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Stem Cell Biology and Regenerative Medicine

Fikrettin Şahin
Ayşegül Doğan
Selami Demirci Editors

Dental
Stem Cells
Stem Cell Biology and Regenerative Medicine

Series Editor
Kursad Turksen, Ph.D.
[email protected]

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

Fikrettin Şahin • Ayşegül Doğan
Selami Demirci
Editors

Dental Stem Cells

Editors
Fikrettin Şahin Ayşegül Doğan
Genetics and Bioengineering Department Genetics and Bioengineering Department
Yeditepe University Yeditepe University
Istanbul, Turkey Istanbul, Turkey

Selami Demirci
Genetics and Bioengineering Department
Yeditepe University
Istanbul, Turkey

ISSN 2196-8985 ISSN 2196-8993 (electronic)

Stem Cell Biology and Regenerative Medicine
ISBN 978-3-319-28945-8 ISBN 978-3-319-28947-2 (eBook)
DOI 10.1007/978-3-319-28947-2

Library of Congress Control Number: 2016931885

© Springer International Publishing Switzerland 2016

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

Printed on acid-free paper

This Springer imprint is published by Springer Nature

The registered company is Springer International Publishing AG Switzerland
Preface

Stem cells are a class of undifferentiated master cells that have robust self-renewal
kinetic and differentiation potential into many specialized cell types in the body.
Stem cell research has been a field of great clinical interest with immense pos-
sibilities of using the stem cells to replace, restore, or enhance the biological func-
tion of damaged tissues and organs due to accidents, diseases, and/or developmental
defects.
Recent studies have demonstrated that mesenchymal stem cells (MSCs) are
found in various tissues in an adult organism. MSCs derived from teeth and support-
ing tissues, called dental stem cells (DSCs), have been mainly characterized into
five different cell types including dental pulp stem cells (DPSCs), dental follicle
stem cells (DFSCs), periodontal ligament stem cells (PDLSCs), stem cells from
human exfoliated deciduous teeth (SHEDs), and stem cells from the apical papilla
(SCAPs).
The knowledge of stem cell technology is moving extremely fast in both dental
and medical fields. Advances in DSC characterization, standardization, and valida-
tion of stem cell therapies and applications have been leading to the development of
novel therapeutic strategies.
Several investigators, especially those who have made significant contribution to
the field of DSC research, have been invited to create this book. With the help of
their intense and substantive efforts, this book reviews different aspects, challenges,
and gaps of basic and applied dental stem cell research, cell-based therapies in
regenerative medicine concentrating on the application and clinical use, and recent
developments in cell programming and tissue engineering. This review will be use-
ful to students, teachers, clinicians, and scientists, who are interested or working in
the fields of biology and medical sciences related to dental stem cell therapy and
related practices.

Istanbul, Turkey Fikrettin Şahin

v
Contents

1 Dental and Craniofacial Tissue Stem Cells: Sources

and Tissue Engineering Applications .................................................... 1
Paul R. Cooper
2 Immunomodulatory Properties of Stem Cells Derived
from Dental Tissues ................................................................................ 29
Pakize Neslihan Taşlı, Safa Aydın, and Fikrettin Şahin
3 miRNA Regulation in Dental Stem Cells:
From Development to Terminal Differentiation .................................. 47
Sukru Gulluoglu, Emre Can Tuysuz, and Omer Faruk Bayrak
4 Signaling Pathways in Dental Stem Cells During
Their Maintenance and Differentiation ................................................ 69
Genxia Liu, Shu Ma, Yixiang Zhou, Yadie Lu, Lin Jin, Zilu Wang,
and Jinhua Yu
5 Genetically Engineered Dental Stem Cells
for Regenerative Medicine ..................................................................... 93
Valeriya V. Solovyeva, Andrey P. Kiyasov, and Albert A. Rizvanov
6 Dental Stem Cells vs. Other Mesenchymal Stem Cells:
Their Pluripotency and Role in Regenerative Medicine ..................... 109
Selami Demirci, Ayşegül Doğan, and Fikrettin Şahin
7 Induced Pluripotent Stem Cells Derived from Dental
Stem Cells: A New Tool for Cellular Therapy...................................... 125
Irina Kerkis, Cristiane V. Wenceslau, and Celine Pompeia
8 Dental Stem Cells in Oral, Maxillofacial
and Craniofacial Regeneration .............................................................. 143
Arash Khojasteh, Pantea Nazeman, and Maryam Rezai Rad
9 Dental Stem Cells: Possibility for Generation of a Bio-tooth.............. 167
Sema S. Hakki and Erdal Karaoz

vii
viii Contents

10 Dental Stem Cells for Bone Tissue Engineering ................................... 197

Zhipeng Fan and Xiao Lin
11 Dental Stem Cells: Their Potential in Neurogenesis
and Angiogenesis ..................................................................................... 217
Annelies Bronckaers, Esther Wolfs, Jessica Ratajczak,
Petra Hilkens, Pascal Gervois, Ivo Lambrichts, Wendy Martens,
and Tom Struys
12 Dental Stem Cell Differentiation Toward Endodermal
Cell Lineages: Approaches to Control Hepatocytes
and Beta Cell Transformation ............................................................... 243
Nareshwaran Gnanasegaran, Vijayendran Govindasamy,
Prakash Nathan, Sabri Musa, and Noor Hayaty Abu Kasim
13 Dental Stem Cells in Regenerative Medicine: Clinical
and Pre-clinical Attempts ....................................................................... 269
Ferro Federico and Renza Spelat
14 Future Perspectives in Dental Stem Cell Engineering
and the Ethical Considerations .............................................................. 289
Naohisa Wada, Atsushi Tomokiyo, and Hidefumi Maeda

Index ................................................................................................................. 309

Contributors

Annelies Bronckaers Group of Morphology, Biomedical Research Institute (BIOMED),

Hasselt University, Diepenbeek, Belgium
Safa Aydın Department of Genetics and Bioengineering, Faculty of Engineering
and Architecture, Yeditepe University, Istanbul, Turkey
Omer Faruk Bayrak Department of Medical Genetics, Yeditepe University Medical
School and Yeditepe University Hospital, Istanbul, Turkey
Paul R. Cooper Oral Biology, School of Dentistry, College of Medical and Dental
Sciences, University of Birmingham, Birmingham, UK
Selami Demirci Genetics and Bioengineering Department, Yeditepe University,
Istanbul, Turkey
Ayşegül Doğan Genetics and Bioengineering Department, Yeditepe University,
Istanbul, Turkey
Esther Wolfs Group of Morphology, Biomedical Research Institute (BIOMED),
Hasselt University, Diepenbeek, Belgium
Zhipeng Fan Capital Medical University School of Stomatology, Beijing, China
Ferro Federico Network of Excellence for Functional Biomaterials, National University
of Ireland, Galway, Ireland
Nareshwaran Gnanasegaran GMP Compliant Stem Cell Laboratory, Hygieia
Innovation Sdn. Bhd, Federal Territory of Putrajaya, Malaysia
Department of Restorative Dentistry, Faculty of Dentistry, University of Malaya,
Kuala Lumpur, Malaysia
Vijayendran Govindasamy GMP Compliant Stem Cell Laboratory, Hygieia
Innovation Sdn. Bhd, Federal Territory of Putrajaya, Malaysia

ix
x Contributors

Sukru Gulluoglu Department of Medical Genetics, Yeditepe University Medical

School and Yeditepe University Hospital, Istanbul, Turkey
Department of Biotechnology, Institute of Science, Yeditepe University, Istanbul,
Turkey
Sema S. Hakki Faculty of Dentistry, Department of Periodontology, Selcuk University,
Konya, Turkey
Ivo Lambrichts Group of Morphology, Biomedical Research Institute (BIOMED),
Hasselt University, Diepenbeek, Belgium
Jessica Ratajczak Group of Morphology, Biomedical Research Institute (BIOMED),
Hasselt University, Diepenbeek, Belgium
Lin Jin Key Laboratory of Oral Diseases of Jiangsu Province and Stomatological
Institute of Nanjing Medical University, Nanjing, Jiangsu, China
Erdal Karaoz Liv Hospital, Center for Regenerative Medicine and Stem Cell
Research & Manufacturing (LivMedCell), Ulus-Beşiktaş, İstanbul, Turkey
Noor Hayaty Abu Kasim Department of Restorative Dentistry, Faculty of Dentistry,
University of Malaya, Kuala Lumpur, Malaysia
Irina Kerkis Laboratory of Genetics, Butantan Institute, São Paulo, Brazil
Arash Khojasteh School of Advanced Technologies in Medicine, Shahid Beheshti
University of Medical Sciences, Tehran, Iran
Faculty of Medicine, University of Antwerp, Antwerp, Belgium
Andrey P. Kiyasov Institute of Fundamental Medicine and Biology, Kazan (Volga
Region) Federal University, Kazan, Russia
Xiao Lin Capital Medical University School of Stomatology, Beijing, China
Genxia Liu Key Laboratory of Oral Diseases of Jiangsu Province and Stomatological
Institute of Nanjing Medical University, Nanjing, Jiangsu, China
Yadie Lu Key Laboratory of Oral Diseases of Jiangsu Province and Stomatological
Institute of Nanjing Medical University, Nanjing, Jiangsu, China
Shu Ma Key Laboratory of Oral Diseases of Jiangsu Province and Stomatological
Institute of Nanjing Medical University, Nanjing, Jiangsu, China
Hidefumi Maeda Department of Endodontology and Operative Dentistry, Division
of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, Fukuoka,
Japan
Sabri Musa Department of Paediatric Dentistry and Orthodontics, Faculty of
Dentistry, University of Malaya, Kuala Lumpur, Malaysia
Contributors xi

Prakash Nathan GMP Compliant Stem Cell Laboratory, Hygieia Innovation Sdn.
Bhd, Federal Territory of Putrajaya, Malaysia
Pantea Nazeman Research Institute of Dental Sciences, School of Dentistry, Shahid
Beheshti University of Medical Sciences, Tehran, Iran
Pascal Gervois Group of Morphology, Biomedical Research Institute (BIOMED),
Hasselt University, Diepenbeek, Belgium
Petra Hilkens Group of Morphology, Biomedical Research Institute (BIOMED),
Hasselt University, Diepenbeek, Belgium
Celine Pompeia Laboratory of Genetics, Butantan Institute, São Paulo, Brazil
Maryam Rezai Rad Research Institute of Dental Sciences, School of Dentistry,
Shahid Beheshti University of Medical Sciences, Tehran, Iran
Renza Spelat Network of Excellence for Functional Biomaterials, National University
of Ireland, Galway, Ireland
Albert A. Rizvanov Institute of Fundamental Medicine and Biology, Kazan (Volga
Region) Federal University, Kazan, Russia
Fikrettin Şahin Genetics and Bioengineering Department, Yeditepe University,
Istanbul, Turkey
Valeriya V. Solovyeva Institute of Fundamental Medicine and Biology, Kazan
(Volga Region) Federal University, Kazan, Russia
Pakize Neslihan Taşlı Department of Genetics and Bioengineering, Faculty of
Engineering and Architecture, Yeditepe University, Istanbul, Turkey
Tom Struys Group of Morphology, Biomedical Research Institute (BIOMED),
Hasselt University, Diepenbeek, Belgium
Atsushi Tomokiyo Division of Oral Rehabilitation, Department of Endodontology
and Operative Dentistry, Faculty of Dental Science, Kyushu University, Fukuoka,
Japan
Emre Can Tuysuz Department of Biotechnology, Institute of Science, Yeditepe
University, Istanbul, Turkey
Naohisa Wada Division of General Dentistry, Kyushu University Hospital, Kyushu
University, Fukuoka, Japan
Zilu Wang Key Laboratory of Oral Diseases of Jiangsu Province and Stomato-
logical Institute of Nanjing Medical University, Nanjing, Jiangsu, China
Cristiane V. Wenceslau Laboratory of Genetics, Butantan Institute, São Paulo, Brazil
Wendy Martens Group of Morphology, Biomedical Research Institute (BIOMED),
Hasselt University, Diepenbeek, Belgium
xii Contributors

Jinhua Yu Key Laboratory of Oral Diseases of Jiangsu Province and Stomatologi-

cal Institute of Nanjing Medical University, Nanjing, Jiangsu, China
Institute of Stomatology, Nanjing Medical University, Nanjing, Jiangsu, China
Yixiang Zhou Key Laboratory of Oral Diseases of Jiangsu Province and
Stomatological Institute of Nanjing Medical University, Nanjing, Jiangsu, China
About the Editors

Fikrettin Şahin received his PhD from the College of Food, Agricultural, and
Environmental Sciences, The Ohio State University, Columbus, Ohio. After com-
pleting his postdoctoral research at the same university and at the Pest Management
Research Centre of Agriculture and Agri-Food Canada, he worked as an assistant
professor in the Department of Plant Pathology, Ataturk University, in Erzurum,
Turkey. Dr. Şahin is now a professor and chair of the Department of Genetics and
Bioengineering, Yeditepe University, İstanbul, Turkey. He is on the advisory board
of several journals, a member of the Technology and Innovation Support Programme
(TEYDEP), and a principal member of the Turkish Academy of Sciences and many
other prestigious scientific committees and initiatives. Prolifically and internation-
ally published, Dr. Şahin’s research focuses on dental stem cells in the contexts of
isolation, maintenance, differentiation, and possible use for particular regenerative
approaches. His other research areas include molecular microbiology, phytopathol-
ogy, stem cell and gene therapy, and cancer.

Ayşegül Doğan received her PhD from Yeditepe University in Istanbul, Turkey,
where she is a postdoctoral researcher in the Department of Genetics and
Bioengineering. She works with the Gene and Cell Therapy group at the University’s
Molecular Diagnostic Laboratory and is a member of the Stem Cell and Cellular
Therapies Society in Turkey. Her research focuses on mesenchymal stem cells, gene
and cell therapy, cancer, and wound healing. Dr. Doğan is currently working with
dental stem cells obtained from wisdom teeth of young adults and the potential use
of these cells in gene and stem cell therapy applications.

Selami Demirci received his PhD from the department of Genetics and
Bioengineering at the University of Yeditepe in Istanbul, Turkey. He is currently a
research fellow at the same department. Dr. Demirci is a member of the Stem Cell
and Cellular Therapies Society, Turkey, and has completed several projects on den-
tal stem cell maintenance and differentiation toward desired cell lineages for a par-
ticular regeneration approach. His ongoing studies include gene functions in stem
cell, wound healing, and regenerative medicine.

xiii
Chapter 1
Dental and Craniofacial Tissue Stem Cells:
Sources and Tissue Engineering Applications

Paul R. Cooper

Abbreviations

ADSCs Adipose stromal/stem cells

BMP Bone morphogenetic protein
BMMSCs Bone marrow stromal cells
DFSCs Dental follicle stem cells
DSCs Dental stem cells
DPSCs Dental pulp stem cells
EGF Epidermal growth factor
DMP1 Dentin matrix protein 1
DSPP Dentin sialophosphoprotein
ESC Embryonic stem cell
FBS Fetal bovine serum
ECM Extracellular matrix
FGF Fibroblast growth factor
GMP Good manufacturing practice
GMSCs Gingiva-derived MSCs
HERS Hertwig’s epithelial root sheath
HS Human serum
IEE Inner enamel epithelium
iPSC Induced pluripotent stem cell
OEE Outer enamel epithelium
OESCs Oral epithelial progenitor/stem cells
PDL Periodontal ligament

P.R. Cooper (*)

Oral Biology, School of Dentistry, College of Medical and Dental Sciences,
University of Birmingham, St. Chad’s Queensway, Birmingham B4 6NN, UK
e-mail: [email protected]

© Springer International Publishing Switzerland 2016 1

F. Şahin et al. (eds.), Dental Stem Cells, Stem Cell Biology
and Regenerative Medicine, DOI 10.1007/978-3-319-28947-2_1
2 P.R. Cooper

PDLSCs Periodontal ligament stem cells

PSCs Periosteum-derived stem cells
SCAPs Stem cells from apical papilla
SGSCs Salivary gland-derived stem cells
SHEDs Stem cells from human exfoliated deciduous teeth
Shh Sonic hedgehog
SR Stellate reticulum
TGF-β Transforming growth factor-β
TGPCs Tooth germ progenitor cells
TMJ Temporomandibular joint
VEGF Vascular endothelial growth factor

1.1 Introduction

Stem cells are present in many tissues throughout the body and at the different
developmental stages of the organism. They are reported to reside in specific areas
within each tissue, in a so called “stem cell niche”. They are also frequently
described as being located within close proximity to the vasculature, i.e. in a peri-
vascular niche [1–3], and this anatomical localisation may facilitate their rapid
mobilisation to sites of injury [4]. Stem cells have been characterised based on their
abilities to self-renew, along with their multi-lineage differentiation capabilities
which enable complex tissue regeneration [5]. They have varying degrees of
potency ranging from totipotent, pluripotent, multipotent through to unipotent.
Totipotent stem cells are derived from the zygote, and can form embryonic and
extra-embryonic tissues, including the ability to generate the placenta [6].
Pluripotent stem cells include embryonic stem cells (ESCs), and are derived from
the inner cell mass of the developing blastocyst. Notably, ESCs can differentiate
into the three main germ layers of the organism including the endoderm, mesoderm
and ectoderm. Postnatal/adult stem cells are regarded as being multipotent and
include populations of hematopoietic and mesenchymal stem cells (MSCs). They
are capable of differentiating toward several germ layer lineages giving rise to cell
types which are necessary for natural organ and tissue turn-over and repair. In addi-
tion, along with these naturally present stem cell types, induced pluripotent stem
cells (iPSCs) have been generated within laboratory settings by transcriptional
reprogramming of somatic cells. Notably, sources of these somatic cells have
included ones of oral and dental origin. iPSCs are reprogrammed to an embryonic-
like state and hence are pluripotent and can differentiate into cells of all three germ
layers [7, 8].
The dental and craniofacial tissues are known to be a rich source of MSCs which
are relatively easily accessible for dentists. Stem cell populations which have been
identified and characterised within these tissues include dental pulp stem cells
(DPSCs) [9], stem cells from the apical papilla (SCAPs) [10–12], dental follicle
1 Dental and Craniofacial Tissue Stem Cells: Sources and Tissue Engineering… 3

Fig. 1.1 The locations of developmental and postnatal stem cell populations in the dental and
craniofacial region indicating sources for isolation from the mandible and teeth. The insert (to the
right) shows the histology of the overlying masticatory mucosa (including oral epithelium, submu-
cosa and bone tissue) and indicates the locations of the stem cell populations within it. Further
details on all the stem cell populations shown are provided in the main text body. Abbreviations
used are: BMMSCs—bone marrow-derived mesenchymal stem cells (MSCs) from mandible (also
maxilla); DPSCs—dental pulp stem cells; SHEDs—stem cells from human exfoliated deciduous
teeth; PDLSCs—periodontal ligament stem cells; DFSCs—dental follicle stem cells; TGPCs—
tooth germ progenitor cells; SCAPs—stem cells from the apical papilla; OESCs—oral epithelial
progenitor/stem cells; GMSCs—gingiva-derived MSCs; PSCs—periosteum-derived stem cells;
SGSCs—salivary gland-derived stem cells

precursor cells (DFSCs) [13–16], periodontal ligament stem cells (PDLSCs) [17,
18], stem cells from human exfoliated deciduous teeth (SHEDs) [19] and tooth
germ progenitor cells (TGPCs) [20]. Furthermore, the presence of other, perhaps as
yet less well characterised stem cell types within the orofacial region have been
reported including oral epithelial progenitor/stem cells (OESCs) [21], gingiva-derived
MSCs (GMSCs) [22, 23], periosteum-derived stem cells (PSCs) [24] and salivary
gland-derived stem cells (SGSCs) [25–27]. In addition, well characterised MSCs
which are not exclusive to the oral and craniofacial tissues, include bone marrow-
derived MSCs (BMMSCs) [28], which can be harvested from maxilla and mandibu-
lar bone, as well as adipose tissue-derived stem cells (ADSCs) [29]. These stem cell
populations and their isolation and application will be discussed in greater detail in
the following sections. Figure 1.1 pictorially shows the dental and craniofacial loca-
tions of these stem cell groups.
The oral and dental stem cell (DSC) populations are defined as MSCs according
to the minimal criteria proposed by the International Society for Cellular Therapy
(ISCT) in 2006 [30]. The criteria defining them, which are tissue independent,
include their ability to adhere to standard tissue cultureware along with their expres-
sion profile of Cluster of Differentiation (CD) and other markers. According to the
ISCT, MSCs should express CD105, CD73 and CD90 but lack expression of CD45,
CD34, CD14 or CD11b, CD79a or CD19, and HLA-DR cell surface molecules.
4 P.R. Cooper

More recently, the expression of other cell surface markers for human MSCs,
including CD271 and MSC antigen-1, have been reported [31, 32]. The presence of
(or lack of) combinations of these markers are not only used to define stem cell
populations but are also used for their isolation, although across species, this may
not be entirely reproducible. Further defining criteria from the ISCT state that MSCs
must be capable of differentiating into osteogenic, adipogenic and chondrogenic
lineages in vitro [33].
The harvesting of MSCs from postnatal dental, craniofacial and other tissues is
not always straightforward and this can be hampered by these cells being present at
relatively low frequencies within tissues, i.e. <1 % of the total cell population. The
simplest approach for isolating postnatal MSCs utilises their ability to adhere to
cultureware which was initially demonstrated for BMMSCs [34]. This approach
has also been used for craniofacial and dental MSCs, and generates a heteroge-
neous population of cells which exhibit the MSC-like properties of clonogenicity
and a high proliferative capacity [9, 19]. However, frequently reported in the litera-
ture is the increasing use of fluorescence-activated cell sorting (FACs) and mag-
netic activated cell sorting (MACs) approaches [35]. These methods enable the
isolation of cells from dissociated tissue which are positive and/or negative for
many of the defining markers previously described. For DPSC isolation, several
studies have applied positive selection for a range of different markers including
STRO-1, CD105, c-kit, CD34 and low-affinity nerve-growth-factor receptor
(LNGFR) with negative selection for CD31 and CD146 [36–40]. These studies
indicate that the dental pulp likely contains several different MSC populations/
niches, and this is also probably true for other dental and craniofacial tissues. It
should, however, be noted that selection of MSCs using STRO-1, CD146 and peri-
cyte-associated antigen also supports the premise that perivascular niches exist in a
variety of tissues throughout the body including those from the dental and cranio-
facial regions [9, 11, 19, 41].
Recent work has also built upon the cultureware adhesion approach initially
reported for BMMSC isolation with studies now demonstrating that several MSC-
types can be derived via selective adhesion to cultureware surfaces coated with
extracellular matrix (ECM) derived molecules. This potentially biomimetic approach
may be based on the in vitro recapitulation of the niche environment whereby MSCs
in vivo are maintained in a quiescent state by the ECM until released and activated
during tissue disease or trauma. This MSC selection technique has been shown to be
successfully applied using ECM-derived proteins such as fibronectin, type I colla-
gen, type II collagen, vitronectin, laminin and poly-L-lysine [42–45].
It is also notable that isolated cells may not always be of a pure population and
may be somewhat heterogeneous in nature, subsequently representing various dif-
ferentiation states. It remains unclear, and is under considerable debate, as to
whether a pure population of cells is indeed needed for therapeutic application, as
within tissues stem cells interact with a variety of other cell types to enable repair.
Further confounding this issue is the fact that MSCs are derived from different
donors, e.g. age range and sexes, and isolated cells may subsequently respond dif-
ferently in vitro and in vivo [28]. Current research, therefore, aims to identify the
1 Dental and Craniofacial Tissue Stem Cells: Sources and Tissue Engineering… 5

most appropriate isolation conditions which will enable predictable clinical applica-
tion and outcomes.
Over the coming years within the dental field, stem cells combined with tissue
engineering strategies are expected to provide novel therapeutic approaches to
regenerate teeth or tooth component tissue and for repair of defects in periodontal
tissues and alveolar bone. Specific oral tissues and organs which are already being
targeted for regenerative medicine strategies include the salivary glands, tongue,
craniofacial skeletal muscles, and component structures of the temporomandibular
joint. The properties and characteristics of craniofacial and dentally relevant MSCs
are subsequently discussed below as is dental tissue development, tissue engineer-
ing and clinical application progress.

1.2 Dental Tissue Development and Repair

In general, the development of many organs requires heterologous cell and tissue
interactions. For tooth development these interactions occur between the
ectodermally-derived enamel organ epithelium and cranial neural crest–derived
ectomesenchyme. These epithelial-mesenchymal interactions also underpin the
development and morphogenesis of many other human organs including hair, mam-
mary gland and salivary glands. Significant work over recent years has shown that
complex growth and transcription factor signalling are critical to coordinate these
cellular events [46]. Gene and protein expression profiles are tightly regulated
throughout all stages of tooth development, and the signalling networks generated
are similar to those found in the development of other organs. Notably, it is these
networks which are reactivated during many repair and regeneration events later on
in life. Indeed, recent studies have now made significant in-roads into the charac-
terisation of these intracellular signalling cascades essentially for coordinating
tooth development [47].
The initiation stage of tooth development is characterized by the formation of the
dental lamina and this occurs at around the fifth week of human gestation [10th
embryonic day (ED 10) of mouse development]. During this stage, a variety of cel-
lular and molecular events occur which determine tooth type, position and orienta-
tion within the developing jaws. Subsequently, the dental epithelium begins to
proliferate to give rise to a narrow horseshoe-like ribbon of cells termed the dental
lamina, and their morphology reflects the future position of the dental arches.
Embryonic epithelial thickenings (ectodermal/dental placodes) of the dental lamina
subsequently develop which are the first morphological indications of teeth and
precede the local appearance of an ectodermal organ. Many growth factors and
signalling molecules such as fibroblast growth factors (FGFs), Paired box’s (PAXs),
WNTs, sonic hedgehog (SHH), msh homeobox’s (MSXs), distal-less homeobox’s
(DLXs) and bone morphogenetic proteins (BMPs) are the main regulators of this
process which provide the relevant positional information for dental placode devel-
opment [48, 49].
6 P.R. Cooper

The dental epithelium continues to proliferate and begins to invaginate into the
ectomesenchyme, and forms tooth buds with the dental placodes continuing to
secrete potent signalling molecules [50–52]. Subsequently, at 20 locations in the
human dental lamina, at around weeks 7–9 of human gestation and mouse (ED
11–11.5), the epithelial cells begin to proliferate and intrude into the mesenchyme
to give rise to an early bud stage structure. The ectomesenchymal cells proliferate
and accumulate around each epithelial bud, and the innermost cells of the epithelial
develops a star-like morphology with the onset of synthesis and secretion of glycos-
aminoglycans. This structure becomes hydrated resulting in the cells becoming
more widely distributed with this internal area of the tooth bud now containing the
stellate reticulum and the intermediate layer. During the bud stage of tooth develop-
ment, the odontogenic potential no longer resides with the epithelium but is driven
by the ectomesenchyme [53].
The tooth bud becomes transformed into a cap-like structure by differential pro-
liferation and infolding of the epithelium. The local mesenchymal cells begin to
secrete a range of ECM molecules, such as tenascin and syndecan, which bind to,
and increase the local concentrations of growth factors. The inductive signalling
results in differential multiplication of the epithelial layer with concomitant trans-
formation of the tooth bud into a pyramid-like structure with the dental lamina at its
tip which marks the future site of the tooth crown. Evidence indicates that BMP4 is
key to the mesenchymal signalling that induces transition from bud to cap stage due
to its regulation of several key transcription factors. Subsequently, an epithelial
mass, the enamel knot, within the central base of this structure develops, and this
reportedly acts as a transient organizer of the morphogenetic signalling for adjacent
cells via its expression of FGFs. The enamel knot is removed via apoptosis at the
end of the cap stage and is entirely lost by the time of the bell stage [54–56]. The
epithelium expands and folds inside the core of the bud in an anterior to posterior
manner and the whole structure begins to resemble an upturned cap. The inner
enamel epithelium (IEE) is found internally within the cap while the outer structure
is covered by the outer enamel epithelium (OEE). Between the IEE and OEE sheets
are vacuolised cells of the stellate reticulum and an intermediate cell layer which is
referred to as the enamel or dental organ. The condensed mesenchymal tissue
within the IEE and between the cervical loop (outer rim of the entire structure) is
the dental papilla which develops into the future dental pulp tissue. The condensed
mesenchyme surrounding the dental papilla and dental organ is the dental follicle
which gives rise to the cementoblasts, osteoblasts and fibroblasts of the periodontal
ligament [57].
Cup position and height are tooth- and species-specific; therefore, correct spac-
ing and size are accurately regulated in multicuspid teeth via primary and secondary
enamel knots. Indeed, secondary knot formation marks the onset of the bell stage of
tooth development and the IEE continues infolding according to the organising sig-
nals that they express. The IEE subsequently displaces the stellate reticulum, and
the structure acquires the form of a bell. At this point, the dental mesenchyme does
not appear to be undergoing cell proliferation, and the enamel organ is separated
from the dental papilla, with the tooth cusps starting to form and the crown height
1 Dental and Craniofacial Tissue Stem Cells: Sources and Tissue Engineering… 7

increasing. Crown morphogenesis and cytodifferentiation occur during the bell

stage with the cells differentiating in situ to give the crown its final shape [58–61].
Subsequently, the mesenchymal cells bordering the dental papilla are attached to the
basement membrane of the IEE, and they take on a polarised columnar form and
differentiate into the odontoblasts which secrete the predentine. Immediately fol-
lowing the deposition of the predentine the basement membrane breaks down and
subsequent signalling leads to cells of the IEE, which are in contact with the preden-
tine, differentiating into polarised columnar ameloblasts which begin their synthesis
of enamel. Mineralization occurs and converts the predentine to dentine, and further
secretion of predentine results in the odontoblasts receding from the dentino-enamel
junction. The odontoblasts leave cellular processes within dentinal tubules as they
traverse towards the pulp core. The two hard tissues of the tooth matrix, the enamel
and dentine, are characterised by their apposition of hydroxyapatite crystal. Notably,
the basal cells of the intermediate layer support the process of enamel formation and
following tooth eruption transform into the junctional epithelium. The dental lam-
ina disintegrates, and the pulp and enamel organ are encased in a condensed mesen-
chyme, which constitutes the dental follicle which ultimately gives rise to
cementoblasts, osteoblasts and fibroblasts [62, 63].
A multitude of genes have been identified as being active during tooth develop-
ment and morphogenesis which indicate the complexity of the process. Our
increased understanding of these molecular and cellular events is necessary to
underpin the development of future stem cell-based therapies for bio-tooth
engineering.

1.2.1 Dentinogenesis

Whilst primary dentinogenesis occurs at a rate of ~4 μm/day during tooth develop-

ment, namely secondary dentinogenesis continues to occur at ~0.4 μm/day follow-
ing tooth root formation throughout the life of the tooth. Tertiary dentinogenesis
refers to the process of repair and regeneration in the dentine–pulp complex which
represents a natural wound healing response. Following relatively mild dental
injury, such as during early stage dental caries, primary odontoblasts are reactivated
to secrete a reactionary dentine which is tubular and continuous with the primary
and secondary dentin. However, in response to injury of a greater intensity, e.g. a
rapidly progressing carious lesion, the primary odontoblasts die beneath the lesion.
Subsequently, if conditions are appropriately conducive, e.g. caries is arrested; the
stem/progenitor cells within the pulp are signalled to home to the site of injury and
differentiate into odontoblast-like cells. These cells deposit a tertiary reparative
dentine matrix resulting clinically in dentine bridge formation walling off the dental
injury. Clearly, the relative complexity of these two tertiary dentinogenic processes
differ with reactionary dentinogenesis somewhat more simply requiring only the
up-regulation of existing odontoblast activity, whereas reparative dentinogenesis
involves recruitment, differentiation, as well as up-regulation of dentine synthetic
8 P.R. Cooper

and secretory activity. It is understood that tertiary dentine deposition rates some-
what recapitulate those of development and are also reported to be ~4 μm/day.
Notably, tertiary dentinogenic events are understood to be signalled by released
bioactive molecules similar to those present during tooth development which were
initially sequestrated within the dentine during its formation [64–66]. Indeed, an
array of molecules are bound within dentine and are known to be released from their
inactive state by carious bacterial acids and restorative materials, such as calcium
hydroxide, and are known to stimulate dentine bridge formation. At the stem cell
level, released dentine matrix components may stimulate cell proliferation and
expansion, recruitment to the site of injury, differentiation into odontoblast-like
cells and the up-regulation of synthetic and secretory activity. Indeed, prime candi-
date signalling molecules for stimulating these events come from the BMP and
transforming growth factor (TGF)-β superfamilies with TGF-β1 alone being shown
to stimulate many of these processes in vitro and in animal models. However, it is
likely that synergistic signalling due to many of the bioactive molecules released
from the dentine ECM are potent regulators of DSC repair processes in vivo
(reviewed in [67, 68]). Notably, however, while it is generally assumed regenerative
processes utilises tissue resident cell sources, a mouse parabiosis model has recently
demonstrated that progenitor cells can be derived externally to the pulp [69]. The
source and properties of stem cells involved in repair and regenerative responses are
discussed in Section 1.3.

1.3 Stem Cell Populations

1.3.1 BMMSCs

Originally in 1970, Friedenstein et al. [34] reported the isolation of adherent colony
forming cells from bone marrow, and demonstrated their ability to differentiate
toward various mesenchymal tissue lineages. In 1999, Pittenger et al. [70] charac-
terized human BMMSCs from the iliac crest, and showed that they could be
expanded in culture, and were able to differentiate down osteogenic, adipogenic and
chondrogenic lineages. More recent work has gone on to demonstrate BMMSCs
also have the capacity to differentiate into non-typical mesenchymal lineages such
as ones involved in neurological repair [71]. Perhaps predictably BMMSCs most
robustly form bone in vitro and in vivo, indicating their utility in bone regenerative
therapy which is frequently exploited clinically in oral and dental procedures. While
BMMSCs are generally isolated from bone marrow aspirates derived from the iliac
crest during a relatively invasive and painful surgery, they can also be isolated from
the maxilla and mandible. These orofacially-derived BMMSCs, derived from cra-
nial neural crest cells, are subsequently likely more applicable for dental treatments
although their safe expansion in numbers is required prior to use in therapeutic
procedures [72–74].
1 Dental and Craniofacial Tissue Stem Cells: Sources and Tissue Engineering… 9

1.3.2 Adipose Tissue-Derived Stem Cells (ADSCs)

ADSCs can be relatively abundantly harvested via lipectomy or from lipoaspirates

from many sites within the adult human body including craniofacial regions.
Notably, their harvest generally results in low donor-site morbidity, and the tissue
isolated is regarded as clinical waste as liposuction is routinely performed during
cosmetic surgery, e.g. cheek and chin reshaping. While intrinsically ADSCs exhibit
some differences compared with BMMSCs, ADSCs appear to exhibit good miner-
alised tissue lineage responses, and therefore have potential for use in bone and
tooth tissue repair including applications in osseointegration [29]. For dental struc-
tures, ADSC transplantation has been used to regenerate pulp tissue and whole teeth
containing dentine, with periodontal ligament and alveolar bone attachments in ani-
mal models [75–78]. Further work characterising the application of ADSCs for
bone, tooth and periodontal tissue regeneration should result in the development of
robust protocols which utilise waste fat tissue for clinical application.

1.3.3 Dental Tissue Stem Cells

1.3.3.1 Postnatal Dental Tissue-Derived Stem Cells

A clonogenic and highly proliferative DPSC population exhibiting phenotypic char-

acteristics similar to those of BMMSCs were initially isolated by enzymatic disag-
gregation of adult dental pulp. Only a few years later, SHEDs were isolated, which
were also shown to exhibit the stem cell properties of self-renewal and multi-lineage
differentiation potential. In animal studies, DPSCs and SHEDs have demonstrated
the ability to generate a mature dentine–pulp-like structure. Further studies using
SHEDs have shown that they can induce bone-like matrix formation which may
relate to processes that occur in deciduous tooth roots, whereby resorption occurs
concurrently with new bone formation. Notably, DPSCs and SHEDs have signifi-
cant clinical application potential for autologous regenerative treatment approaches
as both can be derived from what is regarded as clinical waste tissue. Indeed, DPSCs
can be obtained from teeth extracted for orthodontic reasons, whilst SHEDs are
harvestable from primary teeth which are naturally exfoliated (reviewed in [79]).
Interestingly, up until recently, it was believed that due to the reciprocal interactions
which occur between the embryonic oral epithelium and neural crest-derived mes-
enchyme during tooth morphogenesis, the stem cells from the tooth were derived
from a neural crest origin. However, Kaukua et al. [80] recently demonstrated that a
significant population of MSCs involved in development, self-renewal and repair of
teeth are derived from peripheral nerve-associated glia. While this study was per-
formed in a murine incisor model system, which may limit its relevance to humans,
it does, however, indicate our continued need to better understand both tooth devel-
opment and regeneration events.
10 P.R. Cooper

The periodontal ligament provides another source of postnatal MSCs in the form
of PDLSCs which can also be isolated from extracted waste teeth. Perhaps not sur-
prisingly due to their localisation, PDLSCs have been demonstrated to be able to
regenerate several periodontal tissues including cementum, periodontal ligament and
alveolar bone in animal studies. However, recent work has indicated that the local
derivation of the PDLSCs may significantly influence their differentiation capabili-
ties as PDLSCs from the alveolar bone surface exhibited superior alveolar bone
regeneration properties compared with PDLSCs from the root surface [17, 18, 81].

1.3.3.2 Stem Cells Derived from Developing Dental Tissue

Within the developing dental tissues of the dental follicle, including the dental mes-
enchyme and apical papilla, MSC-like cell populations have been identified. The
dental follicle, also termed the dental sac, contains the developing tooth and within
it, DFSCs with the ability to regenerate several periodontal tissue types are found
[13–16]. At the late bell stage of tooth development, stem cells derived from the
dental mesenchyme of the third molar tooth germ have also been identified and
these are termed as TGPCs [82]. These isolated MSC-like cells demonstrated a high
proliferative capacity along with the requisite capability to differentiate in vitro into
the three germ layer lineages. SCAPs [11, 12] have also been identified in develop-
ing tooth roots. In comparison with DPSCs, SCAPs have demonstrated increased
proliferation rates and enhanced regenerative capabilities for dentine-pulp complex
tissue in animal model studies. Furthermore, as these cells exhibit a developmen-
tally immature phenotype and can be isolated from the clinical waste postnatal or
adult tissue of extracted wisdom teeth, they could provide a valuable source of
autologous stem cells for future regenerative therapies.

1.3.3.3 Oral Mucosal and Periosteum-Derived Stem Cells

The oral mucosa comprises stratified squamous epithelium composed of oral kera-
tinocytes and an underlying connective tissue. The connective tissue consists of a
well vascularised lamina propria and a submucosa which can contain minor salivary
glands, adipose tissue, neuronal structures and lymphatics. Within the oral mucosa
two different types of human postnatal stem cells have been identified; OESCs and
GMSCs [21–23]. OESCs are reportedly relatively small oral keratinocytes (<40 mm
in diameter) and while being unipotent, they can regenerate oral mucosal tissue
ex vivo which may have clinical utility for intra-oral grafts.
GMSCs are reported in the gingival lamina propria which attaches directly to the
periosteum of the underlying bone [21]. In addition, a neural crest stem cell-like
population has also been isolated from the adult human gingival lamina propria
which are termed oral mucosa stem cells (OMSCs) [22]. The relative clinical ease
by which relatively high numbers of both GMSCs and OMSCs could be isolated
makes these cells promising candidates for use in future clinical therapies.
1 Dental and Craniofacial Tissue Stem Cells: Sources and Tissue Engineering… 11

The periosteum of bone comprises two distinct layers; the outer layer which
contains mainly fibroblasts and elastic fibres, while the inner layer contains MSCs
along with other progenitor cell populations. Periosteum-derived cells may have
preferential application for bone regeneration and subsequently may have applica-
tion in craniofacial therapies [83–85]. Indeed, locally derived periosteum cells may
have particular application for bone repair in procedures such as periosteal flap
surgery in conjunction with implant placement along with use in large defect repair
procedures [86–88].

1.3.3.4 Salivary Gland-Derived Stem Cells

Salivary glands develop from the endoderm and when mature comprise of acinar
and ductal epithelial cells with exocrine function. While the existence of salivary
gland stem cells have been proposed following in vivo studies, stem cells that give
rise to the entirety of the epithelial cell types present within the gland have yet to be
identified [25, 27]. MSC-like cells from human salivary glands have, however, been
reported based on their expression of embryonic and postnatal stem cell markers
along with their ability to differentiate toward adipogenic, osteogenic and chondro-
genic lineages [89–91]. Stem cells isolated from this tissue may have particular
application for use in the rescue of dysfunctional gland activity in particular in head
and neck irradiated cancer patients who exhibit salivary gland dysfunction [92].

1.3.3.5 Induced Pluripotent Stem Cells (iPSCs)

The possibility of reprogramming somatic cells to an early embryonic development

stage by introducing the four transcriptional factors, Oct3/4, Sox2, Klf4 and c-Myc,
was initially reported by Takahashi and Yamanaka [93]. Originally, normal mouse
adult skin fibroblasts were used and the resultant reprogrammed cells were termed
as iPSCs. A year later, this work was replicated using human skin cells which sub-
sequently indicated the potential to generate patient-specific cells with ESC-like
characteristics [7, 94]. Indeed, animal studies have demonstrated iPSCs can gener-
ate all the tissues and organs of the body. Notably, it has been shown that iPSCs can
be derived from many cell types derived from oral and dental tissues which can be
relatively easily harvested by dentists. Interestingly, many of these cells have exhib-
ited relative high reprogramming efficiencies which may be explicable as oral and
dental MSCs already express relatively high levels of endogenous multipotent tran-
scription factors [82, 95–99]. In the future, the use of oral and dental waste tissue
may, therefore, provide an ideal cell source for use in iPSC technology in particular
for the regeneration of autologous craniofacial soft and hard tissue structures.
Indeed, recent work utilising iPSCs in a mouse model using enamel matrix derived
molecules demonstrated increased periodontal tissue regeneration, while in vitro
work has demonstrated iPSC application for biotooth-engineering of ameloblast-
and odontoblast-like cells [100–102].
12 P.R. Cooper

Notably, there remain drawbacks with the use of iPSC technology. Much is still
to be learned as to how to optimise their generation and reprogramming efficiency
as well as in controlling their differentiate fate. A major concern also lies with the
risk of tumour formation by iPSCs following clinical implantation. Such a concern
arises due to the use of the c-Myc oncogene as a reprogramming factor along with
the use of the retroviral insertion system for gene transfer. Recent research, how-
ever, may have resolved these issues by using alternative genes for reprogramming
along with the application of small reprogramming molecules. Indeed, the use of
non-viral components such as proteins, microRNAs, synthetic mRNAs and epi-
somal plasmids is being pioneered. A further clinical concern also arises due to
delivery of residual undifferentiated iPSCs remaining amongst the differentiated
target cell population. These cells may proliferate uncontrollably and generate tera-
tomas at the site of implantation. To overcome this issue the use of selective ablation
approaches to remove teratomas via suicide genes and chemotherapy, as well as the
use of antibody-based cell sorting approaches to remove teratoma-forming cells, are
being developed [7, 103–113].

1.4 Scaffolds and Morphogens for Stem Cell Tissue

Engineering

1.4.1 Scaffolds

For dental and oral tissue engineering strategies, along with stem cells, suitable
biomimetic scaffolds and appropriate morphogens/growth factors are required
[114]. Clinically, for periodontal tissue repair, material-based guided tissue regen-
eration (GTR) approaches have been developed. Subsequently, biocompatible or
bioinert scaffolds are used to enable connective tissue and bone regeneration from
local tissue MSC populations [115–118]. Alveolar bone augmentation approaches,
such as guided bone regeneration (GBR), utilise bioactive materials, such as cal-
cium phosphate (CaP)-based biomaterials and collagen-based grafts. While these
materials are bioactive and osteoconductive, they are not osteoinductive; hence,
scaffolds are being developed, which incorporate bone formation promoting growth
factors [119–122].
Fibrous silk protein (fibroin) biomaterial scaffolds are also being developed for
their use in tooth and bone repair. These scaffolds can be generated and harvested
from silkworms and spiders, and can exhibit properties of controllable porosity,
surface roughness and stiffness. They can be further functionally modified to mimic
the natural ECM environment to facilitate stem cell recolonization, differentiation
and tissue regeneration for therapeutic applications [123–125].
Recent studies have demonstrated the utility of hydrogel scaffolds for tooth tis-
sue engineering applications and their promise is likely based on them exhibiting
similar biomechanical properties to pulp tissue. The seeding of pulp derived cells on
1 Dental and Craniofacial Tissue Stem Cells: Sources and Tissue Engineering… 13

collagen scaffolds with subsequent animal implantation has demonstrated the for-
mation of dental tissue structures [126, 127]. Furthermore, DPSCs encapsulated in
collagen hydrogels have been shown to differentiate and deposit a mineralised ECM
in the presence of natural tissue morphogens [40, 128]. Others have generated pulp-
like tissue in vivo following the seeding of SHEDs and human endothelial cells on
biodegradable poly-L-lactic acid hydrogel scaffolds [129]. A peptide-amphiphile
hydrogel scaffold containing bioactive osteogenic supplements has also been shown
to promote differentiation of encapsulated SHED and DPSCs [130]. While chal-
lenges still remain, the development of the most appropriate scaffolds which opti-
mise stem cell responses for clinical application is progressing at a rapid rate.

1.4.2 Role of Growth Factors and Morphogens for Tissue

Regeneration

Our understanding of the molecules involved in signalling tissue development and

repair will underpin the generation of novel naturally inspired clinical therapies.
Current knowledge of this molecular signalling is advancing with the tooth’s hard
and soft tissue ECM being shown to provide both biochemical and biomechanical
regulatory cues. Indeed, comparable with repair processes in other tissues, the regu-
lation of dental tissue regeneration involves signalling derived from its ECM with
inherent growth factors known to coordinate recruitment, proliferation and differen-
tiation of MSC populations [65, 68, 131, 132].
In the periodontal tissues, the application of platelet rich plasma (PRP) enables
the delivery of a cocktail of potent growth factors and morphogens. Indeed cur-
rently, there is significant interest in the use of PRP in combination with bone grafts
and/or stem cells to enable more predictable periodontal regeneration [133]. Enamel
matrix derivatives (EMDs), obtained from porcine tooth buds, also contain a com-
plex cocktail of growth factors and can also stimulate periodontal tissue regenera-
tive events. Indeed both PRP and EMD are morphogenically complex and have
been shown to include BMP-2, platelet derived growth factor (PDGF)-BB and FGF-
2, amongst others [134–136]. These molecules likely act synergistically on MSCs.
However, the action of individual growth factors has been exploited with BMP-2
being used in absorbable collagen sponge scaffolds to induce bone formation for
sinus and alveolar ridge augmentation therapies. Both PDGF-BB and FGF-2 in
combination with CaP or hydrogel scaffolds have also shown some clinically effica-
cious potential based on their ability to stimulate vascular responses which underpin
many MSC-based repair mechanisms [137–145].
The indirect application of MSCs due to their release of growth factor with para-
crine effects has also recently been highlighted. These secretomes contain a
multitude of bioactive molecules such as insulin-like growth factor (IGF)-1 and
vascular endothelial growth factor (VEGF) which promote many tissue repair
mechanisms [146]. Notably, DPSC secretomes exhibit significant neurogenic repair
14 P.R. Cooper

activity as well as being able to immunomodulate T-cell, B-cell, natural killer cell,
and dendritic cell function [147, 148]. Further work is still required to better char-
acterise the active components of the secretomes to determine optimal concentra-
tions for targeted tissue repair and regeneration application.

1.5 Stem Cell Applications for Dental and Craniofacial

Tissue Regeneration

The use of stem cells for regenerative medicine/dentistry is progressing and cur-
rently, the use of adult/postnatal stem cells exhibits the most realistic clinical oppor-
tunity. Regeneration of bone and periodontal tissues using MSCs has received
considerable attention with several studies already reporting clinical application.
Clearly, stem cells used in dental tissue engineering should be; (i) relatively easily
isolated, (ii) straightforward to deliver in a reproducible and clinically simple pro-
cedure, (iii) clear of any patient safety issues, and (iv) ultimately differentiate into
and regenerate the target tissue or organ.
BMMSCs and ADSCs, in particular those derived from the orofacial region, may
provide an appropriate source for craniofacial tissue repair. Other dental and cranio-
facial tissue-derived MSCs may be more appropriate for regenerating dental
mesenchyme-derived hard and soft tissues, including those of the dentine, pulp and
supporting periodontal tissues. The application of MSCs for complete repair of
complex oral organs, such as teeth and salivary glands, which also require cells to
differentiate down epithelial lineages may however be challenging.
Pluripotent embryonic stem cells may, therefore, have utility in these cases; how-
ever, medical and ethical issues associate with their application and the use of iPSCs
still require further technical and safety advancements before they can be applied.
For all stem cell sources, their downstream processing following isolation still
remains an issue for the clinician who would also require onsite specialist equip-
ment and expertise to enable their purification and expansion.

1.5.1 Tooth and Tooth Component Tissue Regeneration

Ultimately, it is aimed that a lost tooth will be replaced by a fully functional bioen-
gineered one; however, current studies indicate that tooth component tissue, such as
root and crown dentine are more realistically clinically achievable. Recent work
using animal models has shown that complex root/periodontal structures can be
regenerated using PDLSCs and SCAPs in conjunction with hydroxyapatite scaf-
folds [11, 149]. The structures regenerated provided suitable abutments for pros-
thetic devices enabling the support of an artificial crown with dental functionality.
Clearly, future work in this area may enable development of the underpinning tech-
nology necessary for human application.
1 Dental and Craniofacial Tissue Stem Cells: Sources and Tissue Engineering… 15

The regeneration of an entire tooth structure is now appearing feasible in the

future based on animal studies utilising several different MSC sources. For tooth
bioengineering, the generation of embryonic tooth primordia has been commonly
used. Initial studies have transplanted pelleted dissociated porcine tooth buds in the
omentum of athymic rats which resulted in the generation of complex tooth struc-
tures which comprised a pulp chamber, dentine, putative Hertwig’s Epithelial Root
Sheath (HERS) and an enamel organ. Transplantation of dissociated rat and mouse
tooth buds have also resulted in the development of similar tooth structures. Notably,
as is described previously, tooth development requires the reciprocal interactions
between embryonic oral epithelial cells and neural-crest derived mesenchyme.
Subsequently, recent work has attempted to determine if mouse-derived ESCs, neu-
ral stem cells and BMMSCs can appropriately respond to mouse embryonic oral
epithelium derived cells. Data indicated that odontogenic differentiation was most
apparent in explants containing BMMSCs although other cell types demonstrated
some potentiality [100, 102, 150–154]. Work conducted by Volponi et al. [155] has
demonstrated tooth tissue regeneration following transplantation of human adult
gingival epithelial cells combined with mouse embryonic tooth mesenchyme cells
in kidney capsules. The tooth structures generated at six weeks of transplantation
contained vascularized pulp-like tissue and signs of root development including the
presence of ameloblast-like cells and epithelial rests of Malassez.
Significantly, a murine model has recently demonstrated that, following the trans-
plantation into the alveolar bone of a bioengineered tooth germ, reconstituted in vitro
in a collagen hydrogel scaffold using epithelial and mesenchymal progenitor/stem
cells, a functioning tooth was formed. Notably, the in vitro step used recapitulated the
developmental events necessary for complex tooth tissue generation and the subse-
quent bioengineered tooth, which when erupted and occluded, exhibited appropriate
mineralised tissue properties [151]. Furthermore, the pulpal tissue was appropriately
innervated and relevantly serviced by a blood supply. The generation of fully func-
tional tooth units in animal models which have utilised MSC and iPSC sources sup-
port the concept that bioengineered structures may one day be routinely generated for
patients. Clearly, significant work is still required to bring this to fruition and to pro-
vide clinically relevant alternatives for patients who require dental implants.

1.5.2 Regeneration of Other Complex Craniofacial Tissues

and Organs

The regeneration of salivary gland function is important in particular for head and
neck oncology patients who have undergone surgery and/or radiotherapy. Recent
mouse model studies using ADSCs, BMMSCs and primitive salivary gland stem
cells have shown that this may one day be clinical feasible. [89, 92, 156, 157]. The
temporomandibular joint (TMJ) disc or condyle can become damaged due to dis-
ease such as arthritis or through trauma. MSCs in conjunction with hydrogels and
ultrasound approaches have been used successfully to reconstruct condylar defects
16 P.R. Cooper

in animal model systems [158–160]. The regeneration of tongue tissue is also

important to many patients. Several animal model systems using MSCs and relevant
scaffold systems has now shown tongue tissue repair is possible [161–163]. Overall
studies are now showing that for many complex tissue and organ systems within the
oro-craniofacial region, bioengineering approaches may one day become a clinical
reality for patient treatment.

1.6 Stem Cell Storage and Processing

While growing evidence demonstrates that dental and oral tissues provide a rich
source of MSCs, their use in regenerative therapies may be limited due to the
requirement to isolate tissue at the time of need, e.g. tooth extraction. The banking
of DSCs or tissues obtained from deciduous and wisdom teeth may, therefore, pro-
vide a practical approach for future stem-cell-based regenerative therapies. Recently,
in several countries worldwide stem cell and tissue banks in the dental field have
been developed, e.g., Advanced Center for Tissue Engineering Ltd., Tokyo, Japan
(http://www.acte-group.com/); Teeth Bank Co., Ltd., Hiroshima, Japan (http://
www.teethbank.jp/); Store-A-ToothTM, Lexington, USA (http://www.store-a-
tooth.com/); BioEDEN, Austin, USA (http://www.bioeden.com/) and Stemade
Biotech Pvt. Ltd., Mumbai, India (http://www.stemade.com/) (reviewed in [164]).
These banking approaches routinely utilise cryopreservation which aims to enable
the long term storage of viable stem cells from tissues such as the PDL, pulp, apical
papilla and whole tooth tissue. Subsequently, it is envisaged that the stem cells will
be retrieved in the future from this cryopreservation and applied in autologous
regenerative therapies for the patient. Much work, however, is still needed to deter-
mine the utility of these biobanks, their longevity, and value for money and the
MSC processing procedures required.
Currently, it is not entirely clear as to how long term cryopreservation affects
MSC viability and phenotype [165]. Therefore, alternative storage approaches are
being developed which may be beneficial. Indeed, recent studies have shown that
MSCs encapsulated in hydrogels may provide a means to decrease archiving costs
while maintaining MSC phenotype and properties. Furthermore, the potential of
tissue engineered product vitrification has also been investigated with studies using
bone constructs consisting of a hydroxyapatite scaffold-cell complexes demonstrat-
ing higher cell survival rates compared with conventional freezing approaches [166,
167]. Further studies of these emerging biobanking approaches are clearly needed.
MSC handling and ex vivo expansion will be required for clinical application due
to the relatively low number of stem cells, <0.1 % of all cell types, present within
tissues. To achieve this, Good manufacturing practice (GMP)-compliant environ-
ments have been developed and are reported to generate clinical-grade MSCs from
several tissue-types, e.g. adipose and bone marrow [168]. Currently, there are mini-
mal published reports evident on GMP-handling and processing for dental MSC-
types. It is proposed that standard GMP procedures should be more routinely applied
1 Dental and Craniofacial Tissue Stem Cells: Sources and Tissue Engineering… 17

across institutes, as previous work has demonstrated significant MSC heterogeneity

which may be due to donor or operator variability. Indeed, data has indicated donor
age may be one key source of this heterogeneity with some studies showing that
proliferative potential and differentiation capability decrease in an age-related man-
ner both in vivo and in vitro, i.e. during culture passage [169–171]. Indeed, our work
using DPSCs has also shown that with higher passages, MSC properties, such as
proliferation rates and differentiation capabilities, diminish [172]. To overcome
these issues, others have supplemented prolonged in vitro cultures with growth fac-
tors to maintain MSC-like properties [173]. Further optimisation of laboratory pro-
cedures may minimise culture differences, and novel techniques which involve
spheroid culturing in the presence of growth factors or the use of relevant hypoxic
conditions [174], which better mimic the MSC niche in vivo, may be exploited.
Cell culture requires several kinds of supportive factors including animal-derived
reagents, such as fetal bovine serum (FBS), and variation in lots have been cited as a
further source of MSC heterogeneity. There are also safety concerns relating to the
use of animal- derived reagents for human MSC expansion due to possible risk of
contaminations such as prions, viruses, and zoonosis, along with the potential for
host immunological reactions against xenogeneic proteins. Subsequently, autolo-
gous human serum (HS) has been proposed for clinical applications as a replace-
ment for FBS. There exist major drawbacks, however, with using autologous HS
due to the need for its harvest in sufficient volumes at the time of need potentially
from patients who may already be compromised with regards to their health.
Alternatively, the utility and safety of other approaches; such as the use of pooled
human platelet lysates, has been proposed [175–179]. To overcome these culture-
related issues, the development of well-defined serum-free media which can support
the growth of MSCs is being explored. While chemically defined media may include
some animal or human serum, efforts are being made to generate a more xeno-free
culture media which would circumnavigate safety issues and improve clinical grade
cell culture consistency [180–183]. However, while initial studies support the poten-
tial GMP application of serum-free media, more work, examining a wider range of
MSC-types, is still required. Furthermore, along with the standardisation of labora-
tory protocols, procedures for aspiration, including harvesting site locations, should
also be undertaken as consistently as possible.
GMP-compliant MSC processing requires multiple complex steps which can
include surgical tissue dissection, cell dissociation, dispersion, expansion and
collection. An expensive clean room is also required along with experienced and
specialist technical staff. Work is generally labour-intensive and the complexity of
the process can result in human error and culture contamination. Subsequently, the
use of automated cell processing approaches has been explored. However, while
such an approach would eliminate human error, the risk of contamination from
operators, and reduce variability within the procedure, robotic approaches are
extremely costly [184–186]. The development and miniaturisation of benchtop
devices and processes through collaborations between biologists, mechanical and
computer engineers may enable automated processes to be more broadly accessible
within clinical practices in the future.
18 P.R. Cooper

Safety issues are a major concern prior to clinical application of bioengineering

approaches and regulation of use of cell and cell-based products differs from coun-
try to country. Safety tests of the cultured cells need to demonstrate that they are free
from infectious agent contamination and tumour formation ability. Indeed, cultures
should be free from bacteria, fungi, mycoplasma, viruses (e.g. hepatitis B, hepatitis
C, and human T-lymphotropic virus), and endotoxin levels should also be moni-
tored [187]. While tumour formation is a common risk for both autologous and
allogeneic cell therapies, the transplantation of MSCs is considered a relatively safe
procedure. However, long-term culture is known to increase the chances of cellular
transformation, therefore, karyotypic analyses and transplantation of the MSCs in
immuno-deficient animals should be routinely used to assess cancer risk [188].
For more global standardisation, characterisation and clinical application, it may
be necessary to safely transport MSCs between sites. The carriage of the MSCs
provides further risks to viability and phenotype, potentially due to needs to main-
tain cells at constant carbon dioxide tension, temperatures and air pressure. There is
also a clear need for appropriate biological safety regulation and associated accom-
panying documentation for clinical materials which can increase administration
processes, time and costs. Minimising the impact of transportation is, therefore,
critical for the clinical and commercial application of cell therapies and devices, and
procedures which enable this are being developed. It is proposed that variations in
temperature during transport might be the most important risk to cell viability and
that the optimal temperature for carriage may depend on cell type. Hydrogel tech-
nology is currently providing a novel means (as previously discussed) to better
maintain MSC phenotype over a longer term at relatively low (<37 °C) and wider
temperature ranges. This approach may be advantageous to ensure consistency in
cell therapies between distant sites.

1.7 Concluding Remarks

While promising data have already been generated in vitro and in preclinical stud-
ies using animals, research remains ongoing to ensure that there is a significant and
sound knowledge-base prior to clinical translation. In order for this translation to
be realised, collaborative work and appropriate communication and dissemination
between researchers, clinicians, industry and healthcare workers worldwide need
to remain ongoing. Indeed, while considerable advancements have already been
made over recent decades, it is imperative that attempts to translate basic science
findings are not made too soon as this may generate risk for the patient. Therefore,
appropriate restraint within the scientific and clinical communities is essential, and
subsequent steps should be approached with caution as the patient’s safety is of
prime importance. Towards this goal, research governance and peer review pro-
cesses need to be firmly in place. It is also important to determine which patient
groups would best benefit from translation of stem cell science advances rather
than incentives for application being driven due to any financial or industrial gain.
1 Dental and Craniofacial Tissue Stem Cells: Sources and Tissue Engineering… 19

Indeed, in terms of dental patient need, it is important to consider whether novel

bio-inspired techniques offer significant advantage over those currently applied.
Such a risk-reward strategy may also help drive which approaches to prioritise
for more rapid clinical fruition. For example, in regenerative endodontics, loss of
pulp vitality in the child or younger adult in which full root formation is also not
complete may provide a more realistic and successful application of pulp tissue
bioengineering approaches which offers a long term patient benefit. Conversely,
tooth bioengineering approaches within significantly older patients may be more
challenging, less likely to succeed and therefore, current treatment approaches may
be more appropriate. Indeed, it will be essential, as is currently the case, for a com-
plete consideration of the patient’s condition including age, need, diet and lifestyle,
to be undertaken before any clinical work is performed which utilise MSC-based
therapies.
In order to ensure coherent and comprehensive advancements within dentistry,
we should also aim to learn from parallel areas of tissue engineering which are well
advanced for repair at other sites of the body. Indeed in the US, stem cell research
has recently become one of the pillars of the health programme and the US Military
are significantly investing (>$250-million) in this area for soldier rehabilitation. It
is, therefore, envisaged that ongoing and increased activity will lead to advances
that will benefit dental medicine and enable clinicians to deliver novel therapies as
part of their routine practice. Within the clinical setting, as has already been dis-
cussed, concomitantly advancements in related technologies will need to occur such
that following tissue isolation, its processing and preparation happen as rapidly and
routinely as possible. It is, therefore, envisaged that intelligent automatic and robotic
systems will be necessary to help the clinician undertake these tasks and standardise
the processes involved. Along with this is the need for the continued education of
the dental team in areas of stem cell biology, biomaterials, and novel clinical proce-
dures and equipment in order to ensure benefits are realised for patients.

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Chapter 2
Immunomodulatory Properties of Stem Cells
Derived from Dental Tissues

Pakize Neslihan Taşlı, Safa Aydın, and Fikrettin Şahin

2.1 Mesenchymal Stem Cell (MSC)-Mediated

Immunomodulation

MSCs are basically characterized by their potential to adhere to plastic surfaces in

cell culture conditions, express cell surface markers of CD105, CD44, CD29, CD73,
and CD90, but not express hematopoietic stem cell surface makers such as CD45,
CD34, CD14, and differentiate into osteo-, adipo-, and chondro-genic cell lines [1].
Along with their high self-renewal and multi-lineage differentiation abilities, they
possess an immune-suppressive activity, which make them promising candidates to
be used in cell and even tissue mediated treatments of a various immune and inflam-
mation dependent diseases.
MSCs can be easily obtained from various human tissues, preserved in culture
conditions, and used in even histocompatibility antigen (HLA) un-matching condi-
tions of transplantation patients. Though they are mainly isolated from bone mar-
row, they can also be obtained from fat tissue [2], cartilage [3], liver [4], amniotic
fluid [5], tooth germ [6], hair follicle [7] and so on [8–10].
Several reports indicate that MSC could be used in vast amount immune disor-
ders. In particular, researchers have found that systematic infusion of allogenic stem
cells may offer a new suitable treatment for Graft versus host disease (GVHD)
patients [11]. In addition, Prasad et al. described a stem cell derived agent to treat
GVHD patients [12]. The ability of MSCs to stimulate activation, proliferation, and

P.N. Taşlı • S. Aydın

Department of Genetics and Bioengineering, Faculty of Engineering and Architecture,
Yeditepe University, 26 Ağustos Campus, Kayisdagi cad. Kayisdagi,
TR-34755 Istanbul, Turkey
F. Şahin (*)
Genetics and Bioengineering Department, Yeditepe University, Istanbul, Turkey
e-mail: [email protected]

© Springer International Publishing Switzerland 2016 29

F. Şahin et al. (eds.), Dental Stem Cells, Stem Cell Biology
and Regenerative Medicine, DOI 10.1007/978-3-319-28947-2_2
30 P.N. Taşlı et al.

function of immune cells may take part on tissue healing and regeneration, which
would also contribute to treatment of several immune disease including GVHD.

2.1.1 Interactions of MSCs with White Blood Cells

2.1.1.1 T Lymphocyte-Mediated Suppression

The basic role of T cells, one main component of the adaptive immunity which
forms immunological memory, is to create specialized immune reactions to particu-
lar pathogens [13]. Interaction between MSCs and T cells is critical for the activity
of immune system. Direct cell to cell contact of MSCs with CD8+ (cytotoxic T cells)
or CD4+ (regulatory T cells) helper T cells moderates the expression of cytokines
for signaling and inhibition of T cell activation [14, 15].
Recent studies have demonstrated that MSCs express Fas ligand (FasL) known
as a death receptor which inhibit T cell migration [13], triggering stimulated T cell
death via direct cell interaction [16]. Even though inhibition of T cell proliferation
via MSCs action has partially known, suppressive effect of MSCs has not been elu-
cidated in the manner of autologous or allogeneic respective [17, 18]. One possible
action of MSCs is suppression of CD8+ cytotoxic T cell proliferation, rather than a
direct suppression of cytolysis [19, 20]. Furthermore, MSCs can enhance secretion
of interferon gamma (IFNγ) and interleukin (IL)-17, while they induce T helper
cells to produce IL-4 [21, 22].
It has been reported that MSCs can promote the proliferation of regulatory T
cells and increase their modulatory ability directly or indirectly. When peripheral
blood mononuclear cell (PBMCs) are triggered by mitogens, modulatory phenotype
presenting CD4+ CD25+ T cells start to proliferate in the existence of MSCs.
Inhibition of T cell proliferation by MSCs is triggered by allogeneic, mitogenic, or
antigen specific stimuli [23].
Notably, scientists found that suppressed T cell proliferation via triggering apop-
tosis when T cells were stimulated by foreign mitogen, had no influence on latent T
cells [23]. However, the proliferation of inactive and separating thymocytes was
repressed via MSCs once treated in absence of systemic elements. These findings
propose that MSCs can be capable of stimulating the endurance of T cells in a latent
state. Furthermore, it has also been revealed that MSCs can repress T cell activation
but not their toxicity by arresting T cells in G0/G1 cell cycle phase through suppres-
sion of cyclin D and increment of p27kip1 expression [24–26]. As a mechanism of
action, it has been reported that MSC-mediated repression of T cell proliferation
was not found to be because of soluble HLA-G5 isoform, but of the surface expres-
sion of HLA G1 [24].
In last decade, many studies using the heading ‘MSCs in Solid Organ
Transplantation (SOT)’ were designed to understand what is unknown about the
MSCs use in the setting of SOT, and how to progress best in clinical trials [27].
Although there is not any clinical studies evaluating the efficiency of MSCs against
Solid Organ Allograft (SOA) rejection, a plentiful and increasing quantity of evi-
2 Immunomodulatory Properties of Stem Cells Derived from Dental Tissues 31

dences obtained from animal models propose that this methodology may be promis-
ing. Based on in vitro and in vivo animal models, a possible role of these cells on
the prevention of acute tissue allograft rejection has been recommended [28–31].
General MSC-based treatment studies have proved that allogenic MSCs can
diffuse into related tissue during SOT. Enhanced allograft persistence or abolition in
the simultaneous suppression is few of the frequently reported conclusions.
Although human clinical trials are not adequate, preclinical studies have shown that
MSCs could be important therapeutic tools for organ transplantation approaches not
just as they have the ability to modulate the host immunity in a way that may pro-
mote tolerance of the transplanted organ, but also their regeneration capability and
trophic factor expression profiles may also help to lessen inflammatory responses to
the allograft. One of the main aim of SOT studies is to inhibit T cell response against
external antigen. In this line, MSCs can suppress the proliferation of cytotoxic T
cells while enhancing the activities of helper and regulatory T cells. Some
immunomodulation-related studies have proposed that these properties of MSCs
may be the key reasons for their ability to prevent the allo-immune reactions in vivo
[32]. More research must be carried out because lack of knowledge about MSCs-
mediated immune suppression; nevertheless, the fewer toxicity and possible long
term immunosuppression effect of MSCs make them a potentially striking thera-
peutic applicant with respect to outmoded T cell modulatory agents. Another advan-
tage of MSCs use for the inhibition of SOA rejection is the infusion of these cells
while SOT may hold the potential to endorse a state of cell chimerism and continu-
ing tolerance of the transplanted organ via host immune system [33, 34].

2.1.1.2 B Lymphocyte-Mediated Suppression

Even though the main player of the immune suppression response is the T cells, B
cells can also secret antibodies to regulate immune response, and they closely coop-
erate with T cells. The exact mechanism of MSCs on B cells still remains unknown.
However, most of the immune suppression related studies have shown that MSCs
can suppress B cell proliferation, differentiation, and cytokine production in in vitro
co-culture assays and in vivo multiple sclerosis models [35–38]. In contrast, it has
also been shown that MSCs increase B cell proliferation and trigger cytokine pro-
ductions from B cells [38, 39].
MSCs can enhance the production of IgG from peripheral blood and spleen orig-
inated B cells, but they can also inhibit IgG secretion if a heavy primary stimulus is
used to trigger B cells. One of the possible actions of B cell dependent MSCs-
mediated suppression can be formed by differentiated B cell or the direct effect of
the local stimulating signals. MSCs can inhibit B cell proliferation when B cells
starts to secret activation signals to the culture medium in co-culture system, sug-
gesting that MSCs need activation indicators derived from B cells to inhibit B cell
stimulation. This cross talk between MSCs and B cells lead to inhibition of B cell
proliferation. Some of the important factors secreted from MSCs are transforming
growth factor (TGF)-β, hepatocyte growth factor (HGF), prostaglandin E2 (PGE2),
32 P.N. Taşlı et al.

and indoleamine 2,3-dioxygenase (IDO), which conduct MSC-derived immunosup-

pressive action on B cells [40].
As a conclusion, important part of B cell stimulation is carried out via T cell
dependent pathways; hence, the effect of MSCs on T cell activity may also indirectly
inhibit B cell actions [37]. Furthermore, MSCs seem to display a direct stimulus on
B cells by direct cell to cell interactions and via production of vital inducer signals
[36, 40]. One other critical issue is that majority of these experiments have been
made in in vitro conditions, not in vivo or ex vivo. Therefore, actual immunosup-
pression effect of DSCs on B lymphocytes in vivo still remains to be determined.

2.1.1.3 Dendritic Cell-Mediated Suppression

MSCs have also been proposed to be effective against monocytes, monocyte deriva-
tive dendritic cells, macrophages, natural killer cells and neutrophils. Several
in vitro works have presented that MSCs can inhibit the evolution of monocytes into
dendritic cells via suppressing the antigen presentation role of these cells [41, 42].
As recently suggested, dendritic cells are an important part of immune reaction in
regards of immune response and tolerance, depending on the stimulation and matu-
ration period and the cytokine environment at locations of inflammation [43]. The
inhibitory effect of MSCs on dendritic cell differentiation, maturation and function
could be via suppressing CD14+ monocyte differentiation into matured dendritic
cells and triggering the production of inducer molecules [44, 45]. In addition, MSCs
can suppress the differentiation of allo-antigen stimulated monocytes toward mature
dendritic cells [45]. Immature dendritic cells can induce cytokine production char-
acterized by a reduced secretion of pro-inflammatory molecules including tumor
necrosis factor alpha (TNFα), IFNγ and IL-12, and an amplified secretion of the
anti-inflammatory molecule such as IL-10, when cultured in the presence of MSCs
[44–47]. Similarly, as MSCs cultured with mature dendritic cells, they started to
display decreased function of antigen-presenting, and down-regulated IL-12 pro-
duction [45].

2.1.1.4 Natural Killer Cell (NKs)-Mediated Suppression

NK cells take crucial roles in intrinsic immune response, particularly in anti-tumor

and anti-viral functions due to their cytotoxic activity, and they have the ability to
produce large amount of pro-inflammatory molecules, including TNFα and IFNγ
[48]. Impulsive cytotoxic function of NK cells especially target the cells displaying
decreased HLA-I expression. Moreover, MSCs suppress IL-2 and IL-15 mediated
NK cell proliferation, IFNγ secretion, and cytotoxic function of both latent and stim-
ulated NK cells [20, 21, 49]. Recently, researches have demonstrated that cell-cell
interaction is the most critical event in the MSC-mediated immunosuppression,
whereas other cells were driven by soluble elements dependent on HLA-I character-
istic. Donor cells isolated from HLA incompatible patients are much more vulnerable
2 Immunomodulatory Properties of Stem Cells Derived from Dental Tissues 33

to be lysed by triggered NK cells via secretion of high amount of IFNγ and TNFα
ex vivo [50]. MSCs suppress the expression of NKp30 and NKG2D surface receptors
which are take part in NK cell function and lysing of targeted cells [51].
Direct cell to cell interaction of MSCs with active immune cells leads inhibition
or limitation of inflammatory responses and enhances the mitigating and anti-
inflammatory pathways. Depending on activation type and expression profile
resulted via danger signals, inhibition or limitation can be chosen by MSCs [52]. In
addition, transition of MSCs into infected and damaged tissue might interfere to
secondary lymphoid organs which are crucial for the immunity. They can also dif-
fuse into the damaged tissue or organ to trigger healing process, resulting in func-
tional tissue or organ regeneration and local immunity. While these studies enlarge
our understanding about MSC-mediated immunosuppression, further studies must
be strictly conducted to fully elucidate exact action of mechanism underlying these
suppressive, regenerative, and anti-inflammatory functions [11, 14].

2.2 Immuno-Modulation of DSCs

Immunomodulation properties of MSCs make them outstanding candidates as

immunosuppressive drugs for the prevention and treatment of various inflammatory
and autoimmune diseases [38, 53]. Starting from the 2000, unique MSC sources
have been obtained from several dental tissues, which exhibit remarkable tissue
regenerative and immunosuppressive properties [54]. These dental tissue derived
cells include dental pulp stem cells (DPSCs), periodontal ligament stem cells
(PDLSCs), gingival mesenchymal stem cells (GMSCs), tooth germ stem cells
(TGSCs), apical papilla stem cells (SCAPs), exfoliated deciduous teeth stem cells
(SHEDs), and dental follicles stem cells (DFSCs) [55–57]. Other than having spec-
tacular self-renewal property and multipotency, DSCs have powerful immunosup-
pressive functions comparable to other stem cell types, making them promising cell
sources for MSC-mediated transplantation treatment [58].

2.2.1 DPSCs

DPSCs have great regeneration capacity which can create pulp and blood vessels
containing fibrous tissue resembling human tooth like structure [55]. Studies explor-
ing the immunosuppressive characteristics of DSCs showed that DPSCs could sup-
press the proliferation of activated T cells better than bone marrow MSCs (BMMSCs)
[17, 57]. This remarkable immunosuppressive function of DPSCs could make them
appropriate cell type for allogenic bone marrow replacement therapy [59]. In addi-
tion, proliferation of PBMCs could be inhibited by TGF-β secreted from DPSCs,
indicating the immunosuppressive role of MSCs triggered by signaling molecules
derived from stimulated DSCs [60].
34 P.N. Taşlı et al.

DPSCs have been used in an important transplantation study to treat Duchenne

Muscular Dystrophy (DMD) of dogs carrying congenitally golden retriever muscu-
lar dystrophy (GRMD). Researchers found that DPSCs displayed immune suppres-
sion in GRMD dogs and helped to relieve disease symptoms [61]. In addition,
DPSCs has been shown to trigger T cell apoptosis in vitro, and improve inflamma-
tion dependent tissue injuries as given to a murine colitis model in vivo. siRNA
mediated inhibition of FasL expression, a trans membrane protein, involves in
induction of the Fas/ FasL apoptotic pathway in DPSCs, leading to partial suppres-
sion of T cell apoptosis in vitro, and reduction in therapeutic effects of DPSCs in
mice colitis models in vivo. Moreover, it has been proposed that FasL expression
levels could regulate duplication rate and differentiation capacity of DPSCs.

2.2.2 PDLSCs

Periodontal ligament (PDL) tissue is originated from neural crest, and it can be
obtained from dental follicle. Mainly, PDL cells connect the cementum to the bone,
and support the tooth organ in the alveolar socket [62, 63]. These cells could also
arrange the tooth nutrient source, homeostasis, restoration and regeneration of
injured tissues [64, 65].
Several comparative studies showed that both PDLSCs and BMMSCs demon-
strated suppressive effects on the production of allogeneic and xenogeneic PBMCs
by blocking the cell doubling via secretion of TGF-β, HGF, and IDO [66]. Another
study exploring the immunosuppressive role of PDLSCs has indicated that the
inflamed PDLSCs displayed considerably reduced suppressor effects on T cell acti-
vation compared to healthy cells. In vitro co-culture studies showed that stimulated
PBMCs displayed significantly less activation of CD4+CD25+Foxp3+ regulatory T
cells, and IL-10 production when inflamed PDLSCs were used. Additionally, inhi-
bition of Th17 differentiation and IL-17 secretion from inflamed PDLSCs was sig-
nificantly less compared to healthy PDLSCs, proving inflamed PDL tissue serve
less immunosuppressive stem cells [67].

2.2.3 TGSCs

TGSCs derived from third molar tooth germs of young adults are MSCs with high
proliferation and differentiation capacity [68]. Besides their availability in adult
body and having no ethical problems associated with embryonic stem cells, human
TGSCs can be converted into osteogenic, adipogenic, myogenic, neurogenic, odon-
togenic [6, 69–71] and endothelial cell linages [72]. Human TGSCs were used as
immunosuppressive agent in rat tooth socket. It has been reported that TGSCs not
only suppressed the immunity but also they regenerated the tooth socket at early
2 Immunomodulatory Properties of Stem Cells Derived from Dental Tissues 35

dental organogenesis [73]. Exact mechanism of immunomodulatory properties of

tooth germ stem cells are still unknown; hence, further studies are required in vitro
and in vivo.

2.2.4 GMSCs

GMSCs were firstly isolated at 2009, and they have been shown to be easily obtained
from waste materials of routine dental treatment. GMSCs exert stem cell-like char-
acteristics and immunosuppressive properties similar to other DSCs [54, 74].
GMSCs have the ability to stimulate a strong inhibitory action against immunity
cells by enhancing the IFNγ-dependent IDO, IL-10, cyclooxygenase (COX)-2 and
inducible nitric oxide synthase (iNOS) synthesis [75].
Zhang et al. showed that when macrophages were co-cultured with GMSCs, they
developed an anti-inflammatory M2 phenotype, which might enhance wound heal-
ing process [76]. Another study reported that systemic infusion of GMSCs intensely
improved contact dermatitis as revealed by a reduced penetration of dental cells,
CD8+ T cells, Th17, and Mast Cells (MCs) [77]. Many in vivo transplantation stud-
ies have shown that stimulated GMSCs exerted stronger regeneration ability than
non-stimulated healthy GMSCs, supporting the idea that GMSCs contribute to the
pathogenesis of drug-stimulated gingival hyperplasia.

2.2.5 SCAPs

SCAP cells are obtained from cervical loop which contributes to tooth formation
and pulp tissue development [78, 79]. SCAP cells showed a two-fold higher cell
doubling rate compared to DPSCs. Moreover, SCAP cells can suppress T cell pro-
liferation in an apoptosis independent way [57]. Further studies are necessary to
explore the potential usage of SCAP cells as an immunosuppressive agent.

2.2.6 SHEDs

SHED cells are obtained from the coronal pulp of exfoliated deciduous teeth [56].
SHED cells exert MSC characteristics but high levels of alkaline phosphatase (ALP)
were detected under osteogenic induction conditions. SHEDs have been shown to
suppress Th17 function proposing that SHEDs could be used to treat systemic lupus
erythematosus (SLE) in vivo [54].
Available data in the literature provides valuable information about MSC-
regulated immune suppression. However, advanced in vivo and clinical studies
should be completed to reveal multiple signals and complicated mechanisms con-
trolling immune regulatory pathways.
36 P.N. Taşlı et al.

2.3 Relation Between Immunosuppression and Stem Cell

Expression Profiles

The discovery of DSCs in the pulp tissue by Gronthos et al. was a milestone for
MSC research and expanded new horizons for the development of alternative treat-
ment strategies [55]. MSCs residing in dental tissues are characterized by differen-
tiation ability into many cell types such as adipocyte, osteocyte, chondrocyte and
myocyte under appropriate culture conditions [80]. In addition to MSC-like behav-
ior at culture conditions, DPSCs are positive for MSC surface markers such as
CD29, CD73, CD105 and CD 90. It has been revealed that stage-specific embryonic
antigen-4 (SSEA-4), a globo-series ganglioside, is also expressed in DPSC popula-
tions, and can be used to sort DPSCs [81]. Moreover, STRO-1, which is a cell sur-
face protein expressed by BMMSCs, is also expressed by DSCs [82]. Although
STRO-1 is used as a specific marker to characterize DSCs, some STRO-1+ cells
express typical hematopoietic stem cell markers such as CD117 and CD34 [83, 84].
Use of DSCs for regenerative medicine could be problematic in some situations.
Inflammation in periapical and pulp tissues could restrict the regeneration potential
of DSCs [85]. In the case of inflammation caused by bacterial infection, several
immune cells such as macrophages, neutrophils, T and B lymphocytes take in
charge as immune protector [86]. In this situation, MSCs express certain cell sur-
face markers providing the interaction with the immune system to suppress the
immunity [86]. The immune system and MSC interactions are well-known for
BMMSCs which express receptors for plenty of cytokines such as IL-4, IL-6, TNFα,
IFNγ, and growth factors including epidermal growth factor (EGF), TGF-β and
bone morphogenetic proteins (BMPs) [87, 88]. Moreover, MSCs express vital mol-
ecules necessary for cell to cell interactions with immune and hematopoietic cells,
particularly various adhesion molecules.
Although similar expression profile of DSCs with other well-studied MSCs is
expected, currently, there is not a comprehensive research for the investigation of
surface proteins of all types of DSCs. Some receptors for distinct mediators
expressed by DSCs are TGF-β, vascular endothelial growth factor (VEGF), fibro-
blast growth factor (FGF) and insulin-like growth factor (IGF) [89–93].
MSCs can receive signals from the inflammatory environment via several recep-
tors. They express some growth factors and cytokines such as IL-11, IL-8 and IL-6,
and involved in the early maturation of T cells [87]. It was shown that MSCs modify
cytokine release of distinct types of adaptive immune cells like T cells or suppress
their proliferation [94–96]. Immunomodulatory potential of MSCs have been
described for BMMSCs, and also have been confirmed for DPSCs. Responses of
activated T cells are prohibited by DPSCs and SCAPs [97, 98]. It has also been
reported that in the existence of DPSCs and PDLSCs, repression of PBMC prolif-
eration has been observed [99]. Similar effects have also been demonstrated for
gingival fibroblast [95].
MSCs derived from dental tissue express some critical receptors for inflam-
matory agents, which can be produced by injured cells and inflammatory cells.
2 Immunomodulatory Properties of Stem Cells Derived from Dental Tissues 37

They can also be released from dental tissues. Released cytokines and growth
factors are able to decrease or increase the proliferation or differentiation potential
of MSCs derived from dental tissues.

2.4 Clinical Applications

Clinical study is the process of experiments and trials, observational studies in med-
ical and other types of research. The aim of a clinical study is to investigate the
activity, safety and mechanism of action of an investigated product, drug or device.
In this regard, MSCs are being currently used in clinical applications for the treat-
ment/prevention of various disorders including immunity related disorders. In par-
ticular, human MSCs derived from adult donors are isolated and cultured in the
laboratory conditions, and they have displayed the ability to find injured tissue,
reduce and control the inflammation [100]. In addition, BMMSCs are evaluated for
the treatment of GVHD [101] and their safety, tolerability and effectivity after liver
or kidney transplantation are also being investigated in clinics [102]. Currently,
there are no clinical studies investigating immunosuppression properties of DSCs.
However, as DSCs have been proven to display comparable immunosuppression
properties with other well-studies MSCs, it is worth to conduct clinical trials using
different DCSs. For instance, DPSCs have exhibited 18 % higher suppression rate of
T lymphocyte growth in comparison with BMMSCs, indicating possible superior
immunosuppression potential of DSCs. In addition to their outstanding immuno-
suppressive activity, DSCs have several advantages which makes them promising
candidates to be used in various clinical trials including;
• They are cost-effective, easily obtainable and do not need ethical and safety con-
cerns as long as they are derived from regular orthodontic procedures [103].
• They can be easily cryopreserved, stored long-term, and combined with many
structural materials [104].
• They have neuro-protective effects on dopaminergic neurons and motor neurons
in spinal cord [105, 106].

2.5 Safety Issues

Absence of clinical studies investigating in vivo survival of MSCs after tissue or cell
transplantation can bring serious doubts to usage of MSCs in human cases. Few
human studies have indicated that long term side effects of MSCs are still unknown.
Not just only immunosuppressive effect of DSCs against immunogenic cells, but
also their anti-inflammatory role, tissue repairing function, and interaction between
these actions must be studied before performing clinical studies. In addition, several
questions including “is this immunogenic suppression mediated by MSCs stable for
long time”, “do DSCs cause malignant transformation in the related or un-related
Other documents randomly have
different content
On the left Eugène had attacked Borodino with Delzons’ division of
the 4th Corps. The attack was made under cover of the mist which
still hung over the field, and the village was carried with a rush. The
Guard Chasseurs lost 30 officers in a quarter of an hour, and were
driven in wild confusion to and across the Kolotza. The bridge was
taken, and the 106th French Regiment poured across it in pursuit.
The garrison would have been destroyed but for the 1st Chasseurs,
under Colonel Karpenko, who hurried up to the rescue. Charged by
them, and smitten by the fire of Ostermann-Tolstoï’s guns from the
farther bank, the 106th lost heavily. General Plauzonne was killed as
he endeavoured to rally it, and its remains were driven back across
the stream. Karpenko’s charge was stopped by the 92nd Regiment,
but he succeeded in destroying the bridge. Eugène left Delzons to
watch the Kolotza north of the village, placed the Royal Guard in
reserve, stationed the cavalry of the 4th Corps and Preising’s division,
now united under General Ornano, to cover the left flank, and turned
Morand’s, Gérard’s and Broussier’s divisions, supported by Grouchy,
against Dokhturov and Raievski.
On the right Poniatowski captured Utitza, held only by the outposts,
without difficulty; but on the knoll beyond Tuchkov had massed a
strong force of artillery, supported by Strogonov’s division, while the
Chasseurs in the wood to the north brought a flanking fire to bear
upon the Poles. Poniatowski ranged 40 guns in advance of the village,
but they failed to silence Tuchkov’s artillery, and for some hours the
action in this quarter was reduced to cannonading and skirmishing.
Kutuzov, seeing that nearly the whole French army was moving
against his centre and left, about 7.30 a.m. ordered Baggohufwudt to
march the bulk of his corps to the support of Bagration. But as the
movement would take some time, and Bagration appeared to need
immediate support, the Ismailovski and Lithuanian Guards, some
Grenadiers, and a brigade of Cuirassiers, were sent forward, much to
the disgust of Barclay, who held strong views about depleting
reserves until the last moment. He hurried to Kutuzov, and begged
him not to use up the Guard until things became critical, and Kutuzov
assented. His action during the greater part of the battle indeed
seems to have been confined to approving his lieutenants’ measures.
Davout and Ney, after being forced from the redans, reformed their
troops for another assault. Friant’s division was called up in support,
and Tharreau’s division of the 8th Corps sent forward by Napoleon
against the wood to the south, from which the Russian light infantry
were keeping up a heavy fire. When the Westphalians began to
penetrate the wood matters appeared critical for Bagration, exposed
to attacks in front and flank at the same time; but at 9 a.m.
Baggohufwudt’s corps arrived to his support. Eugen’s division was
placed in reserve behind Semenovskoï; two of Olsuviev’s regiments
reinforced Tuchkov IV, while the remaining four pushed into the wood
and drove the Westphalians out again.
The attack on the wood had, however, caused the cessation of the
flanking fire which had annoyed the Poles; and Poniatowski attacked
and carried the knoll behind Utitza. The success was but momentary.
Strogonov rallied his broken division; Tuchkov I himself led forward
the Pavlovsk Grenadier regiment; while Olsuviev broke out of the
wood with two regiments of Chasseurs. Attacked in front and flank,
and charged by Olsuviev in the rear, the Poles were unable to stand
and were thrust back to Utitza. The Russian success was achieved at
the cost of the life of Tuchkov, who was mortally wounded as he led
on his Grenadiers.
The arrival of Baggohufwudt enabled Bagration to steady his line
against the renewed advance of Davout and Ney. The struggle on the
low heights was indescribably close and desperate. Behind the
furiously fighting masses of infantry hovered the cavalry, charging
again and again as opportunities presented themselves. By 10 a.m.
the French had once more taken the redans; and the 15th Léger, of
Friant’s division, fought its way into the ruins of Semenovskoï.
Borozdin, charging with four regiments of Grenadiers, drove it out
again past the redans, but was then set upon by Nansouty’s cavalry
and forced back; and the struggle raged more furiously than ever as
the French once more stormed the redans, to be hurled out again by
a counter-attack of Tuchkov IV’s division, led by Konovnitzin. Already
the losses had been fearful. Romœuf, Davout’s chief-of-staff, had
fallen, and on the Russian side General Tuchkov IV, the second of his
family to die for Russia on the field of Borodino.
Eugène, having crossed the Kolotza by the temporary bridges, placed
batteries in position to bombard the Great Redoubt, and formed
Morand’s division opposite that of Paskievich, which held the knoll.
Broussier moved forward in support on Morand’s left, while Gérard
was still crossing the stream. Paskievich’s troops outside the redoubt
were so shattered by the fire of the French batteries that they sought
refuge behind the shoulder of the knoll; and General Bonami, with
the 30th of the Line, saw his chance. As the regiment advanced up
the knoll it suffered fearfully in its close formation, but nevertheless
pressed on dauntlessly and stormed the work, after a furious struggle
with the Russian infantry and gunners, who proved, as Captain
François says, worthy antagonists.
This sudden piercing of the centre of the Russian line produced an
immediate counter-attack, while Morand’s other regiments appear to
have been too busy with Kolubakin’s division to support Bonami. A
message was hurriedly sent to Barclay; but without waiting for orders
all the officers on the spot immediately did the right thing. Yermólov,
who was at hand, picked up a battalion of the Ufa regiment
(Likhachev’s division) and was joined by Colonel Löwenstern,
Barclay’s aide, with one of the Regiment of Tomsk. Likhachev hurried
up the 19th and 40th Chasseurs. Vassilchikov promptly turned a
battalion of Kolubakin’s division against the lost redoubt, and
Paskievich, rallying his broken division, again pushed forward. The
improvised attack was completely successful. Unsupported, except by
one battalion of the 13th Léger, the gallant 30th was lost. The
redoubt was recaptured and Bonami desperately wounded and taken.
As the remains of the regiment streamed away down the knoll,
Barclay came upon the scene, and let loose a brigade of Kreutz’s
Dragoons. The 30th was almost completely destroyed, only 11
officers and 257 men being able to rally. The Russians had not come
off scatheless. Count Kutaïsov was killed as he led the charge with
Yermólov, and the latter was wounded. To cover the escape of the
remains of the 30th Eugène concentrated a tremendous artillery fire
on the redoubt, in which Barclay replaced Paskievich’s shaken division
by Likhachev’s.
The Great Redoubt was retaken at about 11 a.m., and at the same
time Ney, Davout and Murat made a last and determined assault on
Bagration’s position about Semenovskoï. The last reserves of the 1st
and 3rd Corps were thrown into the fight; Napoleon sent up the rest
of the 8th Corps; behind the infantry were ranged Nansouty’s and
Latour-Maubourg’s corps and the Corps Cavalry, and the attack was
covered by the fire of over 250 guns.
This final assault was made by the French and Germans with
magnificent courage. Under the furious fire of the Russian artillery
and musketry the attacking columns pressed steadily on. The sight of
their advance roused Bagration to generous admiration. Believing
that it could not be stayed by artillery and infantry fire, he
determined to make a counter-attack, and sent forward every
available battalion. Again the opposing forces closed in deadly strife—
Frenchmen, Russians and sturdy Germans, Spaniards and Portuguese
enlisted in a quarrel not their own, fighting to the death around the
blood-stained derelict redans. The cavalry joined in the conflict,
individual regiments and squadrons striking in whenever an
opportunity occurred. Bagration was desperately wounded in the leg;
and Löwenstern tells how he found him lying among his staff behind
the line, while Sir James Wylie, Alexander’s surgeon, attended to his
wound. He said to Löwenstern, “How goes it with Barclay? Tell him
that the safety of the army depends upon him. All goes well here at
present”—and then seeing that Löwenstern was himself wounded, he
kindly added: “Get yourself bandaged.” This touch helps one to
understand the personal admiration which Bagration undoubtedly
inspired in nearly everyone who came in contact with him; and it is
certain that his fall caused a serious slackening in the vigour of the
defence. Once more the stubborn Russians were driven from the
redans and past Semenovskoï; and this time they were not to regain
their lost positions.
After Bagration’s wound the temporary command devolved upon the
brave soldier Konovnitzin; but he was unable to check the retreat
which had now definitely set in. The whole of the defending force
gave back towards Tzarévo, and upon it Murat launched his great
masses of cavalry. Still there appear to have been no signs of
demoralisation, and little real disorder; and, though clearly worsted,
the Russian infantry maintained a desperate resistance. Behind
Semenovskoï the Ismailovski and Lithuanian Guards, which had not
yet been seriously engaged, were drawn up in squares; and, their
heavy fire checked the advance of the French cavalry. Murat brought
up some batteries which opened fire against them, while in the
intervals of the cannonade Latour-Maubourg’s corps charged their
shattered battalions. The Guards suffered fearfully, but closed their
ranks and held firm, repelling three charges of the Saxon Cuirassiers,
who were almost annihilated. The two regiments must, however,
have been destroyed also but that Borozdin II came to their rescue
with a Cuirassier brigade, checking the French horsemen by
countercharges and enabling the Guards to follow in the retreat,
leaving their position outlined in squares of dead and dying.
In the rear of Semenovskoï the ground was covered with struggling
hordes of infantry and cavalry. Out of the confusion at length
emerged some sort of order, the Russians taking up position along
the Tzarévo plateau, covered by the fire of batteries from the reserve,
while Davout and Ney reformed in front of Semenovskoï. Davout had
been hit four times, but declined to leave the field, though obliged to
withdraw for a short time, during which Ney and Murat exercised the
command. The King of Naples behaved with all his usual reckless
bravery; and Baron Lejeune speaks with admiration of the splendid
figure presented by Ney, as he stood directing the battle from the
parapet of a redan. And on the other side Barclay, now practically in
sole command of the Russian army, was setting an example no less
heroic, apparently wishing to meet his death on the field of Borodino.
As the stress of battle grew, the Russian generals for the most part
concealed the insignia which made them conspicuous; not so the
slandered War-Minister, who faced the storm wearing full-dress
uniform and all his decorations. It is impossible not to appreciate the
heroic impulse that prompted him, like Nelson and many another
fiery spirit, to expose himself to death decorated with the badges
won on the field of honour. His staff were almost all killed or
wounded, and he had two horses shot under him, but escaped with
the slightest injury.

MARSHAL NEY
Commander of the 3rd French Army Corps. The hero of the Retreat
After the picture by Langlois at Versailles
As he saw Bagration’s line driven in, Kutuzov had ordered the 4th
Corps also to draw in to the centre. By noon the Russian position was
peculiar. Dokhturov on the right and Tuchkov on the left still faced
the French in nearly their old positions, while the rest of the army
stretched in a convex between them. To drive back Dokhturov and
Tuchkov was Napoleon’s next object, and he was about to order
forward part of his Guard when, apparently a little after noon, he
received intelligence that his left was being attacked.
Early in the day Platov, reconnoitring towards the right, had
ascertained that there were comparatively few French troops north of
the Kolotza, and had proposed to the commander-in-chief a cavalry
attack on their flank and rear. Kutuzov assented, and for the purpose
detailed Uvarov’s cavalry corps,[6] some 3500 sabres strong.
Clausewitz, who was then on Uvarov’s staff, criticises the movement
severely. Certainly 3500 regular, and 4000 irregular, horsemen could
not of themselves effect much; but it is a little difficult to concur in
his opinion that the detachment of Uvarov’s corps was a rash
weakening of the line. It is easier to agree with him when he says
that the movement was made too early in the day. Uvarov, however,
was very slow and did not cross the Kolotza until past eleven. About
11.30 he approached the Voïna (a little stream which enters the
Kolotza at Borodino), at Besubovo, about a mile and a half from the
former village. On the Russian side of the stream stood Guyon’s
cavalry brigade and a regiment of the Italian Guard, which withdrew
over a mill dam before the fire of Uvarov’s artillery to join the rest of
the Guard and Delzons’ division, which occupied Borodino. Platov
now came up, and his wild horsemen dashed through a ford and
among the Italian infantry, followed, without orders, by the Cossacks
of the Guard, who lost heavily in charging the squares. Uvarov,
however, would not risk his regulars, halted, sent for orders and
finally withdrew. Clausewitz’s comment is that he was not the man to
lead such an attack. Löwenstern fumes at his slowness and
hesitation. More, undoubtedly, might have been achieved with a
bolder commander. Even so his feeble diversion brought Eugène back
across the Kolotza with Broussier’s division, and delayed the advance
against the Great Redoubt. He finally withdrew about 3 p.m., but
before this Eugène had returned across the Kolotza.
All this time a tremendous and unprecedented cannonade was being
kept up. Between Borodino and Utitza some 900 guns were in action.
The Great Redoubt was being furiously bombarded by Eugène’s
artillery, while Ney’s batteries brought a converging fire to bear upon
it from the southward. To storm it Eugène detailed Gérard’s division,
hitherto but lightly engaged, while Morand and Broussier supported;
and Napoleon ordered Montbrun, with his Cuirassier divisions, to
charge the Russian line on Gérard’s right. Montbrun was killed as he
led forward his men, and General Caulaincourt, brother of the Duke
of Vicenza, came hastily from the Emperor to take up the command.
“Don’t stop to lament!” he said to the dead general’s aides. “Follow
and avenge him!” The mass of mail-clad horsemen broke through the
Russian line south of the redoubt, wheeled to the left and came
thundering upon its rear, just as Eugène’s infantry reached it in front.
For the four regiments of Likhachev’s division which held it there was
no escape—at least as regards the major part. Some of them who
were outside the work succeeded in saving themselves; but those
within were trapped and, after maintaining a desperate resistance
against the charges in front and rear, were almost all cut to pieces.
Likhachev, who was very ill, flung himself among the assailants, and
had almost found the death which he sought when some French
soldiers, attracted by his insignia, took him prisoner, severely
wounded. Caulaincourt was struck down as he led the triumphant
charge of his Cuirassiers—one more victim of the fatal “Battle of the
Generals.”
The capture of the redoubt opened a huge gap in the Russian line,
through which Eugène’s and Grouchy’s cavalry poured to complete
the victory. Against them Barclay hurled all the horsemen whom he
had under his hand—the 2nd and 3rd Cavalry Corps and the
Cuirassiers of the Guard—leading more than one charge in person,
while Ostermann-Tolstoï’s corps, supported by the yet unengaged
portions of the 5th Corps, was ordered to make a counter-attack
towards Semenovskoï. Ostermann-Tolstoï, as he had shown at
Ostrovno, was not the man for an emergency; Löwenstern says that
he appeared to have entirely lost his head. He moved forward so
slowly that Ney, Davout and Murat were able to make preparations to
receive him. The French infantry were almost fought out; the cavalry
had literally “foamed themselves away” in endeavouring to shatter
the resistance of the stubborn Russian infantry; the fire of the
Russian artillery was as steady and effective as ever. The Marshals
sent again and again for some part of the Guard to support their
weary men, but Napoleon refused to risk it. The Russian writers
express astonishment at his caution. All that he would do was to send
forward the reserve of heavy artillery, under Comte Sorbier. Sorbier
swore at Lejeune, who brought the order to advance. “I ought to
have had it an hour ago!” was his comment.
The Marshals had got together 80 guns wherewith to oppose the
Russian advance; and when Sorbier came up the 4th Corps was
overwhelmed with a crushing cannonade, against which it could not
make way. Its losses were terrible. General Bakhmetiev had his leg
carried away; Ostermann-Tolstoï himself and several of his staff were
wounded. The supporting cavalry charged with splendid audacity, and
some of them actually re-entered the Semenovskoï redans. All was in
vain. Sorbier’s battery had turned the scale against the Russians, and
by about 4 o’clock they were in full retreat. The infantry and cavalry
on both sides were fairly fought out, and the struggle was maintained
only by the artillery, except on the extreme left of the Russian line,
where Tuchkov’s force, now commanded by Baggohufwudt, was
practically isolated. The Polish Corps, supported by the Westphalians
on the left, succeeded about 5 p.m. in carrying the Utitza knoll, and
Baggohufwudt, still barring the road, drew back into line with the
centre and left. There was a last flicker of hostility near Semenovskoï,
where the Finnish Guards repulsed an advance of some French
battalions, and then the battle died away in a dwindling cannonade,
until a thick fog shrouded in a merciful veil the awful scene of
slaughter. The Russian line, reformed by Barclay, stretched from
beyond Gorki along the edge of the Tzarévo plateau to the old
Moscow road. Four Chasseur regiments, under Colonel Potemkin,
were on the right with Platov’s Cossacks. The remains of Dokhturov’s
corps, supported by Uvarov, held Gorki. Next came Ostermann-
Tolstoï’s corps; and thence Raievski and Borozdin, with Prince Eugen’s
and Shakovski’s (late Tuchkov IV’s) divisions, continued the line to
Baggohufwudt’s position. The cavalry was in rear, and the 5th Corps
in reserve behind the centre. The French lay opposite, Delzons and
Lecchi north of the Kolotza and, to the south of the river, the 4th,
3rd, 1st, 8th and 5th Corps in succession from Borodino through
Semenovskoï to a point about 1200 yards east of Utitza.
The consensus of opinion of eyewitnesses on the Russian side is that
the spirit of the Russians was unbroken, and that there was little
confusion in the ranks. Löwenstern says that he offered to attack the
Great Redoubt at break of day, and that Barclay approved. Kutuzov,
however, when he learned the extent of the slaughter in his army,
decided to retreat. It is quite certain that he must have retired in a
day or two, since he had no reserves, while Napoleon had 11,000
fresh troops (Laborde and Pino) approaching the field. Barclay,
however, was bitterly angry; and when he received the order to
retreat broke into a fierce invective against Bennigsen, to whose
influence he attributed Kutuzov’s resolution.
Under cover of darkness the Russian army quietly withdrew, and on
the 8th took up a position in front of Mozhaïsk. The retreat was
effectually covered by the Cossacks, who displayed great audacity,
and in the night of the 7th-8th repeatedly disturbed the French
bivouacs. The French cavalry, shattered and exhausted, could do little
or nothing, and the Russians remained all through the 8th at
Mozhaïsk, employing the time in reorganising, and in evacuating
towards Moscow as many as possible of their wounded. Nevertheless
many were left to inevitable death on the field, and thousands more
abandoned at Mozhaïsk to the mercy of the French, who, themselves
in a sorely distressed condition, simply cast them out to die of misery
in the fields.
Regarding the major tactics of the battle of Borodino there is little to
say. Napoleon had deliberately chosen to make a frontal attack upon
the Russian army in place of turning it; and in the practical absence
of his personal supervision the battle almost fought itself. The idea of
taking advantage of the extension of the Russian right by
overwhelming the left was an excellent one. It was foiled by the
determination of Bagration’s resistance, which permitted
Baggohufwudt’s corps to be moved across to his support. On the part
of the Russians the occupation in force of the position north of the
new road proved a blunder, which the remarkable solidity of the
Russian resistance enabled the generals to repair in time.
As regards what is often considered Napoleon’s fatal error in not
throwing in the Guard it is very doubtful whether it was an error at
all. It must be remembered that Ney’s and Davout’s troops were
almost, if not quite, fought out, that the Russians were still solid and
undemoralised, and holding a position quite as strong as that from
which they had been evicted; and that Kutuzov still had some almost
untouched reserves. The Guard would have had no easy victory, and
Napoleon was probably right when he refused to expose it to severe
and perhaps fatal losses. He knew that the Russians were neither
routed nor in disorder; and if they stood to fight again nearer
Moscow he might yet have sore need of his Guard. He was 1200
miles from the frontier of his dominions, and in case of disaster all
must depend upon it.
Of the minor tactics little need be said. On both sides they were
crude and wasteful. There was a deficiency of infantry on the French
side, and the cavalry was freely employed to supplement it, with the
result that it was half destroyed. The infantry formations were dense
and clumsy, it was a case of heavy mass pushing against heavy mass,
with cavalry mingled in the melée, and all under the fire of a
thousand or more pieces of artillery. It is no wonder that the losses
were unprecedented on both sides.
Napoleon gave his loss at 10,000. French writers admit the suspicious
round number of 28,000—6547 killed and 21,453 wounded—but
Martinien’s lists show 49 generals and 1934 officers killed and
wounded, and even allowing for the fact that many of the effectives
were now low, and the proportion of officers to rank and file
therefore higher than usual, this can scarcely imply less than 43,000
casualties. Even the troops which had never been sent forward had
suffered somewhat from the cannonade.
 BATTLE OF BORODINO (September 7th, 1812)
The losses of the First Russian Army from the 4th to the 7th of
September are stated by Bogdanovich at 9252 killed and 19,226
wounded. Those of the Second Army may perhaps, since it contained
only 54 battalions and 52 squadrons as against Barclay’s 122
battalions and 112 squadrons, be estimated at 12,000. Adding the
losses of the militia, a total is obtained of possibly 41,000 or 42,000.
Sir Robert Wilson estimates it at 1500 officers and 36,000 men.
Buturlin gives 15,000 killed and 30,000 wounded.
Prisoners there were few—perhaps 1000 or 1200 French and 2000
Russians. The estimates of guns captured are somewhat vague.
Kutuzov’s official figure of French guns taken and carried off by the
Russians is 8. There may, of course, have been others disabled. The
Russians seem to have lost about 18 in all.
On the field Borodino can scarcely be described as anything but a
drawn battle. Napoleon had gained a little ground, but the Russian
army was unbroken and apparently quite willing to renew the contest
next day. Strategically the French appeared to have obtained a slight
success, since they were able to continue their advance to Moscow.
On the other hand, the battle, which ruined the cavalry and seriously
shattered the infantry, brought ultimate ruin distinctly nearer. Perhaps
its most noteworthy result was the extent to which the morale of the
Napoleonic army was broken.

FOOTNOTES:
[5] This is not absolutely certain, but appears to be proved by the
statements of eyewitnesses of the campaign. De Fezensac, for
example, says that the 3rd Corps had not yet exhausted its
supplies when it entered Moscow.
[6] Uvarov had 32 squadrons in all.

CHAPTER VIII
THE OCCUPATION AND DESTRUCTION OF MOSCOW
THE morning of the 8th of September found the army of Napoleon
bivouacked among the dead and wounded on the field of Borodino.
Only the Guard was really ready for further combat. The corps of
Davout and Ney were terribly cut up; the 17th, 30th and 106th
Regiments were nearly destroyed. The cavalry, which had to
compensate for Napoleon’s comparative weakness in infantry, had
suffered fearfully. Nearly all its corps and divisional commanders were
killed or wounded; several regiments were almost annihilated. The
four corps of the reserves counted some 19,000 men on September
2nd; on the 20th they could muster little more than 10,000.
Thousands of horses had been killed, and there was no present
possibility of being able to replace them, while the wounded animals
were mostly doomed to perish from lack of forage and proper care.
The cavalry of the Guard alone was in a state for serious combat.
The fate of the wounded was horrible. Means of every kind for
tending them were lacking, and fortunate were those whose end was
hastened by the incurable nature of their hurts, or thirst and
starvation. Days elapsed before all had received so much as first aid;
and this was but the commencement of their miseries. The great
monastery of Kolotskoï became the principal hospital, and in its
buildings the victims of Borodino were huddled literally in heaps,
without beds even of straw, without food or fire, and without a tenth
of the medical aid that was needed. Some of the wounded officers
were able to buy food, at enormous prices, from the convoys which
passed these dens of horror; but for the unhappy rank and file, who
possessed little or no money, there was no hope. Sanitation there
was none, and the unfortunate beings died in thousands, amid filth,
pestilence and neglect. François says that in one hospital a dead
officer was found who, in the agonies of starvation, had devoured his
own arm to the bone. It is a painful task even to touch upon these
sickening details, but to fail to do so is to neglect the primary duty of
an historian.
Besides the enormous diminution of the effective strength, the state
of the ammunition-trains was by no means reassuring. There is
reason to believe that the artillery had fired 90,000 rounds during the
5th and 7th; the infantry must have expended millions of cartridges.
It is certain that, immediately after Borodino, Napoleon was anxiously
pressing for fresh supplies of ammunition; and it is doubtful if he
could have delivered another pitched battle before they arrived.
Worse than all, the spirit of the troops was grievously depressed. The
gaiety which commonly characterises Frenchmen, even in untoward
circumstances, had vanished. Gloomy silence reigned during the
march and in the bivouacs. The negative results of the great battle
had completed the discouragement of the troops. The French
soldiers, at any rate, were too intelligent not to have some inkling of
the disasters that might too probably lie before them.
On the whole Kutuzov might perhaps have remained longer on the
field of battle. It is, however, probable that his withdrawal to
Mozhaïsk was wise. He had dealt a tremendous blow at the efficiency
and morale of Napoleon’s army, but in doing so his own forces had
been fearfully shattered. Had he remained in position the
circumstance might have decided Napoleon to use the almost
untouched Guards, and so at the last moment wring a victory from
frowning Fortune.
When the sun dissipated the autumn fog which had enwrapped the
field, the Russian position was seen to be guarded only by the
hovering pulks of Platov’s Cossacks. Against them Murat moved such
of his exhausted horsemen as could be rallied; and before the
advance of regular squadrons the riders withdrew. Behind them,
however, were supports of infantry—the four Chasseur regiments of
the 2nd Corps. They gave back very slowly, and did not reach
Mozhaïsk until 4 p.m. By that time a large number of the Russian
wounded had already been evacuated. The town, however, was still
choked with disabled men, many of whom were in a state to be
moved, and to cover this operation Kutuzov directed Platov to hold it
as long as possible. The Russian main body was in position behind it.
Napoleon, as soon as he was assured that the Russians had really
retired, ordered Murat to press their retreat. The King had the four
reserve cavalry corps and the light horse of Ney and Davout as
before,—a total now of not more than 14,000 lances and sabres—but
Compan’s shattered division was replaced by that of Dufour (vice
Friant wounded). The Emperor apparently at first believed that
Kutuzov was in full retreat, and the head-quarters baggage was
directed on Mozhaïsk; but Murat, as aforesaid, made little or no
headway against Platov; and the head-quarters could not be
transferred. Desultory skirmishing and cannonading went on until
nightfall, when Platov was still in possession of Mozhaïsk.
The firm front shown by Platov must have convinced Napoleon that
the spirit of the Russians was unbroken. He spent a part of the day in
going over the battle-field, examining the positions and reviewing the
troops according to his custom. In the afternoon he went forward to
join Murat, and on the way received another unpleasant reminder of
the unabated courage of his foes, some foragers being driven in by
Cossacks, and an alarm caused. On this day, however, a much
needed reinforcement arrived in the form of Pino’s Italian division.
At about 10 a.m. on the 9th Platov was fiercely attacked by Murat,
expelled, and driven along the Moscow road. Murat’s pursuit was
checked by a reinforcement of twelve battalions and a heavy battery
sent back by Kutuzov, but Mozhaïsk was lost, and Napoleon
transferred his head-quarters thither. Some thousands of the most
seriously injured of the Russian wounded were still there; and there
were hideous scenes as they were cast out of the houses for those of
the French army, who, in carriages and waggons, or dragging
themselves along on foot, streamed in piteous procession in rear of
the leading troops. The Russian main body retired deliberately to
Semlino (or Shelkovka) about 12 miles east of Mozhaïsk.
Napoleon himself remained for three days at Mozhaïsk recovering
from his cold, and transacting arrears of business. Already on August
27th he had sent orders to Victor to bring the 9th Corps from the
Niemen up to Smolensk; and from Mozhaïsk fresh directions were
despatched for him. From Mozhaïsk also was sent the bulletin
announcing the battle of Borodino. As his cold rendered him
speechless Napoleon wrote it with his own hand; and, being at best
an execrable writer, the result may be imagined. The French losses
are stated in it at 10,000. This would, according to Napoleon’s usual
standard, indicate from 40,000 to 50,000 casualties.
Meanwhile the Russian army was still steadily retiring on the high-
road to Moscow, and Murat deliberately following. Eugène, as before,
marched on the north by a track running roughly parallel with the
main road by the towns of Rusa and Zvenigorod, while Poniatowski
formed the right flank guard on the south. Junot remained at
Borodino and Kolotskoï to guard the hospitals. In support of Murat
marched Mortier with the divisions of Roguet and Claparède; and
behind him Davout and Ney in the order named.
Kutuzov was displeased with Platov for abandoning Mozhaïsk
prematurely, as he considered, and superseded him in the command
of the rear-guard by Miloradovich. On the 10th the Russian main
body made another deliberate march of about 8 miles, while
Miloradovich stood to fight at Krymskoië, some 3 miles short of
Kutuzov’s evening position. His force consisted of six weak regiments
of Chasseurs, four line regiments, Uvarov’s nearly intact cavalry
division, and some Cossacks. He occupied a low, partly wooded
ridge; his left was covered by a marsh, his right by woods, while in
the centre the high-road approached the ridge by a narrow gully
which was commanded by the Russian guns. Clausewitz, however,
who was present, does not consider that the position was particularly
advantageous. The twenty defending battalions can hardly have
mustered over 6000 bayonets. Murat came up towards 5 p.m., and
developed a fierce attack by Dufour’s division upon the right of the
position, defended by three Chasseur regiments under Colonel
Potemkin. After a hard struggle Potemkin was forced back from the
summit of the ridge, but he held firm, supported by three regiments
sent to his support by Miloradovich. Uvarov’s horsemen succeeded in
keeping Murat’s broken regiments at bay; and the Russians fought on
doggedly until darkness put an end to the contest. The Russian loss
is stated, probably with some exaggeration, at 2000. As Martinien’s
lists show 71 officers killed and wounded between the 8th and 10th,
the French can scarcely have lost less than 1200.
On the 11th the main Russian army marched 16 miles to Viazema
(Viazma on modern maps). Miloradovich retired to Kubinskoi, 8 miles
from Krymskoië, unpursued by Murat. Eugène and Poniatowski were
nearly level with Murat on the north and south, Mortier and Davout
some distance behind, and Ney only a short way past Mozhaïsk. On
the 12th Kutuzov retrograded to Momonovo, a bare ten miles from
Moscow, while Miloradovich withdrew to Malo Viazema, 12 miles to
the westward, leisurely followed by Murat. On the same day
Napoleon left Mozhaïsk for the front.
 MOSCOW FROM THE SPARROW HILLS
This is practically the scene which Napoleon contemplated in 1812
The question of the fate of Moscow was now imminent. It is at least
possible that Kutuzov would have risked another battle had there
been a fair prospect of success. But it cannot be said that this was
the case. All the way from Mozhaïsk the militia had been steadily
joining, but even so the army mustered less than 90,000 men, and of
these only 65,000 were regulars, as against over 90,000 still under
Napoleon’s hand. Many of the militia were as yet unequipped with
fire-arms, and all were raw and without training. Kutuzov could
expect no further reinforcements of regulars for weeks, whereas
Napoleon would be joined within ten days by Laborde’s division as
well as by some régiments de marche. The defective state of his
ammunition Kutuzov did not know. The spirit of the Russian troops
was indeed excellent, but against it was the greatest military genius
of modern times, backed by an army wearied indeed, and in part
much disheartened, but not yet demoralised, and including 20,000
untouched and undiscouraged veterans.
On June 10th Count Feodor Vasilievich Rostopchin, a former favourite
and confidant of the ill-fated Emperor Paul, and a fanatical opponent
of the French alliance, had been appointed Governor-General of
Moscow. Whether he was the right man for his position must be
questioned. It does not appear that anything was done to organise
and arm the inhabitants or fortify the city. Nevertheless Rostopchin
was furious at the idea of abandoning Moscow. According to Wilson
he never forgave Kutuzov for keeping him in ignorance of the critical
state of affairs.
As a fact he must have known that the evacuation of the city was to
be expected; and he appears to have been steadily clearing it as far
as possible of its inhabitants. This was the more practicable because
in the summer Moscow was considerably less populated than in the
winter, the nobles and their large households of serfs and retainers
being absent on their estates. Otherwise the Governor, who was not
a soldier, seems to have considered a good many rather wild plans of
resistance. It would have been perfectly feasible to defend Moscow
with the 90,000 troops of Kutuzov, but its destruction would thereby
have been rendered inevitable. As regards Rostopchin’s project of
arming the inhabitants, it is certain that such muskets as were
available were obsolete and of bad quality. For the rest, 200,000
human beings cannot abandon their homes in a day, and since the
French found the city nearly deserted it is obvious that the exodus of
the Muscovites had long been in progress.
On the 13th the Russians fell back to Fili, and took up a position on
the east side of Moscow, chosen by Bennigsen, who eulogises it in his
memoirs, saying that its only defect was that it was rather long. On
arriving Barclay proceeded to inspect it, while Kutuzov, who could ill
support the fatigue of the campaign, rested. Dokhturov, who
apparently had a touch of the courtier about him, proceeded to serve
him a meal, but the little picnic was quickly interrupted by Colonel
Löwenstern asking for Dokhturov, with whom Barclay wished to
confer.
“As usual!” said Barclay as he sent off Löwenstern. “There they all
are, dancing attendance on the Prince, and not troubling about what
they (the French) may do. Fetch Dokhturov here, even if his mouth is
still full.”
Kutuzov apparently rather enjoyed Dokhturov’s disappointment on
being thus interrupted. “You must not keep General Barclay waiting,”
he remarked. “I shall manage very well by myself,” and therewith
proceeded with his meal, while poor Dokhturov was obliged to go.
Barclay and he studied the position and came to the conclusion that
it was too weak.
A council of war was called for four o’clock in the afternoon.
Bennigsen, still busy examining the position, kept the other generals
waiting until six. There were present Kutuzov, Barclay, Bennigsen,
Dokhturov, Konovnitzin, Raievski, Uvarov, Ostermann-Tolstoï,
Yermólov, Toll, and, later on, Platov. Bennigsen opened the discussion
by asking whether it was better to give battle or to abandon the
capital. Kutuzov interrupted him, pointing out that the question was
not of Moscow, but the salvation of all Russia, and that this clearly
depended upon the preservation of the army.
Barclay strongly supported Kutuzov, and he was followed by Raievski,
Konovnitzin, and Ostermann-Tolstoï. Barclay appears otherwise to
have had no great confidence in the ability of the Russian troops,
diluted with militia, to manœuvre. When fighting generals such as
Raievski and Konovnitzin ranged themselves with him the question
was practically decided. According to Bennigsen he was supported by
Dokhturov and Platov; but his claim to have had the votes of
Konovnitzin and Yermólov is elsewhere contradicted. To his
suggestion that battle should be delivered Ostermann-Tolstoï replied
with a blunt question as to whether he was ready to answer for
victory. Bennigsen evaded this embarrassing query; but Kutuzov, who
had perhaps already made up his mind, ended the discussion by
deciding upon retreat. As to its direction Barclay appears to have
considered that it should be to the eastward on Vladimir and Nizhnii
Novgorod; but Toll, thinking more of the question of supplies,
suggested a retirement towards Kaluga. A direct retreat on the latter
place would have exposed the army to one of Napoleon’s dreaded
flanking attacks. Strategically Barclay’s suggestion was sound
enough, if perhaps over-cautious. Clausewitz points out that
Napoleon’s offensive power was exhausted, and that he could
scarcely have pursued.
Kutuzov decided that the line of the retreat should be by Kolomna on
Riazan, thus intermediate between that suggested by Barclay and
that proposed by Toll. By taking it no opening was afforded for a
flank attack; and it would be easy to manœuvre on either wing
should occasion arise. The commissariat of the army would be
assured since it would have at its back the fertile “Black Soil”
provinces; and it would furthermore be in easy communication with
the manufactories of arms at Tula and elsewhere. Orders were
therefore issued for the retreat of the army by the Kolomna road.
Bennigsen was extremely discontented, and showed his displeasure
by leaving the council. He attributed it to Barclay, and their relations,
already strained, became more hostile than ever.
There can be no doubt that Kutuzov and Barclay were correct in their
resolution to retreat, but it was no light thing to make it. Kutuzov was
greatly agitated; he passed a sleepless night, and more than once
tears were seen to roll down his cheeks. No Englishman can perhaps
fully understand what it meant to a Russian to leave “White-Walled
Moscow,” the mother of the Russian land, to the mercy of an enemy.
Barclay, though now in bad health, took executive command of the
evacuation. At 2 a.m. on the 14th the army began its passage
through the city. The troops, in the deepest dejection, tramped
through the streets with furled standards and silent bands, many of
them, officers and men, sobbing with rage and despair. The foreign
commandant of the Kremlin garrison regiment began the evacuation
with band playing, according to the usages of war, and there was a
violent outcry among the retreating soldiery. They indignantly
shouted that he was rejoicing, and the music had to cease.
 NAPOLEON’S FIRST VIEW OF MOSCOW
The Emperor is standing on the Sparrow Hills, from which an imposing view of the
old Russian capital is obtained.
From the painting by Verestchagin.
Barclay stationed his staff-officers along the line of retreat to enforce
order. Knowing the especial weakness of the Russian soldiers, he
issued strict orders that anyone found in a beer-shop or intoxicated
was to be summarily punished. He worked himself to death in
directing the march, and was on horseback for eighteen hours. He
complained bitterly of the inefficiency of the staff, which, as usual,
did little or nothing to facilitate the march. There was frequently
disorder among the retiring columns. None the less it must be said
that the operation was remarkably successful. By 9 o’clock in the
evening, after eighteen hours of incessant toil, 90,000 fighting men,
more than 600 guns and thousands of vehicles, had been passed
through the great city and were on the Kolomna road. Kutuzov
himself traversed Moscow in the morning. An eyewitness states that
he saw him near the Kolomna gate sitting in his carriage quite alone,
resting his head on his hands, silent and sad, while before him
troops, guns, and waggons poured in an endless stream. The head of
the army halted for the night at Panki, a village about ten miles from
the city, where Kutuzov established his head-quarters.
Meanwhile, early on the 13th, Napoleon halted his army, fearing that
Kutuzov was manœuvring to attack his right flank. That he could
conceive such an eventuality shows how completely, and not for the
first time in the campaign, his cavalry had failed to keep touch with
the enemy. At 10 a.m., however, he became convinced that the
Russian army was still in his front, and resumed his march. At 1 p.m.
on the 14th Murat’s vanguard crowned the Sparrow Hills, about a
mile and a half west of Moscow, and saw before them in the plain the
Russian rear-guard, and beyond it the widespreading city—the goal
which they had toiled so strenuously to attain.
The Russian army was still pouring through the streets, and
Miloradovich sent an officer to Murat to propose a short armistice,
adding that if it were not granted he should defend the city step by
step, and fire it as he fell back. After a while a sort of informal
suspension of arms until 7 p.m. was made between Miloradovich and
Sebastiani, now commanding the 2nd Cavalry Corps. The Russians
evacuated the Dorogomilov suburb at 3 p.m., and Murat quietly
followed. At about the same time Napoleon reached the Sparrow
Hills. He is said to have gazed long and eagerly upon the goal of his
wishes, now spread out before his greedy eyes; but he may well have
muttered the words attributed to him: “It is full time!”
The Emperor approved of the informal truce concluded by Murat. The
peaceful occupation of Moscow was an end bought cheaply enough
at the price of the quiet withdrawal of the Russian rear-guard. Mortier
was to be Governor, General Durosnel Military Commandant, and M.
Lesseps, who had formerly been French Consul-General at St.
Petersburg, Intendant of the province of Moscow. Orders were issued
to prevent the ingress of plunderers. Eugène and Poniatowski were
ordered to halt some miles short of the city. Mortier was directed to
occupy the Kremlin, and to maintain order by severe methods. As the
day wore on the 1st and 3rd Corps and the old Guard closed up on
Murat. It is a characteristically French touch that the men had decked
themselves out in their parade uniforms to take part in the triumphal
entry.
As Miloradovich evacuated quarter after quarter of the city Murat
advanced, dreading surprise, and taking great precautions against it.
The streets were deserted; silence reigned everywhere. Near the
Kremlin a tumultuous gathering of citizens and stragglers opened a
scattering fire. They were dispersed by cannon shots, and Murat
moved on, only to find silence and apparent desertion. Miloradovich
marched through the city and established himself for the night some
4 miles from the Kolomna gate. Winzingerode’s detachment, which
had been falling back before Eugène, was on the St. Petersburg road,
and another cavalry detachment was escorting the public treasure
and the archives of Moscow to Vladimir.
When at last it became evident that Moscow was indeed deserted by
most of its native inhabitants, a deputation of foreign residents was
collected to be presented to Napoleon. His mortification was extreme.
He quartered himself in the Dorogomilov suburb, and, between his
anxiety and the dirt and vermin of an ill-kept abode, spent a restless
night.
Mortier had duly occupied the Kremlin with Roguet’s division, sending
Claparède to support Murat. The silence of the city impressed even
the reckless soldiery of Napoleon. Sergeant Bourgogne naïvely
expresses it by remarking how disappointed they were to see not
even a pretty girl listening to the regimental bands. With darkness
disorder broke forth everywhere. It was impossible to prevent ill-fed
and ill-clad men from pillaging when all that they needed, not to
speak of wealth, which appeared to their ignorance inconceivable, lay
ready to hand. In the evening fires were already breaking out. In the
morning of the 15th Napoleon, escorted by the Old Guard, took up
his residence in the Imperial Palace in the Kremlin.
It should here be said that the Kreml, or citadel, commonly known in
Western Europe as the “Kremlin,” was the original fortress or walled
town of Moskva, fortified in 1147 by Prince Yurí Dolgorúki (Long-
handed George), the son of the famous Great Prince Vladimir
Monomakh. Around it grew up in the course of ages various suburbs,
and these were in their turn walled. As the streets were wide, and
there were many very large buildings—palaces, monasteries, and the
like—often standing in spacious gardens or enclosures, the city
covered an enormous area. It had the characteristics in general of a
vast country suburb rather than of a city.
A volume might be written concerning the burning of Moscow. The
catastrophe has been described in the works of numerous
eyewitnesses, and lengthy reference to the event itself hardly falls
within the compass of this work. Three points must, however, be
dealt with: the causes of the conflagration; its extent; and its effects
upon the fortunes of Napoleon.
As regards the origin of the fire it may be regarded as certain that it
was not the outcome of Russian patriotic frenzy. The whole evidence
is to the contrary; and the fury and grief of the Russians at the
destruction of their holy city were obviously genuine. It was equally
not due to the deliberate action of the invaders, who had every
motive for preserving the city for their own convenience. It remains
to be considered whether it was the act of Rostopchin or due to mere
accident, assisted by a fortuitous combination of circumstances.
Public opinion at the time attributed the conflagration to Rostopchin.
Two of his own residences were destroyed, and a few weeks later he
deliberately fired his country mansion at Voronovo in order to prevent
its seizure by the French. Sir Robert Wilson, who had means of
knowing, says that Rostopchin’s design was notorious, and that in
order to prevent him from carrying it into execution Kutuzov
repeatedly announced his intention of delivering battle before
Moscow. Buturlin, the contemporary Russian historian, also attributes
the fire to the Governor.
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