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Regenerative
Approaches in
Dentistry
An Evidence-Based Perspective
Sepanta Hosseinpour
Laurence J. Walsh
Keyvan Moharamzadeh
Editors

123
Regenerative Approaches in Dentistry
Sepanta Hosseinpour • Laurence J. Walsh
Keyvan Moharamzadeh
Editors

Regenerative
Approaches in Dentistry
An Evidence-Based Perspective
Editors
Sepanta Hosseinpour Laurence J. Walsh
School of Dentistry School of Dentistry
University of Queensland University of Queensland
Herston, QLD, Australia Herston, QLD, Australia

Keyvan Moharamzadeh
Hamdan Bin Mohammed College of
Dental Medicine (HBMCDM)
Mohammed Bin Rashid University of
Medicine and Health Sciences (MBRU)
Dubai, United Arab Emirates

ISBN 978-3-030-59808-2    ISBN 978-3-030-59809-9 (eBook)

https://doi.org/10.1007/978-3-030-59809-9

© Springer Nature Switzerland AG 2021

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
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the authors or the editors give a warranty, expressed or implied, with respect to the material
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neutral with regard to jurisdictional claims in published maps and institutional affiliations.

This Springer imprint is published by the registered company Springer Nature Switzerland AG
The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
Preface

This book has been written to address the current need for a comprehensive
reference on regenerative approaches in dentistry with a special focus on cur-
rent clinical applications and future potentials. This topic has been evolved
significantly in recent decades, and there is a plethora of papers (experimental
and clinical studies) in various branches of dental science. This book aims to
collect and compare what has been done to provide evidence-based informa-
tion for clinicians and scientists in this emerging field of dentistry.
The book is intended to educate the readers about various therapeutic
modalities used in the reconstruction of hard and soft tissues in the maxillo-
facial region. Different potential laboratory and clinical applications of engi-
neered oral and dental tissue equivalents are discussed in the relevant
chapters.
It is hoped that this book will be useful to students in dentistry (at both
undergraduate and postgraduate levels), as well as scientists and researchers
in the field of biomedical sciences in dentistry, and clinicians.

Queensland, Brisbane, Australia Sepanta Hosseinpour

Queensland, Brisbane, Australia  Laurence J. Walsh
Dubai, United Arab Emirates  Keyvan Moharamzadeh

v
Acknowledgments

This book could not be completed without the valuable contributions of the
authors and their wider research groups and collaborators.
We would like to thank the Springer team, specifically Ms. Deepika Devan,
Ms. Alison Wolf, and We would like to thank the Springer team, specifically
Ms. Deepika Devan, Ms. Alison Wolf, and Mr. Manohar Vignesh, for their
administrative support during the publication process, for their administrative
support during the publication process.
We also thank our colleagues and families for their help and support dur-
ing the preparation of this book.

vii
Contents


The Paradigm of Regenerative Dentistry and Its Future
Perspectives����������������������������������������������������������������������������������������������   1
Laurence J. Walsh and Sepanta Hosseinpour
Dental Tissues Originated Stem Cells for Tissue Regeneration����������   9
Maryam Rezai Rad, Sepanta Hosseinpour, Qingsong Ye,
and Shaomian Yao
Dentine–Pulp Complex Regeneration���������������������������������������������������� 35
Ove A. Peters, Avina Paranjpe, and Alexis Gaudin

Clinical Approach to Regenerative Endodontics���������������������������������� 63
Omid Dianat, Elham Shadmehr, and Yoo Jung Chung
Tooth Bioengineering and Whole Tooth Regeneration������������������������ 89
Ning Cheng, Juan Wen, Rita Hitching, Chang Lei, and Chun Xu
Regenerative Approaches in Periodontics���������������������������������������������� 103
Necla Asli Kocak Oztug, Srinivas Sulugodu Ramachandra,
Cagdas Caglar Lacin, Aya Alali, and Amelia Carr
Regeneration for Implant Dentistry������������������������������������������������������ 133
Tulio Fernandez-Medina and Ashwin Nanda
Regenerative Approaches in Orthodontic
and Orthopedic Treatment���������������������������������������������������������������������� 151
Yan He, Fernando Guastaldi, Chun Xu, and Qingsong Ye
Regenerative Approaches in Oral and Maxillofacial Surgery ������������ 171
Seied Omid Keyhan, Hamid Reza Fallahi, Behzad Cheshmi,
and Shohreh Ghasemi

Regenerative Approaches in Oral Medicine������������������������������������������ 197
Camile S. Farah, Antonio Celentano, Giuseppe Pantaleo,
Kate Shearston, Simon Fox, Naisana Seyedasli,
and Munira Xaymardan
Index���������������������������������������������������������������������������������������������������������� 265

ix
Editors and Contributors

About the Editors

Sepanta Hosseinpour is an Associate Lecturer at the School of Dentistry,

the University of Queensland, Australia. He received his doctorate in dental
surgery (DDS) and Master of Public Health (MPH) in 2017, and he pursued
academic research in tissue regeneration as Research Associate for two years
after graduation and his PhD at the University of Queensland. At the moment,
he has 34 peer-reviewed journal articles in dentistry some of which were
published in high-quality journals.

Laurence J. Walsh is an Emeritus Professor at the University of Queensland

School of Dentistry where has been on the academic staff for over 36 years.
He served for 20 years as research group leader for Advanced Materials and
Technologies at the University of Queensland School of Dentistry. He is also
the program director for the special needs dentistry Doctor of Clinical
Dentistry. He has worked in medical and dental research for over 35 years,
covering advanced aspects of material science, biomedical optics, lasers, and
nanomaterials. Professor Walsh holds patents in six families of advanced
technologies for dental diagnosis and treatment. He has led research into new
technologies, from bench studies through to clinical trials and cost-benefit
studies. His over 900 publications include over 300 refereed journal papers,
over 90 published protocols and guidelines for professional associations, over
190 published technical reports and literature reviews, 5 sole author books,
and a further 30 book chapters or edited books. In January 2018, he was
appointed an Officer of the Order of Australia (AO) in recognition of his dis-
tinguished service to dentistry, and to dental education, as an academic and
author. He is only the fourth dentist in the history of Australia to receive this
national honor.

Keyvan Moharamzadeh is a Professor of Endodontics at Hamdan Bin

Mohammed College of Dental Medicine (HBMCDM), Mohammed Bin
Rashid University of Medicine and Health Sciences (MBRU), Dubai, United
Arab Emirates (UAE). He was previously a Senior Clinical Lecturer at the
University of Sheffield and Honorary Consultant in Restorative Dentistry at
Charles Clifford Dental Hospital, UK. He is a specialist in endodontics, peri-
odontics, prosthodontics, and restorative dentistry and was the program

xi
xii Editors and Contributors

director for postgraduate DClinDent periodontology and prosthodontics

courses at the University of Sheffield. His clinical work has included both
private dental practice at Harley Street Dental Group in London and
­hospital-­based treatment of patients referred by General Dental Practitioners
for advanced restorative rehabilitation. He has authored a clinical reference
book on Diseases and Conditions in Dentistry and coedited a textbook on
Biomaterials for Oral and Dental Tissue Engineering. Professor
Moharamzadeh is a Fellow of the Royal College of Surgeons of England and
the UK Higher Education Academy. He has held an honorary research posi-
tion in Marquette University in the United States, has extensively published
in the literature, and has given many presentations and invited lectures in the
national and international conferences.

Contributors

Aya Alali, BDS, MPhil School of Dentistry, The University of Queensland,

Herston, QLD, Australia
Amelia Carr, BEng (Med) (Hons) School of Dentistry, The University of
Queensland, Herston, QLD, Australia
Antonio Celentano, DDS, LDS, GCUT, PhD Melbourne Dental School,
The University of Melbourne, Carlton, VIC, Australia
Ning Cheng, BDS, DDS, PhD Department of Otolaryngology-Head and
Neck Surgery, University of California San Francisco, San Francisco, CA,
USA
Behzad Cheshmi, DDS, MSc Private Practitioner, Professional Member of
Maxillofacial Surgery and Implantology Research Foundation, Boroujerd,
Iran
Yoo Jung Chung, DDS University of California San Francisco School of
Dentistry, San Francisco, CA, USA
Omid Dianat, DDS, MS, MDS Endodontic Division, University of
Maryland, School of Dentistry, Baltimore, MD, USA
Hamid Reza Fallahi, DDS, MSc Dental Research Center, Research Institute
of Dental Sciences, Shahid Beheshti University of Medical Science, Tehran,
Iran
Oral and Maxillofacial Surgeon, Founder and Director of Maxillofacial
Surgery and Implantology Research Foundation, Ahvaz, Iran
Camile S. Farah, BDSc, MDSc (OralMed OralPath) Australian Centre
for Oral Oncology Research and Education, Nedlands, WA, Australia
Australian Clinical Labs, Subiaco, WA, Australia
Simon Fox, PhD Systems Biology and Genomics, Harry Perkins Institute of
Medical Research, Nedlands, WA, Australia
Editors and Contributors xiii

Alexis Gaudin, DDS, PhD Faculty of Dental Surgery, Department of

Endodontics and Restorative Dentistry, University of Nantes, Nantes, France
Shohreh Ghasemi, DDS, MSc Adjunct Assistant Professor of Oral and
Maxillofacial Surgery of DCG, Georgia, USA
Fernando Guastaldi, PhD Skeletal Biology Research Center, Massachusetts
General Hospital and Harvard School of Dental Medicine, Boston, MA, USA
Yan He, PhD, MDS (Ortho.), DDS Skeletal Biology Research Center,
Massachusetts General Hospital and Harvard School of Dental Medicine,
Boston, MA, USA
Institute of Stem Cells and Tissue Engineering, Wenzhou Medical University,
Wenzhou, China
Rita Hitching, MSc Palo Alto Veterans’ Institute for Research (PAVIR), VA
Palo Alto Health Care System, Palo Alto, CA, USA
Sepanta Hosseinpour, DDS, MPH, PhD candidate School of Dentistry,
The University of Queensland, Herston, QLD, Australia
Seied Omid Keyhan, DDS, MSc Oral and Maxillofacial Surgeon, Founder
and Director of Maxillofacial Surgery and Implantology Research
Foundation, Isfahan, Iran
Cagdas Caglar Lacin, DDS, PhD Department of Periodontology, Faculty
of Dentistry, Istanbul Kent University, Istanbul, Turkey
Chang Lei, BDS, MDS, PhD Australian Institute for Bioengineering and
Nanotechnology, The University of Queensland, Brisbane, QLD, Australia
Tulio Fernandez-Medina, DDS, DClin Dent, PhD School of Dentistry,
The University of Queensland, Herston, QLD, Australia
Keyvan Moharamzadeh, BSc, DDS, PhD, FHEA, FDSRCS Hamdan Bin
Mohammed College of Dental Medicine (HBMCDM), Mohammed Bin
Rashid University of Medicine and Health Sciences (MBRU), Dubai, United
Arab Emirates
Ashwin Nanda, BDS, MDS (Prosthodontics), MPhil School of Dentistry,
The University of Queensland, Herston, QLD, Australia
Necla Asli Kocak Oztug, DDS, PhD Department of Periodontology, Faculty
of Dentistry, Istanbul University, Istanbul, Turkey
School of Dentistry, The University of Queensland, Herston, QLD, Australia
Giuseppe Pantaleo, PhD Oral Surgery, Department of Medicine and
Surgery, University of Salerno, Fisciano, Italy
Avina Paranjpe, BDS, MS, MSD, PhD Department of Endodontics,
University of Washington, School of Dentistry, Seattle, WA, USA
Ove A. Peters, DMD, MS, PhD School of Dentistry, The University of
Queensland, Herston, QLD, Australia
xiv Editors and Contributors

Maryam Rezai Rad, DDS, PhD Research Institute of Dental Sciences,

Dental school, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Srinivas Sulugodu Ramachandra, BDS, MDS School of Dentistry, The
University of Queensland, Herston, QLD, Australia
Elham Shadmehr, DDS, MS UCSF Endodontic Division, University of
California San Francisco School of Dentistry, San Francisco, CA, USA
Kate Shearston, BA/BSc (Hons), PhD UWA Dental School, University of
Western Australia, Nedlands, WA, Australia
Naisana Seyedasli, PhD Faculty of Medicine and Health, University of
Sydney, and Centre for Cancer Research, Westmead Institute for Medical
Research, Westmead, NSW, Australia
Laurence J. Walsh, AO BDSc (Hons), PhD, DDSc, GCEd School of
Dentistry, The University of Queensland, Herston, QLD, Australia
Juan Wen, BDS, MDS (Orthodontics) Department of Orthodontics,
Nanjing Stomatological Hospital, Medical School of Nanjing University,
Nanjing, Jiang Su, China
Munira Xaymardan, BDS, MPhil (Oral Surgery), PhD Faculty of
Medicine and Health, Oral Biosciences, Sydney Dental School, University of
Sydney, Westmead, NSW, Australia
Chun Xu, BDS, MDS (Oral Surgery), PhD School of Dentistry, The
University of Queensland, Herston, QLD, Australia
Shaomian Yao, PhD Department of Comparative Biomedical Sciences,
School of Veterinary Medicine, Louisiana State University, LA, USA
Qingsong Ye, DDS, MOrth, PhD, FICD Skeletal Biology Research Center,
Massachusetts General Hospital and Harvard School of Dental Medicine,
Boston, MA, USA
Center of Regenerative Medicine, Wuhan University, Wuhan, China
Institute of Stem Cells and Tissue Engineering, Wenzhou Medical University,
Wenzhou, China
 The Paradigm of Regenerative
Dentistry and Its Future
Perspectives

Laurence J. Walsh and Sepanta Hosseinpour

1 Regenerative Approaches This makes dental enamel a unique tissue in that

for Treatment it sits at the boundary of hard and soft tissues and
must rely entirely on chemical repair by reminer-
In the human body, there is a continuous renewal alization from ions in the saliva.
of tissues such as bone marrow, epithelia, bone, In the case of bone, the size and shape of
and connective tissue. The potential for regenera- defects created by trauma could be beyond the
tion after injury varies greatly, from tissues that repair potential of the body. Such “critical-sized”
have excellent capabilities for complete regener- bone defects will not heal spontaneously, how-
ation, such as the liver, through to sensory cells ever, in such situations, therapeutic intervention
that give the special senses of hearing and vision, can help the cells to “generate again.” In order to
which do not regenerate when injured. achieve this, an inductive material and scaffold
When considering the response to inflamma- can be used to improve the homing of endoge-
tory diseases, trauma, or malignancy, the pre- nous bone-forming cells and their subsequent
ferred outcome is always true regeneration, rather differentiation. Alternatively, stem cells could be
than repair with scarring. How much any one tis- transplanted into the treatment site [1]. Such ther-
sue can undergo repair, or truly regenerate, is apeutic approaches exploit the principles of tis-
influenced by the type and number of cells that sue engineering to restore the function and
are present, particularly stem cells, which can structure of a specific tissue or organ [2].
differentiate to replace missing tissues. In some Regenerative dentistry is the branch of regen-
sites in the oral cavity, the number of cells with a erative medicine that focuses on regeneration of
high regenerative capacity is limited because of oral and dental tissues. It most often follows the
the small volume of the tissue, e.g., in the dental conceptual triad of tissue engineering, by includ-
pulp, as the pulp chamber reduces in size with ing cells, scaffolds, and bioactive molecules [3].
age to a volume of tens of microliters. The cells could come from any number of stem
Enamel is a unique tissue since the forming cell sources that have been identified, including
cells, the ameloblasts, are no longer present by embryonic stem cells, adult stem cells, and
the time the tooth is erupting into the oral cavity. induced pluripotent stem cells. The choice of cell
type is aligned with the treatment objectives.
L. J. Walsh (*) · S. Hosseinpour Scaffolds can be designed to carry appropriate
School of Dentistry, The University of Queensland, cells, and to deliver signaling molecules to
Herston, QLD, Australia orchestrate tissue healing [4]. In addition, scaf-
e-mail: [email protected];
folds can be used as a carrier or support for cul-
[email protected]

© Springer Nature Switzerland AG 2021 1

S. Hosseinpour et al. (eds.), Regenerative Approaches in Dentistry,
https://doi.org/10.1007/978-3-030-59809-9_1
2 L. J. Walsh and S. Hosseinpour

turing cells ex vivo before the differentiated epithelium). All of these stem cell populations
tissue is transplanted surgically into the defect can be harvested and used in regenerative den-
site. Bioactive molecules, such as growth factors, tistry. They have properties that are broadly simi-
genes, and drugs, can be released by the scaffold, lar to bone marrow-derived mesenchymal cells.
or delivered independently [5]. Once a tooth has formed and then erupted,
It is important that the scaffold mimics the its durability and longevity in the oral cavity
characteristics of the target tissue in terms of bio- can be influenced by many processes includ-
logical activity, mechanical integrity, and func- ing dental caries, periodontal diseases, acceler-
tionality [6, 7]. To achieve this, the optimum ated tooth wear, and traumatic injuries. In oral
design for regenerative treatments will vary, rehabilitation, common methods for replacing a
based on the target tissue. For instance, the tooth that is missing or has been lost include a
requirements of a scaffold for pulpal tissue regen- fixed or removable prosthesis, or a single tooth
eration are totally different from the one used for dental implant that supports a full dental crown
alveolar bone augmentation for dental implant [10]. However, by applying the evolving knowl-
placement. The features must meet the regenera- edge of tooth development and stem cell biology,
tive demands as defined from the target site, and the concept of bioengineered teeth has emerged.
then be optimized to achieve the best outcomes. Although tooth-like structures have been created
successfully in animal models [11–13], the com-
plexity of any one tooth presents a major chal-
2 Therapeutic Targets lenge, because of variables such as tooth type,
in the Field of Dentistry size, color, and occlusal anatomy. These distinct
morphological and functional characteristics pro-
2.1 The Whole Tooth vide a major challenge that causes more complex-
ity in design than replacement of other structures
In humans, tooth development for the primary such as bone. In chapter “Tooth Bioengineering
(deciduous) dentition starts approximately at and Whole Tooth Regeneration,” the most recent
6–8 weeks in utero. The tooth forming organ, the findings regarding this topic will be discussed.
tooth germ or dental follicle, is comprised of an
outer layer of cells from ectoderm (oral epithelium)
and an inner layer of neural crest ectomesenchyme. 2.2 Individual Dental Structures
In the early stages, odontogenic inducing signals
pass from the epithelial cells to the mesenchymal Dental caries is a global public health problem,
cells, which condense to become the dental papilla. and it remains one of the most prevalent micro-
This sequence of tooth formation, which spans the bial diseases around the world [14]. Dental caries
stages from a tooth germ to a completely formed leads to demineralization and proteolytic destruc-
tooth, involves a complex orchestration of cellular tion of the crowns and exposed root surfaces of
activity, with cascades of cytokines and enzymes teeth, with the enamel and dentin being cavitated
that tightly regulate cell arrangement, proliferation, by the action of an aciduric, acidogenic, polymi-
differentiation, and secretion over a period of sev- crobial dental plaque biofilm. If untreated, the
eral years [8, 9]. invasion can reach the dental pulp, causing irre-
As the tooth forms, a range of stem cells par- versible pulpitis and finally necrosis of that
ticipate, including those in the outermost regions tissue.
of the dental follicle where the tooth is forming The current approach to treatment of a cavi-
(dental follicle stem cells), through to stem cells tated tooth is the removal of infected dental tis-
that remain after tooth formation is complete sues, followed by restoration of missing tooth
(e.g., dental pulp stem cells, periodontal ligament structure with synthetic dental materials [15].
stem cells, stem cells from the apical region of However, this method can best be considered
the dental papilla, and stem cells in the gingival a surgical approach involving excision of the
The Paradigm of Regenerative Dentistry and Its Future Perspectives 3

affected area followed by a small extent of repair exceed periods of demineralization during bio-
at the level of the dental pulp. The strategy is film acid production, the surface will be main-
not ideal, however once a tooth surface cavi- tained because of this chemical regeneration
tates, its complete regeneration is not possible. process.
Consequently, promoting healing of affected On the other hand, if cycles of mineral loss
dentin and reversal of incipient lesions of dental dominate, the surface will lose so much mineral
caries that have not yet cavitated are important that its integrity will be compromised. It will
goals of preventive and minimal intervention eventually collapse, and the resulting cavity that
dentistry, so that the tooth is retained as a vital, forms then provides a protected site for the
biologically functional unit. A range of novel microbial biofilm to continue its destruction of
approaches have been used to achieve reversal the tooth, now being located in a more protected
and arrest of early carious lesions where the tooth site. Many dental preventive strategies have been
surface is still intact. developed to tip the balance to favor remineral-
ization over demineralization, and thus break the
2.2.1 Enamel cycle of progressive mineral loss. Regrettably,
Enamel is the hardest tissue in the body due to its once a cavity has formed, the options for arrest-
unique structural properties, its high content of ing the process are far fewer, and focus mostly on
biological apatite minerals (such as hydroxyapa- silver fluoride used in combination with ammo-
tite), and the structural arrangement of enamel nia or with stannous fluoride. These topical treat-
rods and prisms which resists the application of ments can prevent the destruction caused by
external forces [16]. The ameloblasts which form dental caries from progressing further, but they
enamel are highly specialized epithelial cells, and cannot repair the missing tooth structure [18].
only exist during tooth development. They are They also cause dramatic discoloration of the
lost once the tooth crown has fully formed, which tooth.
is some months before the tooth erupts into the Given these limitations, there is interest in the
oral cavity [17]. concept of true regeneration of tooth structure.
Although enamel should be able to withstand For white spot enamel lesions and incipient
the intense forces of mastication during the whole lesions on root surfaces, providing the correct
lifespan, because of the ubiquitous presence of a stoichiometric ratios of calcium, phosphate and
dental plaque biofilm, it is vulnerable to cycles of fluoride ions (5:3:1) can arrest lesions and cause
demineralization. These acid attacks could be subsurface regeneration of mineral, back to nor-
from organic acids produced by a dysbiotic den- mal levels. Casein phosphopeptide-amorphous
tal plaque biofilm that has formed in a low pH calcium phosphate provides such a ratio of ions,
environment, or from inorganic or organic acids releasing these under acidic conditions to drive
found in foods and drinks, or in regurgitated gas- remineralization, but stabilizing these same ions
tric acid contents, as occurs in dental erosion. under alkaline pH conditions.
The stronger the acid and the better it chelates Once a cavity has formed on the crown or
calcium ions, the more rapid crystallites of apa- root surface of a tooth, an operative or restorative
tite mineral on the enamel surface will dissolve approach has been the mainstay of treatment for
into the saliva. many decades. Using techniques that conserve
Initial lesions of dental caries that are still tooth structure and also encourage healing of
contained in the outer half of the enamel are the inner affected dentin is now commonplace.
referred to as white spot lesions because of their A number of biomimetic dental materials (such
unique appearance. When there are sufficient as glass ionomer cements) can have powerful
bioavailable calcium and phosphate ions, these influences on the healing of dentin, and some
lesions can remineralize. The likelihood of this dental materials that are used in deep cavities as
occurring is increased when low levels of fluoride liners (such as alkaline bioceramic cements) are
ions are present. If cycles of remineralization highly antimicrobial. Both material types lack
4 L. J. Walsh and S. Hosseinpour

the physical properties (such as high compres- sues are exposed to chemical and microbial
sive strength) required for large restorations in assaults from oral microorganisms, and this leads
high stress-­bearing areas (such as occlusal sur- to an inflammatory response within the pulp tis-
faces of permanent molar teeth), hence both are sue, and finally to its necrosis.
typically used as a base or foundation, and then Removal of the inflamed/necrotic tissues,
overlaid with tooth-colored materials (such as debridement of the pulp chamber and root canal
resin composites, ormocers, and ceramics) that system, and obturation of the space with root fill-
are inert, from a purely chemical and biological ing materials is the usual method of anterograde
perspective. endodontic treatment. Rather than removing the
There have been attempts to synthesize human pulpal soft tissue, an option that exists in roots
dental enamel or enamel-like materials by chemi- with a partially open apex where there is good
cal reactions using solutions that could be applied blood flow, is regenerative endodontics. With this
topically onto cavities in teeth. Recently, a num- method, the objective is dentine-pulp regenera-
ber of chemical strategies for forming biological tion, and preservation of the vitality of the tooth.
apatites or enamel-like materials in vitro have Several groups have been working on this con-
been studied. Some positive results have been cept, and it has been used in clinical practice in
described for fluorapatite/phosphoric acid pastes recent years [23–25]. Chapters “Dentin Pulp
[19] and for amelogenin-induced hydroxyapatite Complex Regeneration” and “Clinical Approach
[20] (see chapter “Tooth Bioengineering and to Regenerative Endodontics” will explain regen-
Whole Tooth Regeneration”). A major challenge erative endodontics in detail.
with such approaches is that the material formed
on the tooth is relatively thin and lacks the com-
plex reinforced prismatic microstructure of natu- 2.3 Periodontium
ral dental enamel. This makes treating large or
extensive lesions a major challenge. Periodontal diseases and occlusal trauma can
adversely affect the supporting apparatus for
2.2.2 The Dentine-Pulp Complex teeth, known as the periodontium. This encom-
The close interaction between dental pulp tissue passes the periodontal ligament (PDL), cemen-
and surrounding hard tissue (dentine) creates a tum on the root surfaces of teeth, the alveolar
functional unit known as the “dentine-pulp com- bone, and the gingiva. In a healthy PDL, collag-
plex.” This complex arises embryonically from enous fibers from the cementum extend to the
ectomesenchymal cells [21]. The odontoblastic alveolar bone, and anchoring the root of the tooth
cellular layer differentiates in the “bell” stage of into its socket. Plaque accumulation from poor
tooth development, and it secretes dentine as a oral hygiene elicits inflammation, and in suscep-
distinct extracellular matrix (primary dentine). tible individuals, the host inflammatory response
Once the tooth has formed fully, during the causes destruction of the attachment and the alve-
remainder of the lifespan, odontoblasts continue olar bone, in bursts of varying duration. The loss
their synthetic function, but at a greatly reduced of attachment and bone can be so severe that the
pace, forming secondary dentine. tooth is lost as a result.
In response to environmental stimuli such as Regeneration of the periodontium is a chal-
bacterial invasion and trauma, odontoblasts react lenging task. Not only is this a complex tissue
and form reparative dentine, to attempt to wall off with multiple elements, the local environment has
the dental pulp from the noxious stimulus [22]. a high level of microorganisms and the patient
Despite these efforts, the rate or amount of has already shown an inappropriate host immune
destruction is often far greater than the ability of reaction to those microorganisms. Current
the odontoblasts to lay down a sufficient amount treatment modalities for periodontitis include
of protective dentine. Once the defensive line of debridement of teeth, and similar approaches
the dentine has been breached, the pulpal soft tis- are also used for dental implants. Regenerative
The Paradigm of Regenerative Dentistry and Its Future Perspectives 5

surgical procedures are used for advanced cases 2.5 Oral Pathology
[26, 27], with guided tissue regeneration (GTR)
considered the current “gold standard” for treat- The application of stem cells for the treatment of
ment [28]. In this procedure, a barrier membrane oral lesions and conditions is a relatively new con-
is placed at the site against the root surface or cept. Examples include submucosal injection of
implant surface, to support soft tissue regenera- mesenchymal stem cells to promote healing of oral
tion (on its outer surface) and bone regeneration ulcers [34]. Immunomodulatory properties of stem
(on its inner surface). The membrane excludes cells may have a therapeutic benefit for oral vesicu-
invasion of the bony defect by rapidly migrat- lobullous lesions [35, 36]. Moreover, stem cells
ing epithelial cells or fibroblasts. This topic is could also be a delivery vehicle for therapeutic
covered in chapter “Regenerative Approaches in agents, for example, in the setting of treating malig-
Periodontics.” nant lesions [37]. This topic is discussed in the chap-
ter “Regenerative Approaches in Oral Medicine.”

2.4 Bone Defects

3 Dental Tissue-Derived Stem
In oral and maxillofacial surgery, the treatment Cells
of bone defects caused by trauma, infections, or
malignancies is a significant challenge. Bone has Bone marrow mesenchymal stem cell (BMSC)
a considerable capability for self-renewal [29], are the best known and characterized cells used
and as a tissue it must not only provide structural in tissue engineering [38]; however, harvesting
support and protection, but also serve several them from bone is invasive, and there are issues
endocrine and hemopoietic functions [30]. While with age-related differentiative potency [39, 40],
healthy normal bone can respond to strong bio- which impede their usefulness. As a result, there
mechanical forces as well as daily micro-­ has been interest in alternative tissue origins of
damage, around 5–10% of bone fractures and stem cells, such as skeletal muscles [41], dental
most critical-­sized bone defects are caused by tissues [28, 42–44], and adipose tissues [45–47].
trauma, pathology, or congenital malformations, Dental tissues could be a convenient and acces-
do not heal fully despite timely clinical interven- sible source of stem cells. Teeth are extracted due
tions [31]. to various reasons (including as part of orthodon-
In clinical practice, the standard therapeutic tic treatment), and deciduous teeth exfoliate nat-
modality for treating large bony defects is bone urally when the erupting permanent teeth cause
grafting, using autogenous or allogenic bone their roots to resorb. Dental stem cells (DSCs)
grafts. These grafts provide osteoconduction and have demonstrated multipotential differentiation,
induction at the same time. Over 900,000 surgi- being able to differentiate and proliferate to form
cal procedures involving bone grafts are under- osteogenic, odontogenic, neurogenic, and adipo-
taken each year in the United States, and globally genic cell types [48, 49]. DSCs are highly effec-
bone grafting surgery accounts for annual health- tive at forming odontogenic structures, but can
care costs of some $30 billion [32], with bone also form osseous tissues [50]. The precise differ-
being only second to blood as the most com- ences between BMSCs and among the different
monly transplanted tissue [32]. When harvesting types of DSCs remain to be explained fully. DSCs
bone autografts, a significant issue is morbidity are derived from the neural crest (ectomesenchy-
of the donor site, which is a second surgical site mal origin) [51], and so are superior for regen-
in the same patient [33]. In chapters “Regenerative erating a wide variety of tissues [52], compared
Approaches in Periodontics” and “Regenerative to BMMSCs that originate from mesoderm [53].
Approaches in Oral and Maxillofacial Surgery,” DSCs and their regenerative potential will be dis-
current approaches for bone augmentation in cussed in the chapter “Dental Tissues Originated
dental settings will be described. Stem Cells for Tissue Regeneration.”
6 L. J. Walsh and S. Hosseinpour

4 Conclusions and Future 3. Bartold PM, Gronthos S, Ivanovski S, Fisher A,

Hutmacher DW. Tissue engineered periodontal prod-
Direction ucts. J Periodontal Res. 2016;51(1):1–15. https://doi.
org/10.1111/jre.12275.
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cal dental practice rely on restorative materi- M, Motamedian SR, Khojasteh A. Application
of selected scaffolds for bone tissue engineer-
als and prostheses to replace lost tissue loss, ing: a systematic review. Oral Maxillofac Surg.
there is growing interest in using regenerative 2017;21(2):109–29.
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and tissue engineering. Crit Rev Oral Biol Med.
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important to better understand and control the lenges and opportunities for biomedical material
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Odontogenic capability: bone marrow stromal
 Dental Tissues Originated Stem
Cells for Tissue Regeneration

Maryam Rezai Rad, Sepanta Hosseinpour,

Qingsong Ye, and Shaomian Yao

1 Introduction their usefulness. Therefore, several alternative

tissue origins for stem cells have been explored
Stem cell-based therapy as a major field in regen- including skeletal muscle [5], dental tissues [6–
erative medicine has attracted scientists in the 9], and adipose tissue [10–12].
field of tissue engineering [1]. Stem cells pos- In clinical dentistry, teeth are extracted due to
sess a remarkable capability for proliferation and various reasons, such as impacted third molars for
for differentiation into various cell types, and orthodontic reasons. Collecting these extracted
such capabilities can be useful for regenerative teeth does not require additional procedures. In
therapies. Nowadays, although multipotent mes- addition, deciduous human teeth naturally exfoli-
enchymal stem cells derived from bone marrow ate. Hence, harvesting MSCs from dental tissues
(BMMSC) are among the best-known and best-­ of such exfoliated teeth is convenient. Dental
characterized cells for tissue engineering [2], the stem cells (DSCs) have demonstrated the capa-
invasive procedure needed to isolate these from bility to differentiate into various cell lineages,
bone marrow, and variations in their potency including osteogenic, odontogenic, neurogenic,
according to the age of the donor [3, 4] impede and adipogenic pathways [13, 14].
Although because of their origin DSCs seem
M. Rezai Rad to be more efficient for developing odontogenic
Research Institute of Dental Sciences, Dental school structures than other osseous tissues when com-
Shahid Beheshti University of Medical Sciences, pared with BMMSCs [15], the precise differ-
Tehran, Iran ences between DSCs and BMMSCs, and among
S. Hosseinpour each of the different types of DSCs remain
School of Dentistry, The University of Queensland, unclear. These cells are derived from the neural
Brisbane, QLD, Australia
crest (i.e., they have an ectomesenchymal origin)
Q. Ye [16], which gives them a superior capability for
Skeletal Biology Research Center, Massachusetts
General Hospital and Harvard School of Dental regenerating a wide variety of tissues [17], when
Medicine, Boston, MA, USA compared to BMMSCs that originate from meso-
Institute of Stem Cells and Tissue Engineering, derm [18]. Adding to this greater usefulness,
Wenzhou Medical University, Wenzhou, China DSCs can be obtained without any major ethical
S. Yao (*) concerns [19, 20].
Department of Comparative Biomedical Sciences, The first report of MSCs in the dental pulp was
School of Veterinary Medicine, Louisiana State in 1985 by Yamamura [21, 22]. To date, five main
University, LA, USA types of dental tissue-derived stem cells have been
e-mail: [email protected]

© Springer Nature Switzerland AG 2021 9

S. Hosseinpour et al. (eds.), Regenerative Approaches in Dentistry,
https://doi.org/10.1007/978-3-030-59809-9_2
10 M. Rezai Rad et al.

reported. The first type was termed as “postnatal Figure 1 schematically shows the potential
dental pulp stem cell” (DPSC) by Gronthos et al. sources of these various dental tissue-derived
[23] in 2000. These can regenerate a “dentine– stem cells. Although these DSCs have been
pulp complex like” tissue in vitro. Subsequently, investigated in many studies [13, 14, 30, 31], only
in 2003, Miura et al. reported the isolation and limited work has been done to characterize their
characterization of stem cells from exfoliated biological properties and compared their in vivo
deciduous teeth which had proliferated from applications. In this chapter, we have compiled
remnants of living DPSCs. These are known as information regarding the isolation, characteriza-
stem cells from human exfoliated deciduous teeth tion, and potential applications of DSCs in tissue
(SHED) [24]. Morsczeck et al. [25] and Kemoun regeneration and tissue engineering.
et al. [26] reported undifferentiated mesenchymal
progenitor cells from the human dental follicle, in
the connective tissue sac surrounding developing 2  tem Cells Obtained
S
teeth. They named these dental follicle stem cells from Human Permanent
(DFSCs). The dental follicle forms the tooth and Teeth or Exfoliated
the supporting structures, and the follicle devel- Deciduous Teeth
ops into the periodontal ligament when tooth is
erupting. Stem cells have also been reported as As mentioned earlier, the first isolated DSCs
being present in the periodontal ligament [27]. In were dental pulp stem cells (DPSCs). DPSCs
2006, Sonoyama et al. described unique undiffer- were isolated initially from permanent third
entiated stem cells from the dental apical papilla molar teeth. They demonstrated colony forma-
of human immature permanent teeth (SCAP) with tion and a high proliferation rate, as well as the
the ability to generate osteoblasts, odontoblasts, ability to form calcified nodules [23]. DPSCs
and adipocytes in vitro [28, 29]. derived from impacted third molars at the stage

Dental pulp stem cell Periodontal ligament

stem cell
Stem cell from exfoliated
deciduous teeth
Dental follicle stem cell

Stem cell from the apical papilla

Fig. 1 Schematic view of various sources of stem cells from dental tissues
Dental Tissues Originated Stem Cells for Tissue Regeneration 11

of root development can differentiate into active that can be preserved for future applications [24].
migratory odontoblast-like cells, which are able These cells differ from regular DPSCs because of
to produce a three-dimensional dentine-like min- their greater proliferation rate and enhanced pop-
eralized structure [32]. The stem cell properties ulation doubling rate, and their capability to form
of DPSCs vary according to the donor’s age when sphere-like cell clusters [34]. SHED express the
the specimens are obtained (Nakamura et al. minimum essential markers for stem cells defined
2009); i.e., the cells show a reduction in stem cell by the International Society for Cellular Therapy
features as donor age is increased. criteria [15, 35]. They also express some embry-
The transition of deciduous teeth in the pri- onic stem cell markers [24], as listed in Table 1.
mary dentition to the permanent dentition is a While SHED have been isolated using simi-
dynamic and distinctive process, in which the lar methods to those used to isolate DPSCs,
developing permanent teeth buds gradually there are two major differences between SHED
resorb the roots of the overlying deciduous teeth and DPSCs. Firstly, the source of SHED is the
[33]. As mentioned above, a unique population of dental pulp tissue of deciduous teeth, rather than
DSCs can be harvested from the remaining liv- permanent teeth. Secondly, the isolated cells do
ing pulpal cells of exfoliated teeth, and cultivated not grow as proliferative cells, but instead grow
in the laboratory. These cells, known as SHED, as clustered fibroblast-like cells [36]. It has been
provide an easily accessible source of stem cells shown that 69.8% of SHED are in the S and G2

Table 1 Sources, surface markers, multipotentiality, and in vivo applications of dental stem cellsa
Stem cell Surface markers for characterization In vitro Regenerative
type Source Positive Negative multipotentiality in vivo application
Dental pulp Pulpal tissue of ALP, CD 9, CD 10, CD 11b, CD 1. Odontogenic • Bone
stem cell crown/root of CD 13, CD 29, CD 14, CD 19, 2. Osteogenic regeneration
(DPSC) 1. Immature teeth that 44, CD 59, CD 73, CD 24, CD 3. Chondrogenic • Dentin-pulp
their pulp is exposed CD 90, CD 105, CD 31, CD 34, 4. Neurogenic complex
and accessible 146, CD 166, CD CD 45, CD 5. Adipogenic regeneration,
through the root 271, DSPP, DMP1, 117, CD 133, 6. Myogenic regenerative
2. Extracted permanent OPN, BSP, BBX, HLA-DR 7. Melanogenic endodontics,
or deciduous teeth; nestin, Oct4, 8. Endothelial and dentin
dental pulp STRO-1 differentiation regeneration
extraction is 9. Hepatogenic • Periodontal
accomplished regeneration
through the dental • Cornea
crown by cutting the regeneration
cementum–enamel • Angiogenic
junction using regeneration
dental instruments and blood vessel
3. Carious teeth that its • Neural
pulp exposed and reconstruction
removed during • Hair follicle
endodontic repair
treatments
Stem cell Pulpal tissue of crown/ CD 13, CD 29, CD CD 11b, CD 1. Odontogenic • Dentin–pulp
from human root of exfoliated 31, CD 44, CD 73, 14, CD 19, 2. Osteogenic complex and
exfoliated deciduous teeth CD 90, CD 105, CD CD 45, CD 3. Chondrogenic regenerative
deciduous 146, CD 166, nestin, 34, CD 43, 4. Neurogenic endodontics
teeth (SHED) Oct4, STRO-1 CD 45 5. Adipogenic • Cornea
6. Myogenic regeneration
• Hepatocytes
regeneration
(continued)
12 M. Rezai Rad et al.

Table 1 (continued)
Stem cell Surface markers for characterization In vitro Regenerative
type Source Positive Negative multipotentiality
in vivo application
Stem cell Apical papilla of ALP, CD 13, CD CD 14, CD 1. Odontogenic • Dentin–pulp
from apical immature root in 24b, CD 29, CD 44, 18, CD 34, 2. Osteogenic complex
papilla unerupted teeth, CD 53, CD 59, CD CD 45, CD 3. Chondrogenic regeneration
(SCAP) especially impacted 61, CD 73, CD 90, 117, CD 150 4. Neurogenic • Pulpal tissue
third molars CD 105, CD 106, 5. Adipogenic regeneration
CD 146, CD 166, 6. Angiogenic • Neural
nestin, STRO-1 7. Hepatogenic regeneration
• Angiogenic
regeneration
Dental CD 9, CD 10, CD
Dental follicle tissue CD 31, CD 1. Osteogenic • Bone
follicle stem which surrounded the 13, CD 29, CD 44, 34, CD 45, 2. Cementogenic regeneration
cell (DFSC) crown of unerupted CD 53, CD 59, CD CD 133, 3. Chondrogenic • Periodontal
teeth 73, CD 90, CD 105, HLA-DR 4. Neurogenic tissue
CD 106, CD 146, 5. Adipogenic regeneration
CD 166, CD 271, 6. Hepatogenic
nestin, notch-1,
STRO-1
Periodontal Soft connective tissue ALP, CD 10, CD 13, CD 11b, CD 1. Osteogenic • Periodontal
ligament stem which surrounded the CD 29, CD 44, CD 14, CD 34, 2. Cementogenic regeneration
cell (PDLSC) root surface of the 49c, CD 59, CD 73, CD 40, CD 3. Chondrogenic • Angiogenic
tooth between alveolar CD 90, CD 97, CD 45, CD 80, 4. Neurogenic regeneration
bone proper and 105, CD 106, CD CD 86, CD 5. Adipogenic and blood vessel
cementum 146, CD 166, 106, 6. Pancreatic regeneration
SSEA-3 and 4, HLA-DR islet cell • Bone
STRO-1, HLA-A, 7. Endothelial regeneration
HLA-B, HLA-C, differentiation • Tendon and
Oct4, Nanog, cartilage
Scleraxis, Sox2, regeneration
STRO-1
Abbreviations: ALP alkaline phosphatase, BBX bobby sox homolog, BSP, bone sialoprotein, CD cluster differentiation,
DMP dentin matrix protein, DSPP dentin sialophosphoprotein, HLA-DR human leukocyte antigen D related, Oct
octamer-binding transcription factor, OPN osteopontin, PDL periodontal ligament, STRO stromal precursor antigen
a
References were quoted in specific paragraphs in the text
b
Specific marker in comparison to other dental stem cells

stages of the cell cycle; however, 56% of DPSCs tooth) or an outgrowth of a tissue explant [38] are
are in those cell cycle phases. This explains the the major approaches that have been used for
different proliferative capacity of these cells [37]. DPSCs and SHED. Enzymatic digestion usually
In the ensuing sections, the isolation, character- involves placing the source tissues into an appro-
ization, and various applications of DPSCs and priate mixture of enzymes such as collagenase I
SHED will be discussed. and dispase for 30–60 min at 37 °C, to break the
tissue down in order to obtain single-cell suspen-
sions. The tissue remnants are filtered through a
2.1 Isolation and Characterization sieve or strainer. This is followed by colony-­
based cultivation of the stem cells, or by cell sort-
There are many approaches to the collection and ing using magnetic or fluorescent markers [39].
isolation of DPSCs. A key consideration is to On the other hand, in the outgrowth method,
maintain the quality and safety of the isolated the harvested tissue is diced into 1–2 mm pieces
cells. Generally, enzymatic digestion of related and placed into culture plates, to allow outgrowth
tissues (such as the dental pulp of an adult tooth of cells from the tissue pieces [40]. Comparing
or the remaining dental pulp of an exfoliated these two methods of isolation, several studies
Dental Tissues Originated Stem Cells for Tissue Regeneration 13

have shown that DPSCs isolated by enzymatic proliferate in sphere-forming culture systems
digestion have a higher proliferation rate, higher [51, 52], which are also recommended for differ-
expression of stromal precursor antigen (STRO-­ entiation of DSCs [53, 54].
1) and CD 34, and higher rates of osteogenic or Phenotypic markers expressed by DPSCs
odontogenic differentiation [41–43]. are summarized in Table 1. Expression of these
After the isolation of DPSCs and SHED, markers relates to the unique capability of these
the next step is to cultivate the cells to expand cells [28, 55]. For example, STRO-1 positive
their number to reach the amount required for cells show greater odontogenic or osteogenic
cell-­based therapy. In addition, the pathway of differentiation, while CD34 and CD117 positive
differentiation can be adjusted by the choice of DPSCs show a greater ability for cell renewal and
parameters used in the culture system [39, 44, 45]. for mineralization [56]. Low expression of Class
Various culture systems have been used includ- II HLA-DR surface antigen indicates that these
ing serum-free versus serum-rich culture media, cells may be immunologically privileged, which
sphere-forming culture, and co-culture systems. reduces concerns around antigenicity in the set-
Most commonly, a 10–20% concentration of fetal ting of tissue matching between the donor and the
bovine serum (FBS) is included in the culture recipient. If the cells truly are not immunogenic
media for both isolation and expansion of DPSCs then this raises the possibility of creating banks
and SHED, to accelerate cell adhesion during for DPSCs. This aspect is discussed in the last
the first stage of culture. Because of the risk of section of this chapter.
contamination of serum by bovine pathogens The phenotype of SHED differs from that of
(and the presence of bacterial endotoxin) and an DPSCs. Pluripotency markers such as Pou5f1,
altered possibility of malignant transformation of Sox2, Oct3/Oct4, and Nanog are expressed more
the stem cells in a serum-­containing culture sys- strongly in SHED than in DPSCs [57]. Nestin
tem, the use of a chemically defined serum-free as a neuroepithelial marker is expressed less in
culture system has been recommended [46–49]. SHED cells compared to DPSCs [58], which
In this regard, a serum-­free medium (Table 2) explains the reduced ability of SHED cells in
has been reported to be successful for obtaining comparison to DPSCs for neuronal regeneration
DPSCs [50]. Cells from the adherent population [58]. However, due to the greater proliferative
appeared to be more engaged in the odontoblastic capability of SHED cells, they form sphere-like
lineage than the adherent cells. The neural stem clusters in a neurogenic culture medium [24].
cells that are derived from various sources can DPSCs have shown odonto/osteogenic, neuro-
genic, and adipogenic differentiation in preclini-
cal studies [23, 59, 60] (as listed in Table 1). More
Table 2 Serum-free medium for DPSC culture recently, in vitro investigations have revealed
Medium osteogenic, chondrogenic, and myogenic differ-
composition Concentration Source entiation of DPSCs [61–63]. Similar to DPSCs,
Dulbecco’s modified SHED cells have also demonstrated the ability to
Eagle medium
(DMEM)/Ham’s F12
undergo odonto/osteogenic, chondrogenic, neu-
Glucose 33 mM rogenic, adipogenic, and myogenic differentia-
HEPES (pH 7.2) 5 mM tion [13, 14, 30, 31] (Table 1).
N2 supplement Life
Technologies,
Invitrogen
2.2 Regenerative Applications
Human EGF 10 ng/ml
Human bFGF 5 ng/mL Peprotech
Heparin solutiona 0.2% Peprotech The concept of harvesting and banking dental tis-
Streptomycin-­ 5 μg/ StemCell sue MSCs has opened up a new window for stem
penicillin mL–5 UI/mL Technologies cell-based regenerative treatments. As already
Final concentration in the medium is Heparin 5 μg/ml
a
mentioned, DPSCs and SHED can differentiate
14 M. Rezai Rad et al.

effectively to various cell lineages. In this sec- 2.2.2 Bone Regeneration

tion, the regenerative capacity of these cells will Bone formation, including the aggregation of
be discussed further. osteoprogenitor cells, is similar in some ways to
tooth bud development, but without the epithelial
2.2.1 Dentine–Pulp Complex invagination aspect. Intramembranous and endo-
Regeneration chondral bone formation are the two main pro-
Dental pulp is a specialized connective tissue, cesses of bone regeneration. In endochondral
which consists of odontoblasts, endothelial cells, regeneration, the aggregated MSCs first undergo
neurons, fibroblasts, and other cells. The peculiar chondrogenesis followed by ossification of the
internal anatomy of teeth and the unique blood cartilage into bone tissue [73]. In intramembra-
flow may influence the survival of stem cells nous bone formation, MSCs first become osteo-
[64]. When the dental pulp is infected by bacte- progenitor cells and then further differentiate into
rial pathogens, it is hard to remove or inactivate osteoblasts, that create extracellular matrix con-
these pathogens through antibiotics alone. For a taining collagen fibrils and other bone compo-
number of clinical reasons, extirpation of the nents, and this is called osteoid.
whole pulp may need to be undertaken [65]. Bone tissue possesses the intrinsic capabil-
For regeneration, dental pulp stem cells can ity of regenerating itself through adulthood [74].
be expanded in the laboratory. When seeded However, when bone defects or fractures are
onto hydroxyapatite/tricalcium phosphate (HA/ larger than the regenerative capacity of the bone,
TCP), the cells have demonstrated formation other interventions are required to rehabilitate the
of a dentine-­pulp-like complex in mice [23]. In defect site in the bone tissue [74]. One alterna-
addition, SHED implanted into mice can gen- tive is using stem cell-based therapy with stem
erate odontoblast-like cells and dentine-like cells of dental origin [75]. In vitro studies have
mineralized nodules. However, SHED cells do demonstrated the osteogenic and chondrogenic
not seem to be able to form a complete dentine- multipotency of both DPSCs and SHED [75–77].
pulp-like complex like that seen with transplan- An in vivo investigation showed that DPSCs can
tation of DPSCs [24]. In contrast, DPSCs can regenerate osteoblasts and endotheliocytes that
form mineralized nodules, which are covered by eventually formed bone in immunocompromised
a layer of dentine-like reparative tissue in vivo rats [78].
[66]. DPSCs that have been seeded onto calcium The application of SHED in combination with
phosphate [67], hexafluoropropanol silk [68], a hydroxyapatite (HA)/tricalcium phosphate
and polylactic acid [69] scaffolds have demon- scaffold has been shown to regenerate calvarial
strated dentine–pulp complex formation in ani- bone defects in animal models [79]. These stud-
mal models. ies have also shown that a large amount of bone
In addition to dentine–pulp complex regen- and bone marrow-like structures are formed in the
eration, DPSCs have also been used in periodon- regenerated mineralized matrix. Other investiga-
tal regeneration [31]. DPSCs transplanted into tions have reported that the application of DPSCs
immunocompromised mice have been shown to in conjunction with fibroin/collagen scaffolds
differentiate into collagen forming cells, with the can significantly enhance bone formation after
capacity to form a cementum-like tissue [70]. 4–8 months in rats [80–82]. The application of
The expression of particular phenotypic markers DPSCs loaded onto a HA-based hydrogel showed
such as SCD-1, STRO-1, CD44, and CD146 on superior bone healing in rat calvarial defects
DPSCs and SHED cells sensibly linked to their compared to the scaffold only and to untreated
role in periodontal regeneration [71, 72]. In addi- control groups [83]. In addition, DPSCs cultured
tion, both SHED and DPSCs are capable of bone for 13 days on poly(lactide-co-­glycolide) scaf-
regeneration [31]. This aspect will be discussed folds caused significant bone regeneration in the
further in the next section. same types of defects [84]. Other studies have
Dental Tissues Originated Stem Cells for Tissue Regeneration 15

reported the successful application of DPSCs and that systemic administration of SHED was able
SHED in combination with HA/TCP in rats and to enhance bone volume, and promote trabecular
mice in the treatment of calvarial defects [85]. number, thickness, and density [92]. In general,
The research team of d’Aquino et al. demon- SHED transplantation has been found to improve
strated that administration of DPSCs loaded onto cortical bone parameters, including bone area,
a collagen sponge resulted in higher mineraliza- thickness, and cortical bone fraction [93]. Bone
tion after 1 month and complete bone regenera- that has been regenerated using SHED has been
tion with a large amount of cortical bone formed found to express higher levels of Runx2, alka-
after 3 months, in comparison to a scaffold only line phosphatase, and osteocalcin than controls
group [78]. Moreover, an increased level of clini- [94]. At the same time, systemic application of
cal attachment was found in the gingiva overly- SHED markedly downregulates genes associ-
ing the defect site after transplantation of DPSCs ated with bone resorption such as RANKL and
loaded onto a collagen scaffold [86]. C-terminal telopeptide, and upregulates osteo-
Several studies have investigated the use of protegrin (OPG) [79, 92, 95]. This is a powerful
DPSCs or SHED for bone regeneration in maxil- demonstration of the immunomodulatory effects
lary or mandibular bone defects [87–91]. Alkaisi of SHED.
et al. created a mandibular defect (from the first According to a recent systematic review by
premolar to mental foramen) in rabbits, and Leyendecker et al., transplantation of DPSCs into
assessed the formation of bone after applying cranial, maxillary, and mandibular bone defects
SHED without using a scaffold [87]. Radiographic gives superior regenerative outcomes compared
evaluations revealed that in SHED transplanted to controls [93]. However, in one study Annibali
animals, partial bone defects were bridged after et al. found no difference in bone regeneration
only 2. In addition, the regenerated bone in this between DPSCs and control groups [96], while
group showed a bony ridge with higher radiopac- in another study by Behnia et al. there was no
ity than insights treated with a scaffold only after difference after the application of SHED in com-
4 weeks. Finally, after 6 weeks, clear corticaliza- bination with a collagen scaffold compared to
tion and strong radiodensity were observed in a scaffold only for the treatment of mandibular
the defect gap in sites treated with SHED group. defects in dogs [97].
Histomorphometric analysis showed that the The type of scaffold material that is used as
regenerated bone was significantly higher in the the carrier for DSCs plays an important role in
SHED group compared with the scaffold control. bone repair. For example, Zhang et al. did not
Paino et al. created tissue-engineered woven find ectopic bone regeneration in DPSCs loaded
bone tissue using DPSCs in vitro, and then on HA/TCP [98]. However, Kuo et al. demon-
transplanted the manufactured bone tissue into strated that DPSCs in combination with alpha-­
the mandibular defects in rats [88]. Their find- calcium sulfate hemihydrate/amorphous calcium
ings confirmed the remodeling capacity of the phosphate could efficiently promote bone forma-
implanted tissue, and its integration into the sur- tion in comparison to DPSCs + calcium sulfate
rounding normal bone with the passage of time. dihydrate or DPSCs + calcium sulfate dihydrate/
Lamellar bone tissue with vital osteocytes and TCP [90].
vascularized Haversian canals were found after
bone remodeling. 2.2.3 Neural Regeneration
SHED have been administered intravenously Neural regeneration is a challenging objective for
in order to treat osteoporosis in animal models of two major reasons. The first is the lack of neural
this condition [79, 92]. Ma et al. have found that progenitor cells, while the second reason is that
this type of administration route for SHED can the local microenvironment may impede regen-
ameliorate problems of low bone mineral density, erative processes [99]. DPSCs express markers of
and can improve the radiopacity of the trabecular neural progenitors such as nestin and Pax6, due
bone structures [79]. Moreover, Liu et al. showed to the neural crest origin of these cells [100, 101].
16 M. Rezai Rad et al.

Human DPSCs are able to form spheroids under 2.2.4 Other Regenerative
serum-free neuronal stimulating culture condi- Applications
tions [102]. In addition, even without neural
induction, DPSCs express neural markers such as Muscle Regeneration
CDH2, TUBB3, and NFM [103]. Arminan et al. reported the differentiation of
Transplantation of DPSCs and SHED into DPSCs into cardiomyocytes in rats [109], while
defect sites in the central nervous system (CNS) Yang et al. documented that DPSCs can differenti-
has been shown to enhance neural recovery [56, ate into dystrophin-producing muscle cells in a
101, 104]. DPSCs can coordinate axonal regrowth mouse model of cardiac muscle injury [110].
by secreting CXCR-4 and stimulating the SDF-1/ Based on the observation that DPSCs infused into
CXCL12 axis that induces neuroplasticity [105]. cardiac defect sites express dystrophin and myosin
It has been reported that the implantation of [111], it has been proposed that DPSCs can poten-
DPSCs into the hippocampus of immunocompro- tially be applied for muscle regeneration [110].
mised mice can accelerate cell recruitment and
proliferation, and the maturation of endogenous Corneal Regeneration
existing neural cells [118]. In fact, taken together Some experimental evidence indicates that
all these findings suggest that DPSCs may have DPSCs are more similar to epithelial stem cells
an application as a modulator and stimulator in than BMMSCs [112, 113]. In fact, DPSCs can
neural recovery in the CNS [143]. differentiate into keratinocytes and can express
In spinal cord trauma and cerebral ischemia keratinocyte markers [114]. Gomez et al. reported
models, DPSCs can significantly enhance neuro- that eye transparency in a rabbit corneal defect
logical dysfunctions [57, 161]. Furthermore, in model was improved by the implantation of a
the case of a peripheral nerve injury, DPSCs can sheet of tissue-engineered DPSCs into the defect
mediate neural tissue engineering with artificial sites [115]. In another study, DPSCs delivered by
nerve conduits, to regenerate myelinated neural soft contact lenses in a clinical application were
fibers [122]. Luo et al. demonstrated that trans- shown to promote corneal epithelial regeneration
plantation of a heparin–poloxamer hydrogel and [116]. DPSCs transferred from the contact lenses
DPSCs with fibroblast growth factor could sig- to the corneal surface expressed the keratinocyte
nificantly regenerate neurons in spinal cord inju- markers cytokeratin 3 and 12. Moreover, DPSCs
ries, and functionally repair nerve injury defects can impede conjunctival cells from growing into
in rats after 28 days [101]. the center of the cornea [117].
Despite several studies which have demon-
strated that both DPSCs and SHED cells can (1) Cartilage Regeneration
differentiate into functional neural cells which As mentioned earlier, DPSCs can develop into
were voltage-sensitive in vitro, (2) express neural dentine, cartilage, and bone tissues [118]. Yu
markers, and (3) migrate into the CNS in animal et al. showed cartilage formation after 14 days
models [24, 105, 106], many in vivo investiga- after the implantation of pellets containing
tions have reported that DPSCs and SHED are DPSCs into the renal capsule of rats [119]. In
unable to differentiate into functional neurons addition, Morito et al. reported similar results
that the sites of injuries to nerves [102]. The neu- after the subcutaneous implantation of DPSCs in
ral regenerative capacity of DPSCs and SHED combination with FGF in immunocompromised
seems to occur because of their production of mice [120].
neurotrophic products [107]. Furthermore, these
cells impede axon growth inhibitor signals and Hair Follicle and Blood Vessel Regeneration
improve the microenvironment for regeneration DPSCs have been transplanted to the surgically
[108]. Further studies are necessary to clarify compromised hair follicles, where they have been
the neural regeneration capability of DPSCs and shown to cause the formation of new head bolts
SHED. and the regeneration of hair fibers [121].
Dental Tissues Originated Stem Cells for Tissue Regeneration 17

DPSCs can promote angiogenesis and vascu- [132], rheumatoid arthritis [133], autoimmune
logenesis in sites where there is peripheral nerve encephalomyelitis [134], Alzheimer’s disease,
injury [122]. DPSCs can differentiate into endo- and Parkinson’s disease [135, 136].
theliocytes in rat models [78]. There have been few systematic comparisons
between BMMSCs and DSCs in terms of their
Endocrine Regenerative Potential immunophenotype, gene expression profile, and
Previous studies have documented the possibility regenerative potentials [34]. Collectively, in vitro
of using DPSCs and SHED in the treatment of investigations show that DPSCs share a similar
diabetes mellitus using a regenerative approach, pattern of gene expression with BMMSCs [15].
due to their multipotent capabilities, since they In addition, signaling pathways of odontoblastic
can differentiate into insulin-producing cells differentiation of DPSCs are similar to the path-
[13]. DPSCs are capable of differentiating into ways whereby bone marrow-derived stem cells
pancreatic cells and also into insulin-producing take on osteoblastic features [15].
islet-like cells [123, 124]. Kanafi et al. implanted Shi et al. evaluated gene expression in DPSCs
islet-like cell aggregates derived from DPSCs or and BMMSCs, and showed that more than 4000
SHED cells into diabetic mice, and found that the known human genes were similar between these
SHED group was superior to the group treated cells [137]. However, they have found that col-
with DPSCs in terms of maintaining normal lagen type 18, insulin-like growth factor-2, and
blood glucose levels [50]. cyclin-dependent kinase 6 were much more highly
SHED have also been studied for the treat- expressed in DPSCs, while insulin-like growth
ment of injuries to the kidney, where they influ- factor binding protein-7 and collagen types I and
ence the proliferation of tubular epithelial cells II were expressed more in BMMSCs [137].
[125], through a paracrine effect that induce cell Yamada et al. characterized and compared
migration and facilitates the repair of acute kid- DPSCs and BMMSCs using a cDNA microarray
ney damage. system, including 12814 genes and a clustering
algorithm [138]. They demonstrated that after
Regenerative Therapy of Various Systemic osteoinduction DPSCs expressed alkaline phos-
Disease phatase, DSPP, and DMP-1 at levels that were
Previous investigations demonstrated the posi- higher than BMMSCs. However, in the cluster-
tive impact of DPSCs and SHED when used in ing assessment, it became apparent that both cells
the treatment of various systemic diseases [126]. share similar gene regulation pathways for signal-
The enormous capacity of these DSCs makes ing, cell metabolism, and communication [138].
them an attractive source for regenerative thera- Despite DPSCs and BMMSCs having many
pies in medicine. Both DPSCs and SHED can similarities in regulating roles, in signaling fac-
differentiate into hepatic cells [127, 128], which tors, and in their expression profile, these two
makes them promising in the treatment of liver types of stem cells are very different in their pro-
cirrhosis [129]. liferative capacity and their differentiation poten-
As already discussed, due to their myogenic tial, and this has led to distinct patterns of use for
multipotency, DPSCs and SHED cells can be of tissue regeneration in preclinical studies [15]. For
value in the treatment of muscle injuries, includ- instance, the chondrogenic potential of DPSC,
ing to the myocardium. Both cell types have been and the adipogenic capacity of both SCAP and
used experimentally to treat myocardial infarc- DPSCs are weaker than those of BMMSCs.
tion [111] and Duchene muscular dystrophy Conversely, the neurogenic capabilities of DSCs
[130, 131]. Moreover, the immunomodulatory are far more potent than those of BMMSCs. In
and anti-inflammatory effects exerted by these addition, SHED and PDLSCs have a much higher
cells have been applied successfully in the treat- growth potential compared to BMMSCs [139].
ment of a range of inflammatory and autoimmune These differences may be due to the neural crest
diseases including systemic lupus erythematosus origin of DSCs.
18 M. Rezai Rad et al.

3  tem Cells from the Apical

S 3.2.1 Pulp Regeneration
Papilla and Angiogenesis
Regenerative endodontic therapy includes the
The apical part of dental papilla is a cell-rich process of regenerating the dentine–pulp com-
zone containing stem cells (Fig. 1). The apical plex [151]. SCAP are one of the most promis-
papilla can be harvested from an immature ing stem cell sources for such therapy due to
extracted tooth, and used for isolation of SCAP their known odontogenic potency and their
[140]. The main difference between SCAP and expression of dentine-­ related differentiation
DPSCs is that SCAP is the precursor of the markers such as dentine sialphosphoprotein
radicular pulp. The characteristic differences (DSPP) [152].
between these cells are listed in Table 1. In gen- Nowadays, with the application of scaffolds
eral, SCAP derives from the developing dental and the inclusion of growth factors such as vas-
tissues, which consists of early progenitor cells cular endothelial growth factor (VEGF), and
that are distinctive from the cells of mature tis- fibroblast growth factor (FGF), there is increas-
sues (DPSCs) [141]. ing optimism regarding the possibility of regen-
eration of dentin-pulp complex [153–155]. Cell
homing therapies have determined that chemo-
3.1 Isolation and Characterization tactic factors for SCAP include SDF-1, TGF-
β, and granulocyte-colony stimulating factor
After harvesting the apical papilla of a develop- (G-CSF). These not only can improve the migra-
ing root, the tissue is diced into smaller pieces tion of SCAP, but they also can promote their dif-
and subjected to enzymatic digestion with colla- ferentiation [156].
genase and dispase [140] as already described for Sonoyama et al. reported that human SCAP
the isolation of DPSCs and SHED. can develop into a functional root in an animal
Cultivated SCAP possess low immunogenic- model [29]. De novo dentine–pulp complex
ity, as seen by lymphocyte assays in the labora- regeneration by SCAP begins with the migra-
tory [142]. Flow-cytometry analysis shows that tion of these cells onto the dentine surface, fol-
SCAP express typical cell markers including lowed by odontoblastic differentiation and the
CD73, 90, and 105 (Table 1) [143]. In addition, expression of phenotypic markers of dentino-
the perivascular location of SCAP reflected by genesis [157].
their expression of STRO-1 and CD146, which Hikens et al. demonstrated that SCAP are able
gradually fades with extended passaging of the to express a number of markers of angiogenesis
cells [144–146]. Although expressed at a rela- including VEGF), thrombospondin-1, angiopoi-
tively low amount CD24 seems to be exclusively etin-­1, endostatin, matrix metalloproteinases, and
positive in SCAP compared to other DSCs and FGF [153]. Revascularization is a critical require-
other MSCs [147, 148]. Expression of CD24 ment for pulpal tissue engineering [158]. SCAP
expression reduces to zero after the 10th pas- cells appear to be a good candidate for promoting
sage [149, 150]. If CD 24 is a specific marker for revascularization because of their original niche
determining the “stemness” of SCAP, these find- in the perivascular location [159].
ings imply that loss of stemness in SCAP occurs Moreover, in repairing dentine–pulp complex,
after the 10th passage. the administration of scaffolds can be challeng-
ing, as some of the biomaterials that are used (e.g.,
HA and TCP) are osteoinductive, and thus there
3.2 Regenerative Applications is a risk of generalized calcification occurring in
the pulp space [157]. Thus, the properties of the
SCAP can differentiate into various cell lineages scaffold need to be adjusted to suit the require-
[149], which make these cells an attractive source ments of pulp tissue regeneration. Amirkia et al.
for tissue engineering. used a three-dimensional silk fibroin as a natural
Dental Tissues Originated Stem Cells for Tissue Regeneration 19

scaffold, and found that this improved the attach- differentiation patterns. For instance, SCAP iso-
ment of SCAP and their differentiation [160]. In lated from third molars tended to produce diffuse
addition, chitosan-based scaffolds seem to be an mineralization, whereas SCAP isolated from pre-
appropriate carrier for SCAP as they improve molar teeth showed a localized pattern of calcific
their odontogenic potential [161]. deposits [165]. To date, no in vivo investigations
have been conducted to evaluate the bone regen-
3.2.2 Neural Regeneration eration capacity of SCAP.
The neurogenic potential of SCAP has been
exploited for neural tissue engineering [158]. 3.2.4 Other Regenerative
SCAP originated from the neural crest, and they Applications
express high level of the neural marker nestin The chondrogenic potential of SCAP has been
[152]. In addition, SCAP can drive a neuroprotec- assessed using Alcian Blue staining of the chon-
tive mechanism, by decreasing inflammation and drocytes that have formed in vitro [166], but to
inducing differentiation of oligodendrocytes date no study has been carried out to evaluate the
[155]. SCAP also express various markers (genes) molecular evidence for the chondrogenic poten-
for neurogenesis (Table 1). In vitro and in vivo tial of SCAP, such as the expression of chondro-
investigations show SCAP can participate in neu- genic genes in SCAP under chondrogenic
rite outgrowth and in axonal induction [158]. induction. Thus far, no in vivo study of cartilage
Under neurogenic induction, SCAP start to formation by SCAP has been reported.
mimic spindle-shaped neurocytes, with long cel- Several studies have demonstrated the adipo-
lular process [152, 162, 163]. In a study by De genic capacity of SCAP [165]. SCAP can express
Berdt et al., the entire apical papilla was trans- lipoprotein and lipase, suggesting the adipogenic
planted into an area of artificial spinal cord capability of these cells [152]. However, in com-
damage in an animal model. The apical papilla parison to BMMSCs, their adipogenic potential
functioned as a scaffold for SCAP to regener- is low [148, 153, 167].
ate neural tissue in the original niche of the
cells [164]. Interestingly, this study revealed
that hypoxic conditions could stimulate SCAP 4 Dental Follicle Stem Cells
to express neural-specific genes and to secrete
growth factors [164]. The dental follicle is a loose connective tissue sac
surrounding the tooth bud. It plays an important
3.2.3 Bone Regeneration role in tooth development and eruption. The den-
The osteogenic potency of SCAP has been con- tal follicle is involved in tooth eruption by con-
firmed by studies which have shown differentia- trolling osseous remodeling through the timely
tion of these cells into osteoblasts, as determined production of various secreted mediators [168].
by alizarin red staining of calcium deposition and Stem cells have been harvested from dental fol-
the expression of osteogenic markers, including licle of different species in various developmen-
bone sialoprotein, alkaline phosphatase, gamma-­ tal stages [169, 170]. DFSCs are multipotent, and
carboxyglutamate protein, runt-related transcrip- can differentiate to form periodontium, bone, and
tion factor-2 (RUNX2), and bone morphogenetic cementum [169, 171].
proteins (BMPs) [32]. The osteogenic differenti-
ation capability of SCAP is comparable to that of
BMMSCs [148]. 4.1 Isolation and Characterization
The cultivation of SCAP in an osteogenic
medium results in the formation of osteoblast-­ Human extracted third molar teeth are the major
like cells that are able to produce mineralized tissue source that has been used to isolate human
nodules [165]. Nada et al. observed that SCAP DFSCs. The method for DFSCs isolation is simi-
isolated from different teeth showed different lar to that described for other DSCs.
20 M. Rezai Rad et al.

In culture, DFSCs have a typical fibroblast-­ lated DFSCs from the third molar teeth of
like morphology. They express CD9, CD10, 6-month-old pigs [180]. DFCSs were seeded in
CD13, notch-1, and nestin, but they do not the bottom of a tube and DPSCs and the enamel
express CD31, CD34, CD45, and HLA-DR [172, organ epithelium (which originated from same
173] (Table 1). DFSCs also express cementum pigs) were added in order to produce a recombi-
attachment protein and cementum protein-23 nation which mimicked the tooth primordia. The
(CP-23), which are two putative cementoblast whole mixture was then transplanted into the
markers [15]. DFSCs also express STRO-1 and omentum of immunocompromised rats (Fig. 2).
BMP receptors in vivo [26]. A thick layer of dentine with viable odontoblasts
and a layer of cementum-like tissue were found
at 24 weeks post-surgery. Moreover, collagen
4.2 Regenerative Applications fibers were present in a pattern that resembled the
PDL, and they were attached to the cementum-­
DFSCs can differentiate into osteoblasts, cement- like layer.
oblasts [30], and adipocytes in the appropriate Another histological evaluation also con-
inducing culture media [170]. Although experi- firmed the possibility of whole periodontium
mental investigations have revealed mineralized regeneration via expanded DFSCs [182]. A fur-
tissue formation by DFSCs, DPSCs have a ther investigation reported that DFSCs could dif-
greater capacity for all hard tissue formation. ferentiate into PDL cells, and could regenerate
Yagyuu et al. reported hard tissue formation of PDL-like structures including cementum-like
DFSC at preclinical studies [176], while Yokoi tissues [183]. Other researchers have indicated
et al. demonstrated that DFSCs are capable of that DFSCs used in combination with a treated
regenerating soft tissue and PDL in vivo [174]. dentine matrix could regenerate root-like tissues
with a dentine–pulp complex [184].
4.2.1 Bone Regeneration
DFSCs have the capacity to create calcification,
as seen both in cell culture [25] and in animal 5 Stem Cells
[175] studies. Several investigations have from the Periodontal
reported the osteogenic differentiation of DFSCs Ligament
in appropriate osteogenic medium [176–179]. In
vivo bone formation by DFSCs has been demon- Although early studies provided some evidence
strated in critical size bone defects in rat calvaria to support the differentiation capability of peri-
[180]. Moreover, in vitro investigations have odontal ligament (PDL) cells, such as the ability
indicated that BMP-6 and BMP-9 promote the to differentiate into cementum-forming cells and
osteogenic differentiation of DFSCs [176]. osteoblasts [185, 186], Seo et al., conclusively
Rezai Rad et al. showed that a temperature of identified a population of MSCs within the peri-
37–40 °C was optimal for inducing osteogenesis odontal ligament that can express stem cell
of DFSCs in vitro [181]. Honda et al. reported the ­markers and that have the capability to differenti-
results of two different animal studies to evaluate ate into various cell lines [27].
the bone regenerative capacity of DFSCs [180,
182]. The findings of both studies suggest that
DFSCs supported bone regeneration, although it 5.1 Isolation and Characterization
was not clear whether the transplanted stem cells
had in fact differentiated into osteoblasts. To isolate PDLSCs, extracted teeth with an intact
PDL are immersed into a digestion solution con-
4.2.2 Periodontal Regeneration sisting of collagenase and trypsin [187]. The
DFSCs are capable of generating osteoblasts, resultant cells from the enzymatic digestion can
cementoblasts, and PDL [14]. Honda et al. iso- then be cultured in various media such as serum-­
Dental Tissues Originated Stem Cells for Tissue Regeneration 21

Fig. 2 Schematic
diagram of procedure
used to regenerate
engineered dental root porcine mandible
analogue in Honda et al.
study [180]

third molar tooth

bone shaft

dental follicle enamel organ dental pulp

stem cells epithelial cells

pulp cells cavity

transplantation

subcultured
dental follicle
recombination
stem cells

containing media and neurosphere-forming 5.2 Regenerative Applications

medium, according to the intended therapeutic
purpose [53]. The current literature report that PDLSCs can
PDLSCs possess low immunogenicity, and differentiate into cementum-like structures, and
they can modulate behavior of peripheral blood along osteogenic, adipogenic, and chondrogenic
mononuclear cells by secreting TGF-β and HGF cell lineages (Table 1).
[188]. PDLSCs isolated from inflamed periodon-
tium can significantly reduce the activity and pro- 5.2.1 Periodontal Regeneration
liferation of T lymphocytes [189]. They express PDLSCs were firstly applied in animal models
high levels of interleukin (IL)-10, and IL-17 in and used to reconstruct cementum-PDL-like
comparison to PDLSCs derived from healthy tis- structures [27]. Subsequently, several studies
sues [190]. investigated the capability of PDLSCs for peri-
PDLSCs express MSC-related markers and odontal regeneration [191–193]. Ninomiya et al.
tendon specific transcription factor (scleraxis) implanted a HA scaffold loaded with PDLSCs
(Table 1). Scleraxis expression is significantly into the dorsal muscle of rats, and demonstrated
higher in PDLSCs than in DPSCs and in BMMSCs bone-like tissue formation [194]. PDLSCs seeded
[15]. PDLSCs do not express hematopoietic mark- on HA/TCP successfully formed PDL and
ers such as CD14, CD19, CD34, and CD45 [15], cementum-like tissues surrounding the scaffold
or markers associated with hematopoietic cells [195, 196]. Complete PDL regeneration was
including CD40, CD80, and CD86 [188]. achieved, with formation of Sharpey’s fibers
22 M. Rezai Rad et al.

between the newly formed cementum and the [27, 206–208]. Although PDLSCs typically
fibers of the PDL [197]. form cementum-like structures [15], PDLSCs
Likewise, when transplanting PDLSCs that implanted into periodontal defects of animal
were treated with recombinant human plasminogen models appear to accelerate the regeneration of
activator inhibitor-1, and then seeded on HA/TCP trabecular bone next to PDL-like structures, thus
scaffold, into the dorsal region of immunodeficient demonstrating their capacity for alveolar bone
mice, cementum-like tissue surrounded by PDL- regeneration [27, 209].
like tissue was observed after 10 weeks [198]. Transplantation of human PDLSCs encapsu-
Using stem cell all sheets derived from PDLSCs lated in a RGD-modified alginate has been shown
for tissue engineering has been attempted. HA/ to enhance bone formation in critical-sized cal-
TCP wrapped with PDLSCs cell sheets has been varial defects in rats [210]. Several studies have
shown to generate PDL/cementum-like structures observed a lower bone regenerative potential
in rats and mice [199, 200]. Moreover, adding of PDLSCs compared to BMMSCs. By way of
platelet-rich fibrin (PRF) to a HA/TCP scaffold comparison, BMMSCs were reported to be more
wrapped with PDLSCs sheets was shown to effective for alveolar bone repair in canine mod-
regenerate not only cementum and PDL, but also els [211]. Another study confirmed the lower
blood vessels [201]. osteogenic capability of PDLSCs compared to
Transplantation of human PDLSCs sheets into BMMSCs [212]. The reason for this may be the
infra-bony mandibular defects in dogs showed presence of more end-differentiated cells in the
new cementum regeneration, with the formation PDLSCs population [213]. However, there are a
of collagen and nerve fibers around the roots of few contrary reports in the literature suggesting
the teeth after 8 weeks [202]. When PDLSCs and similar or even better osteogenic potentials of
gelatin sponge scaffolds were grafted onto fenes- PDLSCs compared to BMMSCs [214], and other
tration defects in rats, complete healing of bone, DSCs [80, 215]. This point needs further research
cementum, and PDL was seen after only 3 weeks to resolve it.
[203]. These findings suggested that there is con-
siderable potential for regeneration of the peri- 5.2.3 Tendon and Cartilage
odontium using PDLSCs, and that this approach Regeneration
is likely to be successful when used in the clinic Gronthos et al. showed that PDLSCs can express
to manage real bony defects [204]. a tendon-specific marker (scleraxis) in vitro
However, a significant point is that the regen- [206]. A combination of human PDLSCs with an
erative capability of PDLSCs is affected consid- RGD-coupled alginate could form a tendon-like
erably by the presence of inflammation and by the tissue in mice. When used for tendon ­regeneration,
age of the PDL tissue from which the cells are har- compared with BMMSCs, PDLSCs gave a more
vested [200, 204, 205]. Gao et al. compared vari- highly organized tissue with a greater amount of
ous PDLSCs from donors of different ages [200]. collagen fibers [216].
They found that PDLSCs from younger donors Moshavernia et al. showed that cartilage heal-
had greater cementum/PDL formation potential ing could be achieved by applying encapsulated
than those from older donors. Other studies have PDLSCs in an alginate hydrogel [216]. Moreover,
reported that PDLSCs derived from donors with several animal studies have successfully used
periodontitis have a significantly lower capacity PDLSCs for cartilage tissue engineering. Ectopic
to form bone than PDLSCs derived from healthy cartilage formation was observed at PDLSC
sites or healthy donors [205]. transplantation sites [102, 217, 218]. It is well-­
known that cartilage has a very restricted ability
5.2.2 Bone Regeneration for self-renewal and regeneration [219]. In this
The osteogenic potential of PDLSCs has been regard, the chondrogenic potential of PDLSCs is
shown in several in vitro studies, which have noteworthy, and it is likely that they will be of
reported the formation of mineralized nodules interest for cartilage repair [102].
Dental Tissues Originated Stem Cells for Tissue Regeneration 23

Table 3 Licensed dental tissue-derived stem cells bank all around the worlda
Name Website Country
BioEDEN http://www.bioeden.com/ United
Store-A-Tooth http://www.store-atooth.com/ States
StemSave http://www.stemsave.com/
Three brackets (Hiroshima http://www.teethbank.jp/ Japan
University)
Teeth Bank Co. http://www.teethbank.jp/
Advanced Center for Tissue http://www.acte-group.com/
Engineering
MoBaTann: Tooth biobank http://www.uib.no/en/rg/biomaterial/64723/ Norway
mobatann-tooth-biobank
The Norwegian Tooth Bank http://www.fhi.no/morogbarn
Stemade Biotech Pvt. http://www.stemade.com/ India
All information gathered from Chalisserry et al. and Liu et al. systematic reviews
a

5.2.4 Other Regenerative years. Banking of DSCs is one way of easily

Applications maintaining a suitable supply of DSCs to meet
Recently, it has been shown that PDLSCs have the needs of patients later in their life. Such an
both neurogenic and angiogenic differentiation approach can pave a new road for progress in
potentials [53]. PDL-derived spheres are able to healthcare by maintaining a promising source of
differentiate into mesodermal and neural cells. autologous cells for personalized regenerative
MSCs originated from the PDL can form treatments.
Schwann cells by inducing the Erk1 signaling Given these considerations, the ability to har-
pathway [220]. Furthermore, these cells can vest and safely preserve DSCs becomes more
regenerate retinal ganglion-like cells via func- important. Nowadays, DSCs can be cryopre-
tional synapses and respond to calcium [221]. served for a long period of time [224–226]. In
Cen et al. demonstrated that human PDLSCs a number of developed countries, licensed tooth
ameliorate ganglion cells and axonal regenera- banks have been founded (Table 3) [30, 34, 227].
tion when used in the retina of animals with a When such banks have been established there
traumatized optic nerve [222]. are a number of ethical controversies, as well as
Another novel application of PDLSCs is its social, and legal issues that need to be considered.
possible use for cardiogenic differentiation. Consistent and well-documented laboratory pro-
PDLSCs express some cardiac cell markers, cedures are needed to evaluate and preserve the
such as sarcomeric actin and cardiac troponin cells. There is also a need for appropriate regula-
T [223]. tions or legislation regarding stem cell banking.

6 Banking of DSCs 7 Limitations

According to the diversity of DSCs and their Although stem cell-based therapeutic approaches
beneficial aspects, the potential uses of stem have shown much promise with an appealing
cell-­based treatments using DSCs in both den- path to their use in tissue regeneration and func-
tistry and medicine are significant. The adminis- tional repair, multiple factors need to be opti-
tration of a patient’s own DSCs during therapy mized. This will require thorough clinical
may not often be practical, since they may have investigations as well as cell culture studies to
more conditions requiring treatment in their later enhance methods to grow up and maintain a large
years, but have higher numbers of stem cells in quantity of cells. One must bear in mind that the
their tissues when they are in their childhood availability of the dental tissues over a lifetime
24 M. Rezai Rad et al.

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 Dentine–Pulp Complex
Regeneration

Ove A. Peters, Avina Paranjpe, and Alexis Gaudin

1 Introduction ate a functional dentin–pulp complex had been

futile. In a best-case scenario, the pulp stayed
Therapeutic strategies in dentistry are tradition- vital, and a dentine bridge formed. This repara-
ally subtractive rather than additive, for example, tive dentine typically did not regenerate a fully
in case of dental caries, diseased tissue is removed functional dentine–pulp complex; this joint struc-
and replaced with a restoration. In endodontics, ture is characterized by a common embryologic
an irreversibly diseased dental pulp is removed, pathway from the dental papilla and by a tight
and the canal space debrided and obturated with histologic interconnectivity [3].
a root canal filling. The latter practice is well
established and enjoys reasonably high clinical
success rates; however, the procedure leaves the
Box: The Dentine–Pulp Complex
tooth with reduced structural strength [1].
The discovery of dental pulp stem cells two The view that dentine and pulp are embryo-
decades ago [2] opened the door for a regenera- logically, histologically, and functionally
tive approach to the diseased pulp. While the similar tissues have been held for many
healing potential of the pulp had been recognized decades. An intact dentine–pulp complex
for a long time, attempts to predictably regener- serves important purposes:

• Synthesize and secrete dentine

O. A. Peters (*) • Maintaining tissue homeostasis
School of Dentistry, The University of Queensland,
Herston, QLD, Australia • Mediating reparative processes
• Screening for invading pathogens
Department of Endodontics, Arthur A. Dugoni School
of Dentistry, University of the Pacific, • Supporting enamel in force dissipation
San Francisco, CA, USA
e-mail: [email protected]
A. Paranjpe True regeneration of the dentine–pulp com-
Department of Endodontics, University of
Washington, School of Dentistry, Seattle, WA, USA plex would mean “restitutio ad integrum,”
e-mail: [email protected] which must include its histological appearance,
A. Gaudin physiology, and mechanical properties. Current
Faculty of Dental Surgery, Department of clinical approaches have been characterized as
Endodontics and Restorative Dentistry, University of “guided pulpal repair,” which likely offer clini-
Nantes, Nantes, France
cal benefits without fulfilling the set criteria for
e-mail: [email protected]

© Springer Nature Switzerland AG 2021 35

S. Hosseinpour et al. (eds.), Regenerative Approaches in Dentistry,
https://doi.org/10.1007/978-3-030-59809-9_3
36 O. A. Peters et al.

regeneration. Regeneration of the dentine–pulp 2 Dentine–Pulp Complex

complex, in contrast, has been defined as heal- Biology
ing progression starting with inflammation,
using immune signaling, and cellular interac- Human teeth share a similar structure with other
tion toward tissue restoration [4]. Considered by vertebrates. However, there is considerable varia-
some as the “holy grail” of endodontic research, tion in their form and position. The tooth is com-
true regeneration of the dentine–pulp complex posed of both mineralized tissues (enamel,
has yet to be achieved predictably (Fig. 1). This dentine, and cementum) and non-mineralized tis-
chapter will discuss the structure and physiol- sue (dental pulp). Dentine and dental pulp can be
ogy of the dentine–pulp complex and highlight considered as similar tissues, because of their
pathways for regeneration along with associated close embryological, histological, and functional
limitations. similarities. However, while this anatomical

Fig. 1 Dental pulp a Puramatrix rh collagen Puramatrix Human pulp

tissue engineering with SHED SHED (no SHED) (control)
SHED injected into
human root canals and
transplanted into
immunodeficient mice.
(a) Low-magnification
and (b) high-­
magnification images of
tissues formed when
SHED mixed with
scaffolds (Puramatrix™,
rhCollagen type I
groups) were injected
into full-length root
canals of human
premolars. A
vascularized connective
tissue occupied the full
extension of the root
canal. Cell densification
and many blood vessels
were observed along
dentine walls. Scaffolds b Puramatrix rh collagen Human pulp
(Puramatrix™) injected SHED SHED (control)
into the root canals
without cells were used
as controls for SHED.
Freshly extracted human
premolars were used as
tissue controls. Black
arrows point to blood
vessels close to the
odontoblastic layer.
Modified from Rosa
et al., J Dent Res,
92(11):970–975, 2013,
with permission
Dentine–Pulp Complex Regeneration 37

entity is therefore named the “dentine–pulp com- During the cap stage, a new specific structure
plex,” it has no direct analogue from a biological arises due to the epithelial outgrowth: the enamel
point of view, and this terminology may indeed organ. This enamel organ is composed of the
be an oversimplification. outer enamel epithelium, inner enamel epithe-
lium, stellate reticulum, and stratum interme-
dium. The cells of the inner dental epithelium
2.1 Tooth Development will gradually lengthen and form the future ame-
loblasts. Moreover, a particular and transitory
Tooth development or odontogenesis occurs dur- structure appears at the center of the enamel
ing embryonic, fetal, neonatal, and childhood organ: the enamel knot. The enamel knot is an
stages of development. Teeth form from embry- organizing center of the tissue, controlling the
onic cells, then grow, and erupt into the mouth. shape of the crown, and expressing molecules
Odontogenesis is a complex process. The human belonging to the different families of growth fac-
dentition begins to form between the 6th and 8th tors, such as FGF (fibroblast growth factor), BMP
week of prenatal development with primary (bone morphogenetic protein), Hg (hedgehog),
teeth, whereas the permanent teeth begin to form and Wnt (wingless). These growth factors are
in the 20th week. Biomineralization starts during known for their essential role in embryogenesis
the 14th week of gestation [5]. and in the formation of organs [7].
The tooth germ is organized into the enamel The ectomesenchyme condenses in the con-
organ, the dental papilla, and the dental follicle. cavity of the enamel organ and forms the dental
The cells of the tooth germ are derived from the papilla. This dental papilla forms the odonto-
ectoderm of the first pharyngeal arch, and the blasts and the dental pulp. The cap stage evolves
ectomesenchyme of the neural crest. The pro- into the bell stage, during which the dental
cess of odontogenesis is regulated by epithe- crowns acquire its final shape (i.e., morphodiffer-
lial–mesenchymal interactions. Although tooth entiation), and the formation of cusp patterns is
formation occurs as one continuous process, observed.
odontogenesis is classically described by the The outer enamel epithelium and the inner
succession of several stages, i.e., initiation, tooth enamel epithelium join at the cervical loop or
germ morphogenesis, terminal cytodifferen- “zone of reflection.” The growth of cervical loop
tiation, and matrix apposition, resulting succes- cells into the deeper tissues forms Hertwig’s epi-
sively in the following anatomical stages: dental thelial root sheath. This determines the root
lamina, bud stage, cap stage, early bell stage and shape, including root odontoblast differentiation.
late bell stage (with terminal differentiation of The cervical loop progresses in apical direction
odontoblasts and ameloblasts), root formation, due to an increase in cell divisions. The growth of
functional differentiation of cementoblasts, and the germ is consequently amplified, and the pulp
dental eruption. is individualized in relation to the peripheral
At the initial stage of tooth development, a layer of the odontoblasts [8].
basement membrane already separates the epi- In the late bell stage, tooth morphogenesis is
thelium from the underlying ectomesenchyme. followed by a phase of cell differentiation (i.e.,
The epithelium thickens, at the origin of the den- histodifferentiation). These cells will differen-
tal lamina that will later become the dental bud. A tiate in pre-ameloblasts and pre-odontoblasts
more substantial and localized epithelial thicken- in order to become polarized and secreting
ing corresponding to the outlines of future teeth. cells, to ultimately form enamel and dentine
The tooth bud increases in size and then turn into respectively.
dental caps. These are characterized by a concave The condensed ectomesenchyme located at
shape of the epithelial tissue which partially the periphery of the enamel organ and dental
envelops the proliferating underlying mesen- papilla is the dental follicle, and gives rise to
chyme, and the future dental pulp [6]. cementoblasts, osteoblasts, and fibroblasts.
38 O. A. Peters et al.

Thus, the dental follicle is involved and respon- 2.2.1 Odontoblast Life Cycle
sible for the formation of the root and tooth erup- Odontoblasts are postmitotic and highly differen-
tion [9, 10]. tiated cells that originate from the cranial neural
Determining the processes that initiate tooth crest-derived cells of the dental papilla. These
development led to a significant amount of cells produce and regulate an organic matrix
research which has provided the basis for the cur- that will be secondarily mineralized, namely the
rent understanding of the processes involved in dentine.
dentine and dental pulp repair (Fig. 2a).
From Pre-odontoblasts to Polarizing Pre-­
secretory Odontoblasts
2.2 Dentinogenesis The differentiation of odontoblasts from neural
crest cells is a drawn-out process involving a
Details of the basic principles of odontoblast dif- series of changes in the morphology, transcrip-
ferentiation is particularly relevant when consid- tional profile, and expression of proteins secreted
ering the tissue engineering of dentine–pulp by cells in the odontoblast lineage regulated by
complex. the epithelium–dental mesenchyme interactions

a
Bell stage Eruption
Lamina Bud stage Cap stage

Epithelium
Mesenchyme
Dental mesenchyme
Enamel Knot
Enamel
Bone
Dentine

b Ameloblasts Ameloblasts
Pre-ameloblasts
Inner dental epithelium

Basal membrane
Dentine matrix

Ectomesenchymal cells
Odontoblasts
Pre-odontoblasts

Quiescent cells

Fig. 2 Schematic drawings of the development of the differentiate from ectomesenchymal cells that are located
dentine–pulp complex. (a) Stages of tooth development. near the basement membrane. Short, columnar-shaped
The succession of the different anatomical stages: dental pre-odontoblasts elongate and extend cellular processes
lamina, bud stage, cap stage, early bell, and late bell toward the basement membrane where dental epithelium
stages (terminal differentiation of odontoblasts and ame- and ectomesenchyme interface. Secretory odontoblasts
loblasts), root formation, functional differentiation of are fully differentiated polarized columnar cells contain-
cementoblasts and dental eruption. The enamel knot ing numerous organelles in their supranuclear area.
appears before the terminal differentiation of cells and During the last mitosis, the daughter cells in contact with
controls the shape of the crown due to FGF, BMPs, Hg, the basement membrane differentiate into odontoblasts
and Wnt. (b) Terminal events leading to odontoblast dif- while the other cells in the peripheral zone will join the
ferentiation. Odontoblasts originate from the cranial neu- cells of the Höhl layer
ral crest-derived cells of the dental papilla. They
Dentine–Pulp Complex Regeneration 39

mediated by the basement membrane. Initiated at solid permeability membrane limited to mole-
the tip of the cusp in the most peripheral layer of cules of low molecular weight. Finally, amino
the cells of the dental papilla, differentiation con- acids, fatty acids, sugars, and ions cross the space
tinues according to a genetically predetermined between endothelial cells and the basement mem-
temporo-spatial pattern. Inductive signals from brane to be taken up into odontoblasts.
internal epithelial cells involve members of the Although understanding of the molecular
TGF-ß family (BMP-2, BMP-4, and TGF-ß1) events preceding the terminal differentiation of
[11], and other growth factors (IGF: insulin-like odontoblasts has markedly improved, the final
growth factor), which are partially sequestered in determinants of differentiation of odontoblasts
the basement membrane onto which the periph- remain to be characterized [12, 15–18]. The odon-
eral cells of the dental papilla align [12]. toblasts in their terminal cell division are initially
Functional competence of odontoblasts is positioned roughly parallel to the basal lamina, but
achieved after a predetermined number of cell after a short time their major axis becomes per-
divisions and when cells express specific growth pendicular, and they form a palisade-­like structure.
factor receptors. The fixed number of divisions The terminal polarization leads to a partition into a
allows these cells to reach the periphery of the cell body and a long process. The cell body houses
dental pulp. During the last cell division cycle, all the organelles involved in the synthesis of the
only the most peripheral layer of cells underlying extracellular matrix: rough endoplasmic reticu-
the basement membrane (pre-odontoblasts) lum, Golgi apparatus, and immature and mature
responds to signals from the internal dental epi- secretory vesicles, associated with equipment
thelium, to become completely differentiated into lysosomal (smooth endoplasmic reticulum, lyso-
odontoblasts. The other cell resulting from the somal vesicles, multivesicular structures).
cell division cycle, “the daughter cell” that which The cell process, on the other hand, protruding
is not in contact with the basement membrane, a variable distance into the predentine, and adheres
moves away from the previous one, to join the to the dentine walls of the tubules (odontoblast
layer of Höhl [13]. This cell layer has been con- process). To accommodate these organelles and to
sidered as a potential reservoir of cells, contain- prepare for the secretion of the components of the
ing incompletely differentiated cells that could dentine matrix apically and unidirectionally, the
be involved in the healing process of reparative nucleus moves to the opposite pole of the cell, in a
dentinogenesis [14]. position opposite to the internal dental epithelial
cells. Nuclear repolarization is one of the impor-
Odontoblastic Differentiation and Terminal tant characteristics of the differentiation of termi-
Polarization nal odontoblasts, and is a critical step both in the
Once the odontoblasts are differentiated, they formation of primary tubular dentine and in the
undergo a terminal polarization. As the differen- regeneration of dentine tissue (Fig. 2b) [18].
tiation takes place in an apical direction, the cells
change shape, going from round to cuboidal to an 2.2.2 Role, Structure,
increasingly elongated appearance. At the sub- and Composition of Dentine
cellular level, the cells acquire a pronounced syn- Dentine is a calcified tissue that usually is cov-
thetic and secretory apparatus. The Golgi ered on its coronal aspects by enamel, and on its
apparatus migrates from the basal part to a supra- radicular (root) aspect by cementum. It and
nuclear region simultaneously with the develop- houses the entire dental pulp. Dentine is less min-
ment of cytoskeletal proteins, microtubules, eralized and less brittle than enamel. Dentine is
odontoblastic cilium, actin microfilaments, and also necessary for the support of enamel. Dentine
intermediate filaments containing vimentin and rates at approximately “3” on the Mohs scale of
nestin. A distal junction complex appears with mineral hardness.
desmosome-like, communicating junctions (gap As a mineralized connective tissue, dentine
junctions). These junction complexes constitute a constitutes the major part of the tooth, and is
40 O. A. Peters et al.

composed by volume of 40–45% mineral (mainly basolateral and apical compartments, the trans-
hydroxyapatite and some noncrystalline amor- formation of predentine into dentine contributes
phous calcium phosphate), and 30% organic to the formation of primary dentine. It is a tissue
material, of which 90% is collagen type I and the containing canaliculi with an S-curved path, and
remaining 10% is ground substance. The latter is produced by functional odontoblasts at a speed
includes dentine-specific proteins (SIBLINGs: of 4 μm/day. This process ends with the function
small integrin-binding ligand N-linked glycopro- of the tooth in the arch.
tein). Dentine also contains 20–25% water [19]. Secondary dentine is formed when the tooth
Dentine is porous, and contains microscopic becomes functional. The S-shaped trajectory of
channels, called dentinal tubules, which radiate the canaliculi becomes accentuated. In addition,
outward through the dentine from the pulp toward as the number of odontoblasts increases com-
the exterior cementum or enamel border, with pared to a smaller surface, the number of cana-
permanent connections to the odontoblastic layer liculi gradually increases as one gains the
located at the periphery of the pulp due to the innermost layers. In theory, this dentine is formed
penetration of cytoplasmic extensions of odonto- throughout life, without time limits, but its pro-
blasts into the dentinal tubules. duction in the elderly is, however, gradually
Dentine is divided into two areas at the coro- reduced. Primary and secondary dentine are adja-
nal level: the mantle dentine on the periphery, cent, and form in continuity. They are physiologi-
and the circumpulpal dentine near the dental cal types of dentine, and are made up of
pulp. The mantle dentine can be identified by the intercanalicular and pericanalicular dentine.
presence of various characteristics. The collagen Tertiary dentine is formed as a reaction to
fibers here are found perpendicular to the external stimulation such as bacterial attack,
enamel–dentine junction. trauma, and tooth wear. The pulp seeks to pre-
At the root level, two layers are specifically serve its own vitality by synthesizing a scar tissue
observed: the mantle dentine is replaced by the called tertiary dentine. This newly formed tissue
hyaline layer of Hopewell-Smith on the periphery will form a calcified barrier separated from phys-
of dentine, with the granular layer of Tomes iological dentine by a more or less marked calcio-­
beneath this. These superficial layers are to be dis- traumatic line. Depending on the intensity of the
tinguished from circumpulpal dentine both by stimulus and the nature of the lesions induced in
their composition and by their structure. The man- the pulp, there are two types of tertiary dentine
tle dentine and the hyaline layer of Hopewell-­ that may be formed: reactionary, where dentine is
Smith are atubular layers, unlike the granular layer formed from a pre-existing odontoblast, or repar-
of Tomes which contains fine canaliculi [20]. ative dentine, wherein newly differentiated
The innermost layer of dentine is laid down odontoblast-­like cells are formed due to the death
prior to mineralization, and is the predentine. of the original odontoblasts.
This predentine is the initial dentine matrix. The The architecture and structure of tertiary den-
presence of odontoblastic processes here allows tine depend on the intensity and duration of the
the secretion of matrix components. Predentine stimulus. Tertiary dentine is deposited rapidly,
can be 10–40 μm in width, depending on its rate with a sparse and irregular tubular pattern and
of deposition [21, 22]. some cellular inclusions; and is referred to as
“osteodentine.” However, if the stimulus is less
2.2.3 Types of Dentine active, it is laid down less rapidly with a more
There are three types of dentine: primary, sec- regular tubular pattern with minimal cellular
ondary, and tertiary. Primary dentine is the most inclusions [23–25].
prominent dentine in the tooth, and lies between
the enamel and the pulp chamber. As soon as 2.2.4 Dentinal Tubules
the odontoblasts are polarized and the junction One of the characteristics of human dentine is
complexes between the cell bodies delimit the the presence of dentinal tubules that occupy
Exploring the Variety of Random
Documents with Different Content
 CHAPTER II
UNEXPECTED ENTRANCE OF TWO LADIES
For a moment or two Eustace Charliewood did not return his host's
greeting. He was not only surprised by the curious proceeding of
which he had been a witness, but he felt a certain chill also.
"What the deuce are you up to now, Gouldesbrough?" he said in an
uneasy voice. "Another of your beastly experiments? I wish you
wouldn't startle a fellow in this way."
Sir William looked keenly at the big man whose face had become
curiously pallid.
There was a tremendous contrast in the two people in the room.
Gouldesbrough was a very handsome man, as handsome as
Charliewood himself had been in younger days, but it was with an
entirely different beauty. His face was clean shaved, also, but it was
dark, clear-cut and ascetic. The eyes were dark blue, singularly
bright and direct in glance, and shaded by heavy brows. The whole
face and poise of the tall lean body spoke of power, knowledge, and
resolution.
One man was of the earth, earthy; the other seemed far removed
from sensual and material things. Yet, perhaps, a deep student of
character, and one who had gone far into the hidden springs of
action within the human soul, would have preferred the weak, easy-
going sensualist, with all his meannesses and viciousness, to the
hard and agate intellect, the indomitable and lawless will that
sometimes shone out upon the face of the scientist like a lit lamp.
Charliewood sat down in obedience to a motion of his host's hand.
He sat down with a sigh, for he knew that he had been summoned
to Sir William Gouldesbrough's house to perform yet another duty
which was certain to be distasteful and furtive.
Yes! there was no hope for it now. For the last few years the man
about town had been under the dominion of a stronger will than his,
of a more cunning, of a more ruthless brain. Little by little he had
become entangled within the net that Gouldesbrough had spread for
him. And the lure had been then and afterwards a lure of money—
the one thing Charliewood worshipped in the world.
The history of the growth of his secret servitude to this famous man
was a long one. Money had been lent to him, he had signed this or
that paper, he had found his other large debts bought up by the
scientist, and at the end of three years he had found himself willy
nilly, body and soul, the servant of this man, who could ruin him in a
single moment and cast him down out of his comfortable life for ever
and a day.
No living soul knew or suspected that there was any such bond as
this between the two men. Even Charliewood's enemies never
guessed the truth—that he was a sort of jackal, a spy to do his
master's bidding, to execute this or that secret commission, to go
and come as he was ordered.
As yet all the services which Charliewood had rendered to Sir
William, and for which, be it said, he was excellently paid, were
those which, though they bordered upon the dishonourable and
treacherous, never actually overstepped the borders.
Gouldesbrough employed Charliewood to find out this or that, to
make acquaintance with one person or another, to lay the
foundation, in fact, of an edifice which he himself would afterwards
build upon information supplied by the clubman. There was no crime
in any of these proceedings, no robbery or black-mail. And what
happened after he had done his work Charliewood neither knew nor
cared. Of one thing, however, he was certain, that whatever the
scientist's motives might be—and he did not seek to probe them—
they were not those of the ordinary criminal or indeed ever bordered
upon the criminal at all. All that Charliewood knew, and realized with
impotence and bitterness, was that he had allowed himself to
become a mere tool and spy of this man's, a prober of secrets, a
walker in tortuous by-paths.
"What did you wire to me for?" Charliewood said in a sulky voice.
"What do you want me to do now?"
Sir William looked quickly at his guest, and there was a momentary
gleam of ill-temper in his eyes, but he answered smoothly enough.
"My dear Charliewood, I wish you wouldn't take that tone. Surely we
have been associated too long together for you to speak to me in
that way now. It has suited your convenience to do certain things for
me, and it has suited my convenience to make it worth your while to
do them. There is the whole matter. Please let's be friendly, as we
always have been."
Charliewood shrugged his shoulders.
"You know very well, Gouldesbrough," he said, "that I am in your
hands and have got to do anything you ask me in reason. However, I
don't want to insist on that aspect of the question if you don't. What
did you wire to me for?"
"Well," Sir William said, passing a cigar-box over to the other, though
he did not smoke himself, "there is a certain man that I am
interested in. I don't know him personally, though I know something
about him. I want to know him, and I want to know everything I can
about him too."
"I suppose," Charliewood answered, "that there is no difficulty for
you in getting to know anybody you want to?" He said it with a slight
sneer.
"Oh, of course not," Sir William answered, "but still in this case I
want you to get to know him first. You can easily do this if you wish,
you are sure to have some mutual acquaintances. When you get to
know him make yourself as pleasant as you can be to him—and
nobody can do that more gracefully than yourself, my dear boy.
Become his intimate friend, if possible, and let me know as much as
you can about his habits and objects in life. I don't want you to
spare any expense in this matter if it is necessary to spend money,
and of course you will draw upon me for all you require in the
matter."
Charliewood held up his cigar and looked steadily at the crust of
white ash which was forming at the end.
"What's the man's name?" he asked without moving his eyes.
"His name," said Sir William lightly, "is Rathbone, a Mr. Guy
Rathbone. He is a barrister and has chambers in the Temple. A
youngish man, I understand, of about seven and twenty."
At the name Charliewood gave a momentary start. He allowed a
slight smile to come upon his lips, and it was not a pleasant smile.
Gouldesbrough saw it, flushed a little and moved uneasily, feeling
that although this man was his servant there were yet disadvantages
in employing him, and that he also could sting when he liked.
Directly Sir William had mentioned the name of the person on whose
actions and life, not to put too fine a point on it, he was ordering his
henchman to become a spy, Charliewood knew the reason. He
realized in an instant what was the nature of the interest Sir William
Gouldesbrough took in Mr. Guy Rathbone, barrister-at-law.
The famous scientist, long, it was said in society, a man quite
impervious to the attractions of the other sex and the passion of
love, had but a few months ago become engaged.
Wealthy as he was, distinguished, handsome and attractive in his
manner, there had not been wanting ladies who would have very
gladly shared and appropriated all these advantages. Like any other
unmarried man in his desirable position, the scientist had been
somewhat pursued in many drawing-rooms. Of late, however, the
pursuit had slackened. Match-making mothers and unappropriated
daughters seemed to have realized that here was a citadel they
could not storm. Six months ago, therefore, society had been all the
more startled to hear of Sir William's engagement to Miss Marjorie
Poole, the only daughter of old Lady Poole of Curzon Street.
Marjorie Poole was the daughter of a rather poor baronet who had
died some years before, the title going to a cousin. Lady Poole was
left with a house in Curzon Street and a sufficient income for her
own life, but that was all. And among many of the women who hunt
society for a husband for their daughters, as a fisherman whips a
stream for trout, the dowager was one of the most conspicuous.
It was said that she had angled for Sir William with an alertness and
unwearying pursuit which was at last crowned by success. More
charitable people, and especially those who knew and liked Miss
Poole, said that the girl would never have lent herself to any
schemes of her mother's unless she had been genuinely fond of the
man to whom she was engaged. There had been much talk and
speculation over the engagement at first, a speculation which had in
its turn died away, and which during the last few weeks had been
again revived by certain incidents.
Eustace Charliewood, whose whole life and business it was to gather
and retail society gossip, was very well aware of the reason which
made people once more wag their heads and hint this or that about
the Gouldesbrough engagement.
Mr. Guy Rathbone had appeared upon the scene, a young barrister
of good family but of no particular fortune. Several times Mr.
Rathbone had been seen skating with Miss Poole at Prince's. At this
or that dance—Sir William Gouldesbrough did not go to dances—
Rathbone had danced a good deal with Miss Poole. Many envious
and linx-like eyes had watched them for some weeks, and men were
beginning to say in the clubs that "young Rathbone is going to put
the scientific Johnny's nose out of joint."
It was this knowledge which caused the little sneering smile to
appear on Charliewood's face, and it gave him pleasure to detect the
human weakness of jealousy in the inscrutable man who held him so
tightly in his grip.
"Well, all right," Charliewood said at length. "I'll do what you want."
"That's a good fellow," Sir William answered, smiling genially, his
whole face lighting up and becoming markedly attractive as it did so,
"you've always been a good friend to me, Charliewood."
"My banking account is very low just at present," the other went on.
"Then I'll write you a cheque at once," Sir William answered, getting
up from his chair and going to the writing-table in the corner of the
room.
Charliewood's face cleared a little. Then he noticed his cigar had
been burning all down one side. He dropped it into an ash-tray and
put his hand in his coat pocket to find a cigarette.
He took out an ordinary silver case, when his eye fell upon the crest
engraved upon the cover. He started and looked again, turning it so
that the light fell full upon it.
The crest of the Charliewood family was a hand with a battle-axe
and the motto, "Ne Morare," and in the usual custom it was
engraved upon Charliewood's own case.
But this was not the Charliewood crest. It was a wyvern charged on
a shield, and the motto consisted of the single word "GARDEZ."
He gave a startled exclamation.
"What's the matter?" Sir William said, turning round sharply.
"I've got some other fellow's cigarette-case," Charliewood answered
in amazement, opening it as he did so.
There was only one cigarette in the case, but there were several
visiting-cards in one compartment, and moreover the name of the
owner was cut in the inside of the lid.
The case dropped from Charliewood's fingers with a clatter, and he
grew quite pale.
"What is it?" his host inquired again.
"Have you been playing some infernal trick on me, Gouldesbrough?"
Charliewood said.
"No; why?"
"Because this cigarette-case, by some strange chance, is the
cigarette-case of the man we've been talking about, this Guy
Rathbone!"
He stood up, thrusting his hands deep into the pockets of the fur
coat as he did so. Then he pulled out a letter, stamped and
addressed and obviously ready for the post.
"Good heavens!" he said, "here's something else. It's a letter for the
post."
"Who is it addressed to?" Sir William asked in a curious voice.
Charliewood looked at it and started again.
"As I live," he answered, "it's addressed to Miss Poole, 100A, Curzon
Street!"
There was a curious silence for a moment or two. Both men looked
at each other, and mingled astonishment and alarm were on the face
of either. The whole thing seemed uncanny. They seemed, while
concocting something like a plot, to have trodden unawares into
another.
Suddenly Charliewood stamped his foot upon the ground and peeled
off his overcoat.
"I've got it," he cried. "Why, of course I've seen the very man myself
this morning. This is his coat, not mine. I went to a hairdresser's this
morning and left my coat in the ante-room while I was going
through a massage treatment. When I came out there was a man
waiting there for his turn, and I must have taken his coat in
exchange for mine. And the man was this Mr. Guy Rathbone, of
course. You know these dark blue coats lined with astrachan are
quite ordinary, everybody is wearing them this year. And I noticed,
by Jove, that the thing seemed a little tight in the cab! It's about the
oddest coincidence that I've ever come across in my life!"
Sir William bowed his head in thought for a minute or two.
"Well, this is the very best opportunity you could have, my dear
fellow," he said, "of making the man's acquaintance. Of course you
can take him back the coat and the cigarette-case at once."
"And the letter?" Charliewood said swiftly. "The letter to Miss Poole?"
Sir William looked curiously at his guest.
"I think," he said slowly, "that I'll just spend half-an-hour with this
letter first. Then you can take it away with the other things. I assure
you that it will look just the same as it does now."
Charliewood shrugged his shoulders.
"Have it your own way," he said contemptuously, "but don't ask me
to open any letters to a lady, that's all."
Sir William flushed up and was about to make an angry reply, when
the door of the study was suddenly thrown open and they saw the
butler standing there.
There was a rustle of skirts in the passage.
"Lady Poole and Miss Poole, sir," said the butler.
 CHAPTER III
NEWS OF A REVOLUTION
Marjorie and Lady Poole came into the room. For two at least of the
people there it was an agonizing moment. But a second before, Sir
William Gouldesbrough had been proposing to steal and open a
letter written by another man to his fiancée. But a second before,
Mr. Eustace Charliewood, the well-known society man, had sullenly
acquiesced in the proposal. And now here was Marjorie Poole
confronting them.
"We thought we'd come to tea, William," Lady Poole said effusively,
going forward to shake hands with her future son-in-law. "Ah, Mr.
Charliewood, how do you do?" She gave him a bright nod, and he
turned to Marjorie, while her mother was shaking hands with the
scientist.
Charliewood's face was flushed a deep red, and his hand trembled
so that the tall girl looked at him in some surprise.
Marjorie Poole was a maiden for whom many men had sighed. The
oval face with its pure olive complexion, the large brown eyes, clear
as a forest pool, the coiled masses of hair, the colour of deeply
ripened corn, made up a personality of singular distinction and
charm. She was the sort of girl of whom people asked, "Who is
she?" And if younger sons and other people who knew that they
could never win and wear her, said that she was a little too reserved
and cold, it was only a prejudiced way of expressing her complete
grace and ease of manner.
"How are you, Mr. Charliewood?" she said in a clear, bell-like voice.
"I haven't seen you since the Carr's dance."
"Well, to tell the truth, Miss Poole," Charliewood answered with a
voice that had a singular tremor in it, "you startled me out of my
wits when you came in. Just a moment before, Sir William had
mentioned your name, and we were both thinking of you when, as
quick as one of those ridiculous entrances on the stage, pat upon
the very word, the butler threw open the door and you came in."
"Oh, a stage entrance!" Marjorie answered. "I don't like stage
entrances;" and turning away she went up to her fiancé, making it
quite clear that, whatever her opinions about the conventions of the
boards might be, she did not like Mr. Charliewood.
The big, light-haired man stayed for a moment or two, made a few
conventional remarks, and then wished his host farewell.
As he crossed the hall he began mechanically to put on the heavy
astrachan coat upon his arm, then, with a muttered curse which
surprised the butler, he took it off again and hurriedly left the house.
"Well, and how are you, William?" said Lady Poole, sitting down by
the fire. "Are you going to give us some tea? We have been paying
calls, and I told Marjorie that we would just come on and see how
you were, in case you might be in. And how is the electricity going?
Why don't you invent a flying-machine? I'm sure it would be more
useful than the things you do invent. How charming it would be to
step out of one's bedroom window into one's aëriel brougham and
tell the man to fly to the Savoy!"
Gouldesbrough did not immediately reply, but old Lady Poole was
used to this.
She was a tall, florid old thing, richly dressed, with an ample and
expansive manner. Now that Sir William had proposed and the
forthcoming marriage was an accepted thing, the good lady felt her
duty was done. Having satisfied herself of Sir William's position, his
banking account and his general eligibility, she cared for nothing
else, and she had grown quite accustomed to the little snubs she
received from his hands from time to time.
Gouldesbrough was looking at Marjorie. His deep blue eyes had
leapt up from their usual intense calm into flame. The thin-cut lips
were slightly parted, the whole man had become humanized and
real in a single moment.
The sinister suggestion had dropped from him as a cloak is thrown
off, and he remembered nothing of the plot he had been hatching,
but only saw before him the radiant girl he adored with all the force
of his nature and all the passion of a dark but powerful soul, to
which love had never come before.
"How are you, dearest?" he said anxiously. "Do you know that I
haven't heard from you or seen you for nearly four days? Tell me all
that you have been doing, all that you have been thinking."
"Four days, is it?" Lady Poole broke in. "Well, you know, my dear
William, you will have plenty of time with Marjorie in the future, you
mustn't attempt to monopolize her just at present. There have been
so many engagements, and I'm sure you have been entirely happy
with the electricity, haven't you? Such a comfort, I think, to have a
hobby. It gives a real interest in life. And I'm sure, when a hobby like
yours has proved so successful, it's an additional advantage. I have
known so many men who have been miserable because they have
never had anything to do to amuse them. And unless they take up
wood-carving or fretwork or something, time hangs so heavily, and
they become a nuisance to their wives. Poor Sir Frederick only took
up tact as a hobby. Though that was very useful at a party, it was
horribly boring in private life. One always felt he understood one too
well!"
Up to the present Marjorie had said nothing. She seemed slightly
restless, and the smile that played about her lips was faint and
abstracted. Her thoughts seemed elsewhere, and the scrutiny of the
deep blue eyes seemed slightly to unnerve her.
At that moment the butler entered, followed by a footman carrying a
tea-table.
Marjorie sank down with a sigh of relief.
"I'm so tired," she said in a quiet voice. "Mother's been dragging me
about to all sorts of places. William, why do you have that horrid
man, Eustace Charliewood, here? He always seems about the house
like a big tame cat. I detest him."
Gouldesbrough winced at the words. He had put his hand into the
side-pocket of his coat, and his fingers had fallen upon a certain
letter. Ah! why, indeed, did he have Charliewood for a friend?
His answer was singularly unconvincing, and the girl looked at him in
surprise. He was not wont to speak thus, with so little directness.
"Oh, I don't know, dear," he answered. "He's useful, you know. He
attends to a lot of things for me that I'm too busy to look after
myself."
Again Marjorie did not answer.
"What have you been doing, William?" she said at length, stirring the
tea in her cup.
"I've been thinking about you principally," he answered.
She frowned a little. "Oh, I don't mean in that way," she answered
quickly. "Tell me about real things, important things. What are you
working at now? How is your work going?"
He noticed that something like enthusiasm had crept into her voice—
that she took a real interest in his science. His heart throbbed with
anger. It was not thus that he wished to hear her speak. It was he
himself, not his work, that he longed with all his heart and soul this
stately damsel should care about.
But, resolute always in will, completely master of himself and his
emotion, he turned at once and began to give her the information
which she sought.
And as he spoke his voice soon began to change. It rang with power.
It became vibrant, thrilling. There was a sense of inordinate strength
and confidence in it.
While old Lady Poole leant back in her chair with closed eyes and a
gentle smile playing about her lips, enjoying, in fact, a short and
well-earned nap, the great scientist's passionate voice boomed out
into the room and held Marjorie fascinated.
She leant forward, listening to him with strained attention—her lips a
little parted, her face alight with interest, with eagerness.
"You want to hear, dearest," he said, "you want to hear? And to
whom would I rather tell my news? At whose feet would I rather lay
the results of all I am and have done? Listen! Even to you I cannot
tell everything. Even to you I cannot give the full results of the
problems I have been working at for so many years. But I can tell
you enough to hold your attention, to interest you, as you have
never been interested before."
He began to speak very slowly.
"I have done something at last, after years of patient working and
thought, which it is not too much to say will revolutionize the whole
of modern life—will revolutionize the whole of life, indeed, as it has
never been changed before. All the other things I have done and
made, all the results of my scientific work have been but off-shoots
of this great central idea, which has been mine since I first began.
The other things that have won me fame and fortune were
discovered upon the way towards the central object of my life. And
now, at last, I find myself in full possession of the truth of all my
theories. In a month or two from now my work will be perfected,
then the whole world will know what I have done. And the whole
world will tremble, and there will be fear and wonder in the minds of
men and women, and they will look at each other as if they
recognized that humanity at last was waking out of a sleep and a
dream."
"Is it so marvellous as all that?" she said almost in a whisper, awed
by the earnestness of his manner.
"I am no maker of phrases," he replied, "nor am I eloquent. I cannot
tell you how marvellous it is. The one great citadel against which
human ingenuity and time have beaten in vain since our first
forefathers, is stormed at last! In my hands will shortly be the keys
of the human soul. No man or woman will have a secret from me.
The whole relation of society will be changed utterly."
"What is it? What is it?" she asked with a light in her eyes. "Have
you done what mother said in jest? Have you indeed finally
conquered the air?"
He waved his hand with a scornful gesture.
"Greater far—greater than that," he answered. "Such a vulgar and
mechanical triumph is not one I would seek. In a material age it is
perhaps a great thing for this or that scientist to invent a means of
transit quicker and surer than another. But what is it, after all? Mere
accurate scientific knowledge supplemented by inventive power. No!
Such inventions as the steam-engine, printing, gun-powder, are
great in their way, but they have only revolutionized the surface of
things; the human soul remains as it was before. What I now know
is a far, far loftier and more marvellous thing."
In his excitement he had risen and was bending over her.
Now she also rose, and stared into his face with one hand upon his
arm.
"Oh, tell me," she said, "what in life can be so strange, so terrible in
its effects as this you speak of?"
"Listen," he answered once more. "You know what light is? You
know that it can be split up into its component parts by means of
the prism in the spectroscope?"
"Every child knows that to-day," she answered.
"Good!" he replied. And he went on. "I am putting this in the very
simplest possible language. I want you to see the broadest, barest,
simplest outlines. Do you know anything of the human mind? What
should you say hypnotism was, for instance, in ordinary words?"
"Surely," she replied, "it is the power of one brain acting upon
another."
"Exactly," he said, "and in what way? How is a brain, not physically
touching another brain, able to influence it?"
"By magnetism," she replied, "by"—she hesitated for a word—"by a
sort of current passing from one brain to another."
He held out both his hands in front of him. They were clasped, and
she saw that his wrists were shaking. He was terribly excited.
"Yes," he went on, his voice dropping lower and lower and becoming
even more intense, "you have said exactly the truth. The brain is a
marvellous instrument, a sensitive instrument, an electric instrument
which is constantly giving out strange, subtle, and hitherto
uninvestigated currents. It is like the transmitter at the top of Signor
Marconi's wireless telegraphy station. Something unseen goes out
into the air, and far away over the Mother of Oceans something
answers to its influence. That is exactly what happens with the
human brain. Countless experiments have proved it, the scientists of
the world are agreed."
"Then——?" she said.
"Supposing I had discovered how to collect these rays or vibrations,
for that is the better word, these delicate vibrations which come
from the human brain?"
"I think I begin to see," Marjorie said slowly, painfully, as if the
words were forced from her and she spoke them under great
emotion. "I think I begin to see a little light."
"Ah," he answered, "you are always above ordinary women. There is
no one in the world like you. Your brain is keen, subtle, strong. You
were destined for me from the first."
Once more, even in the midst of her excitement, a shade passed
over her face. She touched him on the arm again.
"Go on! Tell me! Not this, not that. Tell me about the work!"
"I," he repeated, "I alone of all men in the world have learnt how to
collect the invisible vibrations of thought itself. Now, remember what
I told you at first. I mentioned Light, the way in which Light can be
passed through a prism, split up into its component parts, and give
the secret of its composition to the eye of the scientist. Not only can
I collect the mysterious vibrations of the human brain, but I can pass
them through a spectroscope more marvellous than any instrument
ever dreamt of in the history of the world. I can take the vibrations
of thought, and discover their consistency, their strength, their
meaning."

She stared at him incredulously. "Even yet," she said, "I fail to see
the ultimate adaptation of all this. I realize that you have discovered
a hitherto unproved truth about the mechanism of thought. That is
an achievement which will send your name ringing down the
avenues of the future. But there seems to be something behind all
you are telling me. You have more to say. What is the practical
outcome of all this, this theoretical fact?"
"It is this," he answered. "I hold in my hands the power to know
what this or that person, be it a king upon his throne, a girl on her
wedding day, or a criminal in the dock, is thinking at any given
moment."
She started from him with a little cry. "Oh no," she said, and her face
had grown very white indeed. "Oh no, God would not allow it. It is a
power only God has."
He laughed, and in his laugh she heard something that made her
shrink back still further. It was a laugh such as Lucifer might have
laughed, who defied a Power which he would not acknowledge to be
greater than his.
"You will never do that," she said, "wonderful as you are."
"Marjorie," he answered, "I am a man with a brain that theorizes,
but never ventures upon a statement that cannot be proved by fact.
If I tell you this, if I hint broadly at the outcome of my life's work, I
am doing so, believe me, because I have chapter and verse for all I
say, because I can prove that it has passed from the dim realms of
theory and of hope into the brilliant daylight of actual achievement!"
She stared at him. His words were too much for her mind to grasp
immediately.
It was an intense moment.
But, as in real life intense moments generally are, it was broken by a
curious interruption.
A voice came thickly from the arm-chair by the fire, where old Lady
Poole had been reclining in placid sleep. It was the strange voice of
one who sleeps, without expression, but perfectly distinct.
"I will not have it, cook—(indistinguishable murmur)—explained
when I engaged you—will not have men in the kitchen!"
Sir William and Marjorie looked at each other for a moment with
blank faces. Then, all overstrung as they were, the absurdity of the
occurrence struck them at the same moment, and they began to
laugh softly together.
It was a little pleasant and very human interlude in the middle of
these high matters, and at that moment the great man felt that he
was nearer to Marjorie than he had been before at any other
moment of the afternoon. She no longer hung entranced upon his
impassioned and wonderful words, she laughed with him quite
quietly and simply.
Lady Poole snored deeply, and no longer vocalized the drama of her
domestic dream.
Suddenly Marjorie turned back once more to Sir William.
"It's only mother dreaming about one of the servants we have had
to send away," she said. "What a stupid interruption! Now, go on, go
on!"
Her voice recalled him to his marvellous story.
"Tell me what is the actual achievement," she said.
"It is this. When you speak into a telephone the vibrations of your
voice agitate a sensitive membrane, and by means of electricity the
vibrations are conveyed to almost any distance. When Madame
Melba sings into the gramophone, her voice agitates the membrane,
which in its turn agitates a needle, which in its turn again makes
certain marks upon a waxen disc."
"Yes, go on, go on!"
"When I put a certain instrument upon the head of a man or a
woman, when I surround the field of emanation by a shield which
captures the vibrations, they are conducted to a receiver more
delicate and sensitive than anything which has ever been achieved
by scientific process before. That receiver collects these vibrations
and can transmit them, just in the manner of a telephone or
telegraph wire, for almost any distance."
"And at the other end?" Marjorie asked.
"It has been a difficulty of ten long, anxious, unwearying years."
"And now?"
"Now that difficulty has been finally overcome."
"Therefore?"
"What a person thinks in London can be sent in vibrations along a
wire to Paris."
"I see. I understand! But when there they can only be transmitted to
another brain, of course. You mean that you have invented a more
marvellous system of telegraphy than has ever been invented
before. For instance, I could sit here in this room and communicate
with you with absolute freedom in Paris. How wonderful that is!
What a triumphant achievement! But—but, William, marvellous as it
is, you do not substantiate what you said just now. The secrets of
thought may be yours, but only when the sender wills it."
"Ah," he answered, with a deep note of meaning coming into his
voice. "If I had only discovered what you say, I should have
discovered much. But I have gone far, far away from this. I have
done much, much more. And in that lies the supreme value of my
work."
Once more they were standing together, strained with wonder, with
amazement and triumph passing between them like the shuttle of a
loom; once more she was caught up into high realms of excitement
and dawning knowledge, the gates of which had never opened to
her brain before.
"To come back to the phonograph," Sir William said. "The marks are
made upon the waxen disc, and they are afterwards reproduced in
sound, recorded upon metal plates to remain for ever as a definite
reproduction of the human voice. Now, and here I come to the final
point of all, I have discovered a means by which thought can be
turned into actual vision, into an actual expression of itself for every
one to read. What I mean is this. I have discovered the process, and
I have invented the machine by which, as a person thinks, the
thought can be conveyed to any distance along the wire, can be
received at the other end by an instrument which splits it up into this
or that vibration. And these vibrations actuate upon a machine by
the spectroscope, by the bioscope, which show them upon a screen
in the form of either pictures or of words as the thoughts of the
thinker are at that moment sent out by the brain in words or
pictures."
"Then what does this mean?"
"It means that once my apparatus, whether by consent of the
subject or by force, is employed to collect the thought vibrations,
then no secrets can be hidden. The human soul must reveal itself.
Human personality is robbed of its only defence. There will be no
need to try the criminal of the future. He must confess in spite of
himself. The inviolability of thought is destroyed. The lonely citadel
of self exists no longer. The pious hypocrite must give his secret to
the world, and sins and sinners must confess to man what only God
knew before."
Marjorie sat down in her chair and covered her face with her hands.
Various emotions thronged and pulsed through her brain. The
stupendous thing that this man had done filled her with awe for his
powers, with terror almost, but with a great exultation also. She did
not love him, she knew well that she had never loved him, but she
realized her influence over him. She knew that this supreme intellect
was hers to do with as she would. She knew that if he was indeed,
as he said, master of the world, she was mistress of his mind, she
was the mistress of him. The mysterious force of his love, greater
than any other earthly force which he could capture or control, had
made him, who could make the minds of others his slaves and
instruments, the slave of her.
Yes! Love! That, after all, was the greatest force in the whole world.
Here was a more conclusive proof than perhaps any woman had
ever had before in the history of humanity.
Love! Even while the inmost secrets of nature were wrested from
her by such a man as this, love was still his master, love was still the
motive power of the world.
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