysmal dilatation was zero A novel application of resorbable materials in vascu-
with or without the outer wrap up to nine months after lar grafting has been reported by Moritz and the Groningen
implant. Again, Dacron appeared to have an inhibitory ef- group.72-75 Moritz applied a constrictive mesh tube of
fect on the cellularity of the luminal surface and inner cap- resorbable mesh to dilated, otherwise unusable veins with
sule.17 Evaluation of an advanced PGA based compound the goal of inducing neoarterial wall growth. He found that
prosthesis is currently underway in our lab. the meshes can effectively reduce the diameter of a venous
Chu’s group at Cornell also investigated the combi- graft by providing external support to the vessel wall, thus
nation of PGA and Dacron. They produced knitted fabric limiting distension.72 The Groningen group has also inves-
grafts composed of PGA and Dacron fibers blended at vari- tigated biodegradable prostheses as external supports for vein
ous compositional ratios. They studied the properties of grafts. They sought to prevent vein graft wall damage due to
these bicomponent fabrics in vitro. They felt that their most the higher arterial pressures encountered after implantation.
important finding was the achievement of increasing water They found that the prostheses can function as a protective
porosity over time without significant losses in the struc- scaffold for vein grafts in the arterial circulation, reducing
tural integrity and strength of the specimens.67 damage to the vein graft wall and allowing gradual arterial-
Having demonstrated the effectiveness of partially ization.73-75
resorbable arterial prostheses in rabbits, we sought to evalu-
ate longer grafts in a higher order species. 4 mm by 5 cm Biopolymers: Resorbable Prostheses
conduits were woven from composite yarns containing 70% of Biologic Origin
PDS/30% polypropylene and implanted into the aorto-iliac Another novel approach has been taken by several
positions of dogs for periods of up to one year. No aneu- groups in Europe and the United States who have sought to
rysms or perigraft hematomas developed. The patency of develop resorbable prostheses through the chemical modi-
the PDS/polypropylene grafts was significantly greater than fication of arterial xenografts.76-81 Vascular prostheses of
that of Dacron or ePTFE control grafts. Inner capsules were biological origin have been shown to develop aneurysms
completely endothelialized by 1 month. IC cellularity and within several years of implantation. It has been hypoth-
thickness were greater than those within Dacron or ePTFE.65 esized that, if properly processed, xenografts can function
as a bioresorbable scaffold. Should this occur, an orderly re-
Compound Grafts: PU/PLLA generation of structural elements during healing could take
The Groningen group investigated compound grafts place, the result being continued mechanical integrity of the
of PTFE fitted around PU/PLLA. They found that these graft.78
grafts, as compared to grafts composed entirely of The biopolymer that has received the most attention
bioresorbable PU/PLLA or biostable but compliant PU, had is aldehyde crosslinked collagen.76 Moczar’s group in France
diminished regeneration of all layers of the arterial wall, most developed a biodegradable microarterial graft from rat aorta.
strikingly in terms of elastic lamina. They attributed this Trypsin treated arterial segments were coated with heparin
or chondroitin sulfate to reduce thrombogenicity. Grafts
Bioresorbable Grafts: A Counterintuitive Approach 501
were implanted into the infrarenal aorta of rats for 3-8 weeks. 15. Audell L, Bowald S, Busch C, Eriksson I. Polyglactin mesh
The grafts were found to undergo resorption in parallel with grafting of the pig aorta. The two-year follow-up in an
re-endothelialization and scar tissue formation. Patency was experimental animal. Acta Chir Scand 1980; 146:97-9.
around 50%.76 Macromolecular repair of the host aortic wall 16. Bowald S, Busch C, Eriksson I. Absorbable material in
vascular prostheses: A new device. Acta Chir Scand 1980;
was documented in one year explants by the incorporation
146:391-5.
pattern of [3H]valine into proteins and the demonstration 17. Greisler HP, Schwarcz TH, Ellinger J, Kim DU. Dacron
of de novo elastin synthesis in the scar replacing the pros- inhibition of arterial regenerative activities. J Vasc Surg
thesis.77 1986; 3:747-56.
18. Greisler HP, Dennis JW, Endean ED, Ellinger J, Buttle
Conclusion KF, Kim DU. Derivation of neointima in vascular grafts.
The resorbable graft represents a paradigm shift in Circulation. 1988; 78:I6-12.
vascular prostheses. Unlike conventional prostheses whose 19. Greisler HP. Arterial regeneration over absorbable pros-
effectiveness depends by and large upon an inherent resis- theses. Arch Surg 1982; 117:1425-31.
tance to a natural healing process, resorbable grafts work 20. Greisler HP, Kim DU, Price JB, Voorhees AB Jr. Arterial
with the body, providing a framework for well established regenerative activity after prosthetic implantation. Arch
Surg 1985; 120:315-23.
healing patterns. It seems intuitive that by augmenting rather
21. Leibovich SJ, Ross R. The role of the macrophage in
than opposing these patterns a more efficacious vascular wound repair. A study with hydrocortisone and
graft should result. Great strides have been made toward this antimacrophage serum. Am J Pathol 1975; 78:71-100.
end. Further research will yield grafts for clinical evaluation 22. Martin BM, Gimbrone MA Jr, Unanue ER, Cotran RS.
in the near future. Stimulation of nonlymphoid mesenchymal cell prolifera-
tion by a macrophage-derived growth factor. J Immunol
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Biomaterials 1994; 15:83-91.
Scaffold Engineering
Material Aspects
CHAPTER 46
Biodegradable Materials
K.J.L. Burg, S.W. Shalaby
Introduction
A bsorbable materials are unique as implants, in that they are absorbed and excreted from
the body at the conclusion of their functional period, thus alleviating the expense and
potential complications of a retrieval surgery. Additionally, as these materials gradually ab-
sorb, they allow a gradual shift in mechanical strength from the biomaterial back to the
healing tissue. The absorption time and therefore loss of mechanical strength of the implant
may be modulated to best match a given application.
Synthetic absorbable materials were first developed as medical implants in the late
1960s and found use in applications such as barriers, scaffolds, and drug delivery systems.1
Prior to this time, naturally derived materials such as collagen, catgut, and silk were used;
however, they were subject to unpredictable, enzymatic degradation and elicited immune
responses due to impurities. The evaluation and application of absorbable materials gradu-
ally expanded from soft tissue devices into bone fracture fixation devices, an area of ongoing
development.2-5 A more recent area of interest is the role of absorbable materials as struc-
tural matrices or supports in tissue engineering.6,7
Criteria for Useful Tissue Engineering Materials
Tissue engineering involves organ development and replacement by the seeding of
cells on or into a polymer matrix which may be either cultivated in vitro or implanted di-
rectly in vivo. The selective use of parenchymal cells minimizes the amount of donor tissue
required and, rather than implanting a large volume of avascular tissue which may not sur-
vive, the revascularization process occurs simultaneously with the tissue growth and mate-
rial bioabsorption. Absorbable materials have found an important role as a matrix material
in such constructs. Indeed, the advent of tissue engineering technology opened yet another
area of absorbable materials research.
Absorbable and biodegradable polymer technology lends itself well to tissue engi-
neering in that it provides a means of creating a temporary, prefabricated tissue template
into or on which cells may be seeded. The absorbable polymer construct is a temporary,
structural support allowing and protecting new tissue growth and gradually absorbing as
the tissue acquires the desired strengths and functions. Development of the tissue may be
begun ex vivo,8 where the polymer supports and maintains the tissue integrity during im-
plantation. The early attempts to grow tissue combined cells with collagenous two-dimen-
sional substrates.9-11 Research has become diverse, with replacement potential in many ar-
eas including liver, skin, cartilage, breast, nerve, and bone repair. Along with the growth in
possible applications came also the development of more suitable forms for three-dimensional
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
506 Tissue Engineering of Prosthetic Vascular Grafts
tissue culture. This included injectable gels and gel formers, sizes larger than 10 micron will allow invasion of fibrovas-
fibrous scaffolds, porous foams, and channeled substrates cular tissue and therefore enhance capillary network devel-
to allow vascularization. The processing techniques also de- opment and subsequent vascularization.16 The rate of the
veloped to allow more custom-made designs and provision tissue infiltration increases with pore size and porosity, and
of more complicated, site-specific shapes. The processing of its survival and integration with the host tissue depends
an absorbable material allows a reproducible end product, largely on the vascularization.
and the use of an absorbable material reduces both the cost An even distribution of cells throughout the matrix is
and the risk of donor and recipient surgery. important to the development and functionality of the tis-
There are many synthetic polymers which are used in sue, as well as the interaction of the device and transplanted
tissue engineering research, among which are polylactide, cells with the surrounding tissue. The hydrophilicity of the
polyglycolide, polyphosphazene, polycaprolactone, and vari- polymeric surface contributes to the wettability of the po-
ous copolymers. Copolymers such as lactide-colysine have rous structure and hence to the distribution of cells through-
been specifically developed in order to provide attachment out the matrix. The crystalline polylactide and polyglycolide
points for cell binding peptide groups.12-14 The synthetic materials are relatively hydrophobic; therefore, aqueous cell
polymers are advantageous in that their properties may be solutions do not adequately infiltrate the polymeric inte-
directly modulated during synthesis and processing to rior. Less polar solvents such as ethanol may be used to
achieve particular requirements. They tend to degrade in a prewet the scaffold before applying the cell solution;17 the
very predictable fashion, largely hydrolytically, which has concern then becomes the minimization of the scaffold-sol-
obvious benefits. vent contact time in order to avoid excessive ester bond cleav-
The natural materials which have found use in tissue age. Another approach to improve cellular distribution
engineering research include alginate, collagen, chitosan and throughout the scaffold without addressing the bulk sub-
coral, as well as similarly peptide modified natural materi- strate properties is to surface coat the device with an ab-
als (Table 46.1). These materials are naturally derived and sorbable, hydrophilic material prior to cell seeding. Low
therefore readily available; their absorption is generally en- molecular weight, soluble polyvinyl alcohol coatings, as well
zymatically driven and subsequently less predictable. Typi- as pluronic coatings, may be successfully applied to these
cally this group of absorbable materials may vary consider- hydrophobic substrates, thus rendering them more attrac-
ably from batch to batch. They do, however, allow gelati- tive for cellular attachment.18 These surfactants do not im-
nous, injectable structures and therefore a broader range of prove the cellular adhesion to the polymeric structure, but
applications. they do allow a much better cell distribution throughout
There are many factors to consider when designing the matrix. Furthermore, they dissolve readily in aqueous
an absorbable substrate for tissue engineering purposes. The environment and are therefore unlikely to interfere with the
amount of exposed polymeric surface area will relate to the intended implant function.
intensity of the inflammatory response; therefore, an im- The criteria for a tissue engineered absorbable vascu-
plant with greater exposed surface will induce a greater re- lar graft material are very focused. The material must be
sponse than a smaller area.7 The polymer purity, crystallin- readily sculpted into an appropriately sized tubular shape,
ity, molecular orientation, molecular weight, and polydis- matching that of surrounding target tissue and, more prob-
persity may all influence the presence and timing of an in- lematic in the case of small diameter vessels, must be able to
flammatory response. The effect of bulk mass loss of the withstand large flow forces without hindering transport. The
polymeric material on the success rate of the newly devel- construct must be a specific porosity in order to optimize
oped tissue is, as a result, an important design issue. the tissue integration and reformation. The tissue ingrowth
The pore size of a three-dimensional scaffold, as well must occur over the entire length of the prosthesis rather
as the means of dispersion and flow rate of cells into the than primarily from a transanastomotic pannus ingrowth.
scaffold, is of importance in determining both the distribu- Most importantly, it must have an anti-thrombogenic lu-
tion of cells and the integrity of the cells.15 Additionally, pore menal cellular lining and must facilitate long term viability.
Table 46.1. Absorbable materials used in tissue engineering
Absorbable Meterial Material Type Typical Forms
polylactide polyester porous sponges
polyglyolide polyester fibrous meshes
alginate polysaccharide hydrogels, spheres
collagen protein porous sponges, knitted fabrics
substituted polyphosphazene polyphosphazene porous sponges
polycaprolactone polyester porous sponges
poly(ethylene glycol-b-propylene glycol) polyether surfactant
Biodegradable Materials 507
Methods of Fabrication of Synthetic Polymeric Gas Dispersion
Materials for Use in Tissue Engineering The pores formed by dispersion are typically uniform
Tissue engineering absorbable graft research has thus and of low size distribution. Gases may be introduced into a
far targeted the synthetic absorbable materials so it is useful polymer melt or a liquid monomer, either directly or as a
to understand the various methods of processing these par- by-product of a chemical reaction. When cooling of the poly-
ticular materials into tissue engineering scaffolds. mer or polymerization of the monomer occurs, the gas
bubbles become entrapped in the material, usually result-
Physical Aggregation ing in a closed pore system. If the melt is extruded, this can
Physical aggregation is the formation of a porous, in- actually result in an open pore system or a “blown” foam.21
terconnected network with no chemical interlocks between These foams typically are a larger pore sized system. Alter-
the polymer matrix components; rather, a physical adhe- natively, a gas may be introduced into a polymeric solid at
sion is formed. The pores formed by aggregation are typi- high pressure. Then when the solid is transferred to atmo-
cally uniform and of low size distribution. This may be ac- spheric conditions, the gas will expand, forming a spongelike
complished in microsphere systems by forming an agglom- material (Fig. 46.1). This does not require use of solvents
erate of particles by adding a biocompatible plasticizing agent and may successfully be accomplished with absorbable
such as triethylcitrate.19 Physical aggregation of this kind polylactide/polyglycolide copolymers and a carbon dioxide
therefore allows an interconnected, biocompatible, mechani- environment.22
cally stable, porous structure.
A second method of physical aggregation is by Solid and Liquid Dispersion
sintering. Tassels and felts have found initial success as trans- Solid or liquid “fillers”, such as thermoset polymer
plantation surfaces in liver and cartilage regeneration stud- spheres21 or water,23 may also be purposefully entrapped in
ies; however, these structures may lack the necessary me- the polymer matrix upon solidification. Subsequent removal
chanical integrity to maintain a specific shape. Fibrous scaf- of the solid or liquid results in an open pore system, the
folds, woven or random arrays, have also found application pore size distribution of which is generally larger than that
in tissue engineering, but their dimensional stability and of the gas dispersion process. Absorbable foams may be
hence retention of shape is questionable. The fibers may be manufactured using this technique.18,24 The polymer can be
sintered together to provide a stable structure in a manner mixed, for example, with a solvent and sodium chloride par-
similar to the processing of the “self reinforced” bone pins;4 ticles of a predetermined size (Fig. 46.2). The solvent is
however, the high temperature required can cause disorien- evaporated, leaving a polymer matrix with entrapped par-
tation of the molecular order and subsequent buckling of ticles. The salt is leached from the system by soaking the
the material.20 The fibers may, rather, be coated with a construct in water, thus yielding a porous structure. The solid
supportive material which maintains the scaffold shape dispersion pore size is variable according to the size of the
during heat treatment, but which may be later dissolved from filler utilized.
the system.20
Supercritical Fluid Extraction
Chemical Aggregation Absorbable polymers, specifically polyglycolides,
Chemical aggregation involves forming a porous, in- polylactides, and polyanhydrides, may be combined with
terconnected network by crosslinking, or chemically bond- supercritical fluids to process open-pore systems.25 The
ing, the polymer matrix components. This may be accom- supercritical fluid is added to the polymer under pressure,
plished with a difunctional agent such as gluteraldehyde.19 the pressure is then dropped to remove the fluid and render
The drawbacks to this include reduced porosity due to in- the material spongelike (Fig.46.3). The percent porosity is
terstitial “filler” material as well as the potential introduc- dependent on the pressure differential between ambient and
tion of a toxic substance (e.g., traces of glutaraldehyde) into processing. One huge benefit of this technique is that no
the biological system. organic solvents are required.
Fig. 46.1. Foam formation by gas
dispersion.
508 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 46.2. Foam formation
by solid dispersion.
Fig. 46.3. Phase dia-
gram indicating path-
way (1-2) of foam for-
mation by supercri-
tical fluid extraction.
Crystallization-Induced Microphase Separation These materials can be copolymerized in order to provide a
This method of foam fabrication26,27 calls for the ad- range of degradation times from weeks to years.
dition of a solid solvent with high melting temperature to The relative lack of biocompatibility of traditional
the polymeric material. The mixture is then heated to form synthetic materials and the persistent dilemma of clinically
an isotropic liquid system. Subsequent cooling causes the unacceptable small diameter (< 6mm) vascular grafts insti-
crystallization-induced microphase separation and the now gated attempts to modify the substrates and provide a more
solidified solvent can be sublimed or removed with a liquid compatible material. One such attempt is the surface modi-
solvent. This process has been successfully applied to the fication of synthetic tubular materials or type I collagen with
formation of open pore absorbable constructs, specifically endothelial cells and smooth muscle cells to provide a more
polydioxanone and poly(>-caprolactone-co-glycolide) po- biologically “friendly” implant.28-30 Other investigators have
rous structures (Fig. 46.4). tried coating 90/10 poly(glycolide-co-L-lactide) based grafts
with absorbable materials such as a mixture of poly-DL-
Biodegradable Polymers for Vascular Grafts— lactide/poly-(2,3-butylene maleate) or poly-(2,3-butylene
Past, Present, and Future fumarate)/poly-DL-lactide.31-33 Fumarate leads to a more
Many attempts have been made at engineering tubu- uniform and predictable absorption rate—there is no evi-
lar vascular grafts by providing a tubular, porous, polymer dence of complications due to aneurysm or rupture. Alter-
support structure. The structure must be mechanically sound natively, the porosity of a tubular implant may be adjusted
so as not to collapse or interfere with blood transport to provide a diminishing array of pore sizes from the exte-
through the conduit. The polylactides and polyglycolides rior to the interior of the implant, thus potentially allowing
have shown favorable responses to such mechanical stresses. increased capillarization and improved biocompatibility.34
Biodegradable Materials 509
Fig. 46.4. Scanning electron micrograph
of absorbable foam formed by crystalli-
zation-induced microphase separation.
Traditionally the ideal vascular graft material was as- to success, as too high a degree causes lack of anastomotic
sumed to be biologically inert. Today, however, research has endothelialization and transinterstitial tissue ingrowth.40
shifted toward creating an active material that will interact The compliance and longevity of a potential vascular
and integrate favorably with the surrounding biological en- graft material can influence the retention of mechanical in-
vironment. The absorbable materials, as dynamic implant tegrity. 41,42 Studies in the polyurethane/polylactide
systems, have this quality and appear to stimulate growth (PU/PLLA) system have shown that a decrease in the per-
factor release through macrophage induction. Both cent polyurethane causes a decrease in compliance and an
polyglycolide (PG), 90/10 glycolide/L-lactide copolymer, and increase in aneurysmal degeneration.43 These studies fur-
polydioxanone (PDS) woven grafts have successfully dem- ther showed that high mechanical compliance of the graft
onstrated, in animal studies, inner capsule development as at implantation stimulates elastin formation and an absorb-
well as capillary invasion, thus resulting in an integrated, able graft material allows maintenance of the mechanical
mechanically stable prosthesis.35-37 The inner capsule con- stability of the newly formed tissue. The PU/PLLA system is
tains layers of myofibroblasts covered by endothelial-like cells initially a compliant one; however, long term incidences of
(presumably from a capillary endothelial source). In similar aneurysmal dilation are significantly greater in this system
studies, Dacron reinforced PGA shows no such capsule de- than the polyester ones. This is possibly because the ongo-
velopment and low amounts of transinterstitial macrophage ing presence of PU leads to fibroplasia, resulting in decreased
migration, presumably due to the inhibitive nature of compliance and elastin deposition. Another important con-
Dacron.38 These types of partially absorbable vascular sideration for scaffold development, as with other applica-
patches, based on woven composite yarn comprising a tions, is that the tissues should gain adequate strength be-
24-82% polyglycolide yarn, the balance being polyethylene fore the material absorbs. If this is not possible, the absorb-
terephthalate yarn, were also evaluated in dogs in terms of able scaffold may be reinforced by an inert (not Dacron)
blood compatibility. 39 Results indicate that the patch component or developed into a composite material whereby
biocompatibility and effect on blood components increased the tissue ingrowth occurs through a series (two or more)
with the increase in the polyglycolide content. This signals of polymers. The multicomponent absorbable vascular pros-
the importance of tissue ingrowth to the construction of theses composed of compound yarns of this type appear to
successful synthetic vascular grafts. be efficacious.44 Studies have shown that, in contrast to the
Studies have also focused on the creation of an opti- absorbable component, the biomechanical properties of the
mal biological component, integral to the prosthesis, for nonabsorbable component modulate the histologic at-
example, tubular nonabsorbable polyester fabrics incorpo- tributes and longevity of the final tissue structure.
rated with bovine serum albumin and a luminal coating of Other attempts at tissue engineering blood vessels have
gelatin heparin sodium solution. These constructs are been made by constructing the vessels ex vivo directly from
crosslinked with a polyepoxy compound. The grafts, once the cellular components. This may be accomplished by
implanted, show no signs of platelet aggregation or fibrin culturing a sheet of human vascular smooth muscle cells in
formation and show development of neointima over the collagen and placing this about a lumenal support to produce
anastomotic graft site. The degree of crosslinking is critical the media.45 A fibroblast sheet is then similarly cultivated
510 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 46.5. Surface
phosphorylation of
polyethylene.
and placed about the media to form the adventitia. The need for a vascular graft implies that the sur-
Endothelial cells are later seeded into the lumen of the vessel, rounding target tissue may be “abnormal” (atherosclerotic,
thus forming a mechanically sound three-dimensional vessel. for example). It has been shown in animal studies that the
quality of such tissues (for example, in the presence of ex-
Newly Tailored Materials cessive amounts of lipoprotein) may have a profound effect
for Tissue Engineering on the development of new tissue.49 It would therefore be
Two recently developed technologies are expected to useful to study the absorbable material in such suboptimal
be of value in tissue engineering applications. First, Shalaby46 conditions or in compromised tissue environments.
developed a series of injectable, absorbable liquid copoly-
mers which undergo gel formation upon contacting moist References
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vitro degradation of poly(l-lactide) used for fracture fixa-
the production of commercial absorbable sutures. The in-
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face phosphonylation of preformed devices, including ternal fracture fixation. Biomater 1987; 8(1):70-73.
microporous open-cell foams that were described by Shalaby 4. Vainionpää S, Kilpikari J, Laiho J et al. Strength and
and co-workers,27,47,48 represents a second area of technol- strength retention in vitro, of absorbable, self-reinforced
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(Fig 46.5). This technology entails the formation of co- 1987; 8(1):46-48.
valently bonded surface phosphonate groups which can: 5. Vert M, Christel P, Garreau H et al. Totally bioresorbable
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1. Introduce hydrophilicity to hydrophobic substrates;
In: Chiellini E, Giusti P, Migliaresi C, Nicolais L, eds.
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tachment; and Press, 1986:263-275.
3. Bind Ca2+ for directing the deposition of hydroxya- 6. Vacanti JP. Beyond transplantation. Arch Surg 1988;
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Among the substrates suitable for surface 7. Vacanti JP, Morse MA, Saltzman WM et al. Selective cell
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Future Prospectives Prevascularization of porous biodegradable polymers.
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9. Boyce ST, Christianson DJ, Hansbrough JF. Structure of
potential vascular graft materials is that, as homopolymers,
a collagen-GAG dermal skin substitute optimized for cul-
they do not have the cell surface receptors required for cel- tured human epidermal keratinocytes. J Biomed Mater Res
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platelets. Surface modification such as that described by sulphate substrate. Surgery 1988; 103(4):421-431.
Shalaby and co-workers may be of value in overcoming some 11. Hansbrough JF, Boyce ST, Cooper ML et al. Burn wound
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development of RGD-peptide-modified poly(lactic acid- tional life of bioresorbable yarns for vascular grafts. Trans
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Biomed Mater Res 1997; 35:513-523. 32. Galletti PM, Trudell LA, Chiu T-H et al. Coated
13. Barrera DA, Zylstra E, Lansbury PT et al. Copolymeriza- bioresorbable mesh as vascular graft material. Trans
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14. Hrkach JS, Ou J, Lotan N et al. Synthesis of poly(L-lactic fully bioresorbable aortic grafts in the dog. Surgery 1988;
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Scaffold Engineering
Material Aspects
CHAPTER 47
The Influence of Porosity and
Surface Roughness on Biocompatibility
J.M. Schakenraad, K.H. Lam
Introduction
V ascular prostheses might solve a lot of clinical problems, if only they would “do their job”
in a functional way. Functional in this respect includes: nonthrombogenicity; compli-
ance, diameter and wall thickness similar to vessel wall; blood compatibility in all its aspects
(complement activation, red and white blood cell damage, thrombocyte activation etc.);
immunological inertness etc. Some of these parameters can be influenced in such a way that
an acceptable functionality is achieved. Especially with regard to small diameter vascular
grafts (< 3 mm), it has proven impossible to solve all problems simultaneously. A vast chal-
lenge still lies ahead for the next generation of scientists.
This chapter will discuss two material surface parameters (roughness and porosity)
having a large influence on biocompatibility and ultimate patency of vascular grafts. A pa-
rameter indicative for short term biocompatibility is the inflammatory response towards an
implanted biomaterial. Upon implantation of a biomaterial, local1,2 and systemic3 effects
can be observed. The local tissue reaction consists of an inflammatory response which serves
to eliminate the cause of an injury, minimize the damage and trigger mechanisms for repair-
ing the tissue damaged by injury. The surgical procedure itself initially determines the type
and intensity of the inflammatory response. However, in addition, implanted biomaterials
themselves provoke an inflammatory response.
Many material characteristics may influence the inflammatory response against a bio-
material (being either a natural polymer, a chemical polymer, metals, ceramics etc.): chemi-
cal composition,1 material toxicity4,5 surface free energy (wettability),6 surface charge7 sur-
face morphology such as porosity,8 roughness of the surface9 and shape,7 implantation site,10
rate of degradation,11 degradation products,12,13 and many more.
Surface morphology is one of the first factors directly influencing the tissue/inflam-
matory response. This is indicated in various studies.7,14 Matlaga et al demonstrated the role
of shape.7 The intensity of the inflammatory response increases when the number of edges
of the implanted materials increases. Also, there are studies indicating that the inflamma-
tory response against implanted porous polymers or biomaterials is more intense when com-
pared to nonporous materials.8
Wettability may influence the tissue reaction to the polymer, because there is a range
in wettability values that is optimal for cell adhesion, growth and spreading. Cells attach and
proliferate less well on polymers having a wettability which is too low15 or too high.16
Another factor is degradability. Degrading polymers provoke a more intense inflam-
matory response compared to nondegrading polymers. A possible cause for this observation
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
514 Tissue Engineering of Prosthetic Vascular Grafts
may be the release of monomers, oligomers and/or fragments Material Characterization
upon degradation.12,13 However, at earlier stages of the deg- Except for PTFE, the final thickness of the films was
radation process, changes (such as increase) in the surface determined with scanning electron microscopy (SEM). All
morphology of a polymer (film) may occur, which in turn films were cleaned by washing in a phosphate buffered sa-
may alter the inflammatory response. line (PBS) for 24 h prior to use.
To illustrate the effect of surface morphology on the
tissue response and ultimate biocompatibility of a material
after implantation, the following series of materials are
compared:
1. Porous versus nonporous;
2. Rough versus smooth;
3. Degradable versus nondegradable.
The inflammatory response was characterized using
semi-quantitative techniques comprising morphological cri-
teria and monoclonal antibodies directed against epitopes
which are specific for the respective cell types.
Such aspects as molecular weight of soluble leachables
and relative surface area are considered to be closely related
to the observed histological parameters and are therefore
also included in this overview.
Evaluation Models
Materials
As a degradable material, the well known and widely
used polylactic acid is used, both as a solid, a porous and a
combination form. As a nondegradable reference material,
PTFE is used, both in a solid shape and as the expanded
PTFE with a pore size of 30 micrometers.
Poly(L-lactic acid) (PLLA) films were cast from PLLA
with a reported Mn of 50,000 (Purac Biochem B.V., The
Netherlands).17 Three types of films were cast: a nonporous
type, a porous type and a “combi” type (porous with a non-
porous layer on one side). The base parameters of the PLLA
films are listed in Table 47.1. All PLLA films were cut in strips
of 15 x 2 mm.
Polytetrafluoroethylene (PTFE) was obtained com-
mercially (Wientjes, The Netherlands). PTFE was cut into
strips of 15 x 2 x 1 mm. Expanded polytetrafluoroenthylene
(ePTFE) was obtained as nonsterile GORE-TEX® ePTFE cell
collector tubing (WL Gore & Associates GMBH, Germany).
The tubing was cut open along the longitudinal axis to ob-
tain films measuring 15 x 2 x 0.25 mm.
Table 47.1. Base parameters of the PLLA films
Parameter Non-porous Porous Combi Fig. 47.1. Scanning electron micrographs of a cross-section of
PLLA film PLLA film PLLA film PLLA films. (A, top.) nonporous, (B, middle.) porous,
(C, bottom.) “combi”; arrowhead indicates the nonporous side.
Mw 98,000 109,000 167,000 The thickness of the films is: nonporous, 33 ∝m; porous, 244 ∝m
Mn 42,000 41,000 53,000 and “combi”, 82 ∝m. The nonporous layer of the “combi” film
Mw/Mn 2.3 2.7 3.1
Tm 176°C 180°C 181°C
was approximately 5 ∝m. The pore size of the porous film var-
Heat of fusion 53 J/g 51 J/g 50 J/g ied from approximately 1-150 ∝m and of the “combi” film from
1-50 ∝m.
The Influence of Porosity and Surface Roughness on Biocompatibility 515
Surface Morphology porous PLLA film, a porous PLLA film, a “combi” PLLA film,
The surface morphology of the different specimens is a PTFE film and an ePTFE film were implanted. The sixth
illustrated in Figure 47.1A-C. Specimens of all films were incision and subcutaneous pocket served as a control (sham
sputter-coated with gold (Balzers 07 120B) and their sur- operation). Three samples per polymer and time interval
face morphology was examined with DS 130 scanning elec- were implanted. The rats had free access to standard rat food
tron microscope (SEM) (ISI), operated at 10 KV. and water. All national rules concerning the care and use of
The thickness of the polymers was calculated using laboratory animals have been observed.
SEM: nonporous 33 ∝ m (Fig.47.1A); porous 244 ∝ m The rats were sacrificed after 1, 3, 7, 14, 40, 90 or 180
(Fig. 47.1B); and the “combi” film 82 ∝m (Fig. 47.1C). The days and the polymer films were removed with excess sur-
nonporous layer of the “combi” film was approximately 5 ∝m. rounding tissue.
The pore size of the porous film varied from approximately
1 to 150 ∝m and of the “combi” film from 1 to 50 ∝m. PTFE Evaluation of the Inflammatory Response
was nonporous, having the same morphological appearance
of the surface as the nonporous PLLA film. The thickness of Light Microscopy
the porous ePTFE film was approximately 250 ∝m with a After harvesting, the samples were immediately fixed
fibril length of approximately 90 ∝m (Fig. 47.2). for at least 24 h at 4°C in a 0.1 M Na-cacodylate buffer,
pH 7.4, containing 2% glutaraldehyde and 0.1 M sucrose.
Wettability The samples were then dehydrated in a graded ethanol se-
Wettability, as a measure for surface free energy (hy- ries and embedded in glycolmethacrylate (Technovit®,
drophobicity) was determined by contact angle measure- Kulzer, Germany), allowing sections to be cut perpendicu-
ments using the sessile drop technique described by Busscher lar to the longitudinal axis of the polymer film. Sections for
et al.18 For PTFE and the nonporous PLLA, the contact angles light microscopy examination were cut on a microtome
were determined using H20 and #%bromonaphthalene as (Jung autocut 1140, equipped with a D knife with a tung-
wetting agents. Five measurements were made for each poly- sten carbide cutting edge), mounted on glass slides and
mer and wetting agent. stained with toluidine blue and alkaline fuchsin.19
The highest value of each set of five measurements is
maximally 5° higher than the lowest value. Therefore, it was Immunohistochemical Staining
not relevant to determine the standard deviation. The H2O After harvesting, samples were snap-frozen at -80°C
contact angles were 101° for PTFE and 72° for nonporous using liquid freon. Cryostat sections of 7 ∝m were cut,
PLLA, and the #-bromonaphthalene contact angles were 66° mounted on glass slides, air-dried and fixed in acetone for
for PTFE and 23° for nonporous PLLA. This shows that 12 minutes. The sections were then again air-dried for 1 h
nonporous PLLA has a less hydrophobic surface than PTFE. and incubated with the first-stage, cell type specific, mono-
clonal antibody (mAb) for 1 h. Subsequently, the sections
Implantation Procedure were washed 3 times in PBS, followed by incubation with
The films were disinfected prior to implantation and the second-stage antibody conjugated to peroxidase, diluted
implanted subcutaneously in 21 female (AO x BN)F1 rats 1:40 in PBS and supplemented with 5% v/v normal rat se-
obtained from our own breeding facility. In each rat a non- rum to prevent nonspecific binding. Swine anti-rabbit Ig
Fig. 47.2. Scanning electron micro-
graph of (porous) ePTFE. The fibril
length (distance between A and B) is
approximately 90 ∝m. Bar indicates
30 ∝m.
516 Tissue Engineering of Prosthetic Vascular Grafts
(Dakopatts, Denmark) was used as second-stage antibody Macroscopically, there were no signs of an infection
to detect the first-stage mAb #-Asialo GM1 (Table 47.2). demonstrated in any of the rats. The results of the semi-
Rabbit anti-mouse Ig (Dakopatts, Denmark) was used to quantitative evaluation of the immunohistochemical stain-
detect the other first-stage mAbs. After incubation with the ing to detect leukocytes (OX 1), neutrophilic granulocytes
second-stage antibody, sections were rinsed 3 times in PBS (HIS 48), macrophages (ED 1, ED 2, ED 3), T lymphocytes
for 5 minutes. Peroxidase activity was demonstrated by ap- (OX 19), vast majority of B lymphocytes (HIS 40), natural
plying 3,3-diaminobenzidine tetrahydrocloride (Sigma) at killer cells (#-Asialo GM1), cells expressing MHC class II
a concentration of 0.5 mg/ml in 0.05 M Tris-HCI buffer (pH antigen—such as activated fibroblasts—(HIS 19) are dem-
7.6) containing 0.01% H2O2 for 10 minutes. After rinsing in onstrated in Figures 47.3A-I respectively. False positive cells
fresh tap water, sections were counterstained lightly with were observed occasionally in PBS controls. This was due to
hematoxylin for 10 seconds. The sections were then dehy- endogenous peroxidase activity expressed only by neutro-
drated using a graded ethanol series and xylene and subse- philic granulocytes, since the staining pattern using HIS 48
quently covered with coverslips using DePeX (Gurr, BDH corresponds with the pattern of endogenous peroxidase ac-
Ltd, England) mounting medium. In controls, PBS was used tivity. Moreover, cells expressing endogenous peroxidase
instead of the first-stage mAb. The first-stage mAbs used, activity can be well distinguished due to their more intense
and their sources, are shown in Table 47.2. staining as compared to cells stained for mAbs.
The concentration of B lymphocytes (Fig. 47.3D) and
Quantification of the Inflammatory Response the concentration of natural killer cells (Fig. 47.3H) was to
The magnification of the light microscope was set at a large extent similar and low for both PLLA, PTFE films
x400 when examining the section stained using mAbs. The and the subcutaneous tissue under the scar of the sham op-
staining patterns of the tissue surrounding or invading the eration.
polymer film was evaluated and the number of positive cells At day3, the concentration of each cell type involved
surrounding or invading the polymer films per field of view in the inflammatory response as a measure for the intensity
counted. Four fields of view per section, two sections per was approximately the same for all the polymer films
sample and three samples for each period of implantation, (Fig. 47.3A to 47.3G). At day 7, macrophages are beginning
polymer film and monoclonal antibody respectively were to play a prominent role in the inflammatory response
examined. The tissue reactions at the edges of the polymer (Fig. 47.3E). Subsets of macrophages surrounding the poly-
films were excluded from evaluation, to avoid artifacts due mer films were found in different locations. ED 1 positive
to mechanical irritation. macrophages are found in the entire area, demonstrating
The number of cells staining positive per field of view inflammatory response against the implant, especially in the
was classified as follows: area in closest approximation to the polymer film
grade 0 = no positive cells, (Fig. 47.4A). In contrast, ED 2 and ED 3 positive macro-
1 = 1 to 5 positive cells per field of view, phages are neither found in the area in closest approxima-
2 = 5 to 10 positive cells per field of view, tion the polymer film, nor in the pores of the PLLA or PTFE
3 = 10 to 25 positive cells per field of view and film. ED 2 and ED 3 positive macrophages remain restricted
4 = more than 25 positive cells per field of view. to the surrounding tissue at some distance from the implant
The average class of the 24 fields of view for each pe- (Fig. 47.4B and 47.4C). The relative ratio between the sub-
riod of implantation, polymer film and monoclonal anti- sets was fairly constant for the respective implants.
body respectively is reported.
Table 47.2. Antibodies (mAbs) used to examine and quantify the inflammatory response against the implanted
polymer films
Monoclonal Antibody Epitope Mainly characterizing cell
type in subcutaneous tissue:
OX 1 CD 45 All leukocytes
HIS 48 probably surface Granulocytes
OX 19 CD 5 T-lymphocytes
HIS 40 IgM heavy chain B-lymphocyte subset likely to react first upon inflammatory response
ED 1 Lysosomal antigen Majority of macrophages. Probably associated with active phagocytosis.
ED 2 Surface antigen Subset macrophages. Probably associated with maturity.
ED 3 Surface antigen Subset macrophages. Probably associated with downregulation of
inflammatory reaction
#-Asialo GM1 Probably surface antigen Large granular lymphocytes natural killer cells
HIS 19 MHC class II Activated tissue cells (fibroblast) IDC, subset macrophage
Source of the mAbs: OX antibodies were a generous gift of the late Dr. A.F. Williams, Department of Biochemistry, University of Oxford,
Great Britain; ED antibodies were a generous gift of Dr. C.D. Dijkstra, Department of Cell Biology. The HIS and a-Asialo antibodies were
generous gifts of Dr. P. Nieuwenhiu’s of the Department of Histology, University of Groningen.
The Influence of Porosity and Surface Roughness on Biocompatibility 517
Fig. 47.3. The average class
of 24 fields of view of the
number of cells involved in
the inflammatory response
against the implanted poly-
mer films, stained with dif-
ferent mAbs. Class criteria:
grade 0 = no positive cells;
grade 1 = 1 to 5 positive
cells; grade 2 = 5 to 10 posi-
tive cells; grade 3 = 10 to 25
positive cells and grade 4 =
more than 25 positive cells
per field of view at an
original magnification of
x400. The following mAbs
were used: (A, top.) OX 1,
all leukocytes. (B, bottom.)
HIS 48, neutrophilic granu-
locytes.
518 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 47.3. (C, top.) OX 19, T-lympho-
cytes, (D, bottom.) HIS 40, vast major-
ity of B lymphocytes.
The Influence of Porosity and Surface Roughness on Biocompatibility 519
Fig. 47.3. (E, top.) ED 1, vast majority of
macrophages, including multinuclear gi-
ant cells; (F, bottom.) ED 2, subset (ma-
ture/resident) macrophages.
520 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 47.3. (G, top.) ED 3, subset mac-
rophages. (H, bottom.) # -Asialo
GM1, natural killer cells/large granu-
lar lymphocytes
The Influence of Porosity and Surface Roughness on Biocompatibility 521
Fig. 47.3. (I.) HIS 19, cells expressing MHC
II antigen (e.g., activated fibroblasts,
macrophages, dendritic cells). X-axis
represents implantation time (days), Y-axis
the average number of cells per field of view
as class 0 to 4. Z-axis represents the different
polymer films.
In GMA sections it is observed that the inflammatory flammatory response than PTFE films as demonstrated by
response at day 7 is becoming more intense (Fig. 47.5) for the higher concentration of neutrophils, macrophages/gi-
the porous PLLA and porous side of the “combi” PLLA films. ant cells and T lymphocytes surrounding the PLLA films.
For the nonporous PLLA and the nonporous side of the The concentration of cells against PTFE and ePTFE films is
“combi” PLLA film, the onset of encapsulation by approxi- comparable to the concentration of cells in the subcutane-
mately 3 layers of fibroblasts was observed. Only a minimal ous tissue under the scar of the sham operation. The in-
encapsulation is observed for the porous PLLA film at day flammatory response against the nonporous PLLA film was
7. In contrast to the porous PLLA film almost no cellular mainly localized at the edges of the film or at the edges of
invasion of the ePTFE film was observed. The cell layer sur- the pieces when broken. In contrast, the inflammatory re-
rounding the ePTFE consists mainly of macrophages. The sponse against porous PLLA films was localized in the pores.
PTFE film was surrounded by one or two layers of mac- The “combi” film shows a more pronounced inflammatory
rophages and the onset of encapsulation can be observed. response at the porous side.
At day 14, the inflammatory response becomes chronic At day 90, the difference in the intensity of the inflam-
with predominantly macrophages and T lymphocytes sur- matory response between PLLA and PTFE films and also
rounding the films. In the GMA sections, foreign body gi- between nonporous and porous PLLA films had become
ant cells can be observed surrounding the PLLA fims much more pronounced. This is demonstrated by the in-
(Fig. 47.6). All films are now encapsulated by continuous creased concentration of macrophages, leukocytes and cells
layers of fibrocytes and collagen. The fibrocytes are not expressing MHC class II (HIS 19) antigen, (probably acti-
stained by HIS 19 monoclonal antibody, indicating a de- vated fibroblasts) surrounding or invading the PLLA films.
crease in cell activity. The porous PLLA film and porous side The porous PLLA films provoke a more intense inflamma-
of the “combi” film provoke a more intense inflammatory tory response than the nonporous PLLA film. In contrast,
response than the nonporous PLLA film and nonporous side the inflammatory response against PTFE and ePTFE films
of the “combi” film respectively. However, this observation remained the same as at day 40.
could not be made for the ePTFE film as compared to the At day180, the difference in the intensity of the in-
PTFE film. flammatory response between the porous and the nonpo-
At day 40, in general, the intensity of the inflamma- rous PLLA film was more pronounced. In contrast, the tis-
tory response against the polymer films had decreased fur- sue reaction (“inflammatory response”) against PTFE and
ther. However, PLLA films still provoked a more intense in- ePTFE did not differ much from the tissue reaction in the
522 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 47.4. Immunohistochemical staining of the tissue surround-
ing the porous PLLA film (P) at day 7, with: (A.) ED 1. The
macrophages adjacent to the implant stain positive for ED 1
(arrow heads). No ED 1 positive cells are observed in the poly-
mer film. (B.) ED 2. The macrophages adjacent to the implant
do not stain positive for ED 2. The cells in the lower left corner
stain false positive for ED 2. This is due to endogenous peroxi-
dase activity of neutrophilic granulocytes. (C.) ED 3. The mac-
rophages adjacent to the implant do not stain positive for ED 3.
Bar indicates 40 ∝m.
The Influence of Porosity and Surface Roughness on Biocompatibility 523
Fig. 47.5. “Combi” PLLA film: P 7 days after implanta-
tion. Note the difference in the intensity of the inflam-
matory response between the porous and the nonpo-
rous side. Bar indicates 40 ∝m.
subcutaneous tissue under the scar of the sham operation. The relative polymer surface area in sections (RPSA)
There are almost no cells localized in the pores of ePTFE was determined by morphometrical analysis using light
(Fig. 47.7A). In contrast, the inflammatory response against microscopical sections and a Quantimet 520 (Cambridge
porous PLLA was localized mainly in the large pores Instruments) image analyzer. The RPSA was defined as the
(Fig. 47.7B). ratio between the polymer surface area and measurement
frame area. The boundaries of the measurement frame were
Relative Polymer Surface Area in Sections set at the outer boundaries of the polymer surface area. The
The relative polymer surface area (RPSA) in sections morphometrical analysis was performed on porous and
was determined after in vivo and in vitro procedures. After “combi” films only, because the nonporous film was not
harvesting, samples were immediately fixed by immersion eroded nor did it become porous, not even after an immer-
in a 0.1 M Na-cacodylate buffer, pH 7.4, containing 2% glu- sion or implantation period of 180 days. The RPSA of the
taraldehyde and 0.1 M sucrose, for at least 24 h at 4°C. After nonporous film remained 100%. For each implantation pe-
rinsing with buffer, the samples were dehydrated in graded riod or immersion period ten measurements of the remain-
ethanol series. The samples were then embedded in a posi- ing polymer surface area (of at least two sections) were per-
tion which allowed sections to be cut perpendicular to the formed. The mean value and standard deviation were cal-
longitudinal axis of the polymer films. After embedding in culated from these ten values. All data were normalized to
glycolmethacrylate (GMA) (Technovit, Kulzer, Germany), the initial value (t = 0).
sections of 3 ∝m were cut on a Jung 1140 autocut micro- The results of the morphometrical analyses are pre-
tome, using a D knife with a tungsten carbide cutting edge. sented in Figures 47.8A (in vitro) and 47.8B (in vivo). The
The sections were mounted on glass and stained with tolui- RPSA in vivo shows no significant decrease for the nonpo-
dine blue and alkaline fuchsin. rous, the porous or the “combi” film. Also, no decrease was
524 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 47.6. Nonporous PLLA film: P 14 days after
subcutaneous implantation. Note the large con-
centration of giant cells (arrow heads). Bar denotes
40 ∝m.
observed in vitro for the nonporous and porous film up till sample of 5-10 mg was dissolved in 10 ml chloroform and
day 180 and for the “combi” film, up till day 90. It was not filtered (Spartan 13/20 filter, 0.45 ∝l). The injection volume
possible to obtain the surface area of the “combi” film at day per measurement was 200 ∝l. Chloroform was used as elu-
180, due to fragmentation. ent at a flow rate of 2.0 ml/min. The Mw, Mn and polydis-
During the in vivo experiment with the nonporous persity ratio were calculated using the calibration data of
polymer film, no erosion or pore formation was observed. polystyrene standards of narrow molecular weight distri-
Moreover, after immersion in PBS (in vitro), it was not pos- bution dissolved in tetrahydrofuran (THF).
sible to carry the nonporous film through the embedding The Mw (Fig. 47.9A) and Mn (Fig. 47.9B) decreased
procedures for light microscopy from day 40 on, due to in- for all three films at approximately the same rate. From day
creased brittleness. The surface area of the remaining non- 7 on, the porous film generally retained the highest Mw and
porous film remained 100% over the entire test period. Mn. The initial (t = 0) Mw of the “combi” film was the high-
est (167,000) and of the nonporous film the lowest (98,000;
Mw and Mn porous was 109,000). Before day 7, the curves of the porous
Mw (molecular weight) and MN in vitro were deter- and “combi” film showed large fluctuations for Mw. There-
mined by gel permeation chromatography (GPC) at 20°C after, all values for Mw demonstrated a decreasing trend till
on a Waters Associates GPC apparatus using Waters Associ- the 170th day (Mw nonporous: 24,000, porous: 60,000,
ates columns (bead size of 105, 104, 103 Å). A precolumn “combi”:29,000). The Mn curves roughly approximate the
with a pore size of 500 Å was used. A Waters Associates R same trend as the Mw curves. The fluctuations of the values
403 differential refractometer was used as a detector. A for the “combi” film are larger.
The Influence of Porosity and Surface Roughness on Biocompatibility 525
Fig. 47.7. (A, left.) Porous e-PTFE (P) and (B, right.) porous PLLA (P) after 180 days of implantation. There are almost no inflam-
matory cells localized in the pores of the e-PTFE film. In contrast, the inflammatory response is localized mainly in the pores
F=fibrous encapsulation. Bar indicates 63∝m.
Interpretation of Results blood. SAA may then be converted into amyloid A, which is
deposited in tissues. However, the exact mechanism is yet to
Inflammatory Response and Biocompatibility be fully uncovered, and the relation between the chronic
The results demonstrate that there are two phases in inflammatory response against biomaterials and amyloido-
the inflammatory response against the films. Phase 1 is ob- sis also remains to be investigated.
served upon implantation of the film. It is mainly caused by
the injury sustained by the implantation procedure. This The Role of Macrophages and Giant Cells
uncomplicated inflammatory response, part of the wound ED 1 stains an intracellular antigen, probably associ-
healing reaction, ends after 7-10 days and has been well de- ated with the lysosomal membrane. The antigen probably
scribed.2,20 In this phase, the contribution of the implanted remains present even in the event of fusion of macrophages
polymer film to the intensity of the inflammatory response and formation of foreign body giant cells, since ED 1 posi-
is in most cases minimal, except when leakage of large quan- tive cells are observed on the same location of foreign body
tities of toxic products occurs.5 After one week, any remain- giant cells in conventional light microscopical sections. ED 2
ing inflammatory response can be considered as a tissue re- is a marker which is found on mature tissue macrophages.
action against the implanted biomaterial.11 This chronic It takes about one week for monocytes/macrophages to ex-
inflammatory response (phase 2) is often described as a for- press ED 2 on their cell surface after leaving the vascular
eign body reaction.1,2,5 It mainly consists of macrophages system. The role of ED 2 and ED 3 in the inflammatory re-
and giant cells surrounding the implant. sponse is not well understood. The different staining pat-
A minimal inflammatory response is preferred when terns of the ED antibodies indicate the possibility of either
biomaterials are implanted for a long time span, because a different populations of macrophages which play a role in
persistent (chronic) inflammatory response may predispose the tissue reaction against biomaterials, or macrophages ex-
for amyloidosis,21,22 or carcinogenesis.23 A persistent inflam- pressing different surface receptors in the course of the in-
matory response increases the concentration of both serum flammatory response against a biomaterial.
amyloid A (SAA) and amyloid enhancing factor (AEF) in
526 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 47.8. Remaining polymer surface
area (RPSA) of the PLLA films in a
light microscopical section. (A.) In
vitro: RPSA as a function of immer-
sion time. (B.) In vivo: RPSA. Symbols
represent: ❑ = nonporous, + = porous,
❍ = “combi”. Bars indicate the stan-
dard deviation.
The exact mechanism of foreign body giant cell for- the nonporous PLLA in an aqueous environment. This in-
mations is still to be elucidated. Certain cell types, e.g., T dicates that porosity is an important factor determining the
lymphocytes, may play a pivotal role in this process. T helper intensity of the inflammatory response against implanted
lymphocytes capable of secreting interferon-&, especially, may PLLA films. The small difference in the intensity of the in-
play a role in the fusion of macrophages, forming giant flammatory response between PTFE and ePTFE as compared
cells.24,25 Activation of macrophages may be induced by vari- to porous and nonporous PLLA respectively indicates that
ous pathways. In one pathway, (limited) damage to neutro- porosity as a single factor is not enough to enhance the in-
phils and macrophages may lead to secretion of cytokines flammatory response. The relatively high wettability of PLLA
(IL-1), activating T helper lymphocytes. Another possibility compared to ePTFE allows for better ingrowth of tissue into
is change of shape when a macrophage comes in contact the pores of PLLA and probably more exposure to other fac-
with a boimaterial,26,27 especially when a single macrophage tors, determining the intensity of the inflammatory response.
is not able to phagocytose the polymer(fragment). According to many authors, the surface properties of
an implant have a large influence on the inflammatory re-
Porosity, Wettability, Degradability and Inflammatory sponse. Thus, the difference in wettability as one of the sur-
Response face properties may also be a factor, although Baier et al could
Porous PLLA provokes a more intense inflammatory not demonstrate a difference in the inflammatory response
response from day 7 on, despite a higher degradation rate of against smooth metal pieces having a different wettability.28
The Influence of Porosity and Surface Roughness on Biocompatibility 527
Fig. 47.9. Molecular weight of the
PLLA films as a function of immer-
sion time. (A.) MW. (B.) Mn. Symbols
represent: ❑ = nonporous, + = po-
rous, ❍ = “combi”. Standard deviation
did not exceed 15%.
However, other authors demonstrated that different poly- Also, the size of the pores of the porous PLLA films has in-
mers induce a different level of IL-1 production, correlating creased, possibly with the same effect as fragmentation. In
with the intensity of the inflammatory response,29,30,31 al- the case of PLLA films, the increase in surface area during
though the relation to wettability was not investigated. How- the degradation process as a single factor may be sufficient
ever, the production of IL-1 seems to be less when using for increasing the intensity of the inflammatory response.
relatively hydrophobic materials such as silicon. Further in- It can be concluded that biodegradable PLLA films
vestigation is needed to establish the precise relation between provoke a more intense inflammatory response than non-
wettability and inflammatory response. degradable PTFE films. Also, porosity enhances the inflam-
The differences in inflammatory response against the matory response. However, porosity enhances the inflam-
PLLA (degradable) and PTFE (nondegradable) films became matory response only when the wettability of a biomaterial
apparent from day 40. One reason may be the difference in permits cellular ingrowth.
the rate of degradation and subsequently the difference in
the release of degradation products such as monomers, oli- Relative Polymer Surface Area
gomers and finally fragments. As stated previously, only a The RPSA, which was only determined for the porous
few (mostly nonporous) PLLA films were observed to be and the nonporous polymer film, did not decrease signifi-
broken into two or three pieces. However, there is probably cantly, neither in vitro nor in vivo. However, these results
also increase in surface area which could hardly be observed. must be interpreted with some caution, because RPSA
528 Tissue Engineering of Prosthetic Vascular Grafts
measurements cannot detect release of small amounts of pected to contribute very much to the degradation of poly-
degradation products from the core of the polymer film. The esters in vivo. The role of enzymes in the degradation pro-
loss of weight of the nonporous film could not be detected cess in vivo is not clear. Most enzymes in eukaryotic cells are
with RPSA measurements since there was no detectable loss substrate specific. Therefore, a specific three dimensional
of surface area in cross sections of the PLLA film. There was structure of the substrate (polymer) is required to reach the
also no indication of measurable surface erosion, such as active center of the enzyme. This is not likely at 37°C under
increasing surface roughness and/or decreasing thickness of physiologic enzyme concentrations. The probability is even
the nonporous film. However, this method might be useful smaller for the crystalline parts of the polymer (film). How-
in quantifying the later stages of the degradation and the ever, enhancement of the degradation rate in vitro by en-
resorption process in vivo, when weight measurement has zymes was observed for some polymer/enzyme systems. The
become practically impossible. The results indicate that in role of enzymes may be larger when smaller molecules have
vitro the core of the PLLA films remain largely intact till day been formed by other degradation mechanisms (hydroly-
180, and that in vivo there was no resorption of large sis). This was not investigated in the experiments described
amounts of polymer. These findings support the results of in this paper.
the weight measurements. Degradation of polyesters, such as PLLA, primarily
takes place in the amorphous part of the polymer film.34,35
Molecular Weight This may explain the increasing crystallinity, also observed
The results of the molecular weight measurements by other authors.34 However, in order to explain the differ-
indicate a higher degradation rate for the nonporous film ence in degradation rate between the nonporous and po-
compared to the “combi” and porous film, the latter having rous PLLA film, one must assume other processes taking
the lowest degradation rate. The decrease of MW and MN place during degradation. Vert et al, using different polyes-
is obvious for all three types of film, although results ob- ter “plates” of 2 x 17 x 20 mm, observed the formation of a
tained with GPC must be interpreted with caution when polymer layer around the core of the polymer film, largely
low molecular weight polymers resulting from degradation preventing degradation products (e.g., oligomers) released
processes are examined. in the core, to diffuse freely to the aqueous environment.
Therefore, the differences between the films may not The chemical reactive endgroups of the accumulated oligo-
be significant, but the higher MW and MN of the porous mers in the core then enhance the degradation (autocata-
film compared to the nonporous film, from day 7 till the lytic) process. The nonporous polymer film is more suscep-
end of the experiment at day 180, is very suggestive. The tible to this form of degradation, probably because a larger
initial MW and MN of the different films are not equal inner compartment can be formed compared to the porous
(Figs. 47.9A and 47.9B). A possible explanation may be the and “combi” film. The surface/volume ratio of the latter two
washing out of a larger part of the low molecular weight are probably larger than the cut off value of the polymer
fraction into the media, at the extra rinsing step to remove surface/volume ratio regarding this effect.
the sodium citrate, of the films with a porous component; However, other mechanisms are also possible. There
therefore higher MW and MN were measured. However, it is less flow of the aqueous media in pores of the porous poly-
can be expected that washing out of low molecular weight mer film. Therefore, the concentration of oligomers might
fractions probably also occurs during immersion of the non- be higher in the pores, enhancing degradation. Neverthe-
porous film in the buffer. As a consequence, it can be ex- less, this mechanism apparently has less effect compared to
pected that the difference in MW and MN between the films the mechanism(s) leading to a higher degradation rate of
would disappear in the course of the experiment. This was the nonporous PLLA film.
not the case, indicating another process having an effect on
MW and MN. At day 180 the MW and MN values of the Summary and Conclusions
“combi” film tend to approach the ones of the nonporous In summary, it can be stated that the parameters of
film. This trend corresponds with the erosion of the thin porosity and surface roughness do influence the ultimate
nonporous layer of the “combi” film as observed with SEM: biocompatibility of implanted biomaterials. Potential deg-
The “combi” film was becoming a porous one. Also, the MW radation of a biomaterial will enhance these reactions.
and MN show a lot of fluctuation during the first two weeks
of the experiment. These fluctuations might be caused by Porosity
two phenomena having opposite effects on MW and MN: A high porosity will, as a rule, induce a higher inflam-
washing out of low molecular weight fractions and molecu- matory response as compared to smooth and solid
lar chain fragmentation. However, as stated earlier, this re- biomaterials. The size of pores will, in turn, determine the
mains hypothetical, as the amount of polymer compared to type of foreign body reaction. Small (surface) pores (up to
the amount of buffer in which they were placed did not al- 10 microns) will give rise to more adhesion. In addition,
low for an analysis using GPC. guidance of cells may occur. Ingrowth will not take place.
Larger pores will facilitate tissue ingrowth and therewith
Degradation by Hydrolysis provide a mechanical anchorage of tissue to biomaterial.
Hydrolysis of the ester bonds is the major mechanism Optimal pore sizes have been estimated to be between 50
of PLLA degradation.32 Mechanical stress may enhance the and 200 microns.
degradation process.33 Radiation (UV, IR) or heat is not ex-
The Influence of Porosity and Surface Roughness on Biocompatibility 529
Surface Roughness glycine/DL-lactic acid copolymers. J Biomed Mater Res
As a rule, a high surface roughness will induce a more 1989; 23:1271-1288.
severe foreign body response and also provide better anchor- 12. Rozema FR, Bruin WC de, Bos RRM, Boering G,
age of cells to a biomaterial. The form of the roughness will Nijenhuis AJ, Pennings AJ. Late tissue response to bone
plates and screws of poly L-lactide used for fracture fixa-
determine if other aspects are also involved, e.g., grooved
tion of the zygomatic bone. In: Doherty PJ, Williams RL,
substrata36 will, depending on their dimensions and orien- Williams DF, eds. Advances in Biomaterials, Biomaterial-
tation, improve cell proliferation and orientation. Tissue Interfaces. Amsterdam: Elsevier, 1992:349-355.
13. Schakenraad JM, Oosterbaan JA, Nieuwenhuis P,
Degradation and Biocompatibility Molenaar I, Olijslager J, Potman W, Eenink MJD, Feijin
In general, a degrading biomaterial will evoke a more J. Biodegradable hollow fibers for the controlled release
severe inflammatory response as compared to a nondegrad- of drugs. Biomaterials 1988; 9:116-120.
able material. If this degradable material is porous, we have 14. Spector M. Historical review of porous coated implants.
demonstrated that, in the case of, e.g., collagen, the rate of J Arthroplasty 1987; 2:163-177.
degradation (and thus the degree of foreign body response) 15. Baier RE. Applied chemistry at protein interfaces. Adv
is higher, whereas in the case of polyesters as lactic acid, a Chem Ser 1975; 145:1-25.
16. Wachem PB, Beugling T, Feijen J, Bantjes A, Detmers JP,
porous form will degrade more slowly than a solid form.
Aken WG van. Interactions of cultured human endothe-
In conclusion we can state that porosity and rough- lial cells with polymeric surfaces of different wettabilities.
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J. The influence of substratum free energy on growth and man monocytes. J Immunol 1986; 137:108-113.
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Biomed Mater Res 1990; 24:1203-1219. 28. Baier RE, Meyer AE, Natiella JR, Natiella RR, Carter JM.
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33. Jamshidi K, Hyon S-H, Nakamura T, Ikada Y, Teramatsu 36. Braber ET den, Ruitjer JE de, Smits HTJ, Ginsel LA,
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Scaffold Engineering
Surface Modification
CHAPTER 48
Microgroove Driven Tissue Ingrowth
Edwin T. Den Braber, John A. Jansen
Implants, Tissue Engineering, and Biomaterials
D uring the last two decades the availability and application of medical implants has in
creased dramatically. This concerns a broad variety of medical implants ranging from
knee prostheses to insulin infusion pumps, and from vascular grafts to pacemakers. Some
estimate figures were presented by Ratner1 in his Presidential Address for the Society for
Biomaterials in 1993 (Table 48.1). Although he emphasized that these figures were estimates,
it is clear that the use of implants is considerable. Long term projections even suggest that
implant applications are going to rise in the future. Factors that contribute to this increase
can be ascribed roughly to three major causes. First, the life expectancy of humans increases.
This will inevitably lead to a rise in the demand for implants like hip replacements or artifi-
cial lenses for the treatment of geriatric diseases and defects. Second, more and more medi-
cal treatments are going to include the use of implants in the future. One example of such a
development is the use of percutaneous implants in dialysis.2 Instead of treating patients
with chronic renal failure though intermittent hemodialysis, percutaneous implants enable
continuous ambulatory peritoneal dialysis (CAPD). Third, technologies are evolving that
open new ways of treating specific disorders or defects. This is demonstrated by techniques
that are being developed in the field of tissue engineering.3 Basically, tissue engineering
combines the principles and methods of the life sciences with those of engineering to eluci-
date fundamental understanding of structure-function relationships in normal and diseased
tissues, to develop materials and methods to repair damaged or diseased tissues, and to
create entire tissue replacements.4 Tissue engineering thus spans controlling cellular responses
to implant materials, manipulating the healing environment to control the structure of the
regenerated tissue, producing cells and tissues for transplantation into the body, and devel-
oping a quantitative understanding of many biological equilibrium and rate processes.5
Biomaterials play an important role in many of these activities. Originally, inertness
was thought to be one of the major contributions of the performance of an implant or
biomaterial, but later Williams adapted the definition of biocompatibility to include the
idea that a biomaterial perform with an appropriate host response in a specific application.6
Sadly, most currently used implant materials do not possess these desirable qualities. At his
Presidential Address, Ratner1 voiced this problem very vividly by saying, “For the majority
of our widely used bio-materials, no one sat down in advance and said ‘How can I engineer
the surface of this material to produce the desired biological response?’” He stressed that
most currently used biomaterials, although demonstrating generally satisfactory clinical
performance, were “ad-hoc” biomaterials, developed through a trial and error optimization,
rather than being engineered to produce, for instance, a desired interfacial interaction. The
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
532 Tissue Engineering of Prosthetic Vascular Grafts
devices that closely resemble the original structure, but are
Table 48.1. Selected biomedical implant applications; in no way the best replacement for the original biological
magnitude of usea structure. The development of these kinds of structures
caused Ratner to state during his Presidential Address1 that
Application Numbers Used per Year “existing biomaterials, although demonstrating generally ac-
ceptable clinical success, look like dinosaurs poised for ex-
Ophthalmologic tinction in the light of the winds of change blowing through
Intraocular lenses 1,400,000 the biomedical, biotechnological, and physical sciences”.
Contact lenses 250,000,000b
Retinal surgery implants 50,000 Surface Micropatterns Manipulating Cellular
Prothesis after enucleation 5,000
Behavior
Cardiovascular Among the tools that Ratner identified as having a
Vascular grafts 350,000 great potential for creating engineered biomaterials is the
Arteriovenous shunts 150,000 use of nano- and micropatterned surfaces.1 The possibility
Heart valves 75,000 to influence the behavior of cells by the topographical mor-
Pacemakers 130,000 phology of the surface on which these cells are cultured was
Blood bags 30,000,000
first discovered during the first part of this century. In 1914,
Reconstructive
Breast protheses 100,000 Ross G. Harrison of the Osborn Zoological Laboratory of
Nose, chin 10,000 Yale University cultured cells on spiderwebs.9 In retrospect,
Penile 40,000 this in itself seems quite an achievement, especially when
Dental 200,000 we keep in mind that in 1914 investigators did not have all
the technical equipment that are available in modern cell
Orthopedic culture laboratories nowadays. During his studies, Harrison
Hips 90,000
noticed that the direction of movement of the cells was in-
Knees 65,000
Shoulders, finger joints 50,000 fluenced by the structure of the fragile substrata on which
these cells were incubated. Furthermore, he noticed that the
Other devices cells adapted a shape that seemed to be governed by the lin-
Ventricular shunts 21,500 ear spiderweb threads. Later, this phenomenon was con-
Catheters 200,000,000 firmed by Loeb and Fleisher,10 who introduced the term ste-
Oxygenerators 500,000 reotropism, which was described as the direction in which
Renal dialysers 16,000,000
Wound drains 3,000,000
cells move, governed mainly by the contact with solids or
Sutures 20,000,000 very viscid bodies like fibers or fibrin. In 1945, Weiss con-
a ducted experiments with a wide range of substrata like
Approximate annual usage in United States of America
b plasma clots, fish scales, and engraved glass, and termed the
Worldwide
changes in cell behavior observed by Harrison “contact guid-
ance”.11 Surprisingly, no further attention was paid to this
guidance phenomenon until the early 1970s. It was Rovensky
importance of the issue raised by Ratner is underlined by et al12-13 and Maroudas14-15 who rediscovered that the be-
recent estimates that indicate that biomaterials such as met- havior of cells is affected by the topography of a substratum
als, ceramics, and polymers are found in more than 5,000 surface. Rovensky12-13 studied the behavior of fibroblast-like
different medical devices and almost 40,000 different phar- cells on different kinds of substratum surfaces with an or-
maceutical products, with a collective annual sales approach- derly distribution of 40.0 ∝m deep grooves having a trian-
ing 100 billion US dollars.5 If we include all products that gular profile. He reported that the cells had a bipolar elon-
utilize biomaterials, then the number of people affected gated shape, were orientated after adhesion, and grew par-
worldwide exceeds 1000 million per year.4,7 allel to the grooves. Meanwhile, Maroudas14-15 studied the
As a possible handle for the development of “smart” growth of fibroblasts on small glass beads (diameter
biomaterials, Ratner1 advocated an engineering approach 20-60 ∝m), fibers, and platelets. He observed that cells grown
to achieve the integration between the biomaterial and the on beads with a large diameter tended toward forming
surrounding tissues. He suggested that it is worthwhile to multilayers, while smaller beads progressively failed to sup-
look at how nature handles specificity and rapid-reaction port growth.
kinetics, keeping in mind that three design themes are iter- Since these studies by Rovensky and Maroudas, sev-
ated and exploited throughout biology, i.e., order, recogni- eral investigators have studied the behavior of various types
tion, and mobility. However, as Brunette8 later remarked, of cells to a variety of microtextured substrata materials.
using the principles of design to achieve the optimal struc- These studies have been reviewed very recently by Singhvi
ture differs from attempting to reproduce the original struc- et al,16 von Recum and van Kooten,17 and Brunette.8 In these
ture. According to Brunette, attempting to imitate nature is in vitro and in vivo studies, some specific alterations in cel-
approximate since the complexity and variability of natural lular behavior were seen, while other changes that were sug-
systems make them impossible to replicate with perfect gested by several leading researchers in this field were not.
fidelity. This has led to the production of structures and In the following paragraphs we will attempt to give a com-
Microgroove Driven Tissue Ingrowth 533
prehensive listing of the most obvious and general accepted circular shape and size, washed, sterilized, and inspected as
alterations in cell behavior as a result of cells contacting sur- described elsewhere.18-19
faces with a pattern of parallel microgrooves. We will dem- For the evaluation of the cellular behavior on the
onstrate this by describing various studies that were per- smooth and microtextured silicone substrata, primary cul-
formed at our laboratories. In addition, we will discuss the ture rat dermal fibroblasts (RDFs) were used. These cells were
leading theories that attempt to create a model in which the harvested from ventral skin grafts taken from male Wistar
phenomenon of contact guidance is clarified. rats (100-120 g). After dissociation, these cells were incu-
bated for several days according to a specific protocol.18-19
Changed Cell Shape and Cellular Orientation Subsequently, the fifth generation was used for incubation
If cells are cultured on a surface with parallel micro- on the microtextured surfaces. Therefore, the smooth and
grooves as shown in Figure 48.1, the first thing that can be microtextured substrata were placed in the culture wells of
observed is the change of the shape of the cells. In earlier 24 well plates, after which approximately 1.0 x 104 viable
studies,18-19 we investigated several groove and ridge dimen- RDFs ml-1 were added to each substratum. The RDFs were
sions to study and quantify the influence of these groove incubated on a specific substratum up to 7 days under static
patterns on the size, shape, and orientation of the cells cul- conditions.
tured on these substrata. The effect of the surface microgeometry on the cellu-
In order to obtain a microgrooved substratum, we first lar morphology was quantified by digital image analysis.18-19
produced silicon oxide molds in a class 100 clean room us-
ing photolithography.20-21 Photolithography is a technique
that is developed for the production of microelectronic com-
ponents like, for example, computer microcircuits. In our Table 48.2.Dimensions of the micro features on the
experiments, these mold surfaces were produced by coating substrata surfaces (Gd=groove depth, Gw=groove
silicon oxide masks with high reflective chrome, after which width, Rw=ridge width, and P=pitch).
the chrome was coated with a thin (0.5 ∝m) layer of positive
photoresist. Subsequently, the photoresist was exposed and Gd Gw Rw P
developed, uncovering the underlying chrome, which was (∝
∝m) (∝
∝m) (∝
∝m) (∝
∝m)
etched. Finally, the unexposed photoresist was stripped off,
thus creating a parallel groove pattern. The substrata were 1.00 1.00 1.00 2.00
finally obtained by covering the molds with polydimethyl- 1.00 1.00 2.00 3.00
1.00 1.00 4.00 5.00
siloxane, thus producing negative surface replicas which
1.00 1.00 8.00 9.00
possessed either a smooth surface or a surface with parallel 1.00 4.00 1.00 5.00
grooves, showing a wide variety of groove and ridge widths 1.00 8.00 1.00 9.00
(Table 48.2). During our experiments, the groove depth was 0.45 2.00 2.00 4.00
either 0.5 ∝m or 1.0 ∝m. After polymerization, the silicone 0.45 5.00 5.00 10.00
rubber castings were removed, cut into their appropriate 0.45 10.00 10.00 20.00
Fig. 48.1. Three dimensional repre-
sentation of the results of AFM
measurements on a microtextured
substratum surface with parallel
grooves of 5.0 ∝ m, separated by
5.0 ∝m ridges. The grooves are ap-
proximately 0.5 ∝m deep. Different
X- and Y-axis magnifications were
used to clarify the conformation of
the substratum surface.
534 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 48.2. Schematic representation
of a rat dermal fibroblast (RDF) on
a microtextured substratum. The
parameters measured during digital
image analysis (DIA) were the RDF
surface area (area within perimeter),
the longest length of the cell (L), the
cellular breadth (B), perimeter (P),
circularity (not shown), angle of
cellular orientation (# ), and the
number of pitches spanned by the
cell (N).
In short, RDFs were photographed by phase contrast mi- the microtextured substratum surfaces and the overlaying
croscopy on every day of the incubation period. These pho- fibroblasts. During these additional experiments, we cultured
tographs were scanned digitally and analyzed with an image the RDFs similarly as in the earlier studies,18-19 but visualized
analysis program. This program made it possible to mea- specific cytoskeletal components and proteins interacting
sure several cell parameters, i.e., the cellular surface area, in the attachment of the RDFs with the following mono-
cellular perimeter, cellular circularity, maximum cell length, clonal and polyclonal antibodies:22
cell breadth perpendicular to the maximum length, the angle 1. The mouse monoclonal antibody hVIN-1, specific
of cellular orientation relative to the surface grooves (#), and for vinculin;23
number of pitches spanned by a single cell (Fig. 48.2). 2. The rabbit monoclonal anti-fibronectin antibody
These studies provided a lot of interesting data, but FN-3E2;24
most of all clearly showed that the RDFs on surfaces with a 3. A rabbit polyclonal antiserum raised against bovine
ridge width smaller than 4.0 ∝m were highly orientated fibronectin;25
(# < 10°) and elongated along the surface grooves (Figs. 48.3 4. The rabbit polyclonal antiserum against bovine
and 48.4). However, if the ridge had a width larger than vitronectin;26
4.0 ∝m, then the cellular orientation was random (Χ45°) and 5. A polyclonal antiserum raised against human
the shape of the RDFs became significantly more circular vitronectin in rabbits, which has been shown to have
(Fig. 48.5). The appearance and orientation of the cells on a good crossreactivity with rat vitronectin, but not
the surfaces with ridge widths larger than 4.0 ∝m in many with bovine vitronectin.27
cases proved to be not significantly different from those on After incubation, these primary antibodies were
the smooth substratum surfaces (Fig. 48.6). Furthermore, it incubated with the appropriate secondary antibody, i.e., fluo-
became apparent that the ridge width was the most impor- rescein isothiocyanate (FITC)-conjugated goat anti-mouse
tant parameter, since varying the groove width and groove IgG or FITC-conjugated goat anti-rabbit IgG. For the
depth did not affect the RDF size, shape, or the angle of staining of the RDF filamentous actin no antibodies were
cellular orientation (#) significantly. used, since F-actin was visualized with thiorhodamine
isothiocyanate (TRITC)-labeled phalloidin. Immediately
Cell Attachment on Microtextured Surfaces after performing the double stain procedures, the RDFs were
During these studies, we found after careful examina- examined with a confocal laser scanning microscope
tion of the phase contrast images that the fibroblasts showed (CLSM), which was equipped with a krypton/argon mixed
indications of attaching specifically to the ridges of the sur- gas laser. This type of laser offers separate, well spaced wave-
face patterns (Fig. 48.4). If the fibroblasts did actually attach lengths for the excitation of FITC (ϑ = 488 nm) and TRITC
solely to the surface ridges, this would mean that the cells (ϑ = 568 nm).28 Next to the fluorescence mode, the reflection
displayed a specific preference of attachment location. In- mode of the CLSM was used to visualize the underlying sub-
trigued by the phase contrast microscopy/digital image stratum surface.22 The resulting digital images were captured
analysis results, we performed experiments which were de- and overlay images were created, thus making it possible to
signed to show us more of the intricate interaction between capture the fluorescent and reflection data in one 24 bit RGB
Microgroove Driven Tissue Ingrowth 535
Fig. 48.3. Phase contrast image of RDFs
on a B substratum (Gw 1.0 ∝m, Rw
1.0 ∝m, Gd 1.0 ∝m, bar = 100 ∝m) on
day 1. The cells are highly aligned and
elongated along the surface grooves.
Fig. 48.4. Phase contrast image of
RDFs on a substratum with grooves
of 8.0 ∝m wide and 1.0 ∝m deep,
while the ridge has a width of 1.0 ∝m
(bar = 100 ∝m) after 2 days of incu-
bation. These substrata are a nega-
tive replica of the substrata in Fig-
ure 48.5. The RDFs are clearly ori-
entated. Cell protrusions attach to
the ridges (arrows ).
(Red-Green-Blue) picture. Creation of these 24 bit images vinculin relative to the grooves and ridges of the
enabled the composition of 1 digital image with 3 different microtextured surface was charted and analyzed.22
information levels, each within one color segment, and of- The results of the CLSM observations and additional
fered the possibility to investigate the stained objects in con- image and statistical analysis showed that the microfilaments
junction with each other and the surface patterns. First, the and vinculin aggregates of the RDFs on the 2.0 ∝m grooved
(acute) angle of orientation of F-actin, vinculin, fibronectin, substrata were orientated along the surface grooves, while
vitronectin, and the surface grooves relative to a virtual X-Y these proteins were significantly less orientated on the 5.0
axis were measured. Second, the relative position and the and 10.0 ∝m grooved surfaces (Fig. 48.7). In contrast, bo-
angle of the linear components of vinculin, fibronectin, and vine and endogenous fibronectin and vitronectin were ori-
vitronectin were compared with the position and angle of entated along the surface grooves on all textured surfaces.
orientation of the actin filaments. Finally, the location of These extracellular proteins did not seem to be hindered by
536 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 48.5. Phase contrast image of
RDFs on a substratum with grooves
of 1.0 ∝m wide and 1.0 ∝m deep, and
8.0 ∝m wide ridges (bar = 100 ∝m) on
day 2. Appearance of and orientation
of the fibroblasts does not differ sig-
nificantly from the RDFs on a smooth
substratum surface.
Fig. 48.6. Phase contrast image of
RDFs on a smooth substratum after 2
days of incubation (bar = 100∝m). The
well spread RDFs have a characteris-
tic multipolar appearance and are ran-
domly orientated.
the surface grooves, since many groove spanning filamen- vealed that these focal adhesion points were occasionally
tous deposits were found on all microgrooved surfaces wrapped around the edges of the ridges. Attachment of the
(Fig. 48.8). Vinculin was located mainly on the surface ridges cells on the silicone rubber and titanium microtextured sub-
on all textured surfaces (Fig. 48.9). strata was never observed on the surfaces with 1.0 or 2.0 ∝m
These findings were later confirmed in additional stud- grooves. Only the RDFs on the 5.0 ∝m and 10.0 ∝m grooved
ies.29-30 During these investigations we made ultrathin sec- surfaces protruded into the grooves, while attachment to the
tions of RDFs cultured on silicone and titanium groove floor was observed only on the 10.0 ∝m textures. In
microgrooved surfaces with parallel grooves ranging from addition, only the RDFs on the titanium 5.0 ∝m and 10.0
1.0 ∝m up to 10.0 ∝m. The depth of these grooves varied ∝m grooved surfaces possessed focal adhesion points on the
between 0.45 ∝m and 2.2 ∝m. Transmission electron micros- walls of the grooves (Fig. 48.10). Comparison between the
copy (TEM) again showed that the focal adhesion points of observations of the cells on the microtextured silicone rub-
the RDFs on both the silicone rubber and titanium ber and titanium substrata suggested that material specific
microtextured surfaces were located mainly on the surface properties did not influence the orientational effect of the
ridges (Fig. 48.10). On the titanium surfaces, TEM also re- surface texture on the observed RDF cellular behavior.30
Microgroove Driven Tissue Ingrowth 537
Fig. 48.7. Digital overlay image of a
reflection micrograph of a 5.0 ∝m
grooved silicone surface and the cor-
responding RDF actin after a incuba-
tion of 3 days. Fibronectin filaments
at the ventral side of this fibroblast can
be seen in Figure 48.8. Note the simi-
larity in the orientation of the mi-
crofilaments and fibronectin fila-
ments in Figure 48.8.
Fig. 48.8. Immunofluorescence
micrograph of a rat fibronectin stain-
ing on a surface with 5.0 ∝m grooves
(incubation period of 3 days)
showing thin fibronectin filaments at
the leading edge of the cell. The main
fibronectin filaments possess similar
orientational vectors, which are
directed roughly parallel to the
groove pattern.
Fig. 48.9. Overlay image of the location
of vinculin (bright spots) in relation
to the 10.0 ∝ m grooves after an
incubation of 5 days. Vinculin is
located mainly on the surface ridges
and often attaches to the edges of
these ridges.
538 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 48.10. Transmission electron
micrograph of a RDF on a 5.0 ∝ m
grooved titanium surface after 3 days of
incubation. The cell protrudes into the
groove, but does not contact the bottom
of the groove. Focal adhesion points
(arrowheads) can be seen on the edge of
the ridge and the wall of the surface
groove. Larger magnification showed
that the focal adhesion point on the
lefthand ridge edge was wrapped around
this edge.
Other Surface Topography Induced Cell changes in cellular differentiation, DNA/RNA transcription,
Behavior Alterations In Vitro cell metabolism, and cellular protein production.16-17,34-35
Apart from changes in cell size, shape, orientation, and For example, increased F-actin content and increased per-
attachment, investigators have suggested and studied many sistence and speed of cell movement of macrophage-like cells
other cell processes that could possibly be influenced by the on grooved surfaces were observed, and changes in the regu-
surface topography of substrata. One of these processes is lation of fibronectin mRNA levels, mRNA stability, and
cell proliferation. On the basis of the results of some of our fibronectin stability and assembly on surfaces with micro-
earlier studies,18,31 we concluded that the presence or dimen- grooves have been reported. Similar results were described
sions of the parallel surface grooves as used in our experi- by Hong and Brunette,36 who saw clear differences in the
ments did not result in a change in RDF proliferation rate. fibronectin mRNA and proteinase levels between oral epi-
This conclusion was, however, in contrast to the findings of thelial cells that were cultured on smooth and microgrooved
Green et al and Ricci et al. For example, Green et al32 re- surfaces. Consequently, they remarked that these surface
ported that especially abdomen fibroblasts (CCD-969sk) topography induced changes could be considered as “good
cultured on surfaces with 2.0 and 5.0 ∝m square pillars news/bad news”. According to these investigators, the good
showed increased proliferation rates. In addition, Ricci et news was the fact that specific surface topographies could
al33 evaluated the in vitro growth of rat tendon fibroblasts be used to enhance the production of a specific protein, while
and rat bone marrow colonies on unidirectional (grooved) the bad news might be that the production/secretion of other
surface microgeometries. They found that the overall colony proteins might also be enhanced. If the production/secre-
growth rate was changed, and concluded that surface tion of these “other proteins” would be elevated, this might
microgeometry could be used to control the growth rate at have a deleterious effect on the integration of the implant.
implant surfaces. However, this study by Ricci et al also For example, a rise in the production/release of proteinases
showed that the response to surface topography is depen- could result in a large scale degradation of the connective
dent on cell type, which could account for the discrepancy tissue. This simple example demonstrates that, at the mo-
in the results that we found between the proliferation rates lecular level, the regulation of cell function by altering the
of our rat dermal fibroblasts, the results of Ricci et al, and surface topography of an implant or substratum could be a
the data of Green et al. A point of discrepancy could be the very complex affair.
use of different surface textures. Although Ricci et al used a
pattern of parallel grooves, Green et al used a texture con- Microtextured Surfaces In Vivo
sisting of square pillars. This could mean that we not only Up to this point, we have only described the effect of
have to consider the possibility of cell type dependent dif- microtextured surfaces on cells in vitro. However, the re-
ferences in cell proliferation, but also have to contemplate sults of these in vitro studies have led to the idea that sur-
the effect that different surface textures could trigger. face microtexturing could be used deliberately to achieve
Another process that seems to be affected by the to- certain desired end results in processes like morphogenesis,
pography of the surface that the cell contacts is that of the cell invasion, repair, and regeneration.4 If this hypothesis
cell metabolism. Several publications have reported behav- proves to be true, it is obvious that surface texturing can be
ioral differences of cells on microtextured surfaces, such as a very important tool in designing successful implants.17,37
Microgroove Driven Tissue Ingrowth 539
Sadly, in vivo studies with microtextured implants challeng- gradually after cell-cell contacts are formed.18,37,43 In tissues,
ing these ideas are scarce. In addition, review of these in vivo these contacts with other cells are already present, which
studies shows that the design of the used textured implants could mean that the orientational effect of the textured sur-
is very diverse. But even with this large diversity, it is pos- faces is overruled by stronger tissue related signals or cues.
sible to perceive the possible potential of microtextured im- Three dimensional reconstruction of CLSM images
plant surfaces on several implant related processes. For ex- and normal light microscopy showed no significant differ-
ample, some studies38-39 have reported on the reduction of ences between the thickness of the capsule surrounding the
epithelial downgrowth with microgrooved skin penetrating smooth and microgrooved implants. Since differences be-
devices. Other investigators, who implanted microporous tween the 2.0, 5.0, and 10.0 ∝m grooved implants were not
or pillared surfaces subcutaneously, found tightly adherent detected, we concluded that the depth of the grooves used
fibrous capsules without inflammatory cells,40 reduced fi- was not sufficient to facilitate “mechanical interlocking”. This
brosis,41 and improved blood vessel proximity.41 interlocking would reduce the stress and movement at the
In order to investigate the effect of microtextured im- implant interface and limit the consequential “mechanical
plants on the surrounding tissue and to be able to compare irritation” of the surrounding tissues, which is supposed to
our in vitro and in vivo data, we implanted the smooth and induce tissue damage, fibrosis, and severe inflammatory re-
microtextured silicone rubber substrata with 2.0, 5.0, and sponses.16-17,40-41 Other investigators have indeed reported
10.0 ∝m grooves (Table 48.2) subcutaneously in a total of 12 reduction of the capsule size due to microtextured surfaces.
female New Zealand White rabbits for periods of 3, 7, 42, However, review of these studies40-41,44-45 shows that the sur-
and 84 days.42 Scanning electron microscopy (SEM) showed face texture of the implants in these studies differs signifi-
fibroblasts, erythrocytes, lymphocytes, macrophages, fibrin, cantly from our implants, both in terms of microfeature
and collagen on all implant surfaces after 3 and 7 days appearance (pores, pillars, tapered pits, or V-shaped grooves)
(Fig. 48.11). After 42 and 84 days only a little collagen and a and dimensions (feature depth, size, and pitch). As a result,
small number of fibroblasts, but no inflammatory cells, were the mechanical irritation of the smooth and grooved sur-
seen on the implant surfaces. In contrast with the RDFs in faces would be comparable, resulting in capsules of equal
the in vitro experiments, the fibroblasts that were observed thickness. Furthermore, our textured implants possessed one
on the microtextured implant surfaces, were not orientated smooth and one textured side. Although this opened up the
along the surface grooves. A possible explanation for these possibility for intraimplant evaluation, it did not enhance
differences between in vitro and in vivo orientational cell possible mechanical interlocking between the implant and
behavior could be that the cells that are used in in vitro stud- the surrounding tissues. Therefore, it can be questioned
ies are isolated cells, which have no contact with other cells, whether the capsule thickness would have been less if both
cell types, or ECM. Previous studies18,37,43 have shown that sides of the implant had been textured.
prolonged in vitro incubation on microtextured surfaces re- Furthermore, normal light microscopy did show a sig-
sults in the formation of cell-cell contacts, an increase of the nificantly lower number of inflammatory cells, and a sig-
spread area, and a decrease of the orientation of the cells on nificantly higher number of blood vessels, in the capsules
these surfaces. Consequently, it was supposed that the ob- surrounding the microgrooved implants. The cause of these
served guidance phenomenon is an initial response of cells differences remains unclear. However, it is possible to specu-
in vitro to certain microtextured surfaces, which is lost late that the differences in the number of blood vessels were
Fig. 48.11. On this SEM image (3 days
of implantation), the 2.0 ∝m grooved
silicone surface is visible underneath
the collagen fibers. Several punctured
erythrocytes (E) and a macrophage
(M) were located within the
collagen matrix.
540 Tissue Engineering of Prosthetic Vascular Grafts
part of the proliferation phase of the wound healing pro- energy have an influence on the displayed cellular behavior.
cess.46 This proliferation phase is a part of the formation of The vinculin location and orientation however, pleas in fa-
granulation tissue, which is characterized by high fibroblast vor of the “ridge width” theory, although warped focal ad-
densities, the formation of new blood vessels, and a new hesion points that were found in the TEM results30 seem to
connective tissue matrix.46 After repair, the number of the contradict this theory. Finally, whether the cells orientate to
vessels decreases generally, marking the end of the wound the microtextured surfaces as a result of the force distribu-
healing process and the start of a steady state. The fact that tion that is created by the texture of these surfaces is impos-
more vessels were observed around the textured implants sible to determine since no (known) studies have been per-
during our study could indicate a higher rate of tissue re- formed to map the force distribution within cells cultured
pair around these implants. Further research is, however, on microgrooved substratum surfaces. Recognizing the fact
required to determine if this in fact is the case. that these three hypotheses can even be integrated into one
overall model contributes to the intriguing phenomena of
The Hypotheses cellular behavior on microtextured surfaces.
In the previous paragraphs we have presented several
studies with microgrooved (implant) surfaces. In spite of Future Perspectives: Vascular Grafts
the fact that several publications like these and some excel- and Microtextured Surfaces
lent reviews 3,16-19,31,37 have reported on the effects of As mentioned before, many investigators have already
microtextured surfaces, little is known about the exact speculated on the benefits of microtextured implants. For
mechanism whereby surface topography exerts its effects. example, Ratner1 speculated on the benefits of an implant
Several theories have been suggested, however. First, it has surface that would not cause the formation of a fibrous cap-
been hypothesized that wettability plays a role in these phe- sule. According to Ratner, such an implant would not “be
nomena. A microtextured surface could possess local differ- walled off ”, but the cells contacting the implant surface would
ences in surface free energy which promote a specific depo- respond “as if they are not seeing and interacting with the
sition pattern of the substratum-bound attachment pro- biomaterial”. This wound healing reaction would be prefer-
teins.16-17,47-49 In addition, the spatial arrangement of the able for the clinical success of several frequently used im-
adsorbed proteins50-52 and the conformation of these pro- plants. For example, reduction of the capsule thickness
teins53-54 would be influenced by these substratum surface around an implant would mean a reduction of the capsule
properties. Second, it has been suggested that the specific contraction that is often observed with, for example, sili-
geometrical dimensions of the focal adhesion plaques force cone breast implants.66 Furthermore, capsule reduction
a cell on a surface with small grooves and ridges to orientate would enhance the performance of many implanted
itself parallel to these ridges.55-56 This hypothesis is based biosensors, pacemakers, and infusion pumps.17 These de-
on the observation that a minimum length of 2.0 ∝m is re- vices all benefit from an optimal contact between the tissues
quired for focal contacts to establish adhesion.57 This im- and the implant for the transduction of signals. For instance,
plies that, if the ridge width increases, multiple vectors of the necessary electrical pulse of a pacemaker would be bet-
adhesion plaque orientation are possible, enabling less ori- ter conducted to the heart muscle if the capsule around the
entated cell attachment. Finally, a third hypothesis58-59 sup- electrode of this device were minimized. Another good ex-
poses that the orientation and alignment of cells on ample is the sensor of an implanted insulin infusion pump.
microtextured surfaces are a part of the cellular efforts to For optimal detection of insulin levels, maximal contact
reach a biomechanical equilibrium with the net sum of forces between the sensor of this device and the surrounding tis-
minimized. This phenomenon has, for instance, been de- sues is required. However, if a fibrous capsule shields the
scribed extensively in the so-called tensegrity models.58-60 sensor from the crucial signal, the performance of the im-
According to these models, it is possible that the anisotropic plant will be insufficient.
geometry of substratum surface grooves and ridges estab- Additional applications of microtextured biomaterials
lishes stresses and shear-free planes that influence the direc- have been reported in the discipline of tissue engineering.
tion of microtubule61 and microfilament growth62-63 in or- For example, microtextured surfaces have been used in in
der to create a force economic situation. vitro experiments to decrease hepatocyte dedifferentia-
Given the current available information, it is impos- tion67-68 or to induce guided nerve regeneration.42,69-70 Skin
sible to express an opinion on which or whether one of these autografts already have been generated out of individual
hypotheses is true. On the other hand, several separately keratinocytes by using orienting scaffolds,71 while specula-
performed studies have reported that surface microtextrues tions are voiced that guided tissue regeneration could per-
can have a profound effect on specific elements of the cy- haps reduce the formation of scarring tissue and enhance
toskeleton like, for example, the microfilament bundles,62-65 the repair of highly orientated structures like tendons.46,72-73
focal contacts,55 and microtubules,61 making it safe to say Furthermore, attempts have been made to produce large
that microtextured surfaces influence the orientation of the tubular morphologies with the help of tissue engineering
intracellular and extracellular proteins. Although results of that could function as intestine or ureter segmental replace-
our studies corroborate with all three hypotheses, they do ments.74 In addition, many publications have reported on
not justify a specific choice for one of these hypotheses. The efforts to produce blood vessels.1,3,75-82 Microtextured
differences in deposition patterns and the appearances of biomaterials could be very useful as scaffolds in this field of
the ECM proteins during the CLSM study for example, make tissue engineering research.
it possible to suggest that surface properties like surface free
Microgroove Driven Tissue Ingrowth 541
Figure 48.12 shows the basic anatomy of a blood ves- possibly be obtained by applying longitudinal or circular
sel. Large blood vessels have a thick, tough wall of connec- groove patterns. Not only would the overall orientation of
tive tissue and smooth muscle, which is lined by an exceed- the cell be influenced, but also the orientation of the cytosk-
ingly thin, single layer of endothelial cells, separated from eleton, thus contributing to the mechanical stiffness of the
the surrounding outer layers by a basal lamina. The thick- cells.84 By using microgrooved scaffolds, endothelial orien-
ness of the connective tissue component of the vessel wall tation could be achieved without exposing the cells to flow
varies with the vessel’s diameter and function, but the en- and cyclic stretch, phenomena that have a comparable ef-
dothelial lining is always present.83 Study of embryos has fect on the orientation of endothelial cells.85-86 This com-
revealed that large blood vessels have all developed from parison also concerns the level of gene expression. It has been
small simple vessels constructed solely out of endothelial reported that flow and cyclic stretch can influence the regu-
cells and a basal lamina.83 Therefore, the endothelial cells lation of messenger RNA,87-88 an effect on the cellular me-
are often referred to as the pioneers, preceding the develop- tabolism that is also supposed for cells on microtextured
ment of connective tissue and smooth muscle around the surfaces. What kind of effect the combination of the effects
vessels when required. This is why early studies have con- of factors like surface texture, flow, and cyclic stretch will
centrated on the formation of capillary tubes out of colo- have remains to be seen, since no study investigating this
nies of endothelial cells.76 However, the tissue engineering combination has been performed up to this date.
of a functional, well developed substitute blood vessel does Another interesting option concerns the extracellular
not depend solely on the presence of vascular cells and ex- matrix proteins. The endothelial cells and smooth muscle
tracellular matrix (ECM) components. Several cellular sig- cells in vivo are surrounded by an intricate mixture of pro-
nals are equally important to processes such as cell growth teins like collagens, elastin, laminin, fibronectin, and
and cellular differentiation. Ziegler and Nerem79 identified glycoaminoglycans.79 Several studies have shown that these
these signals as originating from three sources, i.e.: proteins can affect the growth, differentiation, and choles-
1. Chemical signals, derived from the fluid (blood) terol metabolism of both the endothelial and smooth muscle
flowing through the vessel; cells. Endothelial derived ECM can affect smooth muscle
2. Signals associated with the ECM—the ECM proteins cell growth, depending on its composition.89 Collagen and
not only hold the vascular wall together, but also par- fibronectin containing matrices promote the growth of
ticipate in regulating the biology of the vascular wall; smooth muscle cells, while matrices containing heparan sul-
3. The mechanical environment of the vascular wall, fate proteoglycans selectively inhibit identical smooth muscle
imposed by the hemodynamics of the vascular sys- cell populations.89 Furthermore, ECM proteins can change
tem. the response of smooth muscle cells to low density lipopro-
If we speculate on the roles that microtextured scaf- teins (LDL). Earlier study90 has shown that collagen type I,
folds could play in meeting these demands, one is evident. for example, induces a decrease in smooth muscle cell
The orientation of the endothelial cells in the direction of growth, while endothelial derived ECM induces an increase
the flow, and circularly orientated smooth muscle cells, could in smooth muscle cell growth following incubation with
Fig. 48.12. Schematic drawing of the
anatomy of a blood vessel. The lon-
gitudinal orientation of the endothe-
lial lining (1) and the circular orien-
tation of the smooth muscle cells
(2) can be seen clearly.
542 Tissue Engineering of Prosthetic Vascular Grafts
LDL.90 The ability of microtextured surfaces to enhance the 13. Rovensky YA, Slavnaya IL. Spreading of fibroblast-like
production of specific proteins as mentioned earlier8 could, cells on grooved surfaces. Exp Cell Res 1974; 84:199-206.
if true, prove a powerful tool in the creation of blood vessels 14. Maroudas NG. Anchorage dependence: Correlation be-
through tissue engineering. tween amount of growth and diameter of bead, for single
cells grown on individual glass beads. Exp Cell Res 1972;
These few examples show that microtextured surfaces
74:337-342.
could contribute in the study of, and the creation of, vascu- 15. Maroudas NG. Growth of fibroblasts on linear and pla-
lar grafts. This ranges from in vitro study of smooth muscle nar anchorages of limiting dimensions. Exp Cell Res 1973;
cells, where these surfaces could stop/slow down the change 81:104-110.
from a nongrowing, contractile phenotype to a proliferat- 16. Singhvi R, Stephanopoulos G, Wang DIC. Review: Effects
ing, protein secreting mode,91-92 to the induction of endothe- of substratum morphology on cell physiology. Biotech-
lial and smooth muscle cell orientation in artificial grafts.1,75 nology and Bioengineering, 1994; 43:764-771.
Therefore, it is recommended that research of microtextured 17. von Recum AF, van Kooten TG. The influence of micro-
surfaces be exploited further in order to develop “smart” topography on cellular response and the implications for
manipulative implants with higher clinical success rates. In silicone implants. J Biomater Sci Polymer Edn 1995;
addition, these new studies could also contribute to a better 7:181-198.
18. den Braber ET, de Ruijter JE, Smits HTJ et al. Quantita-
understanding of the mechanisms that cause cellular con-
tive analysis of cell proliferation and orientation on sub-
tact guidance and guidance related processes. Such insight strata with uniform parallel surface micro grooves.
would not only enlarge the general knowledge of these Biomaterials 1996; 17:1093-1099.
processes, but also offer implant designers a tool for design- 19. den Braber ET, de Ruijter JE, Ginsel LA et al. Quantita-
ing a wider variety of more successful implants with tive analysis of fibroblast morphology on microgrooved
predicable qualities. surfaces with various groove and ridge dimensions.
Biomaterials 1996; 17:2037-2044.
Acknowledgments 20. Schmidt JA, von Recum AF. Texturing of polymer sur-
This study is supported by the Technology Founda- faces at the cellular level. Biomaterials 1991; 12:385-389.
tion (STW). 21. Schmidt JA, von Recum AF. Surface characterization of
microtextured silicone. Biomaterials 1992; 13:675-681.
22. den Braber ET, de Ruijter JE, Ginsel LA et al. Confocal
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Scaffold Engineering
Surface Modification
CHAPTER 49
Surface Bonding of Heparin
Patrick T. Cahalan
n the early 1960s Hufnagel began experiments to form “autogenized” vascular prostheses.1
I This was accomplished by implanting a Teflon rod containing a loosely woven Dacron or
polypropylene cloth surrounding it, and allowing 6-8 weeks for the formation of a collagen
tube. Early experiments involved implantation back into the same animal to avoid immu-
nogenic response. At the start of his work, important knowledge concerning glutaraldehyde
fixation of tissue was awaiting further publications.2 “Autogenized” was replaced with the
term “xenograft” due to parallel efforts by Sparks.3 Hufnagel stayed with his studies over the
next two decades and moved on to attempt heparin incorporation into mandril formed
prostheses as well as commercially available glutaraldehyde-treated human umbilical vein.
Rigorous experimental efforts, including radioactively tagged heparin, allowed him to de-
termine concentration of heparin in solution to effect maximal heparin loading conditions,
and to his surprise he found that continued washing with saline for two hours post “imbib-
ing” into the prosthesis did not significantly diminish the retained heparin, which was about
300 mg/ml. In implant studies the rate of early occlusion was significantly reduced, and
Hufnagel reported that the half life of the heparin was limited to a few days.
While the goal was to prevent thrombotic occlusion, in retrospect the most interesting
observation is the ability of heparin to bind and release from collagen in an unpredicted
manner. The hypothetical paradigms of the time with respect to interactions of biomolecules
and surfaces were still rather narrowly focused in simple ionic charge relationships.4 Pro-
teins (collagen) and polysaccharides (heparin) were widely known in the 70s by carbohy-
drate chemists to possess unique complexing capabilities that could be used to form
viscosifying agents, stabilizers, and precipitation agents in food applications. The synergistic
effect of these biomolecules could not be explained by simple ionic charge relationships.
The tertiary and quaternary structures of biomolecules play an important role in their in-
teractions with other molecules. While this concept is well known to anyone with an intro-
ductory education in biochemistry, it appears to be often forgotten in strategies for immo-
bilizing biomolecules on surfaces. There remain very active efforts at producing a synthetic
ionic analog heparinoid surface,5,6 while at the same time studies are showing how critical is
the dependence of heparin’s bioactivity on its unique structure.7
Early attempts at heparinizing surfaces used fatty quaternary ammonium compounds
as a base coating on polymers in order to present the positive charge of the quaternary
amine for ionic coupling of heparin.8 These early studies clearly showed an impact of hep-
arinized surfaces, but the heparin released rather quickly and was material dependent, sug-
gesting that the quaternary compound (generally considered toxic) also was releasing. In
attempts to make the heparin more stable, or release more slowly at the surface, different
solvent systems were use to attack the base material and entangle the fatty chains with the
polymer surface. Attempts were made to calculate the sustained release rate required to
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
546 Tissue Engineering of Prosthetic Vascular Grafts
maintain thromboresistant surfaces.9 This clearly showed lessons learned should be useful insights into attachment of
that it was impractical to sustain thromboresistance, due to other biomolecules, as cited by Hubbell in attachment of
the large loading required to maintain effectiveness for more peptides to surfaces.23
than a few days. At the same time, Larm published his re- Our group has been studying the performance of he-
sults for “end point” attached heparin on surfaces.10 This parinized surfaces together with the University of Maastricht,
was the first time that an immobilized heparin, with no leach- the Netherlands in attempts to understand the role of cou-
ing or releasing heparin, could demonstrate significant de- pling methodology in the performance of these surfaces in
activation of thrombin. The premise of Larm’s surface was human blood using numerous blood testing systems.24 Us-
that nitrous acid degraded heparin (NAD-Hep) containing ing a rotating disc apparatus as seen in Figure 49.1, it be-
high affinity pentasaccharides for ATIII was covalently im- came evident that deactivation of thrombin at a heparin-
mobilized on the surface by a terminal aldehyde group, thus ized surface is a diffusion limited reaction.
presenting an optimal conformation of the molecule to the The reactor was designed with baffles to assure redi-
blood interface. The clearest evidence of the effectiveness of rection of the flow perpendicular to the test surface, which
Larm’s premise is seen in controlled experiments using hu- was rotating, and thus assured a uniform shear rate profile
man blood in simulated cardiopulmonary bypass circuits on the sample surface. A mathematical model can be hy-
where improvement in platelet protection, platelet release pothesized for the thrombin decay if the rate of deactiva-
products, hemolysis, less white cell activation, and less con- tion is dependent on the diffusion to the surface, and if the
tact activation is demonstrated.11 The group of Wendel has concentrations of thrombin and ATIII added to the reactor,
tested several coatings in the latter system, and nothing to and the speed of the motor, are known. The control line in
date has been reported equal in performance to the co- the graph of Figure 49.1 represents a blank control surface,
valently attached heparin surface. and the limited decay of thrombin is explained by simple
The method of Larm, commercially known as the noncatalytic deactivation of thrombin by ATIII in solution.
Carmeda biologically active surface (CBAS), is in fact a co- The heparinized surface agrees fairly well with the theoreti-
valent attachment of heparin to an adsorbed base, and thus cal decay rate for a diffusion limited reaction. All surfaces
is not completely covalent to the base material. Since Larm’s that were heparinized were tested before the experiment was
invention several additional methods of surface modifica- started to assure that no heparin was releasing.
tion have been used to couple heparin and reported in the Our method of immobilizing heparin consisted of
literature.12-18 The traditional model for the mode of action covalent attachment of hydrogels that were derivatized with
of heparin on a surface is depicted by a heparin molecule amine functionality to which aldehyde-functionalized hep-
with a high affinity pentasaccharide tethered to a surface arin (NAD-Hep, or periodate oxidized) was coupled. By con-
and free to bind circulating ATIII, which it conformation- trolling the amount of amine functionality we were able to
ally alters to make it catalytic in its ability to bind and deac- prepare surfaces with different amounts of heparin coupled.
tivate circulating thrombin. Once this action has occurred, Regardless of how much heparin was coupled per cm2 on
the thrombin-antithrombin complex (TAT) is released, and the surface, or the measured bioactivity the surface possessed
the site is capable of repeating the activity. The relatively to deactivate thrombin, the observed behavior in Figure 49.1
high circulating concentrations of ATIII assists in the dis- was only seen at low concentrations of ATIII. At physiologi-
placement of the TAT complex from the surface.19 The ques- cal concentrations of ATIII (1 U/ml), there was no differ-
tion of protein adsorption and its effect on the heparin ac- ence in thrombin decay between heparinized and uncoated
tivity is a common one. Van Delden showed that the spacer controls. The rate of diffusion to, and deactivation at, the
composition was important for the surface retaining the surface was not as great as the noncatalyzed deactivation in
ability to deactivate thrombin after exposure to plasma.20 In solution with physiological concentrations of ATIII, even
his studies he showed that heparin immobilized via albu- with the most active surfaces. In addition to the latter re-
min-heparin conjugates retain 27% of initial activity after sults, ELISA measurements of TAT and F1+2 fragments from
exposure to blood, whereas heparin immobilized via PEO blood loop experiments showed that very few of the latter
spacers can retain as much as 55% of the initial activity. Most complexes were formed after exposure of the heparinized
interesting from these experiments was the fact that the to- surface to human whole blood in 90 minutes, as compared
tal amount of protein adsorbed onto heparinized surfaces to uncoated control polymers. These results question the
did not correlate with the retention of heparin activity. It model proposed wherein thrombin in flowing blood is de-
has been reported that albumin and fibrinogen adsorbed activated by the heparin-ATIII surface to form TAT com-
onto heparinized surfaces are not conformationally altered.21 plexes that are released, and the heparin repeats this activity
Earlier studies by Sevastianov suggest that it is critical to several times. Further studies in flowing systems developed
properly attach heparin if the proteins adsorbed are to be to study thromboresistance of intravascular stents, where
non platelet adhering and activating.22 During a panel dis- platelet free and platelet rich plasma were used, helped so-
cussion at the Biomaterials conference in New Orleans, May, lidify a refinement of the model (Figs. 49.2, 49.3, and 49.4).
1997, a comment was made to the effect that after all these Figure 49.2 represents the flow through system
years of research on blood-compatible surfaces we are no developed for monitoring thrombin generation for an in-
farther than the heparinized surface. Another perspective travascular stent. Citrated PRP or PFP could be pumped
might suggest that we have finally reached a point where we through the system at controlled flow rates and samples col-
understand how to properly attach heparin to a surface. The lected over time.
Surface Bonding of Heparin 547
Fig. 49.1. Rotating disc apparatus; thrombin decay in rotating disc test.
Fig. 49.2. Flow through system
which monitors Wiktor intravas-
cular stent thrombin generation.
Citrated platelet rich plasma (PRP)
or platelet free plasma (PFP) is
pumped through the system at
controlled flow rates and samples
collected over time.
548 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 49.3. Comparison of contact activation as mea-
sured by factor IXa, and of thrombin generation in
platelet rich plasma (PRP) or platelet free plasma
(PFP). (A) Heparin-coated intravascular stents; (B)
Uncoated stents.
Figures 49.3A and 49.B represent a comparison be- Table 49.1 helps to further explain the importance of
tween heparin coated and uncoated intravascular stents with the quality (ability to bind ATIII) of the attached heparin to
respect to contact activation as measured with Factor IXa, the surface. ATIII uptake to the surface directly correlates
and its comparison to subsequent thrombin generation in with the delay in the onset of thrombin generation, the
PRP or PFP. The black points represent PRP and the gray amount produced at equilibrium and, most importantly, the
represent PFP. The first thing that can be seen is that contact number of adhered platelets. The platelets on the surface
activation is independent of platelets, and that without plate- are determined by the LDH method, and therefore some
lets the even more “thrombogenic” metallic surface does not platelets could be present that are “adhered”, but not acti-
produce significant amounts of thrombin (Fig. 49.3A). Not vated. SEM photos of the surfaces of the heparinized mate-
surprisingly, the anionic heparin stent does cause some rials did not reveal any visible platelets after mild rinsing,
contact activation, but less than the noncoated stent whereas the controls were covered with adhered and spread
(Fig. 49.3B). Clearly, in the presence of platelets the hep- platelets. Much might be learned in the future by study of
arinized surface delays the onset of thrombin generation, platelet adhesion to surfaces such as heparin and collagen.
and at equilibrium is much less thrombogenic than the Exposed subendothelial layers of collagen and GAGs can be
noncoated control material. seen to adhere platelets in dense single cell layers that do not
Surface Bonding of Heparin 549
Fig. 49.4. Contribution of immobilized heparin to clot prevention at blood-material interface by antithrombin (ATIII) deactiva-
tion of platelet-produced thrombin.
Table 49.1. Relation between the AT uptake and the Thrombogenicity of Wiktor stents
Coating AT-Uptake Thrombin stead State Thrombin onset Platelet Adhesion
pmol/cm-2 nM Min
None 0.26±0.36 8.83±0.37 5 287±34
HepamedTM 1.26±0.36 4.98±0.62 20 135±18
HepamedTM 1.99±0.57 3.40±0.25 20 197±45
HepamedTM 7.28±0.21 1.75±0.09 30 42±20
HepamedTM 10.89±0.21 1.48±0.15 34 92±33
Table 49.2 Effects of heparin bonded surface on blood clotting in polyurethane tubing
Surface Surface modification Platelet Adhesion ATIII Adsorption Clotting Time
Plts/cm2 pmol/cm2 seconds
x103 (LDH)
PU55D none 123.1 nd 659
6882-78-1 PU Collagen 234.1 4.45 594
6882-78-2 PU collagen+ 195.4 11.16 1183
heparin
550 Tissue Engineering of Prosthetic Vascular Grafts
appear to be spead or fully activated. This phenomenon has Heparin has several other properties in addition to
led us to further experiments that will be described shortly, acting as an anticoagulant, such as antibacterial and antivi-
but for now suffice it to say that the natural process of wound ral activity, inhibition of several enzymes, and the stimula-
healing of damaged endothelium begs the question as to tion of lipoprotein lipase release from the surface of endo-
whether or not a natural material such as collagen in the thelial cells.26,27 It has been proposed that heparin has a role
presence of certain glycosaminoglycans might produce a in the defense against pathogens outside of the immune sys-
“controlled” and important thrombogenic response. tem.28 This hypothesis seems to have merit based on the fact
Thrombin generation and subsequent fibrin forma- that heparin is predominantly located in organs and tissues
tion is due in a large part to the inability of flow conditions that come in direct contact with the outside environment
to keep the local concentration of thrombin low. Our expe- (lung, skin, and intestine).
rience has been that rapid clot formation takes place when Heparin binding growth factors (HBGFs) represent
the local concentration of thrombin reaches about 2-5 nM. an important family of mediators for the healing response,
Once this point has been reached, the fibrin network is rap- capable of inducing mesenchymal cell proliferation and dif-
idly formed and can entrap platelets and amplify the local ferentiation, tissue regeneration, morphogenesis, and
generation of thrombin. neovascularization.29 Several investigations of the growth
Figure 49.4 is a recent attempt at refining the concep- factors that are found in wound sites use heparin columns
tual model of how heparin works when immobilized on a in separation and identification of these growth factors.30-32
surface. The critical mechanism for preventing clotting is Numerous experiments have been tried by simple addition
that thrombin produced by platelets at the surface is imme- of growth factors to wound sites or to matrices. There are
diately deactivated, thus keeping the local concentration low, also some experiments where the role that heparin might
which in turn minimizes the strongest agent (thrombin) in play has been investigated. Angiogenesis was found to be
platelet activation. When the thrombin level is low, it can- enhanced by the addition of heparin to gels made of base-
not amplify its own production by activating vicinal plate- ment membrane extract that also had added acidic and ba-
lets, and less fibrin will minimize entrapment of additional sic fibroblast growth factors.33 Heparin, along with ECGF, is
platelets. The latter is of great significance for the longer term suggested to play a role as a response modifier of human
maintenance of low thrombogenicity, as thrombin can ad- endothelial cell migration, which may be relevant to tumor
here to fibrin and in this state it is not deactivated by hep- metastasis, wound healing, and atherogenesis.34 Another
arin and ATIII, and can continue to activate platelets.25 study using heparin analogs (chemically substituted dex-
Intramuscular implant studies of immobilized trans) in a collagen plaster was able to show a remarkable
collagen on biomaterials by our group showed immediate effect both on the kinetics and on the quality of the restored
thrombogenic response followed by strong apparent neu- skin.35 This study strongly suggests that endogenous growth
trophil recruitment at the tissue-material interface. This re- factors naturally releasing during the regeneration process
sponse quickly became quiescent, and after 6 weeks the could be trapped, protected and released by the addition of
tissue-material interface had only an occasional macroph- heparin analogs or heparin.
age with mostly fibroblasts (two cell layers) followed by It may be wishful thinking, but perhaps immobilized
adjacent microvascular structures. The control material or surface bonded heparin may play a strong role in tissue
(polyurethane) had a typical foreign body fibrotic response engineering. This may be particularly true if it is properly
with macrophages and foreign body giant cells adjacent to immobilized and capable of trapping, protecting and releas-
the majority of the surface, at least 15 layers of densely packed ing growth factors at locations and rates desired. Realiza-
fibroblasts with no vascular structures, and loose connective tion of this potential probably will require some additional
tissue outside of the fibroblast layers. These results led to thinking beyond heparin by itself. Coupling to biomolecules
preliminary experiments in measuring the coagulation such as collagens and other glycosaminoglycans to polymeric
response to collagen immobilized surfaces, and the surfaces may prove fruitful. From some of the studies men-
potentiating effect that heparin coupled to this collagen layer tioned, it is apparent that gel matrices can be modified to
might have. exhibit angiogenesis and remodeling. It may be entirely pos-
Using the same flow through system developed at the sible to fill porous polymer matrices with such gels and ap-
University of Maastricht and described previously, polyure- proach the ultimate regenerated tissue-like vascular graft.
thane tubings were prepared with immobilized collagen and Still, for those concerned with optimal mechanical proper-
immobilized collagen with heparin covalently bonded to the ties, the proper polymeric scaffold with a more fibrous char-
collagen (type I collagen). acter can have the surfaces modified for covalent attachment
Apparently the fact that more platelets are attached to that may achieve a similar end. The former will require a
the collagen and collagen plus heparin surfaces than the bigger step in regulatory and commercial acceptance, while
polyurethane does not lead to a faster clotting time, and in the latter may lead to incremental improvements. It is hope-
the case of the heparinized collagen the clotting time was ful that efforts in both areas will lead to understanding that
significantly delayed (Table 49.2). These are very prelimi- will result in a vascular graft with complete healing that is
nary experiments, and only suggest additional studies to at- long term patent, and infection resistant.
tempt to look at heparin in a broader context of the total
healing response.
Surface Bonding of Heparin 551
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1. Gomes MN, Hufnagel CA, Dokumaci O. Studies in the Verhoeven M, Cahalan P, Anderson JM. Human mono-
autogenization of prosthesis for samll artery replacement. cyte/macrophage adhesion and cytokine production on
Am Surg 1972; 38:664-666. surface-modified poly(tetrafluoroethylene/hexafluoro-
2. Russel AP, Hopwood D. The biological uses and impor- propylene) polymers with and without protein pread-
tance of glutaraldehyde. Prog Med Chem 1976; sorption. J Biomed Mater Res 1995; 29:257-268.
13:271-299. 19. Elgue G et al. Effect of surface immobilized heparin on
3. Sparks CH. Silicone mandril method for growing rein- the activation of adsorbed factor XII. Art Org 1993;
forced autogenous femoropopliteal artery grafts in situ. 17(8):721-726.
Ann Surg 1973; 177:293-300. 20. van Delden CJ. On the anticoagulant activity of heparin-
4. Bothwell JW, Lord GH, Rosenberg N. Modified arterial ized surfaces. Ph.D. Thesis, University of Twente, The
heterografts: Relationship of processing techniques to in- Netherlands, 1995.
terface characteristics. In: Sawyer PN, ed. Bio-physical 21. Barbucci R, Magnani A. Conformation of human plasma
Mechanisms in Vascular Homeostasis and Intravascular proteins at polymer surfaces: The effectiveness of surface
Thrombosis. Appleton-Century-Crofts, 1965. heparinization. Biomater 1994; 15(12):955-962.
5. Csomor K, Karpati E, Nagy M, Gyorgyi-Edeleny J, 22. Nemets EA, Sevastianov V. The interaction of heparin-
Machovich R. Blood coagulation is inhibited by sulphated ized biomaterials with human serum, albumin, fibrino-
copolymers of vinyl alcohol and acrylic acid under in vitro gen, atntithrombin III, and platelets. Art Org 1991;
as well as in vivo conditions. Thromb Res 1994; 15(5):381-385.
74(4):389-398. 23. Drumheller PD, Hubbell JA. Densely crosslinked polymer
6. Montdargent B, Toufik J, Carreno M, Labarre D, networks of poly(ethylen glycol) in trimethylolpropane
Jozefowicz M. Complement activation and adsorption of triacrylate for cell-adhesion-resistant surfaces. J Biomed
protein fragements by functionalized polymer surfaces in Mater Res 1995; 29:207-215.
human serum. Biomater 1992; 13. 24. Lindhout T, Blezer R, Schoen P, Willems GM, Fouache
7. van Boeckel CAA. From Heparin To A Synthetic Drug: A B, Verhoeven M, Hendiks M, Cahalan L, Cahalan P. An-
Multi-disciplinary Approach. In: Trends in Receptor Re- tithrombin activity of surface-bound heparin studied un-
search. Elsevier Publishers B.V. 1993. der flow conditions. J Biomed Mater Res 1994;
8. Gott VL, Koepke DE, Daggett RL, Zarnstorff W, Young 29:1255-1266.
WP. The coating of intravascular plastic prostheses with 25. Buchanan MR, Liao P, Ofosu FA. Simultaneous inhibi-
colloidal graphite. Surg 1961; 50:382. tion of thrombin by ATIII and HCII and prevention of
9. Bassmadijan D, Sefton MV. Relationship between release thrombus formation and growth: Relative effects of hep-
rate and surface concentration for heparinized materials. arin and sulodexide. Abstract from XIV Congress of
J Biomed Mat Res 1983; 17:509-518. ISTH, 1993.
10. Larm O, Larsson R, Olsonn P. A new nonthrombogenic 26. Wright TC, Castellot JJ, Diamond JR, Karnovsky JJ. Regu-
surface prepared by selective covalent binding of heparin lation of cellular proliferation by heparin and heparan
via a modified reducing terminal residue. Biomat Med Dev sulphate, In: Lane DA, Lindahl U, eds. Heparin, chemical
Art Org 1983; 11:161-173. and bioligical properties, clinical applications. London:
11. Wendel HP, Heller W, Gallimore MJ, Hoffmeister HE. Edward Arnold, 1989.
Heparin-coated oxygenators significantly reduce contact 27. Olivecrona T, Bengtsson-Olivecrona G. Heparin and li-
system activation in an in vitro cardiopulmonary bypass pases. In: Lane DA, Lindahl U, eds. Heparin, chemical
model. Blood Coag Fibrin, 1994; 5. and biological properties, clinical applications London:
12. Grainger DW, Kim SW, Feijen J. Poly(dimethylsiloxane)- Edward Arnold, 1989.
poly(ethylene oxide)-heparin block copolymers. I. Synthe- 28. Björk I, Olson ST, Shore JD. Molecular mechanisms of
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22:231-249. tween antithrombin and clotting proteinases. In: Lane DA,
13. Ebert CD, Kim SW. Immobilized heparin: spacer arm ef- Lindahl U, eds. Heparin, chemical and bioligical proper-
fects on biological interactions. Thromb Res 1982; ties, clinical applications. London: Edward Arnold, 1989.
26:43-57. 29. Lobb RR. Clinical applications of heparin-binding growth
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Kottke-Mrchant K, Marchant RE. Immobilization of high 30. Hamerman D, Taylor S, Kirschenbaum I, Klagsbrun M,
affinity heparin oligosacccharide to radio frequency Raines EW, Ross R, Thomas KA. Growth factors with
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27:811-819. Exp Biol Med 1987; 186(3):384-9.
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16. Engbers GHM. The development of heprinized materials wound fluid as a response to injury. Proc Natl Acad Sci
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552 Tissue Engineering of Prosthetic Vascular Grafts
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Maciag T. Human endothelial cells are chemtactic to en-
Scaffold Engineering
Surface Modification
CHAPTER 50
Covalent Grafting of RGD Peptides to Synthetic Surfaces
Nina M.K. Lamba, S.L. Cooper
Introduction
F abric materials were first used as vascular prostheses in the 1950s, when Voorhees et al
implanted a polymeric vascular graft manufactured from vinyl chloride and acryloni-
trile.1 Since then, a number of polymers have been used to fabricate vascular prostheses, and
today, polyethylene terephthalate (PET, DacronΚ) and polytetrafluoroethylene (PTFE, Gore-
TexΚ) are the most commonly used biomaterials for this application. Vascular grafts made
from either of these materials have an internal diameter usually greater than 6 mm, and are
restricted to use in high shear environments. Currently there is no clinically acceptable syn-
thetic small diameter vascular prosthesis, i.e., one less than 6 mm I.D. Thrombus and
neointima formation will readily occlude synthetic vascular grafts. The resulting loss of pa-
tency remains the greatest obstacle to the development of a small caliber vascular prosthesis.
Thus, there is a need for an engineered material that will provide the necessary mechanical
and thromboresistant properties for a successful synthetic vascular prosthesis, to facilitate
the advancements in vascular surgery. In this chapter we will discuss the motivation for the
endothelialization of luminal surfaces, and describe some of the approaches that have been
taken to achieve this. A discussion of methods to covalently graft Arg-Gly-Asp (RGD) pep-
tides to polymers follows, focusing on methods and results obtained using polymers that are
used to fabricate vascular prostheses.
Methods to Improve Endothelialization
The only nonthrombogenic material known to man is the normal vascular endothelium.
The endothelium actively achieves this nonthrombogenicity through the synthesis of nu-
merous anti-platelet agents and clotting inhibitors. Furthermore, protein adsorption occurs
rapidly onto artificial surfaces from solution, but this is not a feature of endothelial sur-
faces.2-4 There is a great deal of literature detailing various approaches to promote the devel-
opment of a stable, confluent layer of endothelial cells on the inner lumen of a vascular
graft. It is believed that the growth of a confluent monolayer of endothelial cells onto a
synthetic substrate will not only reduce the degree of thrombus formation, but may also
reduce the proliferation of pathogenic organisms on the device. Approaches to encourage
the endothelialization of the inner lumen of vascular grafts have included seeding of sur-
faces with cells, and chemical and physical modification of surfaces to promote cell attach-
ment. Prostheses have been seeded with endothelial cells to promote endothelialization of
the luminal surface. The success of this approach has been limited by problems with har-
vesting endothelial cells, and the subsequent attachment, retention and proliferation of cells
on the surface. Chemical modification of surfaces has included the use of photo discharge
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
554 Tissue Engineering of Prosthetic Vascular Grafts
technology to deposit reactive groups onto polymer surfaces, sequence in a specific protein, whereas other receptors may
or to indirectly influence the proteins adsorbed to the sur- recognize the RGD sequence in more than one protein. The
face. These methods to encourage cell attachment have met specificity of the RGD protein is believed to be modulated
with limited success, owing to the lack of specificity and poor by the conformation of the sequence. This may be deter-
control over protein orientation. Physical methods to im- mined by the amino acids that are immediately adjacent to
prove the development of a pseudointima on the surface the RGD sequence.12-14 Substitution of peptides within the
have involved altering the porosity and texture of surfaces, RGD sequence has been shown to produce a large reduc-
to promote tissue ingrowth into the graft. The neointima tion in the adhesivity of the peptide.15 The presence of a
that forms in the lumen of a graft may not provide compa- peptide adjacent to the sequence has been shown to alter
rable physiological properties of a natural endothelium, lim- the activity of the RGD sequence.10,16,17
iting the success of integration of the implanted device with Covalent grafting of biological molecules to a synthetic
the host by this method.5 Grafts have been coated with ad- substrate prevents the desorption of the agent over time from
hesive proteins or extracellular matrix components in order the surface. An excellent review of the chemistry of covalent
to promote attachment and proliferation of endothelial immobilization of RGD peptides is available.18 Generally,
cells.6-8 An improvement in the extent of endothelialization either the peptide or the surface must be “activated” to pro-
has been observed in animal models. RGD-protein conju- vide suitable sites for immobilization of the ligand to the
gates have also been synthesized,9 but problems in defining substrate. Biological structures are less resistant than syn-
the precise conformation of the protein once it has adsorbed thetic polymers to harsh chemical environments. Prolonged
to the substrate impede interpretation of results. The long exposure of a ligand to a harsh environment may lead to a
term success of materials modified through adsorption of reduction in the biological activity of the peptide. Thus, the
proteins to mediate cellular adhesion is not guaranteed, due polymer substrate is often derivatized or activated prior to
to possible desorption of the protein or proteolysis. peptide coupling. Some of the more commonly used reac-
More recently, there has been an interest in the graft- tion schemes involve the coupling of the peptide sequence
ing of synthetic peptide sequences onto polymeric substrates. to a polymer substrate that contains hydroxyl, thiol, carboxy-
By covalently grafting short peptide sequences onto poly- lic acid or amine groups. Surfaces or ligands can also be
meric substrates, it is believed that cellular adhesion can be derivatized and immobilized using photochemical tech-
mediated directly through receptor-ligand interactions, niques. The efficiency of the coupling reaction between the
rather than through a conditioning layer of adsorbed pro- substrate and the peptide can influence the surface peptide
tein. The covalent grafting of the Arg-Gly-Asp (RGD) pep- density, which has been shown to influence the degree of
tide sequence to synthetic materials has also been shown to cell spreading.19,20 There are also other factors that can af-
promote cell adhesion and attachment for applications such fect the coupling yield and biological activity of the ligand.
as tissue culture substrates and biomaterials for implanta- The RGD sequence can be immobilized by covalent bond-
tion and hybrid artificial organs. Some of the approaches to ing at either end of the sequence, either through the car-
modify biomaterials with cell adhesive peptide sequences boxyl terminus or the amino terminus. The orientation of
containing the RGD sequence relevant to the development the sequence can affect the interactions of the peptide with
of synthetic vascular grafts will be discussed in more detail cell surface receptors. The immobilization of peptide se-
in this chapter. quences on surfaces may also impose steric constraints on
the peptide, affecting the affinity and specificity of the ligand.
Immobilization of RGD Peptides This has been reported with the grafting of other biological
The RGD peptide sequence has been shown to be the molecules, and may be overcome by grafting the ligand onto
minimal cell-recognizable sequence in many adhesive plasma spacer arms, reducing steric hindrance imposed by the prox-
and extracellular matrix proteins.10 The RGD sequence was imity of the ligand to a rigid surface.21 The protection of the
first found in fibronectin, and is also present in vitronectin, peptide during the coupling reaction scheme is also a con-
von willebrand factor, fibrinogen, and collagen. The RGD sideration, to ensure that the activity of the peptide is re-
sequence has been shown to play a crucial role in mediating tained. Competing side reactions such as hydrolysis, poly-
cell attachment and subsequent spreading. It has been shown merization and crosslinking can also affect the grafting yield.
to be adhesive towards platelets and other cells, including The final surface density of ligands can be determined by
fibroblasts and endothelial cells. The RGD tripeptide may radiolableling, photochrome labeling, surface analysis or
also act as a ligand for the integrin superfamily of receptors. gravimetry.18
In particular, many integrin receptors recognize the RGD A number of studies have been performed investigat-
sequence which is present in many adhesive proteins.11 It ing methods to immobilize peptide sequences containing
has been demonstrated that the synthetic RGD peptide in the RGD sequence to polymeric surfaces. With respect to
solution is able to compete with adhesive proteins adsorbed the development of synthetic vascular prostheses, RGD
onto a surface for binding receptors on the cell in solution grafted materials have been studied with the goal of improv-
and prevent cellular adhesion to an adhesive protein ing the integration of the prosthesis into the host cardiovas-
adsorbed to the surface.10 Synthetic surfaces containing im- cular system, with the ability to perform the biological func-
mobilized RGD peptides have been shown to promote cell tions of the endothelium. RGD sequences have been grafted
attachment in a manner similar to fibronectin. Some cell onto polyethylene terephthalate (PET) and polytetra-
surface receptors have been shown to bind with the RGD fluoroethylene (PTFE),22 polyvinyl alcohol (PVA),23 poly-
Covalent Grafting of RGD Peptides to Synthetic Surfaces 555
acrylamide,24 polystyrene,25 and polyurethane.26 It has been Over the years, it has become apparent that the
demonstrated that the presence of RGD peptide sequences thrombogenicity of a synthetic graft is not the only crite-
at the surface increases endothelial cell attachment in vitro. rion by which a vascular graft should be assessed. The me-
The attachment of endothelial cells through specific recep- chanical properties, particularly the compliance and the tex-
tor-ligand interactions should overcome the problems of cell ture of the graft are important factors in promoting tissue
detachment under shear conditions.22 The methods to im- ingrowth, reducing anastomotic hyperplasia and favoring
mobilize RGD peptides to polymers, described below, dem- the long term patency of the graft. Both PET and PTFE are
onstrate that successful grafting can be achieved by either much less distensible when compared with the natural ar-
bulk or surface modification. The main advantages of using tery wall. Natural arteries show an increase in diameter of
surface modification techniques are that smaller quantities about 10% when pressurized to 150 mm Hg (normal arte-
of reagents are required, and the mechanical properties of rial pressure). In comparison, PET and PTFE grafts distend
the material can be retained. However, on a commercial scale, by about 1% under these conditions. Polyurethanes are one
bulk modification of a material requires fewer processing of the strongest candidates for the development of small
steps, which can ease fabrication and reduce costs. diameter vascular prostheses, due to their high tensile
Sugawara and Matsuda have derivatized peptides with strength, compliance and blood compatibility.29,30 Polyure-
4-azidobenzoyloxysuccinimide. The peptides were then thanes have been shown to distend by about 6% under nor-
adsorbed to a polyvinylalcohol surface.27 The surface was mal arterial pressure, 31 and a blended polyurethane-
exposed to UV radiation to covalently bond the peptides to polylactide vascular prosthesis possesses mechanical prop-
the surface. Bovine aortic endothelial cell adhesion was re- erties similar to rat abdominal aorta.32 Arterial substitutes
ported to be enhanced in a biologically specific manner. need to have mechanical properties that match the natural
Ozeki and Matsuda have used a similar strategy to attach vessel wall, to avoid turbulent blood flow, which may result
photochemically peptides containing the RGD sequence to in thrombus formation or destruction of formed blood ele-
polystyrene.25 An increase in the attachment of bovine aor- ments. Lyman et al33,34 have shown that the distensibility of
tic endothelial cells was reported. The specific peptide se- the graft can influence the biocompatibility through the
quence that is grafted may also modulate the adhesivity of degree of anastomotic hyperplasia by comparing vascular
the RGD sequence. Hirano et al16 immobilized RDGS, grafts fabricated from the same material, but with different
RGDV, RGDT, and RGD onto ethylene-acrylic acid copoly- degrees of compliance. Furthermore, a mismatch in com-
mer by coupling the terminal NH2 to the carboxylic resi- pliance between the synthetic graft and the natural vessel
dues in the polymer. The surfaces were evaluated for cell may traumatize the natural vessel and disrupt the endothe-
recognition by cell adhesion activity towards 5 different cell lium, which will in turn initiate thrombosis and stimulate
lines. It was found that the fourth amino acid in the intimal hypertrophy.
tetrapeptides played an important role in determining the Polyurethanes are block copolymers consisting of al-
precise specificity of the RGD sequence. The presence of ei- ternating soft and hard segments. The incompatibility be-
ther serine (S), threonine (T), or valine (V) at the end of the tween these two components of the polymer allows
peptide enhanced cellular adhesion. They postulated that microphase separation to occur within the bulk polymer.
the modulation in the adhesive nature of the RGD sequence The material can be described as being comprised of hard
may arise from a conformational change in the RGD se- domains dispersed within a soft segment matrix. This gives
quence, exerted by the terminal peptide. rise to the superior physical properties of the polyurethanes
Massia and Hubbell have grafted RGD peptides onto and is believed to be a contributory factor to their blood
the surface of polyethylene terephthalate (PET) and and tissue compatibility. Recently, RGD peptide sequences
polytetrafluoroethylene (PTFE) films.22,28 This was achieved have been successfully grafted to polyurethane substrates.35
through the hydroxylation of the polymer followed by sub- Polyurethanes for medical applications are usually synthe-
sequent immobilization of the synthetic peptide sequence sized via a two-step ‘prepolymer’ method. Lin et al synthe-
via tresyl chloride. The RGD could only react via the N-ter- sized a polyurethane from methylene bis (p-phenyl-
minus, ensuring that the orientation of the grafted peptide isocyanate) (MDI) and butane diol (BD), with polytetra-
was the same throughout. Radiolabeling of peptides was used methyleneoxide (PTMO) as the soft segment. Hydrogen at-
to confirm the presence and concentration of the peptide oms on the urethane groups were substituted with carboxyl
sequence. Endothelial cell adhesion was evaluated on the groups, via abstraction of urethane hydrogen by sodium
unmodified, hydroxylated and peptide containing surfaces, hydride, followed by reaction of the polyurethane with
both with and without the presence of serum. In the ab- !-propiolactone. The degree of substitution could be varied
sence of serum, the RGD grafted films were able to support by the quantities of reagents used. This was performed to
the adhesion and spreading of human umbilical vein en- alter the density of the grafted peptides. Peptide sequences
dothelial cells (HUVECs). Neither the unmodified films nor were then coupled to the carboxyl group using 1-(3-
the hydroxylated intermediates showed the same degree of dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
adhesion and spreading. Stress fibers were also observed, (EDCl). The reaction scheme for the carboxylation of the
implying that the cells were attached strongly to the sub- urethane group, followed by coupling of the RGD peptide
strate, and may possess the mechanical strength to resist sequence is shown in Figure 50.1.
detachment by shear forces. Lin investigated the effect of the coupling reaction on
the endothelialization of the substrate. Two coupling
556 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 50.1. Reaction scheme for the synthesis of RGD-containing peptide grafted polyurethane. (a) deprotonation of urethane
group; (b) carboxylation of polyurethane; (c) coupling of RGD peptide.26
methods were used. The first, one step method coupled the where.25 The increased adhesion on the RGD-grafted
free hexapeptide directly to the carboxyl group through the polyurethanes that were hydrated prior to contact with
formation of an amide bond. The second method involved endothelial cells provides further evidence that reorienta-
two steps, coupling a protected peptide to the carboxyl group, tion of the RGD peptides does occur on hydration, and that
followed by deprotection of the peptide. The two step cellular adhesion to RGD grafted materials is mediated
method using the protected peptides appeared to have a through receptor interactions with the peptide. Figure 50.3
higher coupling efficiency of the peptide to the substrate of shows SEMs of endothelial cells grown in vitro, on the base
the unprotected peptide, as coupling could only occur via polyurethane (PEU), carboxylated polyurethane
the N-terminus of the peptide. Furthermore, there were (PEU-COOH), and peptide grafted polyurethanes (PEU-
fewer competing side reactions in the synthetic scheme us- GRGESY, PEU-GRGDSY, and PEU-GRGDVY). The effect
ing the protected peptides, improving the yield of grafting. of substituting one of the amino acids within the RGD se-
Previously, other researchers had reported that the quence reduces the adhesivity of the peptide (PEU-
adhesion and growth of endothelial cells on polyurethane is GRGESY). The substitution of the peptide adjacent to the
relatively poor.36,37 The immobilization of RGD peptides to RGD sequence has the ability to alter the adhesivity, as
a polyurethane as described above increased the number of adhesion was greater on GRGDVY grafted material than
adherent HUVECs after in vitro seeding. A larger number GRGDSY grafted polyurethane.
of cells attached to RGD peptides that had been protected Both Massia22 and Lin35 have investigated the role of
during synthesis than those that had not been protected. In serum proteins in mediating cell adhesion to RGD grafted
addition, greater endothelial cell adhesion to hydrated materials. Endothelial cell adhesion was evaluated on the
samples was observed compared with samples that had not unmodified, intermediate and peptide grafted materials,
been previously hydrated. Polyurethanes contain hydrophilic both with and without the presence of serum. Massia and
and hydrophobic domains that reorient upon hydration, Hubbell found that both the unmodified PET and the hy-
minimizing the interfacial free energy. Cold-stage ESCA droxylated PTFE supported cell adhesion only in the pres-
studies of hydrated and dry samples showed that reorienta- ence of serum proteins, implying that cell adhesion was
tion of the peptides occurs at the hydrated surface,38 lead- mediated by adsorbed proteins. Lin et al35 reached a similar
ing to the conclusion that the RGD peptides on the polyure- conclusion in their study of HUVEC attachment to the RGD
thane backbone do orient towards the surface on hydration. grafted polyurethane. Hubbell and co-workers have also cre-
This reorientation of the functional peptide groups is de- ated highly specific cell adhesive substrates of RGD-contain-
picted schematically in Figure 50.2.38 Reorientation of RGD ing surfaces with low protein adsorption.15,22,39 The low pro-
peptides at an aqueous interface has been reported else- tein adsorbing characteristics of these surfaces has allowed
Covalent Grafting of RGD Peptides to Synthetic Surfaces 557
Fig. 50.2. Schematic of the surface re-orientation of the peptide grafted polyurethanes in the hydrated
and dehydrated states. From: Lin H-B, Lewis KB, Leach-Scampavia D et al. Surface properties of RGD-
peptide grafted polyurethane block copolymers: Variable take-off angle and cold-stage ESCA studies.
J Biomater Sci, Polym Ed 1993; 4:183-198.
558 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 50.3. SEMs of HUVECs at-
tached to polyurethanes. Base
polyurethane (PEU), carboxy-
lated polyurethane (PEU-
COOH), RGE-grafted polyure-
thane (PEU-GRGESY), RGD
grafted polyurethane (PEU-
GRGDSY and PEU-GRGDVY).
From: Lin H-B, Garcia-
Echeverria C, Asakura S et al. En-
dothelial cell adhesion on poly-
urethanes containing covalently
attached RGD peptides.
Biomaterials 1992; 13:905-914.
the study of cell attachment and spreading independently ronment, the physiological properties of the endothelial layer,
of protein adsorption. Using these substrates they have and the interaction of endothelial cells with the formed ele-
shown that the RGD and Tyr-Ile-Gly-Ser-Arg (YIGSR) pep- ments of the blood, such as platelets and white blood cells.
tide sequences promote endothelial cell adhesion indepen-
dently of adsorbed cell adhesion proteins. They have also References
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Matrix Engineering
CHAPTER 51
Hydrogels in Biological Control During Graft Healing
Jeffrey A. Hubbell
Introduction—Why Hydrogels?
H ydrogels are polymeric materials that imbibe a large fraction of water and yet remain
intact, not dissolving even given an infinite period of time. These materials are formed
from polymer chains that have a high affinity for water, either such that the chains would be
individually soluble in water and are restricted from dissolving by virtue of participation in
the polymeric material as a network, or such that the chains are almost, but not quite, soluble
in water. By network, it is meant that the polymer chains in the hydrogel are somehow inter-
acting with each other so as to keep the individual chains from diffusing away into the aque-
ous milieu, either by virtue of being covalently bonded together or by interacting physi-
cally—specific examples will be provided in the section on “Hydrogel Structure and Synthesis”.
Many extracellular structures in the body can be considered as hydrogels. The extra-
cellular matrix of soft tissues and cartilage, for example, exists as a network of glycoproteins
and proteoglycans that both interact with each other biophysically (e.g., by specific biologi-
cal interactions between collagen and a variety of adhesion proteins,1 by specific biological
interactions between glycosaminoglycans in proteoglycans and a variety of adhesion pro-
teins,2 and by hydrogen bonding as in collagen due to a very high content of hydroxypro-
line3) and by covalent interactions (e.g., chemical crosslinking between glutamine residues
on one protein and lysine residues on a neighboring protein under the enzymatic action of
a transglutaminase to catalyze the formation of an amide linkage between the two).4
It is not by coincidence that hydrogels are found extensively in Nature: They possess
distinct biologically useful features, which will likewise be useful in tissue engineering of the
vascular graft and other tissues. Hydrogels are mechanically flexible, are freely permeable to
small molecules such as dissolved gases and low molecular weight nutrients and wastes, and
are controllably permeable to larger molecules such as proteins. Because the gels are highly
swollen with water, the amount of polymer mass per unit volume is relatively low, and this is
of key importance in cell migration in three dimensions. Cell migration is facilitated by
enzymatic remodeling of the three dimensional extracellular matrix, and the amount of
protein and proteoglycan mass to be degraded and moved out of the path of the cell is much
lower in the case of a hydrogel than if the tissue had consisted of less water. Indeed, in cul-
ture models of leukocyte migration through collagen-based gels, cell migration speed was
observed to be reduced by nearly an order of magnitude when the collagen concentration
was doubled.5 Also, because gels are highly swollen with water, their effective surface area is
very high, and this is used in Nature to store large quantities of biologically active molecules
that can be released in a triggerable fashion from the extracellular matrix, e.g., the release of
heparin-binding polypeptide growth factors from heparan sulfate glycosaminoglycan
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
562 Tissue Engineering of Prosthetic Vascular Grafts
components of the proteoglycans in the extracellular ma- trol on shape retention, by freezing the poly(vinyl alcohol)
trix. As engineered hydrogels are developed for specific ap- solution. When ice crystals form, the polymer chains are
plications in tissue engineering, it should be possible to excluded from the water crystal and pushed to the crystal
mimic these biological interactions to achieve these same boundaries, resulting in very highly concentrated regions of
sorts of biological interactions.6-9 polymer. At these highly concentrated domains, when all of
Hydrogels have other potential advantages that are less the water is frozen, segments of the poly(vinyl alcohol) chains
extensively used in Nature. As is addressed in more detail in crystallize due to hydrogen bonding, resulting in an insoluble
the section “Protein and Cell Interactions with Hydrogels”, (without heating far above 37°C) hydrogel in the shape of
there exists the potential to design gels without the the polymer solution that was frozen. Thus, this gel is a net-
incorporation of binding sites for proteins, with the result work polymer where the bonding between polymer chains
being a gel that is highly resistant to cell adhesion. Such is physical by hydrogen bonding.
materials may be useful in preventing the interaction of cells Hydrophobic interactions can also be important in the
with material substrates in a scaffold for tissue engineering formation of hydrogels, and block copolymers of
at sites of application of a gel coating. An additional feature poly(ethylene glycol) and poly(propylene glycol) form a
of hydrogels of potential tissue engineering advantage is that characteristic example.13 Poly(ethylene glycol) is a water-
a solvent for the gel precursors is water; as such, the soluble poly(ether), while poly(propylene glycol) is water-
possibility exists to apply a hydrogel precursor to a tissue insoluble, containing a nonpolar methyl group that the
site, dissolved in physiological saline, and to form the poly(ethylene glycol) repeat unit lacks. Block copolymers of
hydrogel in situ, as is addressed below in the section “In Situ these two compositions in the form of a domain of the hy-
Transformations”. drophobic poly(propylene glycol) flanked on both sides by
domains of poly(ethylene glycol) are water soluble polymers
that, at relatively high concentrations, can form gels by
Hydrogel Structure and Synthesis hydrophobic interactions between the poly(propylene gly-
Hydrogels consist of hydrophilic polymer chains that col) domains. This is to say, the poly(propylene glycol) do-
are held together in a single mass by virtue of either slight mains from several different polymer chains cluster to shield
insolubility or some form of bonding between chains to yield each other from interactions from water, thereby forming
a polymer network.10 The structure of the hydrogel is highly physical crosslinks (within these clusters) based on hydro-
dependent upon the nature of the synthetic procedure that phobic interactions, resulting in a hydrogel. This gel, as do
was used to obtain the gel; for this reason, different means several others based on hydrophobic interactions, has the
of hydrogel synthesis will be illustrated in the paragraphs useful feature that the aqueous mixture is a liquid at cold
below with a few examples. temperatures (below approximately 10°C) but rapidly forms
The nature of some hydrogels is controlled by the slight a hydrogel at 37°C.
insolubility of the polymer chains; a typical example is Electrostatic interactions also play important roles in
hydrogels of poly(hydroxyethylmethacrylate).11 The vinyl hydrogel formation, and gels of alginate with calcium ion14
monomer for this polymer, hydroxyethylmethacrylate, is and polyelectrolyte complexes of alginate and poly(lysine)15
soluble in water. Likewise, oligomers and low molecular form good examples of these principles. Alginate is a natu-
weight polymers of hydroxyethylmethacrylate are soluble in rally occurring polysaccharide product of kelp and several
water, but high molecular weight polymers are slightly bacterial strains. Each sugar residue in the polysaccharide
insoluble in water, leading to a gel that swells to imbibe chain bears a carboxyl group, so the polysaccharide chain is
approximately 50% water by mass. This swelling extent can highly anionic, and these charges can play multiple roles in
be readily controlled by copolymerization with more gel formation. Firstly, divalent cations, such as Ca2+, can form
hydrophobic monomers, such as ethylmethacrylate (i.e., the a bridge between the anionic groups on adjacent alginate
analogous monomer to hydroxyethylmethacrylate, only backbones, resulting in regions of adjacent chains that are
lacking the polar hydroxyl group), to achieve a series of paired, these regions being connected to other paired re-
polymers with swelling ranging from almost nil (for gions by amorphous domains. Practically, dropping the al-
poly(ethylmethacrylate)) to that of poly(hydroxyethyl- ginate solution in Ca2+-free saline into a saline solution con-
methacrylate). These gels have little internal structure, i.e., taining greater than 1 mM Ca2+ results in the rapid forma-
no permanent pores or dense regions, but rather exist as tion of a hydrogel which is stable under physiological con-
dynamic structures with small pores opening and closing in ditions. Secondly, alginate (either solution or as a Ca2+-
equilibrium. crosslinked gel) can be mixed with a polycation in solution,
Hydrogels can also be formed by hydrogen bonding, such as poly(lysine), and these two polymer chains form a
and gels formed from poly(vinyl alcohol) represent a typi- hydrogel, referred to as a polyelectrolyte complex, crosslinked
cal example.12 Poly(vinyl alcohol) is a water-soluble poly- into a network by the electrostatic interactions between the
mer at 37°C. When a solution of poly(vinyl alcohol) is dried oppositely charged polymers. Gels formed in either manner
to form a cast solid, the solid does not dissolve in water when described above can have interesting microscopic structures
it is again placed in an aqueous milieu. In the dry state, re- that are lacking in the other gels described in the paragraphs
gions of the poly(vinyl alcohol) solid crystallize due to hy- above, this microscopic structure deriving from the diffu-
drogen bonding. These regions are so strongly bonded that sive process of gel formation. For example, when the algi-
dissolution of the polymer again at 37°C is infinitesimally nate solution is dropped into a Ca2+-containing bath, Ca2+
slow. This behavior can also be obtained, with greater con- ions begin diffusing into the polymer solution, causing the
Hydrogels in Biological Control During Graft Healing 563
formation of the gel at the interface between the polymer bin cleaves the fibrinogen protein at two points, exposing a
solution and the surrounding Ca2+-containing bath. Because new terminus on two polypeptide chains. This new termi-
the gel region exists at a lower soluble polymer concentra- nus physically binds at a site on other fibrinogen (and cleaved
tion than does the alginate solution, alginate from the core fibrinogen) proteins, this process resulting in a physically
of the droplet diffuses toward the gelled interface; this poly- bonded hydrogel. The coagulation protein factor XIII is also
mer then gels, and more polymer diffuses from the core to cleaved by thrombin to form the active transglutaminase fac-
the periphery, and so on: The net result of this phenom- tor xiiia, which acts to catalyze the formation of an amide
enon is that the periphery of the Ca2+-alginate gel is more bond between glutamine residues and lysine residues on
dense than the core of the particle, owing to the diffusive opposing polypeptide domains in fibrin, resulting in a
nature of the process. The case is likewise with the incorpo- chemically crosslinked fibrin network.
ration of the polyelectrolyte counter-ion poly(lysine). The brief overview provided above should provide the
Poly(lysine) diffuses into the gel particle from the periphery reader with a background understanding of the physical
and interacts first at that periphery, forming denser mem- nature of hydrogels and the vast flexibility in the physical
brane at the periphery, which restricts further poly(lysine) and chemical processes for forming them. Based upon this
diffusion into the core in a self-limiting process. Accordingly, brief background, the reader is well positioned to consider
a thin shell of polyelectrolyte complex can be formed, a mi- the application of hydrogels for tissue engineering of the
crostructure that could be put to good use in tissue engi- vascular graft.
neering.
In addition to the physical interactions described In Situ Transformations
above, hydrogels can also be formed that depend on cova- One interesting advantage in the use of hydrogels in
lent bonding between polymer chains; these gels have a tissue engineering is that they may be delivered in the form
characteristic advantage over those described above in that of a precursor that is dissolved in a physiological saline and
they can be considerably more stable under physiological is then converted into the insoluble hydrogel upon the tar-
conditions. Three examples are presented below, two based get tissue (see Fig. 51.2), in some cases even in direct con-
on synthetic polymers with nonbiological reactions and one tact with cells.19 To perform adequately in such an environ-
based on proteins with enzymatic reactions. The simplest ment, the precursor solution should be lacking in high con-
way to form a covalently crosslinked hydrogel is to centrations of toxic species and low molecular weight mono-
polymerize an otherwise water-soluble polymer in the mers, which may be more likely to cross the cell membrane
presence of a multifunctional crosslinker.16 As an example, than a high molecular weight macromonomer, and the gel
acrylamide is a monomer that polymerizes to form a conversion should occur at physiologically acceptable tem-
water-soluble polymer. If acrylamide is polymerized in the peratures. This has been accomplished with aqueous solu-
presence of a difunctional monomer (i.e., one that has two tions of poly(ethylene glycol) diacrylate and related com-
sites for polymerization, then referred to as a crosslinker), pounds, which may be converted to a hydrogel under
that monomer will participate in two poly(acrylamide) biocompatible reaction conditions by exposure to visible
chains. Accordingly, bis-acrylamido crosslinkers are routinely light in the presence of a visible light sensitizer.20 The
used to crosslink acrylamide hydrogels. Given that each biocompatibility of this process in direct contact with cells
poly(acrylamide) chain may react with more than one can be attributed to the following features:
crosslinker molecule, this polymerization process results in 1. Physiological saline is the solvent;
the formation of a chemically crosslinked hydrogel. In this 2. A very high fraction of the polymerizable precur-
case, the entire macroscopic hydrogel object is a single sors are macromolecular and thus unable to pass the
molecule. cell membrane;
As a second example of the formation of covalently 3. The photoinitiation process does not involve short
crosslinked synthetic hydrogels, hydrogels formed from wa- wavelength ultraviolet light;
ter-soluble polymeric diacrylates form a good example.17 4. The photoinitiation process does not generate large
Poly(ethylene glycol) diacrylates (Fig. 51.1)have been formed amounts of reactive oxygen species; and
by coupling one acrylate moiety to each end of a 5. The polymerization process does not generate large
poly(ethylene glycol) chain. These acrylates are capable of amounts of heat.
polymerizing to form a poly(acrylate), and if a poly(ethylene These principles serve as a design basis for other bio-
glycol) monoacrylate were polymerized the resulting poly- material systems for the formation of hydrogels in situ by
mers would be a comb-like structure, a poly(acrylate) struc- transformation from liquid precursors.
ture forming the back of the comb and poly(ethylene gly- In situ transformation has at least two major advan-
col) chains forming the teeth. If, however, a poly(ethylene tages, for some applications, vs. the implantation of
glycol) diacrylate is polymerized, the acrylates on either end preformed hydrogels. One advantage is that a large hydro-
of the chain can enter into different poly(acrylate) chains, gel implant may be delivered through a very small surgical
resulting in a chemically crosslinked gel that can only be hole, somewhat like assembly of the model ship inside the
broken apart by the cleavage of a covalent bond. bottle. This has been put toward therapeutic development
It is also possible for form covalently crosslinked in the coating of organs in the abdominopelvic cavity
hydrogels using enzymatic crosslinking, and fibrin forms an following surgery to prevent the formation of postoperative
excellent case in point.18 Fibrin is formed by the enzymatic adhesions, engineering the healing response.21 Here is it
action of thrombin and factor xiiia on fibrinogen. Throm- possible to deliver biomaterial implants as organ coatings
564 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 51.1. As an example of designing various mechanisms of degradation and bioactivity of hydrogels, the case of gels formed by
crosslinking poly(ethylene glycol) diacrylates is considered. Gels can be made to be substantially non-degradable (a), to incorpo-
rate nonenzymatic degradation sites by hydrolysis (b), to incorporate oligopeptide domains (c) which could be sites for cleavage by
a protease, e.g. plasmin (d) or which could be sites for binding to adhesion receptors on a targeted cell (e). In each case in this
example, the central block of the macromer is a poly(ethylene glycol) chain, selected for resistance to protein adsorption and for
favorable toxicology of the degradation product (f). These diacrylated poly(ethylene glycol) chains polymerize by free radical
reaction at the acrylate to form oligomers of acrylic acid that are linked to other oligomers of acrylic acid via the poly(ethylene
glycol) chains with the relevant degradation sites (g). Thus, the acrylic acid oligomers form nodes in a three dimensional network,
each connected to other nodes by long strings of poly(ethylene glycol). The permeability of the gels to proteins, and the rate of
release of incorporated protein drugs, can be controlled via manipulation of the density of the oligomeric acrylic acid nodes, by the
number of poly(ethylene glycol) strings emanating therefrom, and by the length of the poly(ethylene glycol) strings. Degradation
products are poly(ethylene glycol) and oligomers of acrylic acid, which are eliminated by glomerular filtration, and lactic acid.
that are many centimeters in diameter, but to deliver these teries, to attempt to engineer a healing response, the ability
implants using laparoscopic instruments to convert the liq- to form gels in situ presents an attractive feature. Such trans-
uid precursor to a hydrogel. A second advantage is that hy- formations can be accomplished even in the presence of in-
drogel implants of very complex shape may be formed in dividual and aggregated cells,24 suggesting that such chemi-
situ; this has been put to advantage in the coating of arterial cal schemes can be adapted for the delivery of cellular thera-
surfaces after deendothelialization to prevent the deposition peutics either within or adjacent to the vascular system.
of platelets on the subendothelium.22,23 In this case, the
photoinitiator was separated from the other components of Protein and Cell Interactions with Hydrogels
the precursor solution and was adsorbed to the arterial sur- Hydrogels can be designed to have minimal interac-
face; when the arterial lumen was flooded with all of the tions with proteins and cells. Cell adhesion to biomaterials
remaining precursors and the artery was irradiated with vis- is mediated by an adsorbed protein overlayer.25 Protein ad-
ible light, the formation of the hydrogel was restricted to sorption to materials is dominated primarily by hydropho-
the interface between the arterial wall and its contents. Thus, bic interactions and secondarily by electrostatic interac-
a hydrogel barrier was formed, only several microns thick, tions. 26 Most insoluble polymers (e.g., poly(ester)s,
that was conformal with the complex shape of the poly(ethylene), poly(propylene), poly(urethane)s, and
deendothelialized artery. poly(tetrafluoroethylene)) achieve their water-insolubility
Given that one may want to employ hydrogels in tis- by interacting poorly with water. These hydrophobic poly-
sue engineering for the delivery of therapeutics and even mers accordingly adsorb large amounts of proteins, which
living cells to polymeric scaffolds and even preexisting ar- act as surfactants to expose more hydrophobic domains to-
Hydrogels in Biological Control During Graft Healing 565
Fig. 51.2. As an example of a transformation that can be carried out in situ, hydrogels can be formed from macromolecular
precursors dissolved in buffered saline, directly on the tissue surface. A tissue surface to be treated (a), such as the anastomotic
region of a vascular graft, is first flushed with a solution of a nontoxic photoinitiator (in this example eosin Y), to stain the tissue
surface with the photoinitiator (b). After the nonadsorbed photoinitiator is flushed away and the artery lumen is filled with the
other components of the initiation system (in this example, the nontoxic buffer triethanolamine) and the polymerizable macromer
(such as a diacrylated poly(ethylene glycol) copolymer with lactic acid, as in Fig. 51.1) (c), the artery is irradiated with light at an
appropriate wavelength (in the case of eosin Y, green light) to cause a wave of polymerization to proceed from the tissue surface out
into the lumen (d). After the nonpolymerized macromer solution is rinsed away, the final result is a crosslinked solid hydrogel,
adherent to and conformal with the tissue surface (e). In the photoinitiation scheme shown in this example, the red eosin Y adsorbs
green light and is excited to a triplet state, from which it participates in a single-electron transfer reaction with triethanolamine,
resulting in a radical on the eosin and the triethanolamine. The free radical on the triethanolamine rearranges and serves as the
actual initiator of the free radical polymerization of the diacrylated macromer.
ward the material surface and thus present a more hydro- Work on the theory of these interactions has been performed
philic new surface to the aqueous surroundings. Electrostatic by Jeon et al.27,28 These investigators determined that when
interactions are secondary (due to the high ionic strength the tethered polymer brush exists at a sufficient density of
of physiological fluids) but remain important in protein in- polymer chains of a sufficient length, compression of the
teraction with materials, especially with regard to adsorp- polymer brush by a potentially adsorbing protein results in
tion of proteins to cationic polymers (since almost all pro- a strong resistance, i.e., steric stabilization, to the adsorp-
teins bear a net anionic charge). Accordingly, one may de- tion of the protein. Thus, one may understand with these
sign a biomaterial to be relatively free of cell adhesion by theoretical analyses based on the physics of polymer chains
making it be both very hydrophilic (e.g., a hydrogel) and that the thermodynamic interactions are unfavorable for
nonionic or anionic. protein adsorption.29 Very well characterized experimenta-
Much of what is understood about protein and cell tion has also been performed with model two dimensional
interactions with hydrogels has been learned from two di- polymer brushes, e.g., by Prime and Whitesides employing
mensional hydrogel models, namely water-soluble polymers self-assembling monolayers of alkane thiols on gold sub-
(which would form hydrogels) tethered to insoluble surfaces. strates, the alkane thiols bearing grafted poly(ethylene glycol)
566 Tissue Engineering of Prosthetic Vascular Grafts
chains.30 These studies demonstrated experimentally that a biomaterials engineering aspects of incorporation of these
high density of poly(ethylene glycol) chains would suffice, signals into hydrogels are addressed.
even if the chains were very short, to dramatically limit pro- In order to be able to engineer a selective adhesion
tein adsorption. response to hydrogel materials, one must devise means by
The physical character of hydrogels is important, in which to incorporate synthetic adhesion ligands, such as
tandem with the chemical character, in determining hydro- synthetic peptides derived from adhesion proteins, into hy-
gel biocompatibility. This is to say, it is not just the chemical drogel networks. Hydrogels derived from poly(ethylene gly-
structure of the repeat unit of the polymer in the hydrogel col) diacrylate again serve as an instructive example. Hern
that determines the biocompatibility of the hydrogel. This and the author34 examined means by which to incorporate
can be considered in the context of crosslinked gels of adhesion peptides into these photopolymerizable gels, us-
poly(ethylene glycol) diacrylate.31 When gels were formed ing the adhesion peptide GRGDY as a model (the N-termi-
from poly(ethylene glycol) of molecular weight 200 or 1000, nal G serving as a spacer, and the C-terminal Y serving as a
these materials were rapidly encapsulated in a typical for- site for radioiodination). The N-terminus of the peptide was
eign body reaction and calcified after subcutaneous implan- acrylated, either with a minimal spacer or with a
tation in the rat; however, gels formed from poly(ethylene poly(ethylene glycol) chain of molecular weight 3400, and
glycol) diacrylate of higher molecular weight remained free these acrylated peptides were incorporated into the hydrogels
of fibrous encapsulation and did not calcify. The calcifica- by copolymerization (i.e., mixing the peptide acrylates and
tion response may be due to a specific interaction between the poly(ethylene glycol) diacrylates in the hydrogel precur-
Ca2+ and poly(ethylene glycol), but the induction of a for- sor solution, and then photopolymerizing the mixture).
eign body reaction almost certainly relates to the difference When the peptides were omitted from the hydrogel, the re-
in water content of these gels (from approximately 75% water sulting material was very resistant to cell adhesion. When
for the poly(ethylene glycol) 200 diacrylate to approx. 90% an acrylated RGD or control inactive RDG peptide was in-
for the higher molecular weight poly(ethylene glycol)-based corporated without a spacer arm, the resulting material sup-
hydrogels. As such, one should understand clearly that all ported cell adhesion in both cases, but only in the presence
hydrogels are not alike, and even that all hydrogels of the of serum proteins; this result can only be explained by the
same chemical composition are not necessarily alike in their peptide being sterically unavailable for biospecific binding
biological responses. to cell adhesion receptors but serving as a site for the non-
specific adsorption of serum proteins. When the RGD and
Designed Cell Adhesiveness of Hydrogels RDG peptides were incorporated with a poly(ethylene gly-
In the above section it was demonstrated that, with col) spacer, the materials with RGD supported cell adhe-
adequate attention to the details of hydrogel design, sion, while those with the inactive RDG supported no adhe-
hydrogels can be formed to effectively resist the attachment sion. Thus, it would seem that for the sequence to be steri-
of cells, even in vivo. Based upon this observation it then cally available for binding to receptors on the cell it must be
becomes attractive to incorporate into such a hydrogel bio- immobilized within the gel via a long spacer; and it would
logical adhesion ligands targeting specific receptors and even further seem that the peptide immobilized without the
perhaps specific cell types. In this manner, one might be able spacer serves as a site for protein adsorption, while the pep-
to develop a hydrogel material for tissue engineering that tide immobilized with the spacer does not. Having these re-
would reject the attachment on one cell type (e.g., the plate- sults in hand, one is then prepared to begin to incorporate
let) but that would accept the attachment of another cell cell type specific adhesion signals (such as the endothelial
type (e.g., the endothelial cell). Tissue engineering with syn- targeting REDV sequence) to engineer the tissue response
thetic adhesion ligands is based on profound advances in to a hydrogel, and thus to a vascular graft.
the molecular biology of cell adhesion over the past several
years, in which a host of extracellular matrix adhesion gly- Nonenzymatic Degradation of Hydrogels
coproteins have been identified, together with their corre- It is clear that one must have in one’s repertoire of
sponding receptors. Moreover, the domains on many of the materials for tissue engineering hydrogels that are degrad-
adhesion proteins that bind to the receptors have been iden- able. Degradation by nonenzymatic hydrolysis can be ob-
tified, and it has been demonstrated that, in many of these tain by incorporating hydrolytically labile bonds within the
cases, synthetic peptide and nonpeptide analogs can be de- gel precursor, either in a crosslinker or within the polymer
veloped with appropriate affinity for the appropriate recep- backbone. The example of poly(ethylene glycol) diacrylate
tors on the cell surfaces. It has also been demonstrated that hydrogels, in this case degradable variations, will be selected
some of these adhesion ligands have affinity for receptors for discussion.
that exist on one cell type do not exist on other cell types A host of degradable polymers are available for use in
present in the same biological environment; e.g., the se- medicine, and these are based on hydrolysis of esters,
quence REDV, which is present in some fibronectin forms, carbonates, and anhydrides, to name only a few of the im-
binds to the integrin receptor #4!1 that is present on human portant approaches (see chapter 47). These compositions
endothelial cells but not on human blood platelets.32,33 The can be incorporated into polymers that form hydrogels, to
biology of adhesion proteins, their receptors and synthetic obtain the physical and biological properties of the hydro-
analogs of the adhesion proteins is the topic of chapter 54 in gel along with the degradability inferred by the water-labile
this book and is not addressed further here; only the bond. To accomplish this in the scheme of photopolymerized
poly(ethylene glycol) diacrylate hydrogels, block copolymers
Hydrogels in Biological Control During Graft Healing 567
have been formed, with poly(ethylene glycol) forming the activity to clear the fibrin from their path. Electron
central block and oligomers of, e.g., lactic acid forming microscopic observation of the morphology of the neurite
flanking blocks; the termini of the lactic acid blocks can be tips and the fibrin near the neurite tips demonstrated that
acrylated, leading to an analog of poly(ethylene glycol) fibrils were present in the immediate vicinity of all aspects
diacrylate with a degradable segment between the of the growth cone, with less than a 50 nm gap between the
poly(ethylene glycol) segments.35 The hydrogel to result from neurite plasma membrane and the fibrin fibrils, suggesting
such a precursor consists of nodes of oligo(acrylic acid) that the neurite did not migrate by finding a pathway that
esterified to poly(ethylene glycol) chains via an oligo(lactic was enzymatically created in a spatially nonlocalized man-
acid) intermediate. Degradation thus yields a low molecular ner, but rather that local enzymatic activity associated with
weight oligo(acrylic acid), lactic acid and its oligomers, and the cell membrane was responsible for local removal of
poly(ethylene glycol) chains of the original molecular weight. material to create a pathway. Furthermore, these studies also
The rates of degradation of hydrogels behave some- demonstrated that there was no dependence in neurite mi-
what differently than those of the poly(ester), gration rate on fibrin fibril morphology, but only on den-
poly(carbonate), or poly(anhydride) parent family. In the sity; i.e., the only feature that mattered was the amount of
parent families, two features control the rate of degradation: material present to be cleared from the pathway of the cell
The intrinsic susceptibility of the water-labile bond to hy- to permit migration.
drolysis, and the rate of transport of water into the hydro- Studies as described above both emphasize the im-
phobic polymer. In hydrogels, by contrast, the entire mac- portance of biologically derived materials for tissue engi-
roscopic material is equally exposed to water, so the intrin- neering and also provide motivation to develop synthetic
sic rate of hydrolysis controls the overall degradation rate. analogs. The fact that, at least in some circumstances, the
This feature can be used to modulate the rate of resorption detailed morphology of the biological structures to be cleared
of such hydrogels: For example, poly(ethylene glycol)- by cell-associated proteolytic activity is less important than
glycolide diacrylate hydrogels degrade faster than the mass of that material encourages one to proceed with
poly(ethylene glycol)-lactide diacrylate hydrogels, which development of such synthetic analogs, in that only the
degrade faster than poly(ethylene glycol)-caprolactone chemical nature need be mimicked, not the morphological
diacrylate hydrogels, in accordance with the expected order nature as well (at least in some cases). With this in mind,
based on chemical kinetics. West et al have examined poly(ethylene glycol)-based hy-
drogel materials that degrade by proteolytic activity.38
Enzymatic Degradation of Hydrogels Poly(ethylene glycol) was used as a substrate for liquid phase
In addition to degradation based on nonenzymatic peptide synthesis, and either plasmin or collagenase-sensi-
hydrolysis, one could contemplate degradation based on tive peptide sequences were synthesized at the termini of
enzymatic processes. Enzymatic degradation may have con- the polymer chains. Upon the termini of these peptide blocks
ceptual advantages over nonenzymatic degradation in some an acrylate group was coupled, to create an analog of the
applications of tissue engineering. For example, cell migra- poly(ethylene glycol)-lactide diacrylate, namely
tion through tissues and through fibrin clots and granula- poly(ethylene glycol)-peptide diacrylate, where the identify
tion tissue in wound healing is dependent upon enzymatic of the peptide was selected for sensitivity to the protease plas-
hydrolysis of glycoproteins and glycosaminoglycans in the min or collagenase. Hydrogels were formed from such pre-
extracellular matrix. Thus, endowing a hydrogel with the cursors and were exposed to either plasmin or collagenase,
ability to be degraded by enzymes would permit the hydro- with the result that the plasmin-sensitive gel degraded in
gel to be remodeled by biological processes associated with the presence of plasmin but not collagenase, and the colla-
cell migration. genase-sensitive gel degraded in the presence of collagenase
It is instructive to consider cell migration through bio- but not plasmin, as designed. Such preliminary studies dem-
logically derived hydrogels, as a prototype for the ultimate onstrate at least the potential for the synthesis of hydrogel
design of synthetic materials that permit cell infiltration by materials that mimic the ability to be remodeled like the
proteolysis. Fibrin serves as one such good prototype, being natural biological hydrogels used in nature.
the primary matrix through which cells migrate in a healing
response. Herbert et al examined the migration of the growth Controlled Release
cones of neurites emanating from aggregated neural and glial Hydrogels have been extensively explored for the con-
cells within a three dimensional fibrin matrix in a culture trolled release of drugs, particularly macromolecular drugs
model.36,37 Fibrin was formed under controlled conditions such as proteins and oligonucleotides. One reason for uti-
around the cell aggregates, and the rate of neurite extension lizing hydrogels for drug delivery is the relative ease in con-
was measured as a function of fibrin fibril density and mor- trolling the permeability of the gel structure to the macro-
phology; additionally, the dependence of the extension pro- molecular drug. While in most gels permanent pore struc-
cess on local protease activity was examined. It was observed tures do not exist, transient pores do exist that can be de-
that either direct inhibition of plasmin activity or inhibi- scribed in terms of molecular weight between crosslinks and
tion of plasminogen activation activity completely inhib- pore dimensions.39 These parameters can be readily con-
ited cell migration, indicating that the neurites did not find trolled, and by adjusting these dimensions relative to the
preexisting pores through which to migrate, and that they radius of gyration of the macromolecular drug one can ei-
did not mechanically push fibrin fibrils out of their path ther restrict drug diffusion to a controlled degree and ob-
during migration, but rather that they expressed chemical tain release from the hydrogel material on a time scale faster
568 Tissue Engineering of Prosthetic Vascular Grafts
than that of degradation, or one can entrap the drug, using Graft Sealing
a gel with a pore dimension much less than the drug size, Knitted and woven grafts must be preclotted to pre-
and obtain drug release that is mediated by the rate of hy- vent excessive blood leakage from the graft, and this process
drogel degradation.40 It is relatively straightforward to syn- of preclotting may predispose the graft to further thrombo-
thetically manipulate the pore dimensions of a hydrogel. For sis in the short term (e.g., due to platelet interactions with
example, with gels formed by crosslinking an unsaturated fibrin in the clot or due to prothrombin activation on the
monomer (refer to the example of acrylamide with a bis- surfaces of cells in the clot) and to the development of an
acrylamido crosslinker) the pore dimensions may be altered excessive neointima in the long term (e.g., due to the growth
via control of the ratio of acrylamide to bis-acrylamide, a factors that are released by activated platelets in the clot). To
high amount of bis-acrylamido crosslinker yielding small reduce the possibility of short and long term graft failure as
pore dimensions and restricted permeability. In the example a result of preclotting, grafts that are based on collagen,44
of poly(ethylene glycol)-diacrylate based hydrogels, one may gelatin,45 and albumin46 preclotting have been developed.
alter pore dimensions via the molecular weight of the These materials are indeed hydrogels, and thus hydrogel-
poly(ethylene glycol) diacrylate, low molecular weights yield- sealed vascular grafts have already been used clinically. One
ing small pore dimensions and low permeability. As an ex- could also employ hydrogels designed more specifically for
ample, Cruise et al41 reported pore dimensions of 19 Å for graft sealing. Such a hydrogel sealing material could either
gels formed from poly(ethylene glycol) 2000 diacrylate, 22 resorb by physical erosion (e.g., with poly(ethylene glycol)-
Å from poly(ethylene glycol) 4000 diacrylate, 34 Å from poly(propylene glycol)-poly(ethylene glycol) block copoly-
poly(ethylene glycol) 8000 diacrylate and 58 Å from mers) or by nonenzymatic hydrolysis (e.g., with a
poly(ethylene glycol) 20,000 diacrylate. It was likewise pos- poly(ethylene glycol)-lactide diacrylate hydrogel). Such a
sible to control pore dimensions via manipulation of the designed hydrogel has the possible benefit that other fea-
reaction conditions with the same poly(ethylene glycol) tures could also be designed into the gel, as described below.
diacrylate precursor; e.g., pore dimensions of 70 Å were
obtained by polymerization of poly(ethylene glycol) Promotion of Endothelialization
diacrylate 20,000 from a 10% precursor solution, 58 Å from It is clear that the rapid establishment of an endothe-
a 20% precursor solution, and 42 Å from a 30% precursor lial cell monolayer atop the neointima within a graft is key
solution. Because of this high flexibility in the control of to success in the tissue engineering of a vascular graft. It is
pore dimensions and gel permeability to macromolecular also clear that thrombosis within the graft lumen is an un-
drugs, hydrogels are extremely attractive for controlled re- desirable outcome. In light of these two observations, one
lease applications in tissue engineering. could consider employing a hydrogel coating or a hydrogel
sealing to both reduce thrombosis and to enhance
Design Principles, Hydrogels for Biological endothelialization, e.g., via a gel that resists fibrinogen ad-
Control During Graft Healing sorption and accordant platelet deposition, that gel also con-
One may consider many possible applications for taining a grafted ligand that demonstrates specificity for an
hydrogels in tissue engineering, some of which have been endothelial cell adhesion receptor. In such a manner, it might
explored already and some of which remain to be experi- be possible to fail to induce platelet adhesion but to succeed
mentally addressed. In this section, some of the more im- in inducing endothelial cell adhesion.32 In the absence of
portant possible applications in vascular tissue engineering seeding of endothelial cells along the entire lumen of the
will be called into focus. These relate to altering the blood graft, one must be concerned with the rate of endothelial
response to a graft, altering the arterial response to the graft, cell migration along the coated graft surface. Lauffenburger
and directing the response of the surrounding tissue to a and colleagues have demonstrated, both based on theoreti-
graft implant. cal considerations and also by experiment, that there exists
an optimum in the surface density of adhesion ligands for
Reduction of Thrombosis fastest cell migration rates.47 At low surface density of adhe-
Because hydrogels can be designed that adsorb rela- sion ligand, cell traction is low and cell migration rates, as
tively low amounts of protein and that thus support rela- well as adhesion strength, are accordingly slow. At high sur-
tively low amounts of platelet adhesion (which has been face density of adhesion ligand, cell traction is very high,
demonstrated to be modulated principally by adsorbed fi- but the rate of binding of an adhesion receptor on the cell
brinogen),42 one obvious, yet important, application of surface with surface-bound adhesion ligand is very high,
hydrogels to vascular graft tissue engineering is the reduc- resulting in very strong adhesion but very low cell migra-
tion of platelet deposition in the hours and days following tion rates. Accordingly, one must design the display of ad-
implantation. Hydrogel coatings have been used to modu- hesion ligands in the hydrogel coating such that adhesion
late the deposition of platelets and other cells on a host of strength is sufficient to resist fluid forces within the vascular
medical devices, most of which are implanted for relatively flow but such that it is not so strong that the rate of cell
short periods of time. Indeed, heparinization of graft sur- migration is unacceptably reduced.
faces may improve graft biocompatibility by both a phar- It has been demonstrated that thrombosis at the anas-
macological effect (the specific biological activity of hep- tomoses of a graft may be well in excess of thrombosis on
arin as it interacts with antithrombin III) as well as a bio- the graft itself.48 Accordingly, efforts at reducing platelet
physical effect (the hydrophilic, anionic heparin hydrogel deposition on the biomaterial per se may be slightly mis-
coating acting to reduce the adsorption of fibrinogen).43
Hydrogels in Biological Control During Graft Healing 569
placed relative to the thrombogenic potential of the anasto- stimulate endothelial cell migration and proliferation, and
moses. One may then consider the use of a hydrogel to pas- another factor to limit perigraft inflammation or smooth
sivate the graft anastomoses. It was demonstrated above that muscle activity.
one advantage of hydrogels over other materials for tissue Stated more generally, Nature’s extracellular matrix
engineering is that they may be formed on the surface of a hydrogel serves to provide a great deal of information to
tissue by an in situ transformation. As an example, it was cells to direct their behavior and organization in space. It
discussed that hydrogel coatings have been formed on the provides information in the form of structural cues, of im-
surfaces of injured arteries to replace one function of the mobilized adhesion molecules, and of immobilized and dif-
endothelium, namely to regulate the deposition of blood fusible polypeptide growth factors. Furthermore, it receives
platelet on the arterial surface.22 Accordingly, it could be information from the cells as they respond. It does this by
envisioned to limit the extent of thrombus deposition on binding to growth factors that are released from cells and by
the anastomoses within a graft that had already been su- degrading in the face of cellularly-displayed enzymes. It
tured in place by the local, in situ deposition of a hydrogel might be possible, and indeed advantageous, in tissue engi-
coating spanning the junction of the native artery and the neering of the vascular graft, to mimic some or all of these
implanted graft. Using such an approach, it might be pos- activities, either in a biologically derived hydrogel matrix or
sible to directly impact one of the most troublesome regions in a completely synthetic biomimetic hydrogel matrix, with
of an implanted graft. the goal of harnessing some of these biological activities for
controlling tissue morphogenesis and function.
Drug Release
Hydrogels have been explored extensively in drug References
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an overall acceleration of the rate of endothelialization and loading, and release. Pharm Res 1992; 9:283-290.
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15. Decher G, Lvov Y, and Schmitt J, Proof of multilayer
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16. Gin H, Dupuy B, Caix J, Baquey C, and Ducassou D, In 34. Hern DL, and Hubbell JA, Incorporation of adhesion pep-
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18. Moser D, Blood coagulation and fibrinolysis. Clin Cardiol romolecules 1993; 26:581-587.
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19. Hubbell JA, In situ material transformations in tissue brinolysis on neurite growth from dorsal root ganglia cul-
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Matrix Engineering
CHAPTER 52
In Vivo Synthesis of Organs Using Collagen-GAG Copolymers
Ioannis V. Yannas
Introduction
A s practiced today, the methodology of tissue engineering emphasizes the reconstruction
of tissues and organs. Such reconstructive activity may be either induced to take place
inside the host organism, at the site of the missing organ (in vivo synthesis), or else it may be
induced outside the organism (in vitro synthesis), typically in a cell culture environment,
prior to being grafted onto the host.
In vivo synthesis of organs has been induced by use of analogs of the extracellular
matrix (ECM). Three organs have been synthesized so far by this method, namely, the skin
(in humans, as well as in swine and guinea pig models), peripheral nerves (rat) and the knee
meniscus (canine). The procedure depends critically on identification of the appropriate
regeneration template, a biologically active ECM analog which can block scar synthesis and
induce instead the desired in situ synthesis of the physiological organ. Convincing evidence
that regeneration has, in fact, occurred must be based on observation of definitive recovery
of both structure and function in an anatomically well defined defect. Furthermore, there
must be evidence that the mass of the regenerated organ which forms in the absence of the
template (spontaneous regeneration) is negligibly small relative to that which is induced in
its presence.
In this chapter, I will first present the evidence supporting the conclusion that organ
regeneration can indeed be reproducibly achieved both in animal models and clinically. The
methodology of induced regeneration will then be briefly summarized, followed by an
introduction to the models which are used to describe the mechanism of induced
regeneration.
Although in vivo synthesis of vascular tissue has not yet been demonstrated, I suggest
that future experimentation in this area could be very fruitful and could benefit directly by
use of the principles described briefly below for other tissues and organs.
Summary of Evidence for In Vivo Synthesis of Organs
There is considerable evidence that the adult mammalian dermis does not regenerate
spontaneously.1,2 In studies which have been conducted since 1970 it has have shown that a
porous graft copolymer of type I collagen and chondroitin 6-sulfate, a glycosaminoglycan
(collagen-GAG copolymer) induces in vivo synthesis (induced regeneration) of the dermis
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
572 Tissue Engineering of Prosthetic Vascular Grafts
in large areas of full-thickness skin loss in the guinea pig.3-10 has an average pore diameter of 5 ∝m, an average molecular
This finding has been independently confirmed repeatedly weight between crosslinks of 30-40 kDa, a preferred orien-
in humans11-13 and recently in swine.14 The evidence for re- tation of pore channel axes in the direction of the nerve axis,
generation was based on histological and ultrastructural and a 98/2 w/w ratio of type I collagen to GAG.16,17 Clearly,
studies, the use of small-angle laser light scattering studies there are significant differences between the network struc-
of histological sections, and functional studies. It was con- ture of skin and nerve regeneration templates.
cluded that the new skin was structurally and functionally Yet a third ECM analog has been reported, capable of
competent; however, it was totally lacking in hair follicles inducing regeneration of the canine meniscus following 80%
and other skin adenexa. transection.21,22 Although the ECM analog used in these
The emphasis in early studies of in vivo synthesis of studies has been stated by the authors to be similar to that
skin was on keratinocyte seeding of the analog prior to graft- described earlier,9 its detailed structure was not reported.
ing, as this procedure led to simultaneous regeneration of There is, therefore, substantial evidence that, under
an epidermis as well as a dermis. It was later appreciated appropriate conditions, the adult mammal can be induced
that cell seeding speeds up epidermal regeneration but it to regrow certain organs which are not regenerated sponta-
appears otherwise not to be required for regeneration ei- neously. This exciting conclusion suggests new therapeutic
ther of the epidermis or of the dermis. avenues. However, there are still many unanswered ques-
The evidence cited above led to identification of a cell- tions about the detailed cell-biological mechanism by which
free macromolecular network with highly specific structure, active ECM analogs modify so spectacularly one or more of
the cell-free skin regeneration template (SRT), which alone the processes involved in conventional wound healing.
possessed this unprecedented morphogenetic activity. Only
one of several collagen-GAG matrices studied as described
above was capable of preventing scar tissue formation and Methodological Principles
promoting dermal regeneration. The active ECM analog was of Induced Regeneration
characterized by a collagen/GAG ratio of 98/2 w/w, average Over the years it has become clear that a number of
pore diameter between 20 and 120 ∝m, random orientation conditions must be in place in order to conduct a reproduc-
of pore channels, and sufficiently high density of covalent ible study of induced regeneration. One of the most impor-
crosslinks (average molecular weight between crosslinks in tant is the choice of an anatomically well-defined lesion (ex-
the template, 12 kDa) to resist degradation by collagenases perimental volume) in which to study the phenomena. This
for about 10 days following grafting. Several other very concept was pioneered in the studies of Billingham and
closely related ECM analogs showed either significantly re- Medawar1 as a means of minimizing animal to animal vari-
duced activity or no activity at all.9 In related studies it was ability in observations. The experimental volume can be iso-
observed that ECM analogs which showed high activity in lated by containing it within anatomically distinct tissues
promoting dermal regeneration also delayed significantly the belonging to a neighboring organ or by a device or (in the
onset of wound contraction.9 The available evidence com- case of skin) by the atmosphere.
pelled the conclusion that the activity of this insoluble net- Since mammals possess a very small but finite poten-
work depended critically on maintenance of a highly spe- tial for regeneration, it is necessary to “correct” the observed
cific three dimensional structure inside the wound bed over regeneration by subtracting the amount which has been con-
a period of time between about 5 and 15 days. The active tributed by spontaneous regeneration. This requirement is
network has been referred to as skin regeneration template equivalent to defining a negative control in every experi-
(SRT). The observed activity of SRT, consisting in drastic mental model, consisting of the observations obtained in
modification of the outcome of the wound healing process, the absence of the presumptive regeneraion template.
has not been duplicated by application on the wound bed Although the mechanistic interpretation of induced
of solutions of one or more growth factors or by application regeneration has not been completed, two arguments have
of suspensions of keratinocytes or fibroblasts. been advanced to support the contention that there is a re-
Regeneration of a partially functional sciatic nerve was quirement for structuring a regeneration template as an
induced across a transected gap of 15 mm in the rat sciatic ECM analog. First, studies of organ development have shown
nerve15-18 by another ECM analog, which was also found to conclusively that the early presence of specific ECM com-
possess a highly specific network structure. In this animal ponents is required for formation of physiological or-
model the nerve stumps at either side of the gap were in- gans.23-25 In contrast, the exudate which flows into a spon-
serted in a silicone tube (tubulation); in the absence of a taneously healing wound after injury does not contain com-
tube, regeneration was decidedly absent and neuroma for- ponents of the insoluble and nondiffusible ECM networks
mation was invariably reported. Although spontaneous re- (although it contains several types of cells and regulators);
generation of nerve through the tube occurred reproduc- the healing process in this case eventually leads to synthesis
ibly at a gap length of 5 mm, regeneration across a 15 mm of scar. Second, the evidence presented in the preceding sec-
gap has not been observed.19,20 The study eventually led to tion clearly supports the contention that specific ECM ana-
identification of an ECM analog which, when filling the sili- logs indeed suffice to lead to regeneration of at least two
cone tube and thereby connecting the two nerve stumps, organs. However, there is clearly insufficient evidence to
induced regeneration across a 15 mm gap. This analog has conclude definitively that regeneration can be induced only
been referred to as nerve regeneration template (NRT). It by specific ECM analogs.
In Vivo Synthesis of Organs Using Collagen-GAG Copolymers 573
Conclusive evidence that an ECM analog has induced mole/cm3/s. Picture the nutrient being transported from the
organ regeneration can only be obtained with a performance solid-like tissue, where the concentration of nutrient is as-
assay which simply compares the structure of the regener- sumed to be a constant C0 due to the presence of vascular
ate to that of a normal control. Assays of this type can be supply, over a distance L through the exudate, until it reaches
destructive (e.g., histology), in which case only one experi- the cell. Prior to the onset of angiogenesis, i.e., during the
mental time point can be obtained per animal, or first few days following template implantation, the nutrient
nondestructive, such that the complete kinetic curve for re- is transported exclusively by diffusion, characterized by a
generation can be obtained from a single animal. The fidel- diffusivity D cm2/s. Dimensional analysis readily yields the
ity of regeneration is a figure of merit which is assigned to cell lifeline number:
each candidate template. The regeneration template is then
the ECM analog which has scored a very high value of the S = RL2/DC0 (2)
fidelity index.
which can be used to compare the relative magnitude
of the rate of nutrient consumption by the cell nutrient (nu-
Models of the Mechanism of Regeneration merator) to the rate of supply of nutrient to the cell by dif-
I summarize below the major physicochemical mod- fusion (denominator). The cell dies if the rate of consump-
els26-28 which have been used to compile a mechanistic in- tion of the critical nutrient exceeds greatly the rate of sup-
terpretation of the process of regeneration. It is evident that ply, S>>1. At steady state the rate of consumption of nutri-
a regeneration template modifies drastically the kinetics and ent by the cell just equals the rate of transport by diffusion
mechanism of spontaneous wound healing which normally over the distance L. Under conditions of steady state,
lead to wound contraction and scar synthesis. However, a S = O(1); at that point, the value of L becomes the critical
view of a template as a classical catalyst is not supported by cell path length, Lc, the distance of migration beyond which
the well known fact that regeneration templates are neces- cells require the presence of a vascular supply. Another way
sarily consumed during the process which they modify. of looking at Lc is that it is the longest distance away from
the wound bed boundary along which the cell can migrate
Micrometer Scale Proximity of Host to Template Surface without requiring nutrient in excess of that supplied by dif-
Cells and cytokines present in the exudate are trans- fusion. For many cell nutrients of low molecular weight, Lc
ferred to the surface of the template following implantation is of order 100 ∝m, an estimate of the maximum template
of the porous template into the wound bed. Surface tension dimension which can support cells.
forces pull the exudate inside the capillaries (pore channels)
of the template, as described by: Template Specific Surface Is Bounded
Following its migration onto the template surface a
P = 2&/r (1) host cell is visualized interacting with binding sites on the
surface. The surface density of binding sites can be expressed
where r is the radius of the pore channel in a template as Λb, equal by definition to the number of sites Nb per unit
undergoing wetting by exudate with an air-liquid surface surface A of template. Λb can also be expressed more use-
tension of & in dyne/cm. Wetting of the opposed surfaces is fully in terms of quantities potentially measurable by opti-
promoted as the suction pressure P in Equation (1) increases, cal microscopy, i.e., in terms of the volume density of bind-
and such increase is occasioned by a decrease in average pore ing sites ∋b (number of sites per unit volume porous tem-
radius. The values reached by P are not negligible; water with plate) and the specific surface ( of the template expressed in
an air-liquid surface tension of & = 72 dyne/cm, for example, units of mm2/cm3:
is pulled inside a pore radius of 100 ∝m with a suction pres-
sure of almost one-hundredth of one atmosphere and P in- Λb = Nb/A = ∋b/( (3)
creases almost to one full atmosphere when the pore radius
decreases to a value as low as 1 ∝m. Under conditions where Assuming that each cell is bound to (an a priori un-
exudate flows inside the pore channels of a template with known number of) 5 binding sites, there will be Nb/5 bound
average pore diameter of 100 ∝m, cells and cytokines are cells per unit surface; the volume density of cells will be ∋c =
within a distance of less than 50 ∝m from the template sur- ∋b/5 and the surface density of cells will be:
face; this distance can then be covered by these components
of the exudate within a few minutes. Λc = Λb/5 = Nb/5A = ∋b/5( = ∋c/( (4)
Maximum Dimension of Template, ~100 ∝m Levels of myofibroblast density inside templates with
Cell migration from the solid-like tissue of the wound pore diameter of about 10 ∝m have been observed to reach
bed into the template requires the availability of adequate typical values of the volume density, ∋c, of order 107
nutrition. The precise nutritional requirements of any cell myofibroblasts per cm3 porous template. For a template of
are too complex to incorporate into a simple model; instead, average pore diameter 10 ∝m the specific surface ( is calcu-
these requirements will be simplified by defining a critical lated to be approximately 8 x 104 mm2/cm3 template; there-
nutrient which is required for normal cell function; such a fore, 1 cm3 porous template is characterized by a cell surface
nutrient is assumed to be metabolized by the cell at a rate R density of Λc = ∋c/( = 107/8 x 104 = 125 cells/mm2. For a
574 Tissue Engineering of Prosthetic Vascular Grafts
template of identical composition but average pore diam- bed in order not to impede cellular processes which lead to
eter as large as 300 ∝m, Λc is the same as above; however, the the emerging organ.
specific surface is calculated to be only about 3 x A steady state model is probably the simplest way to
103 mm2/cm3 template. In this case, the volume density of accommodate these two requirements. It requires synchro-
cells is, accordingly, only ∋c = Λc − ( = 125 x 3 x 103 = 3.75 x nization of the two processes: organ synthesis and template
105 per cm3 porous template. We conclude that the template degradation.5,9 This model leads directly to the hypothesis
which has the smaller average pore diameter (10 ∝m) has a of isomorphous tissue replacement:
volume density of myofibroblasts which is about 27 times
lower than with the template which has the larger pore di- td/ts = O(1) (5)
ameter (300 ∝m). The existence of a maximum pore diam-
eter requirement for the template is suggested by these cal- In Equation (5) td denotes a characteristic time con-
culations, simply to ensure a specific surface which is large stant for degradation of the template at the tissue site where
enough to bind an appropriately large number of cells. a new organ is synthesized with a time constant of ts. The
In addition, it is clear that cells originating in the degradation rate can be estimated by histological observa-
wound bed cannot migrate inside the template and eventu- tion of the decrease in mass of template fragments at vari-
ally reach binding sites on its surface unless the template ous times.10,30 A closer estimate of td has been obtained by
has an average pore diameter large enough to allow for this. measuring the kinetics of disintegration of the macromo-
We conclude that there is, therefore, a requirement for a lecular network using rubber elasticity theory.3 A third pro-
minimum pore diameter for the template, about equal to cedure consists of monitoring the kinetics of mass disap-
the characteristic diameter of the cells (of order 5 ∝m). Thus, pearance of a radioactively labeled template. A rough esti-
the pore diameter of the regeneration template is limited mate of ts can be obtained by observing th, the timescale of
both by an upper and a lower bound. This conclusion is in synthesis of new tissue during healing (in the absence of a
agreement with the experimental evidence which shows that template) at the anatomical site.31 Using the latter approach
ECM analogs, identical in chemical composition but differ- it has been estimated that ts for the regenerating dermis is of
ing only in average pore diameter, show maximum activity order 3 weeks31 and of order 6 weeks for the regenerating
(inhibition of onset of wound contraction, consistent with peripheral nerve.16 These estimates allow adjustment of td
regeneration rather than scar formation) when the average for the template, by adjustment of the crosslink density and
pore diameter lies between 20 and 120 ∝m.9 Further evi- GAG content, to levels which are approximately equal to the
dence has shown that, when other structural parameters of values of ts, as the latter are dictated by the nature of the
the template remain constant, loss of the 20-120 ∝m porous anatomical site.
structure of the template by simple evaporation at room tem- Experimental support for the isomorphous tissue re-
perature (a process which yields an ECM analog with aver- placement hypothesis has been based on observations such
age pore diameter of less than 1 ∝m) leads to synthesis of a that, when the ratio in Equation (5) was adjusted to values
scar capsule at the surface of the grafted analog, evidence of much smaller than one (by implanting a rapidly degrading
a barrier to cell migration inside an implant.29 ECM analog, for which td<<ts), the wound healing process
resulted in contraction and synthesis of scar (as would have
Critical Residence Time of Template been the case if the template was missing). Furthermore,
The residence time of a template is also bounded. A when the ratio in Equation (5) was much larger than one 1
template has to stay in place long enough to induce the ap- (by implanting an ECM analog which degraded very slowly,
propriate synthetic processes to take place; soon after that, so that td<<ts) the ECM analog was surrounded by a cap-
it must disappear in a timely fashion so as not to interfere sule of scar tissue.29,31 Clearly, the available limited evidence
with these same processes which it induces. The time pe- cannot be used to test the hypothesis of Equation (5) con-
riod necessary to induce synthesis is roughly equal to that clusively; nevertheless, such evidence is, at the least, consis-
required to complete the wound healing process at that ana- tent with a template half life which is bounded. Studies of
tomical site. (In general, the rate of wound healing is quite inhibition of wound contraction by several ECM analogs
different in tissues such as, say, the dermis and the sciatic with defined structure have provided direct experimental
nerve.) Since the template is an insoluble (and, therefore, support for this conclusion. These studies have shown that,
nondiffusible) three dimensional network it follows that cells of several ECM analogs studied, the dermal regeneration
which are bound on it become immobilized and their mi- template was the analog which degraded at a rate corre-
gration is, accordingly, arrested. Not only cells are prevented sponding to a half life of about 1.5-2 weeks; ECM analogs
from migrating to locations which are appropriate for syn- which degraded at much slower or much faster rates were
thesis of a new organ but, in addition, the laying down of not active.9
newly synthesized ECM by the cells in the space of the wound From the above it can be inferred that the simplest
bed is probably blocked physically by the presence of the template structure which can degrade and diffuse away with
template. It follows that the persisting insolubility of the tem- minimum harm to the host is one in which the template
plate will increasingly interfere with the synthesis of the new undergoes degradation by enzymes of the wound bed to
organ at that site. To avoid this eventuality, the template is nontoxic low molecular weight fragments.31
required to become diffusible (by degradation to small mo-
lecular fragments) and thereby disappear from the wound
In Vivo Synthesis of Organs Using Collagen-GAG Copolymers 575
Template Chemical Composition sity of covalent bonds between collagen chains and GAG
Developmentally significant interactions are known molecules, i.e., to form a collagen-GAG graft copolymer.34
to involve cells, growth factors and ECM components. The There is evidence that an increase in the fraction of GAG in
latter include the collagens, elastin and several proteoglycans the copolymer increases the resistance of the macromolecu-
as well as cell adhesion molecules such as fibronectin and lar network to degradation by mammalian collagenases3
laminin. Since development and induced regeneration have Such resistance also increases with the density of collagen-
a common end point we will assume quite simply, as a first collagen crosslinks and collagen-GAG crosslinks.29,35 A re-
approximation, that the required cell-matrix binding events view of the effect of each of these structural features of the
in each case are similar; if so, the identity of matrix compo- dermal regeneration template on its activity in the inhibi-
nents in each case must also be similar. This presumptive tion of the onset of wound contraction can be made based
similarity between developmental and regenerative mecha- on the published evidence.9,33 This review suggests that the
nisms has been previously referred to briefly in terms of the chemical composition and the detailed pore structure of the
hypothetical rule: Regeneration recapitulates ontogeny;8 dermal regeneration template contribute about equally to
however, we caution to the lack of detailed evidence for such its activity. A similar study of the relation between structure
an identity. In the dermis, as well as in the connective tissue and activity for the nerve regeneration template has not been
of peripheral nerves, type I collagen is present in greatest made.
abundance, whereas the most prominent glycosaminogly-
cans in the dermis are dermatan sulfate and chondroitin Conclusions
6-sulfate; in peripheral nerves, type I collagen and sulfated There is strong evidence that the adult mammal is
proteoglycans have also been prominently observed.32 capable of almost complete organ regeneration. This sur-
The early exudate of a spontaneously healing skin prising result has been documented with several animal
wound is free of ECM components; although quite richly models and in repeated clinical trials. It is clear that both
endowed with undifferentiated cells and growth factors, it the dermis and the epidermis, as well as the peripheral nerve
is, nevertheless, lacking in components which are known to and the knee meniscus, can be induced to regenerate in le-
be required for development.23-25 As pointed out above, this sions where spontaneous regeneration is well known to be
lack of ECM components is hypothetically associated with negligible. In these instances regeneration has been induced
the absence of synthetic processes which lead to a physio- by analogs of the ECM. A model of the mechanism of re-
logical organ. generation provides an explanation for the observed struc-
This article has been based on the author’s article tural features of the ECM analogs (termed regeneration tem-
“Models of Organ Regeneration Processes Induced by Tem- plates) which have shown this remarkable behavior.
plates”, in Bioartificial Organs, Prokop A, Hunkels D,
Cherrington AD, eds. Ann Ny Acad Sci 1997; 831:280-293. References
The reasoning above is consistent with the choice of 1. Billingham RE, Medawar PB. Contracture and intussus-
type I collagen and at least one of the proteoglycans or gly- ceptive growth in the healing of extensive wounds in
cosaminoglycans (GAGs) as basic structural components of mammalian skin. J Anat 1955; 89:114-123.
2. Peacock Jr EE, Van Winkle Jr W. Wound Repair, Second
regeneration templates. Several efforts have been made to
Edition. Philadelphia: W.B.Saunders 1976.
replace the use of ECM analogs in templates with synthetic 3. Yannas IV, Burke JF, Huang C, Gordon PL. Suppression
polymers; at this time, however, there is no firm evidence of in vivo degradability and of immunogencity by reac-
that synthetic polymers can induce regeneration of the der- tion with glycoaminoglycans. Polymer Repr Am Chem Soc
mis or of a peripheral nerve in lesions where the physiologi- 1975; 16:209-214.
cal structures are not regenerated spontaneously. 4. Yannas IV, Burke JF, Gordon PL, Huang C. Multilayer
Considerable experimental evidence links the biologi- membrane useful as synthetic skin. 1977; US Patent
cal activity of the dermal regeneration template to its de- 4,060,081.
tailed structural features. Two ECM analogs, one of which 5. Yannas IV, Burke JF, Umbreit M, Stasikelis P. Progress
was prepared with a GAG while the other was prepared with in design of an artificial skin. Fed Proc 1979; 38:988.
the corresponding proteoglycan, showed the same activity 6. Yannas IV, Burke JF, Warpehoski M, Stasikelis P, Skrabut
EM, Orgill D, Giard DJ. Prompt, long-term functional
in an in vivo assay (inhibition of onset of wound contraction)
replacement of skin. Trans Am Soc Artif Intern Organs
which predicts dermal regeneration.33 This result suggested 1981; 27:19-22.
that the dermal regeneration template can be constructed 7. Yannas IV, Burke JF, Orgill DP, Skrabut EM. Wound tis-
using a GAG, rather than the corresponding proteoglycan, sue can utilize a polymeric template to synthesize a func-
without loss of activity. A covalently crosslinked network of tional extension of skin. Science 1982; 215:174-176.
collagen and the sulfated GAG appears to be necessary; this 8. Yannas IV, Orgill DP, Skrabut EM, Burke JF. Skin regen-
is inferred by the observation that whereas these two mac- eration with a bioreplaceable polymeric template. In:
romolecules form an ionic complex spontaneously at acidic Gebelein CG, ed. American Chemical Symposium Series,
pH, the complex is dissociated at neutral pH, i.e., under con- Number 256 Washington, D.C. 1984:191-197.
ditions which prevail following implantation.34 To preserve 9. Yannas IV, Lee E, Orgill DP, Skrabut EM, Murphy GF.
Synthesis and characterization of a model extracellular
the chemical composition of the ECM analog in vivo over
matrix that induces partial regeneration of adult mam-
the period suggested by the residence time considerations malian skin. Proc Natl Acad Sci USA 1989; 86:933-937.
discussed above, it is necessary to introduce a certain den-
576 Tissue Engineering of Prosthetic Vascular Grafts
10. Murphy GF, Orgill DP, Yannas IV. Partial dermal regen- 22. Stone KR, Webber RJ, Rodkey WG, Steadman JR. Pros-
eration is induced by biodegradable collagen-glycosami- thetic meniscal replacement: In vitro studies of meniscal
noglycan grafts. Lab Invest 1990; 63:305-313. regeneration using copolymeric collagen prostheses.
11. Burke JF, Yannas IV, Quinby Jr WC, Bondoc CC, Jung Arthroscopy 1989; 5:152.
WK. Successful use of a physiologically acceptable artifi- 23. McPherson JM, Piez KA. In: Clark RAF, Henson PM, eds.
cial skin in the treatment of extensive skin injury. Ann The Molecular and Cellular Biology of Wound Repair.
Surg 1981; 194:413-428. New York: Plenum, 1988:471-496.
12. Heimbach D, Luterman A, Burke J, Cram A, Herndon D, 24. Hay ED, ed. Cell biology of extracellular matrix. New
Hunt J, Jordan M, McManus W, Solem L, Warden G, York: Plenum Press, 1981.
Zawacki B. A multi-center randomized clinical trial. Ar- 25. Loomis WF. Developmental biology. New York:
tificial dermis for major burns. Ann Surg 1988; Macmillan, 1986.
208:313-320. 26. Yannas IV. Models of organ regeneration processes in-
13. Stern R, McPherson M, Longaker MT. Histologic study duced by templates. In: Prokop A, Hunkeler D,
of artificial skin used in the treatment of full-thickness Cherrington AD, eds. Ann NY Acad Sci 1997; 831:
thermal injury. J Burn Care Rehabil 1990; 11:7-13. 280-293.
14. Orgill DP, Butler CE, Regan JF. Behavior of collagen-GAG 27. Yannas IV. Regeneration templates. In: Bronzino JD, ed.
matrices as dermal replacement in rodent and porcine The Biomedical Engineering Handbook. Boca Raton: CRC
models. Wounds 1996; 8:151-157. Press, 1995:1619-1635.
15. Yannas IV, Orgill DP, Silver J, Norregaard TV, Zervas NT, 28. Yannas IV. In vivo synthesis of tissues and organs. In:
Schoene WC. Regeneration of sciatic nerve across 15-mm Lanza RP, Langer RS, Chick WL, eds. Textbook of Tissue
gap by use of a polymeric template. In: Gebelein CG, ed. Engineering. New York: R.G. Landes/Academic Press,
Advances in Biomedical Polymers. New York: Plenum 1996:169-178.
Press,1987:1-9. 29. Yannas IV. Use of artificial skin in wound management.
16. Chang AS, Yannas IV, Perutz S, Loree H, Sethi RR, Krarup In: Dineen P, ed. The Surgical Wound. Philadelphia: Lea
C, Norregaard TV, Zervas NT, Silver J. Electrophysiologi- and Febiger, 1981:171-190.
cal study of recovery of peripheral nerves regenerated by 30. Yannas IV, Burke JF, Huang C, Gordon PL. Correlation
a collagen-glycosaminoglycan copolymer matrix. In: of in vivo collagen degradation rate with in vitro mea-
Gebelein CG, ed. Progress in Biomedical Polymers. New surements. J Biomed Mat Res 1975; 9:623-628.
York: Plenum Press, 1990:107-120. 31. Yannas IV, Burke JF. Design of an artificial skin.Part I.
17. Chang AS, Yannas IV. Peripheral nerve regeneration. In: Design principles. J Biomed Mat Res 1980; 14:65-68.
Smith B, Adelman G, eds. Neuroscience Year (Supplement 32. Rutka JT, Apodaca G, Stern R, Rosenblum M. The extra-
2 to the Encyclopedia of Neuroscience). Boston: cellular matrix of the central and peripheral nervous sys-
Birkhauser, 1992:125-126. tems: Structure and function J Neurosurg 1988;
18. Landstrom A, Yannas IV. Peripheral nerve regeneration. 69:155-170.
In: Smith B, Adelman G, eds. Encyclopedia of Neuro- 33. Shafritz TA, Rosenberg LC, Yannas IV. Specific effects of
science. Boston: Birkhaüser, 1996: In press. glycosaminoglycans in an analog of extracellular matrix
19. Lundborg G, Dahlin LB, Danielson N, Gelberman RH, that delays wound contraction and induces regeneration.
Longo FM, Powell HC, Varon S. Nerve regeneration in Wound Rep Reg 1994; 2:270-276.
silicone chambers: Influence of gap length and of distal 34. Yannas IV, Burke JF, Gordon PL, Huang C, Rubenstein
stump components. Exp Neurol 1982; 76:361-375. RH. Design of an artificial skin. Part II. Control of chemi-
20. Lundborg G. Nerve regeneration and repair: A review. cal composition. J Biomed Mat Res 1980; 14:107-131.
Acta Orthop Scand 1987; 58:145-169. 35. Yannas IV. Regeneration of skin and nerves by use of
21. Stone KR, Rodkey WG, Webber RJ, McKineey L, collagen templates. In: Nimni M, ed. Collagen: Biotech-
Steadman JR. Collagen-based prostheses for meniscal re- nology, Vol. III. Boca Raton: CRC Press, 1988:87-115.
generation. Clin Orthop 1990; 252:129-135.
Matrix Engineering
CHAPTER 53
Artificial Extracellular Matrix Proteins
for Graft Design
Alyssa Panitch, David A. Tirrell
Introduction
ore than 500,000 vascular grafts are implanted annually in the United States.1 Although
M large diameter grafts implanted in regions of high blood flow remain patent for many
years, small and medium caliber prostheses are plagued by unacceptable rates of failure due
to thrombosis and intimal hyperplasia.2
Despite their high failure rates, two materials—expanded polytetrafluoroethylene
(ePTFE) and poly(ethylene terephthalate) (Dacron)—have remained dominant in the tech-
nology of synthetic vascular grafts for more than thirty years. In attempts to reduce leakage,
platelet adhesion, thrombosis and intimal hyperplasia, researchers have modified the sur-
faces of ePTFE and Dacron in many ways, including impregnation with albumin,3 gelatin4
or collagen,5 preclotting with whole blood,6 pretreatment with fibrin glue with or without
growth factors or heparin,7 and preseeding with endothelial cells, either in the operating
room8 or in a preliminary procedure that allows preestablishment of a confluent endothe-
lial cell monolayer.9 Recent reports on clinical trials using endothelialized ePTFE grafts have
been particularly encouraging.10
Nevertheless, the factors leading to the development of thromboresistant,
endothelialized grafts free of intimal hyperplasia remain poorly understood. It is clear that a
confluent layer of endothelial cells is not sufficient; although the endothelial lining of the
healthy artery resists thrombus formation, a procoagulant state can be induced by mechani-
cal trauma, by inflammatory mediators such as interleukin-1,11 by bacterial endotoxin,12
and by other stimuli. Furthermore, endothelial cells are capable of releasing platelet-derived
growth factor-like proteins13-15 and other smooth muscle cell mitogens, and elevated mi-
totic activity in smooth muscle cells located beneath an apparently confluent endothelium
has been reported by Clowes and co-workers in studies of ePTFE grafts implanted in ba-
boons.16 Thus, even confluent endothelial layers can play an active role in graft failure via
thrombosis or intimal hyperplasia.
Our laboratory has adopted a new approach to the problem of preparing vascular
grafts of improved long term patency, an approach based on the synthesis of new artificial
extracellular matrix (ECM) proteins suitable as substrates for the culture of endothelial cells.
The proteins under current study are based on repetitive oligopeptide sequences analogous
to those of mammalian elastin,17 and incorporate ECM-derived cell binding domains, in-
cluding the CS5 region of fibronectin,18 which contains the REDV sequence previously shown
to support attachment and spreading of endothelial cells, but not vascular smooth muscle
cells.19 The potential endothelial cell selectivity of such polymers makes them attractive
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
578 Tissue Engineering of Prosthetic Vascular Grafts
candidates for use in vascular grafts, and it is the prospect of faces on which HUVECs attached and spread, while human
using such materials to culture thromoresistant endothelial foreskin fibroblasts, human vascular smooth muscle cells,
tissue that has attracted our attention. and human blood platelets did not. Subsequent investiga-
The choice of elastin-like sequences to constitute the tions demonstrated that endothelial cell attachment to
structural elements of the artificial ECM proteins was based REDV-grafted substrates is mediated by integrin #4!1,37 and
on several factors. First, elastin is abundant in the walls of that attachment can be inhibited specifically by soluble
small muscular arteries,20 and thus is a critical element in REDV peptides. Endothelial cells also recognize the RGD
the organization and function of the healthy artery. Second, sequence from the central cell-binding domain of fibronectin
Urry and co-workers have demonstrated a remarkably wide (via integrins #5!1 and #v!1), and fibronectin carries at least
range of mechanical properties in repetitive elastin-like poly- two additional recognition sites for #4!1, designated CS1 and
mers, reporting elastic moduli from 104 to more than 108 H1, respectively.34 The integrin binding affinities of CS5 and
dynes/cm2, depending on sequence and water content.17 H1 are comparable, while that of CS1 is approximately
Because the moduli of the arterial wall are of the same or- 20-fold higher.34
der,21 elastin-like polymers offer the prospect of reduced Thus it appears likely that endothelial cell function
compliance mismatch between artery and graft, a recurrent can be regulated by presentation of ECM-derived recogni-
cause of endothelial damage, peri-anastomotic smooth tion sequences, with control exercised by variation in the
muscle cell proliferation, and graft failure.22-25 Elastin-like density and affinity of recognition sites and in the presenta-
polymers are readily processed from aqueous solutions, and tion of multiple sites in combination. The design of artifi-
can be converted in simple operations into films, tubes, and cial ECM proteins allows this approach to be pursued in sys-
surface coatings convenient for evaluation of materials prop- tematic fashion, in that specific recognition sequences, one
erties.26 Finally, the polypentapeptide of repeating unit se- by one or in combination, can be built into structural do-
quence -GVGVP- has been subjected to extensive mains denuded of their intrinsic binding sites. We have be-
biomaterials testing, including determination of mutagenic- gun to test this idea, beginning with elastin-like structural
ity (Ames test), toxicity (mice and rabbits), antigenicity domains, and introducing initially CS5, and subsequently
(Guinea pigs), pyrogenicity (rabbits), and thrombogenicity CS1, as ligands for the #4!1 integrin receptor on the endot-
(dogs), and has performed well in each of these tests.27 helial cell surface.
Poly(GVGVP) is nonadhesive toward human umbilical vein
endothelial cells (HUVECs),28 suggesting that cell attach- Artificial Proteins
ment to such polymers ought to be mediated by specific in- Since 1990, the primary focus of our laboratory has
teractions with recognition sequences built into the chain. been the design and bacterial synthesis of artificial proteins
Urry and co-workers have reported adhesion of HUVECs that exhibit novel and potentially useful material proper-
to variants of poly(GVGVP) that contain periodic GRGDS ties. Our initial efforts addressed strategies for controlling
inserts.28 The same group reported that incorporation of chain folding and supramolecular organization in solid poly-
REDV oligopeptides into poly(GVGVP) did not yield sub- mers, and led to a family of repetitive proteins that adopt
strates capable of supporting adhesion of HUVECs. As chain-folded lamellar architectures of controlled dimensions
shown in the following section, however, insertion of the and surface functionality.38-41 A second set of experiments
longer (20 amino acid) CS5 region does yield adhesive sub- has been directed toward the engineering of novel liquid
strates. Perhaps the short REDV inserts in poly(GVGVP) crystal (LC) phases in solutions of rod-like artificial pro-
are unable to adopt the conformation(s) required for cell teins,42 and has yielded unique smectic LC structures with
surface recognition and attachment. layer spacings subject to precise control on length scales of
The cell-binding domain of primary interest in our tens of nanometers.43 An important, continuing objective
work to date has been the CS5 region of human fibronectin. of this work has been the development of methods for in-
Comprising residues 90-109 of the type III connecting seg- corporating nonnatural amino acids into artificial proteins
ment (IIICS) of fibronectin, CS5 includes the minimal ac- in vivo, and good success has been achieved for selenated,44
tive sequence REDV,18 which is recognized by the integrin fluorinated,45 electroactive,46 olefinic,47 acetylenic and con-
#4!1.29-33 Binding to IIICS appears to be considerably more formationally constrained48 amino acid analogs.
selective than is recognition of the central cell-binding do-
main of fibronectin,34 which carries the cell recognition se- Artificial ECM Proteins
quence RGDS.35,36 In our preliminary experiments, two artificial ECM
Hubbell and co-workers have exploited the selectivity proteins (1a and 1b, see Fig. 53.1) have been prepared and
of the REDV sequence to prepare surfaces that show prefer- subjected to evaluation as substrates for the culture of hu-
ential affinity for endothelial cells.19 Immobilization of the man umbilical vein endothelial cells (HUVECs).
hexapeptide GREDVY on glycophase glass produced sur-
Fig. 53.1. Structure of artificial extracellular matrix
proteins 1a and 1b, 2a and 2b. —[cbd(GVPGI)x]—y 1:cbd=CS5 a: x=40, y=3
2:cbd=CS1 b: x=20, y= 5
Artificial Extracellular Matrix Proteins for Graft Design 579
Synthesis washes; in fact, little or no polymer is removed from the
Proteins 1a and 1b have been expressed in E. coli strain surface after two wash steps. In order to ensure the stability
BL31(DE3)pLysS under control of a bacteriophage T7 pro- of the coating, all samples are washed for 2 hours at 37°C in
moter.49 For batch cultures, 60 mg/L (1a) or 40 mg/L (1b) sterile H2O prior to cell culture experiments, and uniformity
of recombinant protein can be recovered through a simple of coverage is verified by scanning electron microscopy.
procedure involving selective precipitation of contaminat-
ing proteins below the lower critical solution temperature Cell Culture
of the target polymer (vide infra). Human umbilical vein endothelial cells (HUVECs)
have been used for all cell adhesion studies. Harvested cells
Lower Critical Solution Temperatures were resuspended in serum-free M199 medium and diluted
Urry and co-workers have reported extensive studies to a density of 1 x 104 cells/ml (for cell counting) or 5 x104
of the lower critical solution temperatures (or “inverse tem- cells/ml (for determination of spreading behavior). HUVECs
perature transitions”) of elastin-like polypeptides.17 The (1 ml aliquots of the suspensions listed above) were trans-
lower critical solution temperature (LCST) is the tempera- ferred to 24 well dishes containing protein-coated glass cover
ture below which the polymer forms a single-phase solu- slips. Cells were maintained at 37°C and 5% CO2 for 4 h.
tion (e.g., in water); above the LCST, the solution separates Table 53.1 reports the number of cells adherent to each
into polymer-rich and polymer-depleted phases. of the two artificial extracellular matrix proteins 1a and 1b.
Polypentapeptides of repeating unit sequence -VPGVG- Glass and fibronectin (coated on glass) served as controls.
exhibit an LCST at approximately 25°C,50 and substitution Essentially all cells cultured on fibronectin and on 1b were
with increasingly hydrophobic residues shifts the transition adherent, as compared to ~25% of cells plated on glass and
to lower temperatures.50 The choice of -GVPGI- as the elas- on 1a. We attribute the decrease in the number of cells bound
tin-like repeat was based on its predicted LCST of 10°C,50 to 1a as compared to 1b to the lower CS5 density in the
which ensures reduced water solubility (and hence increased former, and perhaps to differences in protein conformation
stability of surface coatings) at physiological temperatures. upon adsorption to the glass surface. Table 53.2 summarizes
The LCSTs of 1a and 1b were determined in doubly the morphologies of cells adherent to these surfaces. Refrac-
distilled H2O at four concentrations ranging from 10 mg/ml tile cells (round and bright under the phase contrast micro-
to 40 mg/ml. The LCST is signaled by an abrupt increase in scope), cells which were round but not highly refractile
the turbidity of the solution, which was measured at a wave- (round and gray), spreading cells (1-2 pseudopodia), and
length of 300 nm. spread cells (more than 3 pseudopodia) were counted.
The results are consistent with the predictions of Urry Although these are preliminary data, the results in
et al,50 in that 1a and 1b both exhibit LCSTs near 10°C Tables 53.1 and 53.2 are consistent with the hypothesis that
(13.4°C for 1a; 12.1°C for 1b). Insertion of the CS5 domain artificial ECM proteins with recognition sites for integrin
causes some perturbation of the LCST, but the objective of #4!1 will support the attachment and spreading of endothe-
depressing the transition to below room temperature has lial cells. Both the number of adherent cells, and the per-
been met for both of these materials. Nevertheless, a centage of spread cells, are increased on 1b as compared to
subambient LCST does not mean a complete lack of water glass. Increasing the density of CS5 sites leads to an increase
solubility, so it is essential to determine the stability of sur- in the number of attached cells; 1b (like fibronectin) sup-
face coatings fabricated from 1a and 1b (and other candi- ports attachment of essentially all cells plated at a density of
date proteins). 104 cells per well. On the other hand, cell spreading is more
complete (or perhaps more rapid) on fibronectin than on
Preparation and Stability of Surface Coatings 1b; essentially all cells have spread on fibronectin after 4 h
Glass cover slips (12 mm diameter) were coated with in serum-free media. Addition of serum-supplemented
1a and 1b from solutions in formamide or water by spread- media to HUVECs cultured on 1b leads to confluent EC
ing 40 ∝L of a 1 mg/ml protein solution over the surface of
the cover slip with the tip of a pipet. Fibronectin was coated
similarly, by using 20 ∝L of a 100 ∝g/ml solution in H2O.
The cover slips were dried in vacuo at 55°C, incubated for
2 h at 37°C in sterile H2O (0.5 ml) to remove loosely bound Table 53.1. Adherent HUVECs after 4 h in serum free
protein, and dried again at 55°C for 2 h. medium
For determination of coating stability, samples of 1a
and 1b were labeled in vivo with [3H]glycine. Labeled Protein/Cast From Adherent Cells per Well
samples were coated on glass cover slips as described above
(without the final wash) and the coatings were subjected to Glass 2,880 ± 1670a
six successive washes with phosphate-buffered saline (PBS, 1a/Formamide 1,150 ± 360
1a/Water 2,590 ± 570
0.5 ml aliquots, 30 min incubation per cycle). Each wash
1b/Formamide 9,170 ± 1800
was analyzed by scintillation counting to determine the 1b/Water 11,700 ± 760
amount of protein removed, and the cover slips were counted Fibronectin 10,140 ± 290
directly after the sixth wash. Such experiments show that a10,000 cells plated per well; adherent cells given as mean ±
substantial amounts of 1a and 1b are removed by successive standard deviation (n=4).
washes. Nevertheless, a stable coating remains even after six
580 Tissue Engineering of Prosthetic Vascular Grafts
Table 53.2. HUVEC morphology on glass and protein surfaces
Protein/Cast From Refractile % Round/Grey % Spreading % Spread %
Glass 49.5 ± 8.6a 12.0 ± 0.4 13.4 ± 2.3 25.1 ± 7.8
1a/Formamide 14.1 ± 1.2 15.9 ± 5.9 21.3 ± 2.5 48.7 ± 2.2
1a/Water 18.7 ± 7.2 16.3 ± 5.5 7.9 ± 4.0 53.7 ± 6.6
1b/Formamide 15.2 ± 5.9 14.6 ± 3.4 14.4 ± 5.0 55.8 ± 3.9
1b/Water 20.9 ± 5.3 11.4 ± 4.9 11.6 ± 1.4 55.8 ± 9.3
aMean ± SD for 4 determinations. Each determination based on scoring of 100-150 cells.
monolayers, with typical cobblestone appearance, within 5. Freischlag JA, Moore WS. Clinical experience with a col-
48 h. lagen-impregnated knitted dacron vascular graft. Ann Vasc
Preliminary measurements of competitive inhibition Surg 1990; 4:449-454.
of EC attachment by soluble CS5 analogs have also been 6. Yates SG, Barros-D’Sa ABB, Berger K et al. The preclotting
of porous arterial prostheses. Ann Surg 1978; 188:611-622.
made. Preincubation of HUVECs with the soluble peptide
7. Gray JL, Kang SS, Zenni GC et al. FGF-1 affixation stimu-
GREDVDY (Research Genetics, Inc.) at concentrations up lates ePTFE endothelialization without intimal hyperpla-
to 2 mg/ml has no observable effect on attachment to sia. J Surg Res 1994; 57:596-612.
fibronectin, as expected. In contrast, attachment to 1b is 8. Williams SK. Endothelial cell transplantation. Cell Trans-
completely inhibited by GREDVDY at concentrations of plantation 1995; 4:401-410.
0.6-0.8 mg/ml, consistent with specific attachment of 9. Zilla P, Deutsch M, Meinhart J et al. Clinical in vitro
HUVECs to CS5 via the #4!1 receptor. endothelialization of femoropopliteal bypass graft: An ac-
tuarial follow-up over three years. J Vasc Surg 1994;
Current and Future Directions 19:540-548.
Our overall objective is the development of a new class 10. Zilla P, Deutsch M, Meinhart M, Fischlein T, Hofmann
of artificial ECM proteins to be used in small diameter vas- G. Long-term effects of clinical in vitro endothelialization
on grafts. J Vasc Surg 1997; 25:1110-1112.
cular grafts of improved long term patency. We believe the
11. Bevilacqua MP, Pober JS, Majeau GR et al. Recombinant
proposed approach to be novel in that genetic engineering, tumor necrosis factor induces procoagulant activity in
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be combined to provide control of both the mechanical and comparison with the actions of interleukin I. Proc
properties and the cell-surface interactions of the new pro- Nat Acad Sci USA 1986; 83:4533.
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of the overall chain length, and introduction of a controlled toxin. J Clin Investi 1983; 71:1893.
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surfaces, and through control of the identity and frequency
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University Press, 1992. IIICS region of plasma fibronectin is mediated by integrin
22. Greisler HP, Joyce KA, Kim DU et al. Spatial and tempo- #4!1. J Biol Chem 1992; 267(20):14019-14026.
ral changes in compliance following implantation of 38. Krejchi MT, Atkins EDT, Waddon AJ et al. Chemical se-
bioresorbable vascular grafts. J Biomed Mat Res 1992; quence control of !-sheet assembly in macromolecular
26:1449-1461. crystals of periodic polypeptides. Science 1994;
23. Kinley CE, Marble AE. Compliance: A continuing prob- 265:1427-1432.
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24. Zwolak RM, Adams MC, Clowes AW. Kinetics of vein the repetitive polypeptide {(AlaGly) 3GluGly(GlyAla) 3
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Surg 1987; 5:126-136. riched analogue. Macromolecules 1993; 26:6691-6693.
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York:Marcel Dekker, 1995. pression of a monodisperse copolypeptide containing
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Matrix Engineering
CHAPTER 54
Cell-Extracellular Matrix Interactions Relevant
to Vascular Tissue Engineering
Stephen P. Massia
Introduction
T he interaction between cells and extracellular surfaces plays a major role in determining
cellular behavior in tissues and on biomaterials. These interactions modulate many aspects
of cell behavior, including adhesion, spreading, migration, proliferation, differentiation, and
metabolism. In the fields of biomaterials and tissue engineering, a fundamental understanding
of cell surface interactions is critical for developing new methods that precisely control cell
interactions at the cell-biomaterial interface. Enhancement of tissue cell adhesion to im-
planted biomaterials would facilitate tissue adhesion/integration at the tissue-biomaterial
interface. In contrast, the reduction of cell adhesion to implanted biomaterials is desirable
for limiting cell-mediated events such as thrombosis and macrophage activation on bioma-
terial surfaces, which often result in biomaterial implant failure. It is also desirable for tissue
engineered implants to promote specific cell-biomaterial interactions which facilitate tissue
regeneration/repair. Tissue engineered devices in the form of prefabricated biohybrid tissue
constructs require the appropriate interactions of transplanted cells with biomaterial scaf-
folds to initiate extracellular matrix (ECM) synthesis and deposition as well as other activi-
ties which promote biohybrid tissue formation in vitro prior to implantation in vivo.
Two major approaches for developing materials which elicit precisely controlled cel-
lular interactions have emerged. One involves fabricating implants, scaffolds, etc. from natural
biomaterials such as collagen where the base material has intrinsic and specific biological
activities which influence cellular interactions. The major disadvantages of using natural
biomaterials is that they generally have a largely unmodifiable and limited range of physico-
chemical properties and can be difficult to isolate and characterize. The second approach
for improving the control of cell-material interactions is to use synthetic materials which
have surface immobilized, biologically active molecules and biologically active surface
microtextrue. This methodology takes advantage of the inexpensive manufacturing costs
for synthetic material-based devices and exploits specific biological activities to modulate
cellular interactions for improved biocompatibility and biohybrid tissue formation. Essen-
tially, these types of surface modifications on synthetic implant materials are attempting to
create a biomimetic surface with surface physicochemical properties that more closely match
those of the ECM in the tissues where the synthetic material will be implanted. This review
will focus on the biology of cell-ECM interactions and how they are utilized in developing
biomimetic surfaces on synthetic biomaterials. The role of these technologies in developing
vascular implants with improved biocompatibility will also be discussed.
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
584 Tissue Engineering of Prosthetic Vascular Grafts
Cell-Extracellular Surface Interactions 3. Cytoskeletal reorganization and a progressive flat-
at the Molecular Level tening or spreading of the cell on the material sur-
face to increase its attachment strength.
Cell Adhesion to Solid Substrates It is well established that cell adhesion and spreading
Mammalian cells in tissues are anchorage dependent, on biomaterials in vitro and in vivo occurs via adsorbed cell
having a fundamental requirement to attach to solid com- adhesion-promoting extracellular matrix (ECM) pro-
ponents in the extracellular matrix to remain viable and teins.5,7-9 Cell culture and implanted materials quickly ad-
grow.1-4 Consequently, cells which are disrupted from tis- sorb proteins, including ECM proteins, from serum, cellu-
sues and cultured in vitro, or cells within tissues or body lar microexudates (cell culture), and tissue fluids (implants).
fluids in vivo which contact implanted materials, readily at- If sufficient ECM cell adhesive proteins adsorb to a material
tach to the available solid substrate (e.g., tissue culture plas- surface in a conformational state that is biologically active
tics, biomaterials) in order to survive.1-6 Following the ad- in promoting cell adhesion, cell interaction and spreading
sorption of serum- or cell-borne proteins, cell adhesion on will occur on that surface. ECM proteins that are important
a solid substrate progresses through the following paradigm for cell adhesion to biomaterials include fibronectin,
(Fig. 54.1):5 vitronectin, and fibrinogen from cell culture serum and
1. Initial contact of the cell with the material; tissue fluids.
2. Formation of adhesive bonds between cell surface
receptors and ligands within adsorbed adhesion pro- Cell-ECM Adhesion Receptors
teins; and Most cell-adhesive interactions with ECM proteins
occur via the family of cell surface receptors known as the
Fig. 54.1. Progression of anchorage-
dependent mammalian cell adhe-
sion. (A) Initial contact of cell with
solid substrate. (B) Formation of
bonds between cell surface receptors
and cell adhesion ligands. (C)
Cytoskeletal reorganization with
progressive spreading of the cell on
the substrate for increased attach-
ment strength.
Cell-Extracellular Matrix Interactions Relevant to Vascular Tissue Engineering 585
integrins. Integrins are a superfamily of transmembrane gly- tein signaling complexes can form within adhesion plaques
coprotein adhesion receptors consisting of a noncovalently and are associated with signal transduction activities.
linked # and ! subunit.10-13 At least 17 # and 8 ! subunits Models of the structural architecture of cytoskeletal
have been described, with resulting combinations of over complexes, which form on the cytoplasmic side of focal ad-
21 functional integrins with varying ligand specificity.13 hesions, have been developed based on in vitro protein bind-
Integrins bind to ECM proteins via cell binding domains, ing assays (Fig. 54.2). These models show how proteins may
and rather small polypeptide sequences within these do- link with each other within cytoskeletal complexes. These
mains promote integrin binding with an affinity that is nearly structural arrangements are considered models because
as high as in the intact molecule.10-13 The tripeptide RGD is many of the protein-protein linkages have not been dem-
a ubiquitous recognition sequence that binds to most onstrated in living cells.20 Talin is a major protein of
integrins and is found within the cell binding domain of cytoskeletal complexes and was the first reported integrin-
many cell adhesion ECM proteins.10-13 Binding of integrins binding cytoplasmic protein.21 Talin also binds to actin22 and
to RGD and other related recognition sequences results in a to vinculin.23 Vinculin apparently serves as a structural hub
cascade of events that promotes cell-matrix adhesion inter- for cytoskeletal complexes, since it can bind to many other
actions that are stable in stationary cells, e.g., epithelial cells cytoskeletal complex proteins. Studies have shown that
on basement membranes, and are transient in motile, mi- vinculin can bind to #-actinin,24 paxillin,25 tensin,26 and
gratory cells. Although this review will focus on integrin- possibly actin via a cryptic binding site.27 #-actinin, which
ECM interactions, there are many nonintegrin cell-ECM crosslinks actin microfilaments to form microfilament
binding receptors including cell surface proteoglycans bundles, has been shown to directly bind to the cytoplasmic
(syndecans),14 hyaluronan receptors (CD44, RHAMM and tails of integrin ! subunits.28 With at least two integrin-bind-
ICAM-1,15 67 kDa laminin-binding proteins,16 annexin II,17 ing cytoplasmic proteins (talin and #-actinin) and other
and receptor type protein tyrosine phosphatase !.18 various interconnecting proteins, e.g., vinculin, an indirect
linkage between ECM-bound integrins and the cytoskeleton
Integrin-Mediated Cytoplasmic Activities is formed via cytoskeletal complexes.
Following ligand-binding, integrins undergo confor- Unlike many other types of transmembrane cell sur-
mational changes, cluster into aggregates, and form adhe- face receptors, the cytoplasmic domains of integrins do not
sion plaques or focal contacts. It is well documented in many have enzymatic activity. Therefore, integrin-mediated intra-
studies that integrin clustering is required for initiating in- cellular signal transduction depends on recruited, enzymati-
tracellular activities.13,19 Integrin-mediated cytoplasmic cally active signal transduction molecules. Integrin cluster-
functions within adhesion plaques can be categorized into ing has been shown to induce phosphorylation of tyrosine
two experimentally separable types: residues of a number of proteins, even though integrins do
1. Cytoskeletal organization and establishment of me- not intrinsically have tyrosine kinase activity.29 One protein
chanical linkages between ligand-bound integrins that is rapidly phosphorylated and localized to focal adhe-
and cytoskeletal structures; and sions is focal adhesion kinase (FAK or pp125FAK).30,31 The
2. Tyrosine kinase-mediated signal transduction. exact mechanism for clustered integrin-mediated FAK phos-
Associated with integrin-cytoskeleton linkages are phorylation is not known. Studies demonstrating direct
cytoskeletal complexes that consist of clustered integrins, binding of FAK to the !1 subunit cytoplasmic domain32 sug-
several cytoplasmic proteins, and actin microfilament gest that changes in integrin aggregation and/or conforma-
bundles. Structurally and biochemically distinct multi-pro- tion alters FAK to promote autophosphorylation, which
upregulates the enzymatic activity of FAK and triggers
Fig. 54.2. Multi-protein cytoskeletal
complexes within focal contacts—me-
chanical linkages between ligand-
bound integrins and cytoskeletal struc-
tures. Three different linkages are de-
picted here: (Left) The integrin cyto-
plasmic domain binds to an unknown
(?) protein that also binds tensin. Tensin
links to actin filaments and vinculin;
(Center) The integrin cytoplasmic do-
main binds to talin which links to ac-
tin-bound vinculin; (Right) The
integrin cytoplasmic domain binds to
#-actinin which crosslinks actin mi-
crofilaments into bundles. V = vinculin;
TEN = tensin; P = paxillin; TAL = talin;
PM = plasma membrane.
586 Tissue Engineering of Prosthetic Vascular Grafts
further downstream signal transduction phosphorylation influence a broad range of cellular activities beyond cell ad-
events.33 Activated FAK phosphorylates many focal contact hesion (Fig. 54.3).
proteins, which generally induces them to form protein-pro-
tein bonds and facilitate multi-protein complex formation.34 Biomimetic Cell-Adhesive Biomaterials
Similar to models of cytoskeletal complexes described in the
previous paragraph, models for an integrin-mediated sig- Controlling Cell Adhesion on Biomaterials
nal transduction complex have been developed from in vitro For both tissue engineers and biomaterial scientists,
studies. Following integrin-mediated autophosphorylation, the precise control of cell interactions with biomaterials is
FAK phosphorylates paxillin, which activates its SH2 and desirable. The tissue engineer requires the interaction of
SH3 (Src homology 2 and 3 binding domains. The signal specific cell types with materials in implants and devices
transducing tyrosine kinase c-Csk readily binds to the designed for tissue ingrowth and regeneration. In biohybrid
paxillin SH2 domain35 and c-Src binds to SH3.36 GRB-2, tissue constructs, the tissue engineer seeds specific cells in
which promotes MAP kinase activation, has been shown to biomaterial scaffolds in vitro and requires cell-biomaterial
bind to phosphorylated FAK and to c-Src.37 This finding interactions which promote appropriate ECM deposition
suggests that GRB-2 can associate with focal adhesion sig- and assembly for tissue formation. The biomaterial scien-
nal transduction complexes to couple MAP kinase-medi- tist requires selective attachment of tissue cells to implanted
ated signal transduction to integrins. In growth factor-me- materials so that tissue adhesion and integration is optimized
diated signal transduction pathways, MAP kinase has been and inflammatory cell-mediated encapsulation is limited at
shown to translocate to the nucleus and play a direct role in the tissue-biomaterial interface. In order to achieve high
gene regulatory pathways.38 Similar signaling mechanisms precision control of cell adhesive and functional responses
may occur in integrin signal transduction complexes. Other to biomaterials, protein adsorption has to be precisely con-
studies have shown that a G protein & subunit and protein trolled as well. Protein adsorption is an exceedingly com-
kinase c type 3 (PKC type 3 ) bind and localize to focal ad- plex phenomenon, and, correspondingly, it is very difficult
hesions, suggesting that activated integrins possibly utilize to control.44-46 The main difficulty is in controlling the speci-
G protein and PKC type 3-mediated signal transduction ficity of protein adsorption, e.g., it is difficult to selectively
pathways coordinately with or independently of tyrosine promote the adsorption of cell adhesion proteins over the
kinase pathways.39,40 Integrin binding has also been shown adsorption of nonadhesive proteins. A second difficulty with
to promote signal transduction-associated events such as the adsorbed protein layer is its lack of stability to denatur-
increased cytoplasmic levels of PIP241 and Ca2+,42 as well as ation and proteolysis, which may affect the long term per-
increased cytoplasmic pH.43 Integrin signal transduction formance of implanted biomaterials and tissue engineered
complexes potentially provide a mechanism where integrin- devices. 44-46 Therefore, a material surface that promotes cell
mediated signaling could be transmitted to the nucleus and adhesion and spreading independently of protein adsorp-
invoke specific changes in gene transcription patterns that tion would be more controllable and beneficial in designing
tissue engineered biomaterials. Essentially, this material sur-
Fig. 54.3. Signal transduction com-
plexes in focal contacts. FAK binds
to integrin cytoplasmic domains and
to paxillin. c-Csk and c-Src bind to
FAK-activated paxillin and GRB-2
binds to c-Src and FAK. Collectively
this protein complex triggers signal
transduction via MAP kinase-depen-
dent pathways. Protein kinase c type
3 can bind to integrin-bound FAK
and potentially promote specific sig-
nal transduction pathways. Similarly
bound G protein & subunits may link
integrin activation with G protein
signal transduction. V = vinculin;
TEN = tensin; P = paxillin; PKC3 =
protein kinase c type 3; G& = G pro-
tein & subunits.
Cell-Extracellular Matrix Interactions Relevant to Vascular Tissue Engineering 587
face would mimic physicochemical features of tissue ECM Surface Immobilized Biologically
adequately enough to intrinsically promote cell interactions, Active Molecules on Biomaterials
circumventing the requirement for adsorbed ECM proteins. Many methodologies for designing biomimetic, ad-
hesion-promoting materials have been developed to circum-
Mimicking the Extracellular Matrix vent the requirement for protein adsorption by covalently
on Biomaterial Surfaces supplying the substrates with stable, synthetic ligands for
The extracellular matrix (ECM) of soft tissues is a cell surface adhesion.47-82 These adhesion ligand peptide-
complex composite material consisting of dense fibrillar grafted substrates have been utilized as simplified models to
cable-like structures (e.g., type I collagen fibers) for rigid investigate molecular aspects of cell-ECM interactions. One
mechanical support and a loose, more compliant gelatinous of the earliest described methods developed polyacrylamide
interstitial matrix consisting of glycoproteins, proteoglycans, substrates with surface-immobilized RGD peptides47 and
and other components which provide a protective cushion quantitatively evaluated tumor cell haptotactic responses to
against harmful external mechanical forces. The complex surface concentration gradients of immobilized RGD pep-
three dimensional arrangement of fibrillar and interstitial tides.48 Silanized glass substrates containing surface immo-
ECM components provides topographical and biochemical bilized adhesion peptides were developed and utilized to
cues for cell orientation and guidance in tissues. Therefore, quantitatively determine the minimal density and spacing
the surfaces of synthetic biomaterials could potentially of surface immobilized RGD peptides for maximal cell
mimic the ECM if biochemical and biologically relevant to- spreading, cytoskeletal development, and focal contact for-
pographical cues were incorporated into the material sur- mation.51,55 Although complete cell spreading was observed
faces (Fig. 54.4). Biochemical cues could be mimicked using at a minimal RGD surface concentration of 1 fmol/cm2, fo-
surface immobilized biologically active molecules, and cal contacts were scarce and the cytoskeletal structures were
biomimetic topographical cues could be provided by high poorly developed. When the RGD surface concentration was
precision surface microtexturing. increased to 10 fmol/cm2, focal contacts and a fully devel-
oped cytoskeleton was observed in all spread cells.55 These
Fig. 54.4. (A) ECM structure in tissues
with a fibrillar web-like network of col-
lagens and other fibrillar ECM proteins.
Interspersed are cell adhesion ECM pro-
teins (solid shapes) which bind to the
ECM fibrils and promote cell adhesion.
(B) A featureless synthetic biomaterial
implant. (C) A synthetic biomaterial
with an ECM-like biomimetic surface.
This surface contains two fundamental
ECM features which influence cell in-
teractions: immobilized cell adhesion
ligands (geometric shapes connected to
the surface) and microtextrue (bumps
on the surface).
588 Tissue Engineering of Prosthetic Vascular Grafts
results suggested that RGD is a sufficient signal for promot- immobilized biological molecule is the predominant deter-
ing a complete and maximal cell adhesive response when minant for cellular responses to these materials.
the ligand is tethered to the material surface. Consequently,
cell surface receptor interactions with tethered RGD ligands Biomaterial Surface Microtexturing to Mimic ECM
generated the contractile force necessary to produce nor- Topography in Tissues
mal focal contact and cytoskeleton formation. The value of The cellular response to attachment substrate topog-
10 fmol/cm2 required for maximal cell spreading, focal con- raphy was first described in 1912 where the migration of
tact, and cytoskeleton formation corresponded to an aver- cultured cells on spider webs was observed to be guided by
age spacing of 140 nm between RGD ligands, or 10 integrin spider web filaments.84 More studies confirmed that surface
widths. It was also observed that covalently immobilized topography influenced cell orientation on solid substrates
RGD peptide promotes cell adhesion and spreading, pre- and the term “contact guidance” was introduced to describe
dominantly via integrin #v!3.55 It was suggested in these stud- this phenomenon.85 Surface topography became an issue in
ies that covalent immobilization of RGD peptide rendered soft tissue biomaterial implant biocompatibility studies be-
it conformationally constrained with highest affinity for cause many researchers have demonstrated a positive corre-
integrin #v!3. Other studies which demonstrated preferen- lation between capsule thickness (biocompatibilty) and tis-
tial binding of cyclic, conformationally constrained RGD sue-implant interfacial motion.86 It was realized that sur-
peptides to integrin #v!3 supported this hypothesis.83 Simi- face topography could be utilized to promote tissue inter-
lar peptide-grafted glass substrates were utilized to charac- digitation or anchorage to reduce interfacial motion. An early
terize nonintegrin interactions to the laminin-based cell study evaluated the effect of surface topography on soft tis-
adhesion ligand YIGSR.63 Cell attachment, spreading, and sue responses to synthetic materials in vivo by implanting
stress fiber formation were observed on these substrates and filter membranes with different uniform pore sizes subcu-
were mediated by the 67 kDa laminin receptor independently taneously in rats and evaluating the tissue response at 6 weeks
of integrins. The 67 kDa receptor was also observed to postimplantation. Excellent tissue attachment was observed
colocalize with vinculin and #-actinin in focal adhesions.63 without signs of inflammation when pore sizes were 1-3 ∝m.
Other studies of cell interactions with peptide-grafted glass Pore sizes above and below this range exhibited symptoms
substrates compared cell migration on glass versus peptide- of a chronic inflammatory response, e.g., diminished tissue
grafted glass substrates and demonstrated that cell motility attachment and thick granulation capsules.87 It was con-
was significantly decreased on RGD peptide-grafted sub- cluded from these studies that the surface topography cre-
strates.81 These results suggested that cell attachment on ated by the 1-3 ∝m pores in membrane filters provided cel-
RGD substrates was stronger and more inhibitory to cell lular cues to promote tissue attachment and improved
migration (more resistant to disruption of cell adhesion sites) biocompatibility. Another study, using the same in vivo
than glass without surface-coupled peptides. Another study model evaluated the effect of the pore size of PTFE mem-
using similar glass substrates quantitated and compared the branes on neovascularization at the membrane-tissue in-
attachment strength of endothelial cells on RGD peptide- terface. This study found that the extent of vascularization
grafted glass versus glass with adsorbed fibronectin and ob- at the membrane-tissue interface was 80-100 times higher
served stronger cell attachment on peptide-grafted sub- in membranes with 5 ∝m pores versus membranes with
strates.82 Overall, substrates containing surface immobilized 0.02 ∝m pores.88 This study provided further evidence of how
cell adhesion peptides have been utilized extensively as sim- surface topographical feature size can influence the
plified models to isolate and study cell adhesive interactions biocompatibility of implanted materials.
at the molecular level. Many in vitro studies have investigated how surface
RGD and other cell-adhesive peptides have been sur- microtextrue influences cellular behavior to correlate with
face immobilized on a wide variety of polymeric biomaterials in vivo studies which have evaluated surface texture effects
to enhance their biocompatibility by promoting cell adhe- on soft tissue biocompatibility. Photo-etching and high pre-
sion in a more controlled manner than relying on random cision milling techniques were developed to precisely con-
protein adsorption. These methods have been developed for trol all geometrical aspects of surface topography down to
many biomedical polymers, including polyethylene tereph- the nanometer range.86 In one report, a simplified model
thalate,54,66 poly tetrafluoroethylene and related polymeric for studying surface topography effects on cell interactions
fluorocarbons,54,77,78 polyurethanes,60,69,75 polyethylene,62 was developed utilizing materials that were fabricated using
cellulose,73 hyaluronic acid,80 poly amino acids,70,76 and high precision methods. On this simple topography, it was
crosslinked dextran.74 Cell-resistant polymeric materials observed that the response of cells in vitro to a single step
which support minimal cell adhesion have been utilized for on a material surface was cell type dependent. Neuronal cell
peptide immobilization to promote highly specific cell- and fibroblast motility were observed to decrease with in-
ligand interactions with a very low background adhesive sig- creasing step height. Neutrophil migration, however, was
nal from adsorbed serum- and cell-borne proteins. These observed to not change in response to step height changes.89
materials include polyethylene glycol,72 nonhydrogel net- Since the cellular response to surface topography is cell type
works of polyacrylate/polyethylene glycol,71 polyacryla- dependent, it is possible that specific microtextured feature
mide,47,48 and polyvinyl alcohol.53 These materials provide sizes may differentially influence tissue cell and inflamma-
maximal control of cell interactions because the surface tory cell interactions with materials to maximize tissue ad-
herence and minimize the inflammatory response. Using
Cell-Extracellular Matrix Interactions Relevant to Vascular Tissue Engineering 589
high precision microtextured surfaces with regular repeated cytoskeletal elements including microtubules, actin mi-
topographical features, the effect of feature dimensions on crofilaments, and focal contacts.96-100 Surface microtexturing
fibroblast contact guidance was assessed in vitro. It was ob- has also been shown to alter cell function, including
served that repeat spacing had a minor and inversely pro- fibronectin mRNA level, mRNA stability, secretion and as-
portional effect on cell alignment, whereas groove depth had sembly by fibroblasts .101 Another study of surface
a more significant role in determining cell alignment, which microtopography and cell function demonstrated that sur-
correlated positively with increased depth.90 In a study ex- face microtextrue promoted osteogenesis.100 These studies
amining cell type dependent responses to repeating alter- further characterize the effects of surface microtextrue on
nating 1 ∝m grooves and 1 ∝m ridges, keratinocytes and neu- cell-material interactions. Overall, surface microtextrue is a
trophils were observed to exhibit no contact guidance, a 20% fundamental surface property that influences cell behavior
response was observed with monocytes and macrophages, and can be exploited to modulate cell-material interactions.
and a 100% response was observed with fibroblasts.91 Simi-
lar studies showed that epithelial cells did not exhibit strong Cell-ECM Interactions in Vascular Biology
contact guidance on microtextured surfaces92 and that they
also responded differently than fibroblasts in vivo as well.93 ECM Modulation of Smooth Muscle Cell Function
In studies comparing fibroblast (model soft tissue cell) and The two major cell types of vascular tissues are smooth
macrophage (model inflammatory cell) responses to muscle and endothelial cells. In the normal adult arterial
microtextured features in vitro, microtextured silicone discs wall, the predominant phenotype or functional state of the
were prepared with multiple surface discontinuities of wells medial smooth muscle cell (SMC) population is predomi-
and pillars with various dimensions, repeat spacings, and nantly nonproliferative and contractile.102-104 These muscle-
90° surface contour angles. These studies revealed that me- like contractile cells contract and relax coordinately in the
tabolism, spreading, and migration of fibroblasts and mac- arterial wall in response to chemical and mechanical stimuli
rophages were altered when they were cultured on substrata to control blood pressure and flow. 102-104 Following me-
with texture feature dimensions ranging from 1-3 ∝m in chanical injury to arterial wall, a large population of medial
width.94,95 Specifically, fibroblast spreading was observed to SMCs will undergo a phenotypic modulation from the con-
increase while macrophage spreading/activation decreased tractile state to a highly proliferative synthetic state in which
in response to this size range of surface feature dimensions extracellular matrix (ECM) proteins are synthesized and
when compared to untextured surfaces. Smaller or larger secreted into the media to repair injury-induced structural
features were observed to have no change in cell responses damage in the arterial wall ECM (Fig. 54.5).105-115 In addi-
when compared to untextured surfaces. When these textured tion to medial repair activity in response to injury, synthetic
silicone materials containing 2 ∝m wide square wells with medial SMCs become motile and migrate from the media
0.5 ∝m depth were implanted subcutaneously in rats, a mini- toward the arterial lumen to form a neointimal layer on the
mal inflammatory response was observed. In contrast, luminal surface of the artery wall. 105-115 Neointima
smooth silicone materials with the same chemical composi- development and progressive neointimal thickening occurs
tion as the textured materials were observed to promote a via endoluminal accumulation of proliferating SMCs with
strong inflammatory response with encapsulation. 86 coordinate ECM deposition.105-115 In the clinical setting,
Therefore, it was concluded that increased fibroblast adhe- neointimal thickening of coronary arteries in response to
sion and decreased macrophage activation in vitro corre- mechanical injury to the artery wall via balloon angioplasty
lated well with the increased tissue attachment and decreased or other percutaneous revascularization procedures is
inflammatory response observed in vivo. Similar results were problematic when the extent of thickening is enough to se-
observed with materials having a wide variety of chemical verely reduce the initial therapeutically beneficial large di-
compositions, including metals, suggesting that surface lated lumen.105-110 To date, restenosis due to extensive
microtextrue effects on cell and tissue responses were inde- neointimal thickening following balloon angioplasty or other
pendent of surface chemistry.86 Overall, these studies indi- catheter-based nonsurgical revascularization procedures
cate a range of topographical parameters that appear to mini- occurs at a rate of 30-50%. 105-110 In vascular and
mize inflammatory responses. Optimal topographical fea- cardiovascular surgery, a synthetic small diameter vascular
tures for maximal cell and tissue attachment include ridges graft that remains patent after implantation has not been
of 1-3 ∝m width with grooves 0.5 ∝m in depth, uniform dis- developed because injury-induced thrombosis and
tribution of features, and sharp feature edges with 90° sur- neointimal hyperplasia causes decreased patency and occlu-
face contour angles. sion at anastomotic sites.111-115
At optimal surface microtextrue dimensions using al- Unlike phenotypically contractile medial SMCs in the
ternating ridge and groove patterns, fibroblast interactions normal arterial wall, synthetic neointimal SMCs in the in-
have been characterized at the subcellular level. Transmis- jured arterial wall synthesize and deposit large quantities of
sion electron microscopy studies have shown that the plasma ECM proteins including collagens,116-119 fibronectin,116,118
membrane of adherent fibroblasts in contact with elastin,119,120 proteoglycans,117,119,121,122 tenascin123,124 and
microtextured surfaces frequently conforms to the textured thrombospondin.125 Tenascin and thrombospondin expres-
feature to interlock with the material surface.96 Several stud- sion in SMCs is greatly increased during postinjury
ies have shown that microtextrue-dependent orientation/ neointima development and accumulates at high levels in
guidance of cells positively correlates with an alignment of the neointimal ECM.123-125 Unlike the other ECM proteins
590 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 54.5. Neointimal hyperplasia
progresses in vascular tissues in re-
sponse to injury. Injury-activated
medial SMCs migrate into the ves-
sel lumen and form a thick
neointimal layer that constricts or
narrows the lumen. When small di-
ameter synthetic vascular grafts are
implanted, mechanical injury to
adjacent vascular tissues promotes
hyperplastic growth into the graft
lumen that results in restricted or
occluded blood flow.
secreted by neointimal SMCs, tenascin and thrombospondin ECM Modulation of Endothelial Cell Function
have cell adhesive and anti-adhesive properties and have been Endothelial cells (ECs) form a monolayer on the lu-
implicated as specialized ECM proteins for events such as minal surfaces of vessels and normally serve as a
tissue regeneration and wound repair.126,127 Tenascin is of nonthrombogenic and selectively permeable boundary be-
particular interest in vascular injury, since its expression in tween the bloodstream and extravascular space.136,137 Fol-
the vessel wall is induced only after mechanical injury or lowing injury and in vascular disease, ECs can become
exposure to growth factors that are released in the vessel wall thrombogenic and promote leukocyte adhesion.136,137
following injury.123,124 It has been speculated that tenascin Throughout the entire vascular system, normal uninjured
facilitates SMC migration and neointima formation in ECs abluminally synthesize, secrete and maintain a basement
vivo124 by creating an ECM of intermediate attachment membrane on their basal surfaces. The subendothelial base-
strength that maximizes SMC migration.129 In vitro studies ment membrane is a complex network of ECM glycopro-
have shown that exogenously added soluble tenascin en- teins including laminin, type IV collagen, heparan sulfate
hances injury-induced SMC migration.130 proteoglycan, nidogen/entactin, and fibronectin.138 Cultured
The ECM plays an active role in the phenotypic modu- endothelial cells have been shown to attach and spread on
lation of SMCs. In vitro studies have shown that SMCs grown various ECM proteins, including fibronectin, laminin, col-
on fibronectin- and RGD peptide-coated materials quickly lagens, vitronectin, and von willebrand factor.139 Similar to
transform from the contractile phenotype to synthetic SMCs, ECM composition has a profound effect on EC phe-
state.131,132 Conversely, similar studies on laminin- and type notype. When ECs are cultured on type I collagen, type III
IV collagen-coated substrates have shown that the contrac- collagen or fibronectin, migration and proliferation were
tile phenotype is conserved.133 observed to be enhanced.140,141 Type IV collagen, laminin,
The interaction of SMCs with the ECM occurs pre- and reconstituted basement membrane (Matrigel) were ob-
dominantly via several members of the integrin superfam- served to inhibit EC proliferation and promote aggregation
ily of extracellular matrix adhesion receptors.12 Ligand af- into capillary-like tubes.138,140,141 Type V collagen has been
finity chromatography and immunoprecipitation analyses observed to selectively inhibit EC attachment and prolifera-
of SMC extracts have identified several !1 integrins which tion without affecting these activities in SMCs and fibro-
bind to a variety of ECM proteins: #5!1, #v!1, and #3!1 bind blasts.142-143
to fibronectin; #1!1 and #7!1 bind to laminin; #v!1 binds to EC-ECM interactions are mediated by several mem-
vitronectin; #1!1 and #2!1 bind to type I collagen; #1!1 binds bers of the integrin superfamily. ECs express several !1
to type IV collagen.128 Integrin #v!3 was observed to bind to integrins, #1!1, #2!1, #3!1, #4!1, #5!1, and #6!1, which pro-
several ECM proteins including fibronectin, laminin, mote adhesion to collagens (#3!1),144,145 LN(#2!1,144-148
vitronectin, type I collagen, and type IV collagen.128 Other #6!1147) and fibronectin (#4!1,149 #5!1).144,145,150,151 Integrin
findings in this study revealed that !1 integrins play a major #v!3, which is present on endothelial cells, recognizes RGD
role in the adhesion and spreading of stationary SMCs, ligands in vitronectin, von Willebrand factor, fibrinogen,
whereas integrin #v!3 is predominant in ECM interactions thrombospondin, and fibronectin.144,145,150,152-159 Like SMCs,
of migrating SMCs (Fig. 54.6). In support of these studies, the expression of specific integrins on ECs is modulated by
TGF-!1 and PDGF, growth factors that are expressed fol- growth factors. For example, TGF-!1 has been observed to
lowing vascular injury and promote SMC migration, were increase surface levels of both !1 and !3 integrins in ECs .134
observed to promote increased surface levels of integrin #v!3 TNF-# and interferon-& have been shown to promote sig-
without subsequent increases in !1 integrins.134-135 These nificant decreases in EC surface levels of !3 integrins with-
findings suggest that growth factor-mediated increased sur- out changing !1 integrin levels.160 Overall, EC and SMC in-
face levels of #v!3 facilitate SMC migration, and further teractions with ECM components contribute significantly
emphasize the role of this integrin in SMC migration. to their overall function in the normal, injured, and diseased
vessel wall. Although not discussed in this review, platelet
and inflammatory cell interactions with the vessel wall play
Cell-Extracellular Matrix Interactions Relevant to Vascular Tissue Engineering 591
Fig. 54.6. Following vascular injury, me-
dial SMCs become migratory as
neointimal hyperplasia progresses.
Integrin function is modulated in this mi-
gratory response. Surface levels of integrin
#v!3 increase, while !1 integrin levels de-
crease or remain the same. Integrin #v!3
also localizes into focal contacts which are
responsible for generating traction for cell
migration.168 !1 integrins are not detect-
able in focal contacts of migratory cells .168
These results suggest that integrin #v!3 is
the predominant mediator of SMC migra-
tion in response to injury.
an important role in vascular healing and during the pro- evaluated in vitro for promoting EC attachment indepen-
gression of disease.137,161 dently of protein adsorption. Surface-grafted RGD- and
YIGSR-containing peptides were observed to promote en-
Modulating Cell-Extracellular Interactions dothelial cell attachment and spreading on polyethylene
for Vascular Tissue Engineering terepthalate (PET) and polytetrafluoroethylene (PTFE), the
two most widely used materials for vascular grafts.54 EC re-
Tissue Engineered Vascular Implants tention on peptide-grafted materials may generally be im-
Common to all endeavors in tissue engineering, the proved over ECM protein-coated materials, since one study
precise control of interactions between cells and extracellu- has shown that EC retention is better on RGD peptide-
lar surfaces is a fundamental goal in vascular tissue engi- grafted glass than on fibronectin-coated glass.82 EC-selec-
neering. Vascular implant materials which promote cell type tive materials containing surface immobilized GREDVY have
selective adhesion and appropriate cellular functions would been observed to promote adhesion and spreading of en-
vastly improve the long term biocompatibility of synthetic dothelial cells, but not fibroblasts, smooth muscle cells or
vascular implants. With such improvements in platelets.56 The cell adhesion ligand REDV is located on some
biocompatibility, synthetic small diameter vascular grafts types of human plasma fibronectin in alternately spliced cell
would no longer fail and would become a viable option for binding domains.163 EC-selective adhesion has been ob-
surgical procedures. The development of biomimetic sur- served on PET films containing surface immobilized UEA-I,
face modifications and controlled release modalities for vas- a lectin that binds ECs with high affinity. These materials
cular implant materials is critical for achieving controllable promoted EC adhesion without significant monocyte, SMC,
modulation of cellular interactions at the tissue-biomate- or fibroblast adhesion.66 Biomimetic, EC-selective materi-
rial interface. als may enhance the long term biocompatibility of vascular
implants, since thrombosis (mediated by platelet attach-
Modulation of EC Interactions ment) and neointimal hyperplasia (mediated by smooth
with Extracellular Surfaces muscle cell attachment and migration) are major modes of
ECs line the luminal surfaces of all components of the failure. Although extensive studies have evaluated the effect
vascular system and actively maintain homeostasis in the of surface microtextrue on cell-material interactions, few
bloodstream and at the vessel wall. Small diameter synthetic have specifically investigated surface microtextrue effects on
vascular grafts fail clinically due, in part, to the lack of a EC-material interactions. One study has shown that ECs
functional endothelium on the luminal surface. For over 20 spread more rapidly on microtextured polymers prepared
years, many research efforts have focused on transplanting from nanoscale surface replicas of subendothelial ECM than
ECs onto synthetic vascular grafts to improve graft function on untextured polymers.164 This finding suggests that EC
and performance.111-115,136,162 Among the many critical is- adhesion and function are altered on the microtextured sur-
sues in developing this technology, modulation of EC-ma- faces. Transplantation of genetically modified ECs in syn-
terial interactions on vascular grafts is very important, since thetic vascular grafts has been considered as an approach
it influences cell retention and function on the graft lumen. for enhancing EC function and improving graft patency
Biomimetic cell-adhesive biomaterials with surface rates. Unfortunately, the postgraft implantation retention
immobilized cell adhesion peptides have been developed and rates of transplanted, genetically modified ECs have been
592 Tissue Engineering of Prosthetic Vascular Grafts
observed to be significantly lower than unmodified injury-induced neointimal hyperplasia.169-171 Local release
ECs.165-167 Therefore, the benefits gained from improved EC of cyclic RGD from a delivery vehicle incorporated in vas-
function are diminished because of increased cell detach- cular grafts may prove to be effective in limiting graft hy-
ment under flow. Biomimetic surface modifications may sig- perplasia. Some ECM components are synthesized and se-
nificantly improve the retention rate of genetically modi- creted in response to vascular injury to facilitate neointimal
fied ECs by increasing their attachment strength to graft hyperplasia development. Local release of agents which spe-
surfaces. Overall, biomimetic material surfaces may be valu- cifically inhibit the synthesis of these molecules could po-
able in promoting well-controlled EC-material interactions tentially limit graft hyperplasia. One study which tested this
and improved biocompatibility of vascular implants. concept observed that local delivery of tenascin mRNA
antisense oligonucleotides to injured arteries significantly
Modulation of SMC Interactions reduces neointimal hyperplasia in vascular grafts.172 As de-
with Extracellular Surfaces scribed earlier, tenascin is synthesized and secreted in the
The SMC population that normally resides within the neointimal ECM by SMCs following injury and has been
media (inside the wall) of uninjured blood vessels is pre- shown to promote SMC migration in vitro. Other
dominantly contractile and functionally regulates blood counteradhesive ECM proteins like thrombospondin and
pressure and flow. Following mechanical injury, medial SMC type V collagen, that are selectively synthesized and depos-
populations convert to a predominantly ECM-secreting, ited following vascular injury, are also potential ECM com-
proliferative phenotype and migrate from the media to the ponents that could be selectively inhibited by local delivery
lumen, forming a thick multilayered neointima (Fig. 54.5). to limit graft hyperplasia. Systems for local delivery of bio-
In small synthetic vascular grafts, neointimal hyperplasia
(progressive neointimal thickening) is a major mode of fail-
ure due to severely reduced patency and blood flow or total
occlusion, especially at anastomoses.111-115,136,162,168 SMC
phenotype conversion and the development of neointimal
hyperplasia results from the initial mechanical injury of graft
implantation surgery and the long term generation of ab-
normal mechanical forces due to compliance mismatch be-
tween the native vessel and the synthetic graft.111-113,168 Since
SMC-ECM interactions play an important role in modulat-
ing SMC phenotype, migration, proliferation, metabolism,
etc., well-controlled, selective modulation of these interac-
tions by biomimetic material surface modifications and lo-
cal delivery of soluble mediators may limit hyperplasia and
improve the long term biocompatibility of small diameter
synthetic vascular grafts.
Since ECM components play an active role in modu-
lating SMC phenotype, biomimetic cell adhesive
biomaterials may provide a way to selectively control SMC
phenotype expression to limit graft hyperplasia. Laminin and
type IV collagen have been identified as ECM components
which promote expression of the contractile phenotype in
cultured SMCs.102,133 Therefore, surface immobilization of
these proteins or biologically active peptides from these pro-
teins on vascular graft biomaterials may promote pheno-
type reversion of injury-activated SMCs that initially con-
tact the implanted graft and limit further development of
neointimal hyperplasia. Currently, the effect of surface
microtextrue on SMC behavior has been unexplored. How- Fig. 54.7. Tissue engineered vascular grafts via modulation of
ever, it could be speculated that surface microtextrue will cell-ECM interactions. The optimal high performance vascu-
influence SMC adhesion/migration and/or phenotype. lar graft must promote complete luminal coverage with a
An alternative approach for limiting vascular graft hy- nonthrombogenic endothelium that adheres strongly to graft
perplasia is to deliver locally from the implanted graft soluble lumen. Biomimetic surface modifications may facilitate the
agents which selectively limit SMC migration. As described design of these characteristics by providing improved control
earlier in this review, in vitro studies have shown that integrin of EC-biomaterial interactions. This idealized graft must also
#v!3 is the predominant receptor in promoting SMC migra- limit neointimal hyperplasia so that long term patency and
tion (Fig. 54.6). Cyclic RGD peptides, which bind to integrin adequate blood flow is maintained. Biomimetic surface modi-
#v!3 with high affinity, have recently been shown to inhibit fications and local drug delivery systems that modulate SMC
SMC migration in vitro; continuous local or systemic deliv- phenotype and restrict SMC migration may provide means for
ery of cyclic RGD peptides has been shown to limit balloon limiting graft hyperplasia.
Cell-Extracellular Matrix Interactions Relevant to Vascular Tissue Engineering 593
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may improve long term graft biocompatibility. affinity receptor for the alternatively spliced segment of
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well-defined ECM-like physicochemical features to promote
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Matrix Engineering
CHAPTER 55
Use of Hydroxypropylchitosan Acetate as a Carrier
for Growth Factor Release
Keiko Yamamura, Toshitaka Nabeshima, Tsunehisa Sakurai
Introduction
T he application of autogenous vein in vascular replacement shows high patency rates as
compared to that of prosthetic vascular graft, because vascular endothelial cells possess
anti-thrombogenic properties through secretion of many biologically active substances such
as heparan sulfate, prostacyclin, thrombomodulin and plasminogen activators. Many inves-
tigators have demonstrated that a large percentage of synthetic graft failures in vascular
reconstruction are due to inadequate regrowth of endothelial cells on the graft surface.1
However, various synthetic prostheses are available when autogenous vein is unsuitable for
use because of venous inflammation and insufficient diameter. If there is confluent
endothelialization at the internal surface of the vascular graft, a long term patency rate would
be expected. As the patency of a vascular graft is the most important factor, many investiga-
tors have tried to seed endothelial cells derived from donor veins into the vascular graft for
this purpose.2,3 However, to become clinically useful, large quantities of inoculating cells
may be required to overcome early cellular loss caused by the shearing forces of blood flow.
In addition, endothelial lining techniques in vitro must be performed aseptically, because
graft sepsis is a disastrous complication.
Basic fibroblast growth factor (bFGF) is a polypeptide mitogen that stimulates the
growth and differentiation of a wide variety of cell types derived from the mesoderm and
neuroectoderm (Fig. 55.1).4 It is also known that bFGF seems to be closely related to stimu-
lation of endothelial cell proliferation.5,6 A recent study suggested that the systemic admin-
istration of bFGF stimulated endothelial regrowth and proliferation in rat denuded arter-
ies.7 Local direct application of such peptides in a vascular regenerating environment may
be more useful than systemic administration because of their short life in vivo. Moreover, it
may be useful to control release of bFGF in order to develop endothelial healing on vascular
prosthetic surfaces. In this work to control the release rate of bFGF, hydroxypropylchitosan
acetate (HPCHA) was also incorporated into the graft as a carrier of bFGF. HPCHA is a
water and alcohol soluble derivative of chitosan, which is known to be a safe and biodegrad-
able polysaccharide from chitin, a marine resource. Tokura and Azuma have reported that
HPCHA, a derivative of acetylated HPCH, is well digested in the presence of lysozyme. 8
It can be used in combination with peptides, as well as many hydrophilic and lipid-
soluble chemicals with poor stability or sensitivity to heat and pH; therefore it has been
employed as a vehicle in pharmaceutical preparations.9,10
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
600 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 55.1. Amino acid sequence of basic fi-
broblast growth factor.
The subject of this article is limited to the possible use function of the initial concentrations of the factor, where
of HPCHA as a carrier controlling the release of growth fac- the increase in protein contents was used as a measure for
tor in vitro and vivo.11 the proliferation of the cells. bFGF content in a bFGF graft
was 2.05 ± 0.32 ∝g (SE, n = 5-6). bFGF content in a bFGF-
Use of HPCHA as a Carrier for bFGF HPCHA graft was 2.71 ± 0.41 ∝g (SE, n = 5). Considering
Acetylation of the amino group in hydroxy- that both Gore grafts were water loaded, this value appears
propylchitosan (HPCH, MW ~800,000; deacetylation de- reasonable. Also, nearly 100% biological activity of the bFGF
gree, 60%; 7200 cps, 5% w/v) was performed following the incorporated into the grafts was maintained. The present
procedure of Tokura et al.8 In brief, 10 g of HPCH was dis- data demonstrates that recombinant bFGF can be incorpo-
solved in 150 ml of deionized water and 100 ml of methanol rated into a graft during aseptic procedures, maintaining
was added to the solution. Then 25 ml of acetic anhydride biological activity.
was added and stirred vigorously overnight. After the re-
sulting solution was dialyzed in deionized water, HPCHA Influence of HPCHA on Release of bFGF
was obtained by lyophilizing the solution. Sixty percent
acetylation was confirmed by IR spectrometry. The chemi- In Vitro
cal structure of HPCHA is shown in Figure 55.2. The bFGF-HPCHA Gore graft was placed into a bo-
The amount of recombinant human basic fibroblast ron coated glass tube containing 1 ml of phosphate buffer
growth factor (bFGF) incorporated in an expanded (pH 5.5) maintained at 37°C, and transferred into new ones
polytetrafluoroethylene graft (Gore-Tex®, inner diameter after 0.5, 1, 2, 4, 6, 8, 16 and 24 h. Samples were stored at 5°C
3 mm, wall thickness 0.64 mm, mean pore size 30 ∝m, po- and assayed within 3 days. The bFGF Gore graft was also
rosity 80%) was predicted from the water accessible volume. studied. Fig. 55.5 shows the cumulative percent of released
Water loading was 0.00116 ml/graft (0.75 cm in diameter) bFGF from bFGF Gore grafts and bFGF-HPCHA Gore
for distilled water and 0.0147 ml/graft (0.75 cm in diam- grafts. bFGF release from the bFGF Gore graft reached 100%
eter) for the HPCHA solution (4%, w/v), respectively. at 24 h. For the bFGF-HPCHA Gore graft, bFGF was re-
bFGF was dissolved in sterilized water at a concentra- leased at about 30% at 8 h and 55% at 24 h. Incorporation
tion of 1800 ∝g/ml. Gore-Tex® loaded with bFGF solution of bFGF with HPCHA can apparently sustain its release as
was freeze-dried by the procedure of Yamamura et al.12 compared with the bFGF Gore graft.
HPCHA was dissolved in bFGF solution (180 ∝g/ml) at a
concentration of 4% (w/v). Specific gravity of the solution In Vivo
was 1.01. Figure 55.3 shows scanning electron micrographs Skin pockets in a single row were made in the left and
(SEM) of inner surfaces of bFGF-HPCHA Gore grafts. the right flanks of Japanese white rabbits under thiopental
To determine the bFGF activity in the Gore grafts, five anesthesia administered intravenously. Six bFGF Gore grafts
bFGF-HPCHA Gore grafts were soaked in 2 ml of a 20 mM were implanted in the left pockets and six bFGF-HPCHA
isotonic phosphate buffer (pH 7.4) in a boron coated screw- Gore grafts in the right pockets; these were removed at 0.5,
cap tube and stored at 5°C for 3 days. The biological activity 1, 2, 4, 6, 8, 16 and 24 h. The removed grafts were placed into
of bFGF was assayed in baby hamster kidney (BHK)-21 cells, phosphate buffer (pH 7.4) at 5°C for 3 days and assayed.
cultured according to Fukunaga et al.13 In brief, BHK-21 Figure 55.6 shows the percent of remaining bFGF in the
cells were seeded at 1 X 103 cells/well with bFGF at various bFGF and bFGF-HPCHA Gore grafts after implantation. In
concentrations. After 3 days in culture, the increase in pro- vivo release of bFGF from the bFGF Gore grafts reached al-
tein contents as a measure for the cell proliferation was de- most 100% at 24 h following implantation, whereas 60% of
termined by the bicinchoninic acid method (BCA Protein bFGF remained in the bFGF-HPCHA Gore grafts at 24 h.
Assay Kit Pierce Co., IL USA) at 595 nm using a microplate Both the animal and in vitro experiments demonstrated that
reader (BIO-RAD model 450). Figure 55.4 shows a typical almost 100% of bFGF was released from the bFGF Gore
proliferation profile of BHK-21 cells treated with bFGF as a grafts after 24 h, while bFGF release from the bFGF-HPCHA
Use of Hydroxypropylchitosan Acetate as a Carrier for Growth Factor Release 601
Fig. 55.2. Chemical struc-
ture of hydroxypropyl-
chitosan acetate, m,n =
integral numbers >1.
Fig. 55.3. Scanning electron micrographs of various surfaces of
Gore grafts: (A) Drug-free Gore graft; (B) bFGF-HPCHA Gore
graft before implantation; (C) bFGF-HPCHA Gore graft (Origi-
nal magnification x1000).
602 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 55.4. Proliferation profile of BHK-
21 cells as a function of initial concen-
trations of bFGF (SE, n = 5).
Fig. 55.5. Release rates of bFGF in grafts
at 37°C. Graft diameter 0.75 cm; Me-
dium: pH5.5 phosphate buffer; empty
circles = bFGF (2.05 ∝g) in bFGF Gore
graft (SE, n = 6); solid circles = bFGF
(2.71 ∝g) in bFGF-HPCHA Gore graft
(SE, n = 6).
Gore grafts was sustained. Although the release rate from Effect of bFGF-HPCHA on the
the HPCHA-free graft in the animal experiment was closely Endothelialization of Grafts
correlated to that in the in vitro experiment, a few differ- To evaluate the effect of bFGF on the
ences in results were observed between these experiments. endothelialization of bFGF-HPCHA Gore grafts, bFGF-
In the animal experiment, the release pattern showed an ini- HPCHA and drug-free Gore (control) grafts were implanted
tial lag period of about 1 h compared with the in vitro ex- between artery and vein in dogs. Four adult mongrel dogs
periment. It is probable that a slight boundary layer will con- were anesthetized with intravenous sodium thiopental, and
trol the release. maintained with nitrous monoxide and halothane. Femoral
artery and vein were exposed on both sides and dogs were
heparinized before clamping. bFGF-HPCHA grafts were
interposed between right femoral artery and vein by double
Use of Hydroxypropylchitosan Acetate as a Carrier for Growth Factor Release 603
Fig. 55.6. Remaining bFGF in grafts
implanted in rabbit abdominal subcu-
taneous tissues removed at various time
intervals. Empty circles = bFGF
(2.05 ∝g) in bFGF Gore graft (SE, n =
5-6); solid circles = bFGF (2.71 ∝g) in
bFGF-HPCHA Gore graft (SE, n = 5-6).
Fig. 55.7. Light microphotographs of the im-
planted site: (Upper), Drug-free Gore graft;
(Lower), bFGF-HPCHA Gore graft at 6 weeks
after implantation. G = graft; RE = regener-
ated endothelial cell. (Hematoxylin-eosin
stain, x20).
604 Tissue Engineering of Prosthetic Vascular Grafts
end to side suture. Control grafts were implanted on the left Further, bFGF-HPCHA Gore grafts are useful for vascular
sides. Specimens were harvested after 6 weeks to investigate reconstruction, because bFGF stimulates regrowth of endo-
proliferation of endothelial cells on the internal surface of thelial cells on the graft surface.
the grafts. Grafts were explanted from anesthetized living
animals, opened longitudinally for observation and fixed for References
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Concluding Remarks and endothelial seeding. J Vasc Surg 1984; 1:877-887.
Our work so far has produced evidence to support 4. Gospodarowicz D, Ferrara N, Schweigerer L et al. Struc-
the use of HPCHA as a biodegradable carrier for controlled tural characterization and biological functions of fibro-
release of bFGF. Coupling bFGF to HPCHA succeeded in a blast growth factor. Endocr Rev 1987; 8:95-114.
stable and reproducible way. 5. Saksera O, Moscatelli D, Rifkin DB. The opposing effects
bFGF-HPCHA solution absorbed into Gore grafts was of basic fibroblast growth factor and transforming growth
about 13-fold in weight greater than that of the bFGF solu- factor beta on the regulation of plasminogen activity in
capillary endothelial cells. J Cell Biol 1987; 105:957-963.
tion. It can be assumed that HPCHA solution is held in the
6. Schweigere L, Neufeld G, Friedman J et al. Basic fibro-
pore network of Gore grafts better than water. Calculations blast growth factor: Production and growth stimulation
of the porosity (~80%) of Gore-Tex® have shown that each in cultured adrenal cortex cells. Endocr 1987; 120:796-800.
Gore-Tex® graft (0.75 cm in diameter) contains pores to- 7. Lindner V, Majak RA, Reidy MA. Basic fibroblast growth
taling 0.0226 cm3; accordingly, the amount of water or factor stimulates endothelial regrowth and proliferation
HPCHA solution that could be held in the pores is estimated in denuded arteries. J Clin Invest 1990; 85:2004-2008.
to be roughly 0.0226 ml. 8. Tokura S, Azuma I. Distribution of acetamide group in
Nevertheless, actual experiments with water or partially deacetylated chitin and its biodegradability. De-
chitosan solution content have shown figures that are sub- rivatives in Life Science 1992; 1:86-92.
stantially lower: For water, roughly one-twentieth of the pro- 9. Machida Y, Banba T, Fujimoto M et al. Synthesis and
antitumor activity of chitosan carrying 5-fluorouracils.
jected volume is held; for chitosan solution, about two-thirds
Macromol Chem 1989; 190:1817-1825.
of the projected volume. 10. Nishioka Y, Kyotani S, Okamura M et al. Release charac-
The explanation for this may be that hydrophobic teristics of cisplatin chitosan microspheres and effect of
materials such as Gore-Tex® repel water, making it difficult containing chitin. Chem Pharm Bull 1990; 38:2871-2873.
for the pores in the graft to retain fluid. A high molecular 11. Yamamura K, Sakurai T, Yano K et al. Sustained release
solution like chitosan has greater inherent viscosity, how- of basic fibroblast growth factor from the synthetic vas-
ever, and is thus more likely to be retained within the Gore- cular graft using hydroxypropylchitosan acetate. J Biomed
Tex® pores than is water. Therefore, one effective way to in- Mater Res 1995; 29:203-206.
crease the amounts of drugs contained within Gore-Tex® 12. Yamamura K, Iwata H, Yotsuyanagi T. Synthesis of anti-
pores is to improve retention by increasing the viscosity of biotic-loaded hydroxyapatite beads and in vitro drug re-
fluids through use of high molecular solutions, and its fur- lease testing. J Biomed Mater Res 1992; 26:1053-1064.
13. Fukunaga K, Hijikata S, Ishimura K et al. Aluminium
ther release rate can be controlled by changing the ratio of
!-cyclodextrin sulphate as a stabilizer and sustained-re-
HPCHA. In conclusion, HPCHA could be used as a carrier lease carrier for basic fibroblast growth factor. J Pharm
to control continuous release of unstable substances such as Pharmacol 1994; 46:168-171.
peptide, hydrophilic and lipid-soluble chemicals in vivo.
Cell Engineering
CHAPTER 56
Transplantation of Transduced Smooth Muscle Cells:
A Vehicle for Local and Systemic Gene Therapy
Randolph L. Geary, Alexander W. Clowes, Monika M. Clowes
Introduction
S ince the advent of techniques to introduce recombinant DNA into mammalian tissues, a
new science of gene transfer has emerged with broad application throughout the life sci-
ences.1-3 With it has come the ability to genetically alter vascular smooth muscle cells (SMC)
and endothelial cells in vitro and in vivo, allowing the vascular biologist to ask and answer
questions previously unanswerable.4-16 Over the past decade an intense enthusiasm for the
clinical application of vascular gene transfer has matured into cautious optimism, as the
limitations of vector systems used to introduce and express recombinant genes in vascular
tissues have been better defined.3 Refinements in vector systems have improved the preci-
sion and safety of vascular gene transfer and proof-of-concept experiments have demon-
strated the potential for both local and systemic therapeutic effects.17-30 While the field is
still in its infancy, the first clinical application of vascular gene transfer has been initiated
and the realization of vascular gene therapy may well be at hand.31,32
The use of prosthetic materials in the treatment of cardiovascular diseases is expand-
ing as new devices are created and the indications for applying existing devices are broad-
ened. A significant limitation of all implantable prosthetic materials is biocompatibility.
Prosthetic vascular grafts have been widely used to reconstruct damaged and diseased arter-
ies and veins. Despite extensive research to improve graft function, thrombosis and intimal
hyperplasia continue to limit graft durability, particularly when used to reconstruct small
arteries or veins. Infections also lead to graft failure and significant morbidity and mortality
in a small but significant number of patients. Application of existing gene transfer technol-
ogy to prosthetic vascular devices has thus been directed toward inhibition of neointimal
growth and thrombus formation and toward improving the extent of graft healing to de-
crease the risk of infection.4,27,33-38 If successful, these strategies will improve graft function
and constitute an important advance in patient care and limb salvage.
In the following discussion we will review the use SMCs as hosts for the expression of
therapeutic recombinant genes. A comprehensive review of vascular gene therapy is beyond
the scope of this chapter and we wish to acknowledge the pioneering work by many investi-
gators in this field without which our studies would not have been possible.39 While our
focus has been on the SMC, others have made extraordinary advances in the application of
endothelial cell-based gene transfer and we refer the reader to those important studies for a
more complete appreciation of the potential for vascular gene therapy.
Tissue Engineering of Prosthetic Vascular Grafts, edited by Peter Zilla and Howard P. Greisler.
©1999 R.G. Landes Company.
606 Tissue Engineering of Prosthetic Vascular Grafts
Smooth Muscle Cells as Targets sertion and promoter sequences essential for integration and
for Gene Transfer expression of the recombinant gene within the host cell ge-
SMCs provide an attractive target for gene transfer, as nome remain (Fig. 56.1). As retroviral vectors integrate their
they comprise the majority of cells within the wall of blood recombinant genes directly into the host cell genome, the
vessels and play a central role in vascular diseases such as transgene is expressed throughout the life of the cell and
atherosclerosis and intimal hyperplasia following arterial can be duplicated during mitosis and passed on to daughter
reconstruction. Modifying SMC behavior by recombinant cells. This provides the potential for stable long term ex-
gene transfer then provides the opportunity to limit SMC pression in contrast to the transient expression (days) of
growth, migration and extracellular matrix elaboration, criti- recombinant genes introduced by plasmid and adenoviral
cal components of atherosclerotic plaque or neointima for- vectors.56,57 The lack of viral coding sequences prevents im-
mation. Vessel wall function could also be improved by lo- mune responses towards foreign proteins, which has been a
cal over-expression of genes encoding factors to inhibit vaso- significant problem with first generation adenoviral vectors
constriction, inflammation, and thrombosis. These and other containing a number of wild type coding sequences.58
modifications could translate into improved outcomes for A major limitation of retroviral vectors is that expres-
patients undergoing surgical or endovascular arterial and sion of the recombinant transgene requires prior integra-
venous reconstruction. tion into the host cell genome. Efficient integration in turn
SMCs have characteristics which make them an at- requires that the host cell be replicating at the time of viral
tractive target tissue in which to express recombinant genes transduction.54,55 As SMCs are generally quiescent within
for systemic therapy. Muscle cells in general have proven to the undisturbed artery wall, direct in vivo transduction is
be durable hosts for recombinant gene expression.40-43 SMCs very inefficient.45 Even at sites of vascular reconstruction,
represent a large population of cells separated from the sys- SMC replication is delayed for hours or days after the injury
temic circulation by a single endothelial cell layer. SMCs are caused by the procedure. Retroviral gene transfer directed
generally quiescent in vivo with replication rates of less than at improving the response to surgical or endovascular re-
one percent,44 and as a result SMCs are very long-lived, pro- construction would require a delayed application of virus
viding the potential for stable long term expression of thera- (24-96 h post-procedure). By that time however, the injury
peutic genes. These attributes have led our group and oth- response is well underway with local thrombus formation,
ers to explore the application of SMCs as hosts for local and release of platelet growth factors, and influx of leukocytes
systemic gene delivery. that may overcome any potential benefits of delayed gene
transfer. Vein grafts present a similar obstacle for retroviral
Vectors for SMC Gene Transfer vectors. For these reasons, alternative vectors capable of gene
As noted above, the practical application of vascular transfer into quiescent SMC in vivo are being explored for
gene therapy has been limited by the vector systems used to gene transfer to SMCs at the time of vascular reconstruc-
introduce recombinant genetic material. Virtually all vector tion (e.g., adeno-associated virus and adenovirus).46,48,56
systems have been used successfully to transduce vascular To accommodate the need for cell replication for
SMCs, including viral vectors (retrovirus,4-6,45 adenovi- retroviral vector integration, ex vivo gene transfer of target
rus,46,47 adeno-associated virus,48 and others3); plasmid cells has been performed in culture. SMCs can be readily
DNA;49 liposomal vectors;50 and hybrid systems.51,52 Each established in culture from small artery or vein biopsies and
vector has its own set of limitations, which generally fall into induced to replicate by serum growth factors.14,45,59 Early
the following categories: inefficient in vivo SMC transduc- passage cells can then be exposed to retroviral vectors
tion; inefficient gene expression following transduction; containing antibiotic resistance genes that allow for selection
transient expression due to a lack of genomic integration; of transduced cells by adding antibiotic to the culture
transient gene expression due to an immune response to medium (Fig. 56.1). Populations of transduced cells can then
products of viral coding sequences; and inability to limit be expanded further in culture for later transplantation back
gene transfer to the target site due to systemic spread of re- into the donor vasculature. This strategy requires the use of
combinant material to nontarget tissues. autologous SMCs to prevent immune rejection of trans-
Our group has employed retroviral vectors13,14,43,45,53 planted cells. It also takes time (weeks) to accumulate the
and adeno-associated virus vectors48 to transduce SMCs ex numbers of transduced cells required to achieve local or
vivo and in vivo, respectively. We will review below our ex- systemic effects.
perience with the retroviral vector system for cell-based vas- We have used this strategy successfully for ex vivo gene
cular gene transfer using transplanted SMCs. transfer and subsequent in vivo transplantation of SMCs
expressing a number of vectors (Fig. 56.1). These include
Retroviral Vectors for Ex Vivo reporter proteins (!-galactosidase (!%gal) and human pla-
Arterial Gene Transfer cental alkaline phosphatase (hAP))13,14,43,45 and proteins with
Retroviruses have been used extensively for recombi- local and systemic biological effects (adenosine deaminase,
nant gene transfer in a wide variety of cell types, both in purine nucleoside phosphorylase, erythropoietin, and oth-
vitro and in vivo.1,2,39,54 This system has a number of ad- ers).14,28,29,43,45,53 Initial experiments were performed using
vantages and disadvantages. Retroviral vectors are made in- SMCs cultured from aortas of the syngeneic Fischer 344 rat
capable of self-replication, as all viral coding sequences es- strain. The cells were transduced with the human adenos-
sential for encapsidation have been deleted.55 Only the in- ine deaminase (ADA) gene which codes for a nonsecreted
Transplantation of Transduced Smooth Muscle Cells: A Vehicle for Local and Systemic Gene Therapy 607
Fig. 56.1. Diagram of retroviral
vectors employed for smooth
muscle cell gene transfer. This
vector system was originally
described by A.D. Miller and
colleagues55 and the vectors are
named based on the order of
genetic elements. L, long terminal
repeat promoter; S, SV40
promoter; N, neomycin resis-
tance gene; PO, polio virus IRES
5' sequences; X, insertion site for
subcloning genes of interest,
including: A, human adenosine
deaminase (ADA); AP, human
placental alkaline phosphatase
(Alk Phos); and Z, lacZ (!-galac-
tosidase).
protein that can be identified and distinguished from rat the neointima from the underlying media. Lesion size and
ADA by protein gel electrophoresis.45 The transduced cells SMC replication were self-limited and the resulting
were then transplanted into Fischer rat carotid arteries neointima was no larger than that of sham-seeded control
following endothelial cell denudation. This strategy results arteries. Stenosis did not occur and transduced cells were
in the immediate formation of a nonocclusive neointima not found outside of the transduced artery wall or in loca-
composed entirely of transduced SMCs (Fig. 56.2). Arteries tions beyond the site of initial transplantation (Fig. 56.2).
were removed at various times following seeding and sig- These results underscore the durability and apparent safety
nificant human ADA expression was documented as late as of SMC-based retroviral vascular gene transfer.
6 months after carotid seeding.45 A follow up study in the Subsequent experiments have demonstrated that this
same model demonstrated effective retroviral vector expres- technique can be used to achieve a potentially therapeutic
sion by seeded SMCs even at one year from the time of trans- systemic response.28,29,53 SMCs were transduced with a
plantation (see below), documenting the durability of the retroviral vector encoding the rat erythropoietin gene and
retroviral approach.43 then transplanted successfully into the rat carotid artery as
While these experiments and those of others4,6 estab- described above. The small number of cells transplanted into
lished the proof-of-concept for ex vivo transduction of carotid arteries (105 cells) produced enough erythropoietin
SMCs, questions regarding the behavior of transduced SMCs to significantly increase erythrocyte production, causing an
following transplantation remained unanswered. For in- increase in reticulocyte counts followed by increases in he-
stance, retroviral transduction preferentially occurs in rep- matocrit and hemoglobin.
licating cells. If cells selected in culture for replication were We have also demonstrated that local gene expression
more prone to replicate in vivo, intimal hyperplasia could within the injured artery wall can inhibit intimal hyperpla-
occur and lead to lumen narrowing in treated vessels or sia. SMCs were transduced with a retroviral vector express-
grafts. Altered migration or matrix elaboration by trans- ing the gene encoding tissue inhibitor of matrix
duced cells would also have adverse effects in vivo. metalloproteinase-1 (TIMP-1). TIMP-1 blocks metallo-
To address these concerns, subsequent experiments proteinase activity, limiting extracellular matrix degradation
were performed using a vector encoding the hAP marker essential for cell migration and replication. TIMP-1
gene to localize transduced cells within seeded rat carotid overexpression impaired migration of transduced cells in
arteries and surrounding tissues (Figs. 56.1 and 56.2).43 culture and, when seeded into rat carotid arteries after bal-
Bromodeoxyuridine was administered to label replicating loon injury, inhibited neointima formation.28 Allaire from
cells, and arteries were removed for analysis at various times our group has used this same TIMP-1 vector to inhibit an-
following transduction. Seeding led to a nonocclusive in- eurysm formation and rupture in rat aortic xenografts
tima composed entirely of transduced SMCs expressing the (guinea pig into rat).29 All grafts transplanted without SMC
AP gene product (Fig. 56.2). Within 2 weeks the neointima seeding developed aneurysms which ruptured between 4 and
also contained nontransduced SMCs that had migrated into 21 days. In contrast, none of the grafts seeded with SMCs
608 Tissue Engineering of Prosthetic Vascular Grafts
Fig. 56.2. Composite photomicrograph of rat carotid artery cross sections removed at 1 week, 2 weeks or 12 months following
transplantation of smooth muscle cells transduced with a retroviral vector encoding the alkaline phosphatase (AP) reporter gene
(dark blue reaction product). At 1 week a nonocclusive intima has been created, with seeded cells expressing the AP gene product.
Within 2 weeks cells have migrated into the neointima from the injured media so that the final intimal lesion is composed of a
mixture of transduced (blue stain) and nontransduced smooth muscle cells. Neointimal area is maximal 4-6 weeks after seeding
and expression of the AP reporter gene is durable, with long term expression shown in an artery removed 12 months following
transplantation.
expressing TIMP-1 developed aneurysms. Grafts seeded with prior to implantation. Autologous SMCs were obtained from
SMCs transduced with a vector encoding only the neo gene baboons via saphenous vein biopsy, and early passage SMCs
(LXSN, Fig. 56.1) all developed aneurysms within 4 weeks were established in culture using standard techniques.14,59
of transplantation, and 2 of 6 ruptured. These results have Cells were then exposed to replication-deficient retroviral
prompted us to extend studies of retroviral vascular gene particles encoding the human placental AP gene coupled to
transfer to other animal models. the neomycin resistance gene (Fig. 56.1). Cells were initially
selected in neomycin, then expanded and within 4-6 weeks
Retroviral Vectors for SMC Gene Transfer of obtaining the vein biopsy, 107 to 109 transduced SMC were
in Prosthetic Vascular Grafts available for PTFE graft seeding (Fig. 56.3). Transduced
As described in the accompanying chapters, prosthetic SMCs were then suspended in a solution of type I collagen
vascular grafts present a unique set of challenges when used and the mixture injected into the lumen of 10 cm long, 4 mm
to reconstruct damaged or occluded arteries and veins. Pros- diameter, PTFE grafts (60 ∝m internodal distance, WL Gore
thetic conduits are more prone to infection than are autolo- Corp., Flagstaff, AZ). Collagen was used to facilitate cell re-
gous grafts and suffer disproportionately from thrombosis tention and adhesion to the PTFE graft material. The cell
and neointima formation at anastomoses. This is particu- suspension was gently infused into the graft lumen and ex-
larly true when grafts are used to reconstruct small arteries truded through graft wall interstices (Fig. 56.3). Seeded grafts
and veins. Prosthetic grafts heal by neointimal ingrowth from were incubated overnight and then grafted end-to-side into
the cut arterial ends at anastomoses. The neointima extends the aorto-iliac position of the original donor baboon.
a limited distance onto the graft lumen surface depending Grafts were allowed to heal for 3-6 weeks, a time pe-
on graft porosity. In more porous grafts, neointima may form riod when an unseeded PTFE graft would have formed a
along the entire lumen surface by ingrowth of granulation significant neointima along its entire length.60-62 In the first
tissue and microvessels directly through graft wall inter- experiments, grafts were more prone to thrombosis due to
stices.60-62 The resulting graft neointima is made up largely exposure of the collagen gel to flowing blood. In subsequent
of SMCs and their surrounding extracellular matrix beneath experiments autologous endothelial cells were cultured from
an endothelial cell monolayer. each vein biopsy. The endothelial cells were not transduced,
SMCs then provide a direct target for gene therapy to but were seeded onto the lumen surface of PTFE grafts fol-
reduce graft intimal hyperplasia and neointimal formation. lowing SMC seeding. This strategy eliminated the problem
Indirect targets include reducing graft thrombogenicity and of increased graft thrombosis. When grafts were removed
improving the resistance of graft material to infection. More- for analysis, a significant population of transduced SMCs
over, given that prosthetic grafts heal by accumulating SMCs, was localized within the graft wall by histochemical staining
we hypothesized that preseeding grafts with large numbers for the hAP gene product (Fig. 56.3). To our surprise, the
of genetically modified SMCs could provide a device for sys- neointima which had formed was devoid of transduced cells,
temic delivery of recombinant gene products capable of indicating that, while they survived the transplantation and
treating inherited or acquired disorders. graft implantation process, cells appeared to stay where they
Using a previously established nonhuman primate were initially seeded and did not migrate into the forming
model of PTFE bypass grafting,60-62 we sought to establish neointima. In addition, transduced SMCs were not recruited
genetically modified autologous vascular SMCs within grafts by microvessels forming within the graft wall granulation
Transplantation of Transduced Smooth Muscle Cells: A Vehicle for Local and Systemic Gene Therapy 609
Fig. 56.3. Composite photomi-
crograph demonstrating PTFE
graft seeding with baboon
smooth muscle cells trans-
duced ex vivo with retroviral
vectors encoding either alka-
line phosphatase (AP, dark blue
staining) or ! -galactosidase
( ! -gal, light blue staining).
PTFE grafts of 60 micron po-
rosity were employed, as these
grafts develop a neointima
along their entire lumen sur-
face in contrast to the less po-
rous clinical graft (Panel A).
Autologous smooth muscle
cells were obtained from
saphenous vein biopsies and
transduced in culture with ei-
ther !%gal (Panel B) or AP vec-
tors (Panel G). Grafts were
seeded 24 hours prior to im-
plantation with 10 6 to 10 9
transduced cells in a collagen gel. Cells became lodged throughout the graft wall as shown in a cross section taken prior to graft
implantation (Panel C). Grafts were removed one month after implantation into the aorto-iliac circulation of baboons, and cross-
sections stained for !%gal (Panels D and F) or AP (Panels H and I). Paired control grafts were placed in each animal, which showed
no staining (Panels F and I) in contrast to the many cells stained within treated grafts. Adjacent sections stained for smooth muscle
#-actin (Panel E) showed a similar pattern of staining. Of note, transduced cells were not found within the graft neointima or
outside of the graft wall in surrounding connective tissue (Panels D and H).
tissue. Perhaps more importantly, transduced SMCs were with specific matrix molecules.63 By implanting cells in
not found outside of the graft wall within the fibrous cap- unique matrix environments, cell growth and adhesion may
sule around the graft or in the vasculature of lower extrem- be enhanced, as it is well established that SMC behavior in
ity muscle beds beyond the seeded interposition grafts.13 vitro and in vivo is altered by adhesion to various matrix
These experiments were repeated using a separate re- components.63,64
porter gene in order to establish that the healing character- Ex vivo techniques, while suitable for elective modifi-
istics of seeded grafts were not influenced by expression of cation of bioprostheses, are not applicable to the majority
the hAP gene. In the follow up study we employed a retroviral of clinical procedures. Furthermore, while retroviral vectors
vector encoding the !-gal reporter gene (Fig. 56.1).14 The have been used in a number of early human gene therapy
same seeding technique was employed and grafts were left trials,2 concerns about their safety remain. Retrovirus vec-
in place for 4-5 weeks and then removed for analysis. The tors integrate randomly into host cell DNA, leading to the
findings were essentially the same. Transduced cells were possibility of latent oncogene activation.2,54 Better vectors
located within the interstices of each seeded graft and cells are being created for direct in vivo vascular gene transfer
were not seen within the neointima or in the surrounding that are more simple to use and more durable than current
perigraft tissues (Fig. 56.3). vectors. Sophisticated systems have been designed to include
transcription initiation sequences that can be turned on and
Unanswered Questions and Future Directions off in the presence or absence of specific cofactors.65 Once
While the results of SMC-based gene transfer in these systems are available, concerns over the lack of control
experimental models are encouraging, a number of ques- of the magnitude and duration of gene expression may be
tions remain to be addressed. Long term expression of alleviated. Device technology is also helping to make clini-
recombinant genes by rat SMCs must be reproduced in cal application of vectors a reality through inventions such
models likely to better represent the human situation. This as through-lumen and spray catheters.66
is critical, as the long term biological behavior of transduced While still in its infancy the first application of vascu-
human SMCs is unknown. Strategies to improve SMC re- lar gene transfer to improve patient care is on the horizon.
tention upon transplantation are also needed in order to Use of this technology to improve the biocompatibility and
apply this strategy effectively to human prosthetic devices.38 function of prosthetic vascular devices has been initi-
This problem may be overcome as we learn more about the ated14,23,27,34,35,37,38 and if successful a revolution in vascular
role of cell surface integrins and their adhesive interactions reconstruction should follow.
610 Tissue Engineering of Prosthetic Vascular Grafts
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Angioplasty, 73, 76, 93, 146, 165, 229-231, 233, 235-240,
Index 242, 249, 251-253, 263, 284, 292, 353, 363, 367, 369,
380, 383, 385, 414, 569, 589, 595, 610-612
Angiotensin converting enzyme (ACE), 158, 232, 233 408
Angiotensin II, 231-233, 238, 239, 242, 253, 255, 257, 265,
Symbols 266, 301, 408, 414
Angiotropin, 280, 302
150.95 protein, 107, 108 Antioxidants, 470, 471, 473
Antithrombin, 63, 115, 117, 389-391, 467, 479, 546, 549,
A 551, 568
#-actinin, 255, 334, 358, 427, 585, 588 Antithrombin complex (TAT), 117, 389-391, 398, 465, 546
#-granule, 28-30, 231, 232, 253, 254 AP-1, 430, 431, 436, 438
#-smooth muscle actin, 105, 407, 408 APAAP technique, 107, 109, 110
#1!1, 272-274, 334, 357, 360, 361, 365, 590 Apoptosis, 44, 122, 164-166, 168, 169, 238, 245, 276, 291,
#2-macroglobulin/low density lipoprotein receptor, 315 324, 335, 341, 411, 416, 593
#4!1, 220, 272, 273, 334, 566, 578-580, 590 Arachidonic acid derivatives, 157
#5!1, 256, 273, 334, 335, 356, 357, 428, 578, 590 Atherectomy, 93, 132, 138, 142, 230, 231, 233, 236,
#6!1, 273, 334, 335, 590 239, 364
Acetylated LDL, 110, 364 Atherosclerotic lesion, 140, 180, 229, 230, 233, 235, 237,
Acidic fibroblast growth factor (aFGF), 32, 158, 159, 162, 251, 252, 254, 410, 415, 433
202, 264, 310, 311, 316, 318, 495 Atherosclerotic plaque, 33, 183, 185, 191, 230, 233, 235,
Acidosis, 279 238, 246, 252, 253, 263, 265, 316, 323, 355, 357, 360,
Actin filaments, 104, 183, 185, 254, 255, 268, 331, 332, 335, 414, 492, 606
354, 535, 585 ATIII, 481, 482, 546, 548-551
Actin stress fiber, 254, 258 Autologous cell(s), 73, 80, 163
Acute inflammatory response, 43, 198, 207-209, 212, 220 #v!3, 233, 239, 272-274, 316, 318, 320, 323, 324, 334, 335,
#d!2, 173 341, 355-357, 360, 361, 559, 588, 590-592, 594,
Adenosine diphosphate (ADP), 116, 255, 258 596, 597
Adenosine monophosphate (AMP), 19, 25, 26, 32, 34-36, #v!5, 434
39, 40, 44, 116, 159, 162, 168, 323, 341, 405, 429, 430,
B
434
Adenovirus, 65, 68, 234, 245, 249, 606 !-galactosidase, 65, 606, 607, 609
Adenylyl cyclase, 159, 429, 430, 434, 435 Balloon angioplasty, 142, 242, 249, 252, 253, 263, 367, 414,
Adipocytes, 95, 96, 102, 103, 105, 107, 108, 110, 111, 124, 569, 589, 610
133, 372, 406 Balloon catheter, 73, 76, 142, 230, 238, 239, 249, 265, 284,
Adipose tissue, 62, 94, 100, 102, 112, 113, 115, 121-124, 292, 364, 595
144, 189, 308 Basic fibroblast growth factor (bFGF), 27, 29, 32, 34, 35,
Adventitia, 6, 9, 10, 12, 20, 22, 23, 25-27, 29, 31, 34, 36, 87, 42-44, 124, 138, 142, 159-164, 168, 190, 202, 203,
197-200, 202, 203, 230, 274, 326, 355, 357, 382, 407, 231-233, 236-239, 242, 243, 248, 252, 253, 257,
408, 411, 425, 441, 443, 452, 458, 490, 497, 498, 510 260-262, 264, 269, 272-275, 277, 285, 294, 299,
Affinity chromatography, 487, 590 310-313, 316-320, 322-324, 335-337, 339, 341, 342,
Aggregation, 40, 42, 61, 82, 116, 158, 172, 231, 233, 256, 351, 356, 364, 415, 416, 435, 479, 495, 496, 502, 550,
267, 334, 339, 480, 502, 507, 509, 585, 590 569, 599-604
Albumin, 31, 199, 207-211, 214-216, 222, 234, 345, 478, Biophilic polymers, 481-483, 487
479, 481, 488, 509, 543, 546, 551, 559, 568, 570, Bioprostheses, 88, 155, 172, 176, 287, 288, 295, 609
577, 580 Bone morphogenetic protein-3, 294, 295, 299
Alginate, 506, 562, 563 Bovine aortic endothelial cell (BAEC), 80-83, 85-89, 176,
Anastomotic hyperplasia, 39, 40, 57, 62, 65, 163, 200, 203, 272, 302, 323, 340, 342, 428-432, 436, 437, 555, 580
248, 482, 555, 558 Burst strength, 482
Aneurysm, 20, 50, 51, 53, 54, 58, 102, 271, 274, 275, 277,
443, 482, 489, 490, 492, 494, 497-500, 508, 509, C
607, 608
c-fos, 238, 243, 245, 248, 431, 436
Angioblast, 373, 374
c-myb, 244, 245, 249, 415, 570
Angiogenin, 302
c-myc, 238, 243-245, 248, 249, 409
Angiography, 47-49, 57, 58, 133, 134, 136, 180, 283,
C3b, 202
284, 295
C3bi, 32, 202, 204
C5a, 199, 208, 216
614 Tissue Engineering of Prosthetic Vascular Grafts
Calf/Calves, 11, 16, 18, 407 Collagen I, 180, 254, 287, 289-293, 296, 298, 330, 332,
Canine model, 18, 40, 41, 76, 77, 89, 96, 97, 100, 131, 338-340, 343,-345, 347, 348, 350, 352, 355, 360, 366,
140-142, 168, 186, 236, 369 367, 408, 414, 494, 545, 550, 575
Capillary sinuses, 10, 12-14, 33, 35 Collagen IV, 80, 180, 254, 340, 360
Cardiac myocytes, 403, 407 Collagen-gag copolymers, 571
Carmeda biologically active surface (CBAS), 546 Collagenase, 33, 35, 42, 62, 67, 96, 100, 102, 108, 110, 122,
Carotid artery, 47, 48, 96, 97, 133, 135, 231, 234-238, 246, 124, 133, 144, 157, 159, 163, 164, 189, 190, 219, 225,
248, 249, 252-254, 265, 284-286, 312, 323, 338, 355, 239, 280, 281, 288, 293, 296, 302, 310, 316, 323, 336,
359, 369, 409, 457, 458, 500, 596, 604, 607, 608, 339, 342, 346, 348, 350, 352, 354, 364, 567, 572, 575
610, 611 Colony stimulating factors (CSF), 202-204, 374, 375, 408
Catecholamines, 231-233 Colony stimulating factor-1 (CSF-1), 374
Cathepsin, 219, 225, 410 Complement activation, 199, 204, 206, 208, 209, 216,
Cd11a, 204, 220 513, 551
Cd11a/cd18, 173, 204 Compliance, 15, 24, 25, 36, 45-48, 50-58, 129, 132, 138,
Cd11b, 31, 32, 42, 43, 204, 205, 213, 217, 220 153, 186, 441, 442, 447, 451-456, 458, 464, 470, 476,
Cd11b/cd18, 32, 42, 204, 205, 213, 217 477, 485, 494, 497, 498, 500-502, 509, 511, 513, 555,
Cd11c/cd18, 204, 205 559, 578, 581, 592
CD14, 393, 405 Compliance mismatch, 36, 45, 46, 50, 51, 56, 58, 132, 456,
CD18, 173, 176, 177, 204, 205, 213, 217, 220, 225, 226 578, 592
CD25, 396 Contact inhibition, 157, 185
CD3, 393, 396, 399 Copolymers, 43, 215, 484, 487, 490, 491, 497, 500, 502,
CD31, 106-109, 111, 113, 124, 158, 173, 174, 180, 272, 273, 506, 507, 510, 511, 529, 551, 555, 557, 559, 562, 566,
275, 373 568, 569, 571
CD32, 405 Corona treatment, 485
CD34, 102, 104-110, 113, 124, 139, 180, 272, 273, 275, 276, Coronary artery bypass graft (CABG), 93, 98-100, 143,
373, 375-378, 384, 388, 390, 392, 393, 397, 401, 146, 180, 247-249, 389
404, 412 Creep, 444, 448, 452, 470
Cd34, 375 Crush resistance, 482
Cd35 receptor, 204 Cyclic adenosine monophosphatase (cAMP), 116, 159,
CD4, 222, 226, 227, 389, 393, 395, 396, 405 160, 162, 263, 358, 429-431, 435, 438
CD40, 389, 395, 397 Cyclic RGD peptides, 592
CD45, 375, 376 Cyclin-dependent kinases, 244, 249
CD68, 31, 182, 393, 399 Cyclooxygenase, 234, 240, 316, 438, 610
Cdk1, 244-246, 249 Cytoplasmic extensions, 101, 104, 105
Cdk2, 244, 245, 249 Cytoplasmic granules, 95, 232
Ceramide sphingosine, 255 Cytoskeletal complexes, 585, 586
Chacma baboon, 5-10, 13, 14, 16-18, 27, 35, 36 Cytoskeletal elements, 75, 101, 589
Chain scission, 484 Cytoskeleton, 65, 75, 140, 175, 233, 254-259, 261, 262,
Chemical vapor deposition, 485 266-268, 272, 290, 291, 323, 334, 335, 341, 352-354,
Chemokinesis, 201, 354 359, 426, 427, 540-543, 585, 587, 588, 593
Chemokinesis, 201, 252, 277, 355, 356
Chemotaxis, 31, 198, 201, 202, 208, 212, 213, 216, 222, 223, D
226, 251-254, 256, 260-262, 264, 265, 269, 271-277, Dacron, 4, 9-22, 24-27, 30-34, 36-41, 50, 51, 62, 67, 69-73,
326, 327, 334, 353-361 75-77, 79, 80, 88, 89, 98-100, 119, 120, 123, 125, 126,
Chloramphenicol acetyltransferase, 65 128, 130, 133, 135, 136, 138, 140-144, 154, 167, 168,
Cholera toxin, 159, 160, 358 172, 187, 193, 197-204, 207, 209, 214, 216, 240, 243,
Cholesterol, 129, 142, 165, 166, 168, 230, 235, 237, 240, 263, 282, 285, 308, 311, 312, 371, 372, 377-386, 388,
248, 254, 265, 363-365, 369, 410, 416, 495, 496, 501, 397, 408, 423, 425, 441, 451, 458, 463, 478, 482,
502, 511, 541 490-496, 499-502, 509, 511, 545, 553, 570, 577, 580,
Chondroitin-sulfate proteoglycans, 337 594, 604
CK18, 107, 110 Degradation, 27, 29-33, 36, 42, 67, 112, 116, 159, 161, 179,
CK19, 107 199-201, 207, 210, 215, 254, 271, 279-282, 287-289,
CK7, 107 302, 304, 313, 315-317, 321-323, 332, 336, 342, 364,
CK8, 107 390, 460-462, 466, 467, 470-479, 482, 484, 486, 489,
Coagulation factor(s), 27, 117, 158, 209, 463 490, 497, 498, 502, 503, 505, 508, 510, 511, 513, 514,
Collagen gel, 272, 281, 289, 290, 293-295, 297, 298, 330, 526-530, 538, 564, 566-569, 572, 574-576, 607
335, 336, 339, 343-352, 433, 569, 608, 609
Index 615
Desmin, 104, 105, 107, 108, 139, 180, 383, 407 Extracellular matrix (ECM), 3, 27-30, 34, 41, 44, 51, 52, 61,
Diabetes, 166, 169, 238, 313, 569 63, 67, 68, 71, 72, 75-77, 80, 81, 84-87, 89, 90, 101,
Diacylglycerol, 358, 429, 437 104, 122, 126, 131, 137, 158, 160, 173, 184, 191, 200,
Diapedesis, 174, 214, 220, 221, 223, 302 220-222, 231-234, 236, 239, 242, 252, 254, 255, 257,
Differential scanning calorimetry, 208, 212, 216 265-267, 269, 271-277, 280, 281, 285, 287, 289, 291,
Dog(s), 7, 10-12, 18, 39, 40, 66, 75, 89, 97, 99, 119, 120, 292, 294-299, 304-307, 311, 316, 320, 322, 323,
130, 132, 133, 140, 141, 159, 162, 168, 187, 193, 198, 326-341, 343, 348, 351-359, 360, 363-370, 372, 394,
236, 238, 240, 243, 254, 272-274, 276, 283, 308, 309, 407-409, 412, 413, 415, 416, 427, 428, 433, 434, 437,
311, 312, 324, 328, 336, 337, 339, 371, 372, 377, 438, 441, 465, 485, 489, 539-542, 544, 554, 558, 559,
379-383, 385, 408, 415, 457, 458, 463, 479, 482, 499, 561, 562, 566, 567, 569, 571-580, 583-594, 596, 597,
500, 502, 509, 511, 516, 522, 529, 535, 544, 550, 578, 606-608, 610, 612
590, 597, 602 Exudation, 198
DOT immunoassay, 110, 111
Duplex sonography, 190 F
Dynabeads, 102, 104, 106, 110, 113, 126, 161 Factor VIII, 13, 14, 68, 95, 107-111, 129, 130, 133, 140, 180,
E 199, 375, 382, 492
Factor VIII-related antigen, 95
E-selectin, 74, 172-174, 272-277, 316, 398, 401 Factor XIIIa, 563
E2F, 244-246, 249 Fallout endothelialization, 18, 40, 236, 240, 372, 377, 380,
ED 1, 33, 516, 519, 522, 525 381, 385
ED 2, 33, 516, 519, 522, 525 Fetal liver kinase-1 receptor (Flk-1), 260, 264, 268, 275,
ED 3, 516, 520, 522, 525 324, 373-375, 378, 405, 406, 413
Elastase, 219, 225, 320, 336 FGF receptor (FGFR), 142, 159, 304-307, 597
Elastic response, 443, 444, 447-449, 458, 503 Fiber density, 28
Elastin, 3, 17, 52, 55, 225, 233, 291, 336, 355, 407, 434, Fibrin, 4-7, 9-16, 18, 25-38, 40-43, 63, 67, 71, 74, 76, 77,
443-445, 492, 497, 498, 501, 541, 575, 577-580, 589 80-83, 85-87, 89, 93, 100, 112, 115, 117-120, 129, 135,
Elastomers, 44, 467, 470, 474-477, 479, 480, 484, 486, 139, 141, 154, 158, 161, 162, 168, 169, 171, 172, 175,
498, 529 176, 179-181, 186, 187, 190, 191, 193, 194, 199, 201,
Electrostatic endothelial cell seeding, 62, 64, 65-67 204-217, 232, 236, 238, 240, 272, 273, 275-277, 280,
ELISAs, 201 281, 283-285, 289, 295, 298, 308, 312-314, 318-324,
EN4, 106, 107, 110, 113, 139 327, 329, 332, 334-337, 339-341, 345, 346, 351, 382,
Endarterectomy, 73, 76, 77, 131, 138, 230, 231, 237, 353, 387-390, 397, 398, 401, 418, 421, 423, 479, 492, 493,
357, 369 497, 499, 509, 532, 539, 543, 546, 550, 551, 554, 563,
Endoglin, 272-274, 276 567-570, 577, 584, 590, 596, 597
Endothelial cell growth supplement (ECGS), 94, 158, Fibrin clots, 41, 324, 337, 339, 567
159, 161 Fibrin coagulum, 5, 26, 30, 492, 493
Endothelial cell seeding, 17, 38, 40, 51, 55, 61-67, 72, 73, Fibrin glue, 67, 71, 76, 80, 81, 89, 100, 154, 168, 176, 180,
75-77, 89, 90, 94, 99, 100, 111, 125, 126, 128-132, 141, 187, 190, 191, 193, 194, 201, 206, 240, 283-285, 295,
152, 154, 161, 167, 168, 176, 194, 369, 377, 467, 511, 308, 312, 324, 499, 577
558, 604 Fibrinogen, 26-32, 34, 36-41, 43, 74, 81-83, 89, 141, 199,
Endothelial cell transplantation, 62, 63, 66, 79, 90-100 201, 204, 207-217, 272, 273, 275-277, 308, 320, 324,
Endothelin-1, 238, 365, 428, 431, 437, 438 334, 335, 340, 390, 543, 546, 551, 554, 563, 568-570,
Endothelium derived relaxing factor (EDRF), 132, 141, 584, 590, 596, 597
159, 162 Fibrinolysis, 29, 31, 42, 169, 171, 175, 238, 314, 321, 323,
Endothelium-specific cytoplasmatic organelles, 140 387-390, 398, 465, 570
Entactin, 272, 339, 590 Fibroblast, 7, 9-13, 15, 17, 25, 27-29, 35, 42-44, 63, 64, 67,
Epidermal growth factor (EGF), 158, 164, 202, 203, 231, 68, 74-77, 86, 87, 89, 96, 102-104, 106, 113, 115, 122,
252, 253, 256, 260, 264, 265, 268, 289, 290, 294, 295, 124, 129, 138, 139, 142, 158-162, 164, 167, 168, 172,
298, 318, 353, 356, 359, 374, 551, 552 175, 183-187, 190, 199-207, 214, 219, 221, 223, 226,
Erythropoietin, 376, 378, 406, 407, 413, 606, 607, 611 231, 232, 237-239, 242, 248, 252, 253, 255-258, 263,
Ethylene oxide, 214, 464, 467, 472, 487, 500, 502, 551, 570 264, 266-269, 272-275, 277, 280, 281, 285, 288, 290,
Expanded polytetrafluoroethylene, 4-13, 15-25, 27, 30-40, 294, 297-299, 301, 303-306, 308-313, 316, 317, 322,
58, 62, 66-68, 76, 88, 90, 100, 113, 119, 122, 126, 128, 323, 324, 330, 332, 335, 338, 339, 341-353, 358, 372,
130, 141, 142, 154, 155, 160, 167, 168, 176, 187, 193, 389, 390, 403, 406-411, 413-423, 443, 466, 479, 485,
207, 208, 237, 308, 311, 312, 370, 386, 478, 525, 570, 490-495, 502, 509, 510, 516, 521, 529, 532-534,
577, 600, 611 536-540, 542, 543, 550, 552, 554, 558, 559, 569, 570,
572-574, 578, 588-591, 593-595, 599, 600, 604
616 Tissue Engineering of Prosthetic Vascular Grafts
Fibronectin, 27, 33, 40, 41, 63, 65-68, 71, 72, 74, 76, 77, 80, Granulocyte-macrophage colony-stimulating factor, 414
85, 89, 99, 104, 106-108, 122, 154, 158, 165, 168, Great saphenous vein, 154, 159, 161, 168, 186
190-193, 199-201, 204, 209, 216, 217, 220, 223, 227, GTP-binding proteins, 255, 257-259, 262
233, 253, 254, 256, 257, 265-267, 272-277, 289-291,
294, 298, 299, 302, 307, 308, 320, 322, 328, 332-337, H
339-341, 350, 354-358, 360, 361, 365-370, 376, 377, Hageman factor, 43, 199, 206
413, 428, 433, 434, 473, 534, 535, 537, 538, 541-543, Haptotaxis, 222, 265, 354-361, 594
554, 559, 566, 569, 570, 575, 577-581, 584, 588-591, Heaprin sulfate, 117, 304, 312, 365
593-597 Heart valves, 153, 155, 171, 172, 175, 176, 295, 459, 460,
Fibronexus, 103, 104 472, 475, 532
Fibrosis, 198, 199, 207, 214, 218-221, 223-225, 227, 284, Helper T cells, 393
285, 352, 407-410, 413, 414, 423, 539 Hematopoiesis, 372, 373, 375, 378, 413
Fibrous bodies, 103 Hemostasis, 31, 41, 63, 83, 119, 171, 212, 217, 238, 277,
Filopodia, 106, 254, 268, 290, 330, 332 285, 321, 323, 338, 466, 484, 558
Finite element method, 442, 449, 450, 453, 454, 456 Heparin binding growth factors (HBGFs), 550
First trimester decidua, 102 Heparin proteoglycans, 132
Flk-1/kdr knockout mice, 374 Heterografts, 482, 551
Flt-1, 260, 264, 265, 324, 378, 405 High endothelial venules, 403, 412
Fluid dynamics, 441 Hill and valley, 367
Fluid flow patterns, 441 Histamine, 77, 116, 172, 177, 213, 214, 217, 256, 266
Fluorescence-activated cell sorting (FACS), 375 Histamine receptor, 213, 214
Fluorocarbon, 460, 473, 588 HLA-DR, 173, 176
Foamy macrophages, 129, 181, 182 Homologous serum, 166
Focal adhesion kinase (FAK), 251, 255-268, 335, 354, 358, Human bone marrow, 102, 113, 377
361, 428, 436, 437, 585, 593, 594 Human placenta, 102, 113, 342, 423, 606-608
Focal adhesion plaques, 254, 426, 428, 540 Human saphenous vein endothelial cells (HSVECs),
Fourier transform, 208 75-77, 125, 162, 174, 187
G Human umbilical vein endothelial cells (HUVECs), 65, 69,
70, 118, 122, 139, 140, 157, 159, 256, 260, 261, 273,
G protein, 267, 430, 436, 437, 586, 594 310, 321, 322, 338, 428, 429-432, 437, 555, 556, 558,
G-CSF, 374 578-580, 589
GAX, 245, 249 Hyaluronan, 257, 267, 288, 290, 297, 298, 332, 355,
Gelatin, 70-73, 76, 124, 158, 165, 168, 174, 175, 233, 280, 360, 585
289, 291, 299, 316, 320, 323, 336, 342, 408, 472, 490, Hyaluronic acid, 290, 298, 320, 339, 588, 595
501, 506, 509, 568, 570, 577, 580, 587 Hydrogel, 465, 467, 479, 486, 487, 506, 511, 546, 561-570,
Gelatinase a, 233, 316, 323, 342 588, 611
Gelatinase b, 233 Hydroxyethylmethacrylate, 43
Gelsolins, 255 Hyperlipidemia, 165, 166
Gene therapy, 3, 68, 80, 89, 99, 160, 175, 239, 247, 249, 284, Hyperplasia, 15, 35, 38-40, 45, 51, 52, 57, 58, 61, 62, 65, 72,
299, 422, 423, 605, 606, 608-611 73, 76, 122, 124, 126-128, 130-138, 140-142, 163, 167,
Genetic manipulation, 75, 112, 160, 245, 593 168, 171, 175, 179, 189, 200, 203, 227, 229-243,
Genetically engineered, 65, 86, 112, 113, 162, 246, 248, 292, 245-249, 251, 254, 263, 264, 282, 285, 286, 295, 301,
581, 611 305, 307, 308, 311, 312, 317, 353, 354, 357, 359, 360,
Glucosaminoglycan, 15 363-369, 414, 426, 436, 441, 457, 464, 482, 489, 492,
Glutaraldehyde, 50, 58, 86, 103, 106, 107, 155, 172, 175, 494, 502, 555, 558, 577, 580, 581, 589-593, 595, 597,
288, 289, 482, 507, 515, 523, 545, 551 605-608, 610, 611
Glycophosphoprotein, 106 Hypoxia, 27, 32, 43, 159, 162, 279, 281, 284, 285, 415
Glycoprotein, 29, 63, 106, 110, 111, 172, 205, 217, 225, 239,
289, 295, 306, 314, 315, 332, 336, 355, 367, 373, 405, I
412, 561, 566, 567, 585, 587, 590, 597 IBMX, 159, 160
GM-CSF, 202, 203, 374, 408 ICAM-1, 52, 74, 172-174, 176, 202, 242, 277, 316, 394, 395,
Golgi apparatus, 103, 104, 303 398, 405, 432, 585
Granulation tissue, 10, 12, 34, 198, 199, 206, 226, 273, 282, ICAM-2, 74, 173, 176
283, 304, 305, 310, 313, 318, 343-346, 350, 351, 414, ICAM-3, 74, 173, 176
490, 497, 540, 567, 608, 609 IFN-&, 44, 174, 203, 220, 227, 323, 394, 397
Granulocyte colony-stimulatin factor, 374 IL-10, 223, 224, 389, 397
Index 617
IL-13, 204, 205 Laminin, 63, 71, 72, 74, 122, 158, 233, 254, 256, 266,
IL-1b, 30, 31, 33, 36, 43, 174, 202, 203, 206, 392 272-276, 280, 291, 298, 302, 328, 333-341, 354-358,
IL-1R, 202, 205 361, 405, 413, 428, 433, 434, 529, 541, 542, 575, 585,
IL-2, 277, 394, 397, 398, 404 588, 590, 594-596
IL-2R, 397 Latency-associated peptide (LAP), 29
IL-3, 374, 376, 378 LFA-1, 173, 176, 220, 529
IL-4, 33, 38, 44, 204, 205, 223, 277, 394, 397 Lipid vacuoles, 183, 185
IL-5, 394, 397 Lipopolysacharide, 172
IL-6, 202, 204, 398 Lipoprotein, 133, 139, 141, 158, 165, 166, 168, 169, 202,
IL-8, 173, 201, 202, 204, 221, 224, 227, 398 203, 254, 314, 315, 321, 322, 416, 418, 510, 541,
Image analysis, 23, 84, 112, 133, 428, 533, 534 544, 550
Immunoglobulins (IgG), 172, 174, 199, 204, 207-209, 216, Liposuction fat, 110, 147
222, 318, 395, 399, 534 Liquid crystalline polymers (LCP), 476
Immunohistochemistry, 103, 106, 108, 202-204, 273, 372, Low-density lipoprotein, 77, 139, 169, 254
423, 542 LPS, 172, 173, 316, 397, 398, 416
In situ hybridization, 33, 112, 113, 202-204, 407, 413, 426 LTB-P, 29
Inner capsule, 11, 12, 15, 16, 32, 198, 202, 485, 49-494, LVAD, 265, 387-401
498-500, 509 Lysophosphatidic acid, 255, 266, 267
Insulin-like growth factor, 158, 160, 162, 202, 203,
231-233, 238, 239, 242, 243, 248, 252, 253, 257, 260, M
262, 264, 267, 269, 294, 301, 339, 356, 360 MAC 387, 107, 108
Interferon-&, 100, 173, 176, 202, 203, 277, 412, 526, 590 Mac-1 receptor, 31, 32
Intermediate filaments, 103-105, 111, 139, 336 Macrophage chemotactic protein 1 (MCP-1), 31, 201-203,
Internal elastic lamina, 230, 242, 302, 354, 490 213, 214, 221, 224, 226, 227, 242, 243, 252, 254,
Internodal distances, 5, 19, 21, 32, 72, 379, 485 261, 432
Interstitial collagenase, 239, 281, 316, 323, 336, 342 Macrophage colony stimulating factor, 202, 203, 374
Intima, 8, 15, 17, 36, 45, 51, 52, 58, 61, 63, 66, 68, 73, 76, Macrophage inflammatory protein-1 (MIP-1), 32, 43, 204,
79, 80, 82, 87-89, 97, 99, 128, 129, 135, 137, 139, 169, 213, 214, 217, 221, 222, 226, 274
180, 181, 185, 232-237, 239, 251, 282, 301, 302, 353, Macrovascular endothelial cells, 116, 117, 122, 123, 140,
355, 357, 371, 382, 383, 414, 426, 443, 463, 497, 498, 153, 161
503, 607, 608 Magnetic particle concentrator, 102
Intimal hyperplasia, 35, 38, 39, 45, 51, 52, 58, 61, 73, 76, Mammary adipose tissue, 102, 112
122, 124, 126-128, 130-138, 140-142, 167, 168, 171, MAP kinase, 260-262, 267, 307, 429, 586, 594
175, 179, 189, 200, 229-249, 251, 254, 263, 264, 282, Mast cells, 214, 217, 218, 256, 302, 306, 410, 411, 415, 416
285, 286, 295, 301, 305, 307, 308, 311, 312, 317, 353, Matrical tracks, 328, 329
354, 357, 359, 360, 363-369, 426, 436, 441, 457, 489, Matricellular protein, 334, 337, 340
492, 494, 502, 577, 580, 589-593, 595, 597, 605-608, Matrigel, 272, 273, 291, 328, 335, 339, 367-369, 405, 590
610, 611 Matrix degradation, 29, 36, 280, 313, 364, 607
Ion beam implantation, 486 Matrix metalloproteinase, 231, 233, 236-239, 254, 266,
Irradiation, 218, 245, 477, 484, 487, 581 280, 281, 316, 317, 320, 323, 336, 342, 354, 359, 409,
Ischemia, 123, 145, 227, 230, 253, 275, 276, 283, 284, 286, 415, 607, 610
302, 309, 312, 384 Matrix synthesis, 112, 223, 363, 364, 366-369
K Mechanotransduction, 430, 436, 543
Membrane-type MMP (MT-MMP), 316, 317
Kaplan-meier analysis, 191 Mesenchymal cells, 35, 96, 289, 295, 297, 405, 412, 413,
Kink resistance, 131, 470, 472, 485 418, 419, 421, 423, 491, 492
Kinking, 24, 54, 470 Mesothelial cells, 67, 76, 95, 100, 103, 104, 107, 112, 113,
KP-1, 181 123, 126, 139, 147, 158, 161
Mesothelium, 62, 95, 102, 144, 406
L MHC class II, 43
L-fucose, 106 Microcystic lacunae, 103-106
L-selectin, 220, 223, 226, 227, 273 Microgrooves, 533, 538
Lamellipodia, 330, 332 Micropatterned surfaces, 532
Laminin, 71, 158, 272, 433, 592, 593 Micropinocytotic vesicles, 111
Microtexture, 485, 511, 532-536, 538-543, 583, 587-589,
591, 592, 595
618 Tissue Engineering of Prosthetic Vascular Grafts
Microvascular endothelial cells (MEC), 62, 66, 67, 76, 95, P
96, 99, 102-113, 115-119, 121, 123, 126, 139, 140, 144,
161, 162, 167, 193, 263, 273, 274, 277, 282, 285, 291, P-selectin, 74, 172, 173, 177, 220, 273, 275
313, 316, 318-320, 322, 323, 339, 341, 352, 372, 377, PAI-1, 29, 233, 238, 280-282, 293, 314-318, 321, 336, 337,
413, 422, 596, 597 342, 428, 430, 431, 437
Microvasculature, 62, 174, 325, 333, 336, 341, 405 PAI-2, 314, 321
Microvilli, 104, 106, 139, 181, 183 PAL-E, 106, 110, 113, 139
Midgraft healing, 4, 5, 11, 15, 35 Pannus, 7, 15-17, 132, 143, 318, 371, 380, 381, 492, 506
MIP-1#, 213, 214, 221, 222, 226 Pannus ingrowth, 7, 143, 371, 380, 382, 384, 492, 506
MIP-2, 213, 221 Para-anastomotic hypercompliance zone, 51
Mitochondria, 103, 104, 409, 418 Paxillin, 255-258, 260, 264-268, 334, 354, 358, 428, 436,
Mitogen-activated protein, 256, 267, 269, 361, 438 585, 586, 593
MMP-1, 316, 323, 336 PCNA, 244-246, 249
MMP-2, 233, 316, 320, 323, 336 PDGF-A, 8, 36, 202-204, 232, 233, 236, 242, 252, 254, 263,
MMP-3, 316, 336 426, 434, 435
MMP-9, 233, 316, 323, 336, 354 PECAM 1, 106
Monocyte-derived macrophages, 42, 204, 397, 416 PEG, 481
Multinucleated giant cells, 6, 180, 206, 390, 529 Percoll, 102, 104, 105, 107-111
Myeloperoxidase, 208-210 Percutaneous transluminal angioplasty (PTA), 167, 237
Myoblasts, 159, 263, 305, 340, 407, 414, 611 Pericyte(s), 7, 35, 44, 102, 103, 105-108, 110, 111, 264, 265,
Myofibroblasts, 15, 75, 103, 104, 183-186, 203, 204, 308, 279, 280, 282, 302, 310, 410, 411, 413, 415, 417,
352, 372, 389, 406-410, 413, 414, 416, 491-494, 509, 419-421
573, 574 Perigraft tissue, 6, 9, 10, 12, 18, 20, 26, 308, 380-382, 609
Myointimal hyperplasia, 45, 76, 132, 140, 239, 307, 597 Phorbol esters, 330, 336, 339
Phosphatidyl, 255, 257, 259, 260, 267-269, 315, 335, 357,
N 358, 361, 429, 465, 482, 483
Photocoupling, 486
Neo-media, 180-182, 184-186 Photoimmobilization, 81
Neointima, 3, 7, 9-13, 15, 26, 38, 39, 41, 61, 63, 68, 73, 76, Photolithography, 533
80, 82, 88, 97, 99, 122, 124, 126, 131-138, 140-142, Physical vapor deposition, 485
171, 175, 185, 198, 201, 230, 237-243, 245-249, Pigs, 18, 40, 198, 288, 375, 380, 409, 414, 480, 490, 498,
251-254, 263, 265, 283, 284, 353, 355, 357, 359-361, 499, 501, 502, 578
369, 383, 385, 387, 400, 409, 426, 436, 441, 457, 463, Pinocytotic vesicles, 104, 105, 111, 140
472, 478, 492, 494, 498, 501, 509, 553, 554, 568, 569, PKA, 429, 430, 434, 435
589-593, 595-597, 605-611 Plasma, 26-28, 31, 32, 43
Neointimal hyperplasia, 73, 76, 122, 124, 126, 131-138, Plasma cryoprecipitate, 81, 82, 87
140, 141, 171, 239-243, 245-247, 251, 254, 263, 360, Plasma polymerization, 484
369, 426, 436, 441, 494, 589-593, 597, 611 Plasmin, 27-29, 31, 41, 44, 210, 211, 232, 233, 236, 254,
Nf-∀b, 351, 352, 398- 401, 430, 431, 438 264, 265, 280-282, 314-318, 320-323, 336, 337,
Nitric oxide (NO), 33, 36, 44, 52, 65, 68, 157, 231, 234, 236, 367, 564
239, 240, 246, 248, 261, 269, 284, 375, 428, 430, 432, Platelet activating factor (PAF), 173, 216
437, 438, 610 Platelet derived endothelial cell growth factor
Nitric oxide synthase (iNOS), 234, 239, 240, 246, 432, 437, (PD-ECGF), 302
438, 610 Platelet derived growth factor (PDGF), 8, 9, 27, 29, 32,
Nitrous acid degraded heparin (NAD-Hep), 546 34-36, 39, 52, 132, 142, 164, 186, 187, 202-204,
No touch technique, 164 231-233, 236, 238, 239, 242, 243, 252-264, 267-269,
Nonintegrin matrix binding protein, 366 294, 297, 301, 341, 351-358, 361, 364-366, 374, 414,
Non-specific esterase (NSE), 208 426, 433-435, 437, 494-496, 590, 594
O Platelet factor, 26, 28, 232, 274
Polyether urethane, 474, 477, 500
Omental cells, 131, 137-141, 167, 263 Poly(ethylene glycol), 465, 467, 488, 559, 562-570, 594
Omental fat, 95, 100, 139, 147, 158, 161 Poly(lysine), 562, 563
Oxidation, 216, 460, 470-473, 475-478, 484, 486 Poly(propylene glycol), 562, 568, 569
Ozonization, 484, 486, 487 Poly(vinyl alcohol), 479, 594
Poly-L-lysine, 106, 291
Polydimethylsiloxane (PDMS), 43, 44, 199-201, 207, 208,
288, 475, 479, 533
Index 619
Polydioxonone (PDS), 308, 491, 493, 494, 498-500, 509 RGD, 34, 80, 83, 89, 217, 254, 273, 274, 318, 340, 511,
Polyethylene (PE), 31, 41, 42, 141, 201, 207-209, 214, 264, 553-559, 578, 581, 585, 587, 588, 590-592, 594,
364, 457, 467, 464, 471, 473, 475, 478, 479, 487, 488, 595, 597
500, 502, 509, 510, 542, 553-555, 580, 588, 591, 594 RGD peptide(s), 80, 554-556, 558, 559, 581
Polyethylene terephthalate (PET), 31, 32, 41, 208-211, 214, Rho, 251, 257, 258-260, 262, 267-269
364, 472, 473, 478, 509, 553-556, 588, 591 RNA-splicing, 409
Polyglactin 910, 479, 490, 491, 494-496, 501, 502, 511 Rough endoplasmic reticulum, 101, 103, 104
Polyglycolic acid (PGA), 377, 49-494, 500, 509, 510 RT-PCR, 373, 394, 396
Polyninyl chloride, 209
Polyolefins, 477, 484 S
Polypropylene (PP), 112, 113, 133, 310, 470, 473, 477-479, Saphenous vein, 44, 69, 131, 138, 140, 141, 180, 186, 187,
481, 484, 499, 500, 502, 511 377, 482, 608
Polytetrafluoroethylene (PTFE), 4, 6-8, 15, 17, 20-22, 24, Scanning angle reflectometry, 208, 215
25, 31, 32, 38-40, 44, 56, 58, 62, 66-68, 75, 76, 88-90, Scanning electron microscopy (SEM), 20, 23-25, 42, 56,
99, 100, 113, 138, 140-142, 167-169, 176, 177, 180, 83-85, 88, 101, 106-108, 110, 112, 133, 139, 142, 180,
181, 187, 207, 208, 229, 232, 236, 237, 240, 242, 243, 181, 199, 206, 365, 480, 514, 515, 528, 539, 542
248, 285, 355, 359, 360, 369, 370, 377, 380, 385, 386, Scanning force microscopy, 208, 215
393, 423, 441, 458, 470, 473, 476, 478, 482, 484, 497, Scar formation, 3, 574
499, 500, 511, 514-516, 521, 523, 525-527, 553-556, Seeded grafts, 65, 72, 73, 132-138, 141, 499
577, 580, 588, 591, 600, 608, 609, 611 Selectins, 74, 272, 273, 375
Polyurethane (PUR), 31, 32, 39, 43, 44, 80-85, 87, 200, 201, Serine proteases, 27-29, 314, 337
205-208, 215, 377, 379, 387-389, 397, 450, 451, 454, Serotonin, 283
455, 457, 470, 471, 473-475, 477-480, 482, 485-488, Severe combined immunodeficiency (SCID), 208, 209, 335
497, 498, 502, 509, 555-559, 588, 594, 595 Shear stress, 15, 28, 38-40, 44, 45, 52, 54, 58, 63, 65, 69, 72,
Pressure sodding, 144 73, 75, 76, 89, 90, 132, 138, 140, 179, 187, 200, 206,
Procoagulant activity, 30, 42, 176, 265, 389, 393, 398 324, 441, 456, 478, 544
Profilin, 255, 260, 261, 266, 358 Sialomucin, 106
Progenitor cells, 106, 185, 186, 275, 372, 373, 375, 378, 390 Sialyl Lewis-A, 273
Prostacyclin, 38, 40, 76, 99, 110, 132, 139, 140, 141, 165, Sialyl Lewis-X, 272, 273, 275, 276
168, 216, 231, 234, 428, 430, 431, 436-438, 497, 498, Signal-transducer receptors, 138
502, 599 Silanization, 484, 486
Prostaglandin metabolite, 494 Silicone, 43, 199, 215, 471, 473, 477, 479, 533, 536, 537,
Protease(s), 27-30, 87, 219, 225, 232, 233, 314, 316, 321, 539, 540, 542, 543
323, 336, 337, 470 Sintering, 507
Protein A-gold, 107, 109, 113 Skirted cells, 106, 111
Protein C, 77, 80, 81, 89, 139, 205, 208, 215, 216, 223, 225, Smooth muscle cells, 3, 7-9, 11, 12, 15, 17, 19, 29, 33-40,
256, 258-260, 268, 336, 435, 554, 579, 581, 585, 44, 61, 62, 68, 74, 77, 86, 87, 179, 180, 185-187, 199,
586, 600 202-204, 364, 369, 370, 441-443, 458, 541, 542
Protein kinase A, 429, 434, 436, 437 Sodding, 67, 88, 96-100, 363
Protein kinase C, 267, 316, 318, 323, 431, 437, 438, Sodium dodecyl sulfate, 208, 212, 215
586, 594 SPARC, 202
Protein phosphatase, 260 Stem cells, 186
Proximal anastomoses, 15 Stereotropism, 532
PTCA, 171, 229, 233, 237 Stress, 15, 19, 24, 27, 28, 38-40, 44-47, 51, 52, 54, 57, 58, 63,
R 65, 69, 72, 73, 75, 76, 89, 90, 104, 116, 118, 122, 123,
125, 130, 132, 138, 140, 153, 154, 174, 175, 177, 179,
Rabbit ear chamber, 327, 417-419, 422 187, 189, 190, 192, 194, 200, 206, 215, 220, 234, 235,
ras, 257, 266, 268, 414, 594 240, 241, 246, 248, 254-260, 262, 266, 268, 292, 304,
Rats, 18, 38, 40, 77, 102, 215, 224, 227, 232-234, 240, 252, 324, 329, 335, 337, 344, 348, 350, 351, 353, 359, 372,
322, 352, 404, 407, 408, 410, 413, 414, 416, 445, 377, 390, 425, 426, 428-433, 436-438, 441-454,
497-499, 501, 502, 511, 515, 516, 533, 588, 589, 611 456-458, 467, 470, 471, 474, 475, 478, 479, 494,
Reactive oxygen intermediates, 33, 219 497-499, 501, 508, 528, 531, 539, 540, 544, 555, 559,
Renin-angiotensin system, 232 581, 588, 594
Reporter proteins, 606 Stress cracking, 467, 470, 475, 479
Resorbable grafts, 282, 489, 490, 494, 498, 499, 501 Stroma cell, 372
Retinoic acid, 404, 405, 411, 412 Stromelysin-1, 316
Retrovirus, 606, 609, 611 Subcutaneous adipose tissue, 62, 121, 122, 124
620 Tissue Engineering of Prosthetic Vascular Grafts
Subcutaneous fat, 95, 99, 102, 103, 105, 107-111, 113, 115, Titanium, 388-390, 397, 460, 529, 536, 538, 542-544, 595
126, 139, 144, 154, 158 TNF-#, 30-32, 172, 173, 202-204, 213, 214, 220, 224, 313,
Subdermal vein, 163, 189 316-320, 389, 392, 396, 398, 417, 421, 422, 590
Supercritical fluid, 507, 511 Topographical morphology, 532
Superoxide, 172, 173, 219, 246, 438, 461 Toxicity, 89, 164, 166, 168, 225, 308, 513, 578, 611
Surface modification, 41, 67, 89, 111, 122, 143, 154, 465, tPA, 27-29, 52, 65, 117-119, 124, 160, 231-233, 238, 280,
466, 469, 470, 473, 482, 483, 484, 486, 487, 488, 508, 281, 314-318, 336, 428, 432, 597
510, 531, 545, 546, 549, 553, 555, 583, 591, 592, 593 Traction, 34, 48, 49, 57, 68, 202, 212, 234, 254, 288-291,
Surface modifying additives, 482 297-299, 322, 326, 328-333, 335, 336, 337-339, 342,
Surface morphology, 106, 183, 513-515 351, 352, 410, 413, 435, 476, 477, 507, 540, 568, 572-
Surface roughness, 513, 528, 529, 595 576, 591
Surface thrombogenicity, 189, 191, 215 Transcription factor, 52, 227, 244, 246, 307, 358, 398, 401,
Syndecans, 409, 585, 593 407, 409, 430, 431, 436, 438
Transdifferentiation, 35, 403-411, 413, 416, 417, 425
T Transduction, 52, 89, 138, 223, 224, 240, 245, 248, 251, 254,
Talin, 255, 256, 258, 260, 261, 268, 334, 354, 358, 427, 585, 256-258, 260, 262, 263, 265, 266, 268, 298, 307, 310,
593, 604 311, 315, 334, 340, 341, 351, 352, 359-361, 373, 378,
Tek, 405 401, 408, 411, 427, 430, 436, 437, 540, 543, 585, 586,
Tenascin, 40, 233, 239, 272, 275, 276, 334, 340, 355, 360, 593-595, 597, 606, 607
405, 409, 412-414, 589, 590, 592, 593, 596, 597 Transfection, 65, 66, 68, 162, 245, 246, 249, 284, 304, 310,
Tensile modulus, 472-475 411, 431
Tensin, 140, 158, 231-233, 237-239, 242, 248, 253, 255, 257, Transformtin growth factor-!, 28-30, 39, 41, 42, 44, 52, 89,
265, 266, 301, 334, 354, 408, 414, 585, 586, 610 168, 202, 203, 219, 225, 239, 242, 252, 263-266, 273-
TGF-!, 27-31, 35-37, 41, 42, 52, 158, 162, 201-203, 219, 275, 277, 280, 285, 289, 292, 294, 295, 298, 299, 302,
220, 224, 227, 233, 238, 242, 243, 252-254, 256, 263, 324, 335, 338, 352, 353, 359, 360, 369, 370, 374, 375,
276, 277, 280, 282, 285, 289-295, 297, 298, 299, 318, 413, 414, 417, 423, 478, 596
335, 337, 339, 341, 343-353, 364, 365, 367, 368, 370, Transmural ingrowth channels, 22-24
374, 375, 405, 407, 408, 410, 411, 415, 417, 421, 422, Transmural microvessel, 19, 379, 385
438, 496, 590, 596 Triglyceride, 165, 166, 168
Th2 lymphocytes, 33 Trypsin, 62, 96, 117, 133, 159, 161, 162, 164, 304, 336,
Thermoplastic, 473, 476, 479, 511 500, 611
Thrombin, 26, 29, 42, 63, 77, 81, 115-119, 139, 155, 161, Tumor necrosis factor alpha, 172
172, 201, 202, 216, 231, 232, 260, 263, 266, 268, 304, Type 1 plasminogen activator inhibitor, 322
308, 311, 320, 387-391, 398, 408, 434, 465, 467, 479, Type I collagen, 203, 266, 272-274, 276, 288-291, 293, 297,
481, 482, 546-551, 563, 568 298, 328-330, 332, 333, 335, 336, 338, 339, 350, 357,
Thrombin generation, 388-390, 398, 465, 546-548, 550 358, 361, 363-370, 433, 434, 508, 550, 569, 571, 572,
Thrombocytes, 8, 28, 116 575, 587, 590, 608
Thromboemboli, 79, 80, 88, 172, 387, 388, 400, 401 Tyrosine kinase, 138, 142, 162, 244, 253, 256, 257, 259, 267,
Thrombomodulin, 116, 117, 139, 162, 393, 394, 599 268, 290, 295, 306, 307, 324, 334, 335, 358, 374, 378,
Thromboplastin, 26, 388 405, 428, 435, 438, 585, 586, 593
Thrombospondin (TSP1), 27-30, 42, 74, 202, 233, 272- U
277, 320, 334, 339, 340, 355, 356, 359, 360, 415, 589,
590, 592, 596, 597 Ulex europaeus Type I Lectin, 106, 107
Thrombotic occlusion, 284, 393, 422, 482, 545 Umbilical vein, 50, 52, 58, 65, 66, 69, 94, 118-120, 122, 125,
Thrombus, 5, 7-10, 16, 27, 30, 40, 62, 63, 68, 80, 82, 88, 139, 140, 157, 160, 168, 175, 266, 273, 291, 310, 316,
128, 129, 133, 135-137, 139, 154, 172, 187, 215, 229, 321-323, 328, 335, 338, 342, 428, 431, 432, 437, 545,
231, 236, 372, 423, 490, 551, 553, 555, 558, 559, 569, 555, 578, 579
577, 605, 606 uPA, 29, 31, 118, 231-233, 238, 280, 313-318, 320, 321,
Thy-1, 110, 374 336, 337
Tight junctions, 103, 104, 140 uPA receptor, 313, 315-318, 336
Tissue factor, 28, 30, 42, 162, 253, 389, 392-394, 397, 398, Urokinase, 27, 29, 42, 65, 117-119, 231-233, 238, 254, 266,
409, 415, 580 280, 313-315, 317, 321-324, 336, 342, 354
Tissue inhibitors of MMPs (TIMP), 231, 233, 254, 316, Urokinase plasminogen activator, 29, 354
317, 336, 607, 608, 610
Tissue plasminogen activator, 27, 29, 52, 65, 68, 77, 154,
161, 266, 321, 610, 611
Index 621
V W
Vascular cell adhesion molecule-1 (VCAM-1), 74, Weibel-Palade bodies, 95, 96, 103-106, 111, 140, 158, 172,
172-174, 202, 273-276, 316, 393-395, 398, 399, 401 176, 426
Vasculogenesis, 268, 279, 281, 302, 324, 335, 341, 373, 379, Wettability, 68, 506, 510, 513, 515, 526, 527, 540
384, 385, 421-423 Wound healing, 29-31, 33, 34, 131, 138, 197, 198, 200-202,
VEGF, 27, 29, 32, 34, 35, 160, 202, 203, 252, 253, 255-257, 206, 212, 220, 221, 223, 225, 253, 256, 271, 272, 274,
260-264, 268, 279-286, 294, 295, 297, 302, 313, 316, 275, 277, 288-290, 294, 298, 302, 305, 311, 314, 318,
318, 320, 322, 337, 374-377, 404, 405 320, 321, 324, 352, 379, 407, 413, 416, 417, 422, 425,
Vein wrap, 380 441, 525, 540, 567, 570, 572-574, 596
Ventricular assist devices, 79, 88, 89, 372, 377, 380, 385,
387, 400, 401, 403 Y
Vimentin, 104, 105, 107, 108, 139 Yellow baboon, 7, 9, 11, 17, 18, 35, 36
Vinculin, 255, 256, 258, 260, 261, 267, 268, 323, 334, 354, YIGSR, 558, 559, 588, 591, 594, 595
358, 427, 534, 535, 537, 540, 542, 585, 586, 588,
593, 594
Vitronectin, 27, 74, 85, 165, 168, 254, 265, 272-275, 277,
314, 315, 318, 320, 322, 324, 335, 337, 355-357, 360,
428, 434, 534, 535, 542, 554, 584, 590, 594, 596, 597
von Willebrand factor, 28, 95, 100, 110, 133-135, 139, 140,
158, 273, 281, 292, 299, 335, 382, 390, 393, 394, 554,
590, 596, 597
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