Stem Cell Biology and Regenerative Medicine

Anna C. Berardi Editor

Extracellular
Matrix for Tissue
Engineering and
Biomaterials
Stem Cell Biology and Regenerative Medicine

Series editor
Kursad Turksen, Ph.D.
e-mail: [email protected]
Our understanding of stem cells has grown rapidly over the last decade. While the
apparently tremendous therapeutic potential of stem cells has not yet been realized,
their routine use in regeneration and restoration of tissue and organ function is greatly
anticipated. To this end, many investigators continue to push the boundaries in areas
such as the reprogramming, the stem cell niche, nanotechnology, biomimetics and
3D bioprinting, to name just a few. The objective of the volumes in the Stem Cell
Biology and Regenerative Medicine series is to capture and consolidate these
developments in a timely way. Each volume is thought-provoking in identifying
problems, offering solutions, and providing ideas to excite further innovation in the
stem cell and regenerative medicine fields.

More information about this series at http://www.springer.com/series/7896

Anna C. Berardi
Editor

Extracellular Matrix
for Tissue Engineering
and Biomaterials
Editor
Anna C. Berardi
Department of Hematology, Transfusional
Medicine and Biotechnology
Hospital Santo Spirito
Pescara
Italy

ISSN 2196-8985 ISSN 2196-8993 (electronic)

Stem Cell Biology and Regenerative Medicine
ISBN 978-3-319-77021-5 ISBN 978-3-319-77023-9 (eBook)
https://doi.org/10.1007/978-3-319-77023-9
Library of Congress Control Number: 2018933515

© Springer International Publishing AG, part of Springer Nature 2018

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part
of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations,
recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission
or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar
methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this
publication does not imply, even in the absence of a specific statement, that such names are exempt from
the relevant protective laws and regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this
book are believed to be true and accurate at the date of publication. Neither the publisher nor the
authors or the editors give a warranty, express or implied, with respect to the material contained herein or
for any errors or omissions that may have been made. The publisher remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.

Printed on acid-free paper

This Humana Press imprint is published by the registered company Springer International Publishing
AG part of Springer Nature
The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
Preface

The extraordinary amount of attention being paid to extracellular matrices, and to

tissue engineering in general, reflects the enormous potential for significant medical
breakthroughs in this field for the foreseeable future.
A vast number of experts from a variety of specialized areas have already
contributed to the understanding of the nature and processes involved in the
extensive field of tissue regeneration and repair and initial steps have been taken in
the attempt to imitate, control, or stimulate some of the ordinary, yet complex,
biological processes, and interactions, in living cell-structures.
This book, which attempts to “photograph” the state of the art of extracellular
matrices and tissue engineering, features contributions from some very eminent
minds involved in this field of research and from those who will most probably be
helping to carry this investigative work forward over the next several decades. The
variety of content aims to provide both detailed specific information on salient
points and a broad context of the range of issues pertinent to the subject area, with
the hope of enhancing understanding and stimulating further interest.
Greater awareness of the specific roles of the extracellular matrix in tissue
growth and regeneration is continually furthering our ability to design and create
artificial matrices and to develop and refine the necessary techniques and materials
to facilitate their use in a wide range of clinical practice.
It is hoped that by perhaps reaching out across neighboring disciplines the
present work might in some small way contribute to the great developments yet to
come.

Pescara, Italy Anna C. Berardi

v
Contents

Part I Extracellular Matrix

1 The Extracellular Matrix, Growth Factors and Morphogens
in Biomaterial Design and Tissue Engineering . . . . . . . . . . . . . . . . . 3
Caterina Bason, Marialucia Gallorini and Anna C. Berardi
2 ECM Hydrogels for Regenerative Medicine . . . . . . . . . . . . . . . . . . . 27
Michael J. Sawkins, Lindsey T. Saldin, Stephen F. Badylak
and Lisa J. White
3 Biologically Relevant Laminins in Regenerative Medicine . . . . . . . . 59
Anna Domogatskaya and Sergey Rodin
4 Extracellular Matrix: Immunity and Inflammation . . . . . . . . . . . . . 83
Amelia Cataldi and Viviana di Giacomo

Part II Material Inspired from Nature

5 Biologically Inspired Materials in Tissue Engineering . . . . . . . . . . . 113
Gianluca Fontana, Luis M. Delgado and Daniela Cigognini

Part III Nanotechnologies and Biomimetic

6 Advances in Nanotechnologies for the Fabrication of Silk
Fibroin-Based Scaffolds for Tissue Regeneration . . . . . . . . . . . . . . . 151
Nicolò Nicoli Aldini and Milena Fini
7 Nanoscale Architecture for Controlling Cellular Mechanoresponse
in Musculoskeletal Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Francesco Oliva, Clelia Rugiero, Umberto Tarantino
and Nicola Maffulli

vii
viii Contents

8 Modular Tissue Engineering: An Artificial Extracellular Matrix to

Address and Stimulate Regeneration/Differentiation . . . . . . . . . . . . . 191
Giovanna Della Porta and Ernesto Reverchon
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Contributors

Nicolò Nicoli Aldini Laboratory of Preclinical and Surgical Studies, Rizzoli Orthopedic
Institute, Bologna, Italy
Stephen F. Badylak Surgery and Bioengineering, University of Pittsburgh, Pittsburgh,
PA, USA
Caterina Bason Department of Medicine, University of Verona, Verona, Italy
Anna C. Berardi Research Laboratory “Stem Cells” U.O.C. Immunohematology,
Transfusion Center and Laboratory of Hematology, Hospital Santo Spirito, Pescara,
Italy
Amelia Cataldi University G. d’Annunzio, Chieti-Pescara, Italy
Daniela Cigognini Network of Excellence for Functional Biomaterials, National
University of Ireland, Dangan, Galway, Ireland
Luis M. Delgado Network of Excellence for Functional Biomaterials, National
University of Ireland, Dangan, Galway, Ireland
Giovanna Della Porta Department of Medicine, Surgery and Dentistry, University
of Salerno, Baronissi, SA, Italy
Viviana di Giacomo University G. d’Annunzio, Chieti-Pescara, Italy
Anna Domogatskaya Division of Transplantation Surgery, Department of Clinical
Science, Intervention and Technology, Karolinska Institute, Huddinge, Sweden
Milena Fini Laboratory of Biocompatibility, Innovative Technologies and Advanced
Therapies, Rizzoli RIT Department, Bologna, Italy
Gianluca Fontana Network of Excellence for Functional Biomaterials, National
University of Ireland, Dangan, Galway, Ireland
Francesco Oliva Department of Orthopaedic and Traumatology, School of
Medicine, University of Rome “Tor Vergata”, Rome, Italy

ix
x Contributors

Marialucia Gallorini Department of Pharmacy, University G. d’Annunzio Chieti-

Pescara, Pescara, Italy
Nicola Maffulli Centre for Sports and Exercise Medicine, Queen Mary University
of London, London, UK; Barts and The London School of Medicine and Dentistry,
Mile End Hospital London, London, UK; Department of Physical and Rehabilitation
Medicine, University of Salerno, Fisciano, Italy
Ernesto Reverchon Department of Industrial Engineering, University of Salerno,
Fisciano, SA, Italy
Sergey Rodin Department of Medical Biochemistry and Biophysics, Karolinska
Institute, Stockholm, Sweden
Clelia Rugiero Department of Orthopaedic and Traumatology, School of Medicine,
University of Rome “Tor Vergata”, Rome, Italy
Lindsey T. Saldin Department of Bioengineering, University of Pittsburgh, Pittsburgh,
PA, USA
Michael J. Sawkins Department of Anatomy, Royal College of Surgeons in Ireland,
Dublin 2, Ireland
Umberto Tarantino Department of Orthopaedic and Traumatology, School of
Medicine, University of Rome “Tor Vergata”, Rome, Italy
Lisa J. White School of Pharmacy, University of Nottingham, Nottingham, UK
Abbreviations

DC Dendritic cell
EAE Experimental autoimmune encephalomyelitis
ECM Extracellular matrix
GAG Glycosaminoglycan
HA Hyaluronan
LPS Lypopolysaccharide
MMP Matrix metalloproteinase
NO Nitric oxide
NOS Nitric oxide synthase
PPAR Peroxisome proliferator-activated receptor
TLR Toll-like receptor
TSP Thrombospondin
TIMP Tissue inhibitor of metalloproteinases

xi
 Part I
Extracellular Matrix
Chapter 1
The Extracellular Matrix,
Growth Factors and Morphogens
in Biomaterial Design and Tissue
Engineering

Caterina Bason, Marialucia Gallorini and Anna C. Berardi

Abstract Cells, morphogens, growth factors, and custom scaffolds are the critical
ingredients for successful tissue regeneration in which morphogens and growth
factors function sequentially. Extensive studies, in vitro and in vivo, have been
made to explore the mechanisms and the roles played by these molecules. As a
consequence, precise, localized control over the signaling of these factors and
appropriate strategy selection, depending on the tissue or organ to be repaired or
regenerated, is known to permit specific management of regenerative processes.
The first part of the chapter examines natural ECMs which are a set of molecules
secreted by cells that provide structural and biochemical support to the surrounding
cells. ECMs also perform many other functions, such as actively regulating cell
function through the control of biochemical gradients, cell density, spatial organi-
zation, and ligand attachment, thus influencing various types of cell processes.
Subsequently, growth factors and morphogens are examined in greater depth to
clarify to what degree progress has been made into improving methodologies and
functionality and, perhaps, to hint at what remains to be done for the future of tissue
engineering.

C. Bason
Department of Medicine, University of Verona, 37134 Verona, Italy
e-mail: [email protected]
M. Gallorini
Department of Pharmacy, University G. d’Annunzio Chieti-Pescara, Pescara, Italy
e-mail: [email protected]
A. C. Berardi (&)
Research Laboratory “Stem Cells” U.O.C. Immunohematology,
Transfusion Center and Laboratory of Hematology,
Hospital Santo Spirito, Pescara, Italy
e-mail: [email protected]; [email protected]

© Springer International Publishing AG, part of Springer Nature 2018 3

A. C. Berardi (ed.), Extracellular Matrix for Tissue Engineering
and Biomaterials, Stem Cell Biology and Regenerative Medicine,
https://doi.org/10.1007/978-3-319-77023-9_1
4 C. Bason et al.

Introduction

Over recent years, the therapeutic approach to repairing, regenerating, or replacing

tissues or organs damaged by disease or injury has evolved, developing a specific
biomedical field called “tissue engineering” (TE) in which cells, scaffolds, and
biologically active molecules are combined to functional tissue and organs [1].
Thus, the TE prototype depends on the successful interaction between three
components: (1) autologous or xenogenic cells from which the tissue itself will be
formed, (2) synthetic scaffolds that hold the cells together and shape the physical
form of the tissue, and (3) biological signaling molecules, such as growth factors,
which instruct cells to express a desired phenotype.
Adult stem cells or induced pluripotent stem cells have most recently been
favored, which can directly provide replacement cells for tissue and organs.
Coupling these with synthetic or natural biomaterials which mimic the tissue/
organ architecture and signaling permits cell adhesion, proliferation, and differ-
entiation into specialized cells. These TE scaffolds should be porous,
three-dimensional structures (3D) so as to match the porosity, pore size, and
interconnectivity of native tissue/organs, allowing and enhancing cellular attach-
ment and proliferation, and providing space in which new tissue growth and
vascularization can occur.
The critical ingredients for tissue regeneration are cells, morphogens, growth
factors, and scaffolds. In tissue regeneration, morphogens and growth factors
function sequentially. Extensive studies, in vitro and in vivo, have been made to
explore the mechanisms and the roles played by these molecules. As a conse-
quence, precise, localized control over the signaling of these factors and appropriate
strategy selection, depending on the tissue or organ to be repaired or regenerated, is
known to permit specific management of regenerative processes.
Examples of engineered tissues are artificial skin and cartilage, and muscu-
loskeletal substitutes that have been approved by the Food and Drug Administration
(FDA); however, currently they have limited use in human patients. Many other
examples have been detailed in the literature including: ligaments, tendons, and
bone, as well as more complex organs like hearts, lungs, kidneys, pancreases, and
liver tissue. All of these exhibit structural, mechanical, or metabolic function [2].
The ultimate goal of tissue engineering is to orchestrate body regeneration by
specifically controlling the biological environment.

Key Molecular ECM Components

Tissue and organ regeneration in the adult are complex processes that often rep-
resent a recapitulation of embryonic development and patterning processes and
which seek to repair tissue and/or to maintain integrity and function. Increasing
1 The Extracellular Matrix, Growth Factors … 5

understanding of how cells and biological systems respond to stimuli continues to

inspire attempts to successfully apply biological concepts in tissue engineering. In
order to optimize the properties of synthetic materials, greater comprehension of
the native microenvironment of the cells is required, since it is the dynamic
interaction of tissue-generating cells with the surrounding microenvironment,
which is largely made up of the extracellular matrix and other cell types (including
fibroblasts, macrophages, and plasma), which regulates tissue formation
characteristics.
Various strategies including a variety of materials and modulating agents, such
as ECM-inspired, biomaterial scaffolds, cells, morphogens, and growth factors,
have been progressively investigated and explored [3], in the attempt to stimulate
regeneration and repair damaged sites [4].
Natural ECMs are a set of molecules secreted by cells that provide structural
and biochemical support to the surrounding cells. Not only do ECMs structurally
support the cells, but they also perform many other functions. They actively
regulate cell function through control of biochemical gradients, cell density,
spatial organization, and ligand attachment, influencing various types of cell
behavior, such as proliferation, adhesion, and migration, and regulating cell dif-
ferentiation and death [5]. They, further, contribute to the mechanical properties of
tissues and even actively participate in the establishment, maintenance, and dif-
ferentiation of tissue and organs, modulating growth factors, the level of hydra-
tion, and the pH of the local environment. These provide a degradable physical
environment which permits neovascularization and remodeling as a response to
dynamic tissue processes such as wound healing [6, 7]. All these varied functions
are achieved by means of complex chemical composition and organization.
Despite the fact that the main constituents of ECMs are water, proteins, and
polysaccharides, ECMs are highly specific in their structural and biochemical
composition for each tissue and organ. All this is a result of the innate properties
of the interaction of those constituent molecules with the activities of resident
cells. Furthermore, the structure and the composition of ECMs are not static but
dynamic in response to environmental stimuli which may be physiological in
origin or stress-related.
An ECM is a complex network composed of fibrous proteins (including the
different types of collagen and elastin), and an interstitial component formed by
glycoproteins of adhesive nature (such as fibronectin, laminins, vitronectin,
thrombospondin, chondronectin, osteonectin, and fibrin). This is embedded in a
well-hydrated, viscoelastic gel, consisting of anionic proteoglycan polymers and
glycosaminoglycans (such as heparan sulfate, chondroitin sulfate, dermatan sulfate,
keratin sulfate, and hyaluronic acid) [8]. Precise categorization into only structural
or functional components of the ECM is impossible because many molecules play a
role in both aspects in healthy functioning processes and in disease states.
6 C. Bason et al.

Prominent Role of the Proteoglycans

and Glycosaminoglycans

Proteoglycans (PG) are composed of a protein axis to which one or more gly-
cosaminoglycan chains (GAGs) are covalently linked. Usually the same protein
binds to a single type of GAG. However, more than one type of GAG exists, such
as those found in cartilage-based proteoglycans, which contain similar quantities of
chondroitin sulfate and keratan sulfate [8]. GAG variety depends on the location of
the ECM, age, and gender of the individual. GAGs promote water retention and
contribute to gel-like properties of the ECM. GAGs also bind cytokines and growth
factors and retain them in the ECM [9]. Around 36 ECM PGs have been identified
in mammals with multiple, diverse functions, which also vary in different types of
tissue. There are three main families of PG: (1) small leucine-rich proteoglycans
(SLRPs), (2) modular proteoglycans, and (3) cell surface proteoglycans [10].
SLRPs, which are ubiquitously expressed in most ECMs, are the largest class of
PGs. They can function as structural components and are involved in multiple
signaling pathways. SLRPs bind with various collagens, tyrosine kinase receptors
(RTK), and innate immune receptors, and, in so doing, they participate in several
biological functions [11], being able to regulate fundamental processes like
migration, proliferation, innate immunity, apoptosis, autophagy, and angiogenesis
[11].
Furthermore, they bind to and activate epidermal growth factor receptors
(EGFR), insulin-like growth factor 1 receptor (IGF1R), and low-density lipoprotein
receptor-related protein 1 (LRP1).
Hyalectans, which are modular PGs, are key structural components of central
nervous systems, cartilage, and blood vessels and can modulate cell adhesion,
migration, and proliferation, by binding hyaluronan to form complexes of high
viscosity. The hyalectan family, found in interstitial membrane matrices, contains
aggrecan, versican, neurocan, and brevican [10]. The essential role of aggrecan, the
principal load-bearing proteoglycan in cartilage, has been confirmed by several
studies. Versican, a large chondroitin sulfate proteoglycan, forms long filaments in
the ECM and has been shown to play an important role in modulating inflammatory
responses to tissue injury. Basement membrane modular PGs (perlecan, agrin, and
collagen type XVIII) have a dual function as pro- and anti-angiogenic factors [12],
mediate ligand, cell–matrix and cell–cell interactions, and interaction with integrins
and RTK.
Heparan sulfate (HS) binds a large number of extracellular proteins. The func-
tions of heparan sulfate-binding proteins range from extracellular matrix compo-
nents to enzymes and coagulation factors, as well as most growth factors, cytokines,
chemokines, and morphogens. For example, heparin sulfate proteoglycans (HSPGs)
bind FGFs and sequester these molecules for storage. HS regulates a wide range of
biological activities, including developmental processes, coagulation, and angio-
genesis [13].
1 The Extracellular Matrix, Growth Factors … 7

The heparan sulfate (HS) proteoglycan, perlecan, is ubiquitously found on the

apical cell surface and in the basement membrane of both vascular and avascular
tissues. Perlecan regulates a variety of biological processes, such as modulating cell
adhesion, thrombosis and cell death, skin and endochondral bone formation, and is
involved in the biomechanics of blood vessels and cartilage [14]. Perlecan binds
and modulates the activity of several growth factors and morphogens. It is a
complex regulator of vascular biology and tumor angiogenesis [11, 15, 16].
Moreover, several studies confirm the role of HS perlecan in modulating
pro-angiogenic factors such as FGF2, VEGF-A, and PDGF.
Cell surface PGs (syndecans and glypicans) connect the surface of cells to the
underlying extracellular matrix and can act as co-receptors, facilitating ligand
encounters with signaling receptors [10].

Fibrous Proteins and Adhesive Glycoproteins

ECMs are critical modulators of connective tissue remodeling by binding to and

activating TGF-b and bone morphogenic proteins (BMPs), which may then influ-
ence health or contribute to disease. TGF-bs, a family that includes TGF-b-1, -2, -3
and BMPs, have a prominent role in ECM metabolism, as a major inducer of
collagen synthesis [17–19]. Collagens are the most abundant proteins in the
mammalian ECM. Collagen consists of molecules that form a triple-stranded helix
to form stretch-resistant fibers that provide tensile strength to tissues. To date, 28
different types of collagen have been identified and described. Type I collagen is the
major protein present in ECM of most tissues, the other collagens being expressed
in lower quantities. For example, in tendons and ligaments, greater than 90% of the
connective tissue is composed of type I collagen. These different compositions
provide distinct mechanical and physical properties to the ECM and contribute to
the interaction between ligands and local cell population. Moreover, collagen is
closely associated with glycoproteins, growth factors, and other structural proteins,
such as elastin and laminin, in the generation of tissue types with specific char-
acteristics and/or properties.
Elastin is another significant component of elastic fibers in matrix tissue and
permits tissues to return to their original form after stretching or contracting. Elastin
is one of the most chemically resistant and durable proteins in the body because of
its high content of hydrophobic amino acids. PGs, including glycosaminoglycan
and in particular the heparin sulfate, have been detected within the elastic core and
they have been reported to regulate the assembly of elastin [20, 21]. Water man-
agement plays an important role in three-dimensional elastin organization and in
determining the degree of hydration and elasticity in tissue.
Fibronectin is second only to collagen within the ECM and may enhance cell
position, and, by spreading, support cell division and migration. Fibronectin is
necessary for the development and morphogenesis of a number of tissues and organs
throughout embryogenesis. Indeed, some studies report that, during cardiac
8 C. Bason et al.

development, precursor cells require fibronectin to complete their migration and in

the absence of this matrix protein epithelial organization is disrupted [22, 23]. It
probably also plays a role in cellular morphology, in directing cellular differentiation
[24], and wound repair. Fibronectin is important in cell adhesion through the
alpha5beta1 integrin, since it is secreted in an inactive soluble form and is activated
by interaction with an integrin [25]. The important role of this protein in matrix
assembly lies in its ability to bind simultaneously to cell surface receptors, such as
integrins, and to collagen, to PGs, and to other adhesion proteins. Fibronectin
domains have been found to bind to a number of different growth factors, including
vascular endothelial growth factor (VEGF) and hepatic growth factor (HGF).
Laminins are made up of a glycoprotein family with a cross-shaped structure, and
their interaction creates a network that provides adhesion between dissimilar tissues.
These proteins are required for the correct healing of tendons and other connective
tissues. Furthermore, laminins are an important, biologically active part of the basal
lamina, and they play important roles in tissue structure and maintenance and in cell
signaling, adhesion, and migration, among other functions [26–28].
Thrombospondins (TSPs) are a group of five modular glycoproteins and are
considered to be “adhesion-modulating” components of the ECM which are able to
modulate cell functions in a variety of tissues [29]. TSP-1 plays an important role in
wound healing by activation of TGF-b during early tissue repair, but in later stages
persistent production of TSP-1 may lead to fibrosis. Moreover, TSP-1 regulates the
activity of several other growth factors, such as VEGF, EGF, and PDGF. TSP-1
binds to a number of cell membrane receptors, integrins, and HS and has been
shown to upregulate type I collagen expression [30]. Contrastingly, TSP-2 is
involved in collagen fibril assembly, but is not able to activate TGF-b.
Fibrinogen is a complex fibrous glycoprotein and is converted by thrombin into
an insoluble fibrin polymer. Fibrinogen and fibrin bind specifically to a variety of
other proteins, like fibronectin, thrombospondin, von Willebrand factor, fibulin,
FGF-2, VEGF, and interleukin-1 (IL-1). This ability to bind to different com-
pounds, particularly to growth factors, gives fibrinogen an important role in car-
diovascular and extracellular matrix physiology [31].
Since it enables cellular attachment, proliferation, and three-dimensional
arrangement, fibrin is generally considered to be a good basic scaffold material.
On one hand, fibrin’s high biocompatibility is advantageous; however, on the other
hand, it suffers from poor biomechanical stability.

Growth Factors

Growth factors (GFs) (soluble signals) are the major regulators of cell behavior;
they promote cell proliferation, migration, and differentiation through specific GF
receptor binding which stimulates cellular signal transduction pathways. GFs are
involved in several physiological and pathological processes, such as tissue repair
and maintenance [32]. GFs are secreted from the cells directly or are sequestered by
1 The Extracellular Matrix, Growth Factors … 9

ECM for presentation to cell surface receptors. Some studies demonstrate that GF
can be released from ECM by the degradation of ECM proteins, of GAGs, or of
PGs. The ability of growth factors to interact with the extracellular matrix is a
dynamic, tissue-specific property [33–35]. GFs, ECM components, and cell surface
receptors form complexes that may lead to additive or synergistic cell signaling
events. The ECM can regulate GF-mediated cell function [36] through many
insoluble (ECM-bound) and soluble (un-bound) cell secretions, through cell surface
proteins and through proteoglycans. Specifically, the ECM regulates GF activity by
sequestering soluble GFs and by cell-demanded release via enzymatic degradation
of the ECM, eliciting a variety of effects on GF signaling that are dependent on the
context and presentation of the GF to cells [36–38]. Both cytokines and growth
factors are present within the ECM in very small quantities, but act as potent
modulators of cell behavior. The list of GFs found within the ECM is substantial
and includes vascular endothelial cell growth factor (VEGF), transforming growth
factor beta (TGF-b), the fibroblast growth factor (FGF) family, insulin-like growth
factor (IGF), epithelial cell growth factor (EGF), keratinocyte growth factor (KGF),
hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), bone
morphogenetic proteins (BMPs), and many others. Multiple isoforms of these
molecules are present, each with its own specific biological activity. VEGF, which
has been the subject of a number of studies, is the major inducer of endothelial cell
homeostasis and of angiogenesis and is an example of context-dependent GF sig-
naling. Soluble and insoluble ECM components regulate VEGF activity in various
ways. Several distinct VEGF genes produce multiple VEGF isoforms which exhibit
variable binding domains for heparan sulfate (HS). Furthermore, every VEGF
isoform plays a distinct role in vascular patterning and arterial development in the
ECM [39, 40]. VEGF induces a1b1 and a2b1 integrin expression in microvascular
endothelial cells, as well as endothelial cell migration and proliferation. Although
the major role of VEGF is related to vascular tissue, several studies have shown
VEGF to also be implicated in bone cell recruitment, activity, and survival [41].
VEGF binding to the cell surface or to ECM and various MMPs and plasminogen
activators can generate diffusible, non-heparin-binding fragments. ECM component
fibronectin can be modified in the presence of heparin or HSPGs which expose a
binding site for VEGF [42–44]. The binding of VEGF to the ECM enhances
endothelial cell proliferation, probably through an increase in mitogen-activated
protein kinase (MAPK) activity [5, 45]. VEGFR-2 and VEGFR-1 tyrosine kinase
receptors mediate VEGF activity [40]. VEGFR-1 has minimal kinase activity, but a
high affinity for VEGF suggesting that it serves as a VEGF sink, preventing VEGF
binding to the active VEGFR-2 receptor [40]. While VEGFR-2 mediates most of
the endothelial growth and survival signals, VEGFR-1-mediated signaling plays an
important role in certain pathological conditions such as inflammation and cancer.
VEGF–VEGFR-2 binding on the cell surface leads to AKT activation and calcium
release, whereas internalization of the receptor complex triggers ERK signaling in
early endosomal compartments [46, 47]. Internalization and trafficking of
VEGFR-2 can be influenced by many factors, including neuropilin-1 (NRP-1),
syndecans, and hypothetically by b1-integrin [46, 47]. The activation of VEGFR-2
10 C. Bason et al.

and its downstream targets leads to increased permeability, proliferation, migration,

and survival of endothelial cells [40]. Matrix metalloproteinases (MMPs) have also
implicated in the release of VEGF from ECM stores. Degradation of VEGF by
MMPs, specifically MMP-1, -3, -7, -9, -16, -19, and MT1-MMP, regulates VEGF
bioavailability. Proteolytic cleavage of insoluble VEGF and FGF by MMPs
mobilizes these molecules, separating them from the ECM and allowing them to
become soluble and spread. Consequently, they are therefore able to interact with
specific cell membrane receptors and thus regulate their bioavailability [48].
TGF-bs family is crucial in development, wound healing, immune response, and
tumor genesis [49]. The ECM helps impose tight regulation over the activation and
activity of TGF-b, and there are multiple levels of posttranslational regulation.
During biosynthesis, pro-TGF-b is initially associated with its pro-peptide,
latency-associated peptide (LAP). This complex also binds to latent TGF-b-binding
protein (LTBP) to form the large latent complex, which then binds to ECM pro-
teins. MMPs and ADAMTSs help regulate TGF-b activity by cleaving ECM fibers
and increasing its bioavailability. It has been demonstrated that MMPs-2, -9, -13,
and MT1-MMP can proteolytically process the latent TGF-b, thus activating a
highly important biological factor. TGF-b availability may also be indirectly af-
fected by MMPs, by MT1-MMP, and MT3-MMP-regulated shedding of the
TGF-b-binding, membrane-anchored, proteoglycan b-glycan [50]. In addition,
several matrix proteases can activate TGF-b by cleaving latency peptides.
Interestingly, there is also evidence suggesting that the mechanical stiffness of the
ECM may lower the activation threshold of TGF-b. In this model, stiff ECM
provides additional resistance to cell pulling and induces a conformational change
in LAP facilitating release. During tissue repair, TGF-b stimulates fibroblasts and
myofibroblasts, signaling through the SMAD pathway, to express numerous
ECM-related genes including those encoding for collagens, TIMPs, and MMPs. In
this way, TGF-b contributes to the deposition of newly synthesized ECM following
tissue damage and remodeling of the ECM. Although TGF-b signaling is critical for
successfully repairing damaged tissues, dysregulation of this pathway can lead to
tissue fibrosis. Persistent inflammation, immune activation, and fibroblast stimu-
lation via TGF-b can lead to excess deposition of ECM proteins and the generation
of fibrotic tissue. TGF-b modulates nearly all stages of the immune response from
early immune to later adaptive response and modulates immune cell activation,
proliferation, and differentiation. Since active TGF-b participates in a number of
important biological processes such as embryogenesis and wound healing, its
deregulation has been correlated with numerous pathological states [50].
Insulin-like growth factor binding proteins (IGFBPs) are a family of proteins
with six distinct subgroups (IGFBP-1 to -6), which bind to insulin-like growth
factors (IGF-I and IGF-II) with high affinity and modulate the biological function of
IGFs. The N-terminus is the primary binding site for IGF. The C-terminus is also
required for IGF binding, as well as for binding to the extracellular matrix [51].
IGFBPs can stimulate the proliferation and migration of a number of cell popula-
tions and also play an important role in bone metabolism. Some studies have shown
a correlation between the local concentration of IGF and new bone formation and
1 The Extracellular Matrix, Growth Factors … 11

mineralization. Two mechanisms of IGF release have been identified: the ECM
binding and the IGFBP proteolysis [52]. These two actions lower the affinity of
IGFBPs for IGFs increasing the local concentration of bioactive IGF. IGFBP-2, -3,
-5, and -6 interact with GAGs [53–55]. IGFBP-2 interacts with heparin-binding
surface-pH-dependent and with GAGs; these process may be relevant where
extracellular pH is low, like in site of wound healing. Interestingly, IGFBP-2 and -6
bind a broad range of GAGs whereas IGFBP-3 and -5 preferentially bind to hep-
arin, heparan sulfate, dermatan sulfate, and minimally to chondroitin sulfates and
hyaluronic acid [56]. These differences in GAG binding specificity confer
tissue-specific actions upon the IGFBPs. GAG binding can also alter IGFBP
interactions with other ligands, thereby modulating the IGF-independent actions of
IGFBPs. For example, activation of plasminogen to plasmin is promoted by the
interaction of IGFBP-5 and plasminogen activator inhibitor-1 (PAI-1), leading to
ECM degradation and remodeling independently of IGF-I. This process is impor-
tant in mammary gland involution. Further interaction with ECM proteins via
IGFBP C-domains has been reported [57]. For example, IGFBP-3 binds fibrin/
fibrinogen, fibronectin, vitronectin [58], and plasminogen, which then can influence
both its IGF-dependent and IGF-independent actions. Also, fibronectin binds
IGFBP-5 and inhibits its ability to promote IGF-dependent cell migration [59].
IGFBPs are well-known MMP substrates. MMPs cleave IGFBPs predominantly
in the linker domain and are not dependent on IGF binding [52, 60]. Proteolysis
provides a mechanism by which the concentration of freely bioavailable IGFs is
increased, leading to subsequent activations of IGF1R. Specifically, the processing
of IGFBP-3 and -5 by MMP-1,-2, -3,-7, and -19 and IGFBP-1 by MMP-3, -9, and -
11 releases active forms of IGFs [50]. The dual effect of IGFs on MMP expression
has also been observed: IGF-1 can upregulate MMP-2 synthesis via PI3-kinase/Akt/
mTOR signaling while concomitantly transmitting a negative regulatory signal via
the Raf/ERK pathway [50].

Protease Activity and Role of Proteolytic Enzyme in ECM

Remodeling enzymes, which are capable of modifying and degrading ECM pro-
teins, are central to the interactions between host immune systems and ECMs.
Migration of immune cells into areas of disease-induced tissue damage is facilitated
by localized ECM breakdown. Additionally, the innate immune system initiates
responses to infection; thanks to products of ECM degradation which can serve as
early signals. In response to injury and infection, ECM remodeling enzymes con-
tribute to modulation of inflammation and tissue repair processes. In this case, most
of these remodeling enzymes are produced by immune cells, and in particular
myeloid lineage immune cells [61].
There are a large number of molecules which play a role in protease activity and
are also involved in proteolytic processes in the ECM, which can be divided into
12 C. Bason et al.

three main groups: (1) serine proteases; (2) matrix metalloproteinases (MMPs),
which are a large family of highly conserved, Zn-dependent endopeptidases;
(3) bone morphogenetic protein 1 (BMP1), which is a member of the tolloid family
of metalloproteinases.
These proteinases have been linked to cellular differentiation and pattern for-
mation through a role in activating latent growth factors in the TGF-b superfamily.
Further, a disintegrin and metalloprotease proteins (ADAMs) are a family of
transmembrane glycoproteins with diverse roles in cell–cell adhesion and proteol-
ysis. MMPs/ADAMs are a family of zinc-dependent, ECM-degrading endopepti-
dases that share common functional domains, activation mechanisms, and,
collectively, have the capability to degrade all types of ECM protein [61]. In
addition to playing a central role in ECM macromolecule turnover, within tissue
microenvironments MMPs/ADAMs can modify extracellular, soluble, or
membrane-bound proteins and induce their rapid release or inactivation. MMPs,
which were initially recognized for collagen remodeling, have now been shown to
play a significant role in the control of immune responses. These enzymes can
proteolytically activate or degrade a variety of non-matrix substrates, including
signaling molecules such as growth factors, chemokines, and cytokines.
Moreover, the main proteolytic enzyme families, MMPs and ADAMs, are
considered to be the predominant proteases in ECM pathophysiological regulation.
Each individual MMP influences the properties of ECMs differently, generating
distinct chemical, biomechanical, and morphological features in the ECM [61]. The
effects produced by a specific MMP through selective ECM degradation uniquely
influences cell behavior, including migration, morphology, gene expression profile,
and activation of intracellular cascades. In addition to degrading ECM proteins,
MMPs modulate a variety of biological factors and non-ECM molecules, directly
influencing tissue homeostasis. Collagenolysis and elastolysis by MMPs occur
during development, wound healing, and in major inflammatory diseases. MMP-2,
MMP-7, MMP-9, MMP-12, and MT1-MMP have been suggested to be elastolytic.
Controlled proteolytic cleavage of ECM protein-release growth factors, such as
FGFs, contributes to localized cell proliferation and differentiation.
Studies have shown that certain cytokines and growth factors, including insulin
growth factors (IGFs), epidermal growth factors (EGFs), vascular endothelial
growth factor (VEGF), transforming growth factor beta (TGF-b), fibroblast growth
factors (FGF), tumor necrosis factor-a (TNF-a), and interleukin-1b (IL-1b), can
stimulate expression of MMPs and ADAMs [61].

Extracellular Vesicles in the ECM Structure/Organization

Extracellular vesicles (EVs) are lipid bilayer membrane-enclosed vesicles released

by virtually all cells types as mediators for intercellular communication. They are
highly heterogeneous in size (ranging from *50 nm to >1 µm, with the vast
1 The Extracellular Matrix, Growth Factors … 13

majority <200 nm) and in molecular composition, carrying functional proteins,

DNA, miRNA, ncRNA, and lipids that they transfer into the target cells. Based on
recent studies, EVs can be considered to be one of the structural and functional
components of the ECM that participate in matrix organization, in the regulation of
cells within the ECM, and in determining the physical properties of soft connective
tissues, bone, cartilage, and dentin. Studies have demonstrated that a variety of
mechanisms regulate EVs-ECM communication: EVs act as key structural and
functional components of the ECM and are able to transport and distribute the
components of the ECM, in terms of both original structure and target tissue
modulation and reorganization, and, furthermore, EVs modulate the matrix com-
position by means of the proteinases and signaling molecules which they carry.
Extracellular vesicles are integral and functional components of the extracellular
matrix [63]. More recently, it has been shown that the structural components of a
decellularized heart have direct effects on the differentiation of stem/progenitor cells
and better preserve the mechanical properties of the heart compared to synthetic
scaffolds [64]. Minae An et al. found EVs with a large amount of microRNA
(miRNA) and exosomal protein derived from decellularized cardiac ECM
(ECM-EVs) [65]. A miRNA is a small noncoding RNA molecule containing about
22 nucleotides that mediates RNA silencing and posttranscriptional regulation of
gene expression [66]. A large number of miRNAs have been reported to play a role
in modulating cardiac regeneration; and a similar range of miRNAs is present in
ECM-EVs from decellularized hearts. An et al. [65] demonstrated that
ECM-derived miRNAs could participate in cardiac regeneration. In the context of
the extracellular matrix, a great deal of further research into EV function and
interaction will be crucial to understanding the range of biological effects and
consequences of EVs and in revealing their full impact on countless potential
applications for a wide range of diagnoses and therapies, including on tissue
engineering.

Importance of ECM–Integrin Interactions

In addition to serving as a reservoir of growth factors and cytokines that modulate

cell functions, the ECM interacts directly with cells and directs cell motility through
integrins expressed on the surface of numerous cell types. The ECM itself provides
a scaffold that cells utilize for their migration. While the ECM serves as a guide for
some cell types, it can also serve as an obstacle, for example, to neutrophils that
migrate to the site of infection. Integrins are a family of heterodimeric, trans-
membrane glycoproteins, are the most abundant cell surface receptors, and are
expressed in all cell types except for erythrocytes. Integrins are composed of two
type I transmembrane glycoproteins, the a and the b subunits, by non-covalent
association. The extracellular domain interacts with ECM proteins such as laminins
and collagens in basement membranes or with fibronectin in connective tissue
14 C. Bason et al.

components, while the intracellular domain interacts with the cytoskeletal proteins
like actin, affecting polarization and motility. For example, some integrins appear to
only be linked to one type of ECM protein (fibronectin or laminin), whereas others
may interact with a number of types (such as collagen, fibronectin, and laminin). It
is also possible that a single integrin may bind to several distinct ligands and that an
individual ligand may be recognized by more than one type of integrin.
A significant number of cytoskeletal and signaling proteins have been reported to
bind to b-cytoplasmic tails, and some have also been found to interact with specific
a tails. Moreover, alternative mRNA splicing, in extracellular, as well as in intra-
cellular, regions, produces a large number of variant forms of a and b subunits.
Differently spliced isoforms of both a and b subunits can differ in their effect on
signal transduction pathways, or in their specificity and affinity for ligands and
interaction with cytoskeletons. The preferential selection of various splice variants
in specific types of cells at precise differentiation stages leads to significant func-
tional differences. For example, a7b1 integrin can be detected in proliferating and
adult myofibers [67], and it is reported that a7A has a minor function in mature
muscle fibers, but a relevant role in regenerating muscle fibers [68, 69].
Furthermore, integrin-mediated adhesion modulates signaling cascades in the
control of cell survival, proliferation, differentiation, and motility. Migration of
adhesive mesenchymal cells depends on ECM proteolysis and the interaction of
integrins with ECM proteins. In addition, integrins are also involved in leukocyte
interactions with the endothelial basement membrane and the transmigration of
these cells to sites of inflammation. Some integrins even bind to receptors present
on other cell types, such as intercellular adhesion molecules (ICAMs) or vascular
cell adhesion molecules (VCAMs) which are expressed on leukocytes and
endothelial cells. There is evidence suggesting that lymphocyte motility and
retention in certain compartments is influenced by integrin–ECM interactions, in
particular, in inflamed tissues with altered ECM composition and integrin expres-
sion, for example in the context of influenza infection. Another important role of
integrins is in the activation of ECM-bound cytokines and growth factors. For
example, the a4b6 integrin activates latent TGF-b regulating the spatial bioavail-
ability of the growth factor. Integrin activation introduces another level of regula-
tion of ECM-bound molecules. The effect of a4b6 activation of TGF-b is likely to
be context dependent. An early study found that mice lacking a4b6 develop airway
hyper-responsiveness due to infiltration of inflammatory cells into the lungs and
skin. Activation of TGF-b by a4b6 may also contribute to fibrotic lung disease
following influenza infection due to increased collagen deposition. Integrins con-
tribute to development and tissue morphogenesis and also play a key role in tissue
homeostasis and tissue repair. The presence of cytokines and growth factors in the
ECM provides a means for host cells to rapidly respond to infection or injury as
these molecules are released and/or activated. In this manner, these ECM-bound
molecules may represent some of the earliest signals to the host immune system to
further rapid responses.
1 The Extracellular Matrix, Growth Factors … 15

Mechanical Signals Inside the ECM

The process of translating mechanical signals into a cellular response is called

mechano-transduction [70]. Our cells actively sense and respond to a variety of
mechanical signals. They experience numerous mechanical stimuli, like shear
stresses from blood flow and stretching forces from tissues associated with mus-
cular activity [71]. ECM stiffening in disease states (e.g., cancer and fibrosis), or
due to aging processes, can adversely affect cell migration, differentiation, and
proliferation [72, 73]. In this light, the ECM is more than just a passive network of
ligands for cell attachment. It carries different types of mechanical signals, and it
provides dimensionality. Focal adhesions (FAs) result from the complex interaction
of hundreds of different trans-plasma membrane integrins and cytoplasmic proteins,
and their exact composition varies in response to physical stimuli
(mechano-transduction). The complexity of the composition and the dynamics of
FA implicate intelligently designed, intricate, molecular interplay. FAs mediate the
strength of cell adhesion, and cell migration, as well as mechano-sensing and
signaling. Moreover, FAs transmit information in a bidirectional manner between
the ECM and cytoplasm [74, 75]. At adhesion sites, integrins connect the ECM to
the F-actin cytoskeleton and transduce the mechanical forces (generated by the actin
retrograde flow and by myosin II) to the ECM through a group of
mechano-sensitive FA proteins, termed “molecular clutch” [76]. Vinculin, which is
prevalent at the distal ends of microfilament bundles on the cell membrane, binds a
variety of FA proteins, thus mediating certain distinct cellular functions [77]. Since
being identified as a component of focal adhesions and adherens junctions, vinculin
has emerged as one of the principal components of the mechano-sensory machinery
[78]. While it is clear that mechanical stimuli can have profound effects on cell
behavior, the mechanisms that translate these forces into biochemical signaling
remain largely unknown.
YAP and TAZ represent a signaling nexus which integrates mechanical and
biochemical signals, influencing the stiffness of the ECM, adhesion ligand density,
and cell–cell contacts. They are powerful regulators of cell proliferation and sur-
vival and play important roles in controlling organ growth, stem cell self-renewal,
and cell differentiation [76, 79, 80].
The sensitivity of cells to forces and substrate stiffness has been recognized as a
powerful tool in tissue engineering, where it can be harnessed to design biomate-
rials that optimally guide stem cells or resident cells toward generating functional
replacement tissue. For instance, cell-derived matrices (CDM) are becoming an
attractive alternative to conventional biological scaffolding platforms due to their
unique ability to closely recapitulate the native extracellular matrix [81].
16 C. Bason et al.

Morphogenesis

Morphogenesis is the developmental cascade of pattern formation, body plan

establishment, and tissue differentiation which culminates in the adult form, and its
principles are applicable to tissue engineering for regenerative medicine and sur-
gery. Responding stem cells, morphogens, (morphogenetic protein signals), and
other bioactive ECM components can be integrated into materials for functional
tissue restoration.
Morphogenesis not only concerns the formation of embryonic tissues and organs
but also maintenance, degeneration, and regeneration in adult tissues.
As mentioned before, ECMs play an important role in many cellular processes,
such as growth, differentiation, and survival, and their precise characteristics can
play a critical role during morphogenesis. The ECM affects cell morphogenesis and
differentiation, providing support, tensile strength, and scaffolding for tissues and
cells, as well as supplying biochemical signals (i.e., growth factors, chemokines,
and cytokines). Cells remodel and reshape the ECM by degrading and reassembling
it, thus playing an active role in sculpting their surrounding environment and
directing their own phenotypes [82].
Integrins contribute to most, if not all, of the morphogenetic events that shape a
complex, multicellular organism in development, in tissue formation, homeostasis,
and repair [82]. For example, during vasculogenesis, endothelial cells migrate,
proliferate, and form 3D tubular structures. This tubular morphogenesis requires
integrin receptor signaling to regulate cell shape through changes in the
cytoskeleton and cell–cell interactions that control the shape of the tubules [83].
Similarly, morphogenesis of branching organs, such as the salivary gland, lung,
breast, and kidney, as well as prostate and pancreas, is dependent on the multiple
downstream activities related to ECM and integrin receptor interactions [84].
Cell adhesions participate in these dynamic rearrangements and maintain tissue
integrity throughout adult life. During collective cell movements that drive changes
in tissue shape, cell–cell adhesions must be remodeled, broken down, or reinforced
depending on the cellular behavior required. For example, cell adhesion to specific
ECM proteins may modify gene expression and alter levels of adhesion molecules
or other regulating proteins [85]. Alternatively, engagement or disengagement of
one type of adhesion might modify the functional activities of another by effecting
changes in membrane trafficking, cytoskeletal association and/or avidity or binding
affinity [86]. For a given morphogenetic movement, the regulation of
cadherin-based adhesions may be implicated in establishing cell polarity,
mechanically coupling neighboring cells, and/or directing cell migration. These
interactions involve the convergence of independently initiated cell signaling
events, often involving downstream effectors that are common to both cadherin
adhesions and integrin. Cadherin adhesions and integrin, which often share sig-
naling effector molecules, are both transmembrane adhesion receptors, both link to
common scaffolding and cytoskeletal elements and can both influence certain
crucial downstream functions, such as cell growth, survival, and transcriptional
1 The Extracellular Matrix, Growth Factors … 17

activity [87]. However, integrin and cadherin interaction may involve not only
shared cytoskeletal linkages, cell–cell, or cell–ECM engagement, but can also
potentially interact with adaptor proteins [i.e., tetraspanin or growth factor receptors
like insulin growth factor I receptor (IGF1R)] which facilitate lateral association of
integrins and cadherin. RhoGTPases are central to cell signaling pathways both
upstream and downstream of cadherin and integrin adhesions [88, 89], making
them prime candidates for mediating integration of adhesion-dependent
signals [90].
Bone morphogenesis is induced by bone morphogenetic proteins (BMPs), which
play a role in pattern formation, cell differentiation, maintenance, and regeneration
of tissues. BMPs are pleiotropic and act on chemotaxis and mitosis and can dif-
ferentiate progenitor cells and stem cells, which may also be programmed by
BMPs. The name is slightly misleading as their range of action also extends beyond
simple bone formation in that they play a role in the development of the teeth, heart,
kidneys, eyes, skin, and brain. The ECM, the native scaffold, can be used to deliver
morphogens such as BMPs for tissue engineering [91].

Morphogenesis of 3D Tissue Architecture in Vivo: Folds,

Tubes, and Branches

Many morphogenetic processes begin with a flat or curved sheet of cells that
eventually gives rise to complex topologies such as folds. Folds can be generated by
a monolayer of cells, by stratified cell sheets, or by multiple interacting tissues.
Consequently, cell–cell adhesions must play different roles depending on the cel-
lular behaviors required. Although the precise mechanism remains unclear,
E-cadherin, for example, is required for fold formation during embryogenesis [92].
It may be involved in transmitting mechanical cell–cell cues and could play a role in
establishing appropriate cell polarity.
Tubulogenesis is generally initiated by tissue folding, usually generating tubes of
larger diameters, and is completed by the formation of new adhesions to seal the
tube [93]. Proper distribution and transmission of tension require precisely con-
trolled adhesion between cells, in blood vessel tubulogenesis; for example, cell–cell
adhesions participate in establishing polarity and may also act as mechano-sensors
linking blood flow to vascular remodeling. Vascular endothelial (VE)-cadherin and
apical–basal polarity are required for endothelial cells to form lumena [94, 95].
Polarity is established through interactions between VE-cadherin and cell polarity
complex proteins Par3 and Pals1 [95]. Without proper localization of cell–cell
junctions and establishment of cell polarity, lumen formation and, therefore, blood
flow are prevented and blood flow is, itself, a major determinant of vascular
morphogenesis, including sprouting and lumen elongation [96, 97]. Pressure and
shear stress cause changes in endothelial cell behavior. These mechanical signals
18 C. Bason et al.

may be transduced (via VE-cadherin), leading to cytoskeletal and junctional

remodeling [98].
Branching morphogenesis requires the coordinated interplay of multiple cell
types with the ECM. Organs morphology is highly varied, ranging from “lobular”
in salivary glands to “tubular” in blood vessels [99]. The morphogenesis of a
branched organ requires both formation of new branches and remodeling of existing
branches. Branch formation in organs occurs through “budding” or “clefting.”
Budding is novel branching from the surface of an epithelium or from the side of a
branch, whereas clefting splits a branch tip into, usually, two or three tips.
Branched organs contain multiple cell types plus their ECM. The core structure
of all branched organs consists of tightly bound epithelial cells. The epithelium is
surrounded by the basement membrane, a dense network of ECM glycoproteins
and proteoglycans, beyond which there is a loosely organized mixture of mes-
enchymal cells. During branching, epithelial cells actively interact, both bio-
chemically and biophysically, with the ECM and other cell types. Although
mechanisms remain unclear, numerous growth factors and regulatory signaling
pathways have been implicated, in branching morphogenesis. The formation of
most branched organs requires one or more tyrosine kinase receptor
(RTK) signaling pathway [40, 100–102]. RTK signaling modulators have been
identified in the mesenchyme (which produces the ligands) and in the epithelium
itself (where the corresponding RTK receptors are expressed) [40, 103–109].

Growth Factors and Morphogens for Tissue Engineering

In the complexity of the ECM, with the vast range of physical, biological, and
chemical interactions, signaling pathways and networks all producing a host of
specific responses to given stimuli, mimicking or recreating these processes through
tissue engineering and regeneration techniques requires a number of different
components. Vital among these are growth factors which, as described above, play
fundamental roles in a variety of tissue and organ formation mechanisms and
processes. The properties of the ECM itself, as a reservoir, have a pivotal role in the
physiological delivery of GFs, crucial to the regenerative process [32]. While the
use of GFs has had some clinical success, their potential as therapeutic agents has
generally been hindered by certain limitations. Many growth factors are intrinsically
unstable, with very brief functional activity [110], while the absence of suitable
delivery methods excludes others. For instance, some GFs like BMP-2 and VEGF
have minimal interaction in their native form with the surrounding ECM, which in
clinical situations requiring repeated dosage due to rapid outward diffusion and fast
proteolysis makes them unsuitable [111, 112]. Therefore, some form of GF engi-
neering may be required in order for regenerative applications to overcome these
limitations [32].
Similarly, given that morphogens are another essential regulatory mechanism in
the creation of improved human tissue models, researchers over the past two
1 The Extracellular Matrix, Growth Factors … 19

decades have used them to develop a variety of hydrogel compositions which can
envelop 3D cell cultures, or modified, synthetic biomatrices, in order to better
deliver both GFs and morphogens into the target tissue [113].

Growth Factor and Morphogen Delivery

Through Engineered ECM

The major issue when a signaling delivery system has to be improved is how to best
mimic naturally occurring, inductive signal sequestering which serves as a template
for the design of synthetic molecules that can sequester GFs and morphogens [36].
“Biomimicry,” which is the underlying mechanism of GF sequestration, was first
used in studying the interaction between a2-M and TGF-b1, a2-M and PDGF-BB,
and TGF-1b and TGF-1b receptor III [114–116] and can be used to identify GF
sequestering moieties. Researchers have demonstrated that moieties engineered to
mimic known proteins, or proteoglycans such as fibronectin and fibrinogen,
exhibited GF or heparin-like sequestering [117]. In addition to soluble approaches,
GFs can be sequestered at 2D and 3D interfaces in the ECM environment. Surfaces
presenting proteoglycans and glycoproteins in a synthetic functionalized monolayer
can sequester GFs and modulate GF-related cell response. As an example, it has
been demonstrated that GFs can be sequestered by a self-assembled monolayer
modified with a HEPpep peptide derived from the heparin-binding domain of
FGF-2 and an Arg-Gly-Glu (RGD), derived from a sequence of fibronectin which
allows integrin-mediated cell adhesion [118].
A 3D interface design, however, requires the optimum choice of an appropriate
biomaterial which must retain GFs. Controlled tailoring of physical properties, like
density, porosity, viscosity, and charge, is essential for the implementation of GF
sequestration inside the scaffold [110]. Additionally, GF activity must be preserved
when incorporated within the biomaterial, which can easily be done by designing
formulations which mimic the ECM, such as hyaluronan, chitosan, collagen/gelatin,
and fibrin-based scaffolds. Moreover, natural biomaterials are cell-growth-friendly
and they avoid limitations such as pathogen transmission or ethical considerations
which can arise using animal or human sources in a clinical situation. Furthermore,
in a 3D matrix scenario, the proximity of the cells to the sequestering event and the
source of the sequestered GF (cell secreted or supplemented into the growth
medium) can influence the final cell response [36]. Hydrogels are excellent sub-
stitutes for native ECM because they can mimic its physical structure and bio-
chemical functions. They are increasingly used for GF sequestering and to control
release for a variety of purposes, such as to increase angiogenesis, and nerve and
bone regeneration [119–121]. However, most organoids are currently formed by
envelopment within Matrigel hydrogels, which are a poorly defined, heterogeneous
mixture of soluble basement membrane proteins from mouse sarcomas. Some
researchers have shown that chemically well-defined hydrogels could be used to
20 C. Bason et al.

support organ morphogenesis, like hyaluronan–chitosan hydrogel blend which has

been used with rudimentary cerebral organoids, or pure PEG hydrogels which can
entrap a single mouse embryonic stem cell for neural tube morphogenesis
[122–124].

Engineering Growth Factors and Morphogens

for Interaction with Exogenous Biomaterials
and for Delivery Through the Native ECM

A large number of strategies permit covalent attachment of GFs to biomaterials,

among which the carbodiimide-mediated conjugation reaction has been used suc-
cessfully, for example, in the cross-linking of VEGF-A/angiopoietin-1 onto colla-
gen scaffolds which has been found to improve vascularization [125]. Instead of
chemical coupling, GFs can be enzymatically conjugated to exogenous matrices
and the incorporation of a substrate sequence in the scaffold allows site-specific
control of the cross-linking location within the GF and minimizes non-specific
interference with other sites of bioactivity [126]. Heparin-binding GFs can associate
with GAGs, leading to the fusion of heparin-binding domains to
non-heparin-binding factors. As an example, the heparin-binding (HB) domain of
EGF has been added to IGF-1 creating the HB-IGF-1 formulation which has been
demonstrated to interact with chondroitin sulfate GAGs within the cartilage matrix
after intra-articular knee injection [127]. Furthermore, abundant collagen fibers are
also a good target for GF binding as demonstrated by the fusion of collagen binding
domain (CBD) and nerve growth factor-b (NGF-b) in order to improve nerve
regeneration after nerve crush injury [128].
Light is beginning to be thrown on the extraordinary role of the ECM in the vast
and complex mechanism underlying tissue and organ generation and in mainte-
nance processes. In the present book we discussed critical aspects of the native
ECM as well as application of innovative engineering methodology which would
get an advancement towards tissue regeneration.

References

1. Katari R, Peloso A, Orlando G. Tissue engineering and regenerative medicine:semantic

considerations for an evolving paradigm. Front Bioeng Biotechnol. 2015;12(2):57.
2. Hellman KB, Johnson PC, Bertram TA, Tawil B. Challenges in tissue engineering and
regenerative medicine product commercialization: building an industry. Tissue Eng Part A.
2011;17(1–2):1–3.
3. Briquez PS, Hubbell JA, Martino MM. Extracellular matrix-inspired growth factor delivery
systems for skin wound healing. Adv Wound Care (New Rochelle). 2015;4(8):479–89.
1 The Extracellular Matrix, Growth Factors … 21

4. Patterson J, Siew R, Herring SW, Lin AS, Guldberg R, Stayton PS. Hyaluronic acid
hydrogels with controlled degradation properties for oriented bone regeneration.
Biomaterials. 2010;31(26):6772–81.
5. Hynes RO. The extracellular matrix: not just pretty fibrils. Science. 2009;326(5957):1216–9.
6. Chan BP, Leong KW. Scaffolding in tissue engineering: general approaches and
tissue-specific considerations. Eur Spine J. 2008;17(Suppl 4):467–79.
7. Labouesse M. Role of the extracellular matrix in epithelial morphogenesis: a view from C.
elegans. Organogenesis. 2012;8(2):65–70.
8. Frantz C, Stewart KM, Weaver VM. The extracellular matrix at a glance. J Cell Sci.
2010;123(Pt 24):4195–200.
9. Sarrazin S, Lamanna WC, Esko JD. Heparan sulfate proteoglycans. Cold Spring Harb
Perspect Biol. 2011;3(7).
10. Schaefer L. Extracellular matrix molecules: endogenous danger signals as new drug targets
in kidney diseases. Curr Opin Pharmacol. 2010;10(2):185–90.
11. Iozzo RV, Schaefer L. Proteoglycan form and function: A comprehensive nomenclature of
proteoglycans. Matrix Biol. 2015;42:11–55.
12. Iozzo RV, Zoeller JJ, Nyström A. Basement membrane proteoglycans: modulators Par
Excellence of cancer growth and angiogenesis. Mol Cells. 2009;27(5):503–13.
13. Ori A, Wilkinson MC, Fernig DG. The heparanome and regulation of cell function:
structures, functions and challenges. Front Biosci. 2008;1(13):4309–38.
14. Boyd DF, Thomas PG. Towards integrating extracellular matrix and immunological
pathways. Cytokine. 2017;98:79–86.
15. Iozzo RV, San Antonio JD. Heparan sulfate proteoglycans: heavy hitters in the angiogenesis
arena. J Clin Invest. 2001;108(3):349–55.
16. Patel VN, Knox SM, Likar KM, Lathrop CA, Hossain R, Eftekhari S, Whitelock JM,
Elkin M, Vlodavsky I, Hoffman MP. Heparanase cleavage of perlecan heparan sulfate
modulates FGF10 activity during ex vivo submandibular gland branching morphogenesis.
Development. 2007;134(23):4177–86.
17. Goldoni S, Iozzo RV. Tumor microenvironment: modulation by decorin and related
molecules harboring leucine-rich tandem motifs. Int J Cancer. 2008;123(11):2473–9.
18. Schaefer L, Iozzo RV. Biological functions of the small leucine-rich proteoglycans: from
genetics to signal transduction. J Biol Chem. 2008;283(31):21305–9.
19. Brinckerhoff CE. Matrix metalloproteinases in health and disease: sculpting the human body.
1st ed. (Republic of Singapore): World Scientific Publishing Co; 2017.
20. Kozel BA, Ciliberto CH, Mecham RP. Deposition of tropoelastin into the extracellular
matrix requires a competent elastic fiber scaffold but not live cells. Matrix Biol. 2004;23
(1):23–34.
21. Gheduzzi D, Guerra D, Bochicchio B, Pepe A, Tamburro AM, Quaglino D, Mithieux S,
Weiss AS, Pasquali Ronchetti I. Heparan sulphate interacts with tropoelastin, with some
tropoelastin peptides and is present in human dermis elastic fibers. Matrix Biol. 2005;24
(1):15–25.
22. Trinh LA, Stainier DY. Fibronectin regulates epithelial organization during myocardial
migration in zebrafish. Dev Cell. 2004;6(3):371–82.
23. Matsui T, Raya A, Callol-Massot C, Kawakami Y, Oishi I, Rodriguez-Esteban C, Izpisúa
Belmonte JC. miles-apart-mediated regulation of cell-fibronectin interaction and myocardial
migration in zebrafish. Nat Clin Pract Cardiovasc Med. 2007;4(Suppl 1):S77–82.
24. Taylor-Weiner H, Schwarzbauer JE, Engler AJ. Defined extracellular matrix components are
necessary for definitive endoderm induction. Stem Cells. 2013;31(10):2084–94.
25. Takahashi S, Leiss M, Moser M, Ohashi T, Kitao T, Heckmann D, Pfeifer A, Kessler H,
Takagi J, Erickson HP, Fässler R. The RGD motif in fibronectin is essential for development
but dispensable for fibril assembly. J Cell Biol. 2007;178(1):167–78.
26. Pankov R, Yamada KM. Fibronectin at a glance. J Cell Sci. 2002;115(Pt 20):3861–3.
27. Singh P, Carraher C, Schwarzbauer JE. Assembly of fibronectin extracellular matrix. Annu
Rev Cell Dev Biol. 2010;26:397–419.
22 C. Bason et al.

28. Riederer I, Bonomo AC, Mouly V, Savino W. Laminin therapy for the promotion of muscle
regeneration. FEBS Lett. 2015;589(22):3449–53.
29. Mosher DF, Adams JC. Adhesion-modulating/matricellular ECM protein families: a
structural, functional and evolutionary appraisal. Matrix Biol. 2012;31(3):155–61.
30. Sweetwyne MT, Murphy-Ullrich JE. Thrombospondin1 in tissue repair and fibrosis:
TGF-b-dependent and independent mechanisms. Matrix Biol. 2012;31(3):178–86.
31. Sahni A, Francis CW. Vascular endothelial growth factor binds to fibrinogen and fibrin and
stimulates endothelial cell proliferation. Blood. 2000;96(12):3772–8.
32. Mitchell AC, Briquez PS, Hubbell JA, Cochran JR. Engineering growth factors for
regenerative medicine applications. Acta Biomater. 2016;30:1–12.
33. Hutchings H, Ortega N, Plouët J. Extracellular matrix-bound vascular endothelial growth
factor promotes endothelial cell adhesion, migration, and survival through integrin ligation.
FASEB J. 2003;17(11):1520–2.
34. Bissell MJ, Hines WC. Why don’t we get more cancer? A proposed role of the
microenvironment in restraining cancer progression. Nat Med. 2011;17(3):320–9.
35. Wu J, Strawn TL, Luo M, Wang L, Li R, Ren M, Xia J, Zhang Z, Ma W, Luo T,
Lawrence DA, Fay WP. Plasminogen activator inhibitor-1 inhibits angiogenic signaling by
uncoupling vascular endothelial growth factor receptor-2-aVb3 integrin cross talk.
Arterioscler Thromb Vasc Biol. 2015;35(1):111–20.
36. Belair DG, Le NN, Murphy WL. Design of growth factor sequestering biomaterials. Chem
Commun (Camb). 2014;50(99):15651–68.
37. Rundhaug JE. Matrix metalloproteinases and angiogenesis. J Cell Mol Med. 2005;9(2):267–
85.
38. Davis GE, Senger DR. Endothelial extracellular matrix: biosynthesis, remodeling, and
functions during vascular morphogenesis and neovessel stabilization. Circ Res. 2005;97
(11):1093–107.
39. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med. 2003;9
(6):669–76.
40. Olsson R, Maxhuni A, Carlsson PO. Revascularization of transplanted pancreatic islets
following culture with stimulators of angiogenesis. Transplantation. 2006;82(3):340–7.
41. Hsiong SX, Mooney DJ. Regeneration of vascularized bone. Periodontology. 2000;2006
(41):109–22.
42. Wijelath ES, Rahman S, Namekata M, Murray J, Nishimura T, Mostafavi-Pour Z, Patel Y,
Suda Y, Humphries MJ, Sobel M. Heparin-II domain of fibronectin is a vascular endothelial
growth factor-binding domain: enhancement of VEGF biological activity by a singular
growth factor/matrix protein synergism. Circ Res. 2006;99(8):853–60.
43. Mitsi M, Hong Z, Costello CE, Nugent MA. Heparin-mediated conformational changes in
fibronectin expose vascular endothelial growth factor binding sites. Biochemistry. 2006;45
(34):10319–28.
44. Mitsi M, Forsten-Williams K, Gopalakrishnan M, Nugent MA. A catalytic role of heparin
within the extracellular matrix. J Biol Chem. 2008;283(50):34796–807.
45. Chen LL, Johansson JK, Hodges RR, Zoukhri D, Ghinelli E, Rios JD, Dartt DA. Differential
effects of the EGF family of growth factors on protein secretion, MAPK activation, and
intracellular calcium concentration in rat lacrimal gland. Exp Eye Res. 2005;80(3):379–89.
46. Eichmann A, Simons M. VEGF signaling inside vascular endothelial cells and beyond. Curr
Opin Cell Biol. 2012;24(2):188–93.
47. Simons M. An inside view: VEGF receptor trafficking and signaling. Physiology (Bethesda).
2012;27(4):213–22.
48. Mettouchi A. The role of extracellular matrix in vascular branching morphogenesis. Cell
Adh Migr. 2012;6(6):528–34.
49. Dong X, Zhao B, Iacob RE, Zhu J, Koksal AC, Lu C, Engen JR, Springer TA. Force interacts
with macromolecular structure in activation of TGF-b. Nature. 2017;542(7639):55–9.
1 The Extracellular Matrix, Growth Factors … 23

50. Levin M, Udi Y, Solomonov I, Sagi I. Next generation matrix metalloproteinase inhibitors—
novel strategies bring new prospects. Biochim Biophys Acta. 2017;1864(11 Pt A):1927–
1939.
51. Parker A, Rees C, Clarke J, Busby WH Jr, Clemmons DR. Binding of insulin-like growth
factor (IGF)-binding protein-5 to smooth-muscle cell extracellular matrix is a major
determinant of the cellular response to IGF-I. Mol Biol Cell. 1998;9(9):2383–92.
52. Firth SM, Baxter RC. Cellular actions of the insulin-like growth factor binding proteins.
Endocr Rev. 2002;23(6):824–54.
53. Booth BA, Boes M, Andress DL, Dake BL, Kiefer MC, Maack C, Linhardt RJ, Bar K,
Caldwell EE, Weiler J, et al. IGFBP-3 and IGFBP-5 association with endothelial cells: role
of C-terminal heparin binding domain. Growth Regul. 1995;5(1):1–17.
54. Kuang Z, Yao S, Keizer DW, Wang CC, Bach LA, Forbes BE, Wallace JC, Norton RS.
Structure, dynamics and heparin binding of the C-terminal domain of insulin-like growth
factor-binding protein-2 (IGFBP-2). J Mol Biol. 2006;364(4):690–704.
55. Forbes BE, Hartfield PJ, McNeil KA, Surinya KH, Milner SJ, Cosgrove LJ, Wallace JC.
Characteristics of binding of insulin-like growth factor (IGF)-I and IGF-II analogues to the
type 1 IGF receptor determined by BIAcore analysis. Eur J Biochem. 2002;269(3):961–8.
56. Fowlkes JL, Serra DM. Characterization of glycosaminoglycan-binding domains present in
insulin-like growth factor-binding protein-3. J Biol Chem. 1996;271(25):14676–9.
57. Bach LA, Headey SJ, Norton RS. IGF-binding proteins–the pieces are falling into place.
Trends Endocrinol Metab. 2005;16(5):228–34.
58. Kricker JA, Hyde CE, Van Lonkhuyzen DR, Hollier BG, Shooter GK, Leavesley DI,
Herington AC, Upton Z. Mechanistic investigations into interactions between IGF-I and
IGFBPs and their impact on facilitating cell migration on vitronectin. Growth Factors.
2010;28(5):359–69.
59. Xu J, Liao K. Protein kinase B/AKT 1 plays a pivotal role in insulin-like growth factor-1
receptor signaling induced 3T3-L1 adipocyte differentiation. J Biol Chem. 2004;279
(34):35914–22.
60. Bunn RC, Fowlkes JL. Insulin-like growth factor binding protein proteolysis. Trends
Endocrinol Metab. 2003;14(4):176–81. Review. PubMed PMID: 12714278.
61. Boyd DF, Thomas PG. Towards integrating extracellular matrix and immunological
pathways. Cytokine. 2017;98:79–86.
62. Yan C, Boyd DD. Regulation of matrix metalloproteinase gene expression. J Cell Physiol.
2007;211(1):19–26.
63. Rilla K, Mustonen AM, Arasu UT, Härkönen K, Matilainen J, Nieminen P. Extracellular
vesicles are integral and functional components of the extracellular matrix. Matrix Biol.
2017.
64. Perea-Gil I, Prat-Vidal C, Bayes-Genis A. In vivo experience with natural scaffolds for
myocardial infarction: the times they are a-changin’. Stem Cell Res. 2015;6(6):248.
65. An M, Kwon K, Park J, Ryu DR, Shin JA, Lee Kang J, Choi JH, Park EM, Lee KE, Woo M,
Kim M. Extracellular matrix-derived extracellular vesicles promote cardiomyocyte growth
and electrical activity in engineered cardiac atria. Biomaterials. 2017;146:49–59.
66. Berardocco M, Radeghieri A, Busatto S, Gallorini M, Raggi C, Gissi C, D’Agnano I,
Bergese P, Felsani A, Berardi AC. RNA-seq reveals distinctive RNA profiles of small
extracellular vesicles from different human liver cancer cell lines. Oncotarget. 2017;8
(47):82920–39.
67. Belkin AM, Zhidkova NI, Balzac F, Altruda F, Tomatis D, Maier A, Tarone G,
Koteliansky VE, Burridge K. Beta 1D integrin displaces the beta 1A isoform in striated
muscles: localization at junctional structures and signaling potential in nonmuscle cells.
J Cell Biol. 1996;132(1–2):211–26.
68. Kääriäinen M, Nissinen L, Kaufman S, Sonnenberg A, Järvinen M, Heino J, Kalimo H.
Expression of alpha7beta1 integrin splicing variants during skeletal muscle regeneration.
Am J Pathol. 2002;161(3):1023–31.
24 C. Bason et al.

69. Tarone G, Hirsch E, Brancaccio M, De Acetis M, Barberis L, Balzac F, Retta SF, Botta C,
Altruda F, Silengo L. Integrin function and regulation in development. Int J Dev Biol.
2000;44(6):725–31. Review. Erratum in: Int J Dev Biol 2001 Sep;45(5–6): following 770.
70. Jansen KA, Donato DM, Balcioglu HE, Schmidt T, Danen EH, Koenderink GH. A guide to
mechanobiology: where biology and physics meet. Biochim Biophys Acta. 2015;1853(11 Pt
B):3043–52.
71. Jaalouk DE, Lammerding J. Mechanotransduction gone awry. Nat Rev Mol Cell Biol.
2009;10(1):63–73.
72. Lu P, Weaver VM, Werb Z. The extracellular matrix: a dynamic niche in cancer progression.
J Cell Biol. 2012;196(4):395–406.
73. Snedeker JG, Gautieri A. The role of collagen crosslinks in ageing and diabetes—the good,
the bad, and the ugly. Muscles Ligaments Tendons J. 2014;4(3):303–8.
74. Cukierman E, Pankov R, Stevens DR, Yamada KM. Taking cell-matrix adhesions to the
third dimension. Science. 2001;294(5547):1708–12.
75. Winograd-Katz SE, Fässler R, Geiger B, Legate KR. The integrin adhesome: from genes and
proteins to human disease. Nat Rev Mol Cell Biol. 2014;15(4):273–88.
76. Sun Z, Guo SS, Fässler R. Integrin-mediated mechanotransduction. J Cell Biol. 2016;215
(4):445–56.
77. Geiger B, Tokuyasu KT, Dutton AH, Singer SJ. Vinculin, an intracellular protein localized
at specialized sites where microfilament bundles terminate at cell membranes. Proc Natl
Acad Sci USA. 1980;77(7):4127–31.
78. Atherton P, Stutchbury B, Jethwa D, Ballestrem C. Mechanosensitive components of
integrin adhesions: role of vinculin. Exp Cell Res. 2016;343(1):21–7.
79. Dupont S. Role of YAP/TAZ in cell-matrix adhesion-mediated signalling and mechan-
otransduction. Exp Cell Res. 2016;343(1):42–53.
80. Elbediwy A, Vincent-Mistiaen ZI, Thompson BJ. YAP and TAZ in epithelial stem cells: a
sensor for cell polarity, mechanical forces and tissue damage. BioEssays. 2016;38(7):644–
53.
81. Kim IG, Gil CH, Seo J, Park SJ, Subbiah R, Jung TH, Kim JS, Jeong YH, Chung HM,
Lee JH, Lee MR, Moon SH, Park K. Mechanotransduction of human pluripotent stem cells
cultivated on tunable cell-derived extracellular matrix. Biomaterials. 2018;150:100–11.
82. Clause KC, Barker TH. Extracellular matrix signaling in morphogenesis and repair. Curr
Opin Biotechnol. 2013;24(5):830–3.
83. Lubarsky B, Krasnow MA. Tube morphogenesis: making and shaping biological tubes. Cell.
2003;112(1):19–28.
84. Hoffman MP, Kidder BL, Steinberg ZL, Lakhani S, Ho S, Kleinman HK, Larsen M. Gene
expression profiles of mouse submandibular gland development: FGFR1 regulates branching
morphogenesis in vitro through BMP- and FGF-dependent mechanisms. Development.
2002;129(24):5767–78.
85. Nam JM, Onodera Y, Bissell MJ, Park CC. Breast cancer cells in three-dimensional culture
display an enhanced radioresponse after coordinate targeting of integrin alpha5beta1 and
fibronectin. Cancer Res. 2010;70(13):5238–48.
86. Avizienyte E, Wyke AW, Jones RJ, McLean GW, Westhoff MA, Brunton VG, Frame MC.
Src-induced de-regulation of E-cadherin in colon cancer cells requires integrin signalling.
Nat Cell Biol. 2002;4(8):632–8.
87. Weber GF, Bjerke MA, DeSimone DW. A mechanoresponsive cadherin-keratin complex
directs polarized protrusive behavior and collective cell migration. Dev Cell. 2012;22
(1):104–15.
88. Huveneers S, Danen EH. Adhesion signaling—crosstalk between integrins, Src and Rho.
J Cell Sci. 2009;122(Pt 8):1059–69.
89. Watanabe T, Sato K, Kaibuchi K. Cadherin-mediated intercellular adhesion and signaling
cascades involving small GTPases. Cold Spring Harb Perspect Biol. 2009;1(3):a003020.
90. Van Aelst L, Symons M. Role of Rho family GTPases in epithelial morphogenesis. Genes
Dev. 2002;16(9):1032–54.
1 The Extracellular Matrix, Growth Factors … 25

91. Reddi AH. Cartilage morphogenetic proteins: role in joint development, homoeostasis, and
regeneration. Ann Rheum Dis. 2003;62(Suppl 2):ii73–8.
92. Bondow BJ, Faber ML, Wojta KJ, Walker EM, Battle MA. E-cadherin is required for
intestinal morphogenesis in the mouse. Dev Biol. 2012;371(1):1–12.
93. Iruela-Arispe ML, Beitel GJ. Tubulogenesis. Development. 2013;140(14):2851–5.
94. Lampugnani MG. Endothelial adherens junctions and the actin cytoskeleton: an ‘infinity
net’? J Biol. 2010;9(3):16.
95. Brinkmann BF, Steinbacher T, Hartmann C, Kummer D, Pajonczyk D, Mirzapourshafiyi F,
Nakayama M, Weide T, Gerke V, Ebnet K. VE-cadherin interacts with cell polarity protein
Pals1 to regulate vascular lumen formation. Mol Biol Cell. 2016;27(18):2811–21.
96. Gebala V, Collins R, Geudens I, Phng LK, Gerhardt H. Blood flow drives lumen formation
by inverse membrane blebbing during angiogenesis in vivo. Nat Cell Biol. 2016;18(4):443–
50.
97. Galie PA, Nguyen DH, Choi CK, Cohen DM, Janmey PA, Chen CS. Fluid shear stress
threshold regulates angiogenic sprouting. Proc Natl Acad Sci USA. 2014;111(22):7968–73.
98. Barry AK, Wang N, Leckband DE. Local VE-cadherin mechanotransduction triggers
long-ranged remodeling of endothelial monolayers. J Cell Sci. 2015;128(7):1341–51.
99. Wang S, Sekiguchi R, Daley WP, Yamada KM. Patterned cell and matrix dynamics in
branching morphogenesis. J Cell Biol. 2017;216(3):559–70.
100. Schuchardt A, D’Agati V, Larsson-Blomberg L, Costantini F, Pachnis V. Defects in the
kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature.
1994;367(6461):380–3.
101. Luetteke NC, Qiu TH, Fenton SE, Troyer KL, Riedel RF, Chang A, Lee DC. Targeted
inactivation of the EGF and amphiregulin genes reveals distinct roles for EGF receptor
ligands in mouse mammary gland development. Development. 1999;126(12):2739–50.
102. De Moerlooze L, Spencer-Dene B, Revest JM, Hajihosseini M, Rosewell I, Dickson C. An
important role for the IIIb isoform of fibroblast growth factor receptor 2 (FGFR2) in
mesenchymal-epithelial signalling during mouse organogenesis. Development. 2000;127
(3):483–92.
103. Hennighausen L, Robinson GW. Information networks in the mammary gland. Nat Rev Mol
Cell Biol. 2005;6(9):715–25.
104. Costantini F, Shakya R. GDNF/Ret signaling and the development of the kidney. BioEssays.
2006;28(2):117–27.
105. Djouad F, Delorme B, Maurice M, Bony C, Apparailly F, Louis-Plence P, Canovas F,
Charbord P, Noël D, Jorgensen C. Microenvironmental changes during differentiation of
mesenchymal stem cells towards chondrocytes. Arthritis Res Ther. 2007;9(2):R33.
106. Lu P, Werb Z. Patterning mechanisms of branched organs. Science. 2008;322(5907):1506–
9.
107. Affolter M, Zeller R, Caussinus E. Tissue remodelling through branching morphogenesis.
Nat Rev Mol Cell Biol. 2009;10(12):831–42.
108. Costantini F, Kopan R. Patterning a complex organ: branching morphogenesis and nephron
segmentation in kidney development. Dev Cell. 2010;18(5):698–712.
109. Blake J, Rosenblum ND. Renal branching morphogenesis: morphogenetic and signaling
mechanisms. Semin Cell Dev Biol. 2014;36:2–12.
110. Lee K, Silva EA, Mooney DJ. Growth factor delivery-based tissue engineering: general
approaches and a review of recent developments. J R Soc Interface. 2011;8(55):153–70.
111. Ferrara N. Binding to the extracellular matrix and proteolytic processing: two key
mechanisms regulating vascular endothelial growth factor action. Mol Biol Cell. 2010;21
(5):687–90.
112. Hankenson KD, Gagne K, Shaughnessy M. Extracellular signaling molecules to promote
fracture healing and bone regeneration. Adv Drug Deliv Rev. 2015;1(94):3–12.
113. Marti-Figueroa CR, Ashton RS. The case for applying tissue engineering methodologies to
instruct human organoid morphogenesis. Acta Biomater. 2017;54:35–44.
26 C. Bason et al.

114. Webb DJ, Roadcap DW, Dhakephalkar A, Gonias SL. A 16-amino acid peptide from human
alpha2-macroglobulin binds transforming growth factor-beta and platelet-derived growth
factor-BB. Protein Sci. 2000;9(10):1986–92.
115. Ezquerro IJ, Lasarte JJ, Dotor J, Castilla-Cortázar I, Bustos M, Peñuelas I, Blanco G,
Rodríguez C, Lechuga Mdel C, Greenwel P, Rojkind M, Prieto J, Borrás-Cuesta F.
A synthetic peptide from transforming growth factor beta type III receptor inhibits liver
fibrogenesis in rats with carbon tetrachloride liver injury. Cytokine. 2003;22(1–2):12–20.
Erratum in: Cytokine. 2006;33(2):119.
116. Serratì S, Margheri F, Pucci M, Cantelmo AR, Cammarota R, Dotor J, Borràs-Cuesta F,
Fibbi G, Albini A, Del Rosso M. TGFbeta1 antagonistic peptides inhibit
TGFbeta1-dependent angiogenesis. Biochem Pharmacol. 2009;77(5):813–25.
117. Martino MM, Briquez PS, Ranga A, Lutolf MP, Hubbell JA. Heparin-binding domain of
fibrin(ogen) binds growth factors and promotes tissue repair when incorporated within a
synthetic matrix. Proc Natl Acad Sci USA. 2013;110(12):4563–8.
118. Terada T, Mizobata M, Kawakami S, Yabe Y, Yamashita F, Hashida M. Basic fibroblast
growth factor-binding peptide as a novel targeting ligand of drug carrier to tumor cells.
J Drug Target. 2006;14(8):536–45.
119. Modaresifar K, Hadjizadeh A, Niknejad H. Design and fabrication of GelMA/chitosan
nanoparticles composite hydrogel for angiogenic growth factor delivery. Artif Cells
Nanomed Biotechnol. 2017;24:1–10.
120. Berkovitch Y, Cohen T, Peled E, Schmidhammer R, Florian H, Teuschl A, Wolbank S,
Yelin D, Redl H, Seliktar D. Hydrogel composition and laser micro-patterning to regulate
sciatic nerve regeneration. J Tissue Eng Regen Med. 2017.
121. Paduano F, Marrelli M, Alom N, Amer M, White LJ, Shakesheff KM, Tatullo M.
Decellularized bone extracellular matrix and human dental pulp stem cells as a construct for
bone regeneration. J Biomater Sci Polym Ed. 2017;28(8):730–48.
122. Hughes CS, Postovit LM, Lajoie GA. Matrigel: a complex protein mixture required for
optimal growth of cell culture. Proteomics. 2010;10(9):1886–90.
123. Lindborg BA, Brekke JH, Vegoe AL, Ulrich CB, Haider KT, Subramaniam S,
Venhuizen SL, Eide CR, Orchard PJ, Chen W, Wang Q, Pelaez F, Scott CM, Kokkoli E,
Keirstead SA, Dutton JR, Tolar J, O’Brien TD. Rapid induction of cerebral organoids from
human induced pluripotent stem cells using a chemically defined hydrogel and defined cell
culture medium. Stem Cells Transl Med. 2016;5(7):970–9.
124. Ranga A, Girgin M, Meinhardt A, Eberle D, Caiazzo M, Tanaka EM, Lutolf MP. Neural
tube morphogenesis in synthetic 3D microenvironments. Proc Natl Acad Sci USA. 2016;113
(44):E6831–9.
125. Boccardo S, Gaudiello E, Melly L, Cerino G, Ricci D, Martin I, Eckstein F, Banfi A,
Marsano A. Engineered mesenchymal cell-based patches as controlled VEGF delivery
systems to induce extrinsic angiogenesis. Acta Biomater. 2016;15(42):127–35.
126. Lorentz KM, Yang L, Frey P, Hubbell JA. Engineered insulin-like growth factor-1 for
improved smooth muscle regeneration. Biomaterials. 2012;33(2):494–503.
127. Miller RE, Grodzinsky AJ, Cummings K, Plaas AH, Cole AA, Lee RT,
Patwari P. Intraarticular injection of heparin-binding insulin-like growth factor 1 sustains
delivery of insulin-like growth factor 1 to cartilage through binding to chondroitin sulfate.
Arthritis Rheum. 2010;62(12):3686–94.
128. Sun W, Lin H, Chen B, Zhao W, Zhao Y, Xiao Z, Dai J. Collagen scaffolds loaded with
collagen-binding NGF-beta accelerate ulcer healing. J Biomed Mater Res A. 2010;92(3):
887–95.
Chapter 2
ECM Hydrogels for Regenerative
Medicine

Michael J. Sawkins, Lindsey T. Saldin,

Stephen F. Badylak and Lisa J. White

Abstract The ECM is a highly complex mix of structural and functional proteins
and other biomolecules. These molecules are secreted by the cells resident in every
tissue in the body but can also influence their behavior through a process of “dy-
namic reciprocity.” As a result, there has been significant interest in utilizing ECM
as a biologic scaffold material in tissue repair and replacement. Numerous pre-
clinical and clinical studies have demonstrated the efficacy of ECM biomaterials,
and more than 4 million patients have now been treated with these scaffold mate-
rials. The discovery that these materials could be formed into hydrogels promised to
further expand their clinical utility by offering minimally invasive delivery and the
ability to fill irregularly shaped defects. This chapter will briefly outline the history
and characterization of ECM biomaterials and their evolution from single sheet to
multisheet, powder, and ultimately hydrogel form. The first studies describing the
production of early-generation ECM hydrogels used well-characterized porcine
small intestinal submucosa and urinary bladder matrix, and these materials will be
discussed in the context of the general methods used to produce and characterize
ECM hydrogels. A detailed consideration of the many second-generation hydrogels
which have since been produced from a wide range of tissues will then be discussed

M. J. Sawkins
Department of Anatomy, Royal College of Surgeons in Ireland,
123 St. Stephen’s Green, Dublin 2, Ireland
L. T. Saldin
Department of Bioengineering, University of Pittsburgh,
Pittsburgh, PA 15219, USA
e-mail: [email protected]
S. F. Badylak
Surgery and Bioengineering, University of Pittsburgh, 450 Technology Drive,
Suite 319, Pittsburgh, PA 15219, USA
e-mail: [email protected]
L. J. White (&)
School of Pharmacy, University of Nottingham, University Park,
Nottingham NG7 2RD, UK
e-mail: [email protected]

© Springer International Publishing AG, part of Springer Nature 2018 27

A. C. Berardi (ed.), Extracellular Matrix for Tissue Engineering
and Biomaterials, Stem Cell Biology and Regenerative Medicine,
https://doi.org/10.1007/978-3-319-77023-9_2
28 M. J. Sawkins et al.

in the context of tissue specificity. The hydrogels discussed in this chapter have
been evaluated in vitro or in small scale in vivo animal studies. More substantial
evaluation is required before these materials can be considered ready for clinical
application, but these hydrogels provide the possibility for minimally invasive
delivery, treatment of irregularly shaped defects in anatomic sites that prove
challenging for invasive surgical procedures, and may provide an ECM formation
that delivers immediate bioactivity as a consequence of its distinctive biomolecular
composition.

Keywords Adipose Brain Cartilage Central nervous system



Cornea Decellularization Demineralized bone matrix Dermis





Extracellular matrix Hydrogel Intervertebral disk Ligament





Liver Myocardium Nucleus pulposus Pericardium Regenerative medicine




Skeletal muscle Skin Spinal cord Small intestinal submucosa




Tendon Tissue engineering Tissue specificity Urinary bladder matrix
Vocal fold

Historical Development of ECM-Derived Materials

Once considered to be a passive bystander in tissue morphology, the extracellular

matrix (ECM) is now understood to instruct cell behavior through “dynamic
reciprocity” [13, 59]. Specifically, cell surface receptors transduce signals from the
microenvironmental niche to the cytoskeleton, the nuclear matrix, and the chro-
matin, thereby affecting gene expression, and then signal back to the niche.
Mechanical dissection and tissue decellularization have been widely used to
generate biologic scaffold materials from the native ECM. These ECM-derived
materials release natural factors upon degradation that alter the default immune
response to injury, and recruit endogenous cells for the formation of site-specific
functional tissue (constructive remodeling). The safety and efficacy of ECM scaf-
folds have been demonstrated in numerous preclinical animal studies and clinical
human studies for a wide variety of tissues, including musculoskeletal, cardio-
vascular, and urogenital. ECM-derived materials represent an attractive tissue
engineering solution because they are FDA approved, “off the shelf,” and obviate
the need to add exogenous growth factors or cells, representing nontrivial regula-
tory hurdles for clinical translation [71]. Moreover, the use of ECM-derived
materials bypasses the issues of cell sourcing and engineering immune acceptance
since the cells populating the scaffold are the patient’s own cells [60]. Currently,
more than 4 million patients have benefited from the use of ECM scaffolds.
2 ECM Hydrogels for Regenerative Medicine 29

Biochemical Content

The ECM consists of the structural and functional proteins secreted by the cells
resident in each tissue and organ and is a primary component of the cell’s
microenvironmental niche. ECM is comprised primarily of fibrillar collagen but
also contains glycoproteins (laminin, fibronectin), proteoglycans, glycosaminogly-
cans (GAGs), and growth factors. The 3D arrangement and biochemical compo-
sition of these proteins are specific to tissue type [59]. Ideally, an ECM-derived
material would preserve the components of native tissue ECM. However, the choice
of decellularization agents, techniques, and post-processing terminal sterilization
steps to produce ECM-derived materials will unavoidably affect the ECM bio-
chemical properties. The balance lies in achieving decellularization and sterility
criteria while best preserving ECM composition and structure [28]. Thin tissues
such as urinary bladder matrix (UBM) and small intestinal submucosa (SIS) may be
effectively decellularized with mechanical scraping, agitation in a dilute acidic
solution (0.1% peracetic acid), and subsequent rinsing with deionized water. UBM
and SIS contain growth factor and GAG levels similar to native ECM after de-
cellularization [3, 88]. Brown et al. [18] reported the urinary bladder matrix con-
tained an intact basement membrane after decellularization.
In contrast, denser tissues such as porcine dermis require a harsher decellular-
ization protocol, usually involving a combination of detergents, organic solvents,
and enzymatic solutions. Reing et al. [67] tested the effect of different processing
steps on the properties of porcine dermal matrix. While a trypsin/Triton X-100
protocol moderately affected ECM properties, the addition of a sodium dodecyl
sulfate (SDS) processing step more severely affected ECM mechanical strength,
growth factor, and GAG content, and the ability of the ECM to support cell growth
in vitro. The results may not be surprising considering Triton X-100 is a relatively
gentle nonionic detergent and trypsin is an enzyme that selectively cleaves cell
adherent proteins to detach cells from the tissue surface. In contrast, SDS is a more
harsh ionic detergent that can disrupt protein covalent bonding.
When processed appropriately, ECM-derived materials have many advantages
compared to their synthetic counterparts. ECM-derived materials recapitulate the
rich natural diversity of proteins, unlike matrices that are manufactured from
individual molecular components of the extracellular matrix (e.g., laminin or col-
lagen gels). Furthermore, the GAGs in the ECM-derived material present growth
factors to cell surface receptors in biologically relevant ratios and 3D conformations
[3]. It is well known that growth factors have an important impact on regulating cell
behavior [83]. Finally, non-chemically cross-linked ECM-derived materials degrade
and release biochemical signals at a similar rate to native tissue ECM.
30 M. J. Sawkins et al.

Bioinductive Properties

ECM-derived materials act as an inductive template for the formation of

site-specific functional tissue through the following identified mechanisms: the
release of sequestered growth factors and cytokines [3] and the release of cryptic
peptide motifs which possess diverse biological activities including antimicrobial
activity [16, 73], chemoattraction of endogenous stem/progenitor cells [2, 68], and
modulation of the host immune response toward an M2/Th2 phenotype [6].
Interestingly, it appears that degradation of the ECM-derived material is essential
for the bioinductive properties of ECM. For example, ECM degradation products
show bactericidal activity, while intact ECM does not show the same antimicrobial
effects. Furthermore, if the scaffold is chemically cross-linked such that it cannot be
degraded, the immune response will be directed toward an M1/Th1 phenotype and a
fibrous capsule will form as opposed to constructive remodeling.

Antimicrobial Properties

ECM-derived materials have displayed consistent resistance to bacterial infection in

clinical applications and even demonstrated resistance to deliberate bacterial
infection in preclinical studies [4, 10, 16, 43, 79]. It has been shown that naturally
occurring gene-encoded antimicrobial peptides (AMPs)/polypeptides such as
defensins [39], cecropins [58], and magainins [12] are found in vertebrates and
invertebrates. These peptides, present in native tissue, act as a potent first line of
defense in the innate immune response to pathogens [21].
At least 18 AMPs have been identified in porcine tissues which are the primary
source of SIS and UBM [15]. Sarikaya et al. [73] showed the antimicrobial peptide
activity of ECM was retained after processing. UBM and SIS degradation products
demonstrated a strong antibacterial effect against gram-negative Escherichia coli
and gram-positive Staphylococcus aureus and completely inhibited the growth of
both bacterial strains after 13 h. Similarly, Brennan et al. [16] tested multiple
fractions of UBM and liver ECM degradation products on their ability to inhibit
S. aureus and E. coli growth. The results suggested multiple antibacterial molecules
are present in ECM degradation products and the profile of these molecules differs
between ECMs derived from different tissues.

Chemoattraction

Badylak et al. [7] used a mouse model to show that a population of bone marrow
cells contributes to constructive tissue remodeling. Briefly, mice deficient for the
b-galactosidase gene were lethally irradiated and received both a transplantation of
b-galactosidase+ bone marrow cells, and one of 4 scaffold implants. The mice
received ECM-derived materials (SIS or urinary bladder submucosa) or control
2 ECM Hydrogels for Regenerative Medicine 31

scaffolds (collagen or degradable synthetic material PLGA). All scaffolds showed

the presence of b-galactosidase+ mononuclear cell infiltration at 7 days. At 56 days,
the ECM-derived material was completely degraded and replaced with highly
organized tissue, with b-galactosidase+ epithelial cells and fibroblasts. In contrast,
the collagen control and PLGA showed chronic inflammatory reaction around the
implants with little to no b-galactosidase+ epithelial cells and fibroblasts present
within each scaffold. This study identified that bone marrow cells were capable of
being recruited and could later differentiate into endothelial and fibroblast cells.
Reing et al. [67] demonstrated that enzymatically digested UBM in vitro pos-
sessed chemotactic and mitogenic activities for human multipotent progenitor cells
with a fivefold increase in migration over control digest buffer and 67% increase in
3-day proliferation over negative control. The digested UBM also inhibited
chemotaxis and proliferation of differentiated endothelial cells.
Chemoattraction by enzymatically digested UBM was illustrated in vivo [1] by
recruitment of a population of multipotent cells (e.g., Sca1+, Sox2+, Rex1+) in a
mammalian model of digit amputation. A single matricryptic peptide with positive
chemotactic potential was subsequently identified [2]. Briefly, ECM degradation
products were serially fractionated by different biochemical properties (i.e., ionic
charge, size, and hydrophobicity) and tested for chemotactic activity in a transwell
assay. The fraction with the greatest chemotactic potential was isolated for sequence
analysis by mass spectroscopy, which identified a single peptide from the alpha
subunit of the collagen III molecule. A synthesized version of the peptide showed
positive chemotactic activity toward several types of stem, progenitor, and differ-
entiated cells in vitro. The peptide was also chemotactic in vivo for the same cell
types in a mouse model of digit amputation [2].

Macrophage Polarization

ECM-derived materials have been shown to facilitate the functional reconstruction

of many tissue types including cardiac, esophageal, dermal, and musculotendinous
structures, among others [48]. Mechanisms underlying the clinical success of
ECM-derived materials are still not fully understood. However, it is logical to
assume the innate immune cells (e.g., neutrophils and macrophages), the first cells
to infiltrate the site of the implanted biomaterial, play a key role in the host response
and subsequent tissue remodeling outcome.
ECM-derived materials polarize macrophages away from the classically acti-
vated pro-inflammatory “M1” macrophages and toward the alternatively activated
immunomodulatory or “M2” macrophages [6]. M1 and M2 represent the extremes
of the continuum for macrophage phenotypic polarization, with markedly different
physiologic roles. M1 macrophages secrete copious amounts of nitric oxide,
reactive oxygen species, and pro-inflammatory cytokines such as interleukin (IL)-
12, IL-6, and tumor necrosis factor alpha. In contrast, M2 macrophages secrete
large amounts of IL-10, TGF-b, and arginase and scavenge debris, promote
angiogenesis, and recruit cells involved in constructive tissue remodeling.
32 M. J. Sawkins et al.

The host remodeling response to SIS and its chemically cross-linked form has
been characterized using autologous graft tissue as a control [8]. SIS elicited a
predominantly “M2” profile (CD163+) when implanted into a rat model, with
long-term constructive remodeling evidenced by vascularization, new skeletal
muscle fiber formation, and organized connective tissue at 16 weeks. Conversely,
the chemically cross-linked SIS elicited a predominantly “M1” profile (CD80+,
CCR7+) with chronic inflammation at 16 weeks evidenced by fibrosis and scarring
around the implant, presence of multinucleated giant cells, and the associated
classic foreign body response. The autologous tissue graft elicited a mixed M1/M2
profile at 16 weeks with moderately well-organized scar tissue, but no detectable
new muscle fibers. A subsequent study showed that the presence of a cellular
component, even an autologous one, polarized macrophages to an M1 profile [20].
Furthermore, the M2:M1 ratio for 14 commercially available ECM-derived mate-
rials was shown to correlate with in vivo constructive remodeling histologic scores
at 14 and 35 days post-implantation, an indication of the broad applicability of this
concept [19].

Types of ECM-Derived Materials

The first ECM scaffolds were intact sheets (60 lm), reflecting “skilled decellular-
ization” [3]. The fragile tissue layer of interest had to be carefully separated from
other layers and withstand the scraping and decellularization process without
tearing. These initial sheets were typically stored as lyophilized sheets and hydrated
before use [5]. ECM sheets are commonly used for wound repair (e.g., partial and
full thickness wounds, ulcers, second-degree burns, trauma wounds). A common
misconception in the use of ECM-derived materials is the requirement of the
material to replace volumetric tissue loss and provide mechanical strength at the
wound site. Instead, the ECM sheets are intended to serve as a bioinductive tem-
plate that degrades as the tissue is restored.
Single-layer ECM sheets may be insufficient for regenerative medicine appli-
cations with load-bearing requirements (e.g., esophagus repair, laryngeal recon-
struction, abdominal wall reconstruction, hernia repair, pelvic floor). ECM-derived
materials were vacuum compressed to form multilayer devices (4–8 layers) to
increase the strength of the material. Collagen fiber orientation has been used
advantageously in sheet stacking to improve the strength of the material device. For
example, a 10-layer ECM device (Restore®, DePuy Orthopedics) was stacked with
collagen fibers at 72° angles every 2 layers resulting in an isotropic construct that
matched the strength of the tendon tissue it was intended to replace [5]. In addition,
vacuum pressing allowed for the creation of a variety of 3D shapes for certain
applications (e.g., tube for trachea, funnel for esophageal anastomosis) [9].
ECM-derived materials can be powdered to apply topically to the wound site and
fill irregular defects (e.g., digit amputation) [40]. The particulates derived from
powdering are of the order of 50–200 um in diameter and retain the surface
2 ECM Hydrogels for Regenerative Medicine 33

topology of the native matrix [5, 72]. The ECM powder may also be suspended in a
pharmacologically approved carrier (e.g., saline, water). High concentrations of
ECM powder suspension (e.g., 500 mg/mL) can form a putty with complex rhe-
ological behavior which has been applied to fill tissue defects [22].

ECM Hydrogel Formation

The discovery that extracellular matrix-derived materials could be formed into

hydrogels was a notable advance that further expanded the clinical utility of
ECM-derived materials [38]. ECM hydrogels retained the inherent bioactivity of
the matrix and provided nanofibrous structural support to guide cellular behavior
[45]. Additionally, key advantages compared to sheets include minimally invasive
delivery, i.e., injection as a viscous pre-gel fluid via catheter, followed by gelation
in situ and the filling of irregularly sized defects. Compared to a suspension of ECM
powder, the ECM hydrogel could be injected with a more homogenous concen-
tration and with greater ease [40].
Hydrogels derived from ECM-derived materials occupy a specific niche as a
diverse class of biomaterials. “Hydrogels” are defined as highly hydrated polymer
materials (>30% water by weight) [34] composed of synthetic or natural polymer
chains with cross-links to maintain the structural integrity of a solid [50]. Hydrogels
from naturally derived materials containing one or more molecules of the extra-
cellular matrix (e.g., collagen, agarose, alginate, chitosan, fibrin, gelatin, and hya-
luronic acid) are often called “ECM mimics” and have been extensively
characterized [34]. The limitation of reducing the complex cellular microenviron-
ment to one or more ECM proteins prompted the development of more complex
hydrogels: in particular Matrigel (reconstituted basement membrane extract) from
Engelbreth–Holm–Swarm tumor, and gels from decellularized tissue. When the
present chapter cites “ECM hydrogels,” the term is applied specifically to ECM
hydrogels derived from decellularized mammalian tissue.
The first ECM hydrogels were composed of porcine small intestinal submucosa
(SIS) (Voytik-Harbin [87] or porcine urinary bladder matrix (UBM) [38]. SIS and
UBM are two of the most well-characterized ECM-derived materials. The first
studies of SIS and UBM hydrogels determined whether the materials retained
bioactivity and the ability to promote constructive remodeling in non-heterologous
tissues; properties characteristic of the 2D sheet forms of SIS and UBM [38]. The
first studies also demonstrated feasibility of ECM hydrogel production and
described test methods for characterization of these materials and thus established a
precedent for the production of the next-generation ECM hydrogels for homologous
tissue applications.
34 M. J. Sawkins et al.

Methods for ECM Hydrogel Production

Gelation of the first SIS and UBM hydrogels was based on well-established pro-
tocols for collagen gelation. Native collagen is a main constituent of the ECM
hydrogel, and its gelation characteristics have been extensively characterized [42,
46, 47, 62]. Although ECM hydrogel preparation has been described with respect to
collagen biochemistry and fibrillogenesis, it is important to note that ECM hydrogel
formation is undoubtedly influenced by the presence of other ECM components
[42]. While bulk mechanical properties of the hydrogel can be described, further
work is required to understand microscale differences that affect collagen poly-
merization and are manifested in distinct viscoelastic properties.
The pepsin enzyme is used in both the SIS and UBM digestion protocols. While
pepsin, derived from the gastric juices, is not a physiologically relevant method for
ECM degradation, biochemists have been using pepsin since 1972 to solubilize a
substantial portion (up to 99%) of the acid-insoluble collagen [33, 57]. It has been
shown that pepsin selectively cleaves telopeptides, the protein domains external to
the triple helix-body of the tropocollagen molecule [42]. These telopeptides form
the intramolecular bonds between the collagen fibril aggregates [33, 70]. The
unraveling of the collagen fibril aggregates by pepsin digestion produces almost
entirely monomeric segments [70]. Visible changes are observed as the pepsin in
dilute acid solubilizes the ECM powder and the suspension transforms into an
opaque, viscous solution (“solubilized ECM”). The solubilized ECM is ready for
gelation when the liquid is homogenous, with no visible particles [45].
Another general method of protein solubilization which has been applied to
ECM materials is homogenization, either by pestle and mortar or high-speed shear
mixer. This is carried out in the presence of a high salt buffer and acts to physically
break down the ECM particles and disrupt the collagen fiber structure [23, 25, 37,
64, 65, 84, 85]. Prior to this physical processing, the material is often treated with
dispase, a specific protease which cleaves fibronectin, collagen IV, and collagen I
[23, 64, 65, 84, 85]. The treatment is used primarily to decellularize the tissue but
will also begin to digest the ECM protein structure. After dispase treatment and
homogenization, the ECM is then typically further processed via one or more urea
extraction steps [23, 37, 64, 65, 84, 85]. Urea in high concentrations acts to disrupt
the non-covalent bonding which is essential to protein folding and structure and
thereby denatures many proteins and increases their solubility. Centrifugation can
be used to remove any remaining insoluble ECM components leaving a viscous,
cloudy solution similar to that obtained by pepsin digestion.
The solubilized ECM is neutralized to physiologic pH and salt concentration and
is referred to as “pre-gel solution.” Gelation is chiefly determined by the poly-
merization of fibrillar collagen as the primary ECM constituent. When the tem-
perature is raised to physiologic conditions (37 °C), solubilized collagen can
self-assemble in an entropy-driven process. It is energetically favorable for collagen
monomers to lose surface water and bind to aggregates in order to bury
hydrophobic residues within the fibril [42, 62]. Self-assembly follows four
2 ECM Hydrogels for Regenerative Medicine 35

characteristic growth phases: a nucleation phase, a lag phase (collagen monomers

form metastable nuclei), a rapid growth phase (fibrils mediate from the nuclei), and
plateau phase [42]. At the end of this process, the solubilized ECM is now an “ECM
hydrogel.”

ECM Hydrogel Characterization

The unique biological, biochemical, mechanical, and topological properties of SIS

and UBM hydrogels have been well characterized using the techniques described
below.

Biocompatibility

At minimum, a biocompatible hydrogel should not cause detrimental effects on

cells interacting with the material and should exist within the body without eliciting
a pro-inflammatory response [53]. Beyond the minimum requirements of biocom-
patibility, the ECM hydrogel should induce or maintain tissue-specific differenti-
ation of cells to promote an appropriate host response.
Cell response to SIS hydrogels has been characterized in vitro and compared to
intact SIS and common tissue culture substrates including tissue culture plastic,
pure collagen gel, and MatrigelTM [87]. Specifically, cell shape, morphology, and
proliferation were characterized for four cell types: squamous epithelial cells
(pulmonary artery), fibroblasts (Swiss 3T3), glandular epithelial cells (adenocarci-
noma), and smooth muscle-like cells (urinary bladder). All cells were able to
adhere, survive, proliferate, and invade the SIS gel. SIS gels promoted similar cell
shapes as the intact SIS. The SIS gel also promoted more cell–cell contact than
collagen and tissue culture plastic, with cells spreading on the surface of the gel and
invading to form three-dimensional multilayers. This cell growth pattern was
markedly different compared to the other substrates where MatrigelTM induced the
formation of aggregates with limited to no cell proliferation for each of the cell
types, tissue culture plastic induced formation of cell monolayers, and collagen gels
displayed mixed 2D and 3D morphologies and restricted matrix penetration.
In another study, SIS hydrogel was characterized as a tissue culture substrate for
cardiac cells and compared to MatrigelTM [30]. Neonatal rat cardiac cells were
combined with SIS gel and MatrigelTM on a porous, elastomeric scaffold. The SIS
gel and MatrigelTM both permitted cell contraction on days 3–4, which continued
for 10 days. Compared to cardiac cells cultured in MatrigelTM, those cultured in SIS
gels displayed significantly higher mean contraction rate (more closely mimicking
native rat myocardium), expression of cardiac-specific marker Troponin-T, and a
more homogenous distribution within the construct. MatrigelTM exhibited greater
cell adhesion compared to SIS gel, likely attributed to the high laminin content of
the former. Overall, the superior performance of SIS gel for tissue-specific cell
36 M. J. Sawkins et al.

differentiation and function is likely attributable to the biochemical composition of

the SIS gel more closely mimicking that of native myocardium, i.e., high content of
collagen I and III.
UBM gels were also characterized for their ability to support growth of rat
aorta-derived smooth muscle cells. Similarly to SIS gels, the smooth muscle cells
were able to adhere, proliferate, form multilayers, and contract. After 10 days,
UBM gels promoted significantly greater cell numbers than collagen gels, but lesser
numbers than intact UBM. The authors proposed the difference between intact
UBM and the UBM gel could be attributed to the loss of certain bioactive peptides
during the hydrogel formation process [38]. UBM gels also have been shown to
support the growth of NIH 3T3 fibroblasts, with almost 100% cell viability after
7 days and gel infiltration [90].

Biochemical Composition

Studies of UBM and SIS gels provided confirmation that the hydrogels possessed
similar biochemical compositions to the intact UBM and SIS scaffolds in terms of
collagen and GAG content [17, 38, 90]. Intact SIS contained 90% collagen, pri-
marily collagen I, with minor amounts of collagen III, IV, V, and VI. Intact UBM
had a similar composition to intact SIS, but with greater amounts of collagen III and
the additional presence of collagen VII [5]. SIS hydrogels were shown to contain at
least three types of collagen (predominantly collagen I and collagen III with minor
amounts of collagen IV), and sulfated GAGs [17]. Gel electrophoresis of the UBM
gel displayed similar bands to purified collagen I, with additional bands present for
other ECM proteins [38].
Recognizing the biochemical complexity of ECM hydrogels, mass spectroscopy
is beginning to be used to determine the proteomic profile of pepsin-solubilized
ECM [14, 76]. Published studies to date have used reversed-phase
high-performance liquid chromatography interfaced with tandem mass spec-
troscopy (LC-MS/MS) and compared the peptide fragments generated with a
protein data bank (Swiss Protein data bank). SIS and UBM hydrogels have yet to be
characterized by LC-MS/MS.

Gelation Kinetics and Mechanical Properties

The delivery of solubilized ECM via catheter or syringe to irregularly shaped

anatomic sites provides increased clinical utility of ECM-derived materials [5]. The
kinetics of gelation and the viscoelastic properties of the gel can be designed to be
similar to those of the tissue being repaired. For instance, desired mechanical
properties important for cell behavior can be achieved by changing the ECM
concentration [90]. The biochemical composition of the ECM (e.g., collagen,
glycoproteins, GAGs, growth factors) is specific to tissue type, and microscale
differences in intermolecular bonding/microstructure are manifested in distinct
2 ECM Hydrogels for Regenerative Medicine 37

viscoelastic properties of the gels. Furthermore, the viscoelastic properties are

affected by any modifications to the gel, including changes in the decellularization
treatment and the addition of exogenous cells or biologics.
The turbidimetric gelation kinetics and rheological behavior of UBM gels have
been well characterized [38, 90]. Gelation kinetics for UBM showed a sigmoidal
shape, similar to that achieved with collagen type I gels. The lag phase and time
required to reach half the final turbidity were greater in the UBM gels than the
collagen type I gels [38]. This difference between the ECM gel and the purified
collagen gel is presumably due to the presence of GAGs and other molecules such
as fibronectin which may modulate self-assembly. The role of GAGs in the gelation
kinetics of ECM-derived gels was investigated by time-lapse confocal reflection
microscopy and spectrophotometry of purified components of SIS hydrogels [17].
A decrease in final turbidimetric absorbance and changes in gelation kinetics,
including increased lag phase, occurred when collagen type I gels were mixed with
different GAGs [17].
ECM hydrogels are thermosensitive viscoelastic materials that display
mechanical properties similar to a viscous liquid at 10 °C and mechanical prop-
erties intermediate between that of a solid and liquid at 37 °C. The mechanical
response of ECM hydrogels can be described as a combination of the elastic (en-
ergy storing) and viscous (energy dissipating) components and can be measured by
rheology. The elastic response of ECM hydrogels may be described by Hooke’s
law, i.e., strain is proportional to applied stress, and the sample quickly returns to its
original shape when the stress is removed. The viscous response of ECM hydrogels
may be described by Newton’s law, i.e., the strain rate is proportional to applied
stress, and the sample will continue to strain with time. Viscoelastic ECM hydro-
gels display intermediate behavior that is time-dependent: The hydrogel is able to
instantaneously strain, but further deformation of the polymer chains is
time-dependent.
Rheological measurements for UBM gels have been reported [38, 90].
Solubilized ECM was loaded onto a parallel-plate rheometer at 10 °C, and defor-
mation in response to constant shear stress was measured to determine viscosity
(“pre-gel creep test”). At 10 °C, UBM gels exhibited low viscosity [38]; the tem-
perature was rapidly increased from 10 to 37 °C to measure the gelation kinetics
and viscoelastic properties of the forming ECM hydrogel (“time sweep test”). An
oscillatory strain at fixed frequency (x) of 1 rad/s was applied to the UBM hy-
drogel, and the stress response characterized. At time zero, UBM hydrogels
exhibited low storage (G′) and loss (G″) moduli. As temperature was raised, G′ and
G″ followed a sigmoidal shape over time and reached a plateau in *12 min,
indicating that gelation was complete. Solid-like behavior was confirmed since the
storage modulus (G′) was greater than the loss modulus (G″) by approximately a
factor of 10 after gelation [38].
Freytes et al. [38] undertook a frequency sweep analysis of UBM hydrogels
which indicated “shear thinning” behavior, i.e., the complex viscosity decreased
with increasing shear rate. This may be a useful property for ECM hydrogels which
are to be directly injected or delivered via catheter. Recently, Wolf et al. [90]
38 M. J. Sawkins et al.

undertook post gelation creep analysis of UBM in comparison with a hydrogel

derived from dermal extracellular matrix. Both hydrogels displayed a viscoelastic
strain profile characterized by a rapid initial strain increase with creep ringing and a
subsequent slower increase over time.

Gel Topology

Upon gelation, SIS, and UBM hydrogels form porous, nanofibrous structures,
which provide cells with a dynamic 3D environment. In contrast to other forms of
ECM-derived materials (sheet and powder), the ECM hydrogels lose the native 3D
structure. However, the complex nanoscale mesh of ECM fibers guides cellular
behavior via contact guidance and fibril-bound proteins that interact with cell
membrane-bound integrin receptors.
Visualization of the SIS and UBM gel topology with scanning electron micro-
scopy (SEM) showed that both hydrogels formed loosely organized, interwoven
networks of fibers [38]. D’Amore et al. [31] developed a MATLAB algorithm that
enables high-throughput, automated analysis of SEM images to more accurately
assess the characteristics of the overall fiber network. Using the image-based
algorithm, UBM hydrogel characteristics were assessed [90]. The UBM hydrogels
had a fiber diameter of 0.074 ± 0.004 lm and pore size of 0.112 ± 0.005 lm,
which is within biological criteria for hydrogel design. Fiber alignment was ran-
domly organized (isotropic) as characterized by fiber alignment close to 0% with
respect to any axis. The described metrics were concentration-independent for
UBM hydrogels, but ECM concentration has been shown to be an important
covariate of fiber diameter and porosity for other ECM hydrogels, such as dermal
matrix hydrogels [90].

Tissue-Specific Hydrogels

Recent studies of ECM-derived biomaterials in various forms have demonstrated

significant tissue specificity in achieving complex biological functions [27, 63, 78,
86]. As a result, having established that SIS and UBM could be processed into
hydrogels, the next step for researchers was to demonstrate hydrogel formation
from a wide range of tissues and establish the efficacy of these materials in tissue
repair. Published studies to date which have contributed to this process will be
discussed in detail in the following sections, grouped according to their target
tissue. The mechanical properties, fiber structures, and gelation kinetics of many of
these novel ECM hydrogels have been characterized using techniques similar to
those described in Section “ECM Hydrogel Characterization,” albeit with
tissue-specific testing conditions and parameters. However, as discussed earlier, the
primary purpose of an ECM hydrogel is to act as a bioinductive template for new
tissue formation, and as a result, the focus of this chapter will be on the biological
2 ECM Hydrogels for Regenerative Medicine 39

performance of the novel gels rather than their physicochemical characteristics. It is

only a matter of a few years since the first publication detailing the production of an
ECM-derived hydrogel. As a result, data published to date in this field have typi-
cally arisen from in vitro experiments and small-scale proof-of-principle studies
in vivo, rather than from fully developed preclinical or clinical evaluations.

Heart

Among the most frequently targeted tissues is cardiac muscle, primarily with the
aim of restoring heart function after myocardial infarction (MI), which is the
leading cause of death in most industrialized countries (Rosamond et al. [69].
Hydrogels from heart tissue have been developed, characterized, and applied to
tissue repair in a number of studies published by Christman and colleagues [44, 45,
75–77, 80, 81]. A protocol was developed for the production of decellularized
matrix from porcine myocardium via a detergent-based technique using sodium
dodecyl sulfate (SDS) and Triton X-100 [80]. Pepsin digestion of this matrix
resulted in a solution which underwent thermoresponsive gelation both in vitro and
in vivo after catheter-based delivery, thus suggesting that the material had good
clinical handling properties. As with many other ECM materials, the myocardial
matrix was shown to be composed predominantly of collagen, but also to contain a
significant amount of GAGs. The most important result of this initial study, how-
ever, was that both smooth muscle cells and endothelial cells migrated toward the
matrix in vitro and in vivo to a greater degree than toward collagen or fetal bovine
serum (FBS). The cell-free material may therefore be sufficient to recruit appro-
priate cell types to defect sites and to ultimately form appropriate new tissue.
Further development of myocardial matrix was shown in a subsequent study which
demonstrated that the fiber size and structure, mechanical properties, and gelation
kinetics could be modulated by alterations of the pH, temperature, ionic strength,
and concentration used for gelation [45]. Modification of the hydrogel in this
fashion could allow the handling properties and cell response to be tailored to
specific applications.
Most recently, the treatment efficacy of the material has been evaluated in both
rat and porcine preclinical models of MI [77, 81]. In the rat model, myocardial
matrix was injected into the infarcted area 1 week after MI and led to significantly
increased numbers of cardiomyocytes and proliferative cells 1 month after injection
when compared to saline-injected controls [81]. Cardiac function was also signif-
icantly increased at this time point as measured by parameters such as the ejection
fraction, end-diastolic dimension, and end-systolic volume. In a later study in the
porcine model [77], myocardial matrix was delivered into the affected tissue
2 weeks after the induction of MI. Treatment contributed to an increased muscle
volume in the affected area when compared to saline-injected controls 3 months
post-MI, and significant increases in cardiac function were again observed. Taken
together the results of these studies suggest great promise for this material in
40 M. J. Sawkins et al.

post-MI cardiac regeneration. This promise is further supported by positive results

obtained during examination of the safety and immune response to myocardial
matrix. In response to injection into healthy rat hearts, no significant increase in
infiltrating inflammatory cell numbers was seen relative to saline-injected controls.
Finally, in vitro hemocompatibility studies showed no effect of myocardial matrix
on platelet activation and clotting.
A recent study sought to evaluate the production of a similar material from
human, rather than porcine myocardium, in order to eliminate the use of xenogenic
material [44]. The efficacy of the two materials was compared in vitro, and it was
shown in principle that the human myocardial matrix could undergo in vivo
gelation. In order to effectively decellularize the human myocardium, the incubation
time in SDS had to be significantly extended relative to the porcine tissue process
and additional DNA and lipid removal steps had to be included. This was attributed
to the accumulation of additional lipids and fibrous tissue in the myocardial matrix
of the human tissue, which was harvested, relatively speaking, from older donors.
As a result of this additional processing, the sulfated GAG content of the
human-derived material was significantly lower than that of the porcine material,
suggesting the complex matrix composition and architecture may be less well
maintained. Both rat aortic smooth muscle cells and human coronary artery
endothelial cells proliferated more rapidly over 5 days on human myocardial matrix
than porcine. However, less than half of the human donors tested gave rise to matrix
solutions which would undergo gelation, and very significant donor-to-donor
variability was observed. As a result, the ability to translate this approach to
large-scale clinical application may be limited.
An alternative material has been developed by the same researchers, but derived
from the pericardium rather than cardiac muscle. A similar approach was taken to
evaluate the potential of this material, with both porcine and human pericardium
decellularized using SDS and solubilized with pepsin [76]. Again, these materials
were composed primarily of collagen, though with significant components of GAG
and other proteins. In vitro chemotaxis assays demonstrated that, as with
myocardial matrix, both endothelial and smooth muscle cells migrated toward
pericardial matrix, with porcine-derived material significantly outperforming human
in this analysis. The in vivo results of injecting these matrices into healthy rat
myocardium also showed significant infiltration by both cell types, and the for-
mation of arterioles, with the human ECM-derived gel outperforming porcine.
Finally, positive staining for c-kit indicated the presence of stem cells in both
materials, suggesting their ability to direct stem cell homing.
The utility of the pericardial matrix in a preclinical MI rat model was evaluated
[75]. In this study, an additional function of the matrix was assessed, in the form of
its ability to sequester and then slowly release basic fibroblast growth factor (bFGF)
in an effort to boost cardiac regeneration. The in vitro release of bFGF from
pericardial matrix was significantly delayed relative to collagen. This delay was
attributed to the ability of GAGs, and more specifically heparin in the matrix to bind
the growth factor, though the specific presence of heparin was not demonstrated at
this stage. Greater retention of bFGF in the infarcted tissue in vivo was shown when
2 ECM Hydrogels for Regenerative Medicine 41

the protein was delivered in matrix than in collagen or saline, and this resulted in
significant increases in arteriole density. Most importantly, the newly formed
vasculature was anastomosed with that preexisting in the host, allowing it to
function to restore oxygen supply to the ischemic tissue. The next stage for the
development of pericardial matrix will be to demonstrate that the observed
improvements in neo-vessel formation result in recovery of cardiac function, as
observed for myocardial matrix.
In addition to the heart-derived materials described thus far, porcine bone
marrow has also been considered as a source tissue for hydrogel biomaterials to
treat MI [66]. A series of nuclease, ethylenediaminetetraacetic acid (EDTA), vortex
and centrifuge steps were used to produce a bone marrow ECM extract which
contained a number of growth factors important in angiogenesis and cardiac repair.
Coating non-tissue culture-treated polystyrene (non-TCPS) with this extract was
found to significantly increase endothelial cell adherence relative to fibronectin.
Fibronectin is an effective single component substrate coating containing many
RGD peptide sequences which can interact with integrins on the cell surface. The
ability of the bone marrow ECM-derived material to support greater cell attachment
is therefore a highly significant result. For three-dimensional hydrogel studies, the
bone marrow extract was reinforced with methylcellulose in order to improve
material handling and retention at the injection site in vivo. When this reinforced
material was delivered into a rat MI model, the infarct area was rapidly reduced
relative to untreated controls, with associated increases in cardiac function.
Furthermore, 3 weeks post-intervention there was a significant reduction in the
numbers of apoptotic cells and macrophages present, alongside increases in stem
cell and blood vessel numbers. This bone marrow-derived ECM material may
therefore be a useful supplement to other biomaterials utilized for cardiac repair
post-MI.
As one of the most heavily targeted tissues for the development of ECM-derived
hydrogel biomaterials, it is perhaps unsurprising that proposed therapies in the area
of post-MI cardiac repair are also among the most well-developed. All three distinct
materials, developed from different source tissues, have shown, in principle at least,
that they can contribute to the generation of functional repair tissue in vivo.

Fat

Like cardiac tissue, fat has also been widely studied as a target for repair by
extracellular matrix biomaterials [23, 65, 85, 92]. If a successful therapy could be
developed, then it could be used in a large number of procedures following trauma
and tumor resection which require the reconstruction of significant volumes of
adipose tissue. The use of ECM hydrogels to support adipogenesis was first
explored in a pair of studies by Brey and coworkers [23, 84]. In the first of these
studies, a combination of dispase digestion, high salt homogenization, and urea
extraction were used to produce rat adipose tissue extracts which underwent
42 M. J. Sawkins et al.

thermo- or pH-responsive gelation [84]. When seeded with pre-adipocytes in vitro

adipose-derived gels supported significantly greater adipogenic differentiation than
MatrigelTM based on histological staining for lipid loading. These results were
echoed in vivo when the matrix was implanted around a rat epigastric pedicle
bundle. Hydrogels formed by changes in temperature or pH were implanted but
only those formed by pH changes supported increased adipogenesis relative to
MatrigelTM at time points greater than 6 weeks post-implantation.
An alternative rat dermis-derived hydrogel, produced by the same protocol, has
also been assessed for its ability to support adipogenesis [23]. As was found for
adipose-derived materials, the dermis-derived gels supported significantly more fat
tissue formation in vivo than MatrigelTM. In vitro experiments, however, showed no
difference between the two in this case. The results of in vitro evaluation in these
two studies suggested that in order to promote adipogenesis, an ECM hydrogel
must be sourced from fat tissue. However, gels from both sources considered here
were highly effective substrates for fat tissue formation in vivo. This highlights the
uncertainty inherent in forming conclusions about tissue specificity based on a
single model and more particularly in relying exclusively on in vitro studies.
Porcine fat tissue extracts have also been prepared using various combinations of
high salt homogenization, dispase treatment, urea extraction, and pepsin digestion.
By combining a number of such extracts, a thermoresponsive gel-forming material
known as adipose-derived matrix (ADM) was produced [65]. A number of relevant
growth factors such as FGF-2 and TGF-b1 were quantified in ADM, which
prompted no significant inflammatory cytokine production by in vitro macrophage
cultures. Adipose-derived stem cells (ASCs) were differentiated on ADM in the
absence of adipogenic medium supplements with greater than 90% efficiency.
Furthermore, when ADM, with or without ASCs, was implanted subcutaneously in
rats, it resulted in significantly more adipose tissue generation than MatrigelTM.
This was the case even when the latter was supplemented with FGF-2 in an effort to
increase adipogenesis. The ADM containing groups also both showed significantly
fewer activated immune cells, echoing the in vitro macrophage results.
More recently, a thermoresponsive matrix has also been generated from human
lipoaspirate by a combination of surfactant and enzymatic treatments, with pepsin
digestion to fully solubilize the protein mixture [92]. This material underwent
gelation upon subcutaneous injection in rats and supported proliferation of
patient-matched ASCs in vitro. However, the growth rate on this novel matrix was
not significantly enhanced relative to collagen or TCPS. It is interesting to note that
the ECM-derived hydrogel was not able to exert a mitogenic effect on relevant stem
cells, but studies of cell differentiation and matrix production may be necessary to
allow comparison with studies of ECM-derived materials from other species.
The results of the studies discussed herein indicate that extracts from both
dermal and subcutaneous adipose tissues may provide useful scaffolds for the
generation of fat tissue in a range of reconstructive surgical procedures.
Nonetheless, it may be instructive to determine whether the lack of benefit seen
with human lipoaspirate was due to the source species or the extraction protocol.
With a readily available tissue such as subcutaneous fat, there is a clear case for
2 ECM Hydrogels for Regenerative Medicine 43

autologous or allogeneic, rather than xenogeneic tissue as the ideal source for
scaffold material as it carries less risk of disease transmission or adverse immune
response.

Skin

In an attempt to generate biomaterials for wound healing and skin repair, hydrogels
have been derived from rat dermis by a combination of dispase treatment, high salt
homogenization, and urea extraction to produce tissue extracts to undergo thermo-
or pH-responsive gelation [64, 85]. In the first of these studies, it was shown that
both endothelial cells and fibroblasts could proliferate on these dermis-derived
biomaterials and that endothelial cells formed tubule networks as an indicator of the
potential for blood vessel formation [85]. However, it is noteworthy that fibroblasts
placed into the gels as spheroids were unable to migrate into the substrate as they
did when cultured in MatrigelTM.
The second study examined the effects of glutaraldehyde cross-linking on the
dermis-derived hydrogels [64]. Cross-linking was shown to significantly increase
the elastic moduli and yield stresses of the hydrogels and to significantly slow
enzymatic degradation in vitro and also after subcutaneous implantation in the rat.
Cross-linked gels exhibited minimal cytotoxicity provided they were adequately
washed prior to exposure to cells or tissue. Furthermore, glutaraldehyde
cross-linking led to significantly increased blood vessel density in the tissue formed
after implantation, suggesting that it has the potential to improve the performance of
these materials in wound healing.
Dermis-derived hydrogels show promise for application in wound healing as
they permit proliferation of relevant cell types and can support new blood vessel
formation both in vitro and in vivo. Nevertheless, it remains to be established that
fibroblasts can migrate into and deposit ECM proteins within these materials. This
is a necessary step if the appropriate structural matrix is to be deposited around the
neovasculature formed in these scaffolds.

Liver

Liver is another tissue in which the potential for ECM-derived hydrogels to support
tissue formation has been established in principle [52, 78]. Initially, a porcine liver-
derived gel was produced using a combined surfactant and enzyme-based approach
to decellularize the tissue, followed by pepsin digestion to solubilize the protein
mixture [78]. It was shown that primary human hepatocytes grown in a sandwich
culture between two layers of liver-derived matrix expressed relevant genes and
proteins as highly as those grown between layers of MatrigelTM, the gold standard
substrate. Subsequently, promising hydrogel materials were generated from rat liver
44 M. J. Sawkins et al.

using Triton X-100 and ammonium hydroxide for decellularization and

pepsin-based solubilization of the ECM proteins [52]. During in vitro culture
experiments, primary rat hepatocytes showed greater cell adhesion and viability on
liver-derived substrate coatings than on collagen I or MatrigelTM. Viability and
hepatocyte functions of albumin secretion and urea synthesis were all significantly
enhanced when cells were grown within three-dimensional ECM gels relative to
collagen controls. More promising from a potential clinical standpoint is that ASCs
were also shown to more highly express hepatocyte genes such as albumin and
hepatocyte growth factor when grown on the ECM-derived hydrogels. This material
was also utilized in an early demonstration of in vivo treatment efficacy. Primary
hepatocytes were delivered subcutaneously into mice in either collagen or liver-
derived ECM pre-gel solutions which underwent gelation post-injection. The results
of this investigation echoed those of the in vitro experiments, with hepatocyte
marker expression and function being significantly enhanced in the tissue-specific
material. This latter study in particular suggests great potential for liver-derived
hydrogels being applied to liver regeneration, but orthotopic, functional in vivo
studies are required before any definitive conclusions can be drawn.

Skeletal Muscle

The use of hydrogels for the repair and regeneration of damaged skeletal muscle has
been investigated using two distinct materials in two distinct models [32, 91].
Firstly, a hydrogel material was developed from porcine skeletal muscle and
evaluated in a rat hindlimb ischemia model [32]. The source tissue was decellu-
larized by the action of SDS, with pepsin digestion used for ECM solubilization.
The solution resulting from this process was found to significantly enhance pro-
liferation of both rat smooth muscle cells and C2C12 myoblasts relative to
pepsin-digested collagen as an in vitro culture supplement. The results of in vivo
experiments in which the muscle-derived hydrogel was injected intramuscularly
into ischemic tissue were also positive. Relative to collagen gels, those derived
from skeletal muscle matrix resulted in significant increases in arteriole density,
endothelial cell numbers, and proliferating muscle cell numbers at all time points
over a 2-week period post-injection.
The second type of hydrogel considered for skeletal muscle repair was devel-
oped from porcine dermis by a combined enzyme, surfactant, and peracetic acid
treatment, followed by pepsin digestion [91]. The performance of this material was
compared to that of a gel derived from UBM both in vitro and in a partial-thickness
rat abdominal wall defect. In vitro results clearly suggested that dermal ECM was a
superior scaffold material for this application. The C2C12 myoblast cell line more
efficiently fused to form myotubes on this substrate, and it was found to be sig-
nificantly more resistant to fibroblast-mediated contraction. The same resistance to
contraction was also found after in vivo implantation, and this is important in
ensuring scar-free wound healing. However, UBM hydrogels contained
2 ECM Hydrogels for Regenerative Medicine 45

significantly more cells expressing muscle markers 5-week post-implantation. It is

interesting to note that, as for studies examining adipogenesis in ECM-derived
hydrogels, tissue specificity was observed in some, but not all, evaluations of
muscle repair. Longer-term studies of functional tissue formation would be needed
to definitively establish the ability of these materials to repair skeletal muscle.
Nevertheless, the results presented thus far demonstrate potential for both dermis-
and muscle-derived ECM hydrogels in promoting recovery after skeletal muscle
injury.

Central Nervous System

A more recent development in the application of ECM-derived hydrogels to tissue

repair is their use to regenerate tissue in the central nervous system (CNS). The first
study to address this used UBM due to its ready availability and history of use [93].
As per previous studies, porcine UBM was produced by peracetic acid treatment
and pepsin digestion was then used to solubilize the material for the formation of
hydrogels. Injection of these hydrogels into healthy rat brain tissue was found to
generate no adverse response. It did not trigger significant activation of microglia,
astrocytosis, or neurodegeneration. Further analysis was conducted in a rat model of
mechanically induced traumatic brain injury. In this setting, the gels were found to
significantly reduce lesion volume relative to PBS injected controls 3-week
post-intervention. More importantly, significant functional recovery of improved
motor function was seen in the treatment group relative to controls. This
improvement was mediated by the ability of UBM hydrogel to prevent myelin
disruption after injury. Although cognitive recovery was not observed, these results
suggest fundamental promise in the application of ECM-derived hydrogels in the
repair of CNS tissue.
A subsequent pair of studies from Badylak and coworkers has sought to build on
this potential by first developing hydrogels from the ECM of the CNS itself and
subsequently comparing their performance to that of UBM [29, 54]. The hydrogels
were produced from the ECM of porcine brain and spinal cord by a combined
surfactant, enzyme, and peracetic acid treatment, followed by solubilization with
pepsin [54]. Rheological measurements and SEM imaging showed significant
structural and mechanical differences between the CNS-derived hydrogels and
those produced from UBM. Nevertheless, when the materials were added as pre-gel
solutions to the culture medium of murine neuroblastoma cells, all three increased
the proportion of cells exhibiting neurite extensions in a dose-dependent manner.
No significant differences between materials were observed based on this measure,
suggesting that all three could promote a neuronal phenotype. However, only brain
ECM gels induced an increase in mean neurite length, indicating that tissue repair
by ECM hydrogels exhibits at least some degree of tissue specificity.
This concept was explored further in a subsequent study examining the effects of
the three matrices on two types of adult human stem cells in vitro [29]. Once again,
46 M. J. Sawkins et al.

the pre-gel solutions were tested as culture medium additives to assess the effects of
ECM-derived soluble peptides. None of the three materials adversely affected the
viability of either neural stem cells (NSCs) or perivascular stem cells at concen-
trations of up to 100 lg mL−1. Furthermore, all three were able to exert positive
chemotactic effects on both cell types relative to FBS controls, though both the
presence and size of these effects were dose- and source tissue-dependent. UBM
pre-gel solutions were also able to significantly increase the proliferation rate of
both stem cell types at all concentrations, while the CNS-derived solutions
exhibited more complex, dose-dependent effects. However, both CNS-derived
solutions were able to direct neuronal differentiation of NSCs with equivalent
efficiency to the positive control differentiation medium. The observed expression
of bIII-Tubulin and neurite extension suggested that the CNS-derived materials
promote a less proliferative, more differentiated, and functional phenotype and
could drive tissue repair by endogenous stem cells. The conclusions of this
investigation suggest that in the case of CNS repair, a match between the source and
target tissues may be important for cell maturation and tissue formation.

Cartilage

The first ECM-derived material considered for cartilage repair was an extract from
human lipoaspirate, produced by homogenization, followed by SDS and nuclease
treatment [25]. A number of relevant growth factors were identified in this tissue
extract, including transforming growth factor-b1 (TGF-b1), an important mediator
of chondrogenic differentiation. Pellet cultures of this material in combination with
ASCs showed that the cells retained high viability across a culture period of more
than 6 weeks. The inclusion of the adipose-derived material in the pellets, with or
without the addition of TGF-b1, led to significant increases in total protein, col-
lagen, and sulfated GAG content of cultures indicating greater new matrix syn-
thesis. Important cartilage markers such as SOX9, aggrecan, and collagen types II
and X were expressed by cells in all ECM-containing pellets, though more strongly
in those cultured in the presence of TGF-b1. This suggests further that the matrix
being synthesized in these constructs had an appropriate, cartilage-like composition.
Although in vivo studies are necessary for progression, the fact that this material
could direct both stem cell differentiation and cartilage-like tissue formation in vitro
is nonetheless promising. It may be instructive to consider whether the positive
results obtained here are specific to the use of ASCs in conjunction with a hydrogel
of the same tissue origin. For a number of practical or biological reasons, it may be
desirable to design therapies using cells from other sources, such as chondrocytes or
mesenchymal stem cells. In these cases, it would be necessary to assess whether the
benefits observed here were retained.
A material derived from cartilage itself has also been considered for use in
cartilage repair, though its evaluation is at an earlier stage than that for the adipose-
derived hydrogel [51]. Porcine articular cartilage was decellularized by the use of
2 ECM Hydrogels for Regenerative Medicine 47

hypotonic buffer, SDS, and nuclease and subsequently solubilized by the action of
pepsin. This study extended the “classical” ECM-derived hydrogel approach by
including lyophilization of the pre-gel solution to form a powder which was sub-
sequently soluble in PBS. Gel formation by subcutaneous injection of reconstituted
material into rats was shown in principle, as was the capability of the ECM-derived
material to bind bovine serum albumin as a model protein and subsequently release
it in vivo over 2–3 weeks. This ability can presumably be attributed to the presence
of GAGs such as heparin in the material, but the exact mechanism has yet to be
conclusively deduced. A dose-dependent inflammatory response was observed for
the cartilage-derived matrix when implanted subcutaneously in rats, but the pro-
portions of activated immune cells within implant sites were low in all cases. This
study demonstrated the fundamental safety of the approach. Future work will be
required to show that endogenous and/or exogenous cells interacting with the
cartilage-derived material can proliferate and deposit appropriate matrix.

Tendon and Ligament

As was the case for cartilage, the first study aiming to demonstrate ECM-derived
hydrogel mediated tendon/ligament repair relied on the use of an already
well-characterized ECM material, namely porcine SIS [36]. This study used a large
animal (goat) model of anterior cruciate ligament (ACL) repair involving the
suturing of a surgically transected ligament with no tissue removal. In an effort to
augment repair, the sutured ligament was wrapped with a bilayered sheet of SIS and
fibrin sponge, and an SIS-derived pre-gel solution, produced by pepsin digestion,
was injected into the defect area. In ECM treated groups, the ACL cross-sectional
area was restored to equivalence with the sham-operated controls 12-week
post-intervention, which was several times the area of ligaments sutured without
augmentation. Furthermore, significant recovery of mechanical strength was seen
relative to suture-only tissue, though these ligaments still only withstood around
half of the load supported by the sham-operated tissues. It is unclear what pro-
portion of the benefit derived in this study is attributable to the gel, rather than the
bilayered sheet scaffold. Additionally, it will be necessary to address more chal-
lenging models before it becomes clear what promise SIS-derived hydrogels may
hold for the repair of damaged tendons and ligaments.
A subsequent study established that human tendon tissue could also be pro-
cessed to form hydrogels [35]. Decellularization using SDS and EDTA was fol-
lowed by pepsin digestion, resulting in a thermoresponsive gelling solution
composed primarily of collagen type I. Pre-gel solutions were delivered subcuta-
neously into rats to demonstrate in vivo gel formation and the host cell response.
Histological staining of explanted gels revealed that they were infiltrated by mac-
rophages and other immune cells around the periphery. Fibroblasts were also seen
throughout the hydrogels, elongated and aligned along the material fiber structure,
indicating the ability of the scaffold to direct cell behavior. Additionally, it was
48 M. J. Sawkins et al.

shown that ASCs could remain viable during in vitro culture either on the surface of
preformed gels or after encapsulation within them. This demonstrates the potential
of this material to be used in cell-seeded form in addition to the off-the-shelf,
cell-free alternative.
A third and final study sought to demonstrate that this human tendon-derived
hydrogel material could contribute to improved functional recovery after tendon
injury [49]. Defects 5 mm in length were created in the Achilles tendons of rats, and
the injection of tendon derived pre-gel solutions compared to contralateral controls
of injected saline. Histological analysis suggested that tendon-derived gel was able
to promote accelerated wound healing, with greater amounts of collagen seen in
gel-treated tendons 2-week post-injection than those injected with saline. At
4 weeks, this difference was still apparent, with a good degree of alignment seen in
the matrix deposited in gel-treated tendons; however, by 8-week post-injection,
there was no discernable difference. Functional mechanical evaluation of the
healing tendons revealed a similar trend, with gel treatment leading to significantly
increased ultimate failure load at the 4-week time point, with no differences seen at
2 or 8 weeks.
As with many other tissues, ECM-derived materials show promise for applica-
tion in tendon and ligament repair and regeneration. They appear to be well tol-
erated in vivo and may be able to accelerate defect healing. However, more
extensive studies are required to determine whether long-term tissue repair is
significantly enhanced by the presence of these hydrogels.

Intervertebral Disk

The nucleus pulposus (NP) of the intervertebral disk (IVD) exists in a naturally
gel-like state and does not therefore require the extensive processing used to gen-
erate hydrogels from other tissues. This has been exploited by Simionescu and
colleagues who have developed decellularized porcine NP as a scaffold for the
reconstitution of the IVD [55, 56]. They showed that both the mechanical properties
and relative concentrations of protein and GAG constituents of the decellularized
NP were unchanged relative to the native tissue [55]. When ASCs were seeded on
the surface of decellularized NP, they remained highly viable over the culture
period of 7 days and were able to migrate into the scaffold. This latter result
suggests that decellularized NP scaffolds could support the infiltration of appro-
priate host cell types for tissue repair after cell-free implantation.
In a second study by the same authors, decellularized NP was further studied to
elucidate the effects of the material on ASC differentiation and tissue repair in vivo
[56]. When ASCs were cultured on decellularized NP scaffolds, they expressed a
number of markers of the NP cell phenotype at significantly higher levels than on
TCPS. This study was conducted in basal growth medium in the absence of soluble
cues, indicating some inherent capacity for the material to direct appropriate stem
cell differentiation. The cells remained predominantly viable across 2 weeks,
2 ECM Hydrogels for Regenerative Medicine 49

contracted the hydrogel scaffold, and deposited a matrix containing collagen and
GAGs, leading to significant increases in scaffold mechanical properties.
Histological staining of scaffolds after subcutaneous implantation in rats indicated
that 4-week post-implantation the material was infiltrated by immune cells and
contained newly formed vasculature and newly deposited collagen. These results
established in principle that the material was well tolerated and the next step would
be to extend the in vivo analysis to include longer-term demonstrations of appro-
priate functional tissue formation.

Others

As described earlier, skeletal muscle-derived ECM hydrogels have been applied in

the reconstruction and regeneration of damaged muscle tissue [32], but their
potential for corneal repair has also been examined [37]. In a proof-of-principle
study, high salt homogenization and urea extraction were used to process human
muscle tissue and form coatings for plastic cell culture substrates. Cells from pri-
mary human limbal epithelial explants were used in this study which aimed to
develop novel substrates for human limbal epithelial stem cell transplantation and
corneal repair. The growth rate of cells on the skeletal muscle extract was equiv-
alent to that on amniotic membrane, the gold standard substrate for this application.
Moreover, this material significantly outperformed MatrigelTM and a corneal stro-
mal extract. Cells grown on the ECM-derived gel also expressed the limbal
epithelial stem cell marker ABCG2, which did not occur on the amniotic mem-
brane. These results must now be extended to thicker gels which can be surgically
manipulated and function as biomaterials, rather than quasi-two-dimensional
coatings. This muscle tissue extract may offer a new and improved substrate for
treating limbal stem cell deficiency which also has the potential to alleviate the
issues of availability and variability encountered in the use of amniotic membrane.
As a mineralized tissue, bone presents a unique challenge from the point of view
of decellularization and subsequent solubilization. Harsh demineralization and lipid
extraction steps are required to expose the underlying protein structure for further
processing. The demineralized bone matrix (DBM) which results from this initial
process is widely used clinically and generally considered to be an acellular bio-
material. However, in the only study to date to address the aim of generating a
hydrogel from mineralized bone tissue it was found that an additional decellular-
ization step was necessary to ensure efficient cell lysis and debris removal [74].
Biochemical characterization showed that both the ECM and DBM gels were
composed predominantly of collagen. Turbidimetric and rheological analysis
demonstrated further that the bone-derived hydrogels had similar gelation kinetics
and mechanical properties to collagen gels. However, when the three materials were
utilized as culture substrates for murine primary calvarial cells only the ECM
hydrogel promoted cell proliferation, leading to significantly higher cell numbers
than on the other substrates. This represents a preliminary study, but nevertheless
50 M. J. Sawkins et al.

this bone ECM hydrogel has potential as a carrier and supplement for existing bone
graft materials such as DBM and allogeneic bone.
The vocal fold (VF) is another tissue, where SIS hydrogel has been evaluated. In
an in vivo proof-of-principle study, SIS gel showed promise, primarily as a carrier
for mesenchymal stem cells rather than as a therapy in its own right [26]. This
publication details the production of an SIS hydrogel by mechanical dissociation
and saline washing of the small intestine, followed by pepsin digestion to solubilize
the resulting material. As in an earlier study targeting cartilage repair (see
Section “Cartilage”), hydrogels formed from this solubilized material were lyo-
philized and milled to form a PBS soluble powder. A rabbit model of VF scar
formation and regeneration was used to evaluate injectable therapies composed of
rabbit MSCs, SIS gel, or both components together. The combined therapy led to
increased MSC retention and engraftment at the injection site, with fluorescently
labeled cells visible 3-week post-injection by in vivo imaging. Significantly, more
cells were observed histologically in defects treated with SIS at both 3- and 8-week
time points, although by this method cells were visible in all MSC-treated groups.
The combined therapy also promoted increased accumulation of sulfated GAGs and
significantly decreased collagen deposition relative to all other groups, indicating
the propensity for more effective regeneration and reduced scar formation. Finally,
these compositional changes were reflected in the assessment of functional regen-
eration of the tissue, as combined treatment led to significantly increased vibratory
capacity relative to other groups. Longer-term large animal studies are needed to
further establish the potential of combined SIS-gel and MSCs as a therapy to
prevent vocal fold scarring, but this study is nevertheless an effective proof of
principle.

Hybrid Hydrogels

A recent evolution in the use of hydrogels derived from decellularized tissue has
been the combination of these materials with other natural or synthetic hydrogels, to
provide mechanical reinforcement and/or enhanced biological activity [41, 82].
Other approaches have utilized particulate ECM materials as additives to synthetic
hydrogels [24, 61] or applied tissue derived hydrogels as coatings for solid bio-
materials [89], but these will not be considered further in this chapter.
The first application of this next-generation concept occurred in liver repair and
utilized extracts from liver cross-linked to the natural ECM components collagen
type I and hyaluronic acid (HyA) [82]. Liver tissue was decellularized using Triton
X-100 and ammonium hydroxide; non-decellularized liver was used as a com-
parator to deduce any adverse effects of the residual cellular and genetic compo-
nent. Both materials were solubilized by the action of pepsin and combined with
collagen or HyA and the cross-linker prior to gel formation. In sandwich culture of
primary human hepatocytes, the addition of liver tissue extracts was found to result
in significantly increased cell numbers. This effect was especially pronounced at
2 ECM Hydrogels for Regenerative Medicine 51

later time points in the 4-week experiment. Decellularized liver extract had a greater
effect than that from non-decellularized tissue, which can be attributed to the higher
concentrations of both structural ECM constituents and growth factors. Although it
is promising that these materials can boost primary cell proliferation, the data in this
study show no significant effect on hepatocyte functions such as albumin and urea
secretion despite the increase in cell numbers. To fully establish their potential, it
must be shown that they can aid functional recovery in damaged tissue.
The second example of this hybrid approach again utilized chemical
cross-linking to conjugate ECM-derived peptides to supplementary polymers, based
on poly(ethylene glycol) (PEG), and intended to offer enhanced mechanical prop-
erties and delayed degradation. These materials were used to reinforce a porcine
myocardial matrix generated by SDS-based decellularization [41]. Incorporation of
PEG-based polymers in the hydrogel network provided significant mechanical
reinforcement alongside structural changes which were dependent on the incor-
poration method and the polymer molecular weight. Experiments with a murine
fibroblast cell line showed that cells seeded on the surface of preformed hydrogels
could migrate into the gels and proliferate on the surfaces regardless of the presence
of the PEG component. This is despite the fact that PEG is an inert synthetic
polymer that generally does not interact with cells as it contains no binding sites for
cell surface receptors such as integrins and cadherins. It was also shown that cells
encapsulated within gels could proliferate rapidly across all conditions, with no
inhibitory effect due to the synthetic component. The fibroblast cell line used has
broad relevance to a range of tissues and the cross-linking methods could be used
with a range of tissue extracts, containing a significant collagen component and
similar functional groups. This technique could therefore potentially be used to
overcome current limitations and tailor the mechanical and degradation properties
of naturally derived hydrogel biomaterials.

Future Directions

The lessons learned from the manufacturing and application of ECM-derived

scaffolds must be applied to ECM hydrogels in order to permit clinical use of these
materials. Since the ECM of each tissue or organ is produced by the unique
combination of cell types resident within it, it is intuitive that a substrate composed
of a tissue-specific ECM may be favorable for regeneration and repair. However, in
some cases the need to use a tissue-specific matrix may be questioned as more
readily available SIS and UBM may have similar effects. A recent comparison of a
tissue-specific muscle ECM and SIS demonstrated constructive remodeling in a
rodent muscle defect model; there was no apparent advantage to using the
tissue-specific ECM despite differences in structure and composition [91]. The use
of non-homologous ECM has many advantages: SIS and UBM are produced by
established standardized protocols, tissue is more accessible, and xenogeneic tissue
is available in large volumes. Furthermore, SIS ECM has been thoroughly
52 M. J. Sawkins et al.

evaluated both in vitro and in vivo and is used clinically [11]. In addition, SIS and
UBM hydrogels have been well characterized in terms of biocompatibility, bio-
chemical composition, gelation kinetics, viscoelastic properties, and gel topology.
The choice to use a tissue-specific ECM hydrogel must therefore be driven by
significantly increased efficacy or additional functionality in specific applications.
The small-scale proof-of-principle studies undertaken to date do not sufficiently
demonstrate tissue specificity in order to justify the additional time and cost
involved in developing clinical grade products for therapeutic application. In all
cases, longer-term preclinical studies are required to fully inform this
decision-making process. It is important that researchers are able to determine the
biochemical and structural factors driving cell responses and tissue formation in
these novel ECM hydrogels. In particular, it is important to consider the effect of
the tissue source (species and anatomical location where appropriate) and the
macrophage polarization response to tissue-specific ECM hydrogels as part of the
global inflammatory and immune response. These factors have been studied for
ECM materials in powder and sheet form but not yet for hydrogels, which have
altered three-dimensional architecture and subtle differences in biochemical
composition.
The knowledge derived from these further characterization studies and pre-
clinical trials may allow the development of optimized homologous and
non-homologous ECM hydrogels for a wide range of applications. These novel
materials may then provide a new hope for the treatment of defects with complex
architecture.

References

1. Agrawal V, Johnson SA, Reing J, Zhang L, Tottey S, Wang G, Hirschi KK, Braunhut S,
Gudas LJ, Badylak SF. Epimorphic regeneration approach to tissue replacement in adult
mammals. Proceedings of the National Academy of Sciences of the USA. 2010;107(8):3351–
5. https://doi.org/10.1073/pnas.0905851106.
2. Agrawal V, Tottey S, Johnson SA, Freund JM, Siu BF, Badylak SF. Recruitment of
progenitor cells by an extracellular matrix cryptic peptide in a mouse model of digit
amputation. Tissue Eng Part A. 2011;17(19–20):2435–43. https://doi.org/10.1089/ten.TEA.
2011.0036.
3. Badylak SE. The extracellular matrix as a scaffold for tissue reconstruction. Semin Cell Dev
Biol. 2002;13(5):377–83. https://doi.org/10.1016/S1084-9521(02)00094-0.
4. Badylak SF, Coffey AC, Lantz GC, Tacker WA, Geddes LA. Comparison of the resistance to
infection of intestinal submucosa arterial autografts versus polytetrafluoroethylene arterial
prostheses in a dog model. J Vasc Surg. 1994;19(3):465–72.
5. Badylak SF, Freytes DO, Gilbert TW. Extracellular matrix as a biological scaffold material:
structure and function. Acta Biomater. 2009;5(1):1–13. https://doi.org/10.1016/j.actbio.2008.
09.013.
6. Badylak SF, Gilbert TW. Immune response to biologic scaffold materials. Sem Immunol.
2008;20(2):109–16. https://doi.org/10.1016/j.smim.2007.11.003.
2 ECM Hydrogels for Regenerative Medicine 53

7. Badylak SF, Park K, Peppas N, McCabe G, Yoder M. Marrow-derived cells populate

scaffolds composed of xenogeneic extracellular matrix. Exp Hematol. 2001;29(11):1310–8.
8. Badylak SF, Valentin JE, Ravindra AK, McCabe GP, Stewart-Akers AM. Macrophage
phenotype as a determinant of biologic scaffold remodeling. Tissue Eng Part A. 2008;14
(11):1835–42. https://doi.org/10.1089/ten.tea.2007.0264.
9. Badylak SF, Vorp DA, Spievack AR, Simmons-Byrd A, Hanke J, Freytes DO, Thapa A,
Gilbert TW, Nieponice A. Esophageal reconstruction with ECM and muscle tissue in a dog
model. J Surg Res. 2005;128(1):87–97.
10. Badylak SF, Wu CC, Bible M, McPherson E. Host protection against deliberate bacterial
contamination of an extracellular matrix bioscaffold versus Dacron mesh in a dog model of
orthopedic soft tissue repair. J Biomed Mater Res. 2003;67(1):648–54.
11. Benders KE, van Weeren PR, Badylak SF, Saris DB, Dhert WJ, Malda J. Extracellular matrix
scaffolds for cartilage and bone regeneration. Trends Biotechnol. 2013;31(3):169–76. https://
doi.org/10.1016/j.tibtech.2012.12.004.
12. Berkowitz BA, Bevins CL, Zasloff MA. Magainins: a new family of membrane-active host
defense peptides. Biochem Pharmacol. 1990;39(4):625–9.
13. Bissell MJ, Hall HG, Parry G. How does the extracellular matrix direct gene expression? J
Theor Biol. 1982;99(1):31–68.
14. Booth AJ, Hadley R, Cornett AM, Dreffs AA, Matthes SA, Tsui JL, Weiss K, Horowitz JC,
Fiore VF, Barker TH, Moore BB, Martinez FJ, Niklason LE, White ES. Acellular normal and
fibrotic human lung matrices as a culture system for in vitro investigation. Am J Respir Crit
Care Med. 2012;186(9):866–76. https://doi.org/10.1164/rccm.201204-0754OC.
15. Brennan BJ, Brown AB, Kolis SJ, Rutman O, Gooden C, Davies BE. Effect of R667, a novel
emphysema agent, on the pharmacokinetics of midazolam in healthy men. J Clin Pharmacol.
2006;46(2):222–8. https://doi.org/10.1177/0091270005283836.
16. Brennan EP, Reing J, Chew D, Myers-Irvin JM, Young EJ, Badylak SF. Antibacterial activity
within degradation products of biological scaffolds composed of extracellular matrix. Tissue
Eng. 2006;12(10):2949–55. https://doi.org/10.1089/ten.2006.12.2949.
17. Brightman AO, Rajwa BP, Sturgis JE, McCallister ME, Robinson JP, Voytik-Harbin SL.
Time-lapse confocal reflection microscopy of collagen fibrillogenesis and extracellular matrix
assembly in vitro. Biopolymers. 2000;54(3):222–34. https://doi.org/10.1002/1097-0282
(200009)54:3<222:aid-bip80>3.0.co;2-k.
18. Brown B, Lindberg K, Reing J, Stolz DB, Badylak SF. The basement membrane component
of biologic scaffolds derived from extracellular matrix. Tissue Eng. 2006;12(3):519–26.
19. Brown BN, Londono R, Tottey S, Zhang L, Kukla KA, Wolf MT, Daly KA, Reing JE,
Badylak SF. Macrophage phenotype as a predictor of constructive remodeling following the
implantation of biologically derived surgical mesh materials. Acta Biomater. 2012;8(3):978–87.
https://doi.org/10.1016/j.actbio.2011.11.031.
20. Brown BN, Valentin JE, Stewart-Akers AM, McCabe GP, Badylak SF. Macrophage
phenotype and remodeling outcomes in response to biologic scaffolds with and without a
cellular component. Biomaterials. 2009;30(8):1482–91. https://doi.org/10.1016/j.biomaterials.
2008.11.040.
21. Bulet P, Stocklin R, Menin L. Anti-microbial peptides: from invertebrates to vertebrates.
Immunol Rev. 2004;198:169–84.
22. Chen TM, Wang HJ. Cranioplasty using allogeneic perforated demineralized bone matrix
with autogenous bone paste. Ann Plast Surg. 2002;49(3):272–7. https://doi.org/10.1097/01.
SAP.0000015488.91165.B8.
23. Cheng M-H, Uriel S, Moya ML, Francis-Sedlak M, Wang R, Huang J-J, Chang S-Y,
Brey EM. Dermis-derived hydrogels support adipogenesis in vivo. J Biomed Mater Res.
2010;92A(3):852–8. https://doi.org/10.1002/jbm.a.32410.
24. Cheung HK, Han TTY, Marecak DM, Watkins JF, Amsden BG, Flynn LE. Composite
hydrogel scaffolds incorporating decellularized adipose tissue for soft tissue engineering with
adipose-derived stem cells. Biomaterials. 2014;35(6):1914–23.
54 M. J. Sawkins et al.

25. Choi JS, Kim BS, Kim JD, Choi YC, Lee HY, Cho YW. In vitro cartilage tissue engineering
using adipose-derived extracellular matrix scaffolds seeded with adipose-derived stem cells.
Tissue Eng Part A. 2012;18(1–2):80–92. https://doi.org/10.1089/ten.tea.2011.0103.
26. Choi JW, Park JK, Chang JW, Kim DY, Kim MS, Shin YS, Kim CH. Small intestine
submucosa and mesenchymal stem cells composite gel for scarless vocal fold regeneration.
Biomaterials. 2014;35(18):4911–8. https://doi.org/10.1016/j.biomaterials.2014.03.008.
27. Cortiella J, Niles J, Cantu A, Brettler A, Pham A, Vargas G, Winston S, Wang J, Walls S,
Nichols JE. Influence of acellular natural lung matrix on murine embryonic stem cell
differentiation and tissue formation. Tissue Eng. 2010;16(8):2565–80. https://doi.org/10.
1089/ten.tea.2009.0730.
28. Crapo PM, Gilbert TW, Badylak SF. An overview of tissue and whole organ decellularization
processes. Biomaterials. 2011;32(12):3233–43. https://doi.org/10.1016/j.biomaterials.2011.
01.057.
29. Crapo PM, Tottey S, Slivka PF, Badylak SF. Effects of biologic scaffolds on human stem cells
and implications for CNS tissue engineering. Tissue Eng. 2014;20(1–2):313–23. https://doi.
org/10.1089/ten.TEA.2013.0186.
30. Crapo PM, Wang Y. Small intestinal submucosa gel as a potential scaffolding material for
cardiac tissue engineering. Acta Biomater. 2010;6(6):2091–6. https://doi.org/10.1016/j.actbio.
2009.10.048.
31. D’Amore A, Stella JA, Wagner WR, Sacks MS. Characterization of the complete fiber
network topology of planar fibrous tissues and scaffolds. Biomaterials. 2010;31(20):5345–54.
https://doi.org/10.1016/j.biomaterials.2010.03.052.
32. DeQuach JA, Lin JE, Cam C, Hu D, Salvatore MA, Sheikh F, Christman KL. Injectable
skeletal muscle matrix hydrogel promotes neovascularization and muscle cell infiltration in a
hindlimb ischemia model. Eur Cells Mater. 2012;23:400–12.
33. Drake MP, Davison PF, Bump S, Schmitt FO. Action of proteolytic enzymes on
tropocollagen and insoluble collagen. Biochemistry. 1966;5(1):301–12.
34. Drury JL, Mooney DJ. Hydrogels for tissue engineering: scaffold design variables and
applications. Biomaterials. 2003;24(24):4337–51.
35. Farnebo SJ, Woon CY, Schmitt T, Joubert LM, Kim M, Pham H, Chang J. Design and
characterization of an injectable tendon hydrogel: a scaffold for guided tissue regeneration in
the musculoskeletal system. Tissue Eng. 2013. https://doi.org/10.1089/ten.TEA.2013.0207.
36. Fisher MB, Liang R, Jung HJ, Kim KE, Zamarra G, Almarza AJ, McMahon PJ, Woo SLY.
Potential of healing a transected anterior cruciate ligament with genetically modified
extracellular matrix bioscaffolds in a goat model. Knee Surg Sports Traumatol Arthrosc.
2012;20(7):1357–65. https://doi.org/10.1007/s00167-011-1800-x.
37. Francis D, Abberton K, Thompson E, Daniell M. Myogel supports the ex vivo amplification of
corneal epithelial cells. Exp Eye Res. 2009;88(3):339–46. https://doi.org/10.1016/j.exer.2008.
06.016.
38. Freytes DO, Martin J, Velankar SS, Lee AS, Badylak SF. Preparation and rheological
characterization of a gel form of the porcine urinary bladder matrix. Biomaterials. 2008;29
(11):1630–7. https://doi.org/10.1016/j.biomaterials.2007.12.014.
39. Ganz T. Defensins: antimicrobial peptides of innate immunity. Nat Rev Immunol. 2003;3
(9):710–20. https://doi.org/10.1038/nri1180.
40. Gilbert TW, Stolz DB, Biancaniello F, Simmons-Byrd A, Badylak SF. Production and
characterization of ECM powder: implications for tissue engineering applications.
Biomaterials. 2005;26(12):1431–5.
41. Grover GN, Rao N, Christman KL. Myocardial matrix-polyethylene glycol hybrid hydrogels
for tissue engineering. Nanotechnology. 2014;25(1). https://doi.org/10.1088/0957-4484/25/1/
014011.
42. Hulmes DJS. Collagen diversity, synthesis, and assembly. In: Fratzl P, editors. Collagen:
structure and mechanics. Berlin: Springer; 2008.
2 ECM Hydrogels for Regenerative Medicine 55

43. Jernigan TW, Croce MA, Cagiannos C, Shell DH, Handorf CR, Fabian TC. Small intestinal
submucosa for vascular reconstruction in the presence of gastrointestinal contamination. Ann
Surg. 2004;239(5):733–8.
44. Johnson TD, Dequach JA, Gaetani R, Ungerleider J, Elhag D, Nigam V, Behfar A,
Christman KL. Human versus porcine tissue sourcing for an injectable myocardial matrix
hydrogel. Biomater Sci. 2014;2(5):735–44. https://doi.org/10.1039/c3bm60283d.
45. Johnson TD, Lin SY, Christman KL. Tailoring material properties of a nanofibrous
extracellular matrix derived hydrogel. Nanotechnology. 2011;22(49):494015. https://doi.org/
10.1088/0957-4484/22/49/494015.
46. Kadler KE, Hill A, Canty-Laird EG. Collagen fibrillogenesis: fibronectin, integrins, and minor
collagens as organizers and nucleators. Curr Opin Cell Biol. 2008;20(5):495–501. https://doi.
org/10.1016/j.ceb.2008.06.008.
47. Kadler KE, Holmes DF, Trotter JA, Chapman JA. Collagen fibril formation. Biochem J.
1996;316(Pt 1):1–11.
48. Keane TJ, Londono R, Turner NJ, Badylak SF. Consequences of ineffective decellularization
of biologic scaffolds on the host response. Biomaterials. 2012;33(6):1771–81. https://doi.org/
10.1016/j.biomaterials.2011.10.054.
49. Kim MY, Farnebo S, Woon CYL, Schmitt T, Pham H, Chang J. Augmentation of tendon
healing with an injectable tendon hydrogel in a rat achilles tendon model. Plast Reconstr Surg.
2014;133(5):645e–53e. https://doi.org/10.1097/PRS.0000000000000106.
50. Kopecek J. Hydrogel biomaterials: a smart future? Biomaterials. 2007;28(34):5185–92.
https://doi.org/10.1016/j.biomaterials.2007.07.044.
51. Kwon JS, Yoon SM, Shim SW, Park JH, Min KJ, Oh HJ, Kim JH, Kim YJ, Yoon JJ,
Choi BH, Kim MS. Injectable extracellular matrix hydrogel developed using porcine articular
cartilage. Int J Pharm. 2013;454(1):183–91. https://doi.org/10.1016/j.ijpharm.2013.06.023.
52. Lee JS, Shin J, Park HM, Kim YG, Kim BG, Oh JW, Cho SW. Liver extracellular matrix
providing dual functions of two-dimensional substrate coating and three-dimensional
injectable hydrogel platform for liver tissue engineering. Biomacromol. 2013;15(1):206–18.
https://doi.org/10.1021/bm4015039.
53. Lee KY, Mooney DJ. Hydrogels for tissue engineering. Chem Rev. 2001;101(7):1869–79.
54. Medberry CJ, Crapo PM, Siu BF, Carruthers CA, Wolf MT, Nagarkar SP, Agrawal V,
Jones KE, Kelly J, Johnson SA, Velankar SS, Watkins SC, Modo M, Badylak SF. Hydrogels
derived from central nervous system extracellular matrix. Biomaterials. 2013;34(4):1033–40.
https://doi.org/10.1016/j.biomaterials.2012.10.062.
55. Mercuri JJ, Gill SS, Simionescu DT. Novel tissue-derived biomimetic scaffold for
regenerating the human nucleus pulposus. J Biomed Mater Res. 2011;96A(2):422–35.
https://doi.org/10.1002/jbm.a.33001.
56. Mercuri JJ, Patnaik S, Dion G, Gill SS, Liao J, Simionescu DT. Regenerative potential of
decellularized porcine nucleus pulposus hydrogel scaffolds: stem cell differentiation, matrix
remodeling, and biocompatibility studies. Tissue Eng. 2013;19(7–8):952–66. https://doi.org/
10.1089/ten.TEA.2012.0088.
57. Miller EJ. Structural studies on cartilage collagen employing limited cleavage and
solubilization with pepsin. Biochemistry. 1972;11(26):4903–9.
58. Moore AJ, Beazley WD, Bibby MC, Devine DA. Antimicrobial activity of cecropins.
J Antimicrob Chemother. 1996;37(6):1077–89.
59. Nelson CM, Bissell MJ. Of extracellular matrix, scaffolds, and signaling: tissue architecture
regulates development, homeostasis, and cancer. Annu Rev Cell Dev Biol. 2006;22:287–309.
https://doi.org/10.1146/annurev.cellbio.22.010305.104315.
60. Nerem R. The challenge of imitating nature. Principles of tissue engineering. 3rd ed.
Burlington: Elsevier; 2007.
61. Ni P, Ding Q, Fan M, Liao J, Qian Z, Luo J, Li X, Luo F, Yang Z, Wei Y. Injectable
thermosensitive PEG-PCL-PEG hydrogel/acellular bone matrix composite for bone regen-
eration in cranial defects. Biomaterials. 2014;35(1):236–48. https://doi.org/10.1016/j.
biomaterials.2013.10.016.
56 M. J. Sawkins et al.

62. Parkinson J, Kadler KE, Brass A. Simple physical model of collagen fibrillogenesis based on
diffusion limited aggregation. J Mol Biol. 1995;247(4):823–31. https://doi.org/10.1006/jmbi.
1994.0182.
63. Petersen TH, Calle EA, Zhao LP, Lee EJ, Gui LQ, Raredon MB, Gavrilov K, Yi T,
Zhuang ZW, Breuer C, Herzog E, Niklason LE. Tissue-engineered lungs for in vivo
implantation. Science. 2010;329(5991):538–41. https://doi.org/10.1126/science.1189345.
64. Pilipchuk SP, Vaicik MK, Larson JC, Gazyakan E, Cheng M-H, Brey EM. Influence of
crosslinking on the stiffness and degradation of dermis-derived hydrogels. J Biomed Mater
Res. 2013;101(10):2883–95. https://doi.org/10.1002/jbm.a.34602.
65. Poon CJ, Pereira E. Cotta MV, Sinha S, Palmer JA, Woods AA, Morrison WA,
Abberton KM. Preparation of an adipogenic hydrogel from subcutaneous adipose tissue.
Acta Biomaterialia. 2013;9(3):5609–20. https://doi.org/10.1016/j.actbio.2012.11.003.
66. Ravi S, Caves JM, Martinez AW, Xiao J, Wen J, Haller CA, Davis ME, Chaikof EL. Effect of
bone marrow-derived extracellular matrix on cardiac function after ischemic injury.
Biomaterials. 2012;33(31):7736–45. https://doi.org/10.1016/j.biomaterials.2012.07.010.
67. Reing JE, Brown BN, Daly KA, Freund JM, Gilbert TW, Hsiong SX, Huber A, Kullas KE,
Tottey S, Wolf MT, Badylak SF. The effects of processing methods upon mechanical and
biologic properties of porcine dermal extracellular matrix scaffolds. Biomaterials. 2010;31
(33):8626–33. https://doi.org/10.1016/j.biomaterials.2010.07.083.
68. Reing JE, Zhang L, Myers-Irvin J, Cordero KE, Freytes DO, Heber-Katz E, Bedelbaeva K,
McIntosh D, Dewilde A, Braunhut SJ, Badylak SF. Degradation products of extracellular
matrix affect cell migration and proliferation. Tissue Eng. 2009;15(3):605–14. https://doi.org/
10.1089/ten.tea.2007.0425.
69. Rosamond W, Flegal K, Friday G, Furie K, Go A, Greenlund K, Haase N, Ho M, Howard V,
Kissela B, Kittner S, Lloyd-Jones D, McDermott M, Meigs J, Moy C, Nichol G, O’Donnell
CJ, Roger V, Rumsfeld J, Sorlie P, Steinberger J, Thom T, Wasserthiel-Smoller S, Hong Y,
American Heart Association Statistics C, Stroke Statistics S. Heart disease and stroke statistics
—2007 update: a report from the American Heart Association Statistics Committee and
Stroke Statistics Subcommittee. Circulation. 2007;115(5):e69–e171. https://doi.org/10.1161/
circulationaha.106.179918.
70. Rubin AL, Drake MP, Davison PF, Pfahl D, Speakman PT, Schmitt FO. Effects of pepsin
treatment on the interaction properties of tropocollagen macromolecules. Biochemistry.
1965;4(2):181–90. https://doi.org/10.1021/bi00878a001.
71. Russell A, Bertram T. Moving into the clinic. Principles of tissue engineering. 3rd ed.
Burlington: Elsevier; 2007.
72. Sampath TK, Reddi AH. Importance of geometry of the extracellular matrix in endochondral
bone differentiation. J Cell Biol. 1984;98(6):2192–7.
73. Sarikaya A, Record R, Wu CC, Tullius B, Badylak S, Ladisch M. Antimicrobial activity
associated with extracellular matrices. Tissue Eng. 2002;8(1):63–71. https://doi.org/10.1089/
107632702753503063.
74. Sawkins MJ, Bowen W, Dhadda P, Markides H, Sidney LE, Taylor AJ, Rose FRAJ,
Badylak SF, Shakesheff KM, White LJ. Hydrogels derived from demineralized and
decellularized bone extracellular matrix. Acta Biomater. 2013;9(8):7865–73. https://doi.org/
10.1016/j.actbio.2013.04.029.
75. Seif-Naraghi SB, Horn D, Schup-Magoffin PJ, Christman KL. Injectable extracellular matrix
derived hydrogel provides a platform for enhanced retention and delivery of a heparin-binding
growth factor. Acta Biomater. 2012;8(10):3695–703. https://doi.org/10.1016/j.actbio.2012.
06.030.
76. Seif-Naraghi SB, Salvatore MA, Schup-Magoffin PJ, Hu DP, Christman KL. Design and
characterization of an injectable pericardial matrix gel: a potentially autologous scaffold for
cardiac tissue engineering. Tissue Eng. 2010;16(6):2017–27. https://doi.org/10.1089/ten.
TEA.2009.0768.
2 ECM Hydrogels for Regenerative Medicine 57

77. Seif-Naraghi SB, Singelyn JM, Salvatore MA, Osborn KG, Wang JJ, Sampat U, Kwan OL,
Strachan GM, Wong J, Schup-Magoffin PJ, Braden RL, Bartels K, DeQuach JA, Preul M,
Kinsey AM, DeMaria AN, Dib N, Christman KL. Safety and efficacy of an injectable
extracellular matrix hydrogel for treating myocardial infarction. Sci Transl Med. 2013;5(173).
78. Sellaro TL, Ranade A, Faulk DM, McCabe GP, Dorko K, Badylak SF, Strom SC.
Maintenance of human hepatocyte function in vitro by liver-derived extracellular matrix gels.
Tissue Eng. 2010;16:1075–82. https://doi.org/10.1089/ten.TEA.2008.0587.
79. Shell DH, Croce MA, Cagiannos C, Jernigan TW, Edwards N, Fabian TC. Comparison of
small-intestinal submucosa and expanded polytetrafluoroethylene as a vascular conduit in the
presence of gram-positive contamination. Ann Surg. 2005;241(6):995–1001.
80. Singelyn JM, DeQuach JA, Seif-Naraghi SB, Littlefield RB, Schup-Magoffin PJ,
Christman KL. Naturally derived myocardial matrix as an injectable scaffold for cardiac tissue
engineering. Biomaterials. 2009;30(29):5409–16. https://doi.org/10.1016/j.biomaterials.2009.
06.045.
81. Singelyn JM, Sundaramurthy P, Johnson TD, Schup-Magoffin PJ, Hu DP, Faulk DM,
Wang J, Mayle KM, Bartels K, Salvatore M, Kinsey AM, DeMaria AN, Dib N,
Christman KL. Catheter-deliverable hydrogel derived from decellularized ventricular
extracellular matrix increases endogenous cardiomyocytes and preserves cardiac function
post-myocardial infarction. J Am Coll Cardiol. 2012;59(8):751–63. https://doi.org/10.1016/j.
jacc.2011.10.888.
82. Skardal A, Smith L, Bharadwaj S, Atala A, Soker S, Zhang Y. Tissue specific synthetic ECM
hydrogels for 3-D in vitro maintenance of hepatocyte function. Biomaterials. 2012;33(18):
4565–75. https://doi.org/10.1016/j.biomaterials.2012.03.034.
83. Streuli C. Extracellular matrix remodelling and cellular differentiation. Curr Opin Cell Biol.
1999;11(5):634–40.
84. Uriel S, Huang J-J, Moya ML, Francis ME, Wang R, S-y Chang, Cheng M-H, Brey EM. The
role of adipose protein derived hydrogels in adipogenesis. Biomaterials. 2008;29(27):3712–9.
https://doi.org/10.1016/j.biomaterials.2008.05.028.
85. Uriel S, Labay E, Francis-Sedlak M, Moya ML, Weichselbaum RR, Ervin N, Cankova Z,
Brey EM. Extraction and assembly of tissue-derived gels for cell culture and tissue
engineering. Tissue Eng. 2009;15(3):309–21. https://doi.org/10.1089/ten.tec.2008.0309.
86. Uygun BE, Soto-Gutierrez A, Yagi H, Izamis M-L, Guzzardi MA, Shulman C, Milwid J,
Kobayashi N, Tilles A, Berthiaume F, Hertl M, Nahmias Y, Yarmush ML, Uygun K. Organ
reengineering through development of a transplantable recellularized liver graft using
decellularized liver matrix. Nat Med. 2010;16(7):814–20. https://doi.org/10.1038/nm.2170.
87. Voytik-Harbin SL, Brightman AO. Small intestinal submucosa: a tissue derived extracellular
matrix that promotes tissue-specific growth and differentiation of cells in vitro. Tissue Eng.
1998;4:157–74.
88. Voytik-Harbin SL, Brightman AO, Kraine MR, Waisner B, Badylak SF. Identification of
extractable growth factors from small intestinal submucosa. J Cell Biochem. 1997;67(4):
478–91.
89. Wolf MT, Carruthers CA, Dearth CL, Crapo PM, Huber A, Burnsed OA, Londono R,
Johnson SA, Daly KA, Stahl EC, Freund JM, Medberry CJ, Carey LE, Nieponice A,
Amoroso NJ, Badylak SF. Polypropylene surgical mesh coated with extracellular matrix
mitigates the host foreign body response. J Biomed Mater Res. 2014;102(1):234–46. https://
doi.org/10.1002/jbm.a.34671.
90. Wolf MT, Daly KA, Brennan-Pierce EP, Johnson SA, Carruthers CA, D’Amore A,
Nagarkar SP, Velankar SS, Badylak SF. A hydrogel derived from decellularized dermal
extracellular matrix. Biomaterials. 2012;33(29):7028–38. https://doi.org/10.1016/j.biomaterials.
2012.06.051.
58 M. J. Sawkins et al.

91. Wolf MT, Daly KA, Reing JE, Badylak SF. Biologic scaffold composed of skeletal muscle
extracellular matrix. Biomaterials. 2012;33(10):2916–25. https://doi.org/10.1016/j.biomaterials.
2011.12.055.
92. Young DA, Ibrahim DO, Hu D, Christman KL. Injectable hydrogel scaffold from
decellularized human lipoaspirate. Acta Biomater. 2011;7(3):1040–9. https://doi.org/10.
1016/j.actbio.2010.09.035.
93. Zhang L, Zhang F, Weng Z, Brown BN, Yan H, Ma XM, Vosler PS, Badylak SF, Dixon CE,
Cui XT, Chen J. Effect of an inductive hydrogel composed of urinary bladder matrix upon
functional recovery following traumatic brain injury. Tissue Eng. 2013;19(17–18):1909–18.
https://doi.org/10.1089/ten.tea.2012.0622.
Chapter 3
Biologically Relevant Laminins
in Regenerative Medicine

Anna Domogatskaya and Sergey Rodin

Abstract Need for biologically relevant coatings in regenerative medicine.

Recent advances in stem cell-based regenerative medicine allow to expand human
cells in almost unlimited amounts. However, expanded cells become devoid of
natural niche cues and, therefore, may become prone to risks of function loss and
malignant transformation. In order to maintain the expanded cells in a safe and
functional way, one has to imitate in vitro the natural niche, comprising biologically
relevant extracellular matrix molecules. For majority of cell types, including neu-
rons, insulin-producing b-cells and vascular endothelial cells, biologically relevant
laminins are essential part of natural niche. Laminins: 16 tissue-specific isoforms
that mediate cell maintenance and behavior. Laminins (LM) are large,
cross-shaped molecules that convey extracellular matrix cues via cell receptors to
cell signaling systems and thus affect cell maintenance and behavior. Laminins can
mediate behavior of associated cells, such as survival, adhesion, migration, pro-
liferation, phenotype maintenance or differentiation. Each of known 16 laminin
isoforms has unique biological function; mutations in laminin-encoding genes often
result in severe pathologies or lethality. Despite molecular structure similarity and
evolutionary homology, certain laminin isoforms may exert antagonistic effects on
cell behavioral patterns. Importantly, biologically relevant laminins act in synergy
with specific growth factors and cell–cell contact molecules, such as E-cadherin.
Lack of either may result in malfunctioning cell culture systems. Lack of biolog-
ically relevant laminins may result in cell apoptosis, phenotype loss, or malignant
transformation. Survival pathways for majority of mammalian cells depend on
niche-specific extracellular matrix anchorage. Lack of such anchorage or irrelevant
anchorage triggers apoptotic pathways and results in anoikis (apoptosis, caused by

A. Domogatskaya (&)
Division of Transplantation Surgery, Department of Clinical Science,
Intervention and Technology, Karolinska Institute, 14152 Huddinge, Sweden
e-mail: [email protected]
S. Rodin
Department of Medical Biochemistry and Biophysics, Karolinska Institute,
Scheeles vagen 2, B1, Division of Matrix Biology, 17177 Stockholm, Sweden
e-mail: [email protected]

© Springer International Publishing AG, part of Springer Nature 2018 59

A. C. Berardi (ed.), Extracellular Matrix for Tissue Engineering
and Biomaterials, Stem Cell Biology and Regenerative Medicine,
https://doi.org/10.1007/978-3-319-77023-9_3
60 A. Domogatskaya and S. Rodin

lack of relevant anchorage). In the absence of biologically relevant matrix cues,

cells may undergo apoptosis or activate malignant pathways of
anchorage-independent anti-apoptotic signaling. Majority of mammalian cell types
depend on interaction with biologically relevant laminins. Biologically relevant
laminins benefit quality cell culture in vitro. In vitro, cell culture systems based on
use of niche-specific laminins are described for human and mouse embryonic stem
cells, hematopoietic stem cells, neurons and Schwann cells, insulin-producing b-
cells, and other cell types. We shall discuss success criteria and possible pitfalls for
generating laminin-based safe, robust, and efficient technologies for culturing other
cell types needed to treat various diseases.

Keywords Laminin Embryonic stem cell Stem cell Extracellular matrix




Regenerative medicine Neuron Pancreatic islets Insulin-producing beta cell





Stem cell technologies Proliferation Survival Apoptosis Differentiation




Adhesion Safety Cell culture Signaling Integrin

Introduction

Since traditional approach of organ transplantation in medicine is limited due to

shortage of donor material, cell therapies technologies based on possibility to
expand donor or autologous cells in vitro are rapidly developing. Recent advances
in stem cell technologies allow to expand the pool of human stem cells to almost
unlimited amounts, which would ease the need for donor material. Ethical concerns
regarding human embryonic stem cell derivation are finally resolved by technolo-
gies that allow to isolate a single blastomere without harming the embryo and
expand it in vitro into a human embryonic stem cell line [67]. Cell banks can be
generated to serve a source of immunohistocompatible stem cell lines. Use of
induced pluripotent stem cells (iPS) that imitate properties of embryonic stem cells
is another attractive alternative, since it allows autologous cells use [67, 68]. Third
alternative is to expand the tissue-specific adult cell lines, stem cells or differenti-
ated, and it is successfully implemented in medicine. In future perspective, cell
therapies approach seems to be more practical alternative compared to traditional
organ transplantation; however, besides obvious benefits, it also bears certain risks.
Mammalian cells, when isolated from tissues, become devoid of their natural niche
cues, become prone to loss of phenotype and function, and, worst of all, may
undergo malignant transformation [7]. Many human embryonic stem (ES) cell lines
develop chromosomal abnormalities after long-term culturing in vitro, some of
which are cancer-related [3, 52].
In order to maintain long-term healthy functionality of the expanded cells, it is
essential to imitate the natural environment, comprising (1) growth factors,
(2) cell–cell contacts, and (3) extracellular matrix contacts, similar to the ones
present in vivo. Laminins are the essential components of basement membranes,
providing niche-specific cues to majority of mammalian cell types, such as
3 Biologically Relevant Laminins in Regenerative Medicine 61

embryonic stem cells, hematopoietic stem cells, peripheral neurons, Schwann cells,
insulin-producing pancreatic b-cells, vascular endothelial cells, keratinocytes,
muscle cells [15]. Laminins can mediate behavior of associated cells in many
different ways, such as: survival, adhesion, migration or stable anchorage, prolif-
eration or quiescence, phenotype stability or dedifferentiation, undifferentiated
status in stem cells, or differentiation. Moreover, laminins are essential for mor-
phogenesis during embryonic/prenatal development and may enable selective
restriction for cell permeability between compartments and cell-guiding function in
regeneration and development. There is evidence that niche-specific laminins act in
concert with specific growth factors and cell–cell contacts to affect cell behavior.
In mammals, at least 16 laminin isoforms are known up to date, and every
isoform has a specific tissue and developmental stage-specific function [15, 16].
Laminins are large cross-shaped heterotrimeric proteins, comprising one a, one b,
and one c chain. Molecular weight of a laminin molecule varies from 400 to
1000 kDa. Laminins convey signals from surrounding extracellular matrix to the
adherent cells via binding to laminin-specific cell receptors, e.g., integrins, and are
essential part of natural mechanotransduction system [25].
Laminins first appear early in evolution, already in hydra, and are essential for
ability of hydra to regenerate [70, 72, 97]. In primitive species, such as
Caenorhabditis elegans and Drosophila melanogaster, every laminin isoform is
essential; lack of any laminin chain results in early lethality [28, 32, 46, 85, 92]. In
zebrafish, mouse and human laminin chains mutations often result in either
embryonic/postnatal lethality or diseases. Notably, knockout phenotypes are totally
different for any of a chains, or b chains, or c chains [15, 16, 51]. This indicates that
biological role of every laminin isoforms is unique. It is shown that certain laminins
isoforms, despite visible similarity in structure and amino acid sequence, exert
distinct, and sometimes even opposite influences on certain cell types.
In early mammalian embryos, laminins are the first extracellular matrix molecules
to appear: already at 2–4-cell stage, b- and c-laminin chains are detected, and at 8-cell
stage the whole laminin trimer appears in extracellular space [10, 17, 38]. At the stage
of blastula, at least two laminin isoforms appear: a5 laminins are expressed between
pluripotent cells of inner cell mass and, probably, supporting their pluripotency and
proliferation, while a1 laminins, provided by trophectoderm, cause polarization of
pluripotent cells and subsequent differentiation into ectoderm, endoderm, and
mesoderm [34, 39–41]. At the later stages of embryonic development, tissue-specific
and developmental stage-specific laminins drive morphogenesis; lack of specific
laminins often cannot be compensated by similar laminin isoforms and results in
diseases or sometimes lethality (reviewed in [15, 51]). For example, in kidney
development all the laminin a chains are expressed in a niche-specific manner; in
development of glomerular basement membrane (GBM) early LM-111 is replaced by
intermediate LM-511 and finally by LM-521. Inability to transit from LM-511 to
LM-521 results in renal failures (Pierson syndrome) [50, 76].
Use of niche-specific laminins for expanding functional cells in vitro is an
emerging trend in regenerative medicine-related research. Long-term (up to 150
doublings) and comprehensive (including in-depth analysis of random mutations
62 A. Domogatskaya and S. Rodin

and chromosomal abnormalities) studies were performed for mouse and human
embryonic stem cells [14, 67, 68]; short-term studies demonstrated obvious benefits
of biologically relevant laminins for in vitro cultures of adult stem cells and dif-
ferentiated cells, such as neurons, insulin-producing b-cells, and even whole pan-
creatic insulin-producing islets [53, 64, 82, 86]. However, for many mammalian cell
types that could be used in regenerative medicine, knowledge of biologically rel-
evant laminins in vivo and in vitro is scarce. Therefore, we shall discuss not only
existing laminin-based technologies, but rather the approaches, success criteria, and
possible pitfalls in implementing concept of biologically relevant laminins use into
generation of new cell culture systems. In conclusion, we shall discuss the existing
challenges in laminin research, regarding regenerative medicine needs, and
potential possibilities to exploit more sophisticated functions of laminins in tech-
nologies that are yet to be invented.

Cell Niche and Extracellular Matrix

Expanding Cells in Vitro: Future of Regenerative Medicine

Whole organ transplantation is a traditional approach in medicine. Techniques of

organ transplantations are well-established and enable high survival rates. For
example, one-year survival rate in liver transplantations is 85% and five-year sur-
vival rate is 70% [12]. However, there is a drastic shortage in donor material
appropriate for use in medicine [12]. Abilities of whole human organs or tissues to
regenerate are limited; therefore, the very approach of organ transplantation does
not allow to resolve the needs of all patients.
Regenerative medicine is a rapidly developing field, aiming to be able to expand
cells and/or tissues in sufficient amounts to serve every patient in need [58].
Technologies based on use of human embryonic stem cells (hESC) or induced
pluripotent cells (iPS cells) give promise to provide unlimited amounts of human
cells for medical use. Major ethical problems, concerning human embryonic cell
derivation from human embryos, have been recently resolved. Now, human em-
bryonic stem cell lines can be established from single-cell biopsies that do not
jeopardize the human embryo survival [35, 67]. It also becomes possible to create
GMP-compliant HLA-matched human ES cell banks to provide patients with
immunocompatible cells. Possibility to expand cells and assemble them into
functional organs in vitro seems a far more promising approach in a long run,
compared to traditional organ transplantation. Technologies that allow expansion of
mature cell cultures for transplantation are also used in medicine.
However, mammalian cells, both stem cells and mature differentiated cells, when
deprived of the natural niche cues, have high risk to undergo transformation, such
as loss of function, loss of phenotype, and even malignant transformations [7]. The
risk increases with number of cell culture passages. Human vascular endothelial
cells, when cultured in vitro, lose phenotype within less than 15 doublings [6].
3 Biologically Relevant Laminins in Regenerative Medicine 63

Human embryonic stem cells, when cultured in vitro for long passages, may
undergo not only random mutagenesis, but also non-random gains of chromosomes
or large chromosome fragments, similar to what happens during malignant trans-
formation [3, 52]. Though such cells may have typical phenotypic features and high
proliferation rate, they can be dangerous in transplantation.
Prolonged cell culture in vitro, unlike organ transplantation, requires use of
culture media, specific supplements, growth factors, and other molecular sub-
stances, often undefined chemical compositions. One should consider risks of
introducing pathogens to the cultured cells. For example, bacterial collagenase
preparations used for clinical derivation of insulin-producing pancreatic islets aimed
for transplantation were subject to bovine spongiform encephalopathy contamina-
tion risk; therefore, measures were taken to exclude it [80]. Human embryonic stem
cells, when cultured in the presence of animal-derived substances, have been shown
to acquire immunogenicity [47]. Therefore, FDA and EU regulatory agencies
suggest that all the compounds in contact with the cells aimed for medical use
would better be chemically defined and xeno-free [26, 27, 84].
Human laminins are chemically defined, xeno-free, non-immunogenic, and
biologically relevant molecules [67, 68]. Specific laminins, as essential part of
natural niches for majority of mammalian cell types, such as insulin-producing b-
cells, vascular endothelial cells, or embryonic stem cells, can contribute to gener-
ation of safe and robust in vitro culture systems that would imitate the natural cell
niches and maintain healthy phenotypes.

Three Pillars of Cell Niche: Growth Factors, Cell–Cell

Contacts, and Niche-Specific Extracellular Matrix

Cell behavior is guided by signals that cell receives from its environment.
Influenced by environmental cues, a cell may survive or undergo apoptosis, migrate
in scattered or directed fashion, form a stable anchorage, proliferate or remain
quiescent, maintain its phenotype, differentiate, dedifferentiate, or lose phenotype.
Understanding the nature of cues received by cell from its microenvironment is
essential to design a solution to safe and robust regenerative medicine protocols.
Mimicking natural niches by providing a set of appropriate molecular cues to
mammalian cells in vitro could allow to expand them in healthy and functional way
[67]. On the contrary, unnatural molecular cues may provoke loss of function and
phenotype and even genetic mutations [7].
Usually, a cell receives three types of cues from its microenvironment:
(1) growth factors, hormones, and other soluble molecules that activate cell
receptors by binding them, (2) cell–cell contacts, being essential for cells like
embryonic stem cells or insulin-producing pancreatic b-cells, and (3) niche-specific
extracellular matrix. The latter modulates cell signaling not merely by binding to
the cell receptors, but also by mechanotransduction [25].
64 A. Domogatskaya and S. Rodin

Effect of extracellular matrix on cell behavior is often underestimated.

Extracellular matrix is usually considered a neutral, passive substrate with mere
function of keeping cultured cells well adherent. However, multiple studies
demonstrated that different extracellular matrix coatings can exhibit strikingly
distinct effects on cultured cells. We have demonstrated that use of different laminin
isoforms on same culture of embryonic stem cells with the same medium, pas-
saging, and other culture procedures can facilitate such different events as: (1) rapid
detachment and following apoptosis, (2) rapid differentiation into quiescent cells,
(3) continuous proliferation in undifferentiated state for over 150 doublings [14].
Not only the binding and dissociating constants of laminin/receptor interactions, but
also mechanical properties of extracellular milieu incorporating the laminins such as
the matrix stiffness [2, 13, 18] can affect cell behavior.

Laminins: Sixteen Niche-Specific Extracellular Matrix

Molecules with Unique Biological Function

Idea of extracellular matrix being a neutral, passive substance—sort of biological

adhesive for 2D and gel for 3D cell cultures—arises from early studies when
niche-specific extracellular matrix molecules were not available for wide scientific
community. Laminin-111 (i.e., often referred to as just “laminin”) and Matrigel (a
tumor-derived gel containing mostly laminin-111 and collagen IV) were widely
available, but since they are not natural substrata for majority of mammalian cell
types, they could not exert strong positive influence on majority of niche-sensitive
mammalian cells. However, in early 90s it became clear that several laminin iso-
forms exist and, as further experiments showed, tissue-specific laminins facilitated
cell effects such as proliferation, adhesion, migration, differentiation.
Evidently, laminins have tissue-specific and even niche-specific expression
patterns and unique biological functions. Laminins, serving as a natural bridge
between extracellular matrix and cell surface receptors, are the very molecules that
provide the niche-specific cues to majority of mammalian cell types, including
epithelial and endothelial cells. In combination with niche-specific growth factors
and cell–cell adhesion molecules, niche-specific laminins can provide a close
imitation of natural environment for many specific mammalian cell types in vitro.

Laminins: Molecular Aspects and Cell Signaling

In this chapter, we shall briefly review common knowledge about the laminin
family: (1) molecular structure of laminins, (2) functional interactions with other
molecules, such as cell receptors and other extracellular matrix molecules, (3) effect
3 Biologically Relevant Laminins in Regenerative Medicine 65

of proteolytical processing on laminin function, (4) in vivo biological functions of

laminins, and knockout phenotypes/mutations.
Then, we shall discuss influence of laminin–cell receptors interaction on cell
signaling pathways and, consequently, cell behavior patterns: (1) principles of cell
signaling, mediated by laminin interaction with cell receptors, (2) co-signaling,
which is synergetic effect of biologically relevant laminin and growth factor
interaction with appropriate receptors, (3) cell behavior that requires biologically
relevant laminin signaling.

Laminins: Chains and Trimers

Laminins are large, heterotrimeric molecules that comprise one a, one b, and one c
chain. Size of laminin trimer varies from 400 to 1000 kDa. Laminin trimers have
either cross-like, or Y-like, or rodlike shape (see Fig. 3.1). Short arms of laminins
(N-terminal parts of a, b, and c chains) can bind other laminins short arms and other
extracellular matrix proteins. Long arm consists of (1) a, b, and c chains inter-
twined together into a trimeric spiral via coil-coiled domains and (2) C-terminal
fragment of a chain, comprising of five globular LG domains (LG1-LG5) that
interact with cell receptors.
Five a (a1-a5), four b (b1-b4), and three c (c1-c3) chains are known in
mammals; a5, b1, b2, and c1 are expressed ubiquitously, and other chains are more
tissue-restricted [4]. Up to date, 16 trimeric laminin isoforms present in vivo have
been characterized in mammals; however, theoretically 60 combinations are pos-
sible. Modern nomenclature describes laminin isoforms according to their chain
composition [4]. For example, ubiquitous laminin-511 (LM-511) consists of a5, b1,
and c1 chains.
It is shown by Macdonald et al. [43] that not all the combinations of a, b, and c
chains can assemble into trimeric forms. Sixteen laminin trimers have been iden-
tified in vivo and two more have been predicted by Macdonald et al. [43].

Fig. 3.1 Schematic representation of typical laminin trimer forms. a Cross-shaped. b Y-shaped.
c Rod-shaped
66 A. Domogatskaya and S. Rodin

Associations of c1 with b1 or b2 form trimers with all the five a chains. The a1, a2,
a3B, a5 trimers are cross-shaped, while the a3A, a4 trimers are Y-shaped or
rod-shaped.
Extensive body of data has been generated on single laminin chain expression
and distribution in various mammalian tissues, and yet very little is known about
specific trimers localizations in vivo. However, information on single laminin
chains expression patterns and knockout animal models can give us some insights
into in vivo function of certain laminin trimers.
a1 laminins. Vast majority of laminin studies in twentieth century involved
either LM-111 (called “laminin”) or Matrigel, a tumor-derived gel comprising
LM-111. Lack of a1 results in early embryonic lethality, presumably, due to inner
cell mass polarization failure [49, 71]. However, expression pattern of LM-111 in
mature mammalian organisms is restricted (compared to ubiquitous LM-511/521)
and biological function clearly known for embryonic events, such as blastocyst
inner cell mass polarization [39, 41, 49] and early kidney glomerular basement
membrane development [50, 76].
a2 laminins play important roles in differentiation and maintenance of muscular
and neural cells; mutations in LAMA2 (gene encoding a2 laminin) result in
muscular dystrophy and neural system disorders [8, 31, 78, 90]. a2 laminins are the
major laminins in muscle [61, 69] and play an important role in smooth muscle
myogenesis [66]. a2 laminins are an important part of neural system and are
essential for thymocytes maturation [29, 44]. a2 laminins are expressed in testis
[37] and deficiency of those results in infertility [23].
a3 laminins are specific for epithelial basement membranes, for example
underlying skin epidermis and intestinal epithelia. Two splice variants of LAMA3
gene exist: a3A, the short chain, and a3B, the long chain.
a4 laminins, Y-shaped, are supposed to be “not very important” since no lethal
or early severe phenotype is observed. However, mutations in LAMA4 cause
microcirculation development disorders [81], cardiomyopathy [36, 87], chronic
kidney disorders [1], neurodisorders [62, 86], and impairment of leukocyte
recruitment [33]. It appears that a4 laminins attenuate fine guidance of cells, such as
tip cells Dll4/Notch signaling in angiogenesis [77], or high-precision spatial
matching of active zones of motor axon terminus with junctional folds on muscle
end plate within developing neuromuscular junctions [62].
a5 laminins, cross-shaped molecules capable of self-polymerization, are ubiq-
uitously expressed. They first appear in earliest embryonic development stages; for
example, they are expressed by blastocyst inner cell mass to provide themselves
vital cues; a5 laminins are essential part of vascular basement membranes, skin,
hair follicle niches, and many others. Detailed review of knockout phenotypes and
list of references are available in [15, 16]. Lack of a5 laminins causes late-stage
embryonic lethality in mice [48] and cannot be compensated by other isoforms.
b1 laminins are ubiquitously expressed. They are essential for organism
maintenance from the earliest stages of embryonic development to complete mat-
uration of mammalian organism. b1 laminins are present in almost every organ
3 Biologically Relevant Laminins in Regenerative Medicine 67

comprising basement membranes. Lack of b1 results in early embryonic lethality,

already on the stage of blastocyst [49].
b2 laminins are also expressed in many organs and yet are more restricted in
expression patterns. Though b2 laminin chain is closely evolutionary related to b1,
some biological roles of b2-laminins are quite opposite of analogous b1 isoforms.
b1 and b2 isoforms may coexist within same tissue, but share different functional
niches. For example, surface of motor axon and of muscle fiber is coated with
b1-laminins except for the neuromuscular junctions, which specifically contains b2,
but not b1-laminins. As b1-laminins support Schwann cell spreading upon axon
surface, b2-laminins restrict Schwann cell migration into the neuromuscular junc-
tion so that neurotransmitter, released by motor axon end plate, could reach
receptors on the muscle end plate [59, 60]. Mature b2 isoform LM-521 replaces
embryonic b1 isoform LM-511 within kidney glomerular basement in order to
provide durable, stress-resistant GBM. If it fails, LM-511 is not able to resist stress
and renal failure occurs [56]. b2-laminins mediate Ca ++ signaling within neuro-
muscular junction by binding Ca++ channels, while b1-laminins do not [54].
b3 laminins are mostly epithelial-specific. Laminin b3 chain can only associate
with c2 [43] and both of them can only associate with a3 to form LM-332. This is
the only known up-to-date rod-shaped laminin isoform. LM-332 is specific for
epithelial basement membranes and driving epithelial cells stable anchoring or
migration. LM-332 plays an important role in wound healing. Mutations in any of
LM-332 chains (LAMA3, LAMB3, or LAMC3) result in severe, often lethal
phenotype of junctional epidermolysis bullosa, associated with skin blistering.
b4 laminin chain, encoded by LAMB4 gene, exists in human, but not in mouse.
Number of laminin b chains differs in vertebrates. For instance, zebrafish has six b
chains. Function in b4 in human is unknown, and mouse knockout model cannot be
established, since no LAMB4 exists in mouse [15]. It has not described any trimers
comprising b4 chain yet.
c1 laminins are ubiquitously expressed. They are essential for organism
maintenance from the earliest stages of embryonic development to complete mat-
uration of mammalian organism and are major laminins in almost every basement
membrane, except for c2-rich epithelial basement membranes. Lack of c1 results in
early embryonic lethality, already at the stage of blastocyst [75]. Laminin c1 in
cooperation with either b1 or b2 can form trimers with any of a chains, thus giving
rise to LM-111, 121, 211, 221, 311, 321, 411, 421, 511, and 521.
c2 laminin chain associates with a3 and b3 to form epithelial-specific LM-332.
c3 laminins have restricted expression pattern in tissues. LM-213 is present in
testis basement membrane [37]; LM-423 and LM-523 are present in central nervous
system and eye retinal structures [42]. c3 laminins may exist in mammalian tissues
in non-basement membrane forms. LAMC3 (gene encoding c3) was first not
considered to have important biological role, since knockout model in mouse
lacked obvious defects in phenotype [11] except for retinal defects [63]. However,
it has been recently shown that mutations in human LAMC3 affect brain structure
and function [5].
68 A. Domogatskaya and S. Rodin

Macdonald et al. predicted that two additional isoforms LM-312 and LM-422
may exist since coil-coiled domains of the named chains can associate as trimers
[43].

Molecular Interactions of Laminins

Unique function of laminins is ability to bind both extracellular matrix molecules

and cell receptors and thus transmit signals from extracellular matrix to the cells.
The knowledge concerning these molecular interactions is vast, and we address our
readers to specific articles devoted to molecular interactions of laminins for more
details, like [83], [57, 79, 93]. Hereby, we give very short and generalized overview
of laminins interactions.

Interactions with Cell Receptors and Co-Signaling

Laminins interact with cell receptors mostly via C-terminal LG domains of a

chains. Integrin receptors such as a3b1, a6b1, a6b4, and a7b1 are well-known
receptors to bind laminins. Integrin receptors are known to bind LG1-3 domains,
while dystroglycan and syndecans are known to bind LG45 domains. Shift from
LG45 binding via integrin a3b1 to LG1-3 via integrin a6b4 affects epithelial cell
behavior changing from migration to stable anchoring via hemidesmosomes for-
mation [45]. Different laminins have been shown to interact with same integrin
receptors, however, with different affinities (for complete review, see (Yurchenco
[93]).
Laminin signaling is more complex as mere activation of certain integrin
receptors by binding them. For instance, laminins that bind same integrin receptor
may exhibit different effects on the cells, while two different laminins acting via two
different integrin receptors may induce same cell phenotypes [64]. Below we shall
discuss effects of laminin co-signaling, receptor clustering, and other complex
laminin–cell interaction patterns.
Co-signaling in mammary epithelial cells, Xu et al. have demonstrated [91]
that only synergy between (1) LM-111 binding the integrin receptors and (2) pro-
lactin binding prolactin receptor allows to activate a STAT5-driven signaling
pathway that leads to b-casein secretion by mammary epithelial cells (essential for
lactation). In vivo, mammary endothelial cells respond to prolactin stimulation
during nursing period by expression of b-casein, the milk protein. However, when
the cells are cultured upon LM-111 in vitro and are stimulated by prolactin, no
b-casein expression occurs. Thus, the cells fail to respond to naturally stimulating
growth factor. However, when LM-111 polarity is inverted, that is, LM-111 and
prolactin stimulation occur from the same basal side of the cell, the STAT5 pathway
is activated and b-casein is produced. It has been hypothesized that LM-111
3 Biologically Relevant Laminins in Regenerative Medicine 69

binding the mammary epithelial receptors on the basal side of the cell attracts the
prolactin receptor to the same side, as it occurs in vivo. If prolactin is provided from
the same basal side, it meets the prolactin receptor and signaling pathway occurs
[91].
Co-signaling in vascular endothelial cells. Co-signaling from LM-411 and
VEGF to vascular endothelial tip cell allows to switch on the Notch/Dll pathway,
thus restricting the occurrence of vessel branching [77]. In the absence of LM-411,
the mechanism fails, and pathological vessel branching occurs and results in
pathologies.
Clustering calcium channels in motor nerve terminals. Laminin b2 binds and
clusters together voltage-gated calcium channels within synaptic cleft, which is
essential for neurotransmitter release from motor nerve terminals [54].
Neuromuscular junctions in mice lacking laminin b2 suffer abnormalities: synaptic
cleft being blocked by invading Schwann cell processes and reduced number of
active zones in nerve terminals [55, 60]. Apparently, b1-laminins present in the
synaptic cleft cannot compensate for the missing b2 isoforms.

Extracellular Matrix Interactions

Self-assembly and interaction with other laminin isoforms. Laminins are cap-
able of self-assembly via N-terminal short arms. N-terminal parts of non-truncated
a, b, and c chains can interact with each other. Cross-shaped laminins, like
LM-111, can form 3D gels, for which they require calcium ions [94]. It is important
to note that Y-shaped laminins, like LM-311/321 or 411/421, as such cannot form
3D gels without help of cross-shaped laminins or basement membranes molecules.
Possible combinations of interaction between N-terminal short arms of a1, a2, a5,
b1, b2, b3, c1, and c3 laminin chains and respective Kd values are described in
[57].
Other extracellular matrix molecules. Laminins constitute basement mem-
branes together with collagen IV. Collagen IV and laminins can self-assemble;
however, those two intertwining networks are not connected directly, but via
smaller connector molecules, such as nidogens [95, 96].

Mechanotransduction

Biological function of laminins relies on ability to interact with cell receptors and
extracellular matrix scaffold simultaneously. It is shown that cell function, such as
differentiation or activation, depends on surrounding extracellular scaffold
mechanical properties (stiffness or elasticity modulus) [13, 18–20]. Laminins are
capable of outside-in signaling.
Important implications for biotechnology are: (1) laminin peptides or single
domains, such as whole or partial LG domains, often lack an ability to couple
70 A. Domogatskaya and S. Rodin

extracellular scaffold to cell receptors, and (2) it is important to consider the

mechanoelasticity message the laminin transmits to the cultured cells. Plastic cul-
ture dish surface is abnormally stiff, compared to majority of mammalian tissues
except for bone, while 3D gels like Matrigel are more relevant in respect of
mechanical properties.

Proteolytically Degraded Forms of Laminins

Proteolytically degraded forms of laminin isoforms used in research are often

named same as their full-size natural trimmers. However, the degraded forms may
lack functionally important domains responsible either for interaction with cell
receptors or with extracellular matrix molecules. Wondumi et al. [88] have
demonstrated structural difference between complete trimeric laminin molecules
comprising all the functional domains that are produced in cultured mammalian
cells and laminins that are produced by non-specific proteolytical degradation of
tissues, for example human placenta. The proteolytically degraded laminins that are
lacking some functional domains cannot be considered as chemically defined,
because the degradation is batch-to-batch difference and cannot be controlled. It is
also important to note that laminin single chains, domains, peptides, and
peptide-based hydrogels may affect the cultured cells in totally different way in
comparison with biologically relevant and intact trimeric molecules. For practical
applications in cell biology, it is always important to know if all the functional
domains are present in the laminin molecule used.

Possible Antagonistic Functions of Related Laminins

Certain laminin isoforms are highly homological and are expressed in same tissues.
One may assume that they would exert similar influence on the cells, which are in
contact with them. Indeed, it sometimes such as laminins impose similar effects on
the cells; for example, LM-511 and LM-521 support embryonic stem cells
self-renewal in vitro [67, 68]. However, sometimes closely related laminin isoforms
exert antagonistic impact on cells.
Example 1: b1- versus b2-laminins within neuromuscular junction. Laminin
b1 isoforms, such as LM-211, 411, and 511, are enveloping surface of muscular
fibers and motor nerves that innervate them, in exception for neuromuscular
junction: the very spot where signal is transmitted from nerve terminal to the
muscle. The neuromuscular junction contains b2-analogues of the same laminins:
LM-221, 421, and 521. In LAMB2-/knockout mice, the b1 isoforms LM-211, 411,
and 511 appear within synaptic cleft, in order to compensate for the absence of
b2-laminins. The Schwann cells are repelled by b2-laminin LM-521 and thus
prevented from spreading processes into synaptic cleft in healthy mice.
3 Biologically Relevant Laminins in Regenerative Medicine 71

On contrary, the Schwann cells are attracted by b1-laminins to invade and block the
synaptic cleft in LAMB2-/knockout mice that results in severe neural system dis-
orders [55, 60].
Example 2: Processed versus unprocessed 3 chain in LM-332 in cell adhe-
sion. LM-332 affects epithelial cells behavior differently depending on whether a3
chain is processed or not. Unprocessed a3 chain binds integrin a3b1 and facilitates
cell migration, wherein cells form temporary contacts with the LM-332 surface.
However, processed a3 LM-332 binds integrin a6b4 and forms hemidesmosomes
that enable stable anchorage of the cells.

Biologically Relevant Laminins for In Vitro Cell

and Organoid Cultures

In this part, we shall present several examples where biologically relevant laminins
are used for establishing functional cell cultures in vitro.

Human and Mouse Embryonic Stem Cells

Laminins are the first extracellular matrix molecules emerging in embryonic

development. Laminin chains are first detected at two-cell stage and full-size trimer
at eight-cell stage of embryonic development. Blastocyst consists of trophectoderm
that will give rise to extraembryonic tissues and inner cell mass (ICM) that will give
origin to all three embryonic germ layers: ectoderm, mesoderm, and endoderm.
Embryonic stem cells, derived from inner cell mass of blastocyst, have unlimited
proliferative capacity and potential to differentiate into any mammalian cell type,
from neurons to insulin-producing b-cells.
Some stem cells, due to their high proliferation potential, are especially prone to
malignant transformations [3, 52], which are a major concern in regenerative
medicine. Therefore, it is most essential (1) to imitate a healthy, natural niche of
inner cell mass: natural origin of embryonic stem cells and (2) ensure that chro-
mosomal abnormalities and mutations do not occur after long-term passaging.
Two major laminin types are detected in blastocysts. a5-laminins are present
between cells of the inner cell mass (ICM), so that every ICM cell in vivo is in
contact with a5-laminins, while a1-laminins form outer basement membrane [34].
a1-laminins are essential for the very first event of ICM cells differentiation:
polarization following differentiation toward ectoderm, mesoderm, and endoderm
[39, 41, 71]. However, would a5-laminins support the pluripotency state of
embryonic stem cells in vitro?
72 A. Domogatskaya and S. Rodin

Mouse embryonic stem cells that were cultured on a1-, a3-, a4- and a5-lami-
nins, respectively, in the absence of differentiation inhibitors underwent four dif-
ferent scenarios [14]. Cells, cultured on LM-111, differentiated within 2 weeks, and
proliferation abruptly reduced (which is consistent with natural role of LM-111
[40]). Cells cultured on LM-411 suffered lack of adhesion contacts, detached and
died. Cells cultured on LM-332 and LM-511 proliferated for over 150 doublings
with approximately same rate, while continuously expressing the pluripotency
markers: Oct-4, Sox2, and Nanog. However, the functional test—ability to form
three germ layers and give rise to whole functional organism—revealed the drastic
difference between LM-511 and LM-332. As latter gave rise to very weakly chi-
meric and/or sick chimeric animals which failed to undergo germ line transfer, the
former (LM-511) gave rise to healthy animals with strong chimerism [14] that in
turn gave rise to germ line transfer, healthy animals (Domogatskaya, Rodin,
Tryggvason: unpublished manuscript).
Due to unlimited proliferative capacity and ability to differentiate into practically
all adult cell types, human embryonic stem (ES) cells may be a valuable source of
cells for regenerative medicine. Nevertheless, only a few clinical trials have been
approved for human ES therapies. One of the major reasons of that was lack of
xeno-free (animal substance-free) and chemically defined environments that sup-
port self-renewal of human pluripotent stem (PS) cells (human ES and induced
pluripotent stem cells). Using a5-laminins, we have been able to develop such
human cell culture systems. Indeed, LM-511 and especially LM-521 allow robust
proliferation of human PS cells for more than 6 months in culture. The number of
cultured cells has been multiplied more than 1020 times, which is enough to develop
enough cells to treat hundreds of people. During the culturing, the cells express
stable levels of markers of pluripotency Oct-4, Nanog, Sox2, SSEA-4, etc. After six
months in culture, human PS cells can be differentiated into all three germ lineages
of the human embryo both in in vivo and in vitro assays. In-depth genotyping assay
has revealed that the cells cultured on LM-521 acquire little number of genetic
abnormalities and can be used in regenerative medicine. Importantly, LM-521
allows passaging of human PS cells in single-cell suspensions, which facilitates
automation of the culturing.
Individualized human PS cells die from anoikis that is a form of apoptosis
caused by unnatural milieu. That feature of human PS cells complicates manipu-
lations with their genomes, which can be used in regenerative medicine, e.g., for
correction of hereditary monogenic diseases. Since relevant integrin-mediated ex-
tracellular matrix signaling and cadherin-mediated cell–cell signaling prevent
anoikis, we sought to identify molecular cues that are sufficient to mimic natural
environment of human PS cells. E-cadherin, which is abundantly expressed on
human ES cell membranes, and LM-521 taken at 1:9 w/w ratio and used as cell
culture substratum have been able to prevent anoikis and permit survival of indi-
vidualized human PS cells. LM-521/E-cadherin substratum also allows derivation
of new hES cell lines from single blastomeres acquired through a single-cell biopsy
3 Biologically Relevant Laminins in Regenerative Medicine 73

from an eight-cell in vitro fertilization (IVF) embryo. This enables development of

cells without a need to destroy the parental embryo and confirms that LM-521 and
E-cadherin can mimic human ES cell niche.

Bone Marrow-Derived Hematopoietic Stem Cells

Many early in vitro studies with hematopoietic stem cells have been performed on
Engelbreth-Holm-Swarm sarcoma-derived LM-111; however, it is not a natural
laminin for this type of cells. LM-511 and LM-411 are expressed in human and
bone marrow, and LM-211 expression is weak and restricted to arterioles and
LM-111 not expressed at all. b1-laminins are present in adult bone marrow, but not
b2-laminins [21, 73].
In vitro, human CD34+ cells adhere strongly to LM-511/-521, but not to
LM-211 or LM-111. LM-511 exerts mitogenic activity in human hematopoietic
progenitor cells. LM-511/-521, unlike LM-111, are strongly adhesive for multi-
potent hematopoietic FDCP cells. LM-521/-511, unlike other isoforms, enable
robust adhesion for variety of hematopoietic lineages [22, 65, 73].

Insulin-Producing Pancreatic Islets

Insulin-producing pancreatic islets (islets of Langerhans) are small endocrine

organoids formed by several endocrine cell populations, including
insulin-producing b-cells. In order to regulate glucose blood levels, islet b-cells
have to function properly, and their function depends on receiving proper cues from
their niche. b-cells in vivo are in direct contact with underlying vascular basement
membrane, containing specific laminin isoforms (reviewed in [15]). As we have
previously demonstrated, the basement membrane contains a4- and a5-laminins,
but not a1-, a2-, or a3-laminins [53]. In vitro, experiments have shown that b-cells
depend on contact with those natural laminins in order to produce insulin. Notably,
b-cells are not capable of producing any laminins themselves; they depend on
vascular endothelial cells producing the laminins [15, 53].
We have demonstrated that a5-laminins have unique effect on islet culture
in vitro. Isolated islets, which are used for transplantation purposes and diabetes
research, have extremely high affinity toward natural a5-laminins, but not
a1-laminin (LM-111), which is not part of natural islet niche. The adhesion force
between the islet cells and natural a5-laminins is so strong that islets attain flat
shape and yet remain cohesive. Since contact with a5-laminins is part of natural
b-cell niche, it allows islet survival, islet cell proliferation, and insulin secretion by
b-cells after 3–4 weeks in culture (Tryggvason et al., US Patent U.S. Patent
9,499,794, [82].
74 A. Domogatskaya and S. Rodin

Neurobiology

Major laminin isoforms in the peripheral nervous system are: a2, a4, and a5.
b1-laminins are ubiquitous, while b2 are restricted to specific locations, like neu-
romuscular junctions. Deficiency in either of those chains results in severe neural
pathologies (reviewed in [15]).
Different cells of peripheral nervous system have specific preferences regarding
laminin substrate in vitro. Spinal motoneurons prefer to sprout long axons on
LM-211 compared to LM-411, while Schwann cells, in the opposite, form longer
sproutings on LM-411 compared to LM-211 [86]. Adult dorsal root ganglion
neurons formed longed neuritis on LM-511 and LM-111, but not on LM-411 or
LM-211. Notably, though neurons spread axons on LM-511 as well as on LM-111,
they engage different integrin receptors [64]. It is possible that different signaling
pathways are activated and cells would differ in receptiveness from growth factor
cues. We suggest therefore that one has not only considered mere adhesiveness and
morphological phenotype as sole evaluation criteria of cell culture system, but also
whether the extracellular matrix coating is biologically relevant and whether it can
provide relevant niche cues to the cell.

Evaluation Criteria in Developing Cell Culture System

As we have discussed above, synergetic action of specific laminin (or several

laminins) with specific growth factor(s) is required to provide the cultured cells
imitation of in vivo-like environment. Concentration range for every compound in
the system should be estimated experimentally. High-throughput-automated
screening with automated quantitative evaluation is often used to find the best
possible combination.
When automated screening of numerous samples is performed, the key impor-
tance is a set of selection criteria. Biased criteria may result in false positive errors
(that would generate cells that would not be able to function correctly after trans-
plantation or even undergo malignant transformation) and false negative errors
(good cell culture systems, most closely imitating the healthy natural environment
being discarded as “not proliferating with sufficiently high rate”).
Hereby, we discuss several possible pitfalls that should be avoided.
1. Adhesion as sole success criteria for extracellular matrix coating. Extracellular
matrix is often perceived as a neutral substance that does not affect cell phe-
notype, only function of it being able to keep cells attached. However, different
extracellular matrix molecules engage different signaling pathways and can
drive cell behavior in very distinct ways.
2. High proliferation rate as a success criterion. In the absence of biologically
relevant extracellular matrix majority mammalian cells would react by anoikis
(apoptosis) or phenotype loss. In the latter case, the cells often attain
3 Biologically Relevant Laminins in Regenerative Medicine 75

fibroblast-like appearance and behavior and, sometimes, high proliferation rate.

However, function is lost and after transplantation such cells would be useless.
Worst case scenario, however, would be if lack of relevant extracellular matrix
would trigger malignant transformation processes in the cultured cells.
3. Expression of specific markers, determined by immunohistochemistry or
western blot being a sole criterion of cell functionality. Only the functional
assays, specific for each cell type, would be a sufficient proof of the cell identity.
Functional assays should be designed for each cell or organotypic culture with
regard to their biological role. For example, embryonic stem cells function is to
give rise to all the three germ layers. For insulin-producing pancreatic b-cells, it
would be to release insulin in glucose-dependent manner.
4. Screening for different growth factors in the absence of biologically relevant
extracellular matrix, or screening for different laminins in the absence of bio-
logically relevant growth factors. In vivo, the niche cues are in many cases
enabled only by co-signaling from both specific laminin and specific growth
factor binding relevant cell receptors. Another pitfall would be to apply stim-
ulatory growth factors and laminins without regard to natural cell polarity [91].

Future Challenges

Use of biologically relevant laminins in regenerative medicine is a new area of

research. We have reviewed above some examples of how the “niche-based”
concept can be successfully translated into biomedical technologies. We believe
that biologically relevant laminins may happen to be a useful tool in:
1. Generating new technologies of
• In vitro cell culture systems to expand niche-sensitive primary cell types
• Robust differentiation protocols for embryonic stem cells and adult stem
cells
• 3D artificial tissues and organs with sophisticated architecture
2. Improve efficacy of existing technologies
• Increase number of doublings that a primary cell culture can undergo without
loss of phenotype.
• Increase rate of proliferation (however, within healthy natural range).
• Increase functionality of cells cultured in vitro.
3. Improve safety of existing technologies
• Replace non-defined extracellular scaffolds with defined ones.
• Replace systems that bear potential source of pathogens, such as feeder cells
or xeno compounds, to pathogen-free.
76 A. Domogatskaya and S. Rodin

In this chapter, we have focused on laminins’ ability to provide general main-

tenance for specific mammalian cell types, such as to enable survival, healthy
proliferation and responsiveness to growth factor stimuli. In vivo, however, lami-
nins have many other advanced biological in vivo roles, which are not yet translated
into biomedical technologies. For example, in vivo certain laminins can regulate
and maintain organogenesis: e.g., regulate diameter of blood vessels and extent of
branching in vasculogenesis and angiogenesis [30, 77], and enable neural crest and
brain compartments formation [9, 24]. Laminins can regulate permeability of viable
tissue structures, from biomolecules (such as in glomerular basement membrane in
kidney [56]) to cells [74, 89]. Laminins may enable mechanosensing by conveying
stiffness signals from extracellular matrix to residing cells [25], thus regulating cell
behavior [18]. Laminins can guide cell-to-cell contacts with high precision, such as
in neuromuscular junction [62]. It is possible that laminins could allow not only to
expand isolated cells in vitro, but to design highly organized artificial 3D tissues
with natural-like architecture, hosting heterogenic cell communities.

Summary

1. At least 16 laminin isoforms exist in mammals. Each isoform has a unique

function. Laminin isoforms are not “similar.” In fact, two different isoforms may
exert opposite influences on a certain cell type.
2. Golden standard in regenerative medicine technologies would be to expand specific
cells in a culture system specifically tailored to mimic their in vivo microenvi-
ronment. Such culture would necessarily require niche-specific (1) growth factors,
(2) cell contacts (or imitation of those), and (3) tissue-specific extracellular matrix
cues.
3. Knowledge of biological roles for laminins in vivo (tissue-specific expression,
knockout or mutant models, laminin-associated pathologies, etc.) may provide
valuable clues to choose laminin isoforms that would be relevant for culturing
specific cell type in vitro. However, knowledge of laminin trimers distribution
and action in vivo is limited up to date.
4. Specific laminins act in concert with specific growth factors (co-signaling). Use
of specific laminin in the absence of specific growth factor, or vice versa, may
result in negative result.
5. Criteria for successful in vitro cell culture system should not only include
phenotype analysis (such as adhesion, proliferation, and specific markers
expression), but also (1) functional assays and (2) side-effects monitoring, such
as malignant transformation of the cell lines. Golden standard for establishing a
cell culture system should be the in vivo cells within natural environment.
6. Expanding cells approach in regenerative medicine has advantages, compared to
traditional organ transplantation approach, but also carries hidden hazards. An
isolated cell, when deprived on natural cues, may undergo apoptosis (anoikis),
loss of phenotype, or loss of function. One more possibility, which is probably
3 Biologically Relevant Laminins in Regenerative Medicine 77

the worst, is malignant transformation of the cells. Such cells may exhibit
abnormally high survival, adhesion, and proliferation properties, which would
give them a competitive advantage over non-malignant, healthy cells.
7. Use of tissue-specific laminins for expanding tissue-specific cell cultures is a
new trend in regenerative medicine. Natural properties of laminins potentially
can enable not only expansion of a specific cell type, but many other advanced
technologies, like generation of complex, natural-like 3D tissues.

Acknowledgements This work was supported by Swedish Research Council (project

2016-01831, A.D.), Foundation for Assistance to Small Innovative Enterprises (project 24026, A.D.)
and Russian Science Foundation (project 14-15-00712, A.D.).

References

1. Abrass CK, Hansen KM, Patton BL. Laminin alpha4-null mutant mice develop chronic
kidney disease with persistent overexpression of platelet-derived growth factor. Am J Pathol.
2010;176:839–49.
2. Akhmanova M, Osidak E, Domogatsky S, Rodin S, Domogatskaya A. Physical, spatial, and
molecular aspects of extracellular matrix of in vivo niches and artificial scaffolds relevant to
stem cells research. Stem Cells Int. 2015;167025.
3. Amps K, Andrews PW, Anyfantis G, Armstrong L, Avery S, Baharvand H, Baker J, Baker D,
Munoz MB, Beil S, Benvenisty N, Ben-Yosef D, Biancotti JC, Bosman A, Brena RM,
Brison D, Caisander G, Camarasa MV, Chen J, Chiao E, Choi YM, Choo AB, Collins D,
Colman A, Crook JM, Daley GQ, Dalton A, de Sousa PA, Denning C, Downie J, Dvorak P,
Montgomery KD, Feki A, Ford A, Fox V, Fraga AM, Frumkin T, Ge L, Gokhale PJ,
Golan-Lev T, Gourabi H, Gropp M, Lu G, Hampl A, Harron K, Healy L, Herath W, Holm F,
Hovatta O, Hyllner J, Inamdar MS, Irwanto AK, Ishii T, Jaconi M, Jin Y, Kimber S,
Kiselev S, Knowles BB, Kopper O, Kukharenko V, Kuliev A, Lagarkova MA, Laird PW,
Lako M, Laslett AL, Lavon N, Lee DR, Lee JE, Li C, Lim LS, Ludwig TE, Ma Y, Maltby E,
Mateizel I, Mayshar Y, Mileikovsky M, Minger SL, Miyazaki T, Moon SY, Moore H,
Mummery C, Nagy A, Nakatsuji N, Narwani K, Oh SK, Olson C, Otonkoski T, Pan F,
Park IH, Pells S, Pera MF, Pereira LV, Qi O, Raj GS, Reubinoff B, Robins A, Robson P,
Rossant J, Salekdeh GH, Schulz TC, et al. Screening ethnically diverse human embryonic
stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage. Nat
Biotechnol. 2011;29:1132–44.
4. Aumailley M, Bruckner-Tuderman L, Carter WG, Deutzmann R, Edgar D, Ekblom P,
Engel J, Engvall E, Hohenester E, Jones JC, Kleinman HK, Marinkovich MP, Martin GR,
Mayer U, Meneguzzi G, Miner JH, Miyazaki K, Patarroyo M, Paulsson M, Quaranta V,
Sanes JR, Sasaki T, Sekiguchi K, Sorokin LM, Talts JF, Tryggvason K, Uitto J, Virtanen I,
von der Mark K, Wewer UM, Yamada Y, Yurchenco PD. A simplified laminin nomenclature.
Matrix Biol. 2005;24:326–32.
5. Barak T, Kwan KY, Louvi A, Demirbilek V, Saygi S, Tuysuz B, Choi M, Boyaci H,
Doerschner K, Zhu Y, Kaymakcalan H, Yilmaz S, Bakircioglu M, Caglayan AO, Ozturk AK,
Yasuno K, Brunken WJ, Atalar E, Yalcinkaya C, Dincer A, Bronen RA, Mane S, Ozcelik T,
Lifton RP, Sestan N, Bilguvar K, Gunel M. Recessive LAMC3 mutations cause
malformations of occipital cortical development. Nat Genet. 2011;43:590–4.
6. Baudin B, Bruneel A, Bosselut N, Vaubourdolle M. A protocol for isolation and culture of
human umbilical vein endothelial cells. Nat Protoc. 2007;2:481–5.
78 A. Domogatskaya and S. Rodin

7. Chiarugi P, Giannoni E. Anoikis: a necessary death program for anchorage-dependent cells.

Biochem Pharmacol. 2008;76:1352–64.
8. Chun SJ, Rasband MN, Sidman RL, Habib AA, Vartanian T. Integrin-linked kinase is
required for laminin-2-induced oligodendrocyte cell spreading and CNS myelination. J Cell
Biol. 2003;163:397–408.
9. Coles EG, Gammill LS, Miner JH, Bronner-Fraser M. Abnormalities in neural crest cell
migration in laminin alpha5 mutant mice. Dev Biol. 2006;289:218–28.
10. Cooper AR, Macqueen HA. Subunits of laminin are differentially synthesized in mouse eggs
and early embryos. Dev Biol. 1983;96:467–71.
11. Denes V, Witkovsky P, Koch M, Hunter DD, Pinzon-Duarte G, Brunken WJ. Laminin
deficits induce alterations in the development of dopaminergic neurons in the mouse retina.
Vis Neurosci. 2007;24:549–62.
12. Dienstag JL, Cosimi AB. Liver transplantation–a vision realized. N Engl J Med.
2012;367:1483–5.
13. Discher DE, Janmey P, Wang YL. Tissue cells feel and respond to the stiffness of their
substrate. Science. 2005;310:1139–43.
14. Domogatskaya A, Rodin S, Boutaud A, Tryggvason K. Laminin-511 but not -332, -111, or
-411 enables mouse embryonic stem cell self-renewal in vitro. Stem Cells. 2008;26:2800–9.
15. Domogatskaya A, Rodin S, Tryggvason K. Functional diversity of laminins. Annu Rev Cell
Dev Biol. 2012;28:523–53.
16. Durbeej M. Laminins. Cell Tissue Res. 2010;339:259–68.
17. Dziadek M, Timpl R. Expression of nidogen and laminin in basement membranes during
mouse embryogenesis and in teratocarcinoma cells. Dev Biol. 1985;111:372–82.
18. Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage
specification. Cell. 2006;126:677–89.
19. Engler AJ, Sweeney HL, Discher DE, Schwarzbauer JE. Extracellular matrix elasticity directs
stem cell differentiation. J Musculoskelet Neuronal Interact. 2007;7:335.
20. Georges PC, Hui JJ, Gombos Z, McCormick ME, Wang AY, Uemura M, Mick R,
Janmey PA, Furth EE, Wells RG. Increased stiffness of the rat liver precedes matrix
deposition: implications for fibrosis. Am J Physiol Gastrointest Liver Physiol. 2007;293:
G1147–54.
21. Gu Y, Sorokin L, Durbeej M, Hjalt T, Jonsson JI, Ekblom M. Characterization of bone
marrow laminins and identification of alpha5-containing laminins as adhesive proteins for
multipotent hematopoietic FDCP-Mix cells. Blood. 1999;93:2533–42.
22. Gu YC, Kortesmaa J, Tryggvason K, Persson J, Ekblom P, Jacobsen SE, Ekblom M. Laminin
isoform-specific promotion of adhesion and migration of human bone marrow progenitor
cells. Blood. 2003;101:877–85.
23. Hager M, Gawlik K, Nystrom A, Sasaki T, Durbeej M. Laminin {alpha}1 chain corrects male
infertility caused by absence of laminin {alpha}2 chain. Am J Pathol. 2005;167:823–33.
24. Halfter W, Dong S, Yip YP, Willem M, Mayer U. A critical function of the pial basement
membrane in cortical histogenesis. J Neurosci. 2002;22:6029–40.
25. Hallmann R, Horn N, Selg M, Wendler O, Pausch F, Sorokin LM. Expression and function of
laminins in the embryonic and mature vasculature. Physiol Rev. 2005;85:979–1000.
26. Heinonen M, Oila O, Nordstrom K. Current issues in the regulation of human
tissue-engineering products in the European Union. Tissue Eng. 2005;11:1905–11.
27. Hewitt ZA, Amps KJ, Moore HD. Derivation of GMP raw materials for use in regenerative
medicine: hESC-based therapies, progress toward clinical application. Clin Pharmacol Ther.
2007;82:448–52.
28. Huang CC, Hall DH, Hedgecock EM, Kao G, Karantza V, Vogel BE, Hutter H,
Chisholm AD, Yurchenco PD, Wadsworth WG. Laminin alpha subunits and their role in
C. elegans development. Development. 2003;130:3343–58.
29. Iwao M, Fukada S, Harada T, Tsujikawa K, Yagita H, Hiramine C, Miyagoe Y, Takeda S,
Yamamoto H. Interaction of merosin (laminin 2) with very late activation antigen-6 is necessary
for the survival of CD4+ CD8+ immature thymocytes. Immunology. 2000;99:481–8.
3 Biologically Relevant Laminins in Regenerative Medicine 79

30. Jakobsson L, Domogatskaya A, Tryggvason K, Edgar D, Claesson-Welsh L. Laminin

deposition is dispensable for vasculogenesis but regulates blood vessel diameter independent
of flow. FASEB J. 2008;22:1530–9.
31. Jones KJ, Morgan G, Johnston H, Tobias V, Ouvrier RA, Wilkinson I, North KN. The
expanding phenotype of laminin alpha2 chain (merosin) abnormalities: case series and
review. J Med Genet. 2001;38:649–57.
32. Kao G, Huang CC, Hedgecock EM, Hall DH, Wadsworth WG. The role of the laminin beta
subunit in laminin heterotrimer assembly and basement membrane function and development
in C. elegans. Dev Biol. 2006;290:211–9.
33. Kenne E, Soehnlein O, Genove G, Rotzius P, Eriksson EE, Lindbom L. Immune cell
recruitment to inflammatory loci is impaired in mice deficient in basement membrane protein
laminin alpha4. J Leukoc Biol. 2010;88:523–8.
34. Klaffky E, Williams R, Yao CC, Ziober B, Kramer R, Sutherland A. Trophoblast-specific
expression and function of the integrin alpha 7 subunit in the peri-implantation mouse
embryo. Dev Biol. 2001;239:161–75.
35. Klimanskaya I, Chung Y, Becker S, Lu SJ, Lanza R. Human embryonic stem cell lines
derived from single blastomeres. Nature. 2006;444:481–5.
36. Knoll R, Postel R, Wang J, Kratzner R, Hennecke G, Vacaru AM, Vakeel P, Schubert C,
Murthy K, Rana BK, Kube D, Knoll G, Schafer K, Hayashi T, Holm T, Kimura A, Schork N,
Toliat MR, Nurnberg P, Schultheiss HP, Schaper W, Schaper J, Bos E, den Hertog J, van
Eeden FJ, Peters PJ, Hasenfuss G, Chien KR, Bakkers J. Laminin-alpha4 and integrin-linked
kinase mutations cause human cardiomyopathy via simultaneous defects in cardiomyocytes
and endothelial cells. Circulation. 2007;116:515–25.
37. Koch M, Olson PF, Albus A, Jin W, Hunter DD, Brunken WJ, Burgeson RE,
Champliaud MF. Characterization and expression of the laminin gamma3 chain: a novel,
non-basement membrane-associated, laminin chain. J Cell Biol. 1999;145:605–18.
38. Leivo I, Vaheri A, Timpl R, Wartiovaara J. Appearance and distribution of collagens and
laminin in the early mouse embryo. Dev Biol. 1980;76:100–14.
39. Li L, Arman E, Ekblom P, Edgar D, Murray P, Lonai P. Distinct GATA6- and
laminin-dependent mechanisms regulate endodermal and ectodermal embryonic stem cell
fates. Development. 2004;131:5277–86.
40. Li S, Edgar D, Fassler R, Wadsworth W, Yurchenco PD. The role of laminin in embryonic
cell polarization and tissue organization. Dev Cell. 2003;4:613–24.
41. Li S, Harrison D, Carbonetto S, Fassler R, Smyth N, Edgar D, Yurchenco PD. Matrix
assembly, regulation, and survival functions of laminin and its receptors in embryonic stem
cell differentiation. J Cell Biol. 2002;157:1279–90.
42. Libby RT, Champliaud MF, Claudepierre T, Xu Y, Gibbons EP, Koch M, Burgeson RE,
Hunter DD, Brunken WJ. Laminin expression in adult and developing retinae: evidence of
two novel CNS laminins. J Neurosci. 2000;20:6517–28.
43. Macdonald PR, Lustig A, Steinmetz MO, Kammerer RA. Laminin chain assembly is
regulated by specific coiled-coil interactions. J Struct Biol. 2010;170:398–405.
44. Magner WJ, Chang AC, Owens J, Hong MJ, Brooks A, Coligan JE. Aberrant development of
thymocytes in mice lacking laminin-2. Dev Immunol. 2000;7:179–93.
45. Marinkovich MP. Tumour microenvironment: laminin 332 in squamous-cell carcinoma. Nat
Rev Cancer. 2007;7:370–80.
46. Martin D, Zusman S, Li X, Williams EL, Khare N, Darocha S, Chiquet-Ehrismann R,
Baumgartner S. wing blister, a new Drosophila laminin alpha chain required for cell adhesion
and migration during embryonic and imaginal development. J Cell Biol. 1999;145:191–201.
47. Martin MJ, Muotri A, Gage F, Varki A. Human embryonic stem cells express an
immunogenic nonhuman sialic acid. Nat Med. 2005;11:228–32.
48. Miner JH, Cunningham J, Sanes JR. Roles for laminin in embryogenesis: exencephaly,
syndactyly, and placentopathy in mice lacking the laminin alpha5 chain. J Cell Biol.
1998;143:1713–23.
80 A. Domogatskaya and S. Rodin

49. Miner JH, Li C, Mudd JL, Go G, Sutherland AE. Compositional and structural requirements
for laminin and basement membranes during mouse embryo implantation and gastrulation.
Development. 2004;131:2247–56.
50. Miner JH, Patton BL, Lentz SI, Gilbert DJ, Snider WD, Jenkins NA, Copeland NG, Sanes JR.
The laminin alpha chains: expression, developmental transitions, and chromosomal locations
of alpha1-5, identification of heterotrimeric laminins 8-11, and cloning of a novel alpha3
isoform. J Cell Biol. 1997;137:685–701.
51. Miner JH, Yurchenco PD. Laminin functions in tissue morphogenesis. Annu Rev Cell Dev
Biol. 2004;20:255–84.
52. Narva E, Autio R, Rahkonen N, Kong L, Harrison N, Kitsberg D, Borghese L, Itskovitz-Eldor
J, Rasool O, Dvorak P, Hovatta O, Otonkoski T, Tuuri T, Cui W, Brustle O, Baker D,
Maltby E, Moore HD, Benvenisty N, Andrews PW, Yli-Harja O, Lahesmaa R.
High-resolution DNA analysis of human embryonic stem cell lines reveals culture-induced
copy number changes and loss of heterozygosity. Nat Biotechnol. 2010;28:371–7.
53. Nikolova G, Jabs N, Konstantinova I, Domogatskaya A, Tryggvason K, Sorokin L, Fassler R,
Gu G, Gerber HP, Ferrara N, Melton DA, Lammert E. The vascular basement membrane: a
niche for insulin gene expression and Beta cell proliferation. Dev Cell. 2006;10:397–405.
54. Nishimune H, Sanes JR, Carlson SS. A synaptic laminin-calcium channel interaction
organizes active zones in motor nerve terminals. Nature. 2004;432:580–7.
55. Noakes PG, Gautam M, Mudd J, Sanes JR, Merlie JP. Aberrant differentiation of
neuromuscular junctions in mice lacking s-laminin/laminin beta 2. Nature. 1995;374:258–62.
56. Noakes PG, Miner JH, Gautam M, Cunningham JM, Sanes JR, Merlie JP. The renal
glomerulus of mice lacking s-laminin/laminin beta 2: nephrosis despite molecular compen-
sation by laminin beta 1. Nat Genet. 1995;10:400–6.
57. Odenthal U, Haehn S, Tunggal P, Merkl B, Schomburg D, Frie C, Paulsson M, Smyth N.
Molecular analysis of laminin N-terminal domains mediating self-interactions. J Biol Chem.
2004;279:44504–12.
58. Orlando G, Wood KJ, Stratta RJ, Yoo JJ, Atala A, Soker S. Regenerative medicine and organ
transplantation: past, present, and future. Transplantation. 2011;91:1310–7.
59. Patton BL. Laminins of the neuromuscular system. Microsc Res Tech. 2000;51:247–61.
60. Patton BL, Chiu AY, Sanes JR. Synaptic laminin prevents glial entry into the synaptic cleft.
Nature. 1998;393:698–701.
61. Patton BL, Connoll AM, Martin PT, Cunningham JM, Mehta S, Pestronk A, Miner JH,
Sanes JR. Distribution of ten laminin chains in dystrophic and regenerating muscles.
Neuromuscul Disord. 1999;9:423–33.
62. Patton BL, Cunningham JM, Thyboll J, Kortesmaa J, Westerblad H, Edstrom L,
Tryggvason K, Sanes JR. Properly formed but improperly localized synaptic specializations
in the absence of laminin alpha4. Nat Neurosci. 2001;4:597–604.
63. Pinzon-Duarte G, Daly G, Li YN, Koch M, Brunken WJ. Defective formation of the inner
limiting membrane in laminin beta2- and gamma3-null mice produces retinal dysplasia. Invest
Ophthalmol Vis Sci. 2010;51:1773–82.
64. Plantman S, Patarroyo M, Fried K, Domogatskaya A, Tryggvason K, Hammarberg H,
Cullheim S. Integrin-laminin interactions controlling neurite outgrowth from adult DRG
neurons in vitro. Mol Cell Neurosci. 2008;39:50–62.
65. Qian H, Georges-Labouesse E, Nystrom A, Domogatskaya A, Tryggvason K, Jacobsen SE,
Ekblom M. Distinct roles of integrins alpha6 and alpha4 in homing of fetal liver
hematopoietic stem and progenitor cells. Blood. 2007;110:2399–407.
66. Relan NK, Yang Y, Beqaj S, Miner JH, Schuger L. Cell elongation induces laminin alpha2
chain expression in mouse embryonic mesenchymal cells: role in visceral myogenesis. J Cell
Biol. 1999;147:1341–50.
67. Rodin S, Antonsson L, Niaudet C, Simonson OE, Salmela E, Hansson EM, Domogatskaya A,
Xiao Z, Damdimopoulou P, Sheikhi M, Inzunza J, Nilsson AS, Baker D, Kuiper R, Sun Y,
Blennow E, Nordenskjold M, Grinnemo KH, Kere J, Betsholtz C, Hovatta O, Tryggvason K.
3 Biologically Relevant Laminins in Regenerative Medicine 81

Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined

and xeno-free environment. Nat Commun. 2014;5:3195.
68. Rodin S, Domogatskaya A, Strom S, Hansson EM, Chien KR, Inzunza J, Hovatta O,
Tryggvason K. Long-term self-renewal of human pluripotent stem cells on human
recombinant laminin-511. Nat Biotechnol. 2010;28:611–5.
69. Sanes JR, Engvall E, Butkowski R, Hunter DD. Molecular heterogeneity of basal laminae:
isoforms of laminin and collagen IV at the neuromuscular junction and elsewhere. J Cell Biol.
1990;111:1685–99.
70. Sarras MP Jr, YAN L, Grens A, Zhang X, Agbas A, Huff JK, St John PL, Abrahamson DR.
Cloning and biological function of laminin in Hydra vulgaris. Dev Biol. 1994;164:312–24.
71. Scheele S, Falk M, Franzen A, Ellin F, Ferletta M, Lonai P, Andersson B, Timpl R,
Forsberg E, Ekblom P. Laminin alpha1 globular domains 4-5 induce fetal development but
are not vital for embryonic basement membrane assembly. Proc Natl Acad Sci U S A.
2005;102:1502–6.
72. Shimizu H, Zhang X, Zhang J, Leontovich A, Fei K, Yan L, Sarras MP Jr. Epithelial
morphogenesis in hydra requires de novo expression of extracellular matrix components and
matrix metalloproteinases. Development. 2002;129:1521–32.
73. Siler U, Seiffert M, Puch S, Richards A, Torok-Storb B, Muller CA, Sorokin L, Klein G.
Characterization and functional analysis of laminin isoforms in human bone marrow. Blood.
2000;96:4194–203.
74. Sixt M, Engelhardt B, Pausch F, Hallmann R, Wendler O, Sorokin LM. Endothelial cell
laminin isoforms, laminins 8 and 10, play decisive roles in T cell recruitment across the
blood-brain barrier in experimental autoimmune encephalomyelitis. J Cell Biol. 2001;153:
933–46.
75. Smyth N, Vatansever HS, Murray P, Meyer M, Frie C, Paulsson M, Edgar D. Absence of
basement membranes after targeting the LAMC1 gene results in embryonic lethality due to
failure of endoderm differentiation. J Cell Biol. 1999;144:151–60.
76. Sorokin LM, Pausch F, Durbeej M, Ekblom P. Differential expression of five laminin alpha
(1–5) chains in developing and adult mouse kidney. Dev Dyn. 1997;210:446–62.
77. Stenzel D, Franco CA, Estrach S, Mettouchi A, Sauvaget D, Rosewell I, Schertel A, Armer H,
Domogatskaya A, Rodin S, Tryggvason K, Collinson L, Sorokin L, Gerhardt H. Endothelial
basement membrane limits tip cell formation by inducing Dll4/Notch signalling in vivo.
EMBO Rep. 2011;12:1135–43.
78. Sunada Y, Bernier SM, Kozak CA, Yamada Y, Campbell KP. Deficiency of merosin in
dystrophic dy mice and genetic linkage of laminin M chain gene to dy locus. J Biol Chem.
1994;269:13729–32.
79. Suzuki N, Yokoyama F, Nomizu M. Functional sites in the laminin alpha chains. Connect
Tissue Res. 2005;46:142–52.
80. Szot GL, Lee MR, Tavakol MM, Lang J, Dekovic F, Kerlan RK, Stock PG, Posselt AM.
Successful clinical islet isolation using a GMP-manufactured collagenase and neutral
protease. Transplantation. 2009;88:753–6.
81. Thyboll J, Kortesmaa J, Cao R, Soininen R, Wang L, Iivanainen A, Sorokin L, Risling M,
Cao Y, Tryggvason K. Deletion of the laminin alpha4 chain leads to impaired microvessel
maturation. Mol Cell Biol. 2002;22:1194–202.
82. TRYGGVASON K., TRYGGVASON K.K. & DOMOGATSKAYA A. U.S. Patent
9,499,794, issued November 22, 2016.
83. Tzu J, Marinkovich MP. Bridging structure with function: structural, regulatory, and
developmental role of laminins. Int J Biochem Cell Biol. 2008;40:199–214.
84. Unger C, Skottman H, Blomberg P, Dilber MS, Hovatta O. Good manufacturing practice and
clinical-grade human embryonic stem cell lines. Hum Mol Genet. 2008;17:R48–53.
85. Urbano JM, Torgler CN, Molnar C, Tepass U, Lopez-Varea A, Brown NH, de Celis JF,
Martin-Bermudo MD. Drosophila laminins act as key regulators of basement membrane
assembly and morphogenesis. Development. 2009;136:4165–76.
82 A. Domogatskaya and S. Rodin

86. Wallquist W, Plantman S, Thams S, Thyboll J, Kortesmaa J, Lannergren J, Domogatskaya A,

Ogren SO, Risling M, Hammarberg H, Tryggvason K, Cullheim S. Impeded interaction
between Schwann cells and axons in the absence of laminin alpha4. J Neurosci.
2005;25:3692–700.
87. Wang J, Hoshijima M, Lam J, Zhou Z, Jokiel A, Dalton ND, Hultenby K, Ruiz-Lozano P,
Ross J, Jr, Tryggvason K, Chien KR. Cardiomyopathy associated with microcirculation
dysfunction in laminin alpha4 chain-deficient mice. J Biol Chem. 2006;281:213–20.
88. Wondimu Z, Gorfu G, Kawataki T, Smirnov S, Yurchenco P, Tryggvason K, Patarroyo M.
Characterization of commercial laminin preparations from human placenta in comparison to
recombinant laminins 2 (alpha2beta1gamma1), 8 (alpha4beta1gamma1), 10 (alpha5be-
ta1gamma1). Matrix Biol. 2006;25:89–93.
89. Wu C, Ivars F, Anderson P, Hallmann R, Vestweber D, Nilsson P, Robenek H,
Tryggvason K, Song J, Korpos E, Loser K, Beissert S, Georges-Labouesse E, Sorokin LM.
Endothelial basement membrane laminin alpha5 selectively inhibits T lymphocyte extrava-
sation into the brain. Nat Med. 2009;15:519–27.
90. Xu H, Wu XR, Wewer UM, Engvall E. Murine muscular dystrophy caused by a mutation in
the laminin alpha 2 (Lama2) gene. Nat Genet. 1994;8:297–302.
91. Xu R, Nelson CM, Muschler JL, Veiseh M, Vonderhaar BK, Bissell MJ. Sustained activation
of STAT5 is essential for chromatin remodeling and maintenance of mammary-specific
function. J Cell Biol. 2009;184:57–66.
92. Yarnitzky T, Volk T. Laminin is required for heart, somatic muscles, and gut development in
the Drosophila embryo. Dev Biol. 1995;169:609–18.
93. Yurchenco PD. Basement membranes: cell scaffoldings and signaling platforms. Cold Spring
Harb Perspect Biol.2011;3.
94. Yurchenco PD, Cheng YS. Self-assembly and calcium-binding sites in laminin. A three-arm
interaction model. J Biol Chem. 1993;268:17286–99.
95. Yurchenco PD, Cheng YS, Colognato H. Laminin forms an independent network in basement
membranes. J Cell Biol. 1992;117:1119–33.
96. Yurchenco PD, O’Rear JJ. Basement membrane assembly. Methods Enzymol. 1994;245:
489–518.
97. Zhang X, Fei K, Agbas A, Yan L, Zhang J, O’reilly B, Deutzmann R, Sarras MP JR. Structure
and function of an early divergent form of laminin in hydra: a structurally conserved ECM
component that is essential for epithelial morphogenesis. Dev Genes Evol. 2002;212:159–72.
Chapter 4
Extracellular Matrix: Immunity
and Inflammation

Amelia Cataldi and Viviana di Giacomo

Abstract The extracellular matrix (ECM) is the non-cellular component of any

tissues and organs. It provides not only support, tensile strength, and scaffolding for
tissues and cells, but also biochemical signals and specialized proteins. The
destabilization or alteration of the ECM structural and chemical composition affects
growth, morphogenesis, differentiation, migration, communication, survival of all
cells as well as inflammation and immune response. Inflammation is a complex
defense mechanism characterized by leukocyte migration from the vasculature to
control tissue damage induced by pathogenic (bacterial or viral), traumatic, or toxic
injury with subsequent deposition of extracellular matrix resulting in tissue repair.
At sites of injury, phagocytic cells, namely macrophages and neutrophils, provide
innate cell-mediated immunity, and immune cells are influenced in their migration
by the topography and composition of the matrix architecture. The physical and
biochemical ECM properties are also able to modulate a number of processes in
immune cells, especially lymphocytes that can ultimately lead to inefficient immune
response. Among the large number of molecules responsible for ECM homeostasis,
matrix metalloproteinases, versican, hyaluronan, and thrombospondins are the most
involved in inflammation and immunity.

The Extracellular Matrix

The extracellular matrix (ECM) is the non-cellular component of any tissue and
organ [30]. Depending on the origin and on the condition (injury, inflammation,
tumor) of the tissue, the three-dimensional (3D) matrix architecture can assume a
variety of conformations [131]. In some tissues, the ECM can be dense and stiff,
while, in others, it is soft and more porous with gap size of different diameters.

A. Cataldi
V. di Giacomo (&)

University G. d’Annunzio, Chieti-Pescara, Italy
e-mail: [email protected]
A. Cataldi
e-mail: [email protected]

© Springer International Publishing AG, part of Springer Nature 2018 83

A. C. Berardi (ed.), Extracellular Matrix for Tissue Engineering
and Biomaterials, Stem Cell Biology and Regenerative Medicine,
https://doi.org/10.1007/978-3-319-77023-9_4
84 A. Cataldi and V. di Giacomo

ECM fibers also display highly variable thickness, straightness, and spatial
arrangements. The fibers can be relaxed and non-oriented or, in contrast, linearized
and oriented in a specific direction. These physical characteristics determine the
architecture of the tissue but, as reported below, they are also essential for regu-
lating immune cell migration and behavior [92].
The extracellular matrix consists of a complex assembly of many proteins and
polysaccharides whose specific composition varies from tissue to tissue. The pri-
mary components include insoluble fibrous structural proteins (i.e., collagens,
laminins, fibronectin, vitronectin, and elastin) and proteoglycans. These large and
negatively charged sugars efficiently bind water and fill the space between the
fibers. The ECM primarily fulfills a structural role by maintaining an insoluble
scaffold, which ultimately defines the shape and stiffness of organs [92].
The extracellular matrix consists in a reinforced composite of collagens and
elastic fibers embedded in a viscoelastic gel of proteoglycans, hyaluronan (HA),
and water, together with a wide variety and arrangement of assorted glycoproteins
[40, 46, 54, 78]. These molecules interact by entanglement and cross-linking to
form a bioactive polymer which, in part, regulates the biomechanical properties of
tissues and the phenotype of the cells belonging to those tissues. This regulation
involves molecular interactions that govern the attachment of cells to their ECM
scaffolds through integrin and non-integrin receptors, detachment of cells from
those scaffolds, and molecular rearrangements in the matrix that allow cells to
change shape during morphogenetic and remodeling events that occur in devel-
opment and disease. The amount and composition of the ECM is controlled by the
coordinated and differential regulation of synthesis and turnover of each of the
ECM components. It is becoming increasingly evident that matrix individual
components can exert dramatic effects on cell behavior and tissue response to
endogenous and exogenous stimuli [129].
The ECM provides not only support, tensile strength, and scaffolding for tissues
and cells, but also biochemical signals and specialized proteins (i.e., growth factors,
chemokines, cytokines, small matricellular proteins and small integrin-binding
glycoproteins) [18].
The destabilization or alteration of the structural and chemical matrix compo-
sition affects cell growth, morphogenesis, differentiation, migration, communication
and survival [33], along with inflammation and immune response [82].
In addition to providing structural integrity, the extracellular matrix is recognized
to play critical roles in regulating progenitor and reparative cell behaviors such as
migration, differentiation, proliferation, and survival. The ECM dictates these
activities through its binding to adhesion receptors as well as its ability to regulate
growth factor bioavailability and signaling. More recently, a key role for mechanical
control of cell fate through their interaction with the ECM has emerged [121].
Both mechanical and biochemical molecules influence extracellular matrix
dynamics in multiple ways, by releasing small bioactive signaling molecules and
growth factors stored within the ECM, by eliciting structural changes to matrix
proteins which expose cryptic sites, and by degrading matrix proteins directly [18].
4 Extracellular Matrix: Immunity and Inflammation 85

ECM remodeling takes place throughout different phases of disease progression

as part of an injury and/or inflammatory response. These phases involve breakdown
and disassembly of various matrix components and reassembly of specific com-
ponents as part of the pathogenesis of these diseases. The sequence of changes is
not unlike what is seen during wound repair in which the early ECM changes are
characterized by deposits which create a loose, open, and watery matrix (referred to
as a “provisional ECM”) [17, 130] which allows for cellular invasion and repair.
This provisional matrix is then replaced by a more fibrous ECM enriched in col-
lagens and assorted glycoproteins [128].
Among the large number of molecules responsible for ECM homeostasis, matrix
metalloproteinases (MMPs), versican, hyaluronan (HA), and thrombospondins are
the most involved in inflammation and immunity.

Matrix Metalloproteinases (MMPs)

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes involved in

physiological processes including tissue homeostasis and repair, and host defense.
There is also evidence that MMPs play a role in the pathogenesis of inflammatory
diseases with focal tissue destruction, such as rheumatoid arthritis, osteoarthritis, and
chronic cutaneous ulcerations, as well as in cancer progression [13, 56, 105]. The
expression and activity of metalloproteinases are under strict control in physiological
situations, whereas excessive activity of MMPs is often observed in pathological
conditions [91]. MMPs were initially characterized as extracellular matrix
(ECM) cleaving proteolytic enzymes, but during the past years, a growing number of
non-matrix substrates for MMPs have been identified. Metalloproteinases can
orchestrate the inflammatory functions at various levels [14, 91], regulating the
transmigration of inflammatory cells from vasculature to the site of inflammation in
tissue by processing ECM components, growth factors, cytokines, and chemokines.
MMPs belong to the metzincin superfamily, which is characterized by the
presence of a highly conserved motif containing three histidine residues, which
chelate a zinc ion in the catalytic site [35]. Other families in the metzincin super
family are ADAMs (proteinases with a disintegrin and a metalloprotease domain)
and ADAM-TSs (ADAM with thrombospondin-like motif), astacins, and ser-
ralysins. Metalloproteinases are ubiquitously expressed zinc-dependent endopepti-
dases with wide substrate specificities. They are produced either as soluble or cell
membrane-anchored proteinases that cleave proteins and proteoglycan components
of the extracellular matrix. In addition, MMPs cleave a multitude of non-matrix
substrates including cytokines, chemokines, growth factors, growth factor recep-
tors, and cell surface adhesion receptors. The members of the MMP family display
marked differences in their tissue-specific expression, and substrate specificity
makes these proteins a group of proteolytic enzymes with multiple physiological
functions [86].
86 A. Cataldi and V. di Giacomo

MMPs comprise a family of endopeptidases, which can cleave almost every

component of the extracellular matrix proteins. It is documented that many
non-ECM proteins can also be cleaved by selected metalloproteinases. Structurally,
they all have a zinc ion in the catalytic domain, and their activity is dependent on
divalent ions, mainly Zn2+ and Ca2+ [28, 108]. There are about 27 different MMPs
discovered so far, and they are subdivided into groups according to substrate
specificity and structural integrity. Induction and expression of these proteins are
regulated at the level of transcription and translation, respectively. Further com-
plexity of metalloproteinases is the activation from zymogen to active enzyme and,
secondly, the mRNA stability of few MMPs plays a critical role. ProMMPs are
converted into active enzymes by intramolecular cleavage of cysteine bridge
between thiol group at the prodomain and Zn2+ near the catalytic site. The overall
activity depends on substrate availability as well as on the presence of inhibitors in
pericellular space, though a high concentration of MMPs exists near the plasma
membrane [114].
The first metalloproteinase (collagenase) was identified in tadpole tails during
metamorphosis by Gross and Lapière [38]. Most MMPs have been studied in
vertebrates (25 members), but are also found in lower animals and plants. In
humans, these proteins comprise 24 genes, encoding 23 proteins, as one MMP
(MMP-23) is coded by two identical genes at chromosome 1 (MMP-23A and
MMP-23B). Mammalian metalloproteinases are classified according to:
(I) their localization, soluble (secreted into ECM) or insoluble (anchored to cell
membrane);
(II) their similarities in tridimensional structure and substrate affinity, being
usually divided into six subgroups: collagenases (MMPs-1, -8, and -13),
gelatinases (MMPs-2 and -9), stromelysins (MMPs-3, -10, and -11),
matrilysins (MMPs-7 and -26), membrane-type metalloproteinase (MMPs-14,
-15, -16, -17, and -24 or MMP-MT1, MT2, MT3, MT4, MT5, and MT6,
respectively), and others (MMPs-12, -18, -19, -20, -21, -22, -23, -27, and -28);
(III) numerically listed according to chronological discovery. MMPs-4, -5, and -6
are missing in the list since they were shown to be identical to other members
of the family [84].
Metalloproteinase regulation occurs at multiple levels, according to the cell type
involved, in a temporal and spatial manner and quantities, by intra- and extracel-
lular mechanisms. Inductive or suppressive signaling from the extracellular matrix
(cytokines, growth factors, EMMPRIN, signals from integrins, ECM proteins,
cellular stress, morphological changes, etc.) and intracellular signal transduction
induce the activation or repression of the MMP genes. In the nucleus, the genes
may be transcriptionally controlled by genetic alterations (polymorphisms or
mutations, particularly in promoter regions) and by epigenetic control (DNA
methylation status and remodeling of chromatin by histone acetylation) as well as
post-transcriptionally through mRNA processing. In the cytoplasm, metallopro-
teinases may be post-transcriptionally regulated by mRNA stability (microRNAs
4 Extracellular Matrix: Immunity and Inflammation 87

action and degradation pathway), intracellular activation of furin-susceptible

MMPs, insertion of prosthetic groups (N- and O-glycosylation and GPI-anchor)
or specific domains in the pro-MMP structure, and, finally, by inducible and con-
stitutive pro-enzyme secretion into the ECM. Several metalloproteinases may be
stored in the cytoplasm within granules in specific cell types prior to stimuli, such
as inflammatory stimuli, and then secreted while their counterpart on the cellular
membrane may be regulated by their localization on specialized membrane
microdomains (lipid rafts or caveolae), by endocytosis/recycling (clathrin or
caveolin-dependent) and intracellular degradation. In the extracellular matrix, the
MMPs may be controlled by proteolytic processing and inactivation, proteolytic
activation of pro-MMPs, binding of pro and active forms to inhibitors, and inter-
action with specific ECM components, leading to specific localization (pericellular
perimeter or far from cell secretion point within the matrix), and allosteric control
[16, 32, 39, 41, 68, 94, 95, 97, 111, 120, 135] (Table 4.1).
The balance between metalloproteinases and their inhibitors is required for
physiological extracellular matrix remodeling, and imbalance in these enzymes
leads to pathological states. In the tissues, MMPs are mainly reversibly inhibited in
the ECM by their physiologic tissue inhibitors (TIMPs), while the cell surface
MMPs are inhibited by the RECK glycoprotein [107].

Versican

Versican, whose production was found increased in many different diseases, is an

extracellular matrix (ECM) proteoglycan observed in the pericellular environment
of most tissues. Versican, produced by either stromal or myeloid cells, interacts
with cells to influence their ability to proliferate, migrate, adhere, assemble, and
remodel an ECM and can exert a major role in immunity and inflammation.
Versican contributes to the structural integrity of tissues and interacts with cells
through direct and indirect means to regulate, in part, cellular events that represent

Table 4.1 MMPs regulation

Step Regulated by
Genes activation Cytokines, growth factors, integrins, ECM proteins, cellular stress,
morphological changes
Transcription Polymorphisms, mutations, DNA methylation, histone acetylation
Translation miRNA degradation, insertion of specific domains
Storage within Localization, endocytosis, recycling, intracellular degradation
granules
Zimogen secretion Proteolytic activation/inactivation, inhibitors binding, interaction with
ECM components, allosteric control
88 A. Cataldi and V. di Giacomo

the basis of various diseases. Hence, targeting versican as a way to control cell
phenotype offers a novel approach in the treatment of many diseases [129].
Versican is negatively charged due to its glycosaminoglycan (GAG) chains and
attracts water, contributing to the viscoelasticity of the pericellular microenviron-
ment [29]. In addition, versican interacts with a number of matrix components near
the cell surface including hyaluronan (HA), tenascin-R and -C, thrombospondin 1,
fibronectin, and fibrillin [49, 62, 74, 133] to create a mechanically active
biopolymer around cells which influences their ability to change shape, adhere,
proliferate, migrate, assemble other ECM components, and survive. Versican and
extracellular matrix-related molecules may modify the mechanical stiffness around
cells contributing to alterations in mechanotransduction influencing cell behavior
and phenotype [23, 26, 125]. Versican can also act as a reservoir for cytokines and
growth factors to be released at various times, establishing a further subtle control
over cell activity and behavior [34, 75, 126].
Versican interacts with several different ECM molecules and, in part, plays a
central role in matrix assembly. The domain structure of versican lends itself to
multiple types of interactions through either protein–protein or protein–carbohy-
drate interactions. The best known of these interactions is the binding between the
amino-terminal domain of versican (G1) and hyaluronan (HA) [64]. This interac-
tion is stabilized by another protein—link protein—which exhibits selective bind-
ing specificity for both HA and versican [20] (Fig. 4.1).
In addition to hyaluronan, versican interacts with other extracellular matrix
molecules controlling their organization: it interacts with tenascin-R through its

Fibrillin

Hyaluronan Tenascins Fibulins

1 and 2
Link
protein Lectin binding domain

NH 3
Versican Thrombo-
spondin-1
Chemokines,
Cytokines,
Growth
factors Fibronectin
Cell
receptors

Fig. 4.1 Versican interactions

4 Extracellular Matrix: Immunity and Inflammation 89

lectin-binding domain and involves protein–carbohydrate interactions [8]. The

lectin-binding domain participates in other ligand interactions as well. For example,
versican interacts with fibulin-1 and fibulin-2 [9, 88], a growing family of extra-
cellular matrix proteins highly expressed in the developing heart valve. In adults,
however, fibulin-1 and -2 are found associated with microfibrils that are part of
elastic fibers. Versican can also interact with proteins associated with elastin, like
fibrillin [83, 88], and has been shown to co-localize with elastic fibers in the skin
[49]. Furthermore, fibrillins bind fibulin-2, and fibulin is preferentially localized to
the elastin/microfibril interface in some tissues, but not in others [96]. Fibulin may
represent a bridge between versican and fibrillin, forming high-ordered multi-
molecular structures important in the assembly of elastic fibers (Fig. 4.1). The
relationship between versican and assembly of elastic fibers is interesting and
unusual. In fact, elastic fibers are conspicuously absent in newly remodeled ex-
tracellular matrix as well as from atherosclerotic and restenotic lesions. The
importance of elastic fibers in regulating vascular disease is highlighted by studies
of the elastin KO mouse [128].

Hyaluronan

Hyaluronan (HA) is an atypical and relatively simple glycosaminoglycan (GAG); in

fact, it is an unsulfated and unbranched polysaccharide not linked to any PG-core
proteins. HA is ubiquitously expressed in the extracellular matrix of mammals and
is composed of D-glucuronic acid (GlcUA) and N-acetyl- D-glucosamine (GlcNAc)
bound together through b 1,3 and b 1,4 glycosidic bonds, respectively [77]. This
disaccharide moiety is repeated thousands of times generating a linear polymer with
a molecular mass ranging from 5  105 to 4–5  106 Da and more. Due to its
hydrophilic properties, hyaluronan is very hydrated and makes the extracellular
matrix an ideal environment in which cells can move and proliferate. Hyaluronan is
an important space-filling molecule as it is evident in humor vitreous, derma and at
joint level. Besides its molecular sieving properties related to the chemical and
biomechanical characteristics, this polymer interacting with specific proteins called
hyaladerins, such as TSG6, and membrane receptors like CD44, RHAMM, HARE,
and toll-like receptor (TLR) 4/2, modulates development, morphogenesis, tumori-
genesis, migration, apoptosis, cell survival, and inflammation [19, 116–119].

Thrombospondins

Thrombospondins (TSPs) are secreted extracellular matrix proteins from TSP

family, which consists of five homologous members. They share a complex domain
structure and have numerous binding partners in the extracellular matrix and
90 A. Cataldi and V. di Giacomo

multiple cell surface receptors. Information emerged over the past decade identifies
TSPs as important mediators of cellular homeostasis, assigning new important roles
in cardiovascular pathology to these proteins [102].
Recent studies of the functions of TSP in the cardiovascular system, diabetes,
and aging, which placed several thrombospondins in a position of critical regula-
tors, demonstrated the involvement of these proteins in practically each aspect of
cardiovascular pathophysiology related to atherosclerosis: inflammation, immunity,
leukocyte recruitment and function, function of vascular cells, angiogenesis, and
responses to hypoxia, ischemia, and hyperglycemia. TSPs play also a major role in
the development and ultimate outcome of the complications associated with
atherosclerosis–myocardial infarction, and heart hypertrophy and failure. Their
expression and significance increase with age and with the progression of diabetes,
two major contributors to the development of atherosclerosis and its complications
[102].
Thrombospondins comprise a conserved family of extracellular, oligomeric,
multidomain, calcium-binding glycoproteins. In general, basal metazoa and pro-
tostomes encode a single TSP in their genomes, and deuterostomes have multiple
TSP genes. The mammal thrombospondins have many complex tissue-specific
roles, including activities in wound healing and angiogenesis, vessel wall biology,
connective tissue organization, and synaptogenesis. These activities derive mech-
anistically from interactions with cell surfaces, growth factors, cytokines, or com-
ponents of the extracellular matrix that collectively regulate many aspects of cell
phenotype. Emerging evidence on the functions of TSPs in invertebrates suggests
that ancient functions include bridging activities in cell–cell and cell–ECM inter-
actions. Knowledge of TSP domain structures provides a rational basis for under-
standing their roles in vivo and associations with human disease and is assisting
ongoing translational applications [2].

Cells

Cells can remodel and reshape the extracellular matrix by degrading and
reassembling it, thus playing an active role in sculpting their surrounding envi-
ronment and directing their own phenotypes. The dynamic reciprocal communi-
cation between cells and the ECM plays a fundamental role in tissue development,
homeostasis, and wound healing [18].
Cells, in order to change shape during division and migration, must modify their
pericellular environment by first degrading the existing matrix and replacing it with
new components. Hyaluronan and versican drive the changes leading to expansion
of the pericellular matrix and to modifications in the mechanical properties of the
ECM that influence cell phenotype [129].
Most matrix components are produced by fibroblasts that also play a role in their
assembly into fibers and their spatial disposition. Collagen cross-linking is almost
exclusively mediated by an enzyme, the lysyl oxidase (LOX) [134]. The synthesis
4 Extracellular Matrix: Immunity and Inflammation 91

and cross-linking of ECM fibers is balanced by the action of matrix metallopro-

teinases that degrade collagen and other ECM proteins [56].
Fibroblasts are the most common cell type of the connective tissues found
throughout the body and the main source of the extensive extracellular matrix
characteristic of the tissues. They are also central mediators of pericellular matrix
pathological fibrotic accumulation and of cellular proliferation and differentiation
which occur in response to prolonged tissue injury and chronic inflammation [55].

Inflammation

Inflammation is triggered when innate immune cells detect infection or tissue

injury. Surveillance mechanisms involve pattern recognition receptors (PRRs) on
the cell surface and in the cytoplasm. Induction of genes encoding enzymes,
chemokines, cytokines, adhesion molecules, and regulators of the extracellular
matrix promotes the recruitment and activation of leukocytes, which are critical for
eliminating foreign particles and host debris [85].
Inflammation is a complex defense mechanism characterized by leukocyte
migration from the vasculature to control tissue damage induced by pathogenic
(bacterial or viral), traumatic, or toxic injury [132] with subsequent deposition of
extracellular matrix resulting in tissue repair. The inflammatory process is generally
categorized into an acute, rapid response, and a persistent but slowly evolving
chronic condition, which may progress into inflammatory diseases. An excessive
deposition of ECM leads to overgrowth, hardening, and/or scarring of tissues,
defined as fibrosis [100].
Fibrosis is the excessive deposition of extracellular matrix proteins into tissues
leading to scar formation, disruption of normal tissue architecture, and organ fail-
ure. Despite the large clinical impact of fibrosis, treatment options are limited.
Adhesion molecules, in particular avb6 and a3b1 integrins and cadherin-11, have
been demonstrated to be important mediators of tissue fibrosis. Fibrosis is the final
common pathway of a tissue response to injury, including chemical exposures,
infections, and autoimmunity [3].
Inflammation is frequently accompanied by capillary permeability and induces
endothelial activation, which, when persistent, results in capillary sprouting. The
occurrence of such events is in large part mediated by extracellular matrix proteins
and proteases. ECM turnover by proteases, in fact, allows the invasion of tissue by
specialized endothelial cells and provides specific mechanical forces to expose
cryptic adhesion sites and to release factors involved in vascular morphogenesis.
Moreover, matrix remodeling and vascular regression contribute to the resolution of
inflammation and facilitate tissue repair. In addition, a topic that has recently
became of great interest is the degradome, i.e., the battery of proteases and inhi-
bitors express by a cell type under specific conditions, or the protease substrate
repertoire of a specific cell type. Its characterization in endothelial, mural, and
accessory cells activated in distinct inflammatory contexts promises to identify new
92 A. Cataldi and V. di Giacomo

targets for the inhibition or promotion of angiogenesis. Normalization of aberrant

vasculature, rather than inhibition of its formation, could offer a better prospect for
treating inflammatory diseases characterized by uncontrolled vascularization [6].
Throughout the body, the extracellular matrix provides structure and organiza-
tion to tissues and also helps regulate cell migration and intercellular communi-
cation. In all tissues, inflammation can be induced and propagated by ECM
disruption. Extracellular matrix molecules newly liberated by injury or inflamma-
tion include hyaluronan fragments, tenascins, and sulfated proteoglycans. These act
as “damage-associated molecular patterns” or “alarmins,” i.e., endogenous proteins
that trigger and subsequently amplify inflammation. Activated inflammatory cells,
in turn, further damage the extracellular matrix by releasing degradative enzymes
including matrix metalloproteinases [33].
Fibrotic disorders are multistage progressive processes that often arise from
different causes and are commonly associated with chronic inflammation. Excessive
deposition of extracellular matrix is the hallmark of many fibrotic diseases. This
may be due to an excess of fibroblast recruitment and activation, as well as to their
differentiation in myofibroblasts. These events may be triggered by cytokines,
chemokines, and growth factors released by lymphocytes or macrophages. The
excessive production of extracellular matrix is apparently due to alterations of
metabolic pathways in activated fibroblasts. It has been suggested that a defective
autophagy, an important subcellular process with multiple homeostatic roles, also
recognized as a key component of both innate and acquired immunity, could play a
role [21].
Normally, the amount of ECM in the tissue is controlled through the balance
between its production and degradation. A key role in this balance is played by
matrix metalloproteinases.
The ability of MMPs to modify the structural integrity of tissues is essential for
certain aspects of normal physiology, such as cell migration, proliferation, growth,
and development, as well as for the occurrence of several pathological events (heart
remodeling, metastasis, etc.) [73, 113]. Under healthy conditions, their proteolytic
activity is precisely regulated by tissue inhibitor of metalloproteinases (TIMP) [16].
The regulation of the ratio MMPs: TIMPs plays an important role in wound healing,
and the disruption of this ratio can result in pathological processes [5].

Metalloproteinases and Inflammation

Recent observations provide evidence that matrix metalloproteinases modulate

various features of inflammation. They can regulate the integrity of physical barriers
and the transmigration of leukocytes from vasculature to tissue as well as the
availability and activity of inflammatory mediators, such as cytokines and che-
mokines. Metalloproteinases also generate chemokine gradients in tissue to recruit
inflammatory cells to the site of injury or inflammation and can also regulate
survival of inflammatory cells [14, 91]. Studies with MMP knockout mice have
4 Extracellular Matrix: Immunity and Inflammation 93

elucidated the specific roles of distinct metalloproteinases in different experimental

models involving inflammation. Interestingly, the only inflammatory phenotype
without challenge is detected in MT1-MMP-deficient mice, which spontaneously
develop arthritis [43]. Among challenges with different experimental models,
MMP-9 has been shown to play a role in mouse models of myocardial aging and
infarction, cerebral ischemia, asthma, and multiple sclerosis (experimental
autoimmune encephalomyelitis (EAE)) [7, 24, 25, 109, 115]. In addition, elevated
levels of MMP-2, -7, and -8 have been reported in mice with EAE [36, 58, 87]. The
mechanistic role of MMP-12 in a mouse model of emphysema has been elucidated,
indicating that defective activation of latent TGF-b regulates the expression of
MMP-12 in macrophages [81]. MMP-8 has been shown to regulate inflammation in
skin, and MMP-9 may also function as an anti-inflammatory mediator in skin
inflammation and in glomerulonephritis [65, 122]. The role of MMP-8 in the
regulation of inflammation appears complex. Recruitment of neutrophils is
impaired in chemically induced epidermal carcinomas in MMP-8 knockout mice, as
compared to wild-type mice [10]. However, in MMP-8-null mice, neutrophil
recruitment into alveolar space after lipopolysaccharide stimulation in mouse model
of acute lung injury is increased, suggesting an anti-inflammatory potential of
MMP-8 [89]. MMP-3 and -9 have also been shown to play an important role in
cutaneous inflammation in the mouse model for contact hypersensitivity.
Specifically, MMP-3 appears to be required for cutaneous inflammation while
MMP-9 may function as an anti-inflammatory mediator in skin. Expression of
MMP-9 and -13 is potently upregulated in murine antigen-induced arthritis model,
and the expression pattern correlates with the course of synovial inflammation [52].
Furthermore, expression of MMP-13 in mouse synovial tissue induces the onset
of inflammation characterized by increased cytokine and chemokine production and
inflammatory cell influx [53]. Additional evidence for the pro-inflammatory role of
MMP-13 was provided by a recent study, showing that activation of TNF-a by
MMP-13 plays an important role in the mouse model of inflammatory bowel dis-
ease, suggesting also a possible mechanistic role for MMP-13 in pathogenesis of
intestinal ulcerations [112].
The discovery of the disintegrin and metalloproteinase 17 (ADAM17), originally
identified as tumor necrosis factor-a converting enzyme (TACE) for its ability as
sheddase of TNF-a, inspired scientists to attempt to elucidate the molecular mech-
anisms underlying ADAM17 implication in diseased conditions. In recent years, it
has become evident that this protease can modify many non-matrix substrates, such
as cytokines (e.g., TNF-a), cytokine receptors (e.g., IL-6R and TNF-R), ligands of
ErbB (e.g., TGF-a and amphiregulin), and adhesion proteins (e.g., L-selectin and
ICAM-1). Several recent studies have described experimental model system to better
understand the role of specific signaling molecules, the interplay of different signals,
and tissue interactions in regulating ADAM17-dependent cleavage of most relevant
substrates in inflammatory diseases [70].
Recent literature suggests that ADAM-17 not only is the main protease
responsible for the release of TNF-a during the inflammatory response, but it also
94 A. Cataldi and V. di Giacomo

appears to be the most important sheddase in terms of the range of its targets. The
inflammatory condition is characterized by an increased accumulation of leukocytes
in tissue mediated by the transmembrane protein selectins. ADAM-17 also has a
role in the shedding of L-selectin, a cellular adhesion molecule, from macrophages,
and this process is promoted by leukocyte attachment to endothelial or E-selectins.
Thus, ADAM-17 was shown to shed several factors contributing to successful
recruitment of leukocytes to the inflammation site. Interestingly, there is some
evidence that ADAM17 controls not only pro- but also anti-inflammatory signals.
The enzyme was reported to down-regulate the macrophages activation cleaving
colony-stimulating factor-1 (CSF-1) from their surface [123]. In addition, a corre-
lation was reported between ADAM-17 expression and IL-15 receptor a activation,
which is a soluble form of the receptor implied in the collagen-induced arthritis and
cardiac allograph rejection. Also, different stimuli activate ADAM17-mediated IL-6
receptor-a cleavage, contributing to the decline of neutrophil infiltration and to
promotion of monocyte recruitment, essential for resolution of the inflammation.
Among the tissue inhibitors of matrix metalloproteinases, TIMP-3 is the only one
that binds to the extracellular matrix and contains an amino acid sequence
(PFG) required to inhibit ADAM-17 [66]. TIMP-3 is induced by molecules
involved in inflammation, such as the pro-inflammatory agent PMA and the
anti-inflammatory cytokine TGF-b, and has the potential to impact many different
branches of haematopoiesis, immunity, and inflammation, primarily as a regulator
of ADAM-17, but also as an inhibitor of other proteases which target growth
factors, cytokines, and adhesion molecules [70].

Versican and Inflammation

An emerging body of evidence indicates that secreted proteoglycans act as sig-

naling molecules, in addition to their canonical function in maintaining and regu-
lating the architecture of various extracellular matrices. Proteoglycans interact with
a number of receptors that regulate growth, motility and immune response. In part,
as a consequence of their complex structure, proteoglycans can induce cross talk
among various families of receptors and can also interact with natural receptor
ligands, often blocking and sequestering their bioactivity. In their soluble form,
originating from either partial proteolytic processing or through de novo synthesis
by activated cells, some proteoglycans can become potent danger signals, denoting
tissue stress and injury. Recently, it has been shown that proteoglycans, especially
those belonging to the small leucine-rich and hyaluronan-binding gene families as
well as the glycosaminoglycan hyaluronan, act as endogenous ligands of the
toll-like receptors, a group of central receptors regulating innate immunity.
Furthermore, proteoglycans can activate intracellular inflammasomes and trigger
sterile inflammation [31].
A number of studies have identified upregulation of versican gene in monocytes
in a number of pro-inflammatory states [34, 127, 129].
4 Extracellular Matrix: Immunity and Inflammation 95

Based on its structural complexity, versican is capable of interacting with other

ECM components, cell surface proteins (e.g., receptors), proteases, chemokines,
and growth factors (Fig. 4.1). Thus, several different mechanisms of controlling
inflammation by this proteoglycan have been described [126]. Versican has been
suggested to act as a ligand to the toll-like receptors 2/6 heterodimer and to its
adaptor CD14, thereby activating tumor-infiltrating myeloid cells to elicit the
production of pro-inflammatory cytokines such as TNF-a and IL-6, and promoting
tumor metastasis [124]. It is of note that in various inflammatory diseases and in
response to lipopolysaccharide, activated macrophages synthesize and secrete
versican [126]. Under similar conditions, de novo synthesis of biglycan and decorin
by macrophages also occurs. Thus, it is conceivable that macrophages, upon
stimulation with pro-inflammatory factors, start to synthesize several proteoglycans
that act as endogenous TLR ligands and promote the inflammatory response in an
autocrine and paracrine manner. The process is reminiscent of the very late acti-
vation antigen in B cells, where immune cells synthesize specific integrins de novo,
facilitating their binding to extracellular matrix constituents during inflammation
[45].
Furthermore, there is evidence that versican might be involved in M1/M2
macrophage polarization, thereby regulating the phenotype of macrophages. The
binding of versican to hyaluronan changes the structure and functional properties of
the matrix, which becomes capable of promoting leukocytes adhesion and retention
and of stabilizing CD44 signaling [31].

Hyaluronan and Inflammation

Hyaluronan, a major component of the extracellular matrix, together with its

receptors and binding partners, is a “keystone molecule” in the inflammatory
milieu. HA and its binding proteins regulate the expression of inflammatory genes,
the recruitment of inflammatory cells, the release of inflammatory cytokines, and
can modulate the course of inflammation. The ability of HA to function as either a
pro- or anti-inflammatory molecule is dependent upon its size, microenvironment,
localization, and availability of specific binding partners [93].
Accumulation and turnover of extracellular matrix components are the hallmarks
of tissue injury. Fragmented hyaluronan stimulates the expression of inflammatory
genes by a variety of immune cells at the injury site. Hyaluronan fragments signal
through both toll-like receptor (TLR) 4 and TLR-2 as well as CD44 to stimulate
inflammatory genes in inflammatory cells. Hyaluronan binds to a number of cell
surface proteins on various cell types, and it is also present on the cell surface of
epithelial cells to provide protection against tissue damage from the environment by
interacting with TLR-2 and TLR-4. Hyaluronan and hyaluronan-binding proteins
regulate inflammation, tissue injury, and repair through regulating inflammatory
cell recruitment, release of inflammatory cytokines, and cell migration [51].
96 A. Cataldi and V. di Giacomo

HA plays a key role in regulating inflammation, being at the center of a complex

network of ECM molecules that together exert decisive effects on the nature of
inflammation. Hyaluronan has several unique features that qualify it as a keystone
molecule in the inflammatory response:
(I) First, HA is known to play important roles in most aspects of the tissue
response to injury, including wound healing, angiogenesis, cell trafficking,
and proliferation [50].
(II) Second, HA physically interacts with a large and diverse network of ECM
molecules present in inflamed tissues. These include HA-binding molecules
called hyaladherins such as tumor necrosis factor-stimulated gene (TSG-6),
versican, inter-a-inhibitor (I a I), CD38, and others [19]. HA-binding
interactions with its receptor CD44 are also impacted by other extracellular
matrix molecules, including osteopontin and fibronectin.
(III) Third, hyaluronan is highly abundant in inflamed tissue, and its production
can increase as much as 200-fold following injury [27].
(IV) Fourth, fluid shifts caused by HA are responsible for many of the physiologic
changes associated with inflammation. Because of the repeating disaccha-
rides of N-acetyl glucosamine and glucuronic acid that make up hyaluronan
carry strong negative charges, its synthesis generates oncotic forces that
result in edema, vascular permeability changes, and leukocyte egress at sites
of injury [63]. Studies utilizing magnetic resonance imaging (MRI) to detect
edema associated with islet inflammation [22] may well be tracking the
effects of HA.
(V) Fifth, the length of hyaluronan strands in the ECM is a sensitive barometer of
the inflammatory milieu. Uninjured tissues are characterized by modest
amounts of high molecular weight HA (HMW-HA, > 1000 kDa). Following
tissue injury, there is an accumulation of low molecular weight HA
(LMW-HA, < 250 kDa) and short HA oligomers (sHA, < 30 kDa) gener-
ated through enzymatic degradation of HMW-HA by endogenous hyalur-
onidases as well as catabolism by a diverse group of microbial
hyaluronidases, mechanical forces, and oxidative stress [104]. Normally,
LMW-HA is cleared within 14 days of injury, and enhanced hyaluronan
production ceases as collagen production increases in conjunction with
wound healing. However, chronic wounds are characterized by large
amounts of LMW-HA. Both the size and the amount of HA in the tissue
environment are therefore tightly linked to the stage of an injury response
and to its resolution [51, 103].

Thrombospondins and Inflammation

Inflammation is a defensive process against tissue injury. Once this self-protective

strategy is initiated, an effective resolution of the process is crucial to avoid major
4 Extracellular Matrix: Immunity and Inflammation 97

and unnecessary tissue damage. If the underlying event inducing inflammation is

not addressed and homeostasis is not restored, this process can become chronic and
lead to angiogenesis and carcinogenesis. Thrombospondin-1 (TSP-1) is a matri-
cellular protein involved in angiogenesis, cancer, and inflammation [60]. The
effects of TSP-1 have been studied in many preclinical tumor models, and mimetic
peptides are being tested in cancer clinical trials. However, the molecular mecha-
nisms explaining its role in inflammatory processes are not well understood.
TSP-1 is transiently released early during the acute phase of inflammation, and
multiple factors seem to modulate the release of TSP-1 during this process. This
protein is strongly expressed in neutrophils, inducing an intense chemotactic
response to injured tissues. TSP-1 is secreted in response to inflammation, pro-
moting the resolution of the inflammatory process and facilitating phagocytosis of
damaged cells [37]. Thus, enhanced production of TSP-1 could be a compensatory
mechanism for controlling the immune response and protecting tissues from
excessive damage.
TSP-1 mediates macrophage phagocytosis of apoptotic cells via CD36. This
receptor is coexpressed with TSP-1 in macrophages and endothelial cells, and its
binding to thrombospondin-1 induces apoptosis in endothelial cells. By activating
CD36, TSP-1 also controls blood flow and leukocyte infiltration modulating the
action of the nitric oxide (NO) pathway in injured tissues (Fig. 4.2). NO is a gas
produced when L-arginine is converted to L-citrulline by the enzyme nitric oxide
synthase (NOS). There are four different isoforms of NOS, neuronal (nNOS),
endothelial (eNOS), mitochondrial (mtNOS), and the inducible isoform (iNOS).
The first two are secreted during normal physiological events, but only iNOS is
expressed upon inflammatory stimuli. The effects of nitric oxide in inflammation
have been extensively recognized in a variety of studies. NO can modulate
leukocyte adhesion in a dose-dependent manner [48]. At low doses, nitric oxide is
anti-inflammatory and anti-angiogenic but, after inflammatory stimuli, high levels
of NO are secreted promoting angiogenesis and leukocyte adhesion to the
endothelium. TSP-1 could inhibit the soluble guanylyl cyclase system in
endothelial cells, and consequently the activation of NO by interacting with CD36
and CD47. Through this mechanism, TSP-1 inhibits inflammation by blocking
adhesion and activation of leukocytes to the endothelium and by diminishing
angiogenesis [47] (Fig. 4.2).
Another factor interacting with TSP-1 during early inflammation is the peroxi-
some proliferator-activated receptor (PPAR), a member of the nuclear hormone
receptor superfamily of transcription factors. When PPAR is absent in leukocytes,
the leukocytes secrete high levels of TSP-1. PPAR-c, one of the isoforms of this
receptor, greatly enhances the proapoptotic effects of the TSP-1-derived peptide
ABT510. This peptide corresponds to the second type 1 repeat (TSR) of TSP-1 and
induces vascular apoptosis in vitro and in vivo through its interaction with CD36.
By using a PPAR-c agonist, the expression of CD36 in endothelial cells is
enhanced, improving the anti-angiogenic effects of ABT510 in a CD36-dependent
manner [44]. The TSP-1 receptor CD47 is critical for the migration of leukocytes
through endothelial and epithelial barriers. CD47 is strongly expressed in
98 A. Cataldi and V. di Giacomo

Phagocytosis
TSP-1 CD47

Leukocytes
NO Leukocytes infiltration
apoptosis

Angiogenesis
ABT
CD36
510

PPAR γ
Endothelial cell sapoptosis

Fig. 4.2 TSP-1 control of inflammation. NO nitric oxide

polymorphonuclear cells, and its activation enhances the expression of TSP-1 in

leukocytes. TSP-1 also induces leukocytic apoptosis through the CD47 pathway.
CD47 can directly cause apoptosis through mitochondrial mechanisms, or by
activation of the Fas/CD95 pathway. Expression of CD47 in apoptotic granulocytes
can influence the phagocytic functions of the macrophages in inflammatory sites
suggesting a critical role of this factor in the resolution of the process [72]
(Fig. 4.2).

Immunity

At sites of injury, phagocytic cells, namely macrophages and neutrophils, provide

innate cell-mediated immunity and initiate the inflammatory response.
Macrophages secrete cytokines that attract neutrophils to leave the blood stream and
enter the injured area. The arrival of neutrophils initiates the inflammatory response,
by which cells and molecules of innate immunity are recruited into sites of
wounding or infection [100].
Immune cells other than T cells are also influenced in their migration by the
topography and composition of the matrix architecture. For instance, studies per-
formed in 3D collagen matrices have demonstrated that macrophages can adopt
distinct migratory mechanisms according to the extracellular environment. In a
recent study, it was argued that the ability of T cells to mount an anti-tumor
response is dependent on the matrix structure, more precisely on the balance
between pro-migratory reticular fiber networks and unfavorable migration zones
composed of dense and aligned ECM structures. Thus, the matrix architecture, that
has long been considered to merely provide the structural framework of connective
4 Extracellular Matrix: Immunity and Inflammation 99

tissues, can play a key role in facilitating or suppressing the anti-tumor immune
surveillance. It is now well accepted that a deregulated ECM favours tumor pro-
gression and metastasis. Recent progress in imaging technologies has also high-
lighted the impact of the matrix architecture found in solid tumor on immune cells
and especially T cells. Besides the effect exerted on tumor cells, the physical and
biochemical properties of the ECM are also able to modulate a number of processes
in immune cells, especially lymphocytes, that can ultimately lead to inefficient
tumor killing. Extracellular matrix determinants can indeed promote tumor evasion
from the immune system, both by inhibiting the anti-tumor effector activity of T
cells, either directly or through the recruitment of immunosuppressive cells, and by
limiting cell contact with tumor cells [92].
Along with collagen and fibronectin, some non-structural ECM proteins are
often upregulated during tumor development and can modulate cell–cell and cell–
ECM interactions, eventually restraining T cell activation [15, 98]. For instance,
tenascin C seems to directly inhibit T cell proliferation and IFN-c production in
lung cancer [90] while the glycoprotein SPARC seems to influence the trafficking
and the function of immune cells, as SPARC-knockout mice display an increased
number of macrophages and neutrophils in tumors and higher cytotoxicity in
polymorphonuclear cells [4, 99]. In addition, several ECM fragments are able to
recruit and activate macrophages and neutrophils to sites of inflammation, where
they could regulate inflammation and adaptive responses [1, 101].
The innate immune system down-regulates effector mechanisms and restores
homoeostasis in injured tissue via cytokines from the IL-10 and TGF (transforming
growth factor) families mainly released from macrophages, preferentially the M2
subset, which have a capacity to induce regulatory T cells, inhibit the production of
pro-inflammatory cytokines, and induce healing of the tissue by regulating extra-
cellular matrix protein deposition and angiogenesis [106]. Proteoglycans are also
synthesized by leukocytes and may play a role in the inflammatory response [110].

Metalloproteinases and Immunity

Matrix metalloproteinases are members of the metzincin group of proteases which

share the conserved zinc-binding motif in their catalytic active site. It was originally
thought that their main function is to degrade the various components of the ex-
tracellular matrix, yet recent studies have led us to appreciate their significance as
regulators of extracellular tissue signaling networks. Due to the broad spectrum of
their substrate specificity, MMPs contribute to the homeostasis of many tissues and
participate in several physiological processes, such as bone remodeling, angio-
genesis, immunity, and wound healing. Metalloproteinase activity is tightly con-
trolled at the level of transcription, pro-peptide activation, and inhibition by tissue
inhibitors of MMPs.
Dysregulation of the activity of these proteins leads to pathological conditions
such as arthritis, inflammation, and cancer, thus highlighting MMPs as promising
100 A. Cataldi and V. di Giacomo

therapeutic targets. Analysis of MMP mutant mice has proved to be an essential

tool for the identification of novel functions and interactions of single matrix
metalloproteinase members. Advancing our understanding of the MMP contribu-
tion to tissue homeostasis will lead us to identify causal relationships between their
dysregulation and the development of disease pathologies, thus guiding us to
successful MMP-directed therapies [71].
Over the past 50 years, steady growth in the field of metalloproteinase biology
has shown that the degradation of extracellular matrix components represents only a
fraction of the functions performed by these enzymes and has highlighted their
fundamental roles in immunity. Metalloproteinases regulate aspects of immune cell
development, effector function, migration, and ligand–receptor interactions. They
carry out ectodomain shedding of cytokines and their cognate receptors. Together
with their endogenous inhibitors TIMPs, these enzymes regulate signaling down-
stream of the tumor necrosis factor receptor and the interleukin-6 receptor, as well
as that downstream of the epidermal growth factor receptor and Notch, which are all
pertinent for inflammatory responses [57].
Metalloproteinases and their inhibitors affect immunity as early as the first stages
of haematopoietic cell development. In adult tissues, specific deficiencies of Mmp,
Adam, or Timp genes in mice culminate in a range of spontaneous or induced
inflammatory phenotypes.
Efficient neutrophil migration along chemotactic gradients and extravasation
through blood vessels and tissues to sites of infection is important for establishing
effective immunity, and metalloproteinases have been shown to contribute to these
functions through the modification of chemotactic agents. Chemokine processing
by MMPs was simultaneously discovered by two groups; the first reported that
MMP-9 processes an amino-terminal fragment of IL-8 (functionally similar to
CXCL6 in mice) to increase its potency by a factor of ten; and the second showed
that MMP-2 inactivates monocyte chemotactic protein 3 (MCP3; also known as
CCL7), which is a mononuclear cell-attracting chemokine 7. Mmp7 −/− mice are
protected from acute lung injury, and histological analysis linked this protection to a
decrease in pulmonary neutrophil infiltration. MMP-7-mediated cleavage releases
the heparan sulfate proteoglycan syndecan 1, along with its associated
CXC-chemokine ligand 3 (CXCL3; also known as KC), which attracts neutrophils
to the site of infection. Thus, the absence of MMP-7 compromises the CXCL3
chemokine gradient [69]. Similarly, neutrophils failed to migrate to sites of LPS
administration in Mmp8 −/− mice, as MMP-8 is required for the activation of the
neutrophil-recruiting chemokine CXCL6.
Further metalloproteinase-dependent events that contribute to neutrophil
chemotaxis and function have been recently described using proteomics, genetic
mouse models, or human primary cell cultures; for example, using a
double-knockout mouse model, MMP-2 and MMP-9 were shown to function
together to cleave CXCL6 and to increase neutrophil migration to the peritoneum
during IL-1b-induced peritonitis. As part of a positive feedback loop, meprins can
cleave pro-MMP-9 to sensitize it to activation by MMP-3 (also known as stro-
melysin 1); an identical N-terminal MMP-9 cleavage product has been found in the
4 Extracellular Matrix: Immunity and Inflammation 101

conditioned media of neutrophil and monocyte cultures, which suggests that

meprins are involved in MMP-9 activation during an immune response [57].
Consistent with the function of TIMPs as metalloproteinase inhibitors, neu-
trophils migrated more rapidly to the site of infection in Timp1 −/− mice, and
TIMP-1 deficiency amplified the immune response in a mouse model of acute lung
injury. Moreover, neutrophil inflammation was sustained in the absence of TIMP-3
in a mouse model of bleomycin-induced lung injury, which indicates that TIMP-3
suppresses neutrophil entry and/or promotes the resolution of inflammation [59].
Once they are proximal to the site of infection, neutrophils extravasate through
blood vessels and tissues by making a series of molecular contacts with the vascular
endothelium. L-selectin (also known as CD62L) is expressed by neutrophils and
participates in their adhesion to vascular endothelial cells. The final step in the
neutrophil response is the elimination of the foreign invaders, mainly through
phagocytosis, the generation of reactive oxygen species (ROS) and the release of
microbicidal substances. As mentioned above, MMP-9 is the major component of
gelatinase granules that are secreted from neutrophils following stimulation with
IL-8. As MMP-9 cleaves the N-terminal fragment of IL-8 to increase its activity, a
positive feedback loop for effective neutrophil degranulation and pathogen clear-
ance seems to be established at the site of infection. In certain pathological settings,
this neutrophil-derived metalloproteinase activity has deleterious effects. Chronic
obstructive pulmonary disorder (COPD) is characterized by prominent neutrophilic
inflammation, and the associated metalloproteinase release is refractory to gluco-
corticosteroid treatment [57].
Neutrophils normally undergo apoptosis and clearance by phagocytosis, which
leads to the resolution of acute inflammation. Recent studies highlight novel
functions for metalloproteinases in these processes. Peritoneal neutrophils from
Adamts12 −/− mice showed a reduction in annexin-V staining, which indicates that
this protease is associated with neutrophil apoptosis [80]. These examples reiterate
the importance of metalloproteinase function in neutrophil-mediated immune
responses.

Versican and Immunity

Versican has been suggested to contribute to hyaluronan fragment activation of

macrophages, and enhanced cancer metastasis through induction of the hyalur-
onidases. Several inflammation-associated cytokines, including transforming
growth factor-b1, -2, -3, and platelet-derived growth factor (PDGF), have been
shown to increase biosynthetic levels of both versican and HA, while IL-1b, and
IFN-c have been shown to reduce levels of versican [11]. Leukocyte trafficking and
localization to regions of inflammation mediated by interaction with cell-adhesion
receptors functions as a critical initiating step in the inflammatory cascade. Specific
chondroitin sulfate (CS) chains on versican preferentially bind to chemokines
known to attract mononuclear leukocytes. Versican itself is capable of binding to a
102 A. Cataldi and V. di Giacomo

number of cell surface receptors present on leukocytes through interaction also

mediated by CS chains, including both L- and P-selectins and CD44 [42]. Direct
binding of P-selectin glycoprotein ligand-1 (PSGL-1) by the G3 domain of versican
has also been shown to cause aggregation of leukocytes. Together, these
HA-binding proteins contribute to the maintenance of tissue integrity and to direct
cell–ECM interactions in normal and pathological conditions. Many of the adhesive
properties of hyaluronan polymers depend upon the presence of HA-binding pro-
teins, and together with IaI, HCs, TSG-6, and versican contribute to a dynamic
extracellular environment capable of directing cell adhesion and the production of
inflammatory cytokines [93].

Hyaluronan and Immunity

Within the last year, the role of hyaluronan as an endogenous activator of innate
alloimmunity has been investigated [67]. Recent studies have described that the
hyaluronan receptor, CD44, modulates these innate immune responses by aug-
menting regulatory T cell function, and the expression of negative regulators of
toll-like receptor signaling. Hyaluronan activates innate alloimmune responses and
subsequently influences adaptive alloimmunity.
In regulatory T cells, HMW-HA stimulates STAT5 signaling through CD44
cross-linking, promoting their maintenance and thereby inhibiting their prolifera-
tion. Conventional T cell precursors stimulated with HMW-HA produce IL-10, and
infusion of these cells attenuates the disease course in a murine model of colitis
[12].
CD44 is a type I transmembrane glycoprotein and is widely regarded as the
major cell surface HA-binding protein. Widely studied in several contexts, CD44
interactions with HA have important roles in tumor metastasis, lymphocyte adhe-
sion, T cell signaling, angiogenesis, and inflammation. CD44 is expressed on many
cell types that contribute to inflammation including leukocytes, neutrophils, mac-
rophages, chondrocytes, fibroblasts, epithelial, and endothelial cells. While the
glycosaminoglycans (GAGs) of CD44 can bind to cytokines, growth factors, and
extracellular matrix proteins such as fibronectin, the majority of the functions of
CD44 depend upon its ability to bind to hyaluronan [93].
Toll-like receptors function as surveillance receptors, interacting with a number
of microbial-derived molecules and activating the innate immune system in
response to pathogen-associated molecular patterns. Increasingly, the TLRs are also
shown to sense damage-associated molecular patterns in response to injury as well.
The idea that endogenous matrix degradation products act as regulators of cellular
processes is not a new one, but with respect to GAG fragments, the role of HA is
the best studied. Hyaluronan is a component of the cellular coat on some pathogens
and many of them also express hyaluronidases. The presence of a HA coat likely
assists in evasion by the immune system, while the hyaluronidase enzymes may aid
in colonization of the host [76].
4 Extracellular Matrix: Immunity and Inflammation 103

Thrombospondins and Immunity

During the early stages of injury and inflammation, high levels of

thrombospondin-1 increase the tolerance of dendritic cells (DCs) to antigens,
ending the inflammatory response. TSP-1 can modulate inflammation by inhibiting
or enhancing the secretion of the cytokine interleukin 10; by this way, TSP-1 can
also regulate the functions of DCs. Thrombospondin-1 has been recently shown to
induce regulatory T cells and impair dendritic cell maturation in a melanoma model
[61]. In addition, after adding IL-6, IL-10, or TGF-b1 to cultured DCs, they become
immune tolerant and show upregulation of intracellular TSP-1. This protein also
inhibits the function of antigen-presenting cells by suppressing their capacity to
sensitize T cells in the host. CD47 has also a crucial role in T cell activation.
Interaction of TSP-1 with CD47 promotes the activation of thymus-derived
CD4+ CD25+ T regulatory cells (Tregs). Through this mechanism, CD47 helps to
maintain self-tolerance inducing a suppressive phenotype [37].
It has been recently reported that bacterial pathogenesis may be mediated by
CD47. Suppression of CD47 or TSP-1 expression in dendritic cells by using small
interfering RNA (siRNA) technique actually protects newborn mice against bac-
terial (Escherichia coli) meningitis. Again, the loss of CD47 activity prevents the
maturation of the DCs and the production of inflammatory cytokines [79]. In
conclusion, CD47 seems to have pivotal functions in inflammation and immunity
and provides a major mechanistic pathway for the functions of TSP-1 in that
process.
Finally, the deficiency of CD36 enhances the severity of bacterial and malaria
infection. Cd36 −/− mice exhibit an impaired early pro-inflammatory response to
infection, elevation of cytokines, and higher mortality. These findings suggest that
CD36 is quite critical for the recognition and clearance of pathogens in acute and
chronic infections. By binding to this receptor, TSP-1 could modulate the inflam-
matory process by activating macrophages and favoring phagocytosis. During
chronic inflammation, these adaptive immune mechanisms provide defense against
diseases and are regulated by cellular interactions and cytokines. B lymphocytes
secrete antibodies that bind to infectious agents and label them for destruction or
elimination. Once inside a cell, a pathogen becomes inaccessible to those antibodies
and cytotoxic T cells could destroy them by inducing apoptosis of the cell host.
Regulatory T cells can modulate the secretion of cytokines enhancing the functions
of macrophages and B lymphocytes. Thrombospondin-1 has been reported to
decrease immune responses by inhibition of T cell effectors, or by directly inducing
T cell apoptosis. In addition, by binding to a4b1 integrin, TSP-1 promotes T cell
adhesion and chemotaxis [72].
104 A. Cataldi and V. di Giacomo

References

1. Adair-Kirk TL, Senior RM. Fragments of extracellular matrix as mediators of inflammation.

Int J Biochem Cell Biol. 2008;40:1101–10.
2. Adams JC, Lawler J. The thrombospondins. Cold Spring Harb Perspect Biol. 2011;3(10):
a009712.
3. Agarwal SK. Integrins and cadherins as therapeutic targets in fibrosis. Front Pharmacol.
2014;5:131.
4. Alvarez MJ, Prada F, Salvatierra E, et al. Secreted protein acidic and rich in cysteine
produced by human melanoma cells modulates polymorphonuclear leukocyte recruitment
and antitumor cytotoxic capacity. Cancer Res. 2005;65:5123–32.
5. Amălinei C, Căruntu ID, Giuşcă SE, et al. Matrix metalloproteinases involvement in
pathologic conditions. Rom J Morphol Embryol. 2010;51:215–28.
6. Arroyo AG, Iruela-Arispe ML. Extracellular matrix, inhflammation, and the angiogenic
response. Cardiovasc Res. 2010;86:226–35.
7. Asahi M, Wang X, Mori T, et al. Effects of matrix metalloproteinase-9 gene knock-out on
the proteolysis of blood–brain barrier and white matter components after cerebral ischemia.
J Neurosci. 2001;21:7724–32.
8. Aspberg A, Binkert C, Ruoslahti E. The versican C-type lectin domain recognizes the
adhesion protein tenascin-R. Proc Natl Acad Sci U S A. 1995;92:10590–4.
9. Aspberg A, Adam S, Kostka G, et al. Fibulin-1 is a ligand for the C-type lectin domains of
aggrecan and versican. J Biol Chem. 1999;274:20444–9.
10. Balbín M, Fueyo A, Tester AM, et al. Loss of collagenase-2 confers increased skin tumor
susceptibility to male mice. Nat Genet. 2003;35:252–7.
11. Berdiaki A, Zafiropoulos A, Fthenou E, et al. Regulation of hyaluronan and versican
deposition by growth factors in fibrosarcoma cell lines. Biochim Biophys Acta.
2008;1780:194–202.
12. Bollyky PL, Wu RP, Falk BA, et al. ECM components guide IL-10 producing regulatory
T-cell (TR1) induction from effector memory T-cell precursors. Proc Natl Acad Sci U S A.
2011;108:7938–43.
13. Burrage PS, Mix KS, Brinckerhoff CE. Matrix metalloproteinases: role in arthritis. Front
Biosci. 2006;11:529–43.
14. Butler GS, Overall CM. Matrix metalloproteinase processing of signaling molecules to
regulate inflammation. Periodontol. 2000;63:123–48.
15. Chong HC, Tan CK, Huang RL, et al. Matricellular proteins: a sticky affair with cancers.
J Oncol. 2012;2012:351089.
16. Clark IM, Swingler TE, Sampieri CL, et al. The regulation of matrix metalloproteinases and
their inhibitors. Int J Biochem Cell Biol. 2008;40:1362–78.
17. Clark RAF, Henson PM. The molecular and cellular biology of wound repair. New York:
Plenum Press; 1988.
18. Clause KC, Barker TH. Extracellular matrix signaling in morphogenesis and repair. Curr
Opin Biotechnol. 2013;24:830–3.
19. Day AJ, Prestwich GD. Hyaluronan-binding proteins: tying up the giant. J Biol Chem.
2002;277:4585–8.
20. Day AJ, Sheehan JK. Hyaluronan: polysaccharide chaos to protein organization. Curr Opin
Struct Biol. 2001;11:617–22.
21. Del Principe D, Lista P, Malorni W, et al. Fibroblast autophagy in fibrotic disorders.
J Pathol. 2013;229:208–20.
22. Denis MC, Mahmood U, Benoist C, et al. Imaging inflammation of the pancreatic islets in
type 1 diabetes. Proc Natl Acad Sci U S A. 2004;101:12634–9.
23. Discher DE, Mooney DJ, Zandstra PW. Growth factors, matrices, and forces combine and
control stem cells. Science. 2009;324:1673–7.
4 Extracellular Matrix: Immunity and Inflammation 105

24. Dubois B, Masure S, Hurtenbach U, et al. Resistance of young gelatinase B-deficient mice to
experimental autoimmune encephalomyelitis and necrotizing tail lesions. J Clin Invest.
1999;104:1507–15.
25. Ducharme A, Frantz S, Aikawa M, et al. Targeted deletion of matrix metalloproteinase-9
attenuates left ventricular enlargement and collagen accumulation after experimental
myocardial infarction. J Clin Invest. 2000;106:55–62.
26. DuFort CC, Paszek MJ, Weaver VM. Balancing forces: architectural control of mechan-
otransduction. Nat Rev Mol Cell Biol. 2011;12:308–19.
27. Edelstam GA, Laurent UB, Lundkvist OE, et al. Concentration and turnover of
intraperitoneal hyaluronan during inflammation. Inflammation. 1992;16:459–69.
28. Egeblad M, Werb Z. New functions for the matrix metalloproteinases in cancer progression.
Nat Rev Cancer. 2002;2:161–74.
29. Evanko SP, Tammi MI, Tammi RH, et al. Hyaluronan-dependent pericellular matrix. Adv
Drug Deliv Rev. 2007;59:1351–65.
30. Frantz C, Stewart KM, Weaver VM. The extracellular matrix at a glance. J Cell Sci.
2010;123:4195–200.
31. Frey H, Schroeder N, Manon-Jensen T, et al. Biological interplay between proteoglycans
and their innate immune receptors in inflammation. FEBS J. 2013;280:2165–79.
32. Gabison EE, Huet E, Baudouin C, et al. Direct epithelial-stromal interaction in corneal
wound healing: role of EMMPRIN/CD147 in MMPs induction and beyond. Prog Retin Eye
Res. 2009;28:19–33.
33. Gaudet AD, Popovich PG. Extracellular matrix regulation of inflammation in the healthy and
injured spinal cord. Exp Neurol. 2014;258:24–34.
34. Gill S, Wight TN, Frevert CW, et al. Proteoglycans: key regulators of pulmonary
inflammation and the innate immune response to lung infection. Anat Rec (Hoboken).
2010;293:968–81.
35. Gomis-Rüth FX. Structural aspects of the metzincin clan of metalloendopeptidases. Mol
Biotechnol. 2003;24:157–202.
36. Graesser D, Mahooti S, Madri JA. Distinct roles for matrix metalloproteinase-2 and a4
integrin in autoimmune T cell extravasation and residency in brain parenchyma during
experimental autoimmune encephalomyelitis. J Neuroimmunol. 2000;109:21–131.
37. Grimbert P, Bouguermouh S, Baba N, et al. Thrombospondin/CD47 interaction: a pathway
to generate regulatory T cells-from human CD4+ CD25− T cells in response to inflammation.
J Immunol. 2006;177:3534–41.
38. Gross J, Lapiere CM. Collagenolytic activity in amphibian tissues: a tissue culture assay.
Proc Natl Acad Sci U S A. 1962;48:1014–22.
39. Hadler-Olsen E, Fadnes B, Sylte I, et al. Regulation of matrix metalloproteinase activity in
health and disease. FEBS J. 2011;278:28–45.
40. Hay ED. Cell biology of extracellular matrix. New York: Plenum Press; 1991.
41. Hernandez-Barrantes S, Bernardo M, Toth M, et al. Regulation of membrane type-matrix
metalloproteinases. Semin Cancer Biol. 2002;12:131–8.
42. Hirose J, Kawashima H, Yoshie O, et al. Versican interacts with chemokines and modulates
cellular responses. J BiolChem. 2001;276:5228–34.
43. Holmbeck K, Bianco P, Caterina J, et al. MT1-MMP-deficient mice develop dwarfism,
osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnover. Cell.
1999;99:81–92.
44. Huang H, Campbell SC, Bedford DF, et al. Peroxisome proliferator-activated receptor c
ligands improve the antitumor efficacy of thrombospondin peptide ABT510. Mol Cancer
Res. 2004;2:541–50.
45. Hynes RO. The extracellular matrix: not just pretty fibrils. Science. 2009;326:1216–9.
46. Hynes RO, Yamada KM. Extracellular matrix biology. Cold Spring Harbor, NY: Cold
Spring Harbor Laboratory Press; 2012.
47. Isenberg JS, Frazier WA, Roberts DD. Thrombospondin-1: a physiological regulator of
nitric oxide signaling. Cell Mol Life Sci. 2008;65:728–42.
106 A. Cataldi and V. di Giacomo

48. Isenberg JS, Martin-Manso G, Maxhimer JB, et al. Regulation of nitric oxide signalling by
thrombospondin 1: implications for anti-angiogenic therapies. Nat Rev Cancer. 2009;9:
182–94.
49. Isogai Z, Aspberg A, Keene DR, et al. Versican interacts with fibrillin-1 and links extracellular
microfibrils to other connective tissue networks. J Biol Chem. 2002;277:4565–72.
50. Jiang D, Liang J, Noble PW. Hyaluronan in tissue injury and repair. Annu Rev Cell Dev
Biol. 2007;23:435–61.
51. Jiang D, Liang J, Noble PW. Hyaluronan as an immune regulator in human diseases. Physiol
Rev. 2011;91:221–64.
52. Joronen KR, Ala-aho ML, Majuri H, et al. Adenovirus mediated intra-articular expression of
collagenase-3 (MMP-13) induces inflammatory arthritis in mice. Ann Rheum Dis.
2004;63:656–64.
53. Joronen K, Kähäri VM, Vuorio E. Temporospatial expression of matrix metalloproteinases
and tissue inhibitors of matrix metalloproteinases in mouse antigen induced arthritis.
Histochem Cell Biol. 2005;124:535–45.
54. Karamanos N. Extracellular matrix: pathobiology and signaling. Berlin, Boston: Walter de
Gruyter; 2012.
55. Kendall RT, Feghali-Bostwick CA. Fibroblasts in fibrosis: novel roles and mediators. Front
Pharmacol. 2014;5:123.
56. Kessenbrock K, Plaks V, Werb Z. Matrix metalloproteinases: regulators of the tumor
microenvironment. Cell. 2010;141:52–67.
57. Khokha R, Murthy A, Weiss A. Metalloproteinases and their natural inhibitors in
inflammation and immunity. Nat Rev Immunol. 2013;13:649–65.
58. Kieseier BC, Kiefer R, Clements JM, et al. Matrix metalloproteinase-9 and -7 are regulated
in experimental autoimmune encephalomyelitis. Brain. 1998;121:159–66.
59. Kim KH, Burkhart K, Chen P, et al. Tissue inhibitor of metalloproteinase–1 deficiency
amplifies acute lung injury in bleomycin-exposed mice. Am J Respir Cell Mol Biol.
2005;33:271–9.
60. Kirk JA, Cingolani OH. Thrombospondins in the transition from myocardial infarction to
heart failure. J Mol Cell Cardiol. 2016;90:102–10.
61. Kudo-Saito C, Shirako H, Takeuchi T, et al. Cancer metastasis is accelerated through
immunosuppression during Snail-induced EMT of cancer cells. Cancer Cell. 2009;15:
195–206.
62. Kuznetsova SA, Issa P, Perruccio EM, et al. Versican-thrombospondin-1 binding in vitro
and colocalization in microfibrils induced by inflammation on vascular smooth muscle cells.
J Cell Sci. 2006;119:4499–509.
63. Laurent TC, Fraser JR. Hyaluronan. FASEB. 1992;J6:2397–404.
64. LeBaron RG, Zimmermann DR, Ruoslahti E. Hyaluronate binding properties of versican.
J Biol Chem. 1992;267:10003–10.
65. Lelongt B, Bengatta S, Delauche M, et al. Matrix metalloproteinase 9 protects mice from
anti-glomerular basement membrane nephritis through its fibrinolytic activity. J Exp Med.
2001;193:793–802.
66. Lee MM, Yoon BJ, Osiewicz K, et al. Tissue inhibitor of metalloproteinase 1 regulates
resistance to infection. Infect Immun. 2005;73:661–5.
67. Lee-Sayer SS, Dong Y, Arif AA, et al. The where, when, how, and why of hyaluronan
binding by immune cells. Front Immunol. 2015;6:150.
68. Li L, Li H. Role of microRNA-mediated MMP regulation in the treatment and diagnosis of
malignant tumors. Cancer Biol Ther. 2013;14:796–805.
69. Li Q, Park PW, Wilson CL, et al. Matrilysin shedding of syndecan-1 regulates chemokine
mobilization and transepithelial efflux of neutrophils in acute lung injury. Cell.
2002;111:635–46.
70. Lisi S, D’Amore M, Sisto M. ADAM17 at the interface between inflammation and
autoimmunity. Immunol Lett. 2014;S0165-2478(14):00178-3.
4 Extracellular Matrix: Immunity and Inflammation 107

71. Löffek S, Schilling O, Franzke CW. Series “matrix metalloproteinases in lung health and
disease”: Biological role of matrix metalloproteinases: a critical balance. Eur
Respir J. 2011;38:191–208.
72. Lopez-Dee Z, Pidcock K, Gutierrez LS. Thrombospondin-1: multiple paths to inflammation.
Mediat Inflamm. 2011;2011:296069.
73. Lu P, Takai K, Weaver VM, et al. Extracellular matrix degradation and remodeling in
development and disease. Cold Spring Harb Perspect Biol. 2011;3(ii):a005058.
74. Lundell A, Olin AI, Morgelin M, et al. Structural basis for interactions between tenascins
and lectican C-type lectin domains: evidence for a crosslinking role for tenascins. Struct
(Camb). 2004;12:1495–506.
75. Macri L, Silverstein D, Clark RA. Growth factor binding to the pericellularmatrix and its
importance in tissue engineering. Adv Drug Deliv Rev. 2007;59:1366–81.
76. Matzinger P. Tolerance, danger, and the extended family. Annu Rev Immunol.
1994;12:991–1045.
77. McDonald JA, Camenisch TD. Hyaluronan: genetic insights into the complex biology of a
simple polysaccharide. Glycoconj J. 2002;1:331–9.
78. Mecham RP. The extracellular matrix: an overview. Berlin: Springer; 2011.
79. Mittal R, Gonzalez-Gomez I, Prasadarao NV. Escherichia coli K1 promotes the ligation of
CD47 with thrombospondin-1 to prevent the maturation of dendritic cells in the pathogenesis
of neonatal meningitis. J Immunol. 2010;185:2998–3006.
80. Moncada-Pazos A, Obaya AJ, Llamazares M, et al. ADAMTS-12 metalloprotease is
necessary for normal inflammatory response. J Biol Chem. 2012;287:39554–63.
81. Morris DG, Huang X, Kaminski N, et al. Loss of integrin avb6-mediated TGF-b activation
causes MMP12-dependent emphysema. Nature. 2003;422:169–73.
82. Morwood SR, Nicholson LB. Modulation of the immune response by extracellular matrix
proteins. Arch Immunol Ther Exp (Warsz). 2006;54:367–74.
83. Murasawa Y, Watanabe K, Yoneda M, et al. Homotypic versican G1 domain interactions
enhance hyaluronan incorporation into fibrillin microfibrils. J Biol Chem. 2013;288:29170–81.
84. Nagase H, Visse R, Murphy G. Structure and function of matrix metalloproteinases and
TIMPs. Cardiovasc Res. 2006;69:562–73.
85. Newton K, Dixit VM. Signaling in innate immunity and inflammation. Cold Spring Harb
Perspect Biol. 2012;4:a006049.
86. Nissinen L, Kähäri VM. Matrix metalloproteinases in inflammation. Biochim Biophys Acta.
2014;1840:2571–80.
87. Nygårdas PT, Hinkkanen AE. Up-regulation ofMMP-8 andMMP-9 activity in the BALB/c
mouse spinal cord correlates with the severity of experimental autoimmune encephalomyeli-
tis. Clin Exp Immunol. 2002;128:245–54.
88. Olin AI, Morgelin M, Sasaki T, et al. The proteoglycans aggrecan and Versican form networks
with fibulin-2 through their lectin domain binding. J Biol Chem. 2001;276:1253–61.
89. Owen CA, Hu Z, Lopez-Otin C, et al. Membrane-bound matrix metalloproteinase-8 on
activated polymorphonuclear cells is a potent, tissue inhibitor of metalloproteinase-resistant
collagenase and serpinase. J Immunol. 2004;172:7791–803.
90. Parekh K, Ramachandran S, Cooper J, et al. Tenascin-C, over expressed in lung cancer down
regulates effector functions of tumor infiltrating lymphocytes. Lung Cancer. 2005;47:17–29.
91. Parks WC, Wilson CW, Lopez-Boado YS. Matrix metalloproteinases as modulators of
inflammation and innate immunity. Nat Rev Immunol. 2004;4:617–29.
92. Peranzoni E, Rivas-Caicedo A, Bougherara H, et al. Positive and negative influence of the
matrix architecture on antitumor immune surveillance. Cell Mol Life Sci. 2013;70:4431–48.
93. Petrey AC, de la Motte CA. Hyaluronan, a crucial regulator of inflammation. Front
Immunol. 2014;5:101.
94. Piperi C, Papavassiliou AG. Molecular mechanisms regulating matrix metalloproteinases.
Curr Top Med Chem. 2012;12:1095–112.
95. Ra HJ, Parks WC. Control of matrix metalloproteinase catalytic activity. Matrix Biol.
2007;26:587–96.
108 A. Cataldi and V. di Giacomo

96. Reinhardt DP, Sasaki T, Dzamba BJ, et al. Fibrillin-1 and fibulin-2 interact and are
colocalized in some tissues. J Biol Chem. 1996;271:19489–96.
97. Rietz A, Spiers J. The relationship between the MMP system, adrenoceptors and
phosphoprotein phosphatases. Br J Pharmacol. 2012;166:1225–43.
98. Sangaletti S, Colombo MP. Matricellular proteins at the crossroad of inflammation and
cancer. Cancer Lett. 2008;267:245–53.
99. Sangaletti S, Stoppacciaro A, Guiducci C, et al. Leukocyte, rather than tumor-produced
SPARC, determines stroma and collagen type IV deposition in mammary carcinoma. J Exp
Med. 2003;198:1475–85.
100. Silini A, Parolini O, Huppertz B, et al. Soluble factors of amnion-derived cells in treatment
of inflammatory and fibrotic pathologies. Curr Stem Cell Res Ther. 2013;8:6–14.
101. Sorokin L. The impact of the extracellular matrix on inflammation. Nat Rev Immunol.
2010;10:712–23.
102. Stenina-Adognravi O. Thrombospondins: old players, new games. Curr Opin Lipidol.
2013;24:401–9.
103. Stern R, Asari AA, Sugahara KN. Hyaluronan fragments: an information-rich system. Eur J
Cell Biol. 2006;85:699–715.
104. Stern R, Jedrzejas MJ. Hyaluronidases: their genomics, structures, and mechanisms of
action. Chem Rev. 2006;106:818–39.
105. Sternlicht MD, Werb Z. How matrix metalloproteinases regulate cell behavior. Annu Rev
Cell Dev Biol. 2001;17:463–516.
106. Striz I, Brabcova E, Kolesar L, et al. Cytokine networking of innate immunity cells: a
potential target of therapy. Clin Sci (Lond). 2014;126:593–612.
107. Takahashi C, Sheng Z, Horan TP, et al. Regulation of matrix metalloproteinase-9 and
inhibition of tumor invasion by the membrane-anchored glycoprotein RECK. Proc Natl
Acad Sci U S A. 1998;95:13221–6.
108. Tezvergil-Mutluay A, Agee KA, Hoshika T, et al. The requirement of zinc and calcium ions
for functional MMP activity in demineralized dentin matrices. Dent Mater. 2010;26:1059–67.
109. Toba H, Cannon PL, Yabluchanskiy A, et al. Transgenic overexpression of macrophage
matrix metalloproteinase-9 exacerbates age-related cardiac hypertrophy, vessel rarefaction,
inflammation, and fibrosis. Am J Physiol Heart Circ Physiol. 2017;312:H375–83.
110. Uhlin-Hansen L, Wik T, Kjellen L, et al. Proteoglycan metabolism in normal and
inflammatory human macrophages. Blood. 1993;82:2880–9.
111. Van den Steen PE, Opdenakker G, Wormald MR, et al. Matrix remodelling enzymes, the
protease cascade and glycosylation. Biochim Biophys Acta. 2001;1528:61–73.
112. Vandenbroucke RE, Dejonckheere E, Van Hauwermeiren F, et al. Matrix metalloproteinase
13 modulates intestinal epithelial barrier integrity in inflammatory diseases by activating
TNF. EMBO Mol Med. 2013;5:932–48.
113. Vargová V, Pytliak M, Mechírová V. Matrix metalloproteinases. EXS. 2012;103:1–33.
114. Verma S, Kesh K, Ganguly N, et al. Matrix metalloproteinases and gastrointestinal cancers:
impacts of dietary antioxidants. J Biol Chem. 2014;5:355–76.
115. Vermaelen KY, Cataldo D, Tournoy K, et al. Matrix metalloproteinase-9-mediated dendritic
cell recruitment into the airways is a critical step in a mouse model of asthma. J Immunol.
2003;171:1016–22.
116. Vigetti D, Karousou E, Viola M, et al. Hyaluronan: biosynthesis and signaling. Biochim
Biophys Acta. 2014;1840:2452–9.
117. Vigetti D, Rizzi M, Moretto P, et al. Glycosaminoglycans and glucose prevent apoptosis in
4-methylumbelliferone-treated human aortic smooth muscle cells. J Biol Chem.
2011;286:34497–503.
118. Vigetti D, Genasetti A, Karousou E, et al. Proinflammatory cytokines induce hyaluronan
synthesis and monocyte adhesion in human endothelial cells through hyaluronan synthase 2
(HAS2) and the nuclear factor-kappaB (NF-kappaB) pathway. J Biol Chem.
2011;285:24639–45.
4 Extracellular Matrix: Immunity and Inflammation 109

119. Vigetti D, Rizzi M, Viola M, et al. The effects of 4-methylumbelliferone on hyaluronan

synthesis, MMP2 activity, proliferation, and motility of human aortic smooth muscle cells.
Glycobiology. 2009;19:537–46.
120. Vincenti MP. The matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase
(TIMP) genes. Transcriptional and posttranscriptional regulation, signal transduction and
cell-type-specific expression. Methods Mol Biol. 2001;151:121–48.
121. Volk SW, Iqbal SA, Bayat A. Interactions of the extracellular matrix and progenitor cells in
cutaneous wound healing. Adv Wound Care (New Rochelle). 2013;2:261–72.
122. Wang M, Qin X, Mudgett JS, et al. Matrix metalloproteinase deficiencies affect contact
hypersensitivity: stromelysin-1 deficiency prevents the response and gelatinase B deficiency
prolongs the response. Proc Natl Acad Sci U S A. 1999;96:6885–9.
123. Wang Y, Herrera AH, Li Y, et al. Regulation of mature ADAM17 by redox agents for
l-selectin shedding. J Immunol. 2009;182:2449–57.
124. Wang W, Xu GL, Jia WD, et al. Ligation of TLR2 by versican: a link between inflammation
and metastasis. Arch Med Res. 2009;40:321–3.
125. Werfel J, Krause S, Bischof AG, et al. How changes in extracellular matrix mechanics and
gene expression variability might combine to drive cancer progression. PLoS ONE. 2013;8:
e76122.
126. Wight TN. The pathobiology of versican. In: Karamanos N, editor. Extracellular matrix:
pathobiology and signaling. KG, Berlin: Walter De Gruyter GMBH & Co.; 2012. p. 154–70.
127. Wight TN, Frevert CW, Debley JS, et al. Interplay of extracellular matrix and leukocytes in
lung inflammation. Cell Immunol. 2017;312:1–14.
128. Wight TN, Kang I, Merrilees MJ. Versican and the control of inflammation. Matrix Biol.
2014;35:152–61.
129. Wight TN, Kinsella MG, Evanko SP, et al. Versican and the regulation of cell phenotype in
disease. Biochim Biophys Acta. 2014;1840:2441–51.
130. Wight TN, Potter-Perigo S. The extracellular matrix: an active or passive player in fibrosis.
Am J Physio Gastrointest Liver Physiol. 2011;301:G950–5.
131. Wolf K, Alexander S, Schacht V, et al. Collagen based cell migration models in vitro and
in vivo. Semin Cell Dev Biol. 2009;20:931–41.
132. Wynn TA. Fibrotic disease and the T(H)1/T(H)2 paradigm. Nat Rev Immunol. 2004;4:
583–94.
133. Wu YJ, La Pierre DP, Wu J, et al. The interaction of versican with its binding partners. Cell
Res. 2005;15:483–94.
134. Xiao Q, Ge G. Lysyl oxidase, extracellular matrix remodeling and cancer metastasis. Cancer
Microenviron. 2012;5:261–73.
135. Yan C, Boyd DD. Regulation of matrix metalloproteinase gene expression. J Cell Physiol.
2007;211:19–26.
 Part II
Material Inspired from Nature
Chapter 5
Biologically Inspired Materials
in Tissue Engineering

Gianluca Fontana, Luis M. Delgado and Daniela Cigognini

Abstract The extracellular matrix (ECM) has unique biochemical, mechanical and
organisational properties through which it provides a physical scaffolding for cells;
a barrier that protects tissues; several signals that affect cell behaviour; and a
reservoir for biologically active molecules. Considering the importance of ECM in
regulating many fundamental cell processes, a myriad of strategies and materials
has been developed to reproduce its properties. The first part of the chapter covers
various approaches aiming to generate scaffolds whose fibre size, orientation and
stiffness could mimic the ECM nanofibrous structure. In particular, the use of
natural fibrous proteins, the application of electrospinning and freeze-drying and
examples of tissue engineering applications are presented. The second part dis-
cusses strategies aiming to address the ECM ligand-binding function and to
reproduce the dynamic, reciprocal, dialogue between cells and their microenvi-
ronment; examples of 3D scaffolds for controlled release of growth factors, drugs
and genetic material are reported. Researchers have also used native ECM com-
ponents to recapitulate the biochemical and biophysical properties of ECM. In the
third part of the chapter, the use of fibrinogen and fibrin is presented as an example
of natural scaffolds recapitulating ECM functions. Fibrinogen and fibrin can be used
as provisional matrix in regenerating tissues; moreover, by varying the fabrication
method and by blending them with other materials, it is possible to produce
biodegradable scaffolds with reasonable control of degradation rate and drug
release.

Keywords Fibrous scaffolds Natural biomaterials Electrospinning




Hydrogels Freeze-drying 3D scaffolds Controlled drug release




Growth factors Gene therapy Fibrinogen Fibrin Crosslinkers

Cell carrier

G. Fontana
L. M. Delgado
D. Cigognini (&)

Network of Excellence for Functional Biomaterials, National University
of Ireland, Biosciences Building, Dangan, Galway, Ireland
e-mail: [email protected]

© Springer International Publishing AG, part of Springer Nature 2018 113

A. C. Berardi (ed.), Extracellular Matrix for Tissue Engineering
and Biomaterials, Stem Cell Biology and Regenerative Medicine,
https://doi.org/10.1007/978-3-319-77023-9_5
114 G. Fontana et al.

Functional Fibrous Scaffolds for Tissue Engineering

Applications

The term of ‘fibrous scaffold’ refers to a 3D fibrous network of molecules that

closely mimics native tissues. Fibrous structures can present two different archi-
tectures: interconnected micropores and nano- or micro-scaled fibres [1, 2]
(Fig. 5.1). Both fibrous architectures are characterised by continuous fibres or thin
layers, high surface to volume ratio, high porosity, adjustable fibre diameter/fibre
distribution/pore size/pore distribution and interconnectivity [3–5]. The attractive-
ness of fibrous scaffolds for tissue engineering is on the broad range of suitable
biomaterials (natural or synthetic) [6]; the possibility of tuning the fibrous structure
[7] and biomechanical strength [8] by varying the fabrication method; the ability to
enhance nutrient transport [9]; and to support cell ingrowth [10].
Fibrous proteins are attractive natural materials because cells can recognise and
attach to specific sites. Moreover, fibrous proteins such as collagen, elastin, keratin
or silk consist of highly repetitive amino acid sequences that form secondary
structures (e.g. triple helices or beta-sheets) during self-assembling, mimicking
native tissue structure. Another advantage is the degradation into non-toxic prod-
ucts [11]. However, some fibrous protein scaffolds display low mechanical prop-
erties and exogenous crosslinking does not induce enough stability; therefore,
fibrous proteins are mixed with synthetic polymers to increase mechanical perfor-
mance [12]. Table 5.1 provides examples of natural materials used to produce
fibrous scaffolds for tissue engineering applications.
Collagens are the most abundant components in the extracellular matrix
(ECM) of connective tissues, constituting 20–30% of total proteins of human body
[13]. To date, 29 different collagen types have been identified [14] and all types
share a common [Gly-X-Y]n sequence, where frequently X is proline and Y is
hydroxyproline [15]. Among all collagen types, collagen type I is the most abun-
dant in the body, and therefore, it is the most widely studied [16, 17], followed by
the denatured form of collagen, gelatine [7]. Collagens are attractive biomaterials

Fig. 5.1 Images obtained by scanning electron microscopy of different fibrous scaffold structure
formed by micropores (sponge, left), nano- or micro-fibres (electrospun film, right)
5 Biologically Inspired Materials in Tissue Engineering 115

Table 5.1 Example of materials, formulations and applications of fibrous scaffolds

Material Formulation Tissue Outcome References
engineering
application
Collagen Hydrogel Intervertebral It maintained NP cells [26]
disc repair phenotype over 14 days
Collagen Electrospun film Wound healing It promoted keratinocytes [27]
adhesion and spreading; it
accelerated wound healing
Collagen/ Electrospun film Wound healing PEUU increased mechanical [12]
PEUU properties, but reduced cell
infiltration and remodelling,
delaying wound healing
Collagen Film Wound healing Crosslinked collagen type I film [21]
with increased mechanical and
enzymatic resistance that
modulates macrophage
response
Elastin Film Wound healing It maintained structural integrity [44]
and prevented hernia recurrence
during wound healing
Elastin Vesicle Wound healing Tuneable elastin vesicles for [45]
growth factor release with
controlled degradation rates
Elastin/ Freeze-dried Wound healing Non-cross-linked sponge [120]
collagen sponge induced wound healing with
minimal foreign body reaction
Keratin Hydrogel Wound healing It promoted in vitro cell [64]
proliferation and accelerated
re-epithelisation in vivo
Keratin Hydrogel Peripheral It improved functional nerve [61]
nerve repair recovery in a rat model
Keratin Hydrogel Wound healing Tuneable elastin vesicles for [62]
drug release with controlled
degradation rates
Silk Gel tubes Vascular grafts It increased cell attachment and [71]
ingrowth of artery smooth
muscle and endothelial cells
Silk Electrospun film Wound healing It promoted keratinocytes [121]
adhesion and spreading,
especially when combined with
collagen coating
Silk Electrospun Vascular grafts Electrospun silk fibroin grafts [73]
conduit enhanced small blood vessels
repair
116 G. Fontana et al.

because they are recognised by the host tissue as normal constituents rather than as
a foreign matter [17]. Other advantages of collagen are its abundance and the ease
of functionalisation with therapeutic moieties. However, the mechanical and
enzymatic stability of collagen produced from animal/human source is minimal
because natural crosslinking does not occur in vitro. For that reason, exogenous
crosslinking (chemical, biological or physical) is required to control stability and
durability in the body [18–21], even though crosslinking has been associated with
cytotoxic issues [22], scaffold calcification [23] and foreign body response [24, 25].
Collagen-based materials can be used in different forms—hydrogels, fibres, films
and sponges—for tissue engineering. For example, self-assembled collagen
hydrogels have been shown to be a promising candidate as cell carrier for adipose
stem cells and nucleus pulposus cells [26]. Electrospun collagen has been able to
enhance wound healing [27, 28]; however electrospinning processing can denature
collagen [29]. Although collagen is mainly extracted from porcine, bovine and
human tissues [30], these sources carry the potential risk of disease transmission
[31], allergic reactions [32], immunogenicity [33] and variability between batches
[30, 34]. In order to avoid these possible issues, fish-extracted collagen and
recombinant human collagen have been proposed as possible alternatives [35, 36].
Recombinant collagen from transgenic tobacco plant, mammalian cells, bacteria or
silkworm have been employed in research [30, 36, 37], whereas fish-extracted
collagen has been rarely used [38] because of its non-mammalian origin.
Elastin is a rubber-like protein that exhibits high elasticity, low stiffness and high
resilience [39]. Therefore, elastin is a crucial protein in tissues requiring large
stretching and shape recovery, such as blood vessels, ligaments, lungs or skin [39].
In the same way, elastin-based scaffolds are particularly interesting for tissue
engineering applications that require large elastic deformation [40, 41]. As for
collagen scaffolds, mechanical and biological stability of elastin-based scaffolds can
be increased by means of chemical crosslinking [40]. Elastin is composed of
approximately 800 amino acids, and it is synthesized from tropoelastin that is
formed by two domains: hydrophobic domains—mainly glycine, valine, alanine
and proline residues—, and hydrophilic domains—predominantly lysine and ala-
nine residues [42]. The principal sources of elastin are solubilised elastin from
mammalian tissues and recombinant elastin-like protein [43]. The main advantage
of recombinant elastin is the better control over the physical and chemical char-
acteristics of the resulting scaffold in comparison to solubilised elastin [40]. For
example, elastin-like proteins have been used to produce films for hernia repair
[44], carrier for growth factor [45] or gene therapy [46].
Elastin has good hemocompatibility, anti-thrombogenic properties and regula-
tory functions over luminal endothelial cells and vascular smooth muscle cells [47].
Consequently, elastin is an excellent biomaterial for vascular applications [48, 49].
Elastin-coated polymeric or metallic devices have been used for increasing
hemocompatibility [50] and for decreasing platelet adhesion, fibrin deposition [51,
52] and implant calcification [53]. Nevertheless, the success of elastin coating may
be affected by the chemical modifications required to covalently bond elastin to the
device surface [54]. Elastin has been also proposed to construct blood vessels;
5 Biologically Inspired Materials in Tissue Engineering 117

however, the use of elastin alone has limited applications due to the low ultimate
tensile strength of the scaffold [54]. Therefore, the combination of elastin with
polyurethane (PU), poly-lactic-co-glycolic acid (PLGA) or polycaprolactone
(PCL) has been explored to improve mechanical properties [55–57]. Finally,
industrial and clinical use of elastin-based scaffolds has been limited by the issues
related to elastin extraction process, which can compromise its biomechanical and
biological properties [54].
Keratin is one of the main constituents of skin, hair and nails [58]. Keratin
contains cell-binding motifs, such as RGD and LDV, that enhance cell attachment,
migration and proliferation [40]. In addition, waste products from the poultry
processing can be used as source of keratin. This fact ensures a cheap and con-
tinuous source, and therefore, keratin extraction has been widely investigated.
Keratin can be extracted by oxidative or reductive methods, but only reductive
extraction with DTT or mercaptoethanol ensures long in vivo stability of recon-
stituted scaffolds [40]. These keratin solutions can be employed to produce hy-
drogels, sponges, films and fibres [59].
Although keratin-based products are well known in cosmetic industry, its clinical
use is limited because of its low mechanical properties and extremely high flexibility
[60]. However, keratin hydrogels and films have been demonstrated to promote
proliferation of endothelial cells, keratinocytes and fibroblasts, and to regulate
Schwann cells activity [61]. Furthermore, keratin-based scaffolds have been suc-
cessfully used as tuneable drug delivery system [62], to improve nerve repair [63] to
accelerate epithelisation and wound remodelling [64] in preclinical models.
Silk is a lightweight, strong and elastic protein produced by several insects and
spiders [65]. The two main components of silk are sericin and fibroin; sericin is a
glue-like protein that links fibroin fibres, while fibroin is a structural protein
commonly used to produce scaffolds [66]. Silk fibroin is composed of glycine-X
sequences, where X is often alanine, serine, threonine and valine [67] and is typ-
ically extracted from silkworm chrysalis [68]. Silk fibroin can be easily processed in
water or other solvents to form hydrogels, fibres, films or sponges [69]. The
degradation rate of silk-based scaffolds can be adjusted from few months to up to
1 year by modifying processing parameters, such as the solvent, fibroin concen-
tration and pore size [70].
Silk fibroin has been used for producing blood vessels with successful attach-
ment and ingrowth of both smooth muscle and endothelial cells [71, 72]. Indeed,
electrospun silk fibroin grafts have been demonstrated to be an encouraging strategy
to repair small blood vessels [73], as they promote endothelial cell and smooth
muscle cells infiltration and the development of an elastic lamina on the internal
surface of scaffolds in a short term [74]. In addition, silk electrospun films have
been studied as potential candidates for wound healing dressings due to their
protective capacity against dehydration and microorganisms and their remarkable
mechanical properties [75–77]. Clinical data concerning silk, that is mainly used for
suturing, suggest that silk is well tolerated at short term with few cases of hyper-
sensitivity associated with sericin residues [69]. However long-term immune
response to this non-mammalian derived material is still unknown [69].
118 G. Fontana et al.

To date, a few different fabrication techniques have been established to produce

fibrous scaffolds. These techniques include electrospinning, self-assembling and
freeze-drying.
Electrospinning is a fabrication method to produce nonwoven films or conduits
with high interconnected porosity and controlled fibre diameter in the range of few
nanometres to microns [78]; however, distance between fibres cannot be controlled
[75]. Electrospinning is a versatile and high cost-efficient technique to mimic nat-
ural and fibrillar structures of native tissues [3]. Additionally, electrospun materials
exhibit a high surface to mass ratio, which increases the availability of material
motifs and the potential effect on cell response. Therefore, it is advocated as a
strong technique to produce fibrous scaffolds for several different tissue regenera-
tion applications, for instance neural [79], tendon [80], cornea [81], wound healing
[12] and cardiovascular [9] applications.
The basic set-up of electrospinning includes a spinneret (e.g. a hypodermic
needle with blunt tip), a syringe pump for ejecting the polymer solution, a power
supply and a collector. The polymer needs to be dissolved in a highly volatile
solvent. During the extrusion, an electric field gradient is applied between the
metallic spinneret and the target collector. This electric gradient induces electro-
static forces that form a jet, accelerate it toward the metallic collector and promote
solvent evaporation. As a result, a fibre with electric charge is deposited onto the
collector. Fibre orientation can be obtained using rotating drum at different speeds
[3, 78, 82]. The modulation of several parameters allows the control over fibre
diameter and alignment. Fibre diameter is controlled by concentration [83], voltage
[83], spinneret diameter [83], extrusion rate [84] and distance between spinneret
and collector [84]. Furthermore, porosity and pore shape have been recently con-
trolled by collectors having a predefined architecture [85]. Although the influence
of process parameters has been widely studied, it is still difficult to predict the ease
of electrospinning for each material and the resulting fibre characteristics, such as
diameter, porosity and stiffness. Moreover, for some materials, it is difficult to
obtain fibres displaying an uniform diameter and not presenting globules [69].
The current trend in electrospun fibrous scaffolds is to mimic the fibre size of
native tissues [6], like collagen fibrils, which have a fibre diameter of 10–500 nm
[18]. Furthermore, fibre orientation has been shown to affect the outcome of the
treatment. In tendon repair, parallel-aligned fibres induced in vitro differentiation of
tendon progenitor cells into tenocytes and promoted tendon-like tissue in vivo,
while random fibres induced mineralisation [86]. Also, parallel-aligned fibre films
promoted peripheral nerve regeneration in rats [87]. On the other hand, radially
aligned fibres have been employed to generate dura mater substitutes [88], while
random fibres have been mainly used for wound healing [12]. Even though the
response to electrospun materials has been widely investigated in vitro, the host
response in vivo is not yet well defined [69].
Self-assembly is defined as the spontaneous organisation of disordered com-
ponents into patterns or structures, without external intervention [89]. It usually
involves the formation of non-covalent interactions such as van der Waals forces,
hydrogen bonds, electrostatic forces and hydrophobic interactions [89]. Some
5 Biologically Inspired Materials in Tissue Engineering 119

natural occurring examples are collagen and phospholipids. An aqueous solution of

atelocollagen, which is collagen solubilised by proteases, self-assembles into
pro-collagen and forms fibrils [90]. By taking advantage of this phenomenon,
several collagen forms can be produced, such as hydrogels, single thick fibres,
bundles of thick fibres and aligned collagen fibrils. Single collagen fibres can be
obtained when collagen solutions are extruded and incubated in polyethylene glycol
buffer; this method allows for the production of fibres with diameter ranging
between 1 and 300 µm, which imitate the structure of native tendon [91]. Also,
bundles of collagen fibres have been used for axonal guidance in nerve repair [92].
Furthermore, in order to control neurite outgrowth, collagen fibrils have been
aligned using isoelectric focusing principle [93]. Self-assembled scaffolds can
present reproducibility issues due to the sensitivity of self-assembling process to
several variables, such as protein purity and concentration, pH, ionic strength and
temperature. For example, the self-assembling rate, fibrils structure and isoelectric
point of collagen type I are directly modulated by pH [94]. The shape of the scaffold
can be controlled by using a mould, and some innovative forms, such as core-shell
fibres, have been used as stem cell delivery system and defect-adjustable tissue
engineering system [95–98].
Also synthetic peptides have shown self-assembling properties. For instance, the
peptide amphiphile molecules are composed by a hydrophobic alkylic tail and a
hydrophilic peptidic head and they can self-assemble into hydrogels in response to
temperature or pH changes [99, 100]. Peptides containing an alternation of polar
and non-polar residues, which form a double-beta sheet structure in water, are
another example of self-assembling. These peptides, such as RADA16-I, have been
widely used for regenerative medicine purposes [101, 102]. Scaffolds formed by
self-assembling sequences can mimick the structure of ECM; however, specific
functional sequences can be added to improve cells adhesion or control cell
behaviour, for example RGD [103], IKVAV [104] or BMHP1 (Bone Marrow
Homing Peptide) [103]. Despite low mechanical properties restrict the use of
self-assembling peptides to treat small tissue defects, they have been successfully
employed for spinal cord [105, 106], bone [100], cartilage [107] or skin [108] repair
in animal models.
Freeze-drying processing, also known as lyophilisation or phase separation, is a
simple dehydratation method for producing porous scaffolds. The material is frozen
in order to induce the formation of ice crystals, and then ice is removed by sub-
limation, creating a 3D porous network. With respect to the previous described
fabrication methods, some advantages of freeze-drying are reproducibility and the
possibility to obtain several engineered shapes, as this technique uses moulds for
processing. Similarly to electrospinning and self-assembling, freeze-dried scaffolds
display low mechanical properties. These materials can be crosslinked to increase
enzymatic resistance, while mechanical properties are slightly improved [18].
Freeze-dried fibrous scaffolds show layers between 50 and 500 nm, pore sizes
over 50 µm, high porosity and interconnected pores, which can enhance cell and
tissue infiltration [109, 110]. The modulation of temperature gradients during
freezing allows the control of pore direction and pore size [111]. Freeze-dried
120 G. Fontana et al.

materials exhibit higher adsorption of protein when compared to bulk/solid mate-

rials; this property is related to the high surface to mass ratio. Due to the benefits of
this fabrication method, freeze-dried scaffolds have been widely used in tissue
engineering, including wound healing [112], spinal cord [113], nerve [114], carti-
lage [115] and tendon [116] repair. For example, freeze-drying was used to produce
sponges with aligned channels that guided linear axonal growth after spinal cord
injury [110, 113]. Freeze-dried collagen sponges with oriented channels supported
axonal regeneration also in peripheral nerve injury [114]. Moreover, functionali-
sation of freeze-dried multichannel collagen conduits has been shown to improve
nerve repair by increasing aligned axonal regeneration [117]. In the same way,
aligned collagen sponges have been used to stimulate tenocytes infiltration and
orientation [118, 119]. In order to improve scaffold stability, collagen sponges were
reinforced with silk and the resulting sponges supported tendon repair over
18 months [116]. Collagen type II sponges promoted in vivo cartilage formation:
chondrocytes infiltrated the sponge and formed new cartilage in 6 weeks [115].
In conclusion, fibrous scaffolds provide structural, biomechanical and bio-
chemical support to cells and new tissue formation; therefore, fibrous scaffolds are a
suitable clinical strategy to enhance functional reconstruction of tissues and organs.
These materials have the advantage of mimicking ECM characteristics, and their
properties can be adapted using different biomaterials and processing techniques.
Although results are encouraging, further research is needed to overtake some
limitations of each material and technique, for example immunogenicity of
biopolymers, reproducibility of some fabrication methods or low mechanical
properties of reconstituted scaffolds. Future investigations should evaluate how to
scale up extraction of biopolymers and scaffold fabrication, controlling homo-
geneity between batches. Further research is also required to correlate scaffold
characteristics and biological response.

3D Drug Releasing Scaffolds

Advances in proteomics and genomics have led to the identification of several

biomacromolecules with therapeutic relevance such as proteins, antibodies, pep-
tides, plasmid DNA and small interfering RNA. This has prompted extensive efforts
to develop efficient in vivo drug delivery systems (Table 5.2). However, the
influence exerted by the microenvironment in which the cells reside remains
underappreciated. The microenvironment includes the ECM and the complex array
of cues that actively participate in the crosstalk between cells and the ECM.
The ECM, in fact, is not only responsible for maintaining the integrity of the tissues
and allowing diffusion of nutrients, but also it provides instructive cues that
influence cell behaviour, dictating cell ability to proliferate, differentiate and to
produce matrix [122–127]. Also, ECM acts as a natural reservoir of growth factors
and other bioactive molecules [128–131].
5 Biologically Inspired Materials in Tissue Engineering 121

Table 5.2 Examples of strategies for controlled drug delivery from 3D scaffolds
Bio-molecule Material Type of strategy Outcome References
VEGF Type I Covalent The covalent incorporation of [152]
collagen incorporation VEGF enhanced the angiogenic
capabilities of the collagen
matrices
VEGF Fibrin Immobilization, The immobilization of VEGF [154]
use of within the scaffold allowed
enzymatically cell-demanded release with
labile sequences increased formation of new
blood vessels with normal
morphological appearance
VEGF PEG Covalent The subcutaneous implantation [156]
incorporation of VEGF-containing scaffolds
in rats resulted in remodelling
of the scaffold and formation of
native, vascularised tissue
bFGF Type I Immobilization Scaffolds in which heparin was [164]
collagen immobilized showed increased
vascularisation for periods up to
3 weeks in vivo
SDF-1a Type I Immobilization Scaffolds containing [166]
collagen immobilized heparin to trap the
stem cell chemo-attractant
SDF-1a were implanted
subcutaneously in mice. Only
few progenitor cells were
recruited early after
implantation
BMP-7 PLLA Particles Scaffolds containing [181]
reservoir system BMP-7-loaded nanospheres
allowed for a prolonged
maintenance of therapeutic
activity of BMP-7 and resulted
in improved bone formation in
rats
BMP-7 Fibrin or Embedding In vitro tests showed that [203]
expressing collagen adenoviruses maintained their
adenovirus therapeutic efficacy for longer
when embedded in fibrin gels.
In vivo, bone formation was
observed in animals implanted
with fibrin or collagen
virus-loaded gels
(continued)
122 G. Fontana et al.

Table 5.2 (continued)

Bio-molecule Material Type of strategy Outcome References
Plasmids HA/PEG Controlled Plasmids were stably [170]
degradation incorporated into hydrogels
with different HA/PEG content.
The cumulative release
increased with a decreasing
PEG or increasing HA content.
The study demonstrated the
dependence of plasmid release
on the physical properties of the
hydrogel
Radiologic b-peptides Enhanced water Supramolecular hydrogels were [177]
tracer diffusion developed by using b-peptides
and loaded with a tracer. For
12 h post-injection in rats, the
tracer was released from the
hydrogel in a uniform and
sustained manner
Lactate PLDLLA Controlled The scaffold was designed with [178]
degradation a high degradation rate in order
to release lactate. 1 year after
implantation into mouse brain,
the scaffold induced robust and
functional vascularisation and
neurogenesis
IGF-1 Bone Adenoviral gene Bone marrow aspirates were [208]
marrow transfer successfully transduced ex vivo.
aspirate 80% of the cells expressed the
transgene over 21 days of
culture

Even though diseases and trauma are often associated with alterations in the
structure and properties of ECM, conventional drug delivery approaches focused on
restoring cell function without repairing the microenvironment that surrounds the
targeted cells [132–134]. This uncontrolled drug delivery often results in poor
therapeutic effect or in severe side effects. An altered microenvironment can affect
the absorption and retention of the drug into the tissue. Moreover, a hostile
microenvironment can accelerate the degradation or denaturation of the bioactive
molecule, resulting in a poor outcome of the therapy. In fact, the pharmacokinetics
in the host tissue is often characterised by an initial excess followed by a sharp
decrease [135–137]. In order to counterbalance this transient effect, biomacro-
molecules are often delivered at high doses, which can lead to severe side effects, as
reported in both animal and clinical studies [138, 139].
Due to these limitations, current research focused on developing 3D drug
releasing scaffolds which can mimic ECM and can also restore the altered
microenvironment [140, 141]. Scaffolds were found not only to preserve and
5 Biologically Inspired Materials in Tissue Engineering 123

prolong the biological activity of biomacromolecules but also to allow controlled

drug release according to the needs of the targeted tissue [142, 143]. Furthermore,
by using ECM-derived biomacromolecules as building blocks, scaffolds can not
only mimic the 3D architecture of the in vivo microenvironment but also influence
and control cell behaviour. These biomimetic scaffolds take advantage of the body’s
inherent ability to heal by guiding the repair process with the delivery of selected
bioactive molecules [144–149]. The major challenge when designing ECM-based
scaffolds is the identification of the appropriate combination of signals and their
spatio-temporal organization to enable formation of functional tissue [143].
An example of such biomimetic approach is given by the delivery of vascular
endothelial growth factor (VEGF) in therapies aiming at increasing tissue angio-
genesis for the treatment of critical limb ischeamia or simply to increase the for-
mation of new blood vessels within scaffold implants [150–153]. A common
problem found in such therapies is the production of malformed and leaky blood
vessels as a result of uncontrolled, often excessive, release of VEGF [154].
Therefore, VEGF release needs to be tightly controlled [139, 155]. In the body,
VEGF is bound to ECM components until it is released by the cellular enzymatic
activity. In an attempt to mimic this phenomenon, Ehrbar et al. used a variant form
of VEGF containing the sequence NQEQVSPL, which is a substrate for the
transglutaminase FXIII [154]. This strategy mimics the transglutaminase
FXIII-mediated incorporation of a a2-plasmin inhibitor in the blood clot during
coagulation. When the modified VEGF was mixed with transglutaminase FXIII,
fibrin and thrombin, VEGF was immobilized in the resulting fibrin network. This
elegant approach not only allowed the maintenance of VEGF activity for a pro-
longed period but also it enabled VEGF release according to the spatial and tem-
poral demand of the tissue, leading to formation of morphologically intact blood
vessels [151, 154, 156–159]. In this process, which is known as ‘dynamic
reciprocity’, the cells and the surrounding scaffold mutually influenced each other,
resulting in scaffold remodelling and progressive release of the immobilized growth
factor.
In general, encapsulation of growth factors in enzymatically labile natural hy-
drogels such as matrigel, fibrin or collagen, enhances their effect on target cells. For
example, this was seen in fibrin matrices, where the incorporation of VEGF facili-
tated the formation of capillary sprouts [151, 156]. The immobilization of heparin to
collagen matrices, crosslinked using N-(3-dimethylaminopropyl)-N′-ethylcarbodii-
mide (EDC) and N-hydroxysuccinimide (NHS), has been successfully employed for
binding and release of growth factors from 3D matrices [160]. These matrices were
employed to trap and deliver VEGF [161–163], fibroblast growth factor-2 (FGF-2)
[160, 164, 165] and stromal cell-delivered factor-1 (SDF-1a) [166].
Although the sustained delivery of tethered growth factors resulted in an
enhanced and prolonged response in vivo [167], 3D scaffolds can be also suc-
cessfully functionalized by harnessing the affinity between growth factors and ECM
components [168]. Since the release of non-immobilized growth factors is not
solely dependent on proteolytic activity, these systems offer a tuneable release
pattern [169]. For example, it is possible, particularly when using hydrogel systems,
124 G. Fontana et al.

to obtain good control over drug release profile by simply varying scaffold prop-
erties such as pore size or swelling ratio, which can be easily modulated by
changing scaffold hydrophilicity, crosslinker molecular weight or the extent of
crosslinking [170, 171]. For instance, an increase in pore size can favour cell
infiltration and proteolytic degradation by immune cells, which in turn accelerates
drug release; also, an increase of scaffold hydrophilicity can enhance drug delivery
by accelerating bulk degradation via hydrolysis [102]; for example, the degrad-
ability of PEG hydrogels can be controlled by varying the length of hydrolytically
degradable units within the polymer crosslink [172].
However, a scaffold with controlled biodegradation must degrade into non-toxic
products [173], and the method chosen to control the degradation rate should not
affect the mechanical properties, in particular the stiffness, of the scaffold. A good
example is the approach proposed by Kong and colleagues that aimed at acceler-
ating the degradation rate of alginate hydrogels without affecting their stiffness.
Alginate degradation can be accelerated by increasing its oxidation degree; how-
ever, it also results in softer gels. To overcome this issue, they adjusted the
molecular weight distribution (MWD) of oxidised alginates without varying the
number of oxidised uronic acids, thus achieving both regulation of the degradation
rate and maintenance of the elastic modulus [174].
Nonetheless, changes in the degradation rate of hydrogels can affect their ability
to retain and transport fluids. This issue is particularly important when hydrogels
are used as cell carrier, which requires controlled pore size to allow the exchange of
nutrients and the release of their paracrine products. Kang et al. [175] developed a
3D hybrid scaffold in which a 3D framework and a hydrogel were combined to
enhance the mechanical properties without chemically altering the transport prop-
erties of the hydrogel. This system consisted in the injection of a mixture of
dopamine-releasing cells and an alginate gel into the internal space of a 3D
framework of Ormocomp®. Release studies revealed that the encapsulated cells
were able to secrete dopamine for over 8 weeks in vitro and the introduction of
RGD peptides into the alginate hydrogel increased the release rate. Moreover, the
performance of the system was tested in a mouse model: the subcutaneous
implantation of the hybrid scaffold mixed with encapsulated cells showed no signs
of immune rejection after 7 days, indicating that the system shielded the exogenous
cells from the host immune system while enabling cells to fulfil their paracrine
function [175].
Another innovation in the field of 3D drug-releasing scaffolds is the develop-
ment of supramolecular hydrogels. Conventional hydrogels have a less flexible pore
size due to the presence of covalent bonds, thereby limiting the responsiveness to
the surrounding environment [176]. Supramolecular hydrogels instead are formed
by small molecule hydrogelators that self-assemble into 3D networks of nanofibers.
These hydrogels show the ability to imbibe large amounts of water, and also to
change their pore size as a result of the reassembly of the hydrogelators during the
shrinkage/swelling processes [176]. These properties favour the diffusion of
molecules from the delivery system in response to microenvironmental changes.
Liang et al. tested the use of supramolecular hydrogels for controlled drug release
5 Biologically Inspired Materials in Tissue Engineering 125

in vivo. The hydrogel was developed by using b-peptides composed of unnatural

amino acids that act as hydrogelator [177]. As a proof of concept, the hydrogel was
loaded with radioactive tracers and implanted in a rat subcutaneous model. The
release of the tracer was tracked for 12 h post-injection. Single photon emission
computed tomography (SPECT) images revealed that the blood concentration of the
tracer was maintained in a narrow range during this period, indicating a uniform and
sustained release from the hydrogel [177].
In an in vivo study [178], a poly(L-lactide-co-D,L-lactide) (PLDLLA) scaffold
was designed with a high degradation rate in order to release lactate, a common
cellular cue that induces angiogenesis [179] and supports neuronal progenitor
metabolism [180]. These scaffolds were also designed with a controlled topography
to mimic the 3D organization of radial glia. Following implantation of these
cell-free scaffolds into a mouse model of injured brain, the animals were monitored
over 15 months. The radial scaffolds induced robust and functional vascularisation
and neurogenesis for more than 1 year due to the delivery of appropriate metabolic
cues, the release of lactate and the controlled biophysical environment [178].
Immobilization or absorption of growth factors on 3D scaffolds can undoubtedly
increase their biological activity. However, this is generally a short-/medium-term
effect as growth factors have a short half-life (in the range of hours/days). If the
therapeutic strategy needs a prolonged delivery of the bioactive proteins, it is
necessary to provide a protective environment to maintain their biological activity.
For instance, poly(L-lactic acid) (PLLA) scaffolds were functionalized by adsorp-
tion of bone morphogenetic protein-7 (BMP-7) or by immobilization of
BMP-7-loaded nanospheres of poly(lactic-co-glycolic acid)(PLGA) [181] and they
were implanted in rats. In PLLA scaffolds loaded with adsorbed BMP-7, a failure in
bone induction was observed, whereas in scaffolds loaded with immobilized
BMP-7-loaded-PLGA nanospheres, new bone formation was found throughout the
scaffold, suggesting that nanospheres shielded BMP-7 from denaturation/
degradation and released it in a sustained manner, thus allowing osteoblast cells
to synthesize functional matrix [181].
Although the encapsulation of growth factors can prolong their effect in vivo,
their intrinsic short half-life still limits potential applications, because high doses or
repeated administrations are required to obtain the desired effect [182]. These
drawbacks can be overcome through the delivery of genetic material to the target
tissue, in order to exploit the cell machinery to produce therapeutic proteins in a
controlled and sustained manner [124, 183, 184]. Gene therapy is a powerful
approach that can stimulate local production of proteins capable of activating
autocrine and paracrine loops that influence tissue development and repair [185].
Hence, 3D scaffolds have been tailored to improve the outcome of gene delivery.
To increase the cell proliferation rate, which is necessary to allow genetic material
to enter the cell nucleus, gene therapy has been associated with delivery of growth
factors. For example, the co-delivery of basic fibroblast growth factor (bFGF) and
fibroblast cells in a PLGA scaffold was found to enhance fibroblasts proliferation
and indirectly, to increase the nucleotide uptake rate with a resulting increase in the
levels and duration of transgene expression [124, 186].
126 G. Fontana et al.

The functionalization of the scaffold with cell adhesion molecules was also
found to influence the outcome of gene delivery [187]. For this reason, the first
scaffold that functioned as gene delivery vehicle was made of collagen, a RGD-rich
biomacromolecule [188]. Such collagen scaffolds have been employed for the
delivery of both viral [189] and non-viral vectors [190, 191]. With these approa-
ches, it was shown that it is possible to induce transgene expression in vivo which
led to tissue repair in a wide range of applications such as bone regeneration [188,
192], wound healing [193–196], muscle repair [197] and optic nerve repair [198].
Other natural polymer-based hydrogels were also used as a platform for gene
therapy. For instance, alginate, which is inert to cell adhesion and protein
adsorption [199], was functionalized with RGD peptides. The spatial distribution of
these cell adhesion peptides influenced the rate of DNA uptake by pre-osteoblasts
cells, and RGD-functionalised alginate hydrogels showed the highest transgene
expression [187, 200].
3D scaffolds were also found to prolong the half-life of viral vectors and to
shield them from the host immune response [201]. Furthermore, delivery of the
vector from the scaffold has the advantage of concentrating the transgene expres-
sion in the implant site [143]. The method used to functionalise the scaffold with the
gene vectors can also determine the gene release profile and the duration of their
biological activity [143]. For example, Schek et al. compared in vitro the bioactivity
of adenoviruses embedded in fibrin hydrogels or simply resuspended in liquid form.
When in solution, the adenoviruses decreased to half-maximal activity after only
15 h, whereas when embedded in fibrin hydrogels the half-life of the adenoviruses
was extended to 45 h [201]. The bioactivity of adenoviruses was tested also in
mice. In this study, adenoviruses bearing the gene for BMP-7 were implanted
intramuscularly: the suspended form of adenoviruses induced bone formation in
50% of the muscles, whereas the efficacy of adenoviruses embedded in fibrin
hydrogels was much higher, with bone formation occurring in 80% of the muscles
treated [201]. A drawback in the use of viral vectors for gene therapy is the
potential damage they can cause when they leak into nearby tissues. Pascher et al.
compared the delivery of viral vectors through collagen/glycosaminoglycan
matrices or via bone marrow clots (BMC) in a rabbit model. Interestingly, BMC
showed superior containment of the vector within the osteochondral defects.
Moreover, high levels of transgenic expression and deposition of healthy matrix
were observed within the clots [202]. Another interesting aspect of this approach is
the use of autologous-derived materials that can overcome the issues related to the
manufacturing process and biocompatibility of scaffolds.
To immobilize gene vectors in biomaterials, a number of techniques mimicking
the binding of viruses to ECM proteins have been developed. For instance, by using
the electrostatic interactions that occur between nucleotides and calcium phosphate
(CaP), plasmids (pDNA) can be complexed into a nanocrystalline form and
immobilized onto a surface [203]. These complexes have been extensively used as
gene vectors for the functionalisation of scaffolds for bone tissue engineering [204,
205].
5 Biologically Inspired Materials in Tissue Engineering 127

There are alternatives to viral vectors for gene therapy. Cationic polymers or
lipids are often used as complexing agents to condense pDNA to form small
particles. These complexes can penetrate the cell membrane and deliver the genetic
material intracellularly. Nonetheless, this approach has lower efficacy than viral
vectors and higher concentrations of complexes are generally required to obtain
substantial biological effects. This can often result in significant toxicity in vitro and
in vivo [206]. The use of reservoir systems, such as microspheres and nanoparticles,
in collagen hydrogels was found to significantly decrease the toxicity of complexed
pDNA [191]. Generally, pDNA delivery is more effective in actively dividing cells
as during mitosis the nucleus disassembles temporarily, facilitating the nuclear
localisation of the transgene in the newly formed cells. However, tissues that most
often are the target of gene therapy are harsh environments in which cell prolif-
eration is often impaired. For this reason, recent research is shifting toward mRNA
delivery, as mRNA does not need to enter the nucleus to be expressed, and
moreover, its structure can be modified to considerably increase its stability and
therapeutic efficacy [207].
Overall, advances in stem cell biology and biomaterial science provide exciting
opportunities for tissue engineering. Biomimetic scaffolds can be rationally
designed to take advantage of the body’s inherent healing capacity and, by incor-
porating appropriate signals, to use the body as a bioreactor and guide the healing
process. However, a more in-depth knowledge of the role of ECM in growth factor
binding/release and cell signalling will lead to more effective implants. One of the
major challenges is presenting the appropriate combination of signals in the
appropriate spatial and temporal patterns. As discussed above, the release pattern
can be controlled by modulating the degradation rate and structure of the scaffold.
Other options are the use of supramolecular hydrogels, which provide a rapid and
uniform release, or the covalent bond of growth factors, which allows a gradual
release. As growth factors present a very short half-life, the incorporation into 3D
scaffolds of genetically engineered cells or gene vectors could offer a prolonged
production in situ. However, improvements in transfection efficiency and bio-
compatibility of non-viral gene vectors will likely enable a wider adoption of gene
therapy in the clinic. Furthermore, a successful tissue engineering strategy will
depend on the development of scaffolds able to influence cell behaviour. Hence, the
integration of on-demand drug delivery systems with instructive matrices is the
future direction of 3D releasing scaffolds in tissue engineering.

Fibrinogen-Based Scaffolds

Fibrinogen and fibrin are attractive polymers for tissue engineering applications
because they can provide a natural environment and they have intrinsic healing
properties.
Fibrinogen is a 340 kDa plasma glycoprotein involved in formation of blood
clots [209]. The molecule is composed of two sets of disulphide-bridged Aa-, Bb-
128 G. Fontana et al.

and c-chains. In response to damage to the vascular system, fibrinogen is converted

to fibrin by the thrombin-mediated cleavage of fibrinopeptide A from the Aa-chains
and fibrinopeptide B from the Bb-chains, thus initiating fibrin polymerization [210].
Fibrin monomers then self-assemble and form an insoluble fibrin network, which is
further stabilized by the factor XIIIa (plasma transglutaminase) that covalently
crosslinks the C-terminal of fibrinogen c-chains. This natural process can be
reproduced in vitro by mixing together fibrinogen and thrombin in the presence of
calcium ions [209]; then, fibrin gel can be reinforced by introducing a chemical
crosslinker, such as genipin [211]. Not only the formation, but also the degradation
rate of fibrin gels can be controlled in vitro and in vivo, by addition of fibrinolytic
inhibitors. Moreover, fibrin scaffolds can be designed to control the release of
growth factors after implantation in response to externally applied stimuli. For
example, ultrasound has been used to actively control growth factors release,
architecture, and stiffness in a fibrin scaffold doped with sonosensitive emulsion
[212]. All these characteristics make fibrin a suitable biomaterial for controlled
delivery of genes and biomolecules [213, 214].
In tissue engineering, fibrinogen and fibrin present many other advantages. Since
they are naturally designed to provide the temporary scaffold required to support
healing and revascularization [215], they can be used as an initial structural support
for regenerating tissues. Moreover fibrinogen contains RGD sequences for cell
attachment and binding sites for bioactive molecules, such as VEGF, fibroblast
growth factor-2 (FGF-2) and interleukin-1 (IL-1) [216].
Furthermore, by varying the fabrication method, fibrinogen and fibrin can be
easily shaped into a variety of scaffolds, such as hydrogels, nanofibrous films and
microporous matrices, which can be used in combination with cells and biomole-
cules in a wide range of tissue engineering applications (Table 5.3). At last but not
least, fibrinogen and thrombin can be extracted from autologous plasma. The use of
autologous fibrin greatly reduces the risk of immune reaction and infection, as fibrin
sealants produced from allogenic pooled plasma—although they are available in
standardised quality—can still have severe adverse effects, such as hypersensitivity,
anaphylactic reaction and transmission of bacteria, viruses and prions [217]. Other
issues related to commercial fibrins are the high concentration of fibrinogen, making
the fibrin scaffold too dense for cell proliferation and migration and the methods for
fibrinogen concentration which can alter the nature of the fibrin. By contrast, an
autologous fibrin scaffold has a physiological concentration of fibrinogen, thus
providing an appropriate matrix for cell ingrowth [218]. Of note, different fibrin
formulations can be used to support different cell functions [219]. For instance,
formulations containing a low concentration of fibrinogen supported human mes-
enchymal stem cells (MSC) growth, while a higher concentration increased their
osteogenic differentiation potential [219, 220].
Hereinafter, the fabrication methods of various fibrinogen- and fibrin-based
scaffolds and their application in tissue engineering are discussed.
Fibrin hydrogels are produced by mixing fibrinogen with thrombin solution in
the presence of calcium ions. Although they have been used in several tissue
engineering applications, they present the disadvantage of having low mechanical
5 Biologically Inspired Materials in Tissue Engineering 129

Table 5.3 Examples of in vitro and in vivo applications of fibrinogen- and fibrin-based scaffolds
Material Formulation Tissue Outcome References
engineering
application
Fibrin/ Hydrogel Spinal cord repair The fibrin/fibrinogen [229]
fibrinogen hydrogel integrated with
the host spinal cord tissue
and supported robust
growth of axon
Fibrin Hydrogel Spinal cord repair Implants of fibrin [236]
containing neurotrophic
factors enhanced axonal
regeneration into
transected rat spinal cord
Fibrin Hydrogel Spinal cord repair Implants of fibrin and [237]
crosslinked MSCs resulted in the
with EDC formation of
longitudinally-aligned
neurites
Platelet-rich Glue Bone repair Glue mixed with MSCs [238]
fibrin and BMP-2 induced bone
tissue formation in dental
implants in dogs
Fibrin Hydrogel Cartilage repair A significant [241]
improvement in knee
function was recorded in
four patients implanted
with fibrin glue and
autologous chondrocytes.
The graft well integrated
with host tissue
Fibrin Glue Wound healing Fibrin glue combined [259]
with fibroblasts and
growth factors enhanced
wound healing in rabbits
Fibrin Glue Wound healing In vitro, human umbilical [247]
and vein endothelial cells
vasculogenesis proliferated and
organised themselves
into capillary-like
structures within the
fibrin matrix. The skin
substitute showed similar
structure to native skin
Fibrinogen Electrospun Wound healing In an in vitro model, [249]
scaffolds fibroblasts migrated into
and remodelled the
electrospun fibrinogen
scaffold with deposition
of collagen
(continued)
130 G. Fontana et al.

Table 5.3 (continued)

Material Formulation Tissue Outcome References
engineering
application
Haemoglobin/ Electrospun Myocardial repair The scaffold supported [251]
gelatin/ scaffold MSC differentiation into
fibrinogen functional
crosslinked cardiomyocytes and
with phytic showed an improved
acid delivery of oxygen
Fibrin Electrospun Tissue In vitro, the scaffold [252]
film engineering of supported the attachment,
soft tissues spreading, and
proliferation of human
umbilical cord
blood-derived MSCs.
The nanostructure
showed the fibre diameter
of native ECM
Fibrin Bio-printed Microvasculature The printed endothelial [254]
channels tissue engineering cells proliferated to form
a tubular structure inside
the fibrin channel
Fibrin Fibrin-coated Orthopaedic Fibrin can be an ideal [256]
substrate tissue engineering bioprinting substrate for
numerous growth factors.
BMP-2 immobilized to
fibrin induced spatially
defined muscle-derived
stem cell differentiation
toward the osteogenic
and myogenic lineages

strength and rapid degradation. Indeed, fibrin and fibrinogen scaffolds lose their
structure within a few days through degradation by proteases in vitro and in vivo
[209]. To overcome these limitations, fibrin and fibrinogen hydrogels can be
(1) blended with synthetic or natural polymers, (2) crosslinked with different
chemical and biological agents or (3) functionalized with plasmin inhibitors.
Examples of materials used to achieve adequate mechanical strength are poly-
urethane (PU), poly(e-caprolactone) (PCL) and polyethylene glycol (PEG). For
instance, a composite construct made from the commercially available fibrin and
PU-based scaffold exhibited increased stability in vitro and in vivo as compared to
fibrin gels alone: in vitro, the fibrin/PU scaffold was seeded with human adipose-
derived stem cells and was shown to be able to maintain its size and weight for
21 days; in vivo, the implantation of fibrin/PU construct resulted in formation of
well-vascularized adipose tissue [221]. The same research group used the fibrin/PU
scaffold also for cartilage tissue engineering applications [222].
5 Biologically Inspired Materials in Tissue Engineering 131

PEG is another material commonly used to increase fibrin stability. Several

studies carried out by Seliktar and colleagues showed the advantages of using a
hybrid scaffold composed of a fibrinogen backbone and crosslinked with PEG. To
include cells in this hydrogel, fibrinogen fragments were PEGylated with
PEG-diacrylates, they were mixed with the photoinitiator and cell suspension and
finally they were exposed to UV light to form a hydrogel. The mechanical prop-
erties were modulated by varying the percent polymeric composition, while the
fibrinogen backbone provided the motifs for cell attachment and proteolytic
degradation [223]. Further modifications of the PEGylated fibrinogen scaffold, such
as blending with a PEGylated albumin hydrogel [224] or incorporation of
Pluronic®F127 micelles [225], lead to the development of composite hydrogels for
3D cell culture and controlled drug release. Overall these studies showed that the
addition of synthetic materials can improve the control over mechanical stiffness of
fibrinogen or fibrin hydrogels. On the other hand, fibrinogen can be used to
functionalise synthetic hydrogels. Thrombin and fibrinogen were loaded within
PEG hydrogels and the resulting fibrin-loaded hydrogel induced higher vasculari-
sation in vivo in comparison to PEG hydrogels without fibrin [226].
Fibrin and fibrinogen can be also combined with natural materials, such as
collagen, alginate and hyaluronic acid, in order to achieve a synergistic effect on
cell behaviour. This strategy offers also the possibility to tune the stiffness and
degradation rate of the hydrogel. A recent study showed that the degree of
vessel-like structure formation was dependent on the collagen/fibrin proportion in a
composite hydrogel seeded with endothelial cells, revealing a correlation between
matrix stiffness and the degree of vasculogenesis [227]. Furthermore, hyaluronic
acid (HA) has been incorporated within fibrin hydrogels to provide structural
reinforcement to the fibrin network, as demonstrated by the improved stability in
the presence of plasmin and cell-contractile forces of a HA-fibrin hydrogel [228].
An interesting study compared the effect of four injectable hydrogels made of
collagen, fibronectin, fibrin and a blend of fibrin/fibronectin in the injured spinal
cord of rats: collagen implants resulted in dense inclusions, whereas fibronectin and
fibrin well integrated with the host tissue and enhanced neurite elongation; how-
ever, the best results in terms of biocompatibility, cell infiltration and axonal growth
were displayed by the blend fibrin/fibronectin [229].
As previously mentioned, another strategy to maintain the size and shape of
hydrogels for prolonged periods is crosslinking. By varying the concentration of
crosslinkers, it is also possible to control the degradation rate. Commonly used
crosslinking agents for natural materials are glutaraldehyde, 1-ethyl-3-
(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and genipin.
In comparison to glutaraldehyde and EDC, genipin presents the advantage to be
a naturally occurring compound with a very low level of cytotoxicity. A genipin
crosslinked human fibrin hydrogel system was seeded with human articular chon-
drocytes and implanted subcutaneously into rats, showing promising applications as
an autologous scaffold for articular cartilage regeneration, indeed genipin
crosslinking improved mechanical strength of the scaffold, enhanced ECM pro-
duction and did not significantly affect cell viability [211].
132 G. Fontana et al.

The third approach to increase the stability of fibrin- and fibrinogen-based

scaffolds is reducing the proteolytic degradation of fibrin by using degradation
inhibitors and their stabilizers, such as factor XIII, aprotinin and e-amino-n-caproic
acid along with sufficient amounts of thrombin and calcium [209]. However,
aprotinin rapidly diffuses out of fibrin matrices and thus do not provide extended
matrix protection. A recent study reported that an engineered aprotinin variant can
be immobilized within fibrin, then providing extended longevity. The recombinant
aprotinin contained the transglutaminase substrate sequence, which allowed to
covalently crosslink the aprotinin variant into fibrin matrices during thrombin- and
factor XIIIa-mediated polymerisation. Subcutaneously implanted matrices con-
taining this aprotinin variant were detectable in mice for more than twice as long as
those containing the wild-type protein [230].
In order to enhance tissue regeneration or replace a damaged organ, cells and
biomolecules can be incorporated into fibrin hydrogels. Many research efforts have
focused on utilising fibrin hydrogels as cell-supporting scaffolds in tissue engi-
neering of adipose, cardiovascular, ocular, liver, skin, neural, cartilage, tendon and
bone tissues.
Recently, a number of preclinical studies have suggested that fibrin patches
seeded with cells hold promise for regenerating post-infarcted myocardial tissue
[231]. Overall, these studies showed that the fibrin patches could keep cells at the
site of injury and improve cardiac function, and they could also reduce the infarct
size. In contrast, implanted cells rarely moved out of the patch, emphasising the
need for a strategy that could enhance also cell migration toward the site of injury.
Of note, the fibrin patches alone, without cells, were able to enhance vascular
growth [231]. The beneficial effect of fibrin patches was further improved by
incorporation of growth factors, such as IGF-1. For example, a fibrin patch loaded
with IGF-encapsulated microspheres and three different types of cells (cardiomy-
ocytes, endothelial cells and smooth muscle cells) derived from human induced
pluripotent stem cells were tested in a porcine model of acute myocardial infarction.
The tri-lineage cell transplantation combined with IGF-1 significantly improved left
ventricular function, myocardial metabolism, and vessel density, while reducing
infarct size [232].
In the field of neural tissue engineering, fibrin hydrogels containing growth
factors have been demonstrated to enhance axonal growth and cell infiltration in
short-gap nerve injuries [233–235]. Implants of fibrin containing neurotrophin-3
(NT-3), brain-derived neurotrophic factor (BDNF) or ciliary neurotrophic factor
(CNTF) enhanced axonal regeneration into transected rat spinal cord, whereas only
a few regenerated axons were found into the implants that lacked neurotrophic
factors [236]. In another study, a novel formulation of fibrin crosslinked with EDC
was used to deliver MSCs to the injured spinal cord. The use of the fibrin scaffold
resulted in the formation of longitudinally aligned layers of MSCs and in the
longitudinally growth of host neurites into this architecture [237].
In bone tissue engineering, fibrin scaffolds have been widely used because of
their good adhesive and heamostatic properties. Fibrin glue is often used as beads
and microbeads, coating agents on other scaffolds, pre-formed scaffolds, or
5 Biologically Inspired Materials in Tissue Engineering 133

injectable hydrogels [219]. In particular, platelet-rich fibrin glue is a promising

autologous scaffold for bone repair because platelets release growth factors and
bioactive proteins that initiate and accelerate tissue repair. Platelet-rich fibrin glue
mixed with bone marrow MSCs and bone morphogenic protein-2 (BMP-2) induced
bone tissue formation in dental implants in dogs [238]. However, the role of pla-
telets in the repair of bone is poorly understood and it needs to be further studied.
Another issue that deserves further investigation is the addition of other materials,
such as osteoconductive ceramics, because it has been shown that fibrin alone is
incapable of healing bone defects [232]. In the field of dental pulp tissue engi-
neering, fibrin appeared to be the most suitable material to support viability of
dental pulp stem cells and tissue formation among other natural (collagen) and
synthetic (PEG-derived scaffolds) materials [239]. Platelet-rich fibrin scaffolds
loaded with MSCs have been also employed in cartilage tissue engineering, for
instance they have been used in a clinical trial to deliver autologous bone marrow
MSCs into patients with full-thickness cartilage defects, and all patients experi-
enced an improvement of symptoms [240]. Fibrin hydrogels have been also used as
chondrocyte carrier in articular cartilage repair [241].
A number of studies showed the successful use of fibrin for skin repair. Fibrin
glue has been already used in clinical practice as a carrier for keratinocytes in deep,
partial and full-thickness wounds. Cell culture studies have shown that the clono-
genic ability, growth rate and long-term proliferative potential of keratinocytes is
maintained when they are cultured on fibrin which is a naturally occurring substrate
in wound healing [242]. In vitro, the production of TGFa which is involved in
dermis regeneration and angiogenesis was dramatically increased when ker-
atinocytes were cultured on fibrin-coated substrates [243]. Poly(L-lactide)
(PLA) nanofibrous membranes modified with fibrin nanocoating significantly
increased cell spreading and expression and synthesis of collagen I in human
dermal fibroblasts, in comparison to nonmodified membranes [244]. Growth fac-
tors, such as FGF-2, epithelial growth factor (EGF) and keratinocyte growth factor
(KGF), can also be added to fibrin scaffolds to improve wound healing, as shown in
animal studies [209]. In clinical trials, it has been shown that extensive burned area
can be covered with a cultured keratinocyte-fibrin suspension [242]. However, in
third-degree burns, the grafts failed to show mechanical stability. To overcome the
low mechanical strength of fibrin-based implants while maintaining adhesion and
proliferation of keratinocytes, synthetic membranes such as polycaprolactone
(PLCL) were coated with fibrin glue [245].
The major drawback of tissue substitutes is the lack of blood supply. One
approach to promote angiogenesis is the use of fibrin matrices, because fibrin is
involved in healing process, provides specific adhesion sequences for endothelial
cells ,and it binds VEGF and FGF-2 with high affinity [246]. In a recent study,
human umbilical vein endothelial cells (HUVECs) were seeded with dermal
fibroblasts and adipose-derived MSCs in a fibrin matrix to produce a novel artificial
skin substitute. This tissue-engineered construct showed a similar structure to native
skin, with endothelial cells proliferating and organising themselves into
capillary-like structures within the fibrin matrix [247].
134 G. Fontana et al.

Another method to produce fibrin- and fibrinogen-based scaffolds is electro-

spinning. This process allows for fabrication of fibrous, nonwoven structures, which
have been applied in the fields of heart and wound repair. Electrospun fibrinogen
scaffolds displayed the geometry of a native provisional wound matrix, and addition
of aprotinin maintained the necessary mechanical integrity for use in wound
dressing [248]. In an in vitro model, fibroblasts migrated into and remodelled the
electrospun fibrinogen scaffold with deposition of collagen. Interestingly, different
concentrations of aprotinin modulated scaffold degradation in a predictable fashion,
whereas glutaraldehyde vapour fixation was less reliable [249]. As glutaraldehyde
vapour crosslinking did not demonstrate any significant improvement in the
mechanical properties of electrospun fibrinogen, authors concluded that the process
of electrospinning does not expose the lysine residues necessary for glutaraldehyde
crosslinking [248, 249]. In a further study, the same group tested three different
crosslinkers on an electrospun fibrinogen scaffold. EDC and genipin in ethanol
significantly enhanced scaffold mechanical properties and slowed degradation rate
in vitro, whereas glutaraldehyde crosslinking failed to produce any significant
improvement. However, crosslinking with EDC and genipin was shown to reduce
scaffold bioactivity, as fibroblasts migration below the surface and scaffold
remodelling were negatively affected [250]. Authors suggested that this poor
bioactivity may have been due to the increased scaffold modulus that could have
made fibres too stiff to allow for subsurface cell migration, or due to the
crosslinking process, that may have masked the integrin binding sites that are
normally exposed on fibrinogen.
In order to overcome the limitations of the above-mentioned crosslinking
methods, the application of a natural crosslinker has been investigated for the
treatment of myocardial infarction. Taking advantage of the high affinity of phytic
acid for 2,3-diphosphoglycerate site in haemoglobin, Ravichandra and colleagues
used phytic acid to crosslink an electrospun construct composed of haemoglobin/
gelatin/fibrinogen, as well as to improve oxygen binding capability: the resulting
scaffold supported MSC differentiation into functional cardiomyocytes and showed
an improved delivery of oxygen even at higher oxygen tensions, that is a desirable
feature in the treatment of ischemic heart [251]. In another study, the need of
synthetic crosslinker agents was overcome by combining fibrinogen and thrombin
within the syringe of an electrospinning set-up: in this fashion fibrin which is
superior to fibrinogen in mechanical properties can be electrospun successfully in a
one-step approach. The characterisation of the fibrin-based electrospun nanofibrous
scaffold revealed the formation of a structure that mimics the native ECM and
enhances cell attachment and proliferation [252].
Fibrin, fibrinogen and thrombin can also be utilised as a bio-ink to produce
specific fibrin 3D patterns. Fibrin gels have a low viscosity allowing easy ejection,
controllable crosslinking mechanism and fast gelation. Due to the short gelation
times and high degradation rates, fibrinogen-/fibrin-based inks are often mixed with
other materials [253]. A bio-ink made of thrombin and human endothelial cells was
printed into a fibrinogen substrate to form fibrin channels. The printed endothelial
cells proliferated to form a tubular structure inside the fibrin channel, suggesting
5 Biologically Inspired Materials in Tissue Engineering 135

that thermal inkjet printing technology can be a promising approach for

microvasculature tissue engineering [254].
Bioprinting is another strategy to produce cell microenvironments with
well-defined spatial patterns of immobilized proteins [255]. In this emerging area of
tissue engineering, fibrin can be an ideal printing substrate because of its capacity to
bind several growth factors. For example, bone morphogenic protein-2 (BMP2)
[256], heparin-binding EGF [257] and FGF-2 [258] were printed and naturally
immobilized onto fibrin-coated substrates in order to evaluate the effect of patterned
growth factor gradients on cell behaviour.
In the light of the data presented, fibrin and fibrinogen stand out as valuable and
versatile natural materials for supplying a provisional matrix and delivering cells,
biomolecules and genes to damaged tissue. In comparison to other natural mate-
rials, fibrin is particularly appealing because of its excellent biocompatibility,
controllable biodegradability, multiple interaction sites for cells and other proteins
and ease of fabrication of completely autologous scaffolds. However, some limi-
tations, such as low mechanical strength and high degradation rate, need to be
addressed.

References

1. Lim EH, Sardinha JP, Myers S. Nanotechnology biomimetic cartilage regenerative scaffolds.
Arch plastic Surg. 2014;41:231–40.
2. Abbah SA, Delgado LM, Azeem A, Fuller K, Shologu N, Keeney M, et al. Harnessing
hierarchical nano- and micro-fabrication technologies for musculoskeletal tissue engineer-
ing. Adv Healthc Mater. 2015;4:2488–99.
3. Fuller K, Pandit A, Zeugolis DI. The multifaceted potential of electro-spinning in
regenerative medicine. Pharm Nanotechnol. 2014;2:23–34.
4. Carbone EJ, Jiang T, Nelson C, Henry N, Lo KWH. Small molecule delivery through
nanofibrous scaffolds for musculoskeletal regenerative engineering. Nanomed Nanotechnol
Biol Med. 2014;10:1691–9.
5. Woo KM, Chen VJ, Ma PX. Nano-fibrous scaffolding architecture selectively enhances
protein adsorption contributing to cell attachment. J Biomed Mater Res A. 2003;67A:531–7.
6. Ma PX. Biomimetic materials for tissue engineering. Adv Drug Deliv Rev. 2008;60:184–98.
7. Barnes CP, Sell SA, Boland ED, Simpson DG, Bowlin GL. Nanofiber technology: designing
the next generation of tissue engineering scaffolds. Adv Drug Deliv Rev. 2007;59:1413–33.
8. Shields KJ, Beckman MJ, Bowlin GL, Wayne JS. Mechanical properties and cellular
proliferation of electrospun collagen type II. Tissue Eng. 2004;10:1510–7.
9. Heydarkhan-Hagvall S, Schenke-Layland K, Dhanasopon AP, Rofail F, Smith H, Wu BM,
et al. Three-dimensional electrospun ECM-based hybrid scaffolds for cardiovascular tissue
engineering. Biomaterials. 2008;29:2907–14.
10. Fleischer S, Shapira A, Regev O, Nseir N, Zussman E, Dvir T. Albumin fiber scaffolds for
engineering functional cardiac tissues. Biotechnol Bioeng. 2014;111:1246–57.
11. Nicodemus GD, Bryant SJ. Cell encapsulation in biodegradable hydrogels for tissue
engineering applications. Tissue Eng Part B Rev. 2008;14:149–65.
136 G. Fontana et al.

12. Hong Y, Takanari K, Amoroso NJ, Hashizume R, Brennan-Pierce EP, Freund JM, et al.
An elastomeric patch electrospun from a blended solution of dermal extracellular matrix and
biodegradable polyurethane for rat abdominal wall repair. Tissue Eng Part C Methods.
2012;18:122–32.
13. Harkness RD. Biological functions of collagen. Biol Rev Camb Philos Soc. 1961;36:399–463.
14. Soderhall C, Marenholz I, Kerscher T, Ruschendorf F, Esparza-Gordillo J, Worm M, et al.
Variants in a novel epidermal collagen gene (COL29A1) are associated with atopic
dermatitis. PLoS Biol. 2007;5:e242.
15. Kielty CM, Grant ME. The collagen family: structure, assembly and organization in
the extracellular matrix. In: Royce PM, Steinmann B, editors. Connective tissue and its
heritable disorders: molecular, genetic and medical aspects. 2nd ed. New York: Wiley; 2002.
p. 159–221.
16. Piez KA. Collagen. In: Kroschwitz JI, editor. Encyclopedia of polymer science and
engineering. New York: Wiley; 1985. p. 699–727.
17. Zeugolis DI, Raghunath M. Collagen: materials analysis and implant uses. In: Ducheyne P,
Healy KE, Hutmacher DW, Grainger DW, Kirkpatrick CJ, editors. Comprehensive
biomaterials. Oxford: Elsevier; 2011. p. 261–78.
18. Friess W. Collagen—biomaterial for drug delivery. Eur J Pharm Biopharm. 1998;45:113–36.
19. Zeugolis DI, Paul GR, Attenburrow G. Cross-linking of extruded collagen fibres—a
biomimetic three-dimensional scaffold for tissue engineering applications. J Biomed Mater
Res A. 2009;89:895–908.
20. Zeugolis DI, Paul RG, Attenburrow G. The influence of a natural cross-linking agent
(Myrica rubra) on the properties of extruded collagen fibres for tissue engineering
applications. Mater Sci Eng C. 2010;30:190–5.
21. Delgado LM, Fuller K, Zeugolis DI. Collagen cross-linking: biophysical, biochemical, and
biological response analysis. Tissue Eng Part A. 2017;23:1064–77.
22. Gough JE, Scotchford CA, Downes S. Cytotoxicity of glutaraldehyde crosslinked collagen/
poly(vinyl alcohol) films is by the mechanism of apoptosis. J Biomed Mater Res.
2002;61:121–30.
23. Levy RJ, Schoen FJ, Sherman FS, Nichols J, Hawley MA, Lund SA. Calcification of
subcutaneously implanted type I collagen sponges. Effects of formaldehyde and glutaralde-
hyde pretreatments. Am J Pathol. 1986;122:71–82.
24. Brown BN, Londono R, Tottey S, Zhang L, Kukla KA, Wolf MT, et al. Macrophage
phenotype as a predictor of constructive remodeling following the implantation of
biologically derived surgical mesh materials. Acta Biomater. 2012;8:978–87.
25. Delgado LM, Bayon Y, Pandit A, Zeugolis DI. To cross-link or not to cross-link?
Cross-linking associated foreign body response of collagen-based devices. Tissue Eng Part B
Rev. 2015;21:298–313.
26. Collin EC, Grad S, Zeugolis DI, Vinatier CS, Clouet JR, Guicheux JJ, et al. An injectable
vehicle for nucleus pulposus cell-based therapy. Biomaterials. 2011;32:2862–70.
27. Rho KS, Jeong L, Lee G, Seo BM, Park YJ, Hong SD, et al. Electrospinning of collagen
nanofibers: effects on the behavior of normal human keratinocytes and early-stage wound
healing. Biomaterials. 2006;27:1452–61.
28. Li M, Mondrinos MJ, Gandhi MR, Ko FK, Weiss AS, Lelkes PI. Electrospun protein fibers
as matrices for tissue engineering. Biomaterials. 2005;26:5999–6008.
29. Zeugolis DI, Khew ST, Yew ESY, Ekaputra AK, Tong YW, Yung L-YL, et al.
Electro-spinning of pure collagen nano-fibres—just an expensive way to make gelatin?
Biomaterials. 2008;29:2293–305.
30. Browne S, Zeugolis DI, Pandit A. Collagen: finding a solution for the source. Tissue Eng
Part A. 2013;19:1491–4.
31. IAEA. Trends in radiation sterilization of health care products. Vienna: International Atomic
Energy Agency; 2008.
5 Biologically Inspired Materials in Tissue Engineering 137

32. Hori H, Hattori S, Inouye S, Kimura A, Irie S, Miyazawa H, et al. Analysis of the major
epitope of the a2 chain of bovine type I collagen in children with bovine gelatin allergy.
J Allergy Clin Immunol. 2002;110:652–7.
33. Lynn AK, Yannas IV, Bonfield W. Antigenicity and immunogenicity of collagen. J Biomed
Mater Res Part B Appl Biomater. 2004;71:343–54.
34. Delgado LM, Shologu N, Fuller K, Zeugolis D. Acetic acid and pepsin result in high yield,
high purity and low macrophage response collagen for biomedical applications. Biomed
Mater. 2017;12:065009.
35. Tang Y, Yang X, Hang B, Li J, Huang L, Huang F, et al. Efficient production of
hydroxylated human-like collagen via the co-expression of three key genes in escherichia
coli origami (DE3). Appl Biochem Biotechnol. 2016;178:1458–70.
36. Ruggiero F, Koch M. Making recombinant extracellular matrix proteins. Methods
(San Diego, Calif). 2008;45:75–85.
37. Yang C, Hillas PJ, Baez JA, Nokelainen M, Balan J, Tang J, et al. The application of
recombinant human collagen in tissue engineering. BioDrugs Clin Immunother Biopharm
Gene Ther. 2004;18:103–19.
38. Parenteau-Bareil R, Gauvin R, Berthod F. Collagen-based biomaterials for tissue engineer-
ing applications. Materials. 2010;3:1863–87.
39. Gosline J, Lillie M, Carrington E, Guerette P, Ortlepp C, Savage K. Elastic proteins:
biological roles and mechanical properties. Philos Trans R Soc Lond B Biol Sci.
2002;357:121–32.
40. Silva R, Fabry B, Boccaccini AR. Fibrous protein-based hydrogels for cell encapsulation.
Biomaterials. 2014;35:6727–38.
41. Miranda-Nieves D, Chaikof EL. Collagen and elastin biomaterials for the fabrication of
engineered living tissues. ACS Biomater Sci Eng. 2017;3:694–711.
42. Daamen WF, Veerkamp JH, van Hest JC, van Kuppevelt TH. Elastin as a biomaterial for
tissue engineering. Biomaterials. 2007;28:4378–98.
43. Werkmeister JA, Ramshaw JA. Recombinant protein scaffolds for tissue engineering.
Biomed Mater. 2012;7:012002.
44. Caves JM, Cui W, Wen J, Kumar VA, Haller CA, Chaikof EL. Elastin-like protein matrix
reinforced with collagen microfibers for soft tissue repair. Biomaterials. 2011;32:5371–9.
45. Devalliere J, Dooley K, Hu Y, Kelangi SS, Uygun BE, Yarmush ML. Co-delivery of a
growth factor and a tissue-protective molecule using elastin biopolymers accelerates wound
healing in diabetic mice. Biomaterials. 2017;141:149–60.
46. Monfort DA, Koria P. Recombinant elastin based nanoparticles for targeted gene therapy.
Gene Ther. 2017;24:610–20.
47. Waterhouse A, Wise SG, Ng MK, Weiss AS. Elastin as a nonthrombogenic biomaterial.
Tissue Eng Part B Rev. 2011;17:93–9.
48. Liu SQ, Tieche C, Alkema PK. Neointima formation on vascular elastic laminae and
collagen matrices scaffolds implanted in the rat aortae. Biomaterials. 2004;25:1869–82.
49. Henry JJD, Yu J, Wang A, Lee R, Fang J, Li S. Engineering the mechanical and biological
properties of nanofibrous vascular grafts for in situ vascular tissue engineering.
Biofabrication. 2017;9:035007.
50. Waterhouse A, Yin Y, Wise SG, Bax DV, McKenzie DR, Bilek MM, et al. The
immobilization of recombinant human tropoelastin on metals using a plasma-activated
coating to improve the biocompatibility of coronary stents. Biomaterials. 2010;31:8332–40.
51. Woodhouse KA, Klement P, Chen V, Gorbet MB, Keeley FW, Stahl R, et al. Investigation
of recombinant human elastin polypeptides as non-thrombogenic coatings. Biomaterials.
2004;25:4543–53.
52. Jordan SW, Haller CA, Sallach RE, Apkarian RP, Hanson SR, Chaikof EL. The effect of a
recombinant elastin-mimetic coating of an ePTFE prosthesis on acute thrombogenicity in a
baboon arteriovenous shunt. Biomaterials. 2007;28:1191–7.
138 G. Fontana et al.

53. Wachi H, Sugitani H, Murata H, Nakazawa J, Mecham RP, Seyama Y. Tropoelastin inhibits
vascular calcification via 67-kDa elastin binding protein in cultured bovine aortic smooth
muscle cells. J Atherosclerosis Thromb. 2004;11:159–66.
54. Almine JF, Bax DV, Mithieux SM, Nivison-Smith L, Rnjak J, Waterhouse A, et al.
Elastin-based materials. Chem Soc Rev. 2010;39:3371–9.
55. Wong CS, Liu X, Xu Z, Lin T, Wang X. Elastin and collagen enhances electrospun aligned
polyurethane as scaffolds for vascular graft. J Mater Sci Mater Med. 2013;24:1865–74.
56. McClure MJ, Sell SA, Simpson DG, Walpoth BH, Bowlin GL. A three-layered electrospun
matrix to mimic native arterial architecture using polycaprolactone, elastin, and collagen: a
preliminary study. Acta Biomater. 2010;6:2422–33.
57. Foraida ZI, Kamaldinov T, Nelson DA, Larsen M, Castracane J. Elastin-PLGA hybrid
electrospun nanofiber scaffolds for salivary epithelial cell self-organization and polarization.
Acta Biomater. 2017;62:116–27.
58. Feughelman M. Natural protein fibers. J Appl Polym Sci. 2002;83:489–507.
59. Shavandi A, Silva TH, Bekhit AA, Bekhit AEA. Keratin: dissolution, extraction and
biomedical application. Biomater Sci. 2017;5:1699–735.
60. Rouse JG, Van Dyke ME. A review of keratin-based biomaterials for biomedical
applications. Materials. 2010;3:999–1014.
61. Sierpinski P, Garrett J, Ma J, Apel P, Klorig D, Smith T, et al. The use of keratin
biomaterials derived from human hair for the promotion of rapid regeneration of peripheral
nerves. Biomaterials. 2008;29:118–28.
62. Ham TR, Lee RT, Han S, Haque S, Vodovotz Y, Gu J, et al. Tunable keratin hydrogels for
controlled erosion and growth factor delivery. Biomacromol. 2016;17:225–36.
63. Pace LA, Plate JF, Mannava S, Barnwell JC, Koman LA, Li Z, et al. A human hair keratin
hydrogel scaffold enhances median nerve regeneration in nonhuman primates: an
electrophysiological and histological study. Tissue Eng Part A. 2014;20:507–17.
64. Blanchard CR, Timmons SF, Smith RA. Keratin-based hydrogel for biomedical applications
and method of production. 1999.
65. Kaplan D, Adams WW, Farmer B, Viney C. Silk: biology, structure, properties, and
genetics. Silk Polymers: American Chemical Society; 1993. p. 2–16.
66. Kasoju N, Bora U. Silk fibroin in tissue engineering. Adv Healthc Mater. 2012;1:393–412.
67. Zhou CZ, Confalonieri F, Jacquet M, Perasso R, Li ZG, Janin J. Silk fibroin: structural
implications of a remarkable amino acid sequence. Proteins. 2001;44:119–22.
68. Rockwood DN, Preda RC, Yucel T, Wang X, Lovett ML, Kaplan DL. Materials fabrication
from Bombyx mori silk fibroin. Nat Protoc. 2011;6:1612–31.
69. Kundu B, Rajkhowa R, Kundu SC, Wang X. Silk fibroin biomaterials for tissue
regenerations. Adv Drug Deliv Rev. 2013;65:457–70.
70. Wang Y, Rudym DD, Walsh A, Abrahamsen L, Kim H-J, Kim HS, et al. In vivo degradation
of three-dimensional silk fibroin scaffolds. Biomaterials. 2008;29:3415–28.
71. Lovett ML, Cannizzaro CM, Vunjak-Novakovic G, Kaplan DL. Gel spinning of silk tubes
for tissue engineering. Biomaterials. 2008;29:4650–7.
72. Zhang X, Wang X, Keshav V, Wang X, Johanas JT, Leisk GG, et al. Dynamic culture
conditions to generate silk-based tissue-engineered vascular grafts. Biomaterials.
2009;30:3213–23.
73. Zamani M, Khafaji M, Naji M, Vossoughi M, Alemzadeh I, Haghighipour N. A biomimetic
heparinized composite silk-based vascular scaffold with sustained antithrombogenicity. Sci
Rep. 2017;7:4455.
74. Cattaneo I, Figliuzzi M, Azzollini N, Catto V, Fare S, Tanzi MC, et al. In vivo regeneration
of elastic lamina on fibroin biodegradable vascular scaffold. Int J Artif Organs. 2013;36:
166–74.
75. Zhang X, Reagan MR, Kaplan DL. Electrospun silk biomaterial scaffolds for regenerative
medicine. Adv Drug Delivery Rev. 2009;61:988–1006.
5 Biologically Inspired Materials in Tissue Engineering 139

76. Song DW, Kim SH, Kim HH, Lee KH, Ki CS, Park YH. Multi-biofunction of antimicrobial
peptide-immobilized silk fibroin nanofiber membrane: Implications for wound healing. Acta
Biomater. 2016;39:146–55.
77. Gil ES, Panilaitis B, Bellas E, Kaplan DL. Functionalized silk biomaterials for wound
healing. Adv Healthc Mater. 2013;2:206–17.
78. Liu W, Thomopoulos S, Xia Y. Electrospun nanofibers for regenerative medicine. Adv
Healthc Mater. 2012;1:10–25.
79. Binan L, Tendey C, De Crescenzo G, El Ayoubi R, Ajji A, Jolicoeur M. Differentiation of
neuronal stem cells into motor neurons using electrospun poly-L-lactic acid/gelatin scaffold.
Biomaterials. 2014;35:664–74.
80. Liu S, Qin M, Hu C, Wu F, Cui W, Jin T, et al. Tendon healing and anti-adhesion properties
of electrospun fibrous membranes containing bFGF loaded nanoparticles. Biomaterials.
2013;34:4690–701.
81. Ortega I, Ryan AJ, Deshpande P, MacNeil S, Claeyssens F. Combined microfabrication and
electrospinning to produce 3-D architectures for corneal repair. Acta Biomater.
2013;9:5511–20.
82. Hu X, Liu S, Zhou G, Huang Y, Xie Z, Jing X. Electrospinning of polymeric nanofibers for
drug delivery applications. J Controlled Release. 2014;185:12–21.
83. Katti DS, Robinson KW, Ko FK, Laurencin CT. Bioresorbable nanofiber-based systems for
wound healing and drug delivery: optimization of fabrication parameters. J Biomed Mater
Res Part B Appl Biomater. 2004;70:286–96.
84. Cramariuc B, Cramariuc R, Scarlet R, Manea LR, Lupu IG, Cramariuc O. Fiber diameter in
electrospinning process. J Electrostat. 2013;71:189–98.
85. Fuller KP, Gaspar D, Delgado LM, Pandit A, Zeugolis DI. Influence of porosity and pore
shape on structural, mechanical and biological properties of poly -caprolactone electro-spun
fibrous scaffolds. Nanomedicine (London, England). 2016;11:1031–40.
86. Yin Z, Chen X, Chen JL, Shen WL, Hieu Nguyen TM, Gao L, et al. The regulation
of tendon stem cell differentiation by the alignment of nanofibers. Biomaterials.
2010;31:2163–75.
87. Lee BK, Ju YM, Cho JG, Jackson JD, Lee SJ, Atala A, et al. End-to-side neurorrhaphy using
an electrospun PCL/collagen nerve conduit for complex peripheral motor nerve regeneration.
Biomaterials. 2012;33:9027–36.
88. Xie J, MacEwan M, Ray W, Liu W, Siewe D, Xia Y. Radially aligned, electrospun
nanofibers as dural substitutes for wound closure and tissue regeneration applications. ACS
Nano. 2010;4:5027–36.
89. Whitesides GM, Grzybowski B. Self-assembly at all scales. Science (New York, NY).
2002;295:2418–21.
90. Friess W. Collagen–biomaterial for drug delivery. Eur J Pharm Biopharm. 1998;45:113–36.
91. Zeugolis DI, Paul RG, Attenburrow G. Extruded collagen-polyethylene glycol fibers
for tissue engineering applications. J Biomed Mater Res Part B Appl Biomater. 2008;85:
343–52.
92. Daly WT, Yao L, Abu-rub MT, O’Connell C, Zeugolis DI, Windebank AJ, et al. The effect
of intraluminal contact mediated guidance signals on axonal mismatch during peripheral
nerve repair. Biomaterials. 2012;33:6660–71.
93. Abu-Rub MT, Billiar KL, van Es MH, Knight A, Rodriguez BJ, Zeugolis DI, et al.
Nano-textured self-assembled aligned collagen hydrogels promote directional neurite
guidance and overcome inhibition by myelin associated glycoprotein. Soft Matter.
2011;7:2770.
94. Li Y, Asadi A, Monroe MR, Douglas EP. pH effects on collagen fibrillogenesis in vitro:
electrostatic interactions and phosphate binding. Mater Sci Eng C. 2009;29:1643–9.
95. Olmos Buitrago J, Perez RA, El-Fiqi A, Singh RK, Kim JH, Kim HW. Core-shell fibrous
stem cell carriers incorporating osteogenic nanoparticulate cues for bone tissue engineering.
Acta Biomater. 2015;28:183–92.
140 G. Fontana et al.

96. Perez RA, Kim HW. Core-shell designed scaffolds for drug delivery and tissue engineering.
Acta Biomater. 2015;21:2–19.
97. Perez RA, Kim JH, Buitrago JO, Wall IB, Kim HW. Novel therapeutic core-shell hydrogel
scaffolds with sequential delivery of cobalt and bone morphogenetic protein-2 for synergistic
bone regeneration. Acta Biomater. 2015;23:295–308.
98. Perez RA, Kim M, Kim TH, Kim JH, Lee JH, Park JH, et al. Utilizing core-shell fibrous
collagen-alginate hydrogel cell delivery system for bone tissue engineering. Tissue Eng
Part A. 2014;20:103–14.
99. Hartgerink JD, Beniash E, Stupp SI. Peptide-amphiphile nanofibers: a versatile scaffold for
the preparation of self-assembling materials. Proc Natl Acad Sci U S A. 2002;99:5133–8.
100. Hartgerink JD, Beniash E, Stupp SI. Self-assembly and mineralization of peptide-amphiphile
nanofibers. Science (New York, NY). 2001;294:1684–8.
101. Loo Y, Zhang SG, Hauser CAE. From short peptides to nanofibers to macromolecular
assemblies in biomedicine. Biotechnol Adv. 2012;30:593–603.
102. Saracino GA, Cigognini D, Silva D, Caprini A, Gelain F. Nanomaterials design and tests for
neural tissue engineering. Chem Soc Rev. 2013;42:225–62.
103. Cunha C, Panseri S, Villa O, Silva D, Gelain F. 3D culture of adult mouse neural stem cells
within functionalized self-assembling peptide scaffolds. Int J Nanomed. 2011;6:943–55.
104. Yang H, Xie Z, Wang P, Bi J. Self-assembling nanofibers alter the processing of amyloid
precursor protein in a transgenic mouse model of Alzheimer’s disease. Neurol Res.
2015;37:84–91.
105. Cigognini D, Satta A, Colleoni B, Silva D, Donega M, Antonini S, et al. Evaluation of early
and late effects into the acute spinal cord injury of an injectable functionalized
self-assembling scaffold. PLoS ONE. 2011;6:e19782.
106. Cigognini D, Silva D, Paloppi S, Gelain F. Evaluation of mechanical properties and
therapeutic effect of injectable self-assembling hydrogels for spinal cord injury. J Biomed
Nanotechnol. 2014;10:309–23.
107. Kisiday J, Jin M, Kurz B, Hung H, Semino C, Zhang S, et al. Self-assembling peptide
hydrogel fosters chondrocyte extracellular matrix production and cell division: implications
for cartilage tissue repair. Proc Natl Acad Sci U S A. 2002;99:9996–10001.
108. Schneider A, Garlick JA, Egles C. Self-assembling peptide nanofiber scaffolds accelerate
wound healing. PLoS ONE. 2008;3:e1410.
109. Ma PX, Zhang R. Synthetic nano-scale fibrous extracellular matrix. J Biomed Mater Res.
1999;46:60–72.
110. Stokols S, Sakamoto J, Breckon C, Holt T, Weiss J, Tuszynski MH. Templated agarose
scaffolds support linear axonal regeneration. Tissue Eng. 2006;12:2777–87.
111. Stokols S, Tuszynski MH. The fabrication and characterization of linearly oriented nerve
guidance scaffolds for spinal cord injury. Biomaterials. 2004;25:5839–46.
112. Garcia Y, Wilkins B, Collighan RJ, Griffin M, Pandit A. Towards development of a dermal
rudiment for enhanced wound healing response. Biomaterials. 2008;29:857–68.
113. Stokols S, Tuszynski MH. Freeze-dried agarose scaffolds with uniaxial channels stimulate
and guide linear axonal growth following spinal cord injury. Biomaterials. 2006;27:443–51.
114. Bozkurt A, Lassner F, O’Dey D, Deumens R, Bocker A, Schwendt T, et al. The role of
microstructured and interconnected pore channels in a collagen-based nerve guide on axonal
regeneration in peripheral nerves. Biomaterials. 2012;33:1363–75.
115. Chen H, Yang X, Liao Y, Zeng X, Liang P, Kang N, et al. MRI and histologic analysis of
collagen type II sponge on repairing the cartilage defects of rabbit knee joints. J Biomed
Mater Res Part B Appl Biomater. 2011;96:267–75.
116. Shen W, Chen X, Hu Y, Yin Z, Zhu T, Hu J, et al. Long-term effects of knitted silk–collagen
sponge scaffold on anterior cruciate ligament reconstruction and osteoarthritis prevention.
Biomaterials. 2014;35:8154–63.
117. Yao L, Daly W, Newland B, Yao S, Wang W, Chen BK, et al. Improved axonal regeneration
of transected spinal cord mediated by multichannel collagen conduits functionalized with
neurotrophin-3 gene. Gene Ther. 2013;20:1149–57.
5 Biologically Inspired Materials in Tissue Engineering 141

118. Caliari SR. Harley BaC. The effect of anisotropic collagen-GAG scaffolds and growth factor
supplementation on tendon cell recruitment, alignment, and metabolic activity. Biomaterials.
2011;32:5330–40.
119. Caliari SR, Ramirez MA, Harley BAC. The development of collagen-GAG
scaffold-membrane composites for tendon tissue engineering. Biomaterials. 2011;32:8990–8.
120. Boekema BK, Vlig M, Olde Damink L, Middelkoop E, Eummelen L, Buhren AV, et al.
Effect of pore size and cross-linking of a novel collagen-elastin dermal substitute on wound
healing. J Mater Sci Mater Med. 2014;25:423–33.
121. Min BM, Jeong L, Nam YS, Kim JM, Kim JY, Park WH. Formation of silk fibroin matrices
with different texture and its cellular response to normal human keratinocytes. Int J Biol
Macromol. 2004;34:281–8.
122. Daley WP, Peters SB, Larsen M. Extracellular matrix dynamics in development and
regenerative medicine. J Cell Sci. 2008;121:255–64.
123. Geiger B, Bershadsky A, Pankov R, Yamada KM. Transmembrane crosstalk between the
extracellular matrix–cytoskeleton crosstalk. Nat Rev Mol Cell Biol. 2001;2:793–805.
124. Kong HJ, Mooney DJ. Microenvironmental regulation of biomacromolecular therapies. Nat
Rev Drug Discovery. 2007;6:455–63.
125. Aszodi A, Hunziker EB, Brakebusch C, Fassler R. Beta1 integrins regulate chondrocyte
rotation, G1 progression, and cytokinesis. Genes Dev. 2003;17:2465–79.
126. Schneiderbauer MM, Dutton CM, Scully SP. Signaling “cross-talk” between TGF-beta1 and
ECM signals in chondrocytic cells. Cell Signal. 2004;16:1133–40.
127. Fontana G, See E, Pandit A. Current trends in biologics delivery to restore intervertebral disc
anabolism. Adv Drug Deliv Rev. 2015;84:146–58.
128. Teti A. Regulation of Cellular Functions by Extracellular-Matrix. J Am Soc Nephrol.
1992;2:S83–7.
129. Kim SH, Turnbull J, Guimond S. Extracellular matrix and cell signalling: the dynamic
cooperation of integrin, proteoglycan and growth factor receptor. J Endocrinol.
2011;209:139–51.
130. Bosnakovski D, Mizuno M, Kim G, Takagi S, Okumura M, Fujinaga T. Chondrogenic
differentiation of bovine bone marrow mesenchymal stem cells (MSCs) in different
hydrogels: influence of collagen type II extracellular matrix on MSC chondrogenesis.
Biotechnol Bioeng. 2006;93:1152–63.
131. DeLise AM, Fischer L, Tuan RS. Cellular interactions and signaling in cartilage
development. Osteoarthritis Cartilage. 2000;8:309–34.
132. Masuda K, An HS. Growth factors and the intervertebral disc. Spine J. 2004;4:330S–40S.
133. Masuda K, Oegema TR, Jr., An HS. Growth factors and treatment of intervertebral disc
degeneration. Spine (Phila Pa 1976). 2004;29:2757–69.
134. Masuda K, Lotz JC. New challenges for intervertebral disc treatment using regenerative
medicine. Tissue Eng Part B Rev. 2010;16:147–58.
135. Eppler SM, Combs DL, Henry TD, Lopez JJ, Ellis SG, Yi JH, et al. A target-mediated model
to describe the pharmacokinetics and hemodynamic effects of recombinant human vascular
endothelial growth factor in humans. Clin Pharmacol Ther. 2002;72:20–32.
136. Lee K, Silva EA, Mooney DJ. Growth factor delivery-based tissue engineering: general
approaches and a review of recent developments. J R Soc Interface. 2011;8:153–70.
137. Saik JE, Gould DJ, Watkins EM, Dickinson ME, West JL. Covalently immobilized
platelet-derived growth factor-BB promotes angiogenesis in biomimetic poly(ethylene
glycol) hydrogels. Acta Biomater. 2011;7:133–43.
138. Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med. 2000;6:389–95.
139. Lee RJ, Springer ML, Blanco-Bose WE, Shaw R, Ursell PC, Blau HM. VEGF gene delivery
to myocardium—deleterious effects of unregulated expression. Circulation. 2000;102:
898–901.
140. Yang WW, Pierstorff E. Reservoir-based polymer drug delivery systems. Jala-J Lab Autom.
2012;17:50–8.
142 G. Fontana et al.

141. Fontana G, Thomas D, Collin E, Pandit A. Microgel microenvironment primes

adipose-derived stem cells towards an NP cells-like phenotype. Adv Healthc Mater.
2014;3:2012–22.
142. Zhang ZP, Hu J, Ma PX. Nanofiber-based delivery of bioactive agents and stem cells to bone
sites. Adv Drug Deliver Rev. 2012;64:1129–41.
143. De Laporte L, Shea LD. Matrices and scaffolds for DNA delivery in tissue engineering. Adv
Drug Deliver Rev. 2007;59:292–307.
144. Li RH, Wozney JM. Delivering on the promise of bone morphogenetic proteins. Trends
Biotechnol. 2001;19:255–65.
145. Chen RR, Mooney DJ. Polymeric growth factor delivery strategies for tissue engineering.
Pharm Res. 2003;20:1103–12.
146. Elisseeff J, Puleo C, Yang F, Sharma B. Advances in skeletal tissue engineering with
hydrogels. Orthod Craniofac Res. 2005;8:150–61.
147. Lutolf MR, Weber FE, Schmoekel HG, Schense JC, Kohler T, Muller R, et al. Repair of
bone defects using synthetic mimetics of collagenous extracellular matrices. Nat Biotechnol.
2003;21:513–8.
148. Place ES, Evans ND, Stevens MM. Complexity in biomaterials for tissue engineering. Nat
Mater. 2009;8:457–70.
149. Thomas D, Fontana G, Chen X, Sanz-Nogues C, Zeugolis DI, Dockery P, et al.
A shape-controlled tuneable microgel platform to modulate angiogenic paracrine responses
in stem cells. Biomaterials. 2014;35:8757–66.
150. Baumgartner I, Pieczek A, Manor O, Blair R, Kearney M, Walsh K, et al. Constitutive
expression of phVEGF(165) after intramuscular gene transfer promotes collateral vessel
development in patients with critical limb ischemia. Circulation. 1998;97:1114–23.
151. Zisch AH, Schenk U, Schense JC, Sakiyama-Elbert SE, Hubbell JA. Covalently conjugated
VEGF-fibrin matrices for endothelialization. J Control Release. 2001;72:101–13.
152. Koch S, Yao C, Grieb G, Prevel P, Noah EM, Steffens GCM. Enhancing angiogenesis in
collagen matrices by covalent incorporation of VEGF. J Mater Sci Mater Med.
2006;17:735–41.
153. Haro H, Kato T, Komori H, Osada M, Shinomiya K. Vascular endothelial growth factor
(VEGF)-induced angiogenesis in herniated disc resorption. J Orthop Res. 2002;20:409–15.
154. Ehrbar M, Djonov VG, Schnell C, Tschanz SA, Martiny-Baron G, Schenk U, et al.
Cell-demanded liberation of VEGF(121) from fibrin implants induces local and controlled
blood vessel growth. Circ Res. 2004;94:1124–32.
155. Post MJ, Laham R, Sellke FW, Simons M. Therapeutic angiogenesis in cardiology using
protein formulations. Cardiovasc Res. 2001;49:522–31.
156. Zisch AH, Lutolf MP, Ehrbar M, Raeber GP, Rizzi SC, Davies N, et al. Cell-demanded
release of VEGF from synthetic, biointeractive cell-ingrowth matrices for vascularized tissue
growth. Faseb J. 2003;17:2260–2.
157. Sakiyama SE, Schense JC, Hubbell JA. Incorporation of heparin-binding peptides into fibrin
gels enhances neurite extension: an example of designer matrices in tissue engineering.
Faseb J. 1999;13:2214–24.
158. Sakiyama-Elbert SE, Panitch A, Hubbell JA. Development of growth factor fusion proteins
for cell-triggered drug delivery. Faseb J. 2001;15:1300–2.
159. Schense JC, Hubbell JA. Cross-linking exogenous bifunctional peptides into fibrin gels with
factor XIIIa. Bioconjugate Chem. 1999;10:75–81.
160. Wissink MJB, Beernink R, Scharenborg NM, Poot AA, Engbers GHM, Beugeling T, et al.
Endothelial cell seeding of (heparinized) collagen matrices: effects of bFGF pre-loading on
proliferation (after low density seeding) and pro-coagulant factors. J Controlled Release.
2000;67:141–55.
161. Grieb G, Groger A, Piatkowski A, Markowicz M, Steffens GCM, Pallua N. Tissue
substitutes with improved angiogenic capabilities: an in vitro investigation with endothelial
cells and endothelial progenitor cells. Cells Tissues Organs. 2010;191:96–104.
5 Biologically Inspired Materials in Tissue Engineering 143

162. Markowicz M, Heitland A, Steffens GCM, Pallua N. Effects of modified collagen matrices
on human umbilical vein endothelial cells. Int J Artif Organs. 2005;28:1251–8.
163. Steffens GCM, Yao C, Prevel P, Markowicz M, Schenck P, Noah EM, et al. Modulation of
angiogenic potential of collagen matrices by covalent incorporation of heparin and loading
with vascular endothelial growth factor. Tissue Eng. 2004;10:1502–9.
164. van Wachem PB, Plantinga JA, Wissink MJB, Beernink R, Poot AA, Engbers GHM, et al.
In vivo biocompatibility of carbodiimide-crosslinked collagen matrices: Effects of crosslink
density, heparin immobilization, and bFGF loading. J Biomed Mater Res. 2001;55:368–78.
165. Wissink MJB, Beernink R, Pieper JS, Poot AA, Engbers GHM, Beugeling T, et al. Binding
and release of basic fibroblast growth factor from heparinized collagen matrices.
Biomaterials. 2001;22:2291–9.
166. Bladergroen BA, Siebum B, Siebers-Vermeulen KGC, Van Kuppevelt TH, Poot AA,
Feijen J, et al. In vivo recruitment of hematopoietic cells using stromal cell-derived factor 1
alpha-loaded heparinized three-dimensional collagen scaffolds. Tissue Eng Part A.
2009;15:1591–9.
167. Lienemann PS, Lutolf MP, Ehrbar M. Biomimetic hydrogels for controlled biomolecule
delivery to augment bone regeneration. Adv Drug Deliver Rev. 2012;64:1078–89.
168. Uebersax L, Merkle HP, Meinel L. Biopolymer-based growth factor delivery for tissue
repair: from natural concepts to engineered systems. Tissue Eng Part B Rev. 2009;15:
263–89.
169. Lin CC, Anseth KS. Controlling affinity binding with peptide-functionalized poly(ethylene
glycol) hydrogels. Adv Funct Mater. 2009;19:2325–31.
170. Wieland JA, Houchin-Ray TL, Shea LD. Non-viral vector delivery from PEG-hyaluronic
acid hydrogels. J Controlled Release. 2007;120:233–41.
171. Kasper FK, Seidlits SK, Tang A, Crowther RS, Carney DH, Barry MA, et al. In vitro release
of plasmid DNA from oligo(poly(ethylene glycol) fumarate) hydrogels. J Controlled
Release. 2005;104:521–39.
172. Bjugstad KB, Lampe K, Kern DS, Mahoney M. Biocompatibility of poly(ethylene glycol)-
based hydrogels in the brain: an analysis of the glial response across space and time.
J Biomed Mater Res Part A. 2010;95A:79–91.
173. Jeon O, Alt DS, Ahmed SM, Alsberg E. The effect of oxidation on the degradation of
photocrosslinkable alginate hydrogels. Biomaterials. 2012;33:3503–14.
174. Kong HJ, Kaigler D, Kim K, Mooney DJ. Controlling rigidity and degradation of alginate
hydrogels via molecular weight distribution. Biomacromol. 2004;5:1720–7.
175. Kang KS, Lee SI, Hong JM, Lee JW, Cho HY, Son JH, et al. Hybrid scaffold composed of
hydrogel/3D-framework and its application as a dopamine delivery system. J Controlled
Release. 2014;175:10–6.
176. Zhou SL, Matsumoto S, Tian HD, Yamane H, Ojida A, Kiyonaka S, et al. pH-Responsive
shrinkage/swelling of a supramolecular hydrogel composed of two small amphiphilic
molecules. Chem Eur J. 2005;11:1130–6.
177. Liang GL, Yang ZM, Zhang RJ, Li LH, Fan YJ, Kuang Y, et al. Supramolecular hydrogel of
a D-amino acid dipeptide for controlled drug release in vivo. Langmuir. 2009;25:8419–22.
178. Alvarez Z, Castano O, Castells AA, Mateos-Timoneda MA, Planell JA, Engel E, et al.
Neurogenesis and vascularization of the damaged brain using a lactate-releasing biomimetic
scaffold. Biomaterials. 2014;35:4769–81.
179. Polet F, Feron O. Endothelial cell metabolism and tumour angiogenesis: glucose and
glutamine as essential fuels and lactate as the driving force. J Intern Med. 2013;273:156–65.
180. Speder P, Liu J, Brand AH. Nutrient control of neural stem cells. Curr Opin Cell Biol.
2011;23:724–9.
181. Wei GB, Jin QM, Giannobile WV, Ma PX. The enhancement of osteogenesis by
nano-fibrous scaffolds incorporating rhBMP-7 nanospheres. Biomaterials. 2007;28:2087–96.
182. Saraf A, Mikos AG. Gene delivery strategies for cartilage tissue engineering. Adv Drug
Deliver Rev. 2006;58:592–603.
144 G. Fontana et al.

183. Shimer AL, Chadderdon RC, Gilbertson LG, Kang JD. Gene therapy approaches for
intervertebral disc degeneration. Spine. 2004;29:2770–8.
184. Woods BI, Vo N, Sowa G, Kang JD. Gene therapy for intervertebral disk degeneration.
Orthop Clin N Am. 2011;42:563–74.
185. Jang JH, Houchin TL, Shea LD. Gene delivery from polymer scaffolds for tissue
engineering. Expert Rev Med Devices. 2004;1:127–38.
186. Riddle KW, Kong HJ, Leach JK, Fischbach C, Cheung C, Anseth KS, et al. Modifying the
proliferative state of target cells to control DNA expression and identifying cell types
transfected in vivo. Mol Ther. 2007;15:361–8.
187. Rowley JA, Mooney DJ. Alginate type and RGD density control myoblast phenotype.
J Biomed Mater Res. 2002;60:217–23.
188. Fang JM, Zhu YY, Smiley E, Bonadio J, Rouleau JP, Goldstein SA, et al. Stimulation of
new bone formation by direct transfer of osteogenic plasmid genes. P Natl Acad Sci U S A.
1996;93:5753–8.
189. Zhang Y, Song J, Shi B, Wang Y, Chen X, Huang C, et al. Combination of scaffold and
adenovirus vectors expressing bone morphogenetic protein-7 for alveolar bone regeneration
at dental implant defects. Biomaterials. 2007;28:4635–42.
190. Fontana G, Srivastava A, Thomas D, Lalor P, Dockery P, Pandit A. Three-dimensional
microgel platform for the production of cell factories tailored for the nucleus pulposus.
Bioconjug Chem. 2014;26:1297–306.
191. Browne S, Fontana G, Rodriguez BJ, Pandit A. A protective extracellular matrix-based
gene delivery reservoir fabricated by electrostatic charge manipulation. Mol Pharm.
2012;9:3099–106.
192. Bonadio J, Smiley E, Patil P, Goldstein S. Localized, direct plasmid gene delivery in vivo:
prolonged therapy results in reproducible tissue regeneration. Nat Med. 1999;5:753–9.
193. Chandler LA, Doukas J, Gonzalez AM, Hoganson DK, Gu DL, Ma CL, et al. FGF2-targeted
adenovirus encoding platelet-derived growth factor-B enhances de novo tissue formation.
Mol Ther. 2000;2:153–60.
194. Chandler LA, Gu DL, Ma CL, Gonzalez AM, Doukas J, Nguyen T, et al. Matrix-enabled
gene transfer for cutaneous wound repair. Wound Repair Regeneration. 2000;8:473–9.
195. Doukas J, Chandler LA, Gonzalez AM, Gu DL, Hoganson DK, Ma CL, et al. Matrix
immobilization enhances the tissue repair activity of growth factor gene therapy vectors.
Hum Gene Ther. 2001;12:783–98.
196. Tyrone JW, Mogford JE, Chandler LA, Ma CL, Xia YP, Pierce GF, et al.
Collagen-embedded platelet-derived growth factor DNA plasmid promotes wound healing
in a dermal ulcer model. J Surg Res. 2000;93:230–6.
197. Doukas J, Blease K, Craig D, Ma CL, Chandler LA, Sosnowski BA, et al. Delivery of FGF
genes to wound repair cells enhances arteriogenesis and myogenesis in skeletal muscle. Mol
Ther. 2002;5:517–27.
198. Berry M, Gonzalez AM, Clarke W, Greenlees L, Barrett L, Tsang W, et al. Sustained effects
of gene-activated matrices after CNS injury. Mol Cell Neurosci. 2001;17:706–16.
199. Smetana K. Cell biology of hydrogels. Biomaterials. 1993;14:1046–50.
200. Kong HJ, Hsiong S, Mooney DJ. Nanoscale cell adhesion ligand presentation regulates
nonviral gene delivery and expression. Nano Lett. 2007;7:161–6.
201. Schek RM, Hollister SJ, Krebsbach PH. Delivery and protection of adenoviruses using
biocompatible hydrogels for localized gene therapy. Mol Ther. 2004;9:130–8.
202. Pascher A, Palmer G, Steinert A, Oligino T, Gouze E, Gouze J, et al. Gene delivery to
cartilage defects using coagulated bone marrow aspirate. Gene Ther. 2004;11:133–41.
203. Shen H, Tan J, Saltzman WM. Surface-mediated gene transfer from nanocomposites of
controlled texture. Nat Mater. 2004;3:569–74.
204. Kumta PN, Sfeir C, Lee DH, Olton D, Choi D. Nanostructured calcium phosphates for
biomedical applications: novel synthesis and characterization. Acta Biomater. 2005;1:65–83.
5 Biologically Inspired Materials in Tissue Engineering 145

205. Olton D, Li JH, Wilson ME, Rogers T, Close J, Huang L, et al. Nanostructured calcium
phosphates (NanoCaPs) for non-viral gene delivery: Influence of the synthesis parameters on
transfection efficiency. Biomaterials. 2007;28:1267–79.
206. Newland B, Moloney TC, Fontana G, Browne S, Abu-Rub MT, Dowd E, et al. The
neurotoxicity of gene vectors and its amelioration by packaging with collagen hollow
spheres. Biomaterials. 2013;34:2130–41.
207. Guan S, Rosenecker J. Nanotechnologies in delivery of mRNA therapeutics using nonviral
vector-based delivery systems. Gene Ther. 2017;24:133–43.
208. Frisch J, Rey-Rico A, Venkatesan JK, Schmitt G, Madry H, Cucchiarini M. Chondrogenic
differentiation processes in human bone marrow aspirates upon rAAV-mediated gene
transfer and overexpression of the insulin-like growth factor I. Tissue Eng Part A.
2015;21:2460–71.
209. Rajangam T, An SS. Fibrinogen and fibrin based micro and nano scaffolds incorporated with
drugs, proteins, cells and genes for therapeutic biomedical applications. Int J Nanomed.
2013;8:3641–62.
210. Ahmed TA, Dare EV, Hincke M. Fibrin: a versatile scaffold for tissue engineering
applications. Tissue Eng Part B Rev. 2008;14:199–215.
211. Dare EV, Griffith M, Poitras P, Kaupp JA, Waldman SD, Carlsson DJ, et al. Genipin
cross-linked fibrin hydrogels for in vitro human articular cartilage tissue-engineered
regeneration. Cells Tissues Organs. 2009;190:313–25.
212. Moncion A, Arlotta KJ, Kripfgans OD, Fowlkes JB, Carson PL, Putnam AJ, et al. Design
and characterization of fibrin-based acoustically responsive scaffolds for tissue engineering
applications. Ultrasound Med Biol. 2016;42:257–71.
213. Raut SD, Lei P, Padmashali RM, Andreadis ST. Fibrin-mediated lentivirus gene transfer:
implications for lentivirus microarrays. J Control Release. 2010;144:213–20.
214. Lei P, Padmashali RM, Andreadis ST. Cell-controlled and spatially arrayed gene delivery
from fibrin hydrogels. Biomaterials. 2009;30:3790–9.
215. Sahni A, Odrljin T, Francis CW. Binding of basic fibroblast growth factor to fibrinogen and
fibrin. J Biol Chem. 1998;273:7554–9.
216. Mosesson MW. Fibrinogen and fibrin structure and functions. J Thromb Haemost.
2005;3:1894–904.
217. Tabele C, Montana M, Curti C, Terme T, Rathelot P, Gensollen S, et al. Organic glues or
fibrin glues from pooled plasma: efficacy, safety and potential as scaffold delivery systems.
J Pharm Pharm Sci. 2012;15:124–40.
218. de la Puente P, Ludena D. Cell culture in autologous fibrin scaffolds for applications in tissue
engineering. Exp Cell Res. 2014;322:1–11.
219. Noori A, Ashrafi SJ, Vaez-Ghaemi R, Hatamian-Zaremi A, Webster TJ. A review of fibrin
and fibrin composites for bone tissue engineering. Int J Nanomed. 2017;12:4937–61.
220. Gasperini L, Mano JF, Reis RL. Natural polymers for the microencapsulation of cells. J R
Soc Interface. 2014;11:20140817.
221. Wittmann K, Storck K, Muhr C, Mayer H, Regn S, Staudenmaier R, et al. Development of
volume-stable adipose tissue constructs using polycaprolactone-based polyurethane scaffolds
and fibrin hydrogels. J Tissue Eng Regen Med. 2016;10:E409–18.
222. Eyrich D, Wiese H, Mailer G, Skodacek D, Appel B, Sarhan H, et al. In vitro and in vivo
cartilage engineering using a combination of chondrocyte-seeded long-term stable fibrin gels
and polycaprolactone-based polyurethane scaffolds. Tissue Eng. 2007;13:2207–18.
223. Almany L, Seliktar D. Biosynthetic hydrogel scaffolds made from fibrinogen and
polyethylene glycol for 3D cell cultures. Biomaterials. 2005;26:2467–77.
224. Oss-Ronen L, Seliktar D. Polymer-conjugated albumin and fibrinogen composite hydrogels
as cell scaffolds designed for affinity-based drug delivery. Acta Biomater. 2011;7:163–70.
225. Frisman I, Seliktar D, Bianco-Peled H. Nanostructuring PEG-fibrinogen hydrogels to control
cellular morphogenesis. Biomaterials. 2011;32:7839–46.
146 G. Fontana et al.

226. Jiang B, Waller TM, Larson JC, Appel AA, Brey EM. Fibrin-loaded porous poly(ethylene
glycol) hydrogels as scaffold materials for vascularized tissue formation. Tissue Eng Part A.
2013;19:224–34.
227. Rao RR, Peterson AW, Ceccarelli J, Putnam AJ, Stegemann JP. Matrix composition
regulates three-dimensional network formation by endothelial cells and mesenchymal stem
cells in collagen/fibrin materials. Angiogenesis. 2012;15:253–64.
228. Lee F, Kurisawa M. Formation and stability of interpenetrating polymer network hydrogels
consisting of fibrin and hyaluronic acid for tissue engineering. Acta Biomater. 2013;9:
5143–52.
229. King VR, Alovskaya A, Wei DYT, Brown RA, Priestley JV. The use of injectable forms of
fibrin and fibronectin to support axonal ingrowth after spinal cord injury. Biomaterials.
2010;31:4447–56.
230. Lorentz KM, Kontos S, Frey P, Hubbell JA. Engineered aprotinin for improved stability of
fibrin biomaterials. Biomaterials. 2011;32:430–8.
231. Roura S, Galvez-Monton C, Bayes-Genis A. Fibrin, the preferred scaffold for cell
transplantation after myocardial infarction? An old molecule with a new life. J Tissue Eng
Regenerative Med. 2017;11:2304–13.
232. Ye L, Chang YH, Xiong Q, Zhang P, Zhang L, Somasundaram P, et al. Cardiac repair in a
porcine model of acute myocardial infarction with human induced pluripotent stem
cell-derived cardiovascular cells. Cell Stem Cell. 2014;15:750–61.
233. Johnson PJ, Parker SR, Sakiyama-Elbert SE. Controlled release of neurotrophin-3 from
fibrin-based tissue engineering scaffolds enhances neural fiber sprouting following subacute
spinal cord injury. Biotechnol Bioeng. 2009;104:1207–14.
234. Wilems TS, Pardieck J, Iyer N, Sakiyama-Elbert SE. Combination therapy of stem cell
derived neural progenitors and drug delivery of anti-inhibitory molecules for spinal cord
injury. Acta Biomater. 2015;28:23–32.
235. Agbay A, Edgar JM, Robinson M, Styan T, Wilson K, Schroll J, et al. Biomaterial strategies
for delivering stem cells as a treatment for spinal cord injury. Cells Tissues Organs.
2016;202:42–51.
236. Iwaya K, Mizoi K, Tessler A, Itoh Y. Neurotrophic agents in fibrin glue mediate adult dorsal
root regeneration into spinal cord. Neurosurgery. 1999;44:589–95.
237. Hyatt AJT, Wang D, van Oterendorp C, Fawcett JW, Martin KR. Mesenchymal stromal cells
integrate and form longitudinally-aligned layers when delivered to injured spinal cord via a
novel fibrin scaffold. Neurosci Lett. 2014;569:12–7.
238. Ito K, Yamada Y, Naiki T, Ueda M. Simultaneous implant placement and bone regeneration
around dental implants using tissue-engineered bone with fibrin glue, mesenchymal stem
cells and platelet-rich plasma. Clin Oral Implan Res. 2006;17:579–86.
239. Galler KM, Brandl FP, Kirchhof S, Widbiller M, Eidt A, Buchalla W, et al. Suitability of
different natural and synthetic biomaterials for dental pulp tissue engineering. Tissue Eng
Part A. 2018;24:234–44.
240. Haleem AM, Singergy AA, Sabry D, Atta HM, Rashed LA, Chu CR, et al. The clinical use
of human culture-expanded autologous bone marrow mesenchymal stem cells transplanted
on platelet-rich fibrin glue in the treatment of articular cartilage defects: a pilot study and
preliminary results. Cartilage. 2010;1:253–61.
241. Handl M, Trc T, Hanus M, Stastny E, Fricova-Poulova M, Neuwirth J. et al [Therapy of
severe chondral defects of the patella by autologous chondrocyte implantation]. Acta Chir
Orthop Traumatol Cechoslovaca. 2006;73:373–9.
242. Horch RE, Kopp J, Kneser U, Beier J, Bach AD. Tissue engineering of cultured skin
substitutes. J Cell Mol Med. 2005;9:592–608.
243. Yamamoto M, Yanaga H, Nishina H, Watabe S, Mamba K. Fibrin stimulates the
proliferation of human keratinocytes through the autocrine mechanism of transforming
growth factor-alpha and epidermal growth factor receptor. Tohoku J Exp Med.
2005;207:33–40.
5 Biologically Inspired Materials in Tissue Engineering 147

244. Bacakova M, Musilkova J, Riedel T, Stranska D, Brynda E, Zaloudkova M, et al. The

potential applications of fibrin-coated electrospun polylactide nanofibers in skin tissue
engineering. Int J Nanomed. 2016;11:771–89.
245. Khor HL, Ng KW, Htay AS, Schantz JT, Teoh SH, Hutmacher DW. Preliminary study of a
polycaprolactone membrane utilized as epidermal substrate. J Mater Sci Mater Med.
2003;14:113–20.
246. Sahni A, Francis CW. Vascular endothelial growth factor binds to fibrinogen and fibrin and
stimulates endothelial cell proliferation. Blood. 2000;96:3772–8.
247. Sanchez Munoz I, Granados R, Holguin Holgado P, Garcia Vela JA, Casares C, Casares M.
The use of adipose mesenchymal stem cells and human umbilical vascular endothelial cells
on a fibrin matrix for endothelialized skin substitute. Tissue Eng Part A. 2015;21:214–23.
248. McManus MC, Boland ED, Koo HP, Barnes CP, Pawlowski KJ, Wnek GE, et al.
Mechanical properties of electrospun fibrinogen structures. Acta Biomater. 2006;2:19–28.
249. McManus MC, Boland ED, Simpson DG, Barnes CP, Bowlin GL. Electrospun fibrinogen:
Feasibility as a tissue engineering scaffold in a rat cell culture model. J Biomed Mater Res
Part A. 2007;81A:299–309.
250. Sell SA, Francis MP, Garg K, McClure MJ, Simpson DG, Bowlin GL. Cross-linking
methods of electrospun fibrinogen scaffolds for tissue engineering applications. Biomed
Mater. 2008;3:045001.
251. Ravichandran R, Seitz V, Venugopal JR, Sridhar R, Sundarrajan S, Mukherjee S, et al.
Mimicking native extracellular matrix with phytic acid-crosslinked protein nanofibers for
cardiac tissue engineering. Macromol Biosci. 2013;13:366–75.
252. Perumcherry SR, Chennazhi KP, Nair SV, Menon D, Afeesh R. A novel method for the
fabrication of fibrin-based electrospun nanofibrous scaffold for tissue-engineering applica-
tions. Tissue Eng Part C Med. 2011;17:1121–30.
253. Wlodarczyk-Biegun MK, Del Campo A. 3D bioprinting of structural proteins. Biomaterials.
2017;134:180–201.
254. Cui XF, Boland T. Human microvasculature fabrication using thermal inkjet printing
technology. Biomaterials. 2009;30:6221–7.
255. Tasoglu S, Demirci U. Bioprinting for stem cell research. Trends Biotechnol. 2013;31:10–9.
256. Phillippi JA, Miller E, Weiss L, Huard J, Waggoner A, Campbell P. Microenvironments
engineered by inkjet bioprinting spatially direct adult stem cells toward muscle- and
bone-like subpopulations. Stem Cells. 2008;26:127–34.
257. Miller ED, Li K, Kanade T, Weiss LE, Walker LM, Campbell PG. Spatially directed
guidance of stem cell population migration by immobilized patterns of growth factors.
Biomaterials. 2011;32:2775–85.
258. Campbell PG, Miller ED, Fisher GW, Walker LM, Weiss LE. Engineered spatial patterns of
FGF-2 immobilized on fibrin direct cell organization. Biomaterials. 2005;26:6762–70.
259. Mogford JE, Tawil B, Jia SX, Mustoe TA. Fibrin sealant combined with fibroblasts and
platelet-derived growth factor enhance wound healing in excisional wounds. Wound Repair
Regeneration. 2009;17:405–10.
 Part III
Nanotechnologies and Biomimetic
Chapter 6
Advances in Nanotechnologies
for the Fabrication of Silk Fibroin-Based
Scaffolds for Tissue Regeneration

Nicolò Nicoli Aldini and Milena Fini

Abstract Silks are protein fibers produced by silkworms whose architecture is

based on two proteins: fibroin and sericin. Because sericin has been recognized as
the main cause of silk’s poor performance due to its antigenicity, fibroin alone has
now remained popular as a biomaterial, also due to its strength and mechanical
properties. Other advantages of this biological product are the water-based pro-
cessing, biodegradability, and the presence of easily accessible chemical groups for
functional modifications. Due to its versatility, fibroin is now widely considered for
use in the manufacture of many biological devices and substitutes in different
medical fields, with very different biological, physiological, and mechanical
properties. In recent years, nanomaterials have gained considerable attention also in
tissue engineering, because they exhibit properties that are significantly different to
corresponding bulk materials, such as large surface area, increased strength, and
enhanced surface reactivity, thus improving material performance. Reviewed
studies, mainly in the regeneration of the musculoskeletal system, have been out-
lined the advantages of fibroin as a scaffold, and the technologies adopted for the
nanostructure development of this protein. Further advancements will open up new
perspectives in the use of this product in tissue regeneration. Silk-based materials
are of particular interest where controlled biodegradation and good mechanical
properties are required, such as in tissue engineering of musculoskeletal tissues.
Their versatility in processing, biocompatibility properties, ease of sterilization,
thermal stability, possibility for surface chemical modifications, and controllable
degradation therefore make silk-derived proteins promising biomaterials for many

N. N. Aldini (&)
Laboratory of Preclinical and Surgical Studies, Rizzoli Orthopedic Institute,
Bologna, Italy
e-mail: [email protected]
M. Fini
Laboratory of Biocompatibility, Innovative Technologies
and Advanced Therapies, Rizzoli RIT Department, Bologna, Italy
e-mail: milena.fi[email protected]

© Springer International Publishing AG, part of Springer Nature 2018 151

A. C. Berardi (ed.), Extracellular Matrix for Tissue Engineering
and Biomaterials, Stem Cell Biology and Regenerative Medicine,
https://doi.org/10.1007/978-3-319-77023-9_6
152 N. N. Aldini and M. Fini

clinical functions. Since research into these applications is quite new, we can expect
interesting future developments, in which the nanotechnologies might play a
decisive role.

Keywords Silk fibroin Nanotechnologies Nanomaterials Biomaterials





Biocompatibility Composite materials Preclinical studies Hard tissues


In vitro study In vivo study

Silks are protein fibers produced by silkworms (Bombyx mori), but also by spiders
and others arthropods. The architecture of silk is based on two proteins: fibroin,
which is the core filament, and sericin, which is a protein that glues fibroin fibers
together.
Although silk was used extensively in surgery for making suture threads, this
application has now been replaced by synthetic materials with greater histocom-
patibility than natural ones. Benefits and drawbacks of silks for biomaterial
applications have been well detailed by Altman, Vepari, and other authors and are
summarized in Table 6.1 [1, 2]. More recently, because sericin has been recognized
as the main cause of silk’s poor performance due to its antigenicity, fibroin alone
has remained popular as a biomaterial.
Fibroin is made of highly organized crystals and semi-crystalline regions that
account for its elasticity. The primary structure is mainly composed of the amino
acids glycine, alanine, serine, valine, and tyrosine with characteristic sequences.
These structural elements produce the strength and resiliency of silk fibroin [3].
Indeed, silk has interesting mechanical properties regarding its use as biomaterial.
Other advantages of this biological product are its water-based processing,
biodegradability, and the presence of easily accessible chemical groups for func-
tional modifications [4].
Nowadays, tissue engineering procedures have become widespread in regener-
ative medicine for the treatment of diseases when standard medical procedures fail.
Regenerative strategies are based on the combination of three main tools: cells
(differentiated and not differentiated), scaffolds, and growth factors. Silk fibroin also

Table 6.1 Benefits and Benefits

drawbacks of silks for Mechanical properties
biomedical applications Prolonged history of use in clinical applications
Manufacturing in water solution and easy insolubilization
Easy chemical modifications
Slow in vivo degradation
Easy functionalization
Drawbacks
Adequate removal of sericin required
Slow degradation of crystalline regions
Possible formation of granuloma
Sensitization and possible allergic response
6 Advances in Nanotechnologies for the Fabrication of Silk … 153

has received much attention as a scaffold material, due to its above-mentioned

biocompatibility, processability, biodegradability, and mechanical and thermal
properties [5]. Scaffolds should mimic tissue extracellular matrix (ECM) in bio-
logical and chemical composition and physical structure [6]. Mimicking the
nanofibrous structures of ECM to achieve better biocompatibility remains a chal-
lenge [7].
In recent years, nanomaterials have gained considerable attention also in tissue
engineering, because they exhibit properties that are significantly different to cor-
responding bulk materials, such as large surface area, increased strength, and
enhanced surface reactivity, thus improving material performance. The definition
adopted by the International Organization for Standardization (ISO/TS 27687:2008)
is: “Material with any external dimension in the nanoscale or having internal
structure in the nanoscale” defining “Nanoscale” as a size range from approximately
1 to 100 nm. Polymeric nanofiber matrix is similar to fibrous ECM proteins and is
thus a candidate as ECM-mimetic biomaterial [6].
Due to its above-mentioned properties, fibroins now being widely considered for
use in the manufacture of many biological devices and substitutes. Table 6.2 shows
several experimental and clinical studies on the possible use of this product in
different medical fields.
Such a wide range of applications, with very different biological, physiological,
and mechanical properties, requires of course an equally wide range of ways to
manage the product to make it suitable for soft and hard tissue substitution.
Following the physicochemical characterization, appropriate preclinical investiga-
tions, with both in vitro and in vivo tests, must be planned to validate novel
production, based on fibroin alone or as a composite. Some studies mainly in the

Table 6.2 Applications of silk fibroinbiomaterials for tissue regeneration

Organ/apparatus Applicationsa Authors
Vascular tissue Flow diverting devices, stents (C) [8, 9]
Neural tissue Peripheral nerve conduits (E) [10]
Skin Composite scaffolding membranes [11]
Bone Composite scaffolds with/without addition of biological [12, 13,
factors (E) 14]
Cartilage Scaffolds to support chondrocytes (E) [15, 16]
Ligament and Scaffolds and scaffold-free ligaments, composites (E) [17, 18]
Tendon
Cardiac tissue Cardiac patch composite + mesenchymal cells (E) [19, 20]
Ocular tissue Cornea replacement (E) [21, 22]
Hepatic tissue Silk fibroin–collagen blended films (E) [23]
Intervertebral Porous scaffolds (E) [24]
tissue
Bladder Wall repair and reconstruction (E) [25]
Eardrum tissue Silk-based membranes (E) [26]
a
C clinical; E experimental
154 N. N. Aldini and M. Fini

regeneration of the musculoskeletal system have been reviewed to focus on the use
of fibroin as a scaffold and the technologies adopted for the nanostructure
development.
Following a preclinical protocol, for example, in an in vitro and in vivo study
Fini et al. [27] evaluated the behavior of an injectable silk fibroin hydrogel through
osteoblast cultures and after implantation in critical size defects of rabbit distal
femurs, using synthetic poly (D, L-lactide–glycolide) copolymer as control material.
In vitro biocompatibility was evaluated by measuring cytotoxicity and cytocom-
patibility on human osteoblast-like cell line (MG 63), whereas in vivo the bone
defect healing rate and quality of the newly formed bone inside the defects were
determined by measuring histomorphometric parameters, such as trabecular bone
volume, trabecular thickness, trabecular number, and trabecular separation. In vitro
tests indicated that both materials significantly increased cell proliferation in
comparison with the negative control. Both materials promoted bone healing when
used to fill critical size defects in rabbit femurs, but the histomorphometry showed
better results in new-formed bone of the silk fibroin hydrogel-treated defects in
comparison with the control gel. The regrown bone of the Silk fibroin hydrogel-
treated defects appeared to be more similar to normal bone than that of the control
synthetic polymeric material-treated defects, in comparison with controls treated
with a synthetic polymeric material, thus suggesting that silk fibroin hydrogel can
accelerate remodeling processes. Like this study, which is aimed at hard tissue
repair, many others describe a range of scaffold preparation procedures and the
tissues to be replaced.
Electrospinning technology, which uses an electrical charge to draw very fine
fibers on the micro- or nanoscale, enables porous nanofibrous scaffolds to be
obtained, which are able to mimic the ECM. Considering the physical–chemical
properties and the structure of the scaffolds, micro- and nanoparticles can be
obtained from silk solutions by various procedures, such as freeze-drying and
grinding procedures, spray drying, jet breaking, self-assembly, and freeze-thawing.
Milling of silk fibers is also an option to obtain silk particles using any chemicals.
According to Kundu et al. [4], these particles can play the dual role of improving
mechanical properties of scaffolds and at the same time act as a carrier of growth
factors for rapid tissue regeneration. Indeed to improve mechanical and biological
performances, inorganic or organic fillers have been incorporated in silk 3D scaf-
folds during or after fabrication to obtain composites. The main advantage is in this
case the compatibility between the components. Consequences of a poor compat-
ibility may result in inhomogeneous mixtures, phase separation, and adverse tissue
reactions. To obtain better compatibility, silk–silk composite scaffolds are made by
incorporating milled silk particles in porous silk sponge, resulting in a significant
improvement in mechanical properties.
With respect to the material porosity, different methods of processing are pre-
sented in the literature. The importance of the processing method is highlighted by
Kuboyama et al. These authors evaluated the porosity of scaffolds prepared using
regenerated Bombyx mori silk fibroin dissolved either in water (AF) orin
hexafluoroisopropanol (HFIP). The two preparations were comparatively analyzed
6 Advances in Nanotechnologies for the Fabrication of Silk … 155

in an animal model in which the formation and growth of new bone in the
implantation site (rabbit femoral epicondyle) was examined by means of micro-CT
and histology. The AF scaffold exhibited significantly greater osteoconductivity
than that obtained by the protein dissolved in HFIP. Micro-CT analysis showed that
the morphology of the newly formed bone differed significantly in the two types of
silk fibroin scaffold. After 4 weeks of implantation, new trabecular bone was seen
inside the pores of the AF scaffold implant, whereas the HFIP scaffold only con-
tained necrotic cells. No trabecular bone was seen within the pores of the latter
scaffolds, although the pores of these did contain giant cells and granulation tissue
[28].
Lin et al. [29] evaluated silk fibroin with different nanostructures, self-assembled
in aqueous solution to improve porous structure formation. In comparison with
scaffolds derived from fresh solution, the nanofibrous silk scaffolds showed better
cell compatibility in vitro. These observations suggest that the control of silk
nanoscale can regulate matrix features including porous structure and nanostructure,
which are important in regulating cell and tissue outcomes.
In the specific field of bone tissue engineering, silk fibroin gained much interest
as a scaffold material and various strategies were developed to create a
three-dimensional (3D) structure with high porosity and osteoconductivity.
Salt-leaching or freeze-drying methods were used to create porous scaffolds.
Moreover, in attempts to mimic bone properties, the incorporation of ceramic
components into silk fibroin scaffolds has also been shown. Fibrous silk fibroin
scaffolds were obtained using the electrospinning technique and the bone regen-
eration in the scaffolds was confirmed; they are considered to be effective in
replacing collagen for many tissue engineering applications [30].
The use of non-mulberry tropical silk fibroin as a bioactive polymer in blended
nanofibrous matrices resulted in osteoconductive scaffolds. The blending of 2 wt%
silk fibroin exhibited higher cell attachment, growth, and ECM formation, when
compared to unmodified polyvinyl alcohol as control and the other blend ratio. The
mechanical robustness of constructs resembled the original bone tissue, thus indi-
cating a promising future for this blend in bone regeneration and reconstruction.
This in vitro study, therefore, lays the foundation for designing clinically relevant
orthopedic grafts in vivo [31]. Considering in particular, the behavior of scaffolds
interacting with cells, Teuschl et al. [32] found that surface modifications of fibroin
with carbohydrate-binding protein lectin enhanced the adhesion of cells, in par-
ticular adipose-derived stromal cells; the proliferation and differentiation in osteo-
genic lineages were however not influenced. Considering that silk fibroin is used to
obtain scaffolds for bone tissue engineering, this possibility may be of practical
interest. Uchida et al. [33] also showed that plasma-irradiated silk fibroin was a
regulator of bone matrix properties in an animal model based on the placement of
scaffolds in critical size defects in rabbit femurs, which increased bone matrix,
mineral concentration, cortical thickness, and trabecular bone volume.
Fibroin fibers can also be used as composites with other polymers. Yang et al.
[34] studied different types of scaffolds based on silk fibroin and poly (L-lactide-co-
caprolactone) blends intended for tendon tissue engineering. Biocompatibility
156 N. N. Aldini and M. Fini

analysis revealed that bone marrow-derived mesenchymal stem cells exhibited a

higher proliferation rate when cultured on nanofibrous scaffolds compared with the
other scaffolds. The mechanical testing results indicated that the tensile properties
of the nanofibrous scaffolds were reinforced in the direction parallel to the
nano-yarns and fulfilled the mechanical requirements for tendon repair.
Teimouri et al. [35] proposed a nanocomposite with silk fibroin–chitosan/Nano
ZrO2 for tissue engineering. The scaffold was found to possess a porous nature with
pore dimensions suitable for cell infiltration and colonization.
Kishimoto et al. [36] observed that the incorporation of montmorillonite, a very
soft crystalline silicate mineral in silk fibers would improve their physical prop-
erties. This nanocomposite might be considered for new biocompatible applica-
tions, such as scaffolds for tissue engineering like bone regeneration, because of the
osteoinductive montmorillonite properties and biodegradable and biocompatible
silk presence.
Hydrogel products constitute a group of polymeric materials, the hydrophilic
structure of which renders them capable of holding large amounts of water in their
3D networks. Hydrogels can be formulated in different physical forms, such as
micro- and nanoparticles, coatings, and films. Kim et al. [37] studied silk fibroin/
hydroxyapatite composite hydrogels obtained with different hydroxyapatite con-
tents in fibroin matrix. Previous studies have shown that fibroin can be easily
manufactured into hydrogel without additional crosslinking reagents or toxic
chemicals. Therefore, the fibroin hydrogel manufacturing process is not only
physiologically safe but also biocompatible. For bone regeneration, this offers
interesting properties for silk-based scaffolds to be used in critical size bone defects
and cartilage.
Chen et al. [5] studied the potential of a Silk Fibroin 3D scaffold produced by
additive manufacturing technology, which can be used to engineer tissues that are
structurally complex, because it is capable of producing precise architectural fea-
tures using a layer-by-layer approach based on computer-aided design, to obtain a
scaffold for cartilage engineering. These authors verified the presence of micropores
and interconnected channels within the scaffold by scanning electron microscopic
observations. In vitro cell culture within the fibroin scaffold using porcine articular
chondrocytes showed a steady increase in cell numbers, thus giving positive
indications for the use of the scaffold for cartilage repair.
Again in the field of osteoarticular apparatus, considering its application as a
tendon and ligament substitute, Farè et al. [38] performed studies on a novel
structure made of silk fibroin able to match the mechanical performance require-
ments of anterior cruciate ligament. In particular, these authors evaluated in vitro
cell ingrowth in sericin-free, silk fibroin knitted sheath with braided core structure.
Micro-CT analysis confirmed that the core was highly porous and had a higher
degree of interconnectivity than that observed for the sheath. The in vivo cell
colonization of the scaffolds is thus expected to penetrate even the internal parts of
the structure. Tensile mechanical tests confirmed the scaffold’s suitability for
anterior cruciate ligament reconstruction. In vitro tests with fibroblasts revealed the
absence of cytotoxic substances in the extracts. ESM obtained with human
6 Advances in Nanotechnologies for the Fabrication of Silk … 157

periodontal ligament Fibroblasts cultured in direct contact with fibroin, compared to

control samples, displayed an increased secretion of aggrecan and fibronectin
(FBN) at 3 and 7 days of culture, and no change in IL-6 and TNF-a secretion, thus
suggesting the usefulness of this novel scaffold for tendon tissue regeneration.
Nanotechnology and tissue engineering are widely involved in the achievement
of constructs for skin tissue regeneration. Gandhimathi et al. [39] performed a study
to evaluate the applications of composite copolymers of polylactic acid and poly-
(e-caprolactone) with silk fibroin, vitamin E, and curcumin, as nanofibrous scaf-
folds, to assess their potential as substrates for the culture of human dermal
fibroblasts for skin tissue engineering. Scaffolds were made by electrospinning and
characterized by fiber morphology, membrane porosity, wettability, mechanical
strength, and chemical properties. Human dermal fibroblasts were cultured on these
scaffolds, and the cell–scaffold interactions were evaluated. The results showed that
human dermal fibroblasts cultured on nanofibrous scaffolds proved to be a potential
support for skin regeneration.
Suganya et al. [40] also evaluated a hybrid biomaterial for dermal substitutes,
based on nanofibrous silk fibroin scaffold. The scaffold was made by the electro-
spinning technique and the physical–chemical characterization showed improved
hydrophilic properties and favorable tensile strain, as well as a favorable cell
proliferation.
Finally, magnetic fibroin scaffolds were also evaluated, by integrating magnetic
materials and fibroin scaffolds, for potential use in magnetic-field-assisted tissue
engineering. Magnetic nanoparticles were introduced into scaffolds at different
strengths of magnetization. The scaffolds were evaluated in vitro and were found
not to be toxic to cells and improved cell adhesion and proliferation. These findings
suggest that magnetized silk-based biomaterials can be engineered with interesting
features for biomaterials and tissue engineering applications [41].
Besides these applications as a support for tissue regeneration, fibroin shows
interesting properties for implantable systems for the controlled release of drugs.
Indeed, nanostructured materials are now frequently used in drug delivery
systems.
Achieving efficient drug delivery systems is a way to improve new routes of
administration of therapeutic agents. Silk fibroin is a suitable material for incor-
poration into a variety of drug delivery vehicles capable of delivering a range of
therapeutic agents. Indeed, silk fibroin matrices have been shown to successfully
deliver anticancer drugs, small molecules, and biomolecules [42]. In a study by
Subia [43], silk fibroin–albumin blended nanoparticles were made using the des-
olvation method and evaluated as carriers for the delivery of methotrexate. They
found promising properties as a nanocarrier for the delivery of drugs and other
bioactive molecules.
158 N. N. Aldini and M. Fini

Conclusions

The manifold investigations that span through very different fields of applications
concur that fibroin is a promising biomaterial. Further advancements will open up
new perspectives in the use of this product in tissue regeneration. Silk-based
materials are of particular interest where slow biodegradation and good mechanical
properties are required, such as in tissue engineering of bone, ligaments, and
musculoskeletal tissues.
Their versatility in processing, biocompatibility properties, ease of sterilization,
thermal stability, possibility for surface chemical modifications, and controllable
degradation therefore make silk-derived proteins promising biomaterials for many
clinical functions. Since research into these applications is quite new, we can expect
interesting future developments, in which the nanotechnologies might play a
decisive role.

References

1. Altman GH, Diaz F, Jakuba C, Calabro T, Horan RL, Chen J, Lu H, Richmond J, Kaplan DL.
Silk-based biomaterials. Biomaterials. 2003;24:401–16.
2. Vepari C, Kaplan DL. Silk as a biomaterial. Prog Polym Sci. 2007;32:991–1007.
3. Meinel L, Hofmann S, Karageorgiou V, Kirker-Heade C, McCoole J, Gronowicz G,
Zichner L, Langer R, Vunjak-Novakovica G, Kaplan DL. The inflammatory responses to silk
films in vitro and in vivo. Biomaterials. 2005;26:147–55.
4. Kundu B, Rajkhowa R, Kundu SC, Wang X. Silk fibroin biomaterials for tissue regenerations.
Advanc Drug Deliv Rev. 2013;65:457–70.
5. Chen CH, Liu JM, Chua CK, Chou SM, Shyu VBH, Chen JP. Cartilage tissue engineering
with silk fibroin scaffolds fabricated by indirect additive manufacturing technology. Materials.
2014;7:2104–19.
6. Ma Z, Kotaki M, Inai R, Ramakrishna S. Potential of nanofiber matrix as tissue-engineering
scaffolds. Tissue Eng. 2005;11(1–2):101–9.
7. Lu G, Liu S, Lin S, Kaplan DL, Lu Q. Silk porous scaffolds with nanofibrous microstructures
and tunable properties. Coll Surf B Biointerfaces. 2014;120:28–37.
8. Causin F, Pascarella R, Pavesi G, Marasco R, Zambon G, Battaglia R, Munari M. Acute
endovascular treatment (48 hours) of uncoilable ruptured aneurysms at non-branching sites
using silk flow-diverting devices. Interv Neuroradiol. 2011;17(3):357–64.
9. Leonardi M, Cirillo L, Toni F, Dall’Olio M, Princiotta C, Stafa A, Simonetti L, Agati R.
Treatment of intracranial aneurysms using flow-diverting silk stents (BALT): a single centre
experience. Interv Neuroradiol. 2011;17(3):306–15.
10. Wang CY, Zhang K-H, Fan CY, Mo XM, Ruan HJ, Li FF. Aligned natural–synthetic
polyblend nanofibers for peripheral nerve regeneration. Acta Biomater. 2011;7:634–43.
11. Yoo CR, Yeo IS, Park KE, Park JH, Lee SJ, Park WH, Min BM. Effect of chitin/silk fibroin
nanofibrous bicomponent structures on interaction with human epidermal keratinocytes. Int J
Biol Macromol. 2008;42:324–34.
12. Meinel L, Betz O, Fajardo R, Hofmann S, Nazarian A, Cory E, Hilbe M, McCool J, Langer R,
Vunjak-Novakovic G, Merkle HP, Rechenberg B, Kaplan DL, Kirker-Head C. Silk based
biomaterials to heal critical sized femur defects. Bone. 2006;39:922–31.
6 Advances in Nanotechnologies for the Fabrication of Silk … 159

13. Li C, Vepari C, Jin HJ, Kim HJ, Kaplan DL. Electrospun silk-BMP-2 scaffolds forbone tissue
engineering. Biomaterials. 2006;27:3115–24.
14. Kweon H, Lee K-G, Chae C-H, Balázsi C, Min S-K, Kim JY, Choi JY, Kim SG.
Development of nano-hydroxyapatite graft with silk fibroin scaffold as a new bone substitute.
J Oral Maxillofacc Surg. 2011;69:1578–86.
15. Wang Y, Blasioli DJ, Kim HJ, Kim HS, Kaplan DL. Cartilage tissue engineering with silk
scaffolds and human articular chondrocytes. Biomaterials. 2006;27:4434–42.
16. Gellynck K, Verdonk PCM, Nimmen EV, Almqvist KF, Gheysens T, Schokens G,
Langenhove LV, Kiekens P, Mertens J, Verbruggen G. Silkwormand spider silk scaffolds for
chondrocyte support. J Mater Sci Mater Med. 2008;19:3399–409.
17. Chen X, Qi YY, Wang LL, Yin Z, Yin GL, Zou XH, Ouyang HW. Ligament regeneration
using a knitted silk scaffold combined with collagen matrix. Biomaterials. 2008;29:3683–92.
18. Sahoo S, LokToh S, Hong JC. Goh PLGA nanofiber-coated silk microfibrous scaffold for
connective tissue engineering. J Biomed Mater Res B Appl Biomater. 2010;95B:19–28.
19. Yang MC, Wang SS, Chou NK, Chi NH, Huang YY, Chang YL, Shieh MJ, Chung TW. The
cardiomyogenic differentiation of rat mesenchymal stem cells on silk fibroin– polysaccharide
cardiac patches in vitro. Biomaterials. 2009;30:3757–65.
20. Patra C, Talukdar S, Novoyatleva T, Velagala SR, Mühlfeld C, Kundu B, Kundu SC,
Engel FB. Silk protein fibroin from Antheraea mylitta for cardiac tissue engineering.
Biomaterials. 2012;33:2673–80.
21. Yang T, Zhang M. Potential of nanofiber matrix as tissue-engineering scaffolds. Int J
Ophthalmol. 2008;8:1557–9.
22. Lawrence BD, Marchant JK, Pindrus MA, Omenetto FG, Kaplan DL. Silk film biomaterials
for cornea tissue engineering. Biomaterials. 2009;30:1299–308.
23. Cirillo B, Morra M, Catapano G. Adhesion and function of rat liver cells adherentto silk
fibroin/collagen blend films. Int J Artif Organs. 2004;27:60–8.
24. Chang G, Kim HJ, Vunjak-Novakovic G, Kaplan DL, Kandel R. Enhancing annulusfibrosus
tissue formation in porous silk scaffolds. J Biomed Mater Res A. 2010;92A:43–51.
25. Cannon GM Jr, Mauney JR, Gong EM, Yu RN, Adam RM, Estrada CR. Silk Asa novel
biomaterial in bladder tissue engineering. J Pediatr Urol. 2010;6(S1):S82.
26. Ghassemifar R, Redmond S, Chirila TV, Zainuddin. Advancing towards a tissue-engineered
tympanic membrane: silk fibroin as a substratum for growing human eardrum keratinocytes.
J Biomater Appl. 2010;24:591–606.
27. Fini M, Motta A, Torricelli P, Giavaresi G, Nicoli Aldini N, Tschon M, Giardino R,
Migliaresi C. The healing of confined critical size cancellous defects in the presence of silk
fibroin hydroge. Biomaterials. 2005;26:3527–36.
28. Kuboyama N, Kiba H, Arai K, Uchida R, Tanimoto Y, Bhawal UK, Abiko Y, Miyamoto S,
Knight D, Asakura T, Nishiyama N. Silk fibroin-based scaffolds for bone regeneration.
J Biomed Mater Res Part B Appl Biomater. 2013;2013(101B):295–302.
29. Lin S, Lu G, Liu S, Bai S, Liu X, Lu Q, Zuo B, Kaplan DL, Zhu H. Nanoscale control of silks
for nanofibrous scaffold formation with improved porous structure. J Mater Chem B Mater
Biol Med. 2014;2(17):2622–33.
30. Kim H, Che L, Ha Y, Ryu W. Mechanically-reinforced electrospun composite silk fibroin
nanofibers containing hydroxyapatite nanoparticles. Mater Sci Eng. 2014;40:324–35.
31. Bhattacharjee P, Kundu B, Naskar D, Maiti TK, Bhattacharya D, Kundu SC. Nanofibrous
Nonmulberry Silk/PVA Scaffold for Osteoinduction and Osseointegration. Biopolymers.
2015;103:271–84.
32. Teuschl AH, Neutsch L, Monforte X, Rünzler D, van Griensven M, Gabor F, Redl H.
Enhanced cell adhesion on silk fibroin via lectin surface modification. Acta Biomaterialia.
2014;10:2506–17.
33. Uchida R, Bhawal UK, Kiba H, Arai K, Tanimoto Y, Kuboyama N, Asakura T, Nishiyama N.
Effect of plasma-irradiated silk fibroin in bone regeneration. J Biosci Bioeng. 2014;118(3):
333e–40e.
160 N. N. Aldini and M. Fini

34. Yang C, Deng G, Chen W, Ye X, Mo X. A novel electrospun-aligned nanoyarn-reinforced

nanofibrous scaffold for tendon tissue engineering. Coll Surf B Biointerfaces. 2014;122:270–6.
35. Teimouri A, Ebrahimi R, Emadi R, Beni BH, Chermahini AN. Nano-composite of silk
fibroin–chitosan/Nano ZrO2 for tissue engineering applications: fabrication and morphology.
Int J Biol Macromol. 2015;76:292–302.
36. Kishimoto Y, Ito F, Usamia H, Togawa E, Tsukada M, Morikawa H, Yamanaka S.
Nanocomposite of silk fibroin nanofiber and montmorillonite: fabrication and morphology.
Int J Biological Macromol. 2013;57:124–8.
37. Kim H, Park JB, Kang MJ, Park YH. Surface-modified silk hydrogel containing
hydroxyapatite nanoparticle with hyaluronic acid–dopamine conjugate. Int J Biological
Macromol. 2014;70:516–22.
38. Farè S, Torricelli P, Giavaresi G, Bertoldi S, Alessandrino A, Villa T, Fini M, Tanzi MC,
Freddi G. In vitro study on silk fibroin textile structure for Anterior Cruciate Ligament
regeneration. Mater Sci Eng C. 2013;33:3601–8.
39. Gandhimathi C, Venugopal JR, Bhaarathy V, Ramakrishna S, Kumar SD. Biocomposite
nanofibrous strategies for the controlled release of biomolecules for skin tissue regeneration.
Int J Nanomed. 2014;9:4709–22.
40. Suganya S, Venugopalc J, Ramakrishnac S, Lakshmib BS, Giri VR. Naturally derived bio
functional nanofibrous scaffold for skin tissue regeneration. Int J Biological Macromol.
2014;68:135–43.
41. Samal SK, Dash M, Shelyakova T, Declercq HA, Uhlarz M, Bañobre-López M, Dubruel P,
Cornelissen M, Herrmannsdörfer T, Rivas J, Padeletti G, De Smedt S, Braeckmans K,
Kaplan DL, Dediu VA. Biomimetic magnetic silk scaffolds. ACS Appl Mater Interfaces.
2015;7(11):6282–92.
42. Mottaghitalab F, Farokhi M, Shokrgozar MA, Atyabi F. Silk fibroin nanoparticle as a novel
drug delivery system. J Controll Release. 2015;206:161–76.
43. Subia B, Kundu SC. Drug loading and release on tumor cells using silk fibroin-albumin
nanoparticles as carriers. Nanotechnology. 2013;24(3):035103 (Epub 21 Dec 2012).
Chapter 7
Nanoscale Architecture for Controlling
Cellular Mechanoresponse
in Musculoskeletal Tissues

Francesco Oliva, Clelia Rugiero, Umberto Tarantino

and Nicola Maffulli

Abstract Cellular mechanoresponse is not very known yet, especially if we con-

sider the function of nanoscale architecture. First, we need understand the tissue
behavior on macroscale and how this feature is transduced in microscale. How the
musculoskeletal system (bone, cartilage, tendons, muscles, and ligaments)
responses to prestress and to external forces still is unknown for several aspects.
Furthermore, focusing the attention to macroscale and microscale changes in the
musculoskeletal system after injuries seems very interesting. Try to clarify this
knowledge; it is very important to create nanoscale scaffolds able to better improve
musculoskeletal tissue healing.

Keywords Musculoskeletal tissues healing Mechanotransduction




Scaffolds Nanoscale engineering Bone Tendon Muscle Ligament

F. Oliva (&)
C. Rugiero
U. Tarantino

Department of Orthopaedic and Traumatology, School of Medicine,
University of Rome “Tor Vergata”, Rome V. le Oxford 81, 00133 Rome, Italy
e-mail: [email protected]
U. Tarantino
e-mail: [email protected]
N. Maffulli
Centre for Sports and Exercise Medicine, Queen Mary University of London,
London, UK
e-mail: [email protected]
N. Maffulli
Barts and The London School of Medicine and Dentistry,
Mile End Hospital London, London, UK
N. Maffulli
Department of Physical and Rehabilitation Medicine, University of Salerno,
Fisciano, Italy

© Springer International Publishing AG, part of Springer Nature 2018 161

A. C. Berardi (ed.), Extracellular Matrix for Tissue Engineering
and Biomaterials, Stem Cell Biology and Regenerative Medicine,
https://doi.org/10.1007/978-3-319-77023-9_7
162 F. Oliva et al.

Nanometric Architecture

Mechanoresponsiveness is a complex feature of all living tissues [1, 2].

Experiments with cultured cells confirm that mechanical stresses can directly alter
many cellular processes, including signal transduction, gene expression, growth,
differentiation, and survival [3–9]. But which is the mechanism by which
mechanical stresses applied on the macroscale are transmitted to individual cells
on the microscale and transduced into a biological response? To understand the
mechanoregulation process, we must consider that living organisms, such as
humans, are composed of several organs (bone, muscle, blood vessels, nerves) that,
in turn, are constructed from tissues (e.g., muscle fibers, vascular endothelium,
connective tissue) which are composed of groups of living cells and their associated
extracellular matrix (ECM). ECMs are macromolecular complexes composed of
different collagens, glycoproteins, proteoglycans, and many others proteins that
represent the in vivo scaffolds for cell anchorage [10]. Each cell contains intra-
cellular organelles, a nucleus, lipid membranes, and a viscous cytosol permeating a
filamentous cytoskeleton [11, 12]. Each of these subcellular components is com-
posed of aggregates of different molecules. In other words, living systems are
neither homogeneous nor isotropic and therefore require the development of
appropriate inhomogeneous and anisotropic engineering models to describe their
behaviors. The question of how the body senses and responds to mechanical
stresses is not simply an issue of the material properties of its components, also it is
a problem related to the architectural arrangement of its microstructure [13].
Recent advances in molecular biology have focused the attention on the
molecular factors in tissue development. There are many regulatory signals, such as
mechanical stresses, that are equally critical for control of tissue form and function.
This is perhaps most evident in orthopedics where it is well-known that muscle and
bone actively remodel in response to changes in exercise or altered gravity as
experienced in spaceflight [14–19].

Nanoscale Engineering

Tissue engineering is aimed at implant or “seeded” living cells as engineering

materials into artificial biopolymer structures capable of supporting
three-dimensional growth. These structures, called scaffolds, are often capable to
influence their own microenvironments. Successful regeneration necessitates the
development of three-dimensional (3D) tissue-inducing scaffolds that mimic the
hierarchical architecture of native tissue extracellular matrix (ECM). Cells in nature
recognize and interact with the surface topography they are exposed to via ECM
proteins. The interaction of cells with nanotopographical features such as pores,
ridges, groves, fibers, nodes, and their combinations has proven to be an important
signaling modality in controlling cellular processes. Integrating nanotopographical
7 Nanoscale Architecture for Controlling Cellular … 163

cues is especially important in engineering complex tissues that have multiple cell
types and require precisely defined cell–cell and cell–matrix interactions on the
nanoscale. Thus, in a regenerative engineering approach, nanoscale materials/
scaffolds play a paramount role in controlling cell fate and the consequent regen-
erative capacity. Advances in nanotechnology have generated a new toolbox for the
fabrication of tissue-specific nanostructured scaffolds. For example, biodegradable
polymers such as polyesters, polyphosphazenes, polymer blends, and composites
can be electrospun into ECM-mimicking matrices composed of nanofibers, which
provide high surface area for cell attachment, growth, and differentiation [20]. It is
also possible to add to the scaffolds different factors that improve the ECM
generation.

Tensegrity

Tensegrity is the physiological tension that is generated in the musculoskeletal

system, and it is due to the internal stress. The recent convergence between physics
and biology has led many physicists to enter the fields of cell and developmental
biology. One of the most exciting areas of interest has been the emerging field of
mechanobiology that investigates how cells control their mechanical properties and
how physical forces regulate cellular biochemical responses, a process that is
known as mechanotransduction. Tensegrity (tensional integrity) architecture, which
depends on tensile prestress for its mechanical stability, has a central role in biol-
ogy. Prestress is the internal stress prior to application of external force, it is a
critical governor of cell mechanics and function, and tensegrity can be used by cells
contributing to mechanotransduction. Tensegrity also predicts both quantitative and
qualitative behaviors of living cells. In addition, tensegrity is used to both stabilize
three-dimensional form and to channel forces from the macroscale to the nanoscale,
thereby facilitating mechanochemical conversion at the molecular level [21].

Architecture and Prestress

The musculoskeletal system supports the weight of our bodies, allows us to rapidly
adjust to resist external forces, and permits us to move freely in our environment.
Selective pressures demand that the construction of such a machine minimizes mass
with flexibility and without compromising its structural integrity to handle unex-
pected forces. The musculoskeletal system has evolved to address these demands
through optimization of both material properties (how the individual support ele-
ments are designed) and architecture (how the different elements are oriented and
connected together).
The human skeleton is actually strongly influenced by the architecture of our
bodies. Their mechanical behavior depends on how the surrounding tensile
164 F. Oliva et al.

muscles, tendons, and ligaments are joined and oriented in. The internal tension
and/or prestress stabilizes the joints: Even when the bones are pulled away from
each other, the joint does not dislocate. Actually, there are multiple muscles, ten-
dons, and ligaments that contribute to the structure of each joint and the number and
position of these tensile elements play a critical role in defining the joint’s potential
strength, power, speed, and range of motion.
The local stress patterns are clearly visualized in sections or radiographs of the
human femur which demonstrate that the network of trabecular bone is organized to
approximate the principal stress directions. This is known by 1892, when Julius
Wolff and others realized that mechanical loads can affect bone architecture in
living beings, but the mechanisms responsible for this effect were unknown. By
2003, we know how this effect occurs and some of its applications. Our
load-bearing bones include tibias, femurs, humeri, vertebrae, radii, mandibles,
maxillae, wrists, hips, etc. The strength of such bones and their trabeculae would
represent their most important physiologic feature but in the special sense of rel-
ative to the size of the typical peak voluntary loads on them. The biologic “ma-
chinery” that determines whole-bone strength forms a tissue-level negative
feedback system called the mechanostat. Two thresholds make a bone’s strains
determine its strength by switching on and off the biologic mechanisms that
increase or decrease its strength. The largest voluntary loads on load-bearing bones
determine most of their strength after birth, and these loads come from muscle. This
process affects, in part, the healing of fractures, bone grafts, osteotomies, and
arthrodeses; the bone’s ability to endure load-bearing joint and dental endopros-
theses; why healthy bones are stronger than the minimum needed to keep voluntary
loads from breaking them suddenly or from fatigue; some general functions and
disorders of bone modeling and basic multicellular unit-based bone remodeling;
some limitations of in vitro data and of pharmaceutical actions; and the fact that
many bone-active humoral and local agents have permissive roles in a bone’s
adaptations and healing, instead of forcing them to occur [22].
This observation suggests that the living cells that continually remodel bone are
able to sense changes in mechanical stresses in their local environment and that they
respond by depositing new ECM where it is needed and removing it from where it
is not. This process results in deposition of bone ECM in specific patterns that
correspond precisely to engineering lines of tension and compression characteristic
for elastic structure of this size and shape with similar loading conditions. It is an
example of the importance of cellular mechanotransduction for regulation of tissue
morphogenesis. The specialized microarchitecture of cancellous bone further
optimizes its structural efficiency (strength/mass ratio). Architectural organization
on a smaller size scale (the molecular level) also contributes significantly to the
mechanical strength of biologic tissue [23]. In the bone, the matrix of each tra-
beculum consists of a composite material containing hydroxyapatite crystals
embedded within a network of collagen fibrils [24]. The collagen augments the
tensile strength of the bone, while the minerals contribute largely to its compressive
stiffness and strength. In the living organism, the stress in the bone ECM is
influenced by the shape of the entire bone, the pull of the surrounding muscles and
7 Nanoscale Architecture for Controlling Cellular … 165

tendons, and its loading conditions. Contractile fibroblasts also prestress the col-
lagen network during the process of tissue development and remodeling, before the
surrounding ECM calcifies. Prestress also plays an important role in determining
the mechanics of cartilage, tendons, and ligaments [23]. In cartilage, the loose
collagen network is stretched open and prestressed by the osmotic force of
hydration of embedded proteoglycan molecules [9, 25, 26]; however, the cellular
components (chondrocytes) and their internal support elements (cytoskeleton,
nucleus) may also bear some mechanical loads [27, 28]. In soft tissues, that are
composed mostly of parallel collagen fibers and elastin [29, 30], such as ligaments
and tendons, the prestress results from the active contraction of living cells (my-
ofibroblasts) that are embedded within its ECM. The cell contractions pull the
collagen into an undulating, buckled structure and keep the ligament under tension
at all times [31]. Thus, these soft tissues remodel and adjust their fiber orientations
to optimize their load-bearing capacity much like bone and cartilage [23].

Tensegrity and Mechanochemical Transduction

Tensegrity structures are characterized by use of continuous tension and local

compression; architecture, prestress, and triangulation play the most critical roles in
terms of determining their mechanical stability. In living organisms, mechanical
stresses applied at the macroscale result in structural rearrangements at the cell and
molecular level. The demonstration of discrete mechanical linkages between cells
and their ECM via integrins suggests how mechanical signals resulting from ECM
deformation may be transferred across cell surface integrin receptors to distinct
structures in the cell and nucleus, including ion channels, nuclear pores, nucleoli,
chromosomes, and perhaps even individual genes, independently of ongoing
chemical signaling mechanisms [8, 9, 27, 28]. In fact, different studies have
demonstrated that signal transduction pathways can be activated within millisec-
onds after cell surface integrins and associated cytoskeletal connections are
mechanically stressed [32]. This type of physical coupling between intracellular
structures, cell surface receptors, and the ECM could serve to coordinate, com-
plement, and constrain slower diffusion-based chemical signaling pathways and
thus explain how mechanical distortion of ECM caused by gravity or other
mechanical stresses can change cell shape, alter nuclear functions, and switch cells
between different genetic programs [8, 33]. The question remains: how could these
tensegrity-based structural rearrangements be transduced into a biochemical
response? Several potential mechanisms can be envisioned. For example, a recent
work suggests that much of the cell’s metabolic and signal transduction machinery
effectively functions in a “solid state” [34]. The enzymes and substrates that
mediate these pathways are physically immobilized on the insoluble molecular
scaffolds that comprise the cytoskeleton and nuclear matrix (nucleoskeleton). In
fact, many signal-transducing molecules that are activated by cell binding to growth
factors and ECM actually appear to be concentrated at the site of integrin binding,
166 F. Oliva et al.

on the cytoskeletal backbone of the focal adhesion [35, 36]. Thus, mechanical
signals may be integrated with other environmental signals and transduced into a
biochemical response through stress-dependent changes in cytoskeletal scaffold
geometry or mechanical deformation [9]. For example, one potential mechanism for
mechanochemical transduction is through stress-dependent cytoskeletal rearrange-
ments that result in changes in proximity between different immobilized enzymes
and substrates. If a protein kinase and its physiological substrate were both
immobilized on the cytoskeleton and physically separated, then no phosphorylation
would result. However, if there is a mechanical deformation of the tissue, ECM,
cytoskeletal composite results in structural rearrangements that bring these two
molecules into direct apposition and then phosphorylation might proceed causing a
downstream signaling cascade to initiate. In addition, the transfer of focused
mechanical energy to soluble molecules in the cytosol will alter their shape and
hence their electrochemical potential through mechanical distortion. Stress-induced
changes in molecular mechanics (stiffness and conformation) can then produce
direct mechanochemical transduction by altering thermodynamic (association and
dissociation constants) or kinetic (molecular motion) parameters. Regulation of
tubulin polymerization (microtubule formation) by mechanical stresses balanced
between microtubules, contractile microfilaments, and ECM provides one example
of a thermodynamic transduction mechanism [37]. Stress-dependent changes in the
frequency of opening and closing “stretch-sensitive” ion channels are an excellent
example of the kinetic form of transduction [33]. Living cells sense multiple
simultaneous inputs and are able to organize a single, concerted response. The
tensegrity structure may be used to focus mechanical energy on critical transducing
molecules and to “tune” the entire cellular response to stress by mechanically
coupling biologic structures at different size scales and in different locations within
living cells, tissues, and organs. This tuning function may be accomplished by
altering the prestress in the system (e.g., by varying cytoskeletal tension), remod-
eling architecture, or modifying the mechanics of individual structural components.
Specificity results from local changes in material properties of the structural ele-
ments (e.g., stress will rapidly dissipate in highly viscous regions), and from how
the different elements are mechanically coupled (e.g., linkage of the cytoskeleton to
the ECM by integrins and to the cytoskeleton of neighboring cells by cell–cell
adhesion molecules, such as cadherins) [13].
Also in tendon studies, it is evident the important role of ECM. Tendon ruptures
are increasingly common, repair can be difficult, and healing is poorly understood.
Tissue engineering approaches often require expansion of cell numbers to populate
a construct, and maintenance of cell phenotype is essential for tissue regeneration.
Total collagen, the ratio of collagen types I and III, and decorin can be used as
indicators of matrix formation in human tendon, and expression of the integrin beta
1 subunit as a marker of cell–matrix interactions, for example in patients receiving
surgery for rupture. During the healing, cells become more rounded and are more
widely spaced at confluence, and confluent cell density declines. No change to total
cell layer collagen is observed, but the ratio of type III to type I collagen increases at
the end. Decorin expression significantly decreases, while integrin expression does
7 Nanoscale Architecture for Controlling Cellular … 167

not change. Thus, the phenotype of tenocytes in culture rapidly drifts during the
healing [38].
This architectural model of biologic organisms may help to explain one of the
most fundamental properties of living creatures: how the parts and the whole
function as a single integrated system. Due to use of tensegrity architecture,
mechanical stress is concentrated and focused on signal-transducing molecules that
physically associate with cell surface molecules that anchor cells to extracellular
matrix, such as integrins, and with load-bearing elements within the internal
cytoskeleton and nucleus. Mechanochemical transduction may then proceed
through local stress-dependent changes in molecular mechanics, thermodynamics,
and kinetics within the cell. In this manner, the entire cellular response to stress may
be orchestrated and tuned by altering the prestress in the cell, just as changing
muscular tone can alter mechanical stability and structural coordination throughout
the whole musculoskeletal system [13].
The finding that cells are not bits of viscous cytoplasm surrounded by an elastic
membrane but instead structured as tensegrities with internal load-bearing struts and
tensed cables also has led to novel insights into developmental control and
pathobiology also in other fields, and not only for the musculoskeletal system. For
example, the finding that microtubules bear compression in living cells is extremely
relevant for heart physiology because an increased density of the microtubule
component of the extramyofilament portion of the cardiocyte cytoskeleton caused
by pressure overload can physically interfere with inward-directed shortening of the
myofibrillar bundle and hence lead to contractile dysfunction associated with car-
diac hypertrophy. The integrated nature of biological architecture also helps to
explain why cardiac diseases and developmental abnormalities can be caused by
mutations in various ostensibly unrelated molecules, including integrins,
cytoskeletal filaments, ion channels, or nuclear components. Forces channeled over
ECMs and to integrins are converted into biochemical changes by producing
changes in deformation of other load-bearing mechanotransducer molecules, such
as stress-sensitive ion channels, protein kinases, G proteins, and other signaling
molecules, inside the cell. Recent advances in mathematics, engineering, and sta-
tistical mechanical models of tensegrity structures, and in the use of nanotechnol-
ogy to create artificial cell–material control interfaces, may provide new ways to
investigate, model, manipulate, probe, and control these fundamental mechan-
otransduction mechanisms in living cells, tissues, and organs, including heart, in the
future [39].

Hormesis

Hormesis is a dose–response phenomenon, characterized by a low-dose stimulation

and a high-dose inhibition. The term hormesis was first introduced into the scien-
tific literature in 1943 by Chester Southam and John Ehrlich, mycology researchers
at the University of Idaho. Reactive oxygen species (ROS) are emerging as
168 F. Oliva et al.

intracellular redox signaling molecules involved in the regulation of bone meta-

bolism, including receptor activator of nuclear factor-jB ligand-dependent osteo-
clast differentiation, but they also have cytotoxic effects that include
lipoperoxidation and oxidative damage to proteins and DNA. ROS generation,
which is implicated in the regulation of cellular stress response mechanisms, is an
integrated, highly regulated, process under control of redox-sensitive gene coding
for redox proteins called vitagenes. Vitagenes, encoding for proteins such as heat
shock proteins (Hsps) Hsp32, Hsp70, the thioredoxin, and the sirtuin protein,
represent a system controlling a complex network of intracellular signaling path-
ways relevant to life span and involved in the preservation of cellular homeostasis
under stress conditions. The biological relevance of dose–response affects those
strategies pointing to the optimal dosing to patients in the treatment of numerous
diseases. Thus, the heat shock response has become an important hormetic target
for novel cytoprotective strategies focusing on the pharmacological development of
compounds capable of modulating stress response mechanisms [40]. Hormesis is a
type of stress often related to reactive oxygen species (ROS) originating from the
mitochondrial respiratory chain (mitochondrial hormesis or mitohormesis). The
accumulation of transient low doses of ROS either through chronic physical activity
or caloric restriction influences signaling from the mitochondrial compartment to
the cell, reduces glucose metabolism, induces mitochondrial metabolism, increases
stress resistance, and increases life span. Mitochondrial formation of presumably
harmful levels (chronic and/or excessive) of ROS within skeletal muscle has been
observed in insulin resistance of obese subjects, type 2 diabetes mellitus and in
impaired muscle function associated with normal aging. Advances in mitochondrial
bioimaging combined with mitochondrial biochemistry and proteome research have
broadened the knowledge of specific cellular signaling and other related functions
of the mitochondrial behavior. Mitochondria are dynamically involved in several
muscle cellular activities including signaling, proliferation, differentiation, autop-
hagy, and death. The dynamics, size, number, and location of mitochondria in
muscle cells vary significantly according to the cellular conditions and energetic
needs. Mitochondria are generally considered as the main source of ROS in skeletal
muscle cells. Several physiological and pathological conditions may result in ROS
production by mitochondria. For example, high intense contractile activity, disuse
muscle atrophy or inflammation increase mitochondrial ROS generation. Although
the excessive ROS production is associated with the etiopathology of several
human disease, low levels of ROS are important mediators for a variety of cellular
process, including cell adhesion, immune response, apoptosis, cell growth, and
differentiation. ROS also act as second messengers in intracellular signaling.
However, chronic and excessive ROS production may lead to muscle cell damage
and death. The final effect of ROS depends on intrinsic and extrinsic factors such as
the level and the duration of ROS targeting muscle cells, the source, the site of ROS
generation, the antioxidant status of target cells, the DNA repair capacity, the
differentiation stage of muscle cells, and the proliferative and myogenic capacity of
satellite cells. Due to their hormetic nature, in muscle tissue mitochondrial ROS
may trigger different signaling pathways leading to different and diverging
7 Nanoscale Architecture for Controlling Cellular … 169

responses, from adaptation to cell death. For example, physical exercises and
chronic contractile activity induce a number of physiological adaptations that
ameliorate muscle function and exercise performance. Trained muscle undergoes a
remodeling toward a more oxidative phenotype altering the ultrastructural and
subcellular organization. One of the most important effects of endurance training is
mitochondrial biogenesis with an increase in mitochondrial content. It is
well-known that physical exercise increases ROS generation in skeletal muscle
cells. During exercise, the increased ROS production is mainly due to the high
oxygen consumption that takes place during increased mitochondrial activity. There
is probably generation of ROS at multiple subcellular locations in response to a
multiplicity of mechanical and metabolic stimuli in muscle cells [41]. Thus,
contraction-induced production of ROS has been shown to cause oxidative stress to
skeletal muscle. As an adaptive response, muscle antioxidant defense systems are
upregulated after heavy exercise. Nuclear factor (NF) kappaB and
mitogen-activated protein kinases (MAPKs) are the major oxidative stress-sensitive
signal transduction pathways. Activation of NF-kappaB signaling cascade has been
shown to enhance the gene expression of important enzymes, such as mitochondrial
superoxide dismutase (MnSOD) and inducible nitric oxide synthase (iNOS).
MAPK activations are involved in a variety of cellular functions including growth,
proliferation, and adaptation. The exercise has effects on NF-kappaB and MAPK
signaling, as well as on the time course of activation, in rat skeletal muscle. In
addition, ROS have a role in the exercise-induced upregulation of MnSOD and
iNOS, and in the potential interactions of NF-kappaB and MAPK in the signaling of
these enzymes. It is clear that ROS may serve as messenger molecules to activate
adaptive responses through these redox-sensitive signaling pathways to maintain
cellular oxidant–antioxidant homeostasis during exercise [42]. Furthermore, phys-
ical exercise shares common metabolic paths with caloric restriction and glucose
restriction increasing mitohormesis and inducing a positive adaptive response that
ends with stress resistance, antioxidant defense, and prolonged life span [41]. In
addition, the mild stress-induced hormesis stimulates maintenance and repair sys-
tems and strengthens the homeodynamic space of cells and organisms. Hormesis
through mild heat shock, natural and synthetic hormetins, and other stressors brings
about several antiaging effects in human fibroblasts, keratinocytes, and
telomerase-immortalized bone-marrow stem cells. Depending on the cell type,
these antiaging hormetic effects include extension of replicative life span, enhanced
proteasomal activities, increased chaperone levels, and improved wound healing,
angiogenesis, and differentiation. The main molecular pathways for achieving such
hormetic effects are through targeting the processes for the repair and removal
of molecular damage, which can slow aging [43].
Hormesis has a role not only in the muscle but also in the bone and in its diseases
such as Alzheimer pathology and osteoporosis [40]. Epidemiological studies also
indicate that patients suffering from atherosclerosis are predisposed to develop
osteoporosis. Atherogenic determinants such as oxidized low-density lipoprotein
(oxLDL) particles have been shown both to stimulate the proliferation and promote
apoptosis of bone-forming osteoblasts. OxLDL particles cause hormesis-like
170 F. Oliva et al.

response with the stimulation of both proliferation and cellular NAD (P)
H-dependent reduction potential by low concentrations, whereas high concentra-
tions are associated with the cell death. The induction of hormesis-like response by
oxLDL in osteoblastic cells is associated with the stimulation of cell proliferation
and ROS production by low concentrations of oxLDL. It is generally accepted that
the stimulation of osteoblastic proliferation may compromise their differentiation
into competent bone-forming cells. In accordance, low concentrations of oxLDL
reduce the alkaline phosphatase activity, a marker of osteoblastic maturity. In
addition, oxLDL compromises the migration of osteoblastic cells. Both functions
have been shown to play a critical role in bone formation, remodeling, and fracture
repair. Therefore, low concentrations of oxLDL may alter the bone metabolism by
reducing osteoblastic differentiation in favor of uncontrolled cell proliferation and
by affecting cell migration. On the other hand, high concentrations of oxLDL cause
osteoblastic cell death that will result in reduced bone formation. In summary,
oxLDL particles alter osteoblastic cell proliferation, migration, and apoptosis rate
and thereby may contribute to alteration of bone metabolism equilibrium and may
be responsible for the reduction of bone mass associated with atherogenic condi-
tions [44].

Bone and Osteoinduction

Mesenchymal stem cells (MSCs) have the potential to replace or restore the
function of damaged tissues and offer much promise in the successful application of
tissue engineering and regenerative medicine strategies [45]. It has been demon-
strated that adult mesenchymal stem cells (MSCs) are capable of giving rise to
several different cell types, including myoblasts, adipocytes, fibroblasts, chondro-
cytes, and osteoblasts [46–49]. MSCs exist in a highly specialized microenviron-
ment in which their maintenance, proliferation, migration, and differentiation
involve a complex interplay of many local and systematic signals between stem
cells and adjacent cells [50–55]. Osteoblasts are derived from MSCs and exist in
close proximity to the bone marrow, which serves as a major source of MSCs and
hematopoietic stem cells. They are located on the surface of bone, express multiple
hormone receptors and various cell surface molecules, and secrete hormones and
enzymes that maintain the balance between bone formation and resorption [56, 57].
However, it remains unclear whether and how osteoblasts affect the self-renewal
and differentiation of another major stem cell population in bone-marrow MSCs.
Co-cultures with MSCs and with a higher density of osteoblasts express greater
mRNA expressions of Runx2, type I collagen, and osteocalcin [58]. Human bone-
marrow-derived MSCs, by virtue of their capacity for self-renewal and multipotent
differentiation, are considered promising candidate cells for regenerative medicine
and tissue engineering applications. The multipotent differentiation capabilities of
MSCs into various types of cell lineage, such as osteoblasts, chondrocytes, and
adipocytes, have been characterized in numerous studies [46, 47]. Osteoblasts,
7 Nanoscale Architecture for Controlling Cellular … 171

which are derived from bone-marrow MSCs, are the main cell type responsible for
bone formation and are one of the neighboring cell types close to the bone marrow.
It is believed that interaction between bone-marrow MSCs and their neighboring
cells may lead to MSC osteogenesis via cell–cell contact or osteoblast-derived
soluble factors [59]. It has been demonstrated that Runx2 is as an essential tran-
scription factor for the induction of early osteogenic differentiation. Studies of the
expression of Runx2 demonstrate that the early osteogenesis of co-cultures is highly
dependent on osteoblast numbers co-cultured in an osteoinductive environment.
Co-cultures with MSCs and osteoblasts demonstrated that direct cell–cell contact is
sufficient to induce osteogenic differentiation by enhancing the gene expressions of
Runx2, type I collagen, and osteocalcin. More significant and distinct osteogenic
differentiation is promoted by additional osteogenic supplements. The quality of
osteogenesis, as evidenced by protein expression, is proportional to the quantity of
osteoblasts in the co-cultures. A recent study has also suggested that primary bone-
derived cells promote the osteogenesis of human embryonic stem cells in a
co-culture model by releasing bone morphogenetic proteins 2 and 4 [60]. These
findings demonstrate that the osteogenesis of stem cells may be induced or pro-
moted in a co-culture system through either direct cell–cell contact or secreted
soluble factors derived from osteoblasts. These findings show that direct cell–cell
contact between MSCs and osteoblasts contributed little to MSC osteogenic dif-
ferentiation. In addition, the co-cultures required at least one osteoinductive factor
to induce or enhance the osteogenic differentiation in MSCs: either an appropriate
number of osteoblasts or treatment with osteogenic supplements. Culture conditions
that included both a large quantity of osteoblasts and osteogenic supplements
resulted in synergistic inhibition during osteogenesis by shrinking the process of
osteogenic differentiation. The direct cell–cell contact and soluble osteogenic fac-
tors might act as synergistic modulators to promote or inhibit osteogenic differ-
entiation in MSCs. Understanding the possible synergistic interactions between
MSCs and osteoblasts might be an alternative approach to manipulating the fates of
stem cells and for differentiating lineages in cell-therapeutic strategies and regen-
erative medicine [58]. Mesenchymal tissues are subjected to mechanical stimuli
in vivo, and terminally differentiated cells from the mesenchymal lineage respond
to mechanical stimulation in vivo and in vitro. MSCs have also been shown to be
highly mechanosensitive, and this may present an ideal method for controlling
MSC differentiation. External mechanical forces are able to induce or enhance MSC
differentiation into a wide variety of tissue-specific cells; however, precisely con-
trolling the timing and outcome of MSC differentiation is still a large challenge.
Characterization of the exact force MSC experience in loading systems is important
to know what mechanisms are actually inducing the observed responses and to help
simplify the design of future loading systems. While most bioreactor systems
employ a common force type (e.g., tension or compression), there are also likely to
be other mechanisms at work causing significant secondary effects. Mathematical
and computer modeling is important for characterizing the forces that are being
experienced by the cells [61].
172 F. Oliva et al.

In conclusion, mechanical stress plays a vital role in maintaining bone archi-

tecture and this was studied for the first time by Julius Wolff: Mechanical loads can
affect bone architecture in living beings [23]. However, the process by which
osteogenic cells convert the mechanical signal into a biochemical response gov-
erning bone modeling is not clear. For example, in a recent study, was investigated
how Atlantic salmon (Salmo salar) vertebra responds to exercise-induced
mechanical loading. Bone formation in the vertebrae was favored through
increased expression of genes involved in osteoid production. Fourier transform
infrared spectroscopy (FT-IR) showed that bone matrix secreted both before and
during sustained swimming had different properties after increased load compared
to control, suggesting that both new and old bones are affected. Concomitantly,
both osteoblasts and osteocytes in exercised salmon showed increased expression of
the receptor nk-1 and its ligand, substance P (SP), both known to be involved in
osteogenesis. The functional role of SP was investigated in vitro using osteoblasts
depleted for SP. The cells showed severely reduced transcription of genes involved
in mineralization, demonstrating a regulatory role for SP in salmon osteoblasts.
Investigation of a-tubulin stained osteocytes revealed cilia-like structures. Together
with SP, cilia may link mechanical responses to osteogenic processes in the absence
of a canaliculi network. These results imply that salmon vertebral bone responds to
mechanical load through a highly interconnected and complex signal and detection
system, with SP as a key factor for initializing mechanically induced bone for-
mation in bone lacking the canaliculi system [62].
Thus, it is probably that the bone development is mediated by neurotransmitters.
In fact, SP and calcitonin gene-related peptide (CGRP) have been also found in the
perichondrium and within the cartilage canals. It is still unknown whether they exert
a direct effect on chondrocytes during joint development. The presence of CGRP
and SP indicates the presence of nerves fibers and precedes the development of
cartilage canals. Nerve fibers may play a role in the development of synovial joints
before and during the presence of cartilage canals. The presence of CGRP and SP in
the cartilage at birth may be involved in the early postnatal maturation of synovial
joints [63]. Substance P is part of the superfamily of tachinins. SP exerts a trophic
influence on neuronal tissue. The release of SP can lead to vasodilatation, plasma
extravasion, and leukocyte recruitment, with a clear proinflammatory action which
continues through the release of collagenase, prostaglandin E2, interleukin 1,
TNFa, and oxygen metabolites [64]. Peripheral nervous fibers containing SP have
been identified in the synovial membrane, in ligaments, in the menisci, the sub-
chondral bone of human or rat and in some feline and horse joints [65]. Recent
studies showed neurofilaments in arthritic cartilage and in osteophytes. This sug-
gests a role of this neuropeptide in pain transmission and during degenerative and
reparative processes occurring in arthritic joints [66]. CGRP is a peptide produced
by tissue-specific alternative processing of the primary RNA transcripts of the
calcitonin gene. Nervous fibers containing CGRP have been identified in spinal
motoneurons [67], in the motor plates of voluntary muscles, in the bone marrow,
close to the epiphyseal plates in contact with osteoblasts, in the periosteum, around
the vessels in Volkman’s channels and the Havers’s channels, in the synovial
7 Nanoscale Architecture for Controlling Cellular … 173

membrane, in ligaments, and in subchondral bone of human joints [68]. CGRP also
seems to be involved in tendon and fracture healing [68, 69]. SP and CGRP have
been identified in the perichondrium and in periarticular neurons [70]. Neuronal
involvement may play an important role in the development of degenerative joint
disease, consequent to progressive age-related loss of joint innervation. Autonomic
nerve fibers may also actively participate in bone repair [71]. It is possible that the
periarticular autonomic nerve fibers, through their neuropeptides, exert a trophic
effect on joint development.

ECM and Tendons

On the other hand, it is necessary to focus the attention on the soft tissues, such as
tendons. Only through the clarification of the microanatomy, pathophysiology,
genetics of ECM, we may be able to improve current therapeutic knowledge on
tendon diseases. Unfortunately, today too often the media and some areas of
medicine offer to the patients new medical therapy based on tissue regeneration of
tendons without scientific certainty of the effectiveness. The main effort of the
research at this time should be focused not only on the in vivo application of new
potential therapies but also and especially on the mechanisms that regulate the
homeostasis of the ECM during exercise and pathologic conditions [72].
Tendon structure. The structure of a tendon is an important example of com-
plexity of ECM three-dimensional organization. The extracellular matrix (ECM) is
a macromolecular network with both structural and regulatory functions. Tendons
are made by a fibrous, compact connective tissue that connects muscle to bone
designed to transmit forces and withstand tension during muscle contraction. The
extracellular matrix (ECM) is a macromolecular network with both structural and
regulatory functions. Indeed, ECM furnishes mechanical and biochemical signals
that cooperate in the integrated control of cell proliferation, survival, migration, and
differentiation. ECM components belong to four major types of macromolecules:
the collagens, elastin, proteoglycans, and noncollagenous glycoproteins (e.g.,
laminins, entactin/nidogen, fibronectin, and tenascins). The most copious proteins
in the ECM are members of the collagen family. The collagens are involved in the
formation of ECM fibrillar and microfibrillar networks and play a key role in
determining tissue-specific mechanical properties.
Tendon’s ultrastructure and ECM. The apparently simple tendonʼs structure
and composition provide both rigidity and flexibility. This ability is based on
tendon nonlinear, viscous-elastic, anisotropic, and heterogeneous mechanical
properties. Similar to other tissues, there is a clear relationship between structure
and function of the tendon [73]. Indeed, different tissue microenvironments provide
specific characteristics to the different three-dimensional organization of the ECM
since embryogenesis [74]. The organization of the ECM in the tendon is peculiar as
well as within different tendons. The dialogues between different cells (fibroblasts,
muscle, and cartilage cells) or better the dialects between different cells mediated by
174 F. Oliva et al.

various signaling cascades dependent on FGFs and TGF beta are used at decisive
stages of development to make specific the phases of induction, organization,
aggregation, or differentiation of cells [75]. The structure of the tendon is so
complex that it is very difficult to maintain its function in the healing since this
process is reparative rather than regenerative in the adult [76, 77]. A tendon consists
of 70% of water and 30% of dry mass, which is composed by 60–80% of type I
collagen and 2% of elastin. Among collagens, the most abundant component is
collagen type I (95%), while type III and type V collagens represent the remaining
5% of the total collagens [77]. There are many types of collagen, and each of these
has a specific function; every year one new type or one new function is discovered.
Tendons in addition to collagen contain proteoglycans, glycosaminoglycans, and
glycoproteins including fibronectin, thrombospondin, and tenascin-C immersed in
different compositions of ECM lying cellular elements represented (90/95%) by
tenoblasts and tenocytes. The remaining 5–10% of the cells consists of chondro-
cytes, synovial and vascular cells [73]. This information can be useful to create
appropriated nanoengineering techniques [78].

Proprioception: Muscle, Tendon, and Ligament

Mechanoreceptors in healthy muscles, ligaments, and tendons procure the sensation

of the joint movement, joint position, and stability. Loss of mechanoreceptors not
only causes mechanical instability, but also leads to a disturbance in the neuro-
muscular control due to the loss of mechanoreceptors. Proprioception is the sen-
sation of the joint movement and joint position [79].
Ligaments and proprioception. Research has shown that four types of nerve
endings can be listed in ligaments: Ruffini corpuscles, Pacinian corpuscles, Golgi
tendon organ-like endings, and free nerve endings [80]. These four types of
receptors were also found in the fibrous joint capsule. Ruffini endings are inter-
twined with collagen fibers, and they seem to be activated by the displacement of
the fibers. Ruffini corpuscles are both static and dynamic mechanoreceptors which
are able to signal static joint position, changes in intra-articular pressure as well as
movement direction, amplitude, and velocity [80, 81]. Pacinian corpuscles are
dynamic, rapidly adapting mechanoreceptors with a low threshold. They are
inactive on immobile joints, though becoming active at the acceleration and
deceleration. Golgi tendon organ endings have an high threshold and are slowly
adapting. They are inactive in immobile joint, and it has been suggested that they
measure the ligament tension. They have been discovered in the cruciate ligaments,
medial collateral ligament, the medial patellar ligament. Finally, free nerve endings
are the articular nociceptor endings [82].
The sensory role of the cruciate ligaments. Knee joint ligaments have been
traditionally considered passive structures only involved in mechanical functions
[83]. A variety of studies concerning the presence of mechanoreceptors in cruciate
ligaments have been reported [84]. Three different types of nerve fibers and of nerve
7 Nanoscale Architecture for Controlling Cellular … 175

endings have been described in normal ACL: (1) fibers of large diameter which are
fast-conducting mechanoreceptive sensory afferents; (2) fibers of small diameter
slow-conducting nociceptive sensory afferents, and (3) sympathetic efferent vaso-
motor fibers. How we said before, four nerve endings have been identified in human
cruciate ligaments: Ruffini corpuscles, Pacinian corpuscles, Golgi tendon organ-like
endings, and free nerve endings [82, 85]. Ligament mechanoreceptors influence
movements via the c-muscle spindle system. They contribute to the stiffness pre-
programming around the joint and are crucial for joint stability maintenance.
Therefore, the knee ligament injury would damage the mechanoreceptors and alter
neuromuscular functions secondary to diminished somatosensory information. ACL
rupture causes considerable changes in stretch reflex excitability, so the “giving
way” of the knee is not simply related to the decrease in mechanical joint stability,
but is closely associated with altered stretch reflex excitability that most probably
takes place on the spinal level. Reduction of mechanoreceptors is not only due to a
lesion of the soft tissue. Mechanoreceptors are not immutable structures, but their
properties change with aging. Aging affects numbers and morphology of
mechanoreceptors. This phenomenon could explain why normal aging process is
associated with deficits in proprioception.
It was left to Abbott et al. [86] to attribute more importance to knee structures, as
they first described the knee ligaments as having rich sensory innervation, which
allowed them to act as the first link in the kinetic chain. Ever since, our under-
standing of this complex functioning has evolved, and it is now widely accepted
that movement or charge in knee position stimulates receptors in knee ligaments
that allow the conscious appreciation of limb position in space. Proprioception is
receptor and neural arc mediated. The stimulation of mechanoreceptors in the knee
ligaments initiates different types of reflex muscle contractions through the neural
arc involving the dorsal root ganglion sensory neurons. Many studies over the last
30 years have demonstrated significant presence of mechanoreceptors in the fibers
of an intact anterior cruciate ligament (ACL). These were first described by Schultz
et al. [87] in 1984, and it was subsequently established that the receptors included
not just Pacinian, Ruffini, or Golgi tendon-type bodies, but also numerous nerve
endings distributed all over the ACL. These receptors play an important role in the
complicated neural network of proprioception. They are capable of detecting
changes not only in tension, speed, acceleration, and direction of movement, but
also allow a subconscious determination of the position of knee joint in space. It
becomes a corollary that damaged mechanoreceptors would alter neuromuscular
functions secondary to diminished somatosensory information (proprioception and
kinesthesia). In modern orthopedics, this has become a key factor in understanding
functional instability after ACL injuries and methods to treat it. Subsequent to an
ACL injury, it has been observed that the relationship between passive stability and
the functional stability of the knee joint is often ambiguous. It is possible that the
functional instability that occurs after an injury to the ACL is due to the combined
effects of excessive tibial translation and a lack of “coordinated muscle activity” to
stabilize the knee joint. This lack of coordinated muscle stabilization of the knee
176 F. Oliva et al.

joint is thought to be due to diminished or absent sensory feedback from the ACL to
the neuromuscular system.
Mechanoreceptors in intact ACL. The first histological demonstration of
mechanoreceptors in the human ACL was done by Schultz et al. [87]. The cruciate
ligaments were obtained at the time of total knee replacement and from autopsy and
amputation specimens. The ultrastructure of nerve endings in a human knee joint
capsule was subsequently described by Halala et al. [88]. These authors found the
three types of nerve endings: free nerve endings, Ruffini corpuscles, and Pacinian
corpuscles. In the joint capsule, free nerve endings were located below the synovial
layer and within the fibrous layer near blood vessels. These nerve terminals are
derived from myelinated A-delta fibers or from unmyelinated C-fibers. Ruffini
corpuscles were present within the fibrous layer and the ligaments of the capsule in
three variations: small Ruffini corpuscles without a capsule, small corpuscles with a
connective tissue capsule, and large Ruffini corpuscles with an incomplete per-
ineural capsule. Their afferent axons were myelinated, and, inside the corpuscle,
nerve terminals were anchored in the connective tissue belonging to the fibrous
layer or to the ligaments, respectively. The presence of an incomplete perineural
capsule depended on the structure of the surrounding connective tissue. In liga-
ments with collagenous fibrils oriented in a parallel fashion, the perineural capsule
was well-developed and the Ruffini corpuscle resembled a Golgi tendon organ; in
areas where the fibrils showed no predominant orientation, Ruffini lacked a capsule.
Small Pacinian corpuscles were situated within the fibrous layer near the capsular
insertion at the meniscus articularis or at the periosteum. Larger Pacinian corpuscles
with one or several inner cores and a perineural capsule were found on the outer
surface of the fibrous layer. By the turn of the century, it was becoming clear that
the mechanoreceptors located in the ACL constitute an afferent source of infor-
mation toward the central nervous system. The ACL deficiency can cause a dis-
turbance in neuromuscular control and affects central programs and consequently
the motor response resulting in serious dysfunction of the injured limb. It was
examined brain activation by using functional magnetic resonance imaging tech-
nique (1.5-T scanner): ACL deficiency can cause reorganization of the central
nervous system, suggesting that such an injury might be regarded as a neuro-
physiologic dysfunction, not a simple peripheral musculoskeletal injury [89]. This
evidence could explain the variation of clinical symptoms that accompany this type
of injury, and the degrees of dysfunctions in different individuals with an
ACL-deficient knee.
Mechanoreceptors in the stump of an injured ACL. In untreated ACL lesions in
humans, morphologically normal mechanoreceptors persisted in the ACL remnant
for about 3 months after injury. Beyond that time, the number of receptors grad-
ually decreased. By the ninth month after injury, only a few nerve endings were
found, and they were totally absent after 1 year. These results indicate that the
proprioceptive potential of the stump may diminish with the passage of time, and
this may have a potential bearing on surgical outcomes in cases where recon-
struction is delayed. Thus, preserving the ACL remnants might improve functional
7 Nanoscale Architecture for Controlling Cellular … 177

outcome after ACL reconstruction as some reinnervation and recovery of propri-

oception are likely in such cases.
Previous studies of ACL anatomy and histology have demonstrated that the
maximum concentration of the nerve endings is at the attachment sites of the
ligament to the bone. This serves as the main tract for proprioceptive feedback.
These are the stumps that are seen at arthroscopy and are routinely removed,
thereby aggravating the sensory damage to the knee joint. In addition, preserving
remnant provided synovium for the reconstructed ACL, and it could accelerate
revascularization of the graft. The remnants not only improved proprioception but
also provided mechanical stability in certain cases. However, there is always a risk
of developing a cyclops lesion if the remnant is preserved and this could lead to
impingement. In conclusion, remnant preservation in ACL reconstruction, although
technically demanding, can provide better clinical results as compared to
remnant-sacrificing techniques.
With presence of degenerative changes, the proprioceptive potential of the
injured stump decreases more, further substantiating the fact that delays in treatment
negatively affect the mechanoreceptor and proprioceptive fibers in the stump. In
ACL-deficient knees of long duration, repeated episodes of giving way and reinjury
due to instability may render the stump prone to degeneration and decrease its
proprioceptive potential.
In conclusion, mechanoreceptors in intact ACL contribute toward functional
stability of the knee joint. Injury to ACL not only causes mechanical instability, but
also leads to a disturbance in the neuromuscular control of the injured knee due to
loss/damage to mechanoreceptors. ACL reconstruction restores proprioceptive
potential of the knee to some extent, but the results vary. Although the remnant
ACL contains residual mechanoreceptors, the number and functionally of these
receptors is dependent, to some extent, on the physical characteristics of the rem-
nant and duration of injury. Nevertheless, these remnants are worth preserving
during ACL reconstruction and can play an important role in restoration of pro-
prioception of knee following ACL reconstruction [90].
Tendinous mechanoreceptors. Golgi tendon organ-like endings have been
found in tendons. Stimulus response suggests they are high-threshold, slowly
adapting mechanoreceptors that are completely inactive in immobile joints. They
become active when joints are at the extreme of their ranges of movement and
considerable stresses are generated in the joint ligaments [80, 82].
Healing of tendon mechanoreceptors. The dynamic response of the Golgi
tendon organs is made physiologically by two components: an initial peak and a
second main component showing correlation between tension rate and discharge
frequency. During first part of the healing, motor units had to come to a full
recovery. Speed contraction and absolute tension were not fully restored yet.
Almost all receptors were reinnervated. Probably after a complete nerve section,
occurs a reinnervation by inappropriate axons. However, abnormal responses seem
more due to an immaturity of the transduction mechanism. After the nerve tran-
section, the structure of the motor units is modified; this can lead to an increase in
178 F. Oliva et al.

the mechanical input to the Golgi tendon organs. Missing of the responses coincides
with an alteration of the dynamic sensitivities [91].
Muscle mechanoreceptors. Golgi tendon organs co-work with another
mechanoreceptor: the muscle spindle, which is the most innervated receptor, with
afferent and efferent fibers. These receptors are aligned parallel to the muscle fibers,
and they primarily detect changes in the length and the speed of muscles [92].
Mechanoreceptors function as transducer devices converting a signal in one form of
energy to another one (i.e., the physical stimulus of tension into a specific nerve
signal). The summation of receptor discharges forms a frequency-modulated code
that the central nervous system uses to analyze joint position, motion, and accel-
eration [93]. The transduction of mechanical stimuli into electrical responses is
essential for audition, kinesthesis, proprioception, and automatic sensation of
pressure and volume. Seems that Pacinian corpuscles and Golgi tendon organs are
more represented than Ruffini endings in ligaments, both Pacinian corpuscles and
Golgi tendon organs are physiologically reflexogenic. Pacinian corpuscles dis-
charge at the onset or cessation of movement and are involved in quick movements;
this afferent feedback to the central nervous system would have a stabilizing effect
on the joints. Golgi tendon organs are found in ligaments, myotendinous and
myoaponeurotic junctions of mammalian skeletal muscle. Their discharges are
responsible for tension and joint position; thus, they are also responsible for joint
stabilization [80, 94].
Healing of muscle mechanoreceptors. Few reports focused the healing of
mechanoreceptors. Some of these showed that muscle spindle afferents reinnervate
their original ending sites with the spindle after a nerve injury but the afferents that
had been regenerated are smaller than the control group. In contrast Haftel and
coworkers showed that not all the regenerated afferences were able to restore a
pathway to central nervous system. Moreover, there were some differences in
muscle spindle responses to stretch, showing that reinnervated afferences had an
increased length in threshold, probably due to an incomplete recovery of gamma
motoneurons drive to reinnervated muscle spindles [95].
In conclusion, mechanoreceptors in normal tissues contribute to stability of the
joints. Injuries of ligaments, tendons, and muscles contribute to the mechanical
instability and to a disturbance in neuromuscular control due to the loss of
mechanoreceptors. This field remains to be investigated more deeply in the way to
clarify the complex relationship between soft tissues and nervous system in normal
and pathological conditions. Further studies need to be conducted on mechanore-
ceptor functions in normal and pathological conditions and in the close future
maybe our surgical procedures; rehabilitation protocols for conservative or post-
surgical treatments will become modified if proprioception sense will be better
addressed [96].
7 Nanoscale Architecture for Controlling Cellular … 179

Nanoscale Biotechnology

Although further studies and new techniques are necessary, there are already dif-
ferent types of nanoscale biotechnology studied and experimented.
Electrospinning. Electrospinning has emerged to be a simple, elegant, and
scalable technique to fabricate polymeric nanofibers. Pure polymers as well as
blends and composites of both natural and synthetics have been successfully
electrospun into nanofiber matrices. Physiochemical properties of nanofiber
matrices can be controlled by manipulating electrospinning parameters to meet the
requirements of a specific application. Such efforts include the fabrication of fiber
matrices containing nanofibers, microfibers, combination of nano- and microfibers,
and also different fiber orientations/alignments. Polymeric nanofiber matrices have
been extensively investigated for diversified uses such as filtration, barrier fabrics,
wipes, personal care, biomedical and pharmaceutical applications. Recently elec-
trospun nanofiber matrices have gained a lot of attention and are being explored as
scaffolds in tissue engineering due to their properties that can modulate cellular
behavior. Electrospun nanofiber matrices show morphological similarities to the
natural extracellular matrix (ECM), characterized by ultrafine continuous fibers,
high surface-to-volume ratio, high porosity, and variable pore-size distribution.
Efforts have been made to modify nanofiber surfaces with several bioactive
molecules to provide cells with the necessary chemical cues and a more in
vivo-like environment. The current paper provides an overlook on such efforts in
designing nanofiber matrices as scaffolds in the regeneration of various soft tissues
including skin, blood vessel, tendon/ligament, cardiac patch, nerve, and skeletal
muscle [97].
Nanofiber scaffolds. Nanofiber scaffolds, produced by the electrospinning
technique, have gained widespread attention in tissue engineering due to their
morphological similarities to the native extracellular matrix. For cartilage repair,
studies have examined their feasibility; however, these studies have been limited,
excluding the influence of other scaffold design features. It evaluated the effect of
scaffold design, specifically examining a range of nano- to micron-sized fibers and
resulting pore size and mechanical properties, on human mesenchymal stem cells
(MSCs) derived from the adult bone marrow during chondrogenesis. MSC differ-
entiation was examined on these scaffolds with an emphasis on temporal gene
expression of chondrogenic markers and the pluripotent gene, Sox2, which has yet
to be explored for MSCs during chondrogenesis and in combination with tissue
engineering scaffolds. Chondrogenic markers of aggrecan, chondroadherin, sox9,
and collagen type II were highest for cells on micron-sized fibers (5 and 9 lm) with
pore sizes of 27 and 29 lm, respectively, in comparison with cells on nano-sized
fibers (300 nm and 600 to 1400 nm) having pore sizes of 2 and 3 lm, respectively.
Undifferentiated MSCs expressed high levels of the Sox2 gene but displayed
negligible levels on all scaffolds with or without the presence of inductive factors,
suggesting that the physical features of the scaffold play an important role in
differentiation. Micron-sized fibers with large pore structures and mechanical
180 F. Oliva et al.

properties comparable to the cartilage ECM enhanced chondrogenesis, demon-

strating architectural features as well as mechanical properties of electrospun fi-
brous scaffolds enhance differentiation [98].
For example, polycystin-1 (PC1) is a large membrane-associated protein that
interacts with polycystin-2 in the primary cilia of renal epithelial cells to form a
mechanosensitive ion channel. Bending of the cilia induces calcium flow into
the cells, mediated by the polycystin complex. Antibodies to polycystin-1 and
polycystin-2 abolish this activation. Based on this, it has been suggested that the
extracellular region of polycystin-1, which has a number of putative binding
domains, may act as a mechanosensor. A large proportion of the extracellular
region of polycystin-1 consists of beta sandwich PKD domains in tandem array.
Atomic force microscopy was used to investigate the mechanical properties of the
PKD domains of polycystin-1. These domains, despite having a low thermody-
namic stability, exhibit a remarkable mechanical strength, similar to that of
immunoglobulin domains in the giant muscle protein titin. Molecular dynamic
simulations performed at low constant force show that the first PKD domain of
polycystin (PKDd1) has a similar unfolding time as titin I27, under the same
conditions. The simulations suggest that the basis for this mechanical stability is the
formation of a force-stabilized intermediate. Thus, these domains will remain folded
under external force supporting the hypothesis that polycystin-1 could act as a
mechanosensor, detecting changes in fluid flow in the kidney tubule [99].
In other studies, PKD1 gene was stably silenced in osteoblastic cell line
MC3T3-E1 by using lentivirus-mediated shRNA technology to investigate the
involvement of PC1 in mechanical strain-induced signaling cascades controlling
osteogenesis. It was showed that mechanical tensile strain sufficiently enhanced
osteogenic gene expressions and osteoblastic proliferation. However, PC1 defi-
ciency resulted in the loss of the ability to sense external mechanical stimuli,
thereby promoting osteoblastic osteogenesis and proliferation. The signal pathways
implicated in this process were intracellular calcium and Akt/b-catenin pathway.
The basal levels of intracellular calcium, phospho-Akt, phospho-GSK-3b, and
nuclear accumulation of active b-catenin were significantly attenuated in
PC1-deficient osteoblasts. In addition, PC1 deficiency impaired mechanical
strain-induced potentiation of intracellular calcium, and activation of
Akt-dependent and Wnt/b-catenin pathways, which was able to be partially
reversed by calcium ionophore A23187 treatment. Furthermore, applications of
LiCl or A23187 in PC1-deficient osteoblasts could promote osteoblastic differen-
tiation and proliferation under mechanical strain conditions. Therefore, these results
demonstrated that osteoblasts require mechanosensory molecule PC1 to adapt to
external mechanical tensile strain, thereby inducing osteoblastic mechanoresponse,
partially through the potentiation of intracellular calcium and downstream Akt/
b-catenin signaling pathway [100].
Scaffolds for chondrogenic differentiation. How we said before, chondrogenic
differentiation of mesenchymal stem cells is strongly influenced by the surrounding
chemical and structural milieu. Since the majority of the native cartilage extracel-
lular matrix is composed of nanofibrous collagen fibrils, much of recent cartilage
7 Nanoscale Architecture for Controlling Cellular … 181

tissue engineering research has focused on developing and utilizing scaffolds with
similar nanoscale architecture. However, the current literature lacks consensus
regarding the ideal fiber diameter, with differences in culture conditions making it
difficult to compare between studies. It was studied how to develop a more thor-
ough understanding of how cell–cell and cell–biomaterial interactions drive in vitro
chondrogenic differentiation of bone-marrow-derived mesenchymal stem
cells (MSCs). Electrospun poly(e-caprolactone) microfibers (4.3 ± 0.8 µm diam-
eter, 90 lm2 pore size) and nanofibers (440 ± 20 nm diameter, 1.2 lm2 pore size)
were seeded with MSCs at initial densities ranging from 1  105 to 4  106 cells
cm−3 -scaffold and cultured under transforming growth factor-b (TGF-b) induced
chondrogenic conditions for 3 or 6 weeks. Chondrogenic gene expression, cellular
proliferation, as well as sulfated glycosaminoglycan and collagen production were
enhanced on microfiber in comparison with nanofiber scaffolds, with high initial
seeding densities being required for significant chondrogenic differentiation and
extracellular matrix deposition. Both cell–cell and cell–material interactions appear
to play important roles in chondrogenic differentiation of MSCs in vitro, and
consideration of several variables simultaneously is essential for understanding cell
behavior in order to develop an optimal tissue engineering strategy [101].
The objective of the scientific studies is to develop a scaffold for mesenchymal
stromal cell (MSC) recruitment, proliferation, and chondrogenic differentiation. The
concept behind the design is to mimic the cartilage matrix and contain stimulatory
agents that make continuous supply of inductive factors redundant.
The use of electrospun extracellular matrix (ECM)-mimicking nanofibrous
scaffolds for tissue engineering is limited by poor cellular infiltration. Cell pene-
tration could be enhanced in scaffolds by using a hierarchical structure where
nanofibers are combined with micron-scale fibers while preserving the overall
scaffold architecture. To assess this, were fabricated electrospun porous poly(lactic
acid) (PLA) scaffolds having nanoscale, microscale, and combined micro/
nanoarchitecture and evaluated the structural characteristics and biological
response in detail. Although the bioactivity was intermediate to that for nanofiber
and microfiber scaffold, a unique result of this study was that the micro/nano
combined fibrous scaffold showed improved cell infiltration and distribution than
the nanofibrous scaffold. Although the cells were found to be lining the scaffold
periphery in the case of nanofibrous scaffold, micro/nanoscaffolds had cells dis-
persed throughout the scaffold. Further, as expected, the addition of nanoparticles of
hydroxyapatite (nHAp) improved the bioactivity, although it did not play a sig-
nificant role in cell penetration. Thus, this strategy of creating a three-dimensional
(3D) micro/nanoarchitecture that would increase the porosity of the fibrous scaffold
and thereby improving the cell penetration can be utilized for the generation of
functional tissue-engineered constructs in vitro [102].
Thus, it is possible to combine nanofibrous (N: *400 nm) and microfibrous (M:
*10 lm) poly-e-caprolactone (PCL) scaffolds; for example, they were combined
with 1% high-molecular-weight sodium hyaluronate (NHA/MHA), 1% hyaluronan
(HA) and 200 ng transforming growth factor beta 1 (TGF-b1; NTGF/MTGF), or
0.1% bovine serum albumin (N/M). Scaffolds were seeded with MSCs from bone
182 F. Oliva et al.

marrow and cultured without growth factors in vitro. Cultures with chondrogenic
medium supplemented with TGF-b1 served as controls. Proliferation, migration,
and release of TGF-b1 were investigated. Cell differentiation was evaluated by
polymerase chain reaction (PCR) and real-time PCR. NTGF and MTGF exhibited
primarily an initial release of TGF-b1. None of the factors released by the scaffolds
recruited MSCs. The expression of aggrecan was dependent on the scaffold ultra-
structure with nanofibers promoting increasing and microfibers decreasing
expression levels. Composites containing HA demonstrated elevated seeding effi-
ciency and lower type I collagen expression. Expression of type II collagen was
dependent on continuous or late supply of TGF-b1, which was not provided by the
scaffold design. The initial release of TGF-b1 induced an expression of type I
collagen and osteogenic marker genes. Thus, nanofibrous PCL scaffolds with or
without augmentation are suitable for chondrogenic initiation of MSCs. Initial
release of HA is sufficient in terms of directing the implanted MSCs toward a
chondrogenic end, whereas a late release of TGF-b1 is preferred to foster type II
and avoid type I collagen expression [103].
Hyaluronic acid (HA) is a molecule of particular interest for producing scaffold
for tissue engineering. It has been reported that bone marrow (BM) is the MSC
source an appropriate in the setting of cartilage regeneration to treat cartilage focal
lesions. Thus, can be used HA membranes reached of BM-MSCs, aspirated by the
posterior iliac crest, as scaffolds. The interaction of MSCs with the scaffolds such as
HA membranes may contribute to positively influence the differentiation process
toward chondrogenesis. Many other factors may help MSC differentiation into
chondrocytes as the spatial conformation of the culture system, additional reagents
as platelet rich plasma (PRP), PL or TGF-b1, but the influence of the structure and
composition of the scaffold is particularly relevant since it could provide favorable
microenvironment for MSC chondrogenesis [104].
PRP and MSCs for tendon regeneration. PRP and mesenchymal stem cells can
be used also for tendon injuries. Tendon tissue shows limited regeneration potential
with formation of scar tissue and inferior mechanical properties. Several growth
factors have the capacity to improve the healing response and decrease scar for-
mation. PRP and MSC work as efficient growth factor carriers. Platelets contained
in PRP produce a number of relevant cytokines participating in physiological
tendon healing (e.g., platelet-derived growth factor (PDGF), epidermal growth
factor (EGF), vascular endothelial growth factor (VEGF)). The angiogenic effect is
vital for tendon and ligament healing, and PRP may contribute to increase it. In
vitro experiments and animal studies showed promising results for the use of PRP;
however, clinical controlled studies have shown a tendency to reduce the
pain-related symptoms but no significant differences in overall clinical scores.
Unfortunately is yet unknown which dose and which type of PRP should be used to
have more significant results. On the other hand, MSCs can contribute to tendon
healing in different ways. First, they can provide tenocytes by direct differentiation
into these cells. Secondly, they can provide a number of anabolic cytokines by their
extraordinary paracrine activity. Thirdly, they show significant anti-inflammatory
activity that may contribute to the healing process. MSCs are not totally arrived in
7 Nanoscale Architecture for Controlling Cellular … 183

clinical use so that there is still a lack of randomized controlled trials. In basic
research experiments, they show an extraordinary paracrine activity,
anti-inflammatory effect, and the possibility to differentiate in tenocytes when
different activating factors are added [105].
PAM and SCF. For some specific tissue regeneration, to overcome problems
such as cell survival, lack of cell differentiation, and integration in the host tissue, a
new tool described as pharmacologically active microcarriers (PAM) has been
described. PAMs are biocompatible and biodegradable microdevices coated with
cell adhesion molecules, conveying cells on their surface and presenting a con-
trolled delivery of growth factor. For other specific tissue regeneration, micro- and
nanoporous scaffolds with a specific 3D-shape serve as temporary support for cells
to grow into a new tissue, before it is transplanted back to the host tissue. PAM
production can be performed by using an innovative continuous process that
involves the use of supercritical fluid (SCF) (mainly supercritical carbon dioxide),
for the treatment of simple or multiple emulsions at relative low temperatures.
Scaffolds with a specific 3D shape require several characteristics, such as high
regular and reproducible 3D structure, porosity exceeding 90% and an open pore
geometry, high internal surface areas, and specific mechanical properties. To pro-
duce interconnected microcells and a nanometric substructure, a new
supercritical-fluid-assisted technique for the formation of 3D scaffolds has been
proposed. It consists of three steps: formation of a polymeric gel loaded with a solid
porogen, drying of the gel using SCF, washing with water to eliminate the porogen.
When gel drying is performed by SCF, the supercritical mixture formed during the
process has no surface tension and can be easily eliminated in a single step. One of
the major problems in gel drying is the possibility of gel collapse; using SCF, the
absence of surface tension avoids this problem preserving the nanoporous structure.
“Injectable scaffold” or temporary scaffold was also tested, respectively, for the
culture of mesenchymal stem cell in order to study their growth and differentiation.
The result showed higher biocompatibility with respect to similar product obtained
by conventional technology [106–110].

Conclusions

The complexity and well-orchestrated structure of musculoskeletal tissues reacts

physiologically and pathologically to mechanical stresses. Thousands of molecular
events occur during these events; understanding both the nanophysiology of cellular
mechanoresponse and how to use this knowledge is the key point of tissue engi-
neering. New information is required to introduce new types of nanotechnology or
to modify those already used, which will be able to improve the reparation of
injuries in various systems such as the musculoskeletal system.
184 F. Oliva et al.

References

1. Ingber DE. Integrins as mechanochemical tranducers. Curr Opin Cell Biol. 1991;3:841–8.
2. Ingber DE. Tensegrity: the architectural basis of cellular mechanotranduction. Ann Rev
Physiol. 1997;59:575–99.
3. Rodan GA, Bourret LA, Harvey A, Mensi T. Cyclic AMP and cyclic GMP: mediators of the
mechanical effects on bone remodeling. Science. 1975;189:467–9.
4. Davies PF, Remuzzi A, Gordon EJ, Dewey CF, Gimbrone MA. Turbulent fluid shear stress
induces vascular endothelial cell turnover in vitro. Proc Nat Acad Sci USA. 1986;83:
2114–7.
5. Jones DB, Scholubbers J-G. Evidence that phospholipase C mediates the mechanical stress
response in bone. Calc Tiss Int. 1987;41(Suppl. 2):4.
6. Brighton CT, Strafford B, Gross SB, Leatherwood DF, Williams JL, Pollack SR. The
proliferative and synthetic response of isolated calvarial bone cells of rats to cyclic biaxial
mechanical strain. J Bone Joint Surg. 1991;73A:320–31.
7. Sah RL-Y, Grodizinsky AJ, Plass AHK, Sandy JD. Effects of static and dynamic
compression on matrix metabolism in cartilage explants. In: Kuettner K, et al. editors.
Articular Cartilage and Osteoarthritis. New York: Raven Press, Ltd; 1992:373–92.
8. Bachrach NM, Valhmu WB, Stazzone E, Ratcliffe A, Lai WM, Mow VC. Changes in
proteoglycan synthesis rates of chondrocytes in articular cartilage are associated with the
time dependent changes in their mechanical environment. J Biomechanics. 1995;28:1561–9.
9. Guilak F, Sah RL-Y, Setton LA. Physical regulation of cartilage metabolism. In: Mow VC,
Hayes WC, editors. Basic orthopaedic biomechanics. Philadelphia: Lippincott-Raven; 1997.
p. 179–208.
10. Comper WD editor. Extracellular Matrix. Amsterdam, The Netherlands: Harwood Academic
Press 1996, Vols I & II.
11. Sung KLP, Sung LA, Crimmins M, Burakoff SJ, Chien S. Dynamic changes in viscoelastic
properties in cytotoxic T-lymphocytes mediating killing. J Cell Sci. 1988;91:179–89.
12. Dong C, Skalak R, Sung KP, Schmid-Schonbien GW, Chien S. Passive deformation analysis
of human leukocytes. J Biomech Eng. 1998;110:27–36.
13. Chen Christopher S, Ingber Donald E. Tensegrity and mechanoregulation: from skeleton to
cytoskeleton. Osteoarthritis Cartilage. 1999;7:81–94.
14. Viidik A. Tensile strength properties of achilles tendon systems in trained and untrained
rabbits. Acta Orthop Scand. 1969;40:261–72.
15. Tipton CM, Matthes RD, Maynard JA, et al. The influence of physical activity on ligaments
and tendons. Med Sci Sports. 1975;7:165–75.
16. Lanyon LE, Rubin CT. Static versus dynamic loads as an influence on bone remodelling.
J Biomech. 1984;17:897–905.
17. Frost HM. Vital biomechanics. General concepts for structural adaptations to mechanical
usage. Calc Tiss Int. 1987;42:145–54.
18. DeLee JC. Tissue remodeling and response to therapeutic exercise [review]. In:
Leadbetter WB, Buckwalter JA, Gordon SL, editors. Sports-Induced Inflammation.
AAOS: American Orthopaedic Society; 1989. p. 547–54.
19. Forwood MR, Turner CH. Skeletal adaptations to mechanical usage: results from tibial
loading studies in rats. Bone. 1995;4(suppl):197S–205S.
20. Deng M, James R, Laurencin CT, Kumbar SG. Nanostructured polymeric scaffolds for
orthopaedic regenerative engineering. IEEE Trans Nanobiosci. 2012;11(1):3–14. https://doi.
org/10.1109/TNB.2011.2179554 Epub 2012 Jan 23.
21. Ingber DE, Wang N, Stamenovic D. Tensegrity, cellular biophysics, and the mechanics of
living systems. Rep Prog Phys. 2014;77(4):046603.
22. Frost HM. A 2003 update of bone physiology and Wolff’s Law for clinicians. Angle Orthod.
2004;74(1):3–15.
7 Nanoscale Architecture for Controlling Cellular … 185

23. Mow VC, Ratcliffe A, Poole AR. Cartilage and diarthrodial joints as paradigms for
hierarchical materials and structures. Biomaterials. 1992;13:67–97.
24. Martin RB, Burr DB. Structure, function, and adaptation of compact bone. New York:
Raven Press; 1989.
25. Silbert JE. Advances in the biochemistry of proteoglycans. In: Uitto J, Perejda AJ, editors.
Connective tissue disease: molecular pathology of the extracellular matrix. New York:
Marcel Dekker; 1987. p. 83–98.
26. Mow VC, Ratcliffe A. Structure and function of articular cartilage. In: Mow VC, Hayes WC,
editors. Basic orthopaedic biomechanics. Philadelphia, PA: Lippincott-Raven; 1997. p. 113–
77.
27. Guilak F. Compression-induced changes in the shape and volume of the chondrocyte
nucleus. J Biomech. 1995;28:1529–41.
28. Guilak F, Ratcliffe A, Mow VC. Chondrocyte deformation and local tissue strains in
articular cartilage: a confocal microscopy study. J Orthop Res. 1995;13:410–21.
29. Nimni ME. Collagen: structure, function, and metabolism in normal and fibrotic tissues. Sem
Arth Rheum. 1983;13:1–86.
30. Woo SL-Y, Gomez MA, Woo YK, Akeson WH. Mechanical properties of tendons and
ligaments. Quasistatic and nonlinear viscoelastic properties. Biorheology 1982;19:385–96.
31. Dickinson RB, Guido S, Tranquillo RT. Biased cell migration of fibroblasts exhibiting
contact guidance in oriented collagen gels. Ann Biomed Eng. 1994;22:342–56.
32. Chen BM, Grinnell AD. Integrins and modulation of transmitter release from motor nerve
terminals by stretch. Science. 1995;269:1578–80.
33. Hamill OP, McBride DW Jr. The cloning of a mechano-gated membrane ion channel.
Trends Neurosci. 1994;17:439–45.
34. Ingber DE, Dike L, Hansen L, Karp S, Liley H, Maniotis A, McNamee H, Mooney D,
Plopper G, Sims J, Wang N. Cellular tensegrity: exploring how mechanical changes in the
cytoskeleton regulate cell growth, migration, and tissue pattern during morphogenesis. Int
Rev Cytol. 1994;150:173–224.
35. Plopper G, McNamee H, Dike L, Bojanowski K, Ingber DE. Convergence of integrin and
growth factor receptor signaling pathways within the focal adhesion complex. Mol Biol Cell.
1995;6:1349–65.
36. Miyamoto S, Akiyama S, Yamada KM. Synergistic roles for receptor occupancy and
aggregation in integrin transmembrane function. Science. 1995;267:883–5.
37. Buxbaum RE, Heidemann SR. A thermodynamic model for force integration and
microtubule assembly during axonal elongation. J Theor Biol. 1988;134:379–90.
38. Yao L, Bestwick CS, Bestwick LA, Maffulli N, Aspden RM. Phenotypic drift in human
tenocyte culture. Tissue Eng. 2006;12(7):1843–9.
39. Ingber DE. Tensegrity-based mechanosensing from macro to micro. Prog Biophys Mol Biol.
2008;97(2–3):163–79. https://doi.org/10.1016/j.pbiomolbio.2008.02.005.
40. Cornelius C, Koverech G, Crupi R, Di Paola R, Koverech A, Lodato F, Scuto M,
Salinaro AT, Cuzzocrea S, Calabrese EJ, Calabrese V. Osteoporosis and Alzheimer
pathology: role of cellular stress response and hormetic redox signaling in aging and bone
remodeling. Front Pharmacol. 2014;5.
41. Barbieri E, Sestili P, Vallorani L, Guescini M, Calcabrini C, Gioacchini AM, Annibalini G,
Lucertini F, Piccoli G, Stocchi V. Mitohormesis in muscle cells: a morphological, molecular,
and proteomic approach. MLTJ. 2013.
42. Ji LL, Gomez-Cabrera MC, Vina J. Exercise and hormesis: activation of cellular antioxidant
signaling pathway. Ann N Y Acad Sci. 2006;1067:425–35.
43. Rattan SI. Targeting the age-related occurrence, removal, and accumulation of molecu-
lar damage by hormesis. Ann N Y Acad Sci. 2010;1197:28–32. https://doi.org/10.1111/j.
1749-6632.2010.05193.x.
44. Hamel P, Abed E, Brissette L, Moreau R. Characterization of oxidized low-density
lipoprotein-induced hormesis-like effects in osteoblastic cells. Am J Physiol Cell Physiol.
2008;294:C1021–33.
186 F. Oliva et al.

45. Delaine-Smith R, Reilly GC. Mesenchymal stem cell responses to mechanical stimuli.
MLTJ. 2012.
46. Baksh D, Song L, Tuan RS. Adult mesenchymal stem cells: characterization, differentiation,
and application in cell and gene therapy. J Cell Mol Med. 2004;8(3):301–16.
47. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA,
Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal
stem cells. Science. 1999;284(5411):143–7.
48. Hao W, Dong J, Jiang M, Wu J, Cui F, Zhou D. Enhanced bone formation in large segmental
radial defects by combining adipose-derived stem cells expressing bone morphogenetic
protein 2 with nHA/RHLC/PLA scaffold. Int Orthop. 2010;34(8):1341–9.
49. Whu SW, Tsai CL, Hsu SH. Evaluation of human bone marrow mesenchymal stem cells
seeded into composite scaffolds and cultured in a dynamic culture system for neocartilage
regeneration in vitro. J Med Biol Eng. 2009;29(2):52–9.
50. Kratchmarova I, Blagoev B, Haack-Sorensen M, Kassem M, Mann M. Mechanism of
divergent growth factor effects in mesenchymal stem cell differentiation. Science. 2005;308
(5727):1472–7.
51. Watanabe T, Sakai D, Yamamoto Y, Iwashina T, Serigano K, Tamura F, Mochida J. Human
nucleus pulposus cells significantly enhanced biological properties in a co-culture system
with direct cell-to-cell contact with autologous mesenchymal stem cells. J Orthop Res.
2010;28(5):623–30.
52. Yu H, Vandevord PJ, GongW WuB, Song Z, Matthew HW, Wooley PH, Yang SY.
Promotion of osteogenesis in tissue-engineered bone by pre-seeding endothelial progenitor
cells-derived endothelial cells. J Orthop Res. 2008;26(8):1147–52.
53. Ozturk AM, Cila E, Kanatli U, Isik I, Senkoylu A, Uzunok D, Piskin E. Treatment of
segmental bone defects in rats by the stimulation of bonemarrow osteo-progenitor cells with
prostaglandin E2. Int Orthop. 2005;29(2):73–7.
54. Chen KY, Chung CM, Kuo SM, Chen YS, Yao CH. Influence of collagen I nanospheres on
the growth and osteogenic difference of rat bone marrow stromal cells. J Med Biol Eng.
2009;29(6):284–9.
55. Jung Y, Song J, Shiozawa Y, Wang J, Wang Z, Williams B, Havens A, Schneider A, Ge C,
Franceschi RT, McCauley LK, Krebsbach PH, Taichman RS. Hematopoietic stem cells
regulate mesenchymal stromal cell induction into osteoblasts thereby participating in the
formation of the stem cell niche. Stem Cells. 2008;26(8):2042–51.
56. Borovecki F, Pecina-Slaus N, Vukicevic S. Biological mechanisms of bone and cartilage
remodelling-genomic perspective. Int Orthop. 2007;31(6):799–805.
57. Li M, Thompson DD, Paralkar VM. Prostaglandin E-2 receptors in bone formation. Int
Orthop. 2007;31(6):767–72.
58. Tsai MT, Lin DJ, Huang S, Lin HT, Chang WH. Osteogenic differentiation is synergistically
influenced by osteoinductive treatment and direct cell–cell contact between murine
osteoblasts and mesenchymal stem cells. Int Orthop (SICOT). 2012;36:199–205.
59. Kim H, Lee JH, Suh H. Interaction of mesenchymal stem cells and osteoblasts for in vitro
osteogenesis. Yonsei Med J. 2003;44(2):187–97.
60. Ahn SE, Kim S, Park KH, Moon SH, Lee HJ, Kim GJ, Lee YJ, Park KH, Cha KY,
Chung HM. Primary bone-derived cells induce osteogenic differentiation without exogenous
factors in human embryonic stem cells. Biochem Biophys Res Commun. 2006;340(2):
403–8.
61. Delaine-Smith R, Reilly GC. Mesenchymal stem cell responses to mechanical stimuli.
MLTJ. 2012.
62. Ytteborg E, Torgersen JS, Pedersen ME, Helland SJ, Grisdale-Helland B, Takle H. Exercise
induced mechano-sensing and substance P mediated bone modeling in Atlantic salmon.
Bone. 2013;53(1):259–68. https://doi.org/10.1016/j.bone.2012.11.025 Epub 2012 Dec 3.
63. Oliva F, Tarantino U, Maffulli N. Immunohistochemical localization of calcitonin
gene-related peptide and substance P in the rat knee cartilage at birth. Physiol Res.
2005;54:549–56.
7 Nanoscale Architecture for Controlling Cellular … 187

64. Niissalo S, Hukkanen M, Imai S, Tornwall J, Kottinen YT. Neuropeptides in experimental

and degenerative arthritis. Ann N Y Acad Sci. 2002;966:384–99.
65. McDougall JJ, Bray RC, Sharkey KA. Morphological and immunohistochemical examina-
tion of nerves in normal and injured collateral ligaments of rat, rabbit, and human knee
joints. Anat Rec. 1997;248:29–39.
66. Fortier LA, Nixon AJ. Distributional changes in substance P nociceptive fiber patterns in
naturally osteoarthritic articulations. J Rheumatol. 1997;24:524–30.
67. Kashihara Y, Sakaguchi M, Kuno M. Axonal transport and distribution of endogenous
calcitonin generelated peptide in rat peripheral nerve. J Neurosci. 1989;9:3796–802.
68. Konttinen YT, Imai S, Suda A. Neuropeptides and the puzzle of bone remodeling. Acta
Orthop Scand. 1996;67:632–9.
69. Ackermann PW, Ahmed M, Kreicbergs A. Early nerve regeneration after Achilles tendon
rupture—a prerequisite for healing? A study in the rat. J Orthop Res. 2002;20:849–56.
70. Salo PT, Seeratten RA, Erwin WM, Bray RC. Evidence for a neuropathic contribution to the
development of spontaneous knee osteoarthrosis in a mouse model. Acta Orthop Scand.
2002;73:77–84.
71. Madsen JE, Hukkanen M, Aspenberg P, Polak J, Nordsletten L. Time-dependent sensory
nerve ingrowth into a bone conduction chamber. Acta Orthop Scand. 2000;71:74–9.
72. Modesti A, Oliva F. All is around ECM of tendons!? MLTJ. 2013.
73. Franchi M, Trirè A, Quaranta M, Orsini E, Ottani V. Collagen structure of tendon relates to
function. Sci World J. 2007;7:404–20.
74. Bei R, Masuelli L, Palumbo C, Tresoldi I, Scardino A, Modesti A. Long-lasting tissue
inflammatory processes trigger autoimmune responses to extracellular matrix molecules. Int
Rev Immunol. 2008;27:137–75.
75. Connizzo BK, Yannascoli SM, Soslowsky LJ. Structure-function relationships of postnatal
tendon development: a parallel to healing. Matrix Biol. 2013;32:106–16.
76. Sharma P, Maffulli N. Tendon injury and tendinopathy: healing and repair. J Bone Joint Surg
Am. 2005;87:187–202.
77. Sharma P, Maffulli N. Biology of tendon injury: healing, modeling and remodeling.
J Musculoskelet Neuronal Interact. 2006;6:181–90.
78. Tresoldi I, Oliva F, Benvenuto M, Fantini M, Masuelli L, Bei R, Modesti A. Tendon’s
ultrastructure. MLTJ. 2013.
79. Proske U, Gandevia SC. The proprioceptive senses: their roles in signaling body shape, body
position and movement, and muscle force. Physiol Rev. 2012;92:1651–97.
80. Zimny ML. Mechanoreceptors in articular tissues. Am J Anat. 1988;182:16–32.
81. Abbot LC, Saunders JB, Dec M, et al. Injuries to the ligaments of the knee. J Bone Joint
Surg. 1944;26:503–21.
82. Michelson JD, Hutchins C. Mechanoreceptors in human ankle ligaments. J Bone Joint Surg
Br. 1995;77:219–24.
83. Hogervorst T, Brand RA. Mechanoreceptors in joint function. J Bone Joint Surg Am.
1998;80:1365–78.
84. Verra WC, van den Boom LG, Jacobs W, Clement DJ, Wymenga AA, et al. Retention
versus sacrifice of the posterior cruciate ligament in total knee arthroplasty for treating
osteoarthritis. Cochrane Database Syst Rev. 2013;11:10.
85. Raunest J, Sager M, Bürgener E. Proprioception of the cruciate ligaments: receptor mapping
in an animal model. Arch Orthop Trauma Surg. 1998;118:159–63.
86. Abbott LC, Saunders JB, Dec M, et al. Injuries to the ligament of the knee joint. J Bone Joint
Surg. 1944;26:503–21.
87. Schultz R, Miller D, Kerr C, et al. Mechanoreceptors in human cruciate ligaments.
A histological study. J Bone Joint Surg Am. 1984;66:1072–6.
88. Halata Z, Rettig T, Schulze W. The ultrastructure of sensory nerve endings in human knee
joint capsule. Anat Embryol. 1985;172:265–76.
188 F. Oliva et al.

89. Kapreli E, Athanasopoulos S, Gliatis J, Papathanasiou M, Peeters R, Strimpakos N, Van

Hecke P, Gouliamos A, Sunaert S. Anterior cruciate ligament deficiency causes brain
plasticity: a functional MRI study. Am J Sports Med. 2009;37(12):2419–26.
90. Dhillon MS, Bali K, Prabhakar S. Differences among mechanoreceptors in healthy and
injured anterior cruciate ligaments and their clinical importance. MLTJ. 2012;2(1):38–43.
91. Scott JJ, Petit J, Davies P. The dynamic response of feline Golgi tendon organs during
recovery from nerve injury. Neurosci Lett. 1996;207:179–82.
92. Sha L, Zhao L. Quantitative study on mechanoreceptors in tibial remnants of ruptured
anterior cruciate ligament in human knees. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi.
2010;24:1318–22.
93. Haftel VK, Bichler EK, Wang QB, Prather JF, Pinter MJ, et al. Central suppression of
regenerated proprioceptive afferents. J Neurosci. 2005;25:4733–42.
94. Jami L. Functional properties of the Golgi tendon organs. Arch Int Physiol Biochim.
1988;96:363–78.
95. Scott JJ, Panesar G. Small-signal sensitivity of muscle spindles reinnervated after long-term
denervation. Brain Res. 1995;671:325–8.
96. Kaya D, editor. Proprioception: the forgotten sixth sense. Chapter: muscle, tendon, ligament
tear and proprioception. OMICS Group eBooks, 2014.
97. Kumbar SG, James R, Nukavarapu SP, Laurencin CT. Electrospun nanofiber scaffolds:
engineering soft tissues. Biomed Mater. 2008;3(3):034002. https://doi.org/10.1088/1748-
6041/3/3/034002. Epub 8 Aug 2008.
98. Shanmugasundaram S, Chaudhry H, Arinzeh TL. Microscale versus nanoscale scaffold
architecture for mesenchymal stem cell chondrogenesis. Tissue Eng Part A. 2011;
17(5–6):831–40. https://doi.org/10.1089/ten.TEA.2010.0409 Epub 2010 Dec 14.
99. Forman JR, Qamar S, Paci E, Sandford RN, Clarke J. The remarkable mechanical strength of
polycystin-1 supports a direct role in mechanotransduction. J Mol Biol. 2005;349(4):
861–71.
100. Wang H, Sun W, Ma J, Pan Y, Wang L, Zhang W. Polycystin-1 mediates mechanical
strain-induced osteoblastic mechanoresponses via potentiation of intracellular calcium and
Akt/b-catenin pathway. PLOS ONE. March 2014.
101. Bean AC, Tuan RS. Fiber diameter and seeding density influence chondrogenic differen-
tiation of mesenchymal stem cells seeded on electrospun poly(e-caprolactone) scaffolds.
Biomed Mater. 2015;10(1):015018. https://doi.org/10.1088/1748-6041/10/1/015018.
102. Shalumon KT, Chennazhi KP, Tamura H, Kawahara K, Nair SV, Jayakumar R. Fabrication
of three-dimensional nano, micro and micro/nano scaffolds of porous poly(lactic acid) by
electrospinning and comparison of cell infiltration by Z-stacking/three-dimensional projec-
tion technique. IET Nanobiotechnol. 2012;6(1):16–25. https://doi.org/10.1049/iet-nbt.2011.
0028.
103. Schagemann JC, Paul S, Casper ME, Rohwedel J, Kramer J, Kaps C, Mittelstaedt H,
Fehr M, Reinholz GG. Chondrogenic differentiation of bone marrow-derived mesenchymal
stromal cells via biomimetic and bioactive poly-e-caprolactone scaffolds. J Biomed Mater
Res A. 2013;101(6):1620–8. https://doi.org/10.1002/jbm.a.34457 Epub 2012 Nov 27.
104. Spoliti M, Iudicone P, Leone R, De Rosa A, Rossetti FR, Pierelli L. In vitro release and
expansion of mesenchymal stem cells by a hyaluronic acid scaffold used in combination with
bone marrow. MLTJ. 2012.
105. Guevara-Alvarez A, Schmitt A, Russell RP, Imhoff AB, Buchmann S. Growth factor
delivery vehicles for tendon injuries: mesenchymal stem cells and platelet rich plasma.
MLTJ. 2014.
106. Della Porta G, Campardelli R, Falco N, Reverchon E. PLGA microdevices for retinoids
sustained release produced by supercritical emulsion extraction: continuous versus batch
operation layouts. J Pharm Sci. 2011. https://doi.org/10.1002/jps.
107. Della Porta G, Reverchon E. Continuous supercritical emulsions extraction: a new
technology for biopolymer microparticles production. Biotechnol Bioeng J. 2011;108
(3):676–86.
7 Nanoscale Architecture for Controlling Cellular … 189

108. Cardea S, Pisanti P, Reverchon E. Generation of chitosan nanoporous structures for tissue
engineering applications using a supercritical fluid assisted process. J Supercrit Fluids.
2010;54:290–5.
109. Della Porta G, Cavalcanti S, Reverchon E. Supercritical Fluid emulsions extraction: a novel
technology for the production of poly-lactic-co-glycolic nanostructured microdevices. In:
Proceedings of II Congresso Nazionale di Bioingegneria; 2010 July 8–10; Turin (Italy):
Patron, p. 637–8, ISBN 978-88-555-3082-8.
110. Reverchon E, Adami R, Cardea S, Della Porta G. Supercritical fluids processing of polymers
for pharmaceutical and medical applications. J Supercrit Fluids. 2009;47:484–92.
Chapter 8
Modular Tissue Engineering:
An Artificial Extracellular Matrix
to Address and Stimulate Regeneration/
Differentiation

Giovanna Della Porta and Ernesto Reverchon

Abstract Tissue engineering uses living cells as engineering materials cultivated

and merged within a tri-dimensional structure in order to develop a tissue-like device;
these artificial extracellular matrixes, called scaffolds, are important in influencing
cells microenvironment and addressing their fate, both ex vivo as well as in vivo.
Indeed, tri-dimensional scaffolds allow cell attachment, enable diffusion of vital cell
nutrients or catabolites and can also deliver mechanical or biological signals. In the
traditional tissue engineering “top-down” approach, cells are seeded onto biopolymer
scaffold and then let them to migrate and colonize the structure; recently, the “bottom-
up” approach has been described to design structural bioengineered microfeatures
that can be used as building blocks to create larger tri-dimensional structures and
build modular tissues. In some specific applications, a mix of the two approaches is
also proposed to generate multiphase matrices. Several technologies for scaffold
production are described in this chapter both using conventional organic solvent and
dense gas or supercritical fluid; some solutions for artificial extracellular matrices
useful for musculoskeletal tissue regeneration are also proposed.

The “Top-Down” or “Bottom-Up” Approach:

Which Is the Best Choice?

Traditional tissue engineering strategies involves the so-called “top-down”

approach (see Fig. 8.1), in which cells are seeded on a biodegradable
three-dimensional (3D) biopolymer structure [1–3] and it is expected that they will

G. Della Porta (&)

Department of Medicine, Surgery and Dentistry, University of Salerno,
Via S. Allende, 84081 Baronissi, SA, Italy
e-mail: [email protected]
E. Reverchon
Department of Industrial Engineering, University of Salerno, Fisciano, SA, Italy
e-mail: [email protected]

© Springer International Publishing AG, part of Springer Nature 2018 191

A. C. Berardi (ed.), Extracellular Matrix for Tissue Engineering
and Biomaterials, Stem Cell Biology and Regenerative Medicine,
https://doi.org/10.1007/978-3-319-77023-9_8
192 G. Della Porta and E. Reverchon

populate the scaffold with the aid of dynamic cultivation, often supplemented with
growth factors, generating an appropriate extracellular matrix (ECM) [4–6]. These
3D scaffolds are still far from leading to successful tissue reconstruction in a clinical
setting because of the absence of specific microarchitecture, a limited mass trans-
port of metabolites or oxygen through the 3D structure, a reduced control over their
biodegradation. Moreover, when bio-signals or growth factors are incorporated by
simple adsorption onto bulk scaffolds, they normally leads to uncontrolled burst
release on implantation with an overdose that may give rise to undesired effects
[7, 8].
More recent tissue engineering approach described as “bottom-up” one or
modular tissue engineering aims to address the challenge of recreating bio-mimetic
structures by designing structural microfeatures that can be used to build modular
tissues such as building blocks to create larger tissues (see Fig. 8.2). These 3D
modules can be fabricated in different ways, such as microfabrication of cell-laden
hydrogels [9, 10], creation of cell sheets [11], or direct tissue printing [12]. By
mimicking native microstructural functional units, these approaches aim to create
more bio-mimetic engineered tissues because these modules can be assembled into
larger tissues-like structure through a number of methods such as random packing
[13, 14], stacking of layers [15], or ordinate assembly [16].
Bottom-up approach is aimed to provide guidance on the cellular level to direct
tissue morphogenesis by creating modular tissues with more physiological
microarchitectural features [17]. In addition, bio-plotting technology can help this
approach by designing a zonal organization into the 3D engineered artificial matric
plotted; for example, one strategy may be to spatially pattern cells with specific
additives from different zones into stratified layers to better mimic the in vivo zonal
organizations of the extracellular matrix. In this sense, bio-plotting additives such as

Fig. 8.1 Top-down approach involves monolithic scaffold on which the cells are seeded; often,
cells do not colonize the interior part of the scaffold
8 Modular Tissue Engineering … 193

Fig. 8.2 Bottom-up approach allows generating scaffolds of higher complexity and functionality
that can be designed with several advantages over conventional monolithic scaffolds

biopolymer microcapsules can further be organized into a 3D pre-designed

microenvironment where they can deliver biomolecule within the continuous matrix
without compromising the properties of the bulk structure; these microdevices
allow generating scaffolds of higher complexity and functionality that can be
194 G. Della Porta and E. Reverchon

designed with several advantages over conventional monolithic bulk scaffolds [18, 19].
Moreover, by introducing microspheres, mechanically weak scaffolds can be sta-
bilized or reinforced providing an improved mechanical behaviour [20, 21].
Biopolymer microspheres with peptides immobilized on the surface have been
also reported as a mouldable scaffold for cartilage tissue engineering [22, 23],
whereas gelatin microspheres with bioactive domains including peptides resulted in
cytocompatible microcarriers suitable to deliver live cells such as chondrocytes for
in vivo chondrogenesis [24, 25]. During the dynamic cultivation, cells adhered,
proliferated and synthesized a thin layer of extracellular matrix (ECM) in and
around the macroporous beads allowing a biological sintering via cell–cell and
cell–matrix interaction after only a few days of dynamic seeding [26].

Biomaterials for Scaffold Fabrication

Natural polymers are of great importance for tissue engineering, basically due to
their intrinsic biocompatibility and biodegradability. This class of polymers is often
favoured more than synthetic ones due to their abundant side groups in their
molecular chains that allow for further functionalization. Further, natural polymers
such as collagen and gelatine contain motifs such as RGD (arginine–glycine–as-
partic acid) sequences, which can modulate cell adhesion and improve their
behaviour. Collagen is the most extensively investigated natural polymer since the
ECM of many mesenchymal tissues (including bone and cartilage) is mainly
composed of collagen as organic phase. Collagen is an attractive candidate material
for bone regeneration due to its excellent biocompatibility, desirable biodegrad-
ability and negligible immunogenicity [27, 28]. As a derivative from collagen,
Gelatin has been also used for biomedical applications; its characteristics include a
controllable degradation, and abundant presence of functional groups that allow for
further functionalization and modification via chemical derivatization. Specifically,
the unique electrical nature of gelatin (commercially available as both positively or
negatively charged polymers at neutral pH) enables gelatin to encapsulate bioactive
molecules by forming polyion complexes [29, 30].
Fibrin can be prepared by combining fibrinogen with thrombin, which are both
derived from the patient’s own blood, thereby fabricating an autologous scaffold
that does not induce an excessive foreign body reaction. However, the rapid
degradation rate and poor mechanical stability of fibrin have been stated as the main
limitation for bone tissue engineering. To overcome this problem, composite
microspheres (alginate/fibrin, poly-hyaluronic acid/fibrin) were developed to sta-
bilize the fibrin matrix, solving the problems of both alginate shortage of bioactive
sequence for cell attachment and fibrin poor capacity for cell encapsulation [31, 32].
Chitosan is a frequently used hydrophilic polysaccharide derived from chitin,
which exhibits favourable physicochemical and biological properties for biomedical
8 Modular Tissue Engineering … 195

applications including biocompatibility and intrinsic antibacterial nature [33],

whereas alginate favourable characteristics including its biocompatibility,
non-immunogenicity, hydrophilicity and cost-effectiveness make it highly suitable
for many applications in drug delivery and tissue engineering. The most pro-
nounced advantage of alginates is related to its gentle gelling behaviour using
crosslinking in the presence of divalent cations, such as Ca2+ [34, 35].
Poly-(glycolic acid) (PGA), poly-(lactic acid) (PLA) and their copolymers are a
family of linear aliphatic polyesters, which are the most frequently used in tissue
engineering because they degrade through hydrolysis of the ester bonds. Because of
its relatively hydrophilic nature, PGA degrades rapidly in aqueous solutions or
in vivo and loses mechanical integrity between two and four weeks. PLA is also
widely used for scaffold fabrication; the extra methyl group in the PLA repeating
unit (compared with PGA) makes it more hydrophobic, reduces the molecular
affinity to water and leads to a slower hydrolysis rate. It takes many months or even
years for a PLA scaffold or implant to lose mechanical integrity. To achieve
intermediate degradation rates between PGA and PLA, various lactic and glycolic
acid ratios are used to synthesize PLGA. Particularly, poly-lactic-co-glycolic acid
(PLGA, with a copolymer ratio of 75:25 or 50:50) has received a great interest in
the development of injectable microparticulate devices [36]. For example, PLGA
microparticles are described for in situ injection to achieve a precise and localized
drug delivery which is more effective in chemotherapy, hormone therapy, DNA/
protein or vaccine delivery [37–40]. Moreover, PLGA microparticles with a specific
size and distribution have also been proposed for the use in tissue engineering as
building blocks of implantable 3D scaffolds offering benefits such as good mor-
phology control and better versatility in the release kinetics of specific growth
factors for cellular function orienting and directing or biodegradable support for cell
culture and/or cell administration [41].
There are other linear aliphatic polyesters, such as poly-(e-caprolactone)
(PCL) and poly-(hydroxy butyrate) (PHB), which are also used in tissue engi-
neering research. PCL degrades at a significantly slower rate than PLA, PGA and
PLGA. The slow degradation makes PCL less attractive for general tissue engi-
neering applications, but more attractive for long-term implants and controlled
release applications [42].

3D Scaffold Fabrication: The Organic Solvent

Can Be Replaced by Supercritical Fluid

Conventional technologies for scaffold and microsphere fabrication use organic

solvents to solubilize the chosen biopolymer or their blend to generate a 3D scaffold
or microcapsules. One largely used method for pre-shaped 3D scaffolds fabrication
is the solvent casting and particulate leaching (see Fig. 8.3). The protocol involves
first mixing water-soluble salt particles in the polymer solution, and then, the
196 G. Della Porta and E. Reverchon

suspension is cast into the mould of a desired shape; after solvent evaporation or
lyophilization, the salt particles are leached out to obtain biopolymer porous 3D
structure with porosity that can be of 90%. Closed cavities in the resulting matrix
and smooth wall are the described drawbacks of these structures as well as complex
post-treatments to remove the residual amount of solvents used [43]; moreover,
poor metabolites mass transfer within the structure during the cell cultivation and
poor cell colonization inside the structure is often described for these scaffolds,
even if cultivated in dynamic conditions [44, 45]. Electrospinning is a more
advanced fabrication technology that uses an electric field to control the formation
and deposition of biopolymer microfibres onto a target substrate (see Fig. 8.4).
A critical voltage is applied in order to overcome the surface tension of the
biopolymer solution and obtain an electrically charged jet; the jet within the electric
field is, then, directed towards the ground target, during which time the solvent
evaporates forming oriented or random fibres with diameters ranging from several
microns down to several hundred nanometres [46]. Limitations described are: low
mechanical strength and difficulty in controlling the 3D structure pore shapes and
sizes. Rapid prototyping is a technology based on the development of computer
science and manufacturing industry. The main advantage of these techniques is
their ability to produce complex products rapidly from a computer-aided design
(CAD) model. One of these rapid prototyping techniques, called 3D printing, has
been used to process biodegradable polymer scaffolds for tissue engineering
applications [47, 48]. All the described technologies lack biological agents incor-
porating within the biopolymer structure; for example, peptides such as growth
factors cannot be easily loaded into the 3D scaffolds because of the extreme con-
ditions (temperature/organic solvents) used in the fabrication process.
Technologies that replace the use of an organic solvent with dense gases or
supercritical fluids are also emerging in fabricating 3D devices for biomedical
applications. Supercritical fluids (SCFs) are defined substances at a temperature and
pressure above their critical point as described by the pressure vs. temperature
diagram for a pure compound (see Fig. 8.5). In a specific pressure and temperature
conditions, a substance becomes supercritical and can be described as an homo-
geneous fluid that can effuse through solids like a gas, but has also the ability to

Fig. 8.3 Solvent casting and particulate leaching method and SEM image of the microstructure of
the 3D scaffold obtainable
8 Modular Tissue Engineering … 197

Fig. 8.4 Schematic representation of electrospinning method and SEM image of the microstruc-
ture of the 3D scaffold obtainable

dissolve materials like a liquid. In addition, close to the critical point, small changes
in pressure or temperature result in large changes in density, allowing a fine tuning
of the supercritical fluid solvent power. As a consequence, the supercritical fluids
seemed suitable to substitute organic solvents in a wide range of industrial pro-
cesses [49]. Particularly, its reduced surface tension coupled with higher diffusivity
(that is similar to gases and of about two orders of magnitude larger than that of
liquid solvents) offers possibilities of fabrication structures not obtainable by using
the conventional liquid solvents. Carbon dioxide is the most commonly used
supercritical fluids (SC-CO2) because it is not toxic, not flammable, cheap and with
critical parameters of pressure and temperature are readily accessible on the
industrial scale (304.1 K and 73.8 bar).
The first use of SC-CO2 for temporary 3D scaffolds production has been sug-
gested with the gas-foaming technique. The process is solventless and very efficient
in producing the porous biopolymer structure because the dense gas can foam a

Fig. 8.5 State diagram for a

pure component in the PT
region
198 G. Della Porta and E. Reverchon

given biopolymer inducing the formation of bubbles within its structure (see
Fig. 8.6). It was also described that the morphology of the foams can be varied by
controlling the depressurization rate of the foamed biopolymer, such as PLA or
PLGA, and the density of the gas; particularly, a rapid depressurization locked in
large numbers of spherical pores within the biopolymer structure, whereas a slower
depressurization reduced pores elongation. Drawbacks of the gas-foaming process
are smooth surfaces and generally closed pores structures [50]. Another technology
described for scaffold production by dense gases is the supercritical assisted phase
inversion method (SAPIM); the process reproduces the traditional phase inversion
method (obtained by liquid–liquid diffusion or by varying the system temperature)
by using SC-CO2. Therefore, the dense gas is used to dry the biopolymer structure
more rapidly due to an enhanced mass transfer caused by a reduced surface tension
and the absence of liquid–vapour interface. In a specific condition of pressure and
temperature and system composition, open pores are obtained and no traces of
organic solvents are described in the resulted scaffold. Moreover, the use of
SC-CO2 by means of pressure introduces an additional variable to influence the
biopolymer/solvent solution de-mixing process and, therefore, the porous structure
morphology. Indeed, it was described that it is possible to control the average pore
size that can decrease with the increase of SC-CO2 density (either by increasing the
pressure or by decreasing the temperature), whereas the average pore diameter
decreased with the increase of the initial polymer concentration [51]. Reverchon &
Cardea [52] explained this behaviour indicating that the phase separation process
can be responsible for the porous cellular structure obtainable in depending on the
liquid/liquid de-mixing by nucleation and growth of droplets of a polymer lean
phase. The subsequent removal of the solvent produces a dry and stable structure.
The proposed process is versatile, and the scaffold characteristics can be continu-
ously modulated varying supercritical CO2 properties. Limitations are again smooth
porous walls and poorly interconnected pores within the 3D structure. To fulfil the
necessity of producing interconnected micropores, a supercritical fluid-assisted
technique named supercritical gel drying has been also proposed and described
[53]. The process involves the formation of a biopolymer alcohol gel loaded with a
solid porogen that is then dried by SC-CO2; the final washing with water eliminates
the porogen fabricating a porous structure. When the gel drying is performed by
supercritical CO2, the supercritical mixture formed during the process (ethanol plus
CO2) has no surface tension and can be easily eliminated in a single step by the
continuous flow of SC-CO2 in the drying vessel. The major problem during gel
drying is the possibility of gel collapse; however, the reduced surface tension of the
supercritical mixture used to avoid this problem preserving the nanoporous struc-
ture in the fabricated 3D aerogel [54]. These scaffolds were successfully tested for
human mesenchymal cells cultivation in a specific dynamic cultivation [55].
8 Modular Tissue Engineering … 199

Fig. 8.6 Schematic representation of supercritical gas-foaming method with SEM image of the
microstructure of the 3D scaffold obtainable

Microdevices and Microcapsules Fabrication:

A Challenge for Supercritical Fluid Technology

Several techniques can be used to prepare biopolymer microcarriers, but the solvent
evaporation/extraction of emulsion (single, double or multiple) is the most widely
used. The process involves single oil-in-water (o/w) or double water-in-oil-in-water
(w/o/w) emulsions to encapsulate biomolecules [56]. The biopolymer is first dis-
solved in a water-immiscible organic solvent. The bioactive molecule is then added
to the polymer solution to produce a solution or dispersion of the substance;
however, more often, a double emulsion is prepared that involves the formulation of
a water-in-oil (w/o) primary emulsion to encapsulate water-soluble molecules like
peptides or proteins, unlike the o/w formulation which is ideal for water-insoluble
compounds [56]. The primary w/o emulsion is, then, emulsified (with appropriate
stirring and temperature conditions) in a larger volume of water in presence of an
emulsifier to yield the final w/o/w emulsion. The emulsion is subjected to solvent
removal by either evaporation or extraction process to harden the oil droplets [57].
The solid microspheres obtained are, then, washed and collected by filtration or
centrifugation and then dried under appropriate conditions or lyophilized. One
of the disadvantages of the emulsification method is the poor encapsulation
efficiencies of moderately water-soluble and water-soluble compounds. Indeed,
these molecules could diffuse out or partition from the dispersed oil phase into the
aqueous continuous phase, and their microcrystalline fragments can deposit
on the microsphere surface and/or outside of the biopolymer matrix [58].
200 G. Della Porta and E. Reverchon

Moreover, the evaporation has several drawbacks because it requires relatively high
temperatures or reduced pressures and shows batch-to-batch reproducibility dis-
turbances, whereas the solvent extraction uses relatively large amounts of a second
solvent with the subsequent problem of solvent mixtures recovery. Both processes,
also, require quite long processing times (several hours) and, as a consequence,
aggregation phenomena may occur between the droplets, producing microspheres
with a larger poly-dispersity with respect to the droplets in the starting emulsion
[59].
Several supercritical technologies were proposed to produce microparticles, and
they are reviewed elsewhere [60]. Recently, the use of SC-CO2 has also been
proposed for biopolymer micro-/nanocarriers manufacturing, replacing the con-
ventional emulsions evaporation/extraction technology. Particularly, SC-CO2 was
proposed as extracting agent of the oily phase of emulsions to lead to solvent-free
microparticles [61–65]. The supercritical extraction of emulsion (SEE) process was
also proposed with a continuous operating mode that allowed greater product
uniformity, higher throughput with smaller plant volumes and elimination of
batch-to-batch repeatability problems (see Fig. 8.7). Indeed, SC-CO2 assured a
faster oily phase extraction with respect to the traditional evaporation/extraction
processes that will have a significant effect on the size distribution of the
biopolymer microcapsules which reproduce exactly the size distribution of the
originally droplets (with a given shrinkage) because any aggregation phenomena is
prevented due to the faster processing [66, 67]. The supercritical technology
achieved the fabrication of microcarriers of several biopolymers such as PLGA,
PLLA, PCL, PMMA with no solvent residues and higher encapsulation efficiencies
with respect to the conventional extraction/evaporation technology, especially when
double emulsions were processed [68, 69].
Micro-/nanocapsules entrapping magnetic iron oxide or gold nanoparticles have
been also fabricated by SEE technology in order to obtain a remote controlled or
triggered drug delivery [70, 71]. SEE-C technology can also generate multiloaded
microsystems with different molecules such as hydroxyapatite (HA), gentamicin
(GE) and teriparatide hormone (TH), for example, to be used in the bone cement
formulation in the treatment of osteoporosis [72].

Micro-/Nanocarriers as Functional Components

for a Bottom-Up Approach to Bioengineered Scaffold

Recent tissue engineering strategies suggested the use of 3D scaffold not only to
simply support the cells growth but also to interact with the cells on-board pro-
viding an additional level of cell environment regulation. In this sense, the locally
and controlled release of specific growth factors and/or bio-signals can be a good
approach in order to mimic more properly the microenvironment that the extra-
cellular matrix provides to the cells in order to address their behaviour.
8 Modular Tissue Engineering … 201

Fig. 8.7 Supercritical emulsion extraction (SEE) process layout description with optical
microscope image of an emulsion (left side) and of an SEM image of the microcapsules produced
(right side)

Current fabrication methods for GF-loaded scaffolds can be divided into two
main categories: attachment of the GF (physical and non-physical) to the scaffold or
entrapment of the GF within the scaffold. The attachment may be achieved by
adsorption or through chemical crosslinking. Fibrin-based matrices are popular
biomaterials for this purpose [73, 74], and the release then occurs passively and
through simultaneous fibrin degradation. Entrapment of GFs has been also
202 G. Della Porta and E. Reverchon

proposed in porous solid scaffolds, through solvent casting/particulate leaching and

supercritical fluid (SCF) processing [75, 76]. SCF processing seemed to be
attractive since it does not require the use of harmful solvents which could
adversely affect the GFs. However, the protein denaturation during scaffold fabri-
cation is the main problem associated with the incorporation of proteins in various
systems and leads to a considerable loss of their activity. Biomolecules have been
also directly incorporated into electrospun nanofibres by mixing the biomolecules
into a polymer solution prior to electrospinning the mixture [77] or by using coaxial
electrospinning wherein a secondary polymer solution containing the biomolecules
is electrospun within the core of the forming nanofibre surrounded by a shell
polymer [78–80]. Again severe protein damage may occur during the scaffold
production.
Loading GFs into hydrogels is also possible by simply mixing the GF into the
hydrogel precursor. As the polymerization occurs, the GFs become entangled in the
polymer chains and are physically trapped within the hydrogel network. However,
the hydrogels pore size is often larger than the size of the GF, so that it often
releases the majority of the contained GF within the first days of cultivation.
However, the direct GF loading into scaffolds does not allow a precise control of
the release kinetics and often when GFs are exposed to the medium; this may result
in loss of bioactivity.
The use of micro- or nanocarriers can overcome this problem by protecting the
GF and allowing the use of various populations of particles with different degra-
dation properties and thus different release kinetics of the encapsulated GFs [81,
82]. Indeed, by incorporating GFs into microspheres and then use these micro-
spheres as a dispersed phase surrounded by a continuous matrix (biopolymer or
hydrogel) allows to establish a skilled 3D scaffolds able to mimic a surrounding
matrix. For example, it has been reported that hBMP delivery from PLGA nano-
spheres incorporated into a PLA scaffold induced significant ectopic bone formation
while failure of bone formation was observed with passive adsorption of hBMP
onto the scaffold, likely due to significant loss of biological activity of the GF and
diffusion away from the implantation site [83].
Microspheres embedded into a continuous matrix can provide a temporal and a
geometrical controlled release of GFs to surrounding cells; an initial burst release or
a sustained release of GFs can be engineered in different part of the bioactive matrix
by tailoring the microcarriers size and distribution, as well as, the biopolymer
compositions or copolymer ratio loading different molecules and by loading dif-
ferent molecules in different micro-/nanodevices population. In the case of bone
engineering, for example, an initial burst release of BMP-2 is actually preferential
for promoting new bone formation. This can be explained by the fact that BMP-2
plays an important role during the healing process to promote cell differentiation but
is equally important at the early stages in the healing processes for the initial
migration of progenitor cells and triggering the fracture healing cascade [20, 84].
Geometrical indications inside a specific scaffold can be also obtained by incor-
porating different microdevices loaded with different bio-signals in different part of
8 Modular Tissue Engineering … 203

the structure or by using magnetic micro-/nanodevices to generate a specific

molecular/enzyme gradient by their orientation with an external magnetic field.
Bio-plotting technology may also have advantage from microdevices designed
for the controlled delivery of bio-signals. Indeed, the bioplotter deposits hydrogel
matrix loaded with living cells in small groups using similar technology to tradi-
tional printing systems. The advantage of this technique is the high potential level
of control in cell and ECM placement and alignment to create engineered tissues
with a wide array of properties and geometries. Indeed, by replacing ink with cells,
therefore named “bio-ink” because suspended in liquid matrix, a specific pattern can
be designed and printed onto a substrate that has to be cultured to allow the cells
and the produced ECM to integrate into a real tissue structures [85, 86]. However,
the bio-plotting technology also allows the loading of additive with the bio-inks;
these additive can be biopolymer microdevices or microcapsules that may influence
not only the mechanical behaviour of the 3D structure but also deliver in a con-
trolled spatio-temporal manner several biomolecules such as growth factor or
enzymes in order to better mimic the specific microenvironment of the extracellular
matrix and give rise of a faster and more appropriate cell addressing versus a
specific fate (see Fig. 8.8). These pre-designed 3D structures may better reproduce
the cells microenvironment that encourage cells differentiation and ECM produc-
tion. Engineered modular tissues, made using these techniques, have often shown
insufficient mechanical properties and on the order of native tissues [87]. However,
these drawbacks can be overcome by a mixed approach with bio-plotting hydrogel
onto a structure with pre-selected mechanical properties.
An example of bioactivated 3D scaffold easy bioplottable can be a continuous
hydrogel matrix of Ca-Alginate (2%w/w) loaded with stem cells and additives such
PLGA microcapsules carrying growth factors such as VEGF and BMP-2 [88].
PLGA-encapsulated GFs were produced by SEE technology, and they assured a
sustained release over 18 days when compared to GFs directly encapsulated into
scaffolds that was released in almost 3 days. Osteogenic differentiation pathway of
hMSCs was clearly observed after few day of cultivation in presence of controlled
release of hBMP-2 and faster with respect to the control cultivation without the
bio-signals delivered within the scaffold structure. Biopolymer micro-/nanocapsules
have been also loaded into collagen [89], gelatin [90], fibrin [91], chitosan [92], and
by functionalizing each type of spheres separately, the structure of the resulting
scaffolds can be precisely controlled at a microscale and/or customized by encap-
sulation of signalling biomolecules or bioactive minerals.
Recently also emerged a common theme of incorporating biopolymer microcap-
sules into densely cellular constructs where microcapsules can offer specific
microenvironment to chondrogenic cells in high-density culture in order to regulate
cell behaviour and tissue formation. As in the case of bone, microspheres may act in a
variety of specialized roles to define the three-dimensional arrangement of neocar-
tilage tissue formation, deliver chondrogenic growth factors in a temporally and
spatially controlled manner and instruct cell–cell and cell–material interactions. In
this particular case, biopolymer microspheres incorporated within densely cellular
tissues can also act as spacers, defining 3D space for tissue formation, while
204 G. Della Porta and E. Reverchon

Fig. 8.8 Schematic representation of the use of micro-/nanospheres for spatio/temporal delivery
of bioactive molecules. These, pre-designed 3D structure may better reproduce the cells
microenvironment that encourage cells differentiation and ECM production

accommodating ECM accumulation and cell infiltration as the microdevice degrades.

The inclusion of polymer microspheres into high-density cell cultivation systems
enables a more uniform cartilaginous matrix assembly with a morphological structure
similar to that of native cartilage tissue when compared to cell-only constructs
without microdevices [93, 94]. Moreover, the use of microcapsule with specific
properties, such us biopolymer degradation rate and size distribution, can influence
cartilage tissue formation, as well as, enabling the formation of a uniform, structurally
appropriate cartilage tissue if microspheres are fabricated with a fine-tuned degra-
dation rate proportional to the rate of ECM elaboration [23]. Microdevices may
contribute to the mechanical stability of the constructs and give a spacing effect that
could be advantageous for creating tissues morphologically similar to native articular
cartilage, which consists primarily of an organized ECM containing water, type II
collagen and proteoglycans with relatively few, disperse chondrocytes.
8 Modular Tissue Engineering … 205

Beyond their utility as structural elements, biopolymer microcapsules can

delivery chondrogenic growth factors such as TGF-b family. The incorporation of
bioactive microspheres in cell culture systems could provide a means to decrease
growth factor diffusion distance within densely cellular constructs, improving
growth factor delivery to central regions of the tissues. Further, these systems
allow growth factor release over a sustained period of time as the microspheres
degrade, enabling localized delivery of chondrogenic factors to the surrounding
cells at controllable rates over a period of days, weeks or months [23, 95–97]. The
sustained delivery of either TGF-b family in implantation models may play a role in
maintaining the chondrocytic phenotype of the cells, preventing dedifferentiation or
hypertrophic progression, as often occurs after the in vivo implantation of in vitro
differentiated MSC pellets [98, 99].

Conclusions and Perspectives

The bottom-up approaches seemed the best tissue engineering approach choice
because it is able to fabricate a better reproduction of the 3D microenvironment that
interacts with the cells to faster and better address their fate. The modular approach
also seemed to rely on the assembly of micron scale structural elements as the basis
for providing defined signals to drive the formation of tissues that can be scaled up
to create larger tissues. Moreover, the use of biopolymer microcapsules inserted
within the 3D scaffold can allow the design of a specific microenvironment able to
instruct the cell behaviour and to organize a pre-designed microenvironment with
an engineered spatio-temporal delivery of multiple bio-signals.
Biopolymer microspheres are also proposed as regulators of the chondrogenic
microenvironment within high-density cell cultures as three-dimensional structural
elements for cell expansion and delivery or vehicles for spatiotemporally controlled
growth factor delivery, to regulate cell behaviour and/or cell material interactions.
These drug-releasing microspheres have the capability not only to overcome dif-
fusion limitations posed by traditional culture techniques, but also to lend tailorable
release kinetics for temporal control of differentiation and extended maintenance of
differentiation state in vivo.
Furthermore, micro-/nanospheres can serve as reinforcement components or
crosslinking agents to provide hydrogels with additional mechanical support.
Indeed, microspheres loaded with inorganic materials of high stiffness can reinforce
the initial mechanical strength of the composites. For example, biopolymer
microspheres can be used as different “bio-ink additives” added to a 3D bioplotted
hydrogel to give specific mechanical or biochemical indications to the cells in the
different layers following the novel concept of 3D organ printing, in which scaffold/
cellularized tissues or organ can be fabricated layer by using tissue spheroids as
building blocks.
206 G. Della Porta and E. Reverchon

References

1. Van Blitterswijk CA, Thomsen P, Lindahl A, Hubbell J, Williams DF, Cancedda R, de

Bruijn J, Sohier J, editors. Tissue engineering textbook. London: Academic Press; 2008.
p. 403–54. ISBN 978-0-12-370869-4.
2. Langer R, Vacanti JP. Tissue engineering. Science. 1993;260(5110):920–6.
3. Hillsley M, Frangos J. Review: bone tissue engineering: the role of interstitial fluid flow.
Biotech Bioeng. 1994;43:575–81.
4. Martin P. Wound healing–aiming for perfect skin regeneration. Science. 1997;276(5309):
75–81.
5. Caplan AI, Elyaderani M, Mochizuki Y, Wakitani S, Goldberg VM. Principles of cartilage
repair and regeneration. Clin Orthop. 1997;342:254–69.
6. Niklason LE, Gao J, Abbott WM, Hirschi KK, Houser S, Marini R, Langer R. Functional
arteries grown in vitro. Science. 1999;284(5413):489–93.
7. Gooch KJ, Blunk T, Courter DL, Sieminski AL, Vunjak-Novakovic G, Freed LE. Bone
morphogenetic proteins modulate in vitro development of engineered cartilage. Tissue Eng.
2002;8(4):91–601.
8. Schaner PJ, Martin ND, Tulenko TN, Shapiro IM, Tarola N, Leichter RF, Carabasi RA,
Dimuzio PJ. Decellularized vein as a potential scaffold for vascular tissue engineering. J Vasc
Surg. 2004;40(1):146–53.
9. Dean DM, Napolitano AP, Youssef J, Morgan JR. Rods, tori, and honeycombs: the directed
self-assembly of microtissues with prescribed microscale geometries. FASEB. 2007;21:
4005–12.
10. Yeh J, Ling Y, Karp JM, Gantze J, Chandawarkard A, Eng G, Blumling J, Langer R,
Khademhosseini A. Micromolding of shape-controlled, harvestable cell-laden hydrogels.
Biomaterials. 2006;27:5391–8.
11. L’Heureux N, Paquet S, Labbe R, Germain L, Auger FA. A completely biological
tissue-engineered human blood vessel. FASEB J. 1998;12(1):47–56.
12. Mironov V, Boland T, Trusk T, Forgacs G, Markwald RR. Organ printing: computer-aided
jet-based 3D tissue engineering. Trends Biotechnol. 2003;21(4):157–61.
13. McGuigan AP, Sefton MV. Design and fabrication of sub-mm-sized modules containing
encapsulated cells for modular tissue engineering. Tissue Eng. 2007;13(5):1069–78.
14. McGuigan AP, Sefton MV. The thrombogenicity of human umbilical vein endothelial cell
seeded collagen modules. Biomaterials. 2008;29:2453–63.
15. L’Heureux N, McAllister TN, de la Fuente LM. Tissue-engineered blood vessel for adult
arterial revascularization. N Engl J Med. 2007;357(14):1451–3.
16. Du Y, Lo E, Ali S, Khademhosseini A. Directed assembly of cell-laden microgels for
fabrication of 3D tissue constructs. Proc Natl Acad Sci. 2008;105(28):9522–7.
17. Nichol JW, Khademhosseini A. Modular tissue engineering: engineering biological tissues
from the bottom up. Soft Matter. 2009;5(7):1312–9.
18. Narayanan LK, Huebner P, Fisher MB, Spang JT, Starly B, Shirwaiker RA. 3D-bioprinting of
polylactic acid (PLA) nanofiber-alginate hydrogel bioink containing human adipose-derived
stem cells. ACS Biomater Sci Eng. 2016;2:1732–42.
19. Shen J, Guvendiren M. Recent advances in bioink design for 3D bioprinting of tissues and
organs. Front Bioeng Biotechnol. 2017. https://doi.org/10.3389/fbioe.2017.00023.
20. Wang H, Leeuwenburgh SCG, Li Y, Jansen JA. The use of micro- and nanospheres as
functional components for bone tissue regeneration. Tissue Eng Part B. 2012;18(1):24–39.
21. Tatard VM, Venier-Julienne MC, Saulnier P, Prechter E, Benoit JP, Menei P, Montero-Menei
CN. Pharmacologically active microcarriers: a tool for cell therapy. Biomaterials. 2005;
26(17):3727–37.
22. Mercier NR, Costantino HR, Tracy MA, Bonassar LJ. Poly (lactide-co-glycolide)
microspheres as a moldable scaffold for cartilage tissue engineering. Biomaterials. 2005;26:
1945–52.
8 Modular Tissue Engineering … 207

23. Kang SW, Jeon O, Kim BS. Poly(lactic-co-glycolic acid) microspheres as an injectable
scaffold for cartilage tissue engineering. Tissue Eng. 2005;11:438–47.
24. Hong Y, Gao CY, Xie Y, Gong YH, Shen JC. Collagen-coated poly-lactide microspheres as
chondrocyte microcarriers. Biomaterials. 2005;26:6305–13.
25. Tan H, Huang D, Lao L, Gao C. RGD modified PLGA/gelatin microspheres as microcarriers
for chondrocyte delivery. J Biomed Mater Res Part B Appl Biomater. 2009;91:228–38.
26. Palmiero C, Imparato G, Urciuolo F, Netti P. Engineered dermal equivalent tissue in vitro by
assembly of microtissue precursors. Acta Biomater. 2010;6(7):2548–53.
27. Lee CH, Singla A, Lee Y. Biomedical applications of collagen. Int J Pharm. 2001;221:1.
28. Li S-T. Biologic biomaterials: tissue-derived biomaterials (collagen). In: Park JB, Bronzino JD,
editors. Biomaterials: principles and applications. Boca Roton, FL: CRC Press; 2003.
29. Schrieber R, Gareis H. Gelatine handbook: theory and industrial practice. Weinheim:
Wiley-VCH Verlag; 2007.
30. Yamamoto M, Ikada Y, Tabata Y. Controlled release of growth factors based on
biodegradation of gelatin hydrogel. J Biomater Sci Polym Ed. 2001;12:77.
31. Ahmed TAE, Dare EV, Hincke M. Fibrin: a versatile scaffold for tissue engineering
applications. Tissue Eng B. 2008;14:199.
32. Perka C, Schultz O, Spitzer R-S, Lindenhayn K, Burmester G-R, Sittinger M. Segmental bone
repair by tissue-engineered periosteal cell transplants with bioresorbable fleece and fibrin
scaffolds in rabbits. Biomaterials. 2000;21:1145.
33. Di Martino A, Sittinger M, Risbud MV. Chitosan: a versatile biopolymer for orthopaedic
tissue-engineering. Biomaterials. 2005;26:5983.
34. Slaughter BV, Khurshid SS, Fisher OZ, Khademhosseini A, Peppas NA. Hydrogels in
regenerative medicine. Adv Mater. 2009;21:3307.
35. Augst AD, Kong HJ, Mooney DJ. Alginate hydrogels as biomaterials. Macromol Biosci.
2006;6:623.
36. Mundargi RC, Babu VR, Rangaswamy V, Patel P, Aminabhavi TM. Nano/micro technologies
for delivering macromolecular therapeutics using poly (D, L lactide-co-glycolide) and its
derivatives. J Control Release. 2008;125:193–209.
37. Verrijk R, Smolde IJ, Bosnie N, Begg AC. Reduction of systemic exposure and toxicity of
cisplatin by encapsulation in poly(lactide-co-glycolide). Cancer Res. 1992;52:6653–6.
38. Meinel L, Illi OE, Zapf J, Malfanti M, Merkle HP, Gander B. Stabilizing insulin-like grow
factor in poly(lactide-co glycolide) microspheres. J Control Release. 2001;70:193–202.
39. Walter E, Dreher D, Kok M, Thiele L, Kiama SG, Gehr P, Merkle HP. Hydrophylic poly
(lactide-co-glycolide) microspheres for the delivery of DNA to human derived macrophages
and dentritic cells. J Control Release. 2001;76:149–68.
40. Luten J, van Nostrum CF, De Smedt SC, Hennink WE. Biodegradable polymers as non-viral
carriers for plasmid DNA delivery. J Control Release. 2008;126:97–110.
41. Jaklenec A, Wan E, Murray ME, Mathiowitz E. Novel scaffolds fabricated from
protein-loaded microspheres for tissue engineering. Biomaterials. 2008;9:185–92.
42. Temenoff JS, Mikos AG, editors. Biomaterials. The intersection of biology and materials
science. NJ: Pearson Prentice Hall; 2008. ISBN 10-0-13-009710-1.
43. Zhang J, Zhang H, Wu L, Ding J. Fabrication of three dimensional polymeric scaffolds with
spherical pores. J Mater Sci. 2006;41:1725–30.
44. Liu X, Ma PX. Polymeric scaffold for bone tissue engineering. Ann Biomed Eng.
2003;32:477–82.
45. Volkmer E, et al. Hypoxia in static and dynamic 3D culture systems for tissue engineering of
bone. Tissue Eng Part A. 2008;14(8):1331–40.
46. Shin M, Yoshimoto H, Vacanti JP. In vivo bone tissue engineering using mesenchymal stem
cells on a novel electrospun nanofibrous scaffold. Tissue Eng. 2004;10(1–2):33–41.
47. Landers R, Pfister A, Hübner U, John H, Schmelzeisen R, Mülhaupt R. Fabrication of soft
tissue engineering scaffolds by means of rapid prototyping techniques. J Mater Sci. 2002;
37(15):3107–16.
208 G. Della Porta and E. Reverchon

48. Hutmacher DW, Sittinger M, Risbud MV. Scaffold-based tissue engineering: rationale for
computer-aided design and solid free-form fabrication systems. Trends Biotechnol. 2004;
22(7):354–62.
49. Brunner G. Gas extraction: an introduction to fundamentals of supercritical fluids and the
application to separation processes. In: Topics in physical chemistry, vol. 4. Ed. Techniques
Ingénieur; 1994. ISBN 3798509441.
50. Mathieu LM, Montjovent MO, Bourban PE, Pioletti DP, Manson JAE. Bioresorbable
composites prepared by supercritical fluid foaming. J Biomed Mater Res A. 2005;75:89–94.
51. Tsivintzelis I, Marras SI, Zuburtikudis I, Panayiotou C. Porous poly(lactic acid) nanocom-
posite scaffolds prepared by phase inversion using supercritical CO2 as antisolvent. Polymer.
2007;48:6311–3.
52. Reverchon E, Cardea S. Formation of cellulose acetate membranes using a supercritical fluid
assisted process. J Membr Sci. 2004;240:187–95.
53. Reverchon E, Cardea S, Rapuano C. A new supercritical fluid-based process to produce
scaffolds for tissue replacement. J Supercrit Fluids. 2008;45:365–70.
54. Della Porta G, Del Gaudio P, De Cicco F, Aquino RP, Reverchon E. Supercritical drying of
alginate beads for the development of aerogel biomaterials: optimization of process
parameters and exchange solvents. Ind Eng Chem Res. 2013;52:12003–9.
55. Pisanti P, Yeatts AB, Cardea S, Fisher JP, Reverchon E. Tubular perfusion system culture of
human mesenchymal stem cells on poly-L-lactic acid scaffolds produced using a supercritical
carbon dioxide-assisted process. J Biomed Mater Res A. 2012;100(10):2563–72.
56. Li WI, Anderson KW, Deluca PP. Kinetic and thermodynamic modelling of the formation of
polymeric microspheres using solvent extraction/evaporation method. J Contr Rel. 1995;37:
187–98.
57. Li M, Rouand O, Poncelet D. Microencapsulation by solvent evaporation: state of the art for
process engineering approaches. Int J Pharm. 2008;363:26–39.
58. Mao S, Shi Y, Li L, Xu J, Schaper A, Kissel T. Effects of process and formulation parameters
on characteristics and internal morphology of poly(D, L-lactide-co-glycolide) microspheres
formed by the solvent evaporation method. Eur J Pharm Biopharm. 2008;68:214–23.
59. Yang YY, Chia HH, Chung TS. Effect of preparation temperature on the characteristics and
release profiles of PLGA microspheres containing protein fabricated by double-emulsion
solvent extraction/evaporation method. J Control Release. 2000;69:81–96.
60. Della Porta G, Reverchon E. Supercritical fluid based technologies for particulate drug
delivery. In: Ravi Kumar MNV, editor. Handbook of particulate drug delivery. American
Scientific Publishers; 2008. p. 35–59. ISBN 978-1-58883-124-8 (Chapter 3).
61. Della Porta G, Reverchon E. Nanostructured microspheres produced by supercritical fluid
extraction of emulsions. Biotechnol Bioeng J. 2008;100(5):1020–33.
62. Kluge J, Fusaro F, Casas N, Mazzotti M, Muhrer G. Production of PLGA micro- and
nanocomposites by supercritical fluid extraction of emulsions: I. Encapsulation of lysozime.
J Supercrit Fluids. 2009;50:327–35.
63. Della Porta G, Falco N, Reverchon E. Antiflammatory drugs release from injectable
microspheres produced by supercritical fluid emulsion extraction. J Pharm Sci. 2010;99(3):
1484–99.
64. Della Porta G, Castaldo F, Scognamiglio M, Paciello L, Parascandola P, Reverchon E.
Bacteria microencapsulation in PLGA microdevices by supercritical emulsion extraction.
J Supercrit Fluids. 2012;63:1–7.
65. Della Porta G, Campardelli R, Falco N, Reverchon E. PLGA microdevices for retinoids
sustained release produced by supercritical emulsion extraction: continuous versus batch
operation layouts. J Pharm Sci. 2011;100(10):4357–67.
66. Della Porta G, Reverchon E. Continuous supercritical emulsions extraction: a new technology
for biopolymer microparticles production. Biotechnol Bioeng J. 2011;108(3):676–86.
67. Falco N, Reverchon E, Della Porta G. Continuous supercritical emulsions extraction: packed
tower characterization and application to poly(lactic-co-glycolic acid) + insulin microspheres
production. Ind Eng Chem Res. 2012;51:8616–23.
8 Modular Tissue Engineering … 209

68. Della Porta G, Campardelli R, Reverchon E. Monodisperse biopolymer nanoparticles by

continuous supercritical emulsion extraction. J Supercrit Fluids. 2013;76:67–73.
69. Della Porta G, Falco N, Giordano E, Reverchon E. PLGA microspheres by supercritical
emulsion extraction: a study on insulin release in myoblast culture. J Biomater Sci Polym Ed.
2013;24(16):1831–47.
70. Campardelli R, Della Porta G, Irusta S, Reverchon E, Santamaria J. Hollow gold
nanoshells-PLA nanocomposites for photothermal controlled delivery. J Mater Chem B.
2014;2:409–17.
71. Govoni M, Berardi AC, Muscari C, Campardelli R, Bonafe F, Guarnieri C, Reverchon E,
Giordano E, Maffulli N, Della Porta G. 3D engineered multiphase three-dimensional
microenvironment to ensure the controlled delivery of cyclic strain and human growth
differentiation factor 5 for the tenogenic commitment of human bone marrow mesenchymal
stem cells. Tissue Eng Part A. 2017. https://doi.org/10.1089/ten.tea.2016.0407.
72. Della Porta G, Campardelli R, Cricchio V, Oliva F, Maffulli N, Reverchon E. Injectable
PLGA/hydroxyapatite/Chitosan microcapsules produced by supercritical emulsion extraction
technology: An in vitro study on teriparatide/gentamicin controlled release. J Pharm Sci.
2016;105(7):2164–72.
73. Whitaker MJ, Quirk RA, Howdle SM, Shakesheff KM. Growth factor release from tissue
engineering scaffolds. J Pharm Pharmacol. 2001;53(11):1427–37.
74. Sakiyama-Elbert SE, Hubbell JA. Controlled release of nerve growth factor from a
heparin-containing fibrin-based cell ingrowth matrix. J Controlled Release. 2000;69(1):
149–58.
75. Kang SW, Kim JS, Park KS, Cha BH, Shimb JH, Kim JY, Cho DW, Rhie JW, Lee SH.
Surface modification with fibrin/hyaluronic acid hydrogel on solid-free form-based scaffolds
followed by BMP-2 loading to enhance bone regeneration. Bone. 2011;48(2):298–306.
76. Kanczler JM, Barry J, Ginty P, Howdle SM, Shakesheff KM, Oreffo ROC. Supercritical
carbon dioxide generated vascular endothelial growth factor encapsulated poly(dl-lactic acid)
scaffolds induce angiogenesis in vitro. Biochem Biophys Res Commun. 2007;352(1):135–41.
77. Murphy WL, Peters MC, Kohn DH, Mooney DJ. Sustained release of vascular endothelial
growth factor from mineralized poly(lactide-co-glycolide) scaffolds for tissue engineering.
Biomaterials. 2000;21(24):2521–7.
78. Sanders EH, Kloefkorn R, Bowlin GL, Simpson DG, Wnek GE. Two-phase electrospinning
from a single electrified jet: microencapsulation of aqueous reservoirs in poly
(ethylene-co-vinyl acetate) fibers. Macromolecules. 2003;36(11):3803–5.
79. Jiang H, Hu Y, Li Y, Zhao P, Zhu K, Chen W. A facile technique to prepare biodegradable
coaxial electrospun nanofibers for controlled release of bioactive agents. J Controlled Release.
2005;108(2–3):237–43.
80. Liao IC, Chew SY, Leong KW. Aligned core-shell nanofibers delivering bioactive proteins.
Nanomedicine. 2006;1(4):465–71.
81. Ungaro F, Biondi M, d’Angelo I, Indolfi L, Quaglia F, Netti PA. Microsphere-integrated
collagen scaffolds for tissue engineering: effect of microsphere formulation and scaffold
properties on protein release kinetics. J Controlled Release. 2006;113(2):128–36.
82. Li B, Yoshii T, Hafeman AE, Nyman JS, Wenke JC, Guelcher SA. The effects of rhBMP-2
released from biodegradable polyurethane/microsphere composite scaffolds on new bone
formation in rat femora. Biomaterials. 2009;30:6768–79.
83. Wei GB, Jin QM, Giannobile WV, Ma PX. The enhancement of osteogenesis by nano-fibrous
scaffolds incorporating rhBMP-7 nanospheres. Biomaterials. 2007;28(12):2087–96.
84. Li B, Yoshii T, Hafeman AE, Nyman JS, Wenke JC, Guelcher SA. The effects of rhBMP-2
released from biodegradable polyurethane/microsphere composite scaffolds on new bone
formation in rat femora. Biomaterials. 2009;30(35):6768–79.
85. Federovich NE, Alblas J, DeWijn JR, Hennink WE, Verbout AJ, Dhert WJA. Hydrogels as
extracellular matrices for skeletal tissue engineering: state-of-the-art and novel application in
organ printing. Tissue Eng. 2007;13(8):1905–25.
210 G. Della Porta and E. Reverchon

86. Roth EA, Xu T, Das M, Gregory C, Hickman JJ, Boland T. Inkjet printing for
high-throughput cell patterning. Biomaterials. 2004;25:3707–15.
87. Adam EJ, Alexandra LR, Ramille NS. Advancing the field of 3D biomaterial printing.
Biomed Mater. 2016;11:014102. https://doi.org/10.1088/1748-6041/11/1/014102.
88. Della Porta G, Nguyen BN, Campardelli R, Reverchon E, Fisher JP. Synergistic effect of
sustained release of growth factors and dynamic culture on osteoblastic differentiation of
mesenchymal stem cells. J Biomed Mater Res Part A. 2014. https://doi.org/10.1002/jbm.a.
35354.
89. Chan BP, Hui TY, Wong MY, Yip KHK, Chan GCF. Mesenchymal stem cell-encapsulated
collagen microspheres for bone tissue engineering. Tissue Eng. 2010;16(2):225–35.
90. Kuroda Y, Akiyama H, Kawanabe K, Tabata Y, Nakamura T. Treatment of experimental
osteonecrosis of the hip in adult rabbits with a single local injection of recombinant human
FGF-2 microspheres. J Bone Miner Metab. 2010;28:608–16.
91. Ben-Ari A, Rivkin R, Frishman M, Gaberman E, Levdansky L, Gorodetsky R. Isolation and
implantation of bone marrow-derived mesenchymal stem cells with fibrin micro beads to
repair a critical-size bone defect in mice. Tissue Eng A. 2009;15(9):2537–46.
92. Jayasuriya AC, Bhat A. Fabrication and characterization of novel hybrid organic/inorganic
microparticles to apply in bone regeneration. J Biomed Mater Res A. 2010;93(4):1280–8.
93. Liu X, Jin X, Ma PX. Nanofibrous hollow microspheres self-assembled from star-shaped
polymers as injectable cell carriers for knee repair. Nat Mater. 2011;10:398–406.
94. Kang SW, La WG, Kim BS. Open macroporous poly(lactic-co-glycolic Acid) microspheres
as an injectable scaffold for cartilage tissue engineering. J Biomater Sci Polym Ed.
2009;20:399–409.
95. Solorio LD, Fu AS, Hernandez-Irizarry R, Alsberg E. Chondrogenic differentiation of human
mesenchymal stem cell aggregates via controlled release of TGF-beta1 from incorporated
polymer microspheres. J Biomed Mater Res A. 2010;92:1139–44.
96. Solorio LD, Dhami CD, Dang PN, Vieregge EL, Alsberg E. Spatiotemporal regulation of
chondrogenic differentiation with controlled delivery of transforming growth factor-b1 from
gelatin microspheres in mesenchymal stem cell aggregates. Stem Cells Translational Med.
2012;1(8):632–9.
97. Han Y, Wei Y, Wang S, Song Y. Cartilage regeneration using adipose-derived stem cells and
the controlled released hybrid microspheres. Joint Bone Spine. 2010;77:27–31.
98. Park JS, Na K, Woo DG, Yang HN, Park KH. Determination of dual delivery for stem cell
differentiation using dexamethasone and TGF-beta 3 in/on polymeric microspheres.
Biomaterials. 2009;30:4796–805.
99. Park JS, Yang HN, Woo DG, Jeon SY, Park KH. The promotion of chondrogenesis,
osteogenesis, and adipogenesis of human mesenchymal stem cells by multiple growth factors
incorporated into nanosphere-coated microspheres. Biomaterials. 2011;32:28–38.
Index

A C
Adhesion, 4–8, 12, 14–17, 19, 35, 44, 59, 61, Cartilage, 4, 6, 7, 13, 20, 46, 47, 50, 119, 120,
64, 71–74, 84, 85, 91, 93–95, 97, 129–133, 153, 156, 161, 165, 172, 173,
101–103, 115, 116, 119, 126, 133, 155, 179–182, 194, 204
157, 166, 168, 183, 194 Cell carrier, 116, 124
Adhesive glycoproteins, 7 Cell-cell contacts, 15, 60, 61, 63
Adipose, 41, 42, 46, 116, 130, 132, 133, 155 Cell culture, 49, 59, 60, 62, 63, 72, 74, 75, 131,
Antimicrobial properties, 30 133, 156, 195, 205
Apoptosis, 6, 59, 60, 63, 64, 72, 74, 89, 97, 98, Cell migration, 9, 11, 15, 16, 67, 71, 84, 92, 95,
101, 103, 168–170 132, 134, 170
Architecture and prestress, 163 Cell niche and extracellular matrix, 62
Cell polarity, 16, 17, 75
B Cell proliferation, 8, 9, 12, 15, 35, 49, 51, 73,
Biochemical composition, 5, 29, 36, 52 99, 115, 125, 127, 128, 154, 157, 170,
Biochemical content, 29 173
Biocompatibility, 8, 35, 52, 126, 127, 131, 135, Cells, 3–5, 7–19, 27–32, 35–51, 59–64, 66–68,
153–155, 158, 183, 194, 195 70–76, 83–85, 87–92, 94, 95, 97–99,
Bioinductive properties, 30 101–103, 113–135, 152, 153, 155–157,
Biomaterials, 15, 20, 27, 33, 38, 41, 43, 49–51, 162–174, 179–183, 191, 192, 194, 198,
114, 120, 126, 153, 157, 158, 201 200, 202–205
Biomaterials for scaffold fabrication, 194 Central nervous system, 45, 67, 176, 178
Bone, 4, 7, 9, 10, 12, 13, 17, 19, 30, 31, 41, 49, Chains and trimers, 65
50, 70, 73, 99, 119, 121, 122, 125, 126, Chemoattraction, 30, 31
129, 132, 133, 135, 153–156, 158, 161, Clustering calcium channels in motor nerve
162, 164, 165, 168–173, 177, 179, 181, terminals, 69
182, 194, 200, 202, 203 Controlled drug release, 123, 124, 131
Bone and osteoinduction, 170 Cornea, 153
Bone marrow-derived hematopoietic stem Co-signaling in mammary epithelial cells, 68
cells, 73 Co-signaling in vascular endothelial cells, 69
Brain, 17, 45, 67, 76, 122, 125, 132, 176 Crosslinkers, 131

© Springer International Publishing AG, part of Springer Nature 2018 211

A. C. Berardi (ed.), Extracellular Matrix for Tissue Engineering
and Biomaterials, Stem Cell Biology and Regenerative Medicine,
https://doi.org/10.1007/978-3-319-77023-9
212 Index

D 152, 154, 165, 182, 192, 195, 196, 200,

Decellularization, 28, 29, 32, 37, 44, 47, 49, 51 203, 205
Demineralized bone matrix, 49 Growth factors and morphogens for tissue
Dermis, 29, 42–45, 133 engineering, 18
Differentiation, 4, 5, 8, 10, 12–17, 35, 36, 42,
46, 48, 59, 61, 64, 66, 69, 71, 72, 75, 83, H
84, 91, 92, 118, 128, 130, 134, 155, 162, Heart, 13, 17, 39, 41, 89, 90, 134, 167
163, 168–171, 173, 174, 179–183, Hormesis, 167–170
202–205 Human and mouse embryonic stem cells, 60
Hyaluronan, 6, 19, 20, 83–85, 88–90, 92,
E 94–96, 101, 102, 181
ECM and tendons, 173 Hyaluronan and immunity, 102
ECM hydrogel characterization, 35 Hyaluronan and inflammation, 95
ECM hydrogel formation, 33, 34 Hybrid hydrogels, 50
Electrospinning, 113, 116, 118, 119, 134, 154, Hydrogel, 19, 20, 27, 28, 33–39, 41–52, 70,
155, 157, 179, 196, 197, 202 115–117, 119, 122–129, 131–133, 154,
Embryonic stem cell, 20, 60, 62 156, 202, 203, 205
Evaluation criteria in developing cell culture
system, 74 I
Extracellular matrix, 7–10, 13, 15, 28, 29, 33, Immunity, 6, 83, 85, 87, 90, 92, 94, 98–100,
38, 41, 59–61, 64, 65, 68–72, 74–76, 103
83–92, 94, 95, 99, 100, 102, 113, 114, Importance of ECM-Integrin interactions, 13
153, 156, 162, 167, 173, 179–181, 192, Insulin-producing pancreatic islets, 73
194, 200, 203 Integrin, 8, 9, 14, 16, 17, 19, 38, 68, 71, 72, 74,
Extracellular matrix interactions, 69 84, 103, 134, 165, 166
Extracellular vesicles in the ECM Intervertebral disc, 48, 115
structure/organization, 3, 5, 7, 8, 12, 13, In vitro study, 155
88, 90, 92, 118, 123, 125, 153, 164, 173, In vivo study, 125, 154
174, 192
K
F Key molecular ECM components, 4
Fat, 41, 42
Fibrin, 5, 11, 113, 123, 126–128, 130–134, L
194, 201 Laminins, 5, 8, 13, 59–76, 84, 173
Fibrinogen, 8, 11, 19, 113, 128, 131, 134 Ligament, 47, 48, 153, 156, 157, 165, 174,
Fibrinogen-based scaffolds, 127, 132, 134 175, 177, 179, 182
Fibrous proteins, 5, 7, 113, 114 Liver, 4, 30, 43, 44, 50, 51, 62, 132
Fibrous scaffolds, 114, 118–120, 156, 157,
180, 181 M
Freeze-drying, 113, 118–120, 155 Macrophage polarization, 31, 52, 95
Future directions, 51 Matrix metalloproteinases (MMPs), 9, 10, 12,
85–87, 92, 99, 100
G Mechanical signals inside the ECM, 15
Gel topology, 38, 52 Mechanotransduction, 15, 61, 63, 88, 163, 164,
Gene therapy, 116, 125–127 167
Glycosamminoglycans, 6 Metalloproteinases and inflammation, 92
Growth factor and morphogen delivery through Methods for ECM hydrogel production, 34
engineered ECM, 19 Molecular interactions of laminins, 68
Growth factors, 3–10, 12–14, 16, 18, 20, Morphogenesis, 7, 14, 16–18, 20, 61, 83, 84,
28–30, 36, 41, 42, 46, 51, 59–61, 63, 64, 89, 91, 164, 192
75, 84–86, 88, 90, 92, 94, 95, 102, 113, Morphogenesis of 3D tissue architecture
120, 123, 125, 127–130, 132, 133, 135, in vivo, 17
Index 213

Muscle, 14, 32, 35, 36, 39, 40, 44, 45, 49, 51, Signaling, 3, 4, 6–19, 59, 60, 63, 65–69, 72,
61, 66, 67, 70, 115–117, 126, 130, 132, 74, 75, 84, 86, 93–95, 102, 162,
162, 164, 168, 169, 173–175, 178, 180 165–169, 174, 180
Musculoskeletal tissues healing, 161 Silk fibroin, 115, 117, 152–157
Skeletal muscle, 32, 44, 45, 49, 168, 169, 178,
N 179
Nanomaterials, 153 Skin, 4, 7, 14, 17, 43, 66, 67, 89, 93, 116, 117,
Nanometric architecture, 162 119, 129, 132, 133, 153, 157, 179
Nanoscale biotechonology, 179 Small intestinal submucosa, 27, 29, 33
Nanoscale engineering, 162 Spinal cord, 45, 119, 120, 129, 131, 132
Nanotechnologies, 158 Stem cell, 15, 40, 41, 46, 48, 49, 59, 60, 119,
Natural biomaterials, 4, 19 121, 127, 130, 170, 183
Neurobiology, 74 Stem cell technologies, 60
Niche-specific extracellular matrix, 59, 63, 64 Survival, 9, 10, 14–16, 59, 61, 62, 72, 73, 76,
Nucleus pulposus, 48, 116 83, 84, 89, 92, 162, 173, 183

O T
Other extracellular matrix molecules, 64, 69, Tendon, 32, 47, 48, 118–120, 132, 153,
96 155–157, 166, 173–179, 182
Tensegrity, 163, 165–167
P Tensegrity and mechanochemical transduction,
Pancreatic islets, 63 165
Pericardium, 40 3D drug releasing scaffolds, 120, 122, 124
Possible antagonistic functions of related Three pillars on cell niche, 63
laminins, 70 3D scaffolds, 113, 123, 125–127, 183, 192,
Preclinical studies, 30, 132 195–197, 202
Proliferation, 4–6, 8–10, 14, 15, 31, 35, 42–44, Thrombospondins and immunity, 103
46, 59, 61, 63, 64, 71, 72, 74–76, 84, 91, Thrombospondins and inflammation, 96
92, 96, 102, 117, 125, 130, 133, 134, Thrombospondins, 89, 90
155–157, 168–170, 180–182 Tissue engineering, 3–5, 13, 15–18, 28,
Prominent role of the proteoglycans, 6 113–116, 119, 120, 126–130, 132, 133,
Protease activity, 11 135, 152, 153, 155–158, 162, 166, 170,
179, 181–183, 191, 192, 194–196, 200
R Tissue-specific hydrogels, 38
Regenerative medicine, 16, 32, 59, 61–63, 71, Tissue specificity, 28, 38, 42, 45, 52
72, 75, 119, 152, 170, 171 Types of ECM-derived materials, 32
Role of proteolic enzyme in ECM, 11
U
S Urinary bladder matrix, 27, 29, 33
Safety, 28, 40, 47, 75
Scaffolds, 3–5, 13, 19, 20, 28, 31, 32, 36, 42, V
43, 48, 49, 51, 75, 84, 113–123, Versican, 6, 83, 85, 87–90, 94–96, 101, 102
125–130, 132–135, 152–157, 161–163, Versican and immunity, 101
165, 179–183, 191, 193–196, 198, Versican and inflammation, 94
201–203 Vocal fold, 50
Self-assembly and interaction with other
laminin isoforms, 69