Bartold Et Al-2019-Journal of Clinical Periodontology

PROF.
PETER MARK BARTOLD (Orcid ID : 0000-0002-5695-3877)

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Article type : Supplement Article
Mesenchymal Stem Cells and Biologic Factors leading to Bone Formation
PM Bartold1, S Gronthos2, D Haynes3, S Ivanovski4
1
School of Dentistry, University of Adelaide, Adelaide, South Australia, Australia
2
Mesenchymal Stem Cell Laboratory, Adelaide Medical School, Faculty of Health and
Medical Sciences, University of Adelaide,
Adelaide, South Australia, Australia
3
Adelaide Medical School, Faculty of Health and Medical Sciences
University of Adelaide, Adelaide, South Australia, Australia
4
School of Dentistry, University of Queensland, Brisbane, Queensland, Australia
Running Title: Stem cells and biologics in osteogenesis
Key Words: Mesenchymal stem cells, growth factors, bone regeneration
Address for Correspondence
Professor PM Bartold
School of Dentistry
This article has been accepted for publication and undergone full peer review but has
not been through the copyediting, typesetting, pagination and proofreading process,
which may lead to differences between this version and the Version of Record. Please
cite this article as doi: 10.1111/jcpe.13053
This article is protected by copyright. All rights reserved.

University of Adelaide
Adelaide, South Australia

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Australia
Email: mark.bartold@adelaide.edu.au
Statement of Sources of Funding:
This review was not funded by any external funding agency
Disclosure of any conflicts of interests:
All authors declare they have conflicts of interest to declare.
Abstract
Background: Physiological bone formation and bone regeneration occurring during
bone repair can be considered distinct but similar processes. Mesenchymal stem
cells and associated biologic factors are crucial to both bone formation and bone
regeneration.
Aim: To perform a narrative review of the current literature regarding the role of
mesenchymal stem cells and biologic factors in bone formation with the aim of
discussing the clinical relevance of in vitro and in vivo animal studies.
Methods: The literature was searched for studies on mesenchymal stem cells and
biologic factors associated with the formation of bone in the mandible and maxilla.
The search specifically targeted studies on key aspects of how stem cells and
biologic factors are important in bone formation and how this might be relevant to
bone regeneration. The results are summarized in a narrative review format.

Results: Different types of mesenchymal stem cells and many biologic factors are
associated with bone formation in the maxilla and mandible.

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Conclusion: Bone formation and regeneration involve very complex and highly
regulated cellular and molecular processes. By studying these processes new
clinical opportunities will arise for therapeutic bone regenerative treatments.
Clinical Relevance:
Scientific Rationale: This is a review regarding the role of mesenchymal stem cells
and biologic factors in bone formation.
Principle Findings
Different types of mesenchymal stem cells and many biologic factors are associated
with bone formation in the maxilla and mandible.
Practical Implications
Although it is widely recognized that a thorough appreciation of the biological basis of

clinical therapies is essential, it is not until we understand the process of formation
that regeneration will become an achievable clinical endpoint for managing disease
and trauma to tissues. This will certainly be the case for bone regeneration.

INTRODUCTION
This review is presented as part of the discussion papers for the XV

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European Workshop in Periodontology on Bone Regeneration. The topic assigned
for this review was “Mesenchymal Stem Cells and Biologic Factors leading to Bone
Formation”. Clearly this is a difficult area to cover in one review as it involves two
philosophies of thinking and thus the scientific and clinical approaches associated
with stem cells and growth factors for bone formation can differ. With this in made
two specific aspects were reviewed namely: how stem cells and biologic factors are
important in bone formation and how this might be relevant to bone regeneration in
both preclinical and clinical settings. Since this is not a systematic review no
conclusions can be drawn concerning clinical outcomes, rather consideration of the
information to date should be helpful in defining future clinical studies.
BONE BIOLOGY, STRUCTURE AND REGENERATION
Bone is a hard connective tissue constituting the various skeletal elements
with a variety of mechanical and structural properties that are derived from its micro-
and macro-structural hierarchical arrangement (Rho et al., 1998). It has a number of
specific roles including providing mechanical support and protection, maintaining
mineral homeostasis, hematopoiesis and endocrine functions (Florencio-Silva et al.,
2015). The hierarchical arrangement of bone can be viewed at both the nano-scale
where structured cross-linked collagen matrices are embedded within a mineralized
matrix and the macro-scale where collagen fibers are aligned according to the
mechanical and functional requirements of the tissue.

Microstructure and Macrostructure of Bone
Bone can be divided into several different types according to its stage of
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development and microstructure as primary (woven), secondary (lamellar),
cancellous (trabecular) and compact (cortical) bone (Buck and Dumanian, 2012)
(Figure 1). Woven bone is a developmentally early form of bone characterized by
poorly organized collagen type I fibers with a high cellular content and large voids
resulting in low mineral content (Garcia-Rodriguez and Martinez-Reina, 2017). It is
the first form of bone to be seen in repairing and regenerating bone defects and is
eventually resorbed and replaced. Secondary, or lamellar bone, is comprised of four
general forms: osteons, trabecular, circumferential and interstitial bone (Buckwalter
et al., 1995). The osteons form the major structural unit of cortical bone and are
formed by concentric lamellae surrounding the central Haversian canal that contains
blood vessels, nerves and lymphatics. Cell processes emanate from the osteon in
the form of canaliculi and these connect the central canal to osteocytes allowing
diffusion of nutrients through the bone. Cortical, or compact, bone is the highly
mineralized, dense outer surface of bone consisting of closely packed osteons. It has
an important structural and weight-bearing role due to its high resistance to bending
and torsion. Cancellous, or trabecular, bone also contains lamellar bone but in a
different arrangement to the osteon and does not have a Haversian canal system but
rather has sinusoids that allow communication between the bone marrow and
mineralized bone tissue. The trabeculae that provide the cancellous bone with a very
high surface area are more cellular and have a higher vascular content than the
cortical bone. Overlying the external cortical bone surface is the periosteum, which is
a thick collagenous layer, comprised of Sharpey’s fibers. This provides a surface for
attachment of ligaments (including the periodontal ligament) as well as providing a
rich blood supply to the outer bone layers.

Bone formation and regeneration
Developmentally bone is derived from the mesoderm although there is also

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some contribution to the craniofacial skeletal tissues from the neural crest. Bone is
formed via two mechanisms know as endochondral and intramembranous bone
ossification. For the purposes of this review only intramembranous bone formation
will be considered since this is the mechanism through most oral osseous defects
repair and regenerate. Interesting embryonic bone formation and bone repair and
regeneration are almost identical processes (Shapiro, 2008). Intramembranous bone
formation is characterized by the formation of bone matrix without the intermediary
step of chondrogenesis (endochondral ossification). Ossification occurs through the
differentiation of mesenchymal stem cells into osteoprogenitor cells and finally into
osteoblasts. All of this is intricately orchestrated by myriad growth, differentiation and
transcription factors (Berendsen and Olsen, 2015). Once formed all bones undergo
continual remodelling dictated predominantly by mechano-transduction signals
(Robling and Turner, 2009). Thus, bone is continually resorbed and reformed
according to the forces placed upon it. These processes occur for developing bone,
newly formed bone and regenerated bone tissues. While primarily designed to
confer optimal strength and function to the osseous structures remodelling can have
significant ramifications for bone grafts or regenerated bone protected from
mechanical forces through excessive resorption resulting in lost architecture and
function.
ALVEOLAR BONE
The alveolar bone, together with the periodontal ligament and root surface
cementum provide the support structures for teeth within the maxilla and mandible
(Figure 2). During development of the maxilla and mandible the alveolar bone

forms via intramembranous bone formation. Structurally alveolar bone is composed
of two parts. One is the alveolar process of the maxilla and mandible that contains
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developing teeth as well as the roots of fully erupted teeth. The other type of alveolar
bone is termed “alveolar bone proper” and this is the portion of the alveolar bone that
houses the tooth roots of erupted teeth and associated periodontal ligament. The
periodontal ligament attaches into the alveolar bone proper via Sharpey’s fibers and
provides a very strong interface between tooth root and bone. Radiographically
alveolar bone proper can be seen as a thick radiopaque line adjacent to the alveolar
socket, termed the lamina dura. The alveolar bone proper is also commonly referred
to as “bundle bone” reflecting the embedded fiber bundles from the periodontal
ligament. Also included within the alveolar bone proper is lamellar bone (also
referred to as the cribriform plate) that is perforated by small holes, or foramina, that
allow egress of nerves and blood vessels from the bone into the periodontal
ligament. These most likely also provide a pathway for mesenchcymal stem cells to
gain access to the periodontal ligament to assist in repair and regeneration. The
alveolar bone proper encases the whole tooth root surface and at it’s most coronal
part merges with the alveolar bone to form the alveolar crest. Once teeth are
extracted there is no need for the alveolar bone proper and it resorbs very rapidly
(Araujo et al., 2015).
STEM CELLS
Stem cells are defined by their capacity to self renew, undergo extensive
proliferation and the potential to reproducibly differentiate into functional cells
indicative of several different lineages (Dominici et al., 2006). Stem/progenitor cells
have been isolated from nearly every tissue and organ of the human body.
Characterization of these cells has shown that there is considerable variability in their

capacity to differentiate with some having considerable pluripotent capacity while
others exhibit more limited differentiation capacity.

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Stem cells may be categorized on the basis of their source and associated
differentiation capacity. In general there are four main sources of stem cells; (i)
embryonic tissues, (ii) foetal tissues, (iii) postnatal tissues and (iv) reprogrammed
differentiated somatic cells (termed induced pluripotent stem cells) (Figure 3)(Lin et
al., 2008).
Embryonic stem cells have been isolated from mammalian embryos and have
a very high proliferative capacity as well as the ability to differentiate into cells with
the features of all three embryonic germ layers (ectoderm, endoderm and
mesoderm). Thus, these pluripotent cells can differentiate into almost all cell types
comprising the human body. While these cells present considerable potential for use
in regenerative medicine their clinical use has been significantly impeded due to the
ethical and legal issues relating to the use of embryos for the acquisition of these
cells. Nonetheless these cells are considered the gold standard for future
regenerative cell-based therapies (Ilic and Ogilvie, 2017).
Just over a decade ago the first reports appeared describing the
reprogramming of somatic cells into pluripotent cells with a differentiation and self-
renewal capacity comparable to embryonic stem cells (Takahashi and Yamanaka,
2006). These cells were termed “induced pluripotent stem cells” (iPSC) are
considered to provide an ethically viable alternative to embryonic stem cells in
regenerative medicine.
Adult, somatic or postnatal stem cells can be isolated from almost all organs
and tissues of the human body. These cells play a crucial role in the maintenance of
tissue integrity as well as responding to injury and restoring damage tissues through
repair and regenerative processes. However, these stem cells are far more

restricted in their proliferative and differentiation capacity compared to embryonic
stem cells and induced pluripotent stem cells. Notwithstanding this, adult stem cells
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have been studied in considerable detail with regards to their use in regenerative
medicine (Kanji and Das, 2017).
Due to their accessibility, high growth capacity and multi-potential,
mesenchymal stem cells hold great promise for tissue regeneration in clinical
applications. However, there are a number of technical issues that still need to be
overcome, including identification and isolation of appropriate precursor cell types,
establishment of optimal growth and differentiation conditions in vitro and efficient
modes of delivery for the successful transplantation of mesenchymal stem cells
(Gronthos et al., 2000). The essential components for generating effective cellular
based therapeutic strategies include a population of multi-potential progenitor cells,
presence of signalling molecules/inductive morphogenic signals and a conductive
extracellular matrix scaffold or appropriate delivery system (Nakashima, 2005,
Nakashima and Reddi, 2003, Srisuwan et al., 2006). One major obstacle that limits
the clinical use of mesenchymal stem cells is the inability to define the initial cell
preparations. The heterogeneity of the starting cell population that may result in poor
reproducibility of therapeutic outcomes, and hence it is clear that comprehensive
characterization of these different cell populations is required (Wagner et al., 2005).
In this review of stem cells we have included both in vitro and in vivo studies
that shed light on the clinical utility of mesenchymal stem cells for bone regeneration.
It is recognized that in vitro and preclinical animal studies can be of limited value but
in the absence of clinical studies they provide the best evidence to date to allow
these cells to be considered for potential clinical utility. Where possible we have
included human clinical trials to substantiate preclinical studies.

MESENCHYMAL STEM CELLS AND BONE REGENERATION
Postnatal stem cells were first derived and cultured from bone marrow and
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were defined as plastic adherent cells with clonal capacity termed colony-forming unit
fibroblasts (Friedenstein et al., 1976, Friedenstein et al., 1966). The incidence of
CFU-F in bone marrow aspirates varies between individuals ranging from 1 to 10
CFU-F per 105 cells plated. Initially, these cells were termed bone marrow stromal
cells or stromal precursor cells, but in recent times they have been generally referred
to as mesenchymal stem cells (Caplan, 1991). In 2006 the International Society for
Cellular Therapy determined that the use of the term mesenchymal stem cell was
appropriate for all multipotent fibroblast-like plastic adherent cells provided they met
specific stem cell criteria (Dominici et al., 2006, Horwitz et al., 2005). These criteria
were listed as: adherence to plastic, a specific surface antigen expression pattern
and multipotent differentiation potential (Dominici et al., 2006). Later, it was
recognized that these parameters were not sufficient to discriminate these cells from
other multipotential, clonogenic, plastic adherent cells that could be isolated from a
variety of stromal tissues. Accordingly the property of an ability to undergo self-
renewal without the loss of development potential was added to the defining features
of mesenchymal stem cells (Bianco et al., 2008).
It is now recognized that there are many defining features of mesenchymal
stem cells that should be met in order to clarify the status of cells being studied either
in vitro or in vivo. While there is considerable variability in the growth and
differentiation potential of mesenchymal stem cells (Gronthos et al., 2000, Seo et al.,
2004), a common defining feature of these cells is their capacity, under appropriate
culture conditions, to differentiate into at least three distinct phenotypic lineages,
such as osteoblasts, chondrocytes and adipocytes (Dominici et al., 2006). In addition
to these properties, cell surface markers (CD73, CD90, CD105 and CD166) have
been used to try to confirm mesenchymal stem cell identity (Dominici et al., 2006).

However, it is now recognized that these surface proteins alone are non-specific
markers of “stemness”, due to their expression by end-differentiated stromal cells

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and fibroblast populations that do not display multi- differentiation capacity. Other
studies have shown that the cell surface antigen, STRO-1 (Gronthos et al., 2003b), is
highly expressed by human bone marrow CFU-F. The STRO-1 antigen was recently
identified as a cell surface epitope on HSC70, expressed on mesenchymal stem cells
and some cancer lines (Fitter et al., 2017). Importantly, the anti-STRO-1 antibody
reacts with less than 10% of adult human bone marrow mononuclear cells,
containing all assayable CFU-F (Gronthos et al., 2003b). Moreover, STRO-1 positive
cultures lack the presence of hematopoietic stem/ progenitor cell populations and
macrophage/ endothelial cells, which are readily detected in primary stromal cultures
derived from unfractionated bone marrow aspirates (Simmons and Torok-Storb,
1991). Adult bone marrow mononuclear cells sorted on the basis of STRO-1
expression possess the capacity for multi-differentiation into various stromal cell
types including osteoblasts, adipocytes, chondrocytes, smooth muscle cells and
hematopoietic supportive stroma (Gronthos et al., 2003b, Dennis et al., 2002).
Interestingly, selection of ex vivo expanded BMSC based on their expression of
STRO-1, exhibit an enhanced capacity for growth, migration, bone formation,
hematopoietic support, immunomodulation and pro-angiogenic properties, compared
to STRO-1 negative cells (Psaltis et al., 2010, Bensidhoum et al., 2004, Goncalves et
al., 2006, Nasef et al., 2009). These developments have opened up new areas of
investigation for evaluating the properties and clinical potency of purified MSC
populations, without the presence of contaminating accessory cell populations with
the potential to influence the growth, paracrine properties and developmental
potential of cultured mesenchymal stem cells.

Thus, it has been recommended that in order for a cell to be labeled a
mesenchymal stem cell rigorous assessments should be carried out (Bartold and
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Gronthos, 2017). These should include demonstration that the individual clonal
populations have the capacity to produce the connective tissue matrix
microenvironment of their tissue of origin when implanted into immunocompromised
mice as well as demonstration of their self-renewal capacity over serial
transplantations in vivo. More recently further criteria have been proposed to define
mesenchymal stem cells and these include assessment of a number of growth
factors and cytokines that promote stem cell survival, angiogenesis, and immune cell
responses (Konala et al., 2016).
Mesenchymal stem cells are amongst the most highly studied types of adult
stem cells. They include cells derived from nearly all tissues comprising the human
body Depending on their source these cells have the capacity to differentiate into
cells with the characteristics of mesoderm (adipocytes, osteoblasts, chondrocytes,
tenocytes, skeletal myocytes and visceral stromal cells) (Gronthos et al., 2003b,
Horwitz et al., 1999, Jiang et al., 2002, Pereira et al., 1995, Pittenger et al., 1999,
Smith et al., 2004), ectoderm (neurons, astrocytes) (Woodbury et al., 2000) and
endoderm (hepatocytes) (Petersen et al., 1999). Therefore, the term mesenchymal
stem cell is very generic, and additional descriptors should be added to better
describe the tissue specificity and origin of these cells (for example: bone marrow
mesenchymal stem cells, adipose stem cells, dental pulp stem cells, periodontal
ligament stem cells).
Mesenchymal stem cells derived from specific tissues retain some “memory”
of those tissues and thus exhibit some tissue specific properties in addition to more
generic multi-potential and these can be defined by their niche environment
(Menicanin et al., 2014). Furthermore, since the first identification of mesenchymal
stem cells in the periodontal ligament (Seo et al., 2004). mesenchymal stem cell-like

populations have been isolated from all dental tissues except enamel (ameloblasts or
other cellular elements following tooth development) (Chen et al., 2012). Because
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dental tissues do not undergo continuous remodelling as do bone tissues, dental-
tissue-derived mesenchymal stem cells are more restricted in their differentiation
capacity compared to bone marrow-derived mesenchymal stem cells (Huang et al.,
2009).
Given the enormous diversity of tissues that harbour mesenchymal stem cell-
like populations this review will be limited to those cells that have been most
extensively studied for their potential to participate in bone formation and
regeneration (Table 1).
Bone Marrow Stromal Cells (BMSC)
Within the bone marrow there are three major stem cell populations:
haematopoietic stem cells that are responsible for the formation of blood cells;
angioblasts that are endothelial cell precursors cells, and bone marrow stromal stem
cell or skeletal stem cell populations responsible for the formation of the
haematopoietic microenvironment that supports and regulates the haematopoietic
stem cells (Friedenstein et al., 1970, Gronthos et al., 2003a, Pittenger et al., 1999,
Owen and Friedenstein, 1988).
These cells are comprised of a mixed population of stromal progenitor cells at
various stages of development, maintained by a minor population of multipotential,
stromal stem cells with the capacity for self-renewal(Owen and Friedenstein, 1988).
Subsequently they were demonstrated to have osteogenic capacity when implanted
into immunodeficient mice and this was controlled by at least four growth factors
including platelet derived growth factor, transforming growth factor-beta, basic
fibroblast growth factor and epidermal growth factor (Kuznetsov et al., 1997a).

These cells have considerable capacity to proliferate and expand in vitro as
well as the ability to differentiate into multiple cell lineages (Gronthos et al., 2003b,
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Kuznetsov et al., 1997b, Menicanin et al., 2010). Therefore, there has been
considerable interest in these cells with regard to their pivotal role in skeletal growth
turnover and potential use in regenerative medicine (Bianco et al., 2001). Thus in
skeletal physiology these cells are crucial in contributing to bone growth and
turnover. These unique features are also critical to their regenerative capacity. In
addition to these functions, bone marrow stromal stem cells help to maintain
hematopoietic stem cells in their niche and in general help to maintain the
hematopoietic surroundings. These properties run in parallel with their regenerative
capacity to modulate tissue function through the release of a number of soluble
mediators instructive for tissue regeneration (Bianco, 2014). This so-called
“secretome” may hold potent regenerative potential and may serve as an alternative
to whole cell transplantation (Katagiri et al., 2017).
As described above an early defining feature of bone marrow stromal stem
cells was the ability to form colonies of cells derived from a single precursor cell
denoted CFU-F (Friedenstein et al., 1974). These cells have a high capacity to
undergo self-replication and can undergo more that 50 cell doublings in vitro. It has
been estimated that billions of cells can be generated from as little as 1 ml of bone
marrow aspirate (Bianco et al., 2001).
The bone forming capacity of bone marrow stromal stem cells is well
recognized and has been extensively reviewed (Arthur et al., 2009, Bianco et al.,
2006, Bruder et al., 1998a, Caplan, 2005, Caplan, 2007, Derubeis and Cancedda,
2004, Kode et al., 2009). Early studies reported the osteoinductive (osteogenic)
capacity of human bone marrow stromal stem cells when they were combined with
an osteoconductive scaffold of hydroxyapatite / tricalcium phosphate (HA/TCP) and
subsequently implanted subcutaneously into the dorsal tissues of

immunocompromised mice (Kuznetsov et al., 1997b). Once excised, histological
assessment of these transplants demonstrated neovascularization, fibrous

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connective tissue and areas of new bone formation. The osseous regenerative
potential of bone marrow stromal stem cell within hydroxyl apatite/tricalcium
phosphate (HA/TCP) scaffolds has also been demonstrated in dogs, sheep and
goats for the repair of critical-sized bone defects (Bruder et al., 1998b, Brodke et al.,
2006, Kon et al., 2000, Viateau et al., 2007) (Bensaid et al., 2005, Petite et al., 2000,
Liu et al., 2008, Liu et al., 2010). Many other studies have assessed and confirmed
the ability of both allogeneic and autologous bone marrow stromal stem cells to
regenerate bone in different animal models (Arinzeh et al., 2003, Dai et al., 2005,
Muraglia et al., 1998, Yuan et al., 2007, Zhu et al., 2006, Field et al., 2011).
Notwithstanding these favourable outcomes, it should be noted that simple
implantation of bone marrow stromal cells into large bone defects has significant
technical difficulties. For example, early studies found that cartilage, rather than
bone, formed due to lack of vascularization (Lerner et al., 2018). These
observations led to efforts to improve the carrier vehicles and scaffolds used to
deliver bone marrow stromal cells to osseous defects using tissue engineering
concepts. A limiting factor in cell transplantation has been the properties of the tissue
engineering delivery vehicle. The fundamental concept underlying tissue engineering
is to combine a scaffold with living cells, and/or biologically active molecules to form
a “tissue engineering construct” that promotes the regeneration of tissues (Bartold et
al., 2000, Bartold et al., 2006). The scaffold should perform various functions,
including support cell colonisation, migration, proliferation and differentiation. The
scaffold design also should consider physico-chemical properties, morphology and
degradation kinetics. External size and shape of the construct are of importance,
particularly if the construct is customised for an individual patient. Scaffolds must
provide sufficient initial mechanical strength and stiffness to substitute for the loss of

mechanical function of the diseased, damaged or missing tissue. Continuous cell and
tissue remodelling is important for stable biomechanical conditions and

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vascularisation at the host site. In addition to these essentials of mechanics and
geometry, a suitable construct will (i) possess a 3D and highly porous interconnected
pore network with surface properties which are optimised for the attachment,
migration, proliferation and differentiation of cell types of interest and enable flow
transport of nutrients and metabolic waste, and (ii) be biocompatible and
biodegradable with a controllable rate to complement cell/tissue growth and
maturation (Bartold et al., 2016, Hutmacher and Cool, 2007). In the early days of
tissue engineering, it was believed that scaffolds should degrade and vanish as the
tissue is growing. Yet, tissue in-growth and maturation differs temporally from tissue
to tissue and, furthermore, tissue in-growth does not equate to tissue maturation and
remodelling, in other words a defect filled with immature tissue should not be
considered “regenerated”. Hence, many scaffold-based strategies have failed in the
past as the scaffold degradation was more rapid than tissue remodelling and/or
maturation. It is now recognized that the onset of degradation should only occur after
the regenerated tissue within the scaffold has remodelled at least once in the natural
remodelling cycle. Work over the last ten years underlines the importance of the
scaffold remaining intact as the tissue matures in the scaffold pores with bulk
degradation occurring later (Vaquette et al., 2018).
A large number of case reports have demonstrated a favourable outcome for
bone regeneration following implantation of bone marrow stromal stem cells
(Killington et al., 2018). Despite these findings, there are surprisingly few
randomised clinical trials or systematic clinical studies (i.e. not just case reports or
case series) using bone marrow stromal stem cells for bone regeneration in humans.
This finding is further highlighted in the review for this Symposium on cell therapies
for orofacial bone regeneration where it is concluded that most studies to date have

used whole bone marrow rather than ex vivo expanded cells (Shanbhag et al., 2018).
A recent systematic review (Killington et al., 2018) reported that 10 clinical trials were
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registered in the clinical trials.gov registry and of these 4 have been completed and
two were published and available for assessment (Dallari et al., 2007, Liebergall et
al., 2013). The two studies evaluated in this systematic review produced equivocal
results with one reporting improved bone formation after implantation of bone marrow
stromal stem cells (Dallari et al., 2007) and one report failing to find any such effect
(Liebergall et al., 2013). A recent study investigating the use of bone marrow stem
cells for the regeneration of alveolar cleft and trauma in adults found that while the
use of these cells is safe, their ability to facilitate complete regeneration of large
alveolar defects is limited (Bajestan et al., 2017). Most recently, significant
regeneration (in both horizontal and vertical dimensions) for severe mandibular
resorption has been reported when bone marrow stromal stem cells were used in
conjunction with biphasic calcium phosphate scaffolds and titanium-reinforced
membranes (Gjerde et al., 2018). Similar results have been reported when using
similar procedures for treating orthopedic non-unions (Gomez-Barrena et al., 2018).
All human clinical studies to date have demonstrated that bone regeneration requires
mesenchymal stem cells to have a suitable environment and this is usually facilitated
by the use of appropriate scaffold as well as biologic factors such as bone
morphogenetic proteins, transforming factor-beta and insulin-like growth factor.
These factors will be discussed later in this review.
In the context of oral reconstruction, bone marrow stromal stem cells have
been used in a number of clinical trials. In the most recent systematic review
concerning the use of mesenchymal stem cells in oral reconstructive surgery 18
studies were included of which 10 involved the use of bone marrow stromal stem
cells mixed with a variety of carriers and biologic factors (Jakobsen et al., 2013).
While these studies generally showed that bone marrow stromal stem cells may be

associated with new bone formation this was not a consistent finding and the
contribution of the scaffolds and biologic agents was not clear due to wide study
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design diversity.
Dental-derived MSC-like cells
As detailed above, mesenchymal stem cells were first isolated from bone
marrow but have since been found in nearly every tissue and organ of the human
body. Stems cells of dental origin were first isolated from the dental pulp (Gronthos
et al., 2000) and subsequently from periodontal ligament (Seo et al., 2004). Since
these early studies, mesenchymal stem cells have been isolated from every dental
tissue except enamel (Chen et al., 2012). This reflects the reparative potential of
dental tissues since following eruption of teeth into the mouth all of the periodontal
and dental structures, with the exception of enamel, have some form of regenerative
capacity presumably mediated by local mesenchymal stem cell-like populations
(Duailibi et al., 2006). Dental mesenchymal stem cells have been well characterized
and demonstrate features originally ascribed to bone marrow stromal stem cells such
as plastic adherence, colony forming unit capacity and multipotent differentiation
capacity (Gronthos et al., 2000, Miura et al., 2003, Seo et al., 2004). At least six
different mesenchymal stem cells have been isolated from dental tissues: dental pulp
stem cell, periodontal ligament stem cells, stem cells from human exfoliated
deciduous teeth, stem cells from apical papilla, dental follicle stem cells and gingival
mesenchymal stem cells.

Dental pulp stem cells (DPSC)
The dental pulp is not dissimilar to bone marrow in that it is a highly

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vascularized soft connective tissue fully encased by mineralized tissue. Thus the
initial discovery of mesenchymal stem cell populations residing within dental pulp
was not surprising, with these cells exhibiting characteristics comparable to those of
bone marrow stromal stem cells such as capacity to form mineralized tissues and
similar cell surface markers associated with endothelium, smooth muscle,
bone/dentine, and fibroblasts (Gronthos et al., 2000). Although the overall
differentiation capacity of dental pulp stem cells is lower than bone marrow stromal
stem cells, they exhibit higher proliferative rates and capacity to form colony forming
units.
The bone forming capacity of dental pulp stem cells has been demonstrated
in animal models and, as for bone marrow stromal stem cells, an appropriate scaffold
and supplementary growth factors seem to be prerequisites for these cells to
participate in bone regeneration in vivo (Wongsupa et al., 2017, Tsukamoto et al.,
2017, Chamieh et al., 2016, Asutay et al., 2015). Notwithstanding the large number
of animal preclinical studies, there are few human in vivo studies addressing the
bone regenerative capacity of dental pulp stem cells. These cells have been
investigated for their potential to assist in tooth extraction socket healing whereby
sites that had been implanted with dental pulp stem cells demonstrated regenerated
compact bone that had a matrix density greater than the controls after three years
(d'Aquino et al., 2009, Giuliani et al., 2013, Monti et al., 2017). While these results
indicate a potential role for dental pulp stem cells in bone regeneration there is need
for more studies to determine the clinical utility of dental pulp stem cells for human
bone regeneration.

Periodontal ligament stem cells (PDLSC)
Before their ultimate identification, the presence of precursor or stem cells

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had long been suspected to be present in the periodontal ligament (McCulloch et al.,
1987). Periodontal ligament stem cells were first isolated from human third molars
and were demonstrated to have mesenchymal stem cell properties similar to those
described for bone marrow stromal stem cells (Seo et al., 2004). Importantly these
cells were shown to have considerable regenerative capacity when implanted into
animals where they generated cementum/periodontal ligament structures (Seo et al.,
2004). Subsequently, these cells were demonstrated to have considerable
osteogenic and bone regenerative properties both in vitro and in vivo (Gay et al.,
2007, Grimm et al., 2011, Seo et al., 2004). As for other mesenchymal stem-like
cells, it is apparent that biocompatible scaffolds are required to maintain the viability
and osteogenic capacity of periodontal ligament stem cells and facilitate bone
formation in vivo (Kim et al., 2009, He et al., 2011, Yu et al., 2014, Tour et al., 2012).
While the periodontal regenerative capacity of periodontal ligament stem cells has
been well demonstrated in animal models and more recently in several small human
clinical case studies (Yamada et al., 2006, Feng et al., 2010, McAllister, 2011, Chen
et al., 2016), studies regarding their role in bone regeneration are limited and
restricted to animal models (Akazawa et al., 2016, Kammerer et al., 2017, Yu et al.,
2014, Kim et al., 2009, Ge et al., 2012).
Stem cells from human exfoliated deciduous teeth (SHED)
Stem cells from human exfoliated deciduous teeth are derived from remnants
of the pulp in the crown of exfoliated human deciduous teeth and represent a very
unique stem cell population (Miura et al., 2003). Interestingly, although derived from
dental pulp, stem cells from human exfoliated deciduous teeth differ from their adult-
derived counterpart dental pulp stem cells with regards to their morphology, colony

formation unit capacity, in vivo osteoconductive capacity, and in contrast to dental
pulp stem cells they do not form a dentin-pulp-like complex (Miura et al., 2003).
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Compared to dental pulp stem cells and bone marrow stromal stem cells, stem cells
from human exfoliated deciduous teeth have been demonstrated to have higher
proliferative capacity and higher expression of genes related to both cell proliferation
and extracellular matrix synthesis (Nakamura et al., 2009). Porcine and canine stem
cells from exfoliated deciduous teeth have been demonstrated to regenerate bone
when implanted into critical sized defects in the mandible and cranium (Yamada et
al., 2011, Zheng et al., 2009, Seo et al., 2008, de Mendonca Costa et al., 2008). No
human studies have been carried out to date.
Stem cells from apical papilla (SCAP)
The apical papilla is a developmental organ associated with tooth root
formation and also contributes to formation of the dental pulp. The cells found at this
site are termed stem cells from apical papilla and are a unique population of
multipotent stem cells that express high levels of survivin and telomerase that are
involved in regulating cell proliferation (Sonoyama et al., 2006). Due to their high
proliferative potential stem cells from apical papilla are considered to have better
regenerative capacity than their dental pulp counterparts, the dental pulp stem cells
(Sonoyama et al., 2006, Sonoyama et al., 2008). In vitro these cells can form
odontoblast-like cells and adipocytes (Sonoyama et al., 2006, Abe et al., 2007). In
addition these cells can express a number of neurologic cell markers (Sonoyama et
al., 2006, Sonoyama et al., 2008, Abe et al., 2007). Stem cells from apical papilla
for transplantation can be obtained from extracted third molars. It has been reported
that sufficient numbers of stem cells from apical papilla can be obtained from one
tooth for transplantation (Sonoyama et al., 2006).

Stem cells from apical papilla have shown osteogenic differentiation potential
in vitro (Park et al., 2009). A population of stem cells from apical papilla with high
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expression of CD-24 is associated with a very high osteogenic differentiation
potential (Sonoyama et al., 2006, Aguilar and Lertchirakarn, 2016). Exposure of stem
cells from apical papilla to insulin-like growth factor has been reported to enhance
osteogenic differentiation (Wang et al., 2012). However, the timing of exposure to
growth factors may be important since basic fibroblast growth factor has been noted
to both increase and decrease osteogenic induction depending on the phase of the
cell cycle at the time of exposure (Wu et al., 2012). Stem cells from apical papilla
have been shown to have regenerative potential for the dentin/pulp complex and bio-
root engineering (Sonoyama et al., 2006, Sonoyama et al., 2008). While animal
studies have demonstrated the osgteogenic potential of implanted stem cells from
apical papilla (Abe et al., 2008) to date, no studies have evaluated the potential for
stem cells from apical papilla to regenerate bone in human defects.
Dental follicle stem cells (DFC)
The dental follicle envelops the developing tooth germ prior to tooth eruption.
Because of this site’s strategic role in tooth development, it is considered to be a
good source of stem cells (Morsczeck et al., 2005). Dental follicle stem cells can be
readily obtained from unerrupted or impacted third molars. In vitro these cells
demonstrate limited osteogenic capacity after induction in osteogenic media
(Morsczeck et al., 2005) and after repeated cell passages they appear to lose their
osteogenic capacity (Yao et al., 2013).
Initial animal studies using dental follicle stem cells showed no evidence of
bone formation (Morsczeck et al., 2005). Later studies have reported bone formation
in vivo when dental follicle stem cells were implanted subcutaneously or into critical

size osseous defects (Tsuchiya et al., 2010, Park et al., 2012, Rezai-Rad et al.,
2015). There are no studies to date investigating the use of dental follicle stem cells
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in human osseous defects.
Gingival Mesenchymal stem cells (GMSC)
Cells from the gingival tissues were first noted to possess significant
heterogeneity and multipotency over 25 years ago (McCulloch and Bordin, 1991).
However, it was not until quite recently that mesenchymal stem cells from gingival
tissues have been characterized (Fournier et al., 2010, Fawzy El-Sayed and Dorfer,
2016). When first described human gingival mesenchymal stem cells were found to
have high proliferative rates, a stable morphology and retained stable karyotype and
telomerase activity over sustained passage in culture (Tomar et al., 2010). These
cells have been repeatedly demonstrated to have osteogenic potential in vitro
(Fournier et al., 2010, Tomar et al., Mitrano et al., 2010, El-Sayed et al., 2015).
While early in vivo studies failed to demonstrate osteogenic capacity of gingival
mesenchymal stem cells when implanted subcutaneously (Zhang et al., 2009),
subsequent studies have shown that these cells can promote bone formation in vivo
when implanted into periodontal defects and calvarial critical size defects (Wang et
al., 2011, Yu et al., 2013). While these cells have been proposed to be a good
source of MSC-like cells for bone regeneration due to their easy availability and
robust in vitro characteristics, no human studies have been carried out to date.

Adipose Tissue Stem Cells (ASC)
Mesenchymal stem cells derived from adipose tissue are emerging as a very
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good source of adult stem cells for regenerative therapies due to their easily
accessible nature and properties comparable to bone marrow stromal stem cells
(Bacakova et al., 2018). Mesenchymal stem cells derived by liposuction from
adipose tissue were first described in 2001 (Zuk et al., 2001). These cells were
demonstrated to have multipotent capacity to differentiate into adipocytes,
osteoblasts chondrocytes and myocytes and can form bone when implanted
subcutaneously into mice (Zuk et al., 2001, Dragoo et al., 2003). More recent studies
have demonstrated that adipose stem cells may be superior to bone marrow stromal
stem cells because they are more easily harvested, undergo later in vitro
senescence, have a higher proliferative rate and have a very high autocrine
production of growth factors and immunomodulatory factors essential for tissue
regeneration (Hsiao et al., 2012, Ding et al., 2013, Varghese et al., 2017).
The ostgeogenic capacity of adipose stem cells has been studied in
considerable detail and has led to the suggestion that these cells are amongst the
most promising for use in cell-based bone regeneration (Lindroos et al., 2011). The
osteogenic effect may arise due to their ability to differentiate into osteoblasts as well
as through paracrine mechanisms facilitating the migration and differentiation of other
precursor osteoblasts (de Girolamo et al., 2007, Tajima et al., 2015).
Adipose tissue stem cells have been found to have considerable osteogenic
bone regenerative capacity in vivo (Parrilla et al., 2011, Pourebrahim et al., 2013).
Enhanced osseous repair has been reported following implantation of these cells into
critical size osseous defects and segmental long bone defects in mice, rats and
rabbits (Levi et al., 2010, Pieri et al., 2010, Kim et al., 2012). Similar findings have
been reported in larger experimental animals such as mandibular bone defects in

pigs (Wilson et al., 2012). As for most mesenchymal stem cells, implantation
protocols for adipose tissue stem cells demonstrate improved efficacy when
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implanted within space maintaining scaffolds (Arrigoni et al., 2013) Further
improvements in the bone regenerative capacity of adipose tissue stem cells have
been reported when combined with growth factors such as bone morphogenetic
protein-2 or platelet rich plasma (Chou et al., 2011, Tajima et al., 2015).
Following successful preclinical studies adipose tissue stem cells have been
used in clinical settings for bone regeneration. A number of single case reports and
case series have demonstrated the potential of adipose tissue stem cells for osseous
regeneration in humans (Lendeckel et al., 2004, Mesimaki et al., 2009, Sandor et al.,
2013, Sandor, 2012, Thesleff et al., 2011). Adipose tissue stem cells have shown
particular promise for craniomaxillofacial osseous regeneration (Paduano et al.,
2017). Since the first case report on the use of adipose tissue stem cells for
augmentation of a post traumatic calvarial defect in a 7-year old female (Lendeckel et
al., 2004), there have been a number of case reports demonstrating the clinical utility
of these cells for craniofacial osseous reconstruction (Paduano et al., 2017). There
is now need for larger controlled clinical trials to confirm that these cells could be a
suitable alternative to bone marrow stromal stem cells.
BIOLOGIC FACTORS IN BONE FORMATION AND REGENERATION
Bone formation repair and regeneration
Osteoinduction, the biologic process of bone formation requires not only
recruitment and expansion of bone forming cells but also stimulation of non-infectious
inflammation and increased vascularization. Therefore, therapeutic adjuncts for bone
healing and regeneration require a sound understanding of the phases of bone
healing which are very complex involving a number of hierarchical and temporal

phases (Hankenson et al., 2014, Araujo et al., 2015). Upon bone injury (e.g. tooth
extraction) there is an immediate vascular response leading to clot formation and

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homeostasis. Next there is inflammatory cell migration into the site followed by a
fibrovascular response with recruitment of mesenchymal stem cells to the site
together with neovascularization. The recruited mesenchymal stem cells can then
differentiate into bone forming cells and facilitate neoossification. Finally the new
bone is remodeled following recruitment of osteoclasts and replacement of woven
bone is replaced by newly formed lamellar bone (Araujo et al., 2015). Each of these
phases is guided and controlled by the release of biologic factors in the form of
growth factors and cytokines with growth factors in particular receiving considerable
attention. A comprehensive list of growth, differentiation and transcription factors
known to be involved in bone formation and regeneration are listed in Table 2. More
specific details of the cellular aspects of bone remodeling and how this is regulated
through specific growth factors is reviewed in this volume in a paper considering the
critical interplay between bone resorbing and bone forming cells (Lerner et al., 2018).
A schematic representation of the cellular and molecular players in bone formation,
resorption and remodelling is shown in Figure 4.
It is important to recognize that much of the knowledge we have regarding
regulation of osteogenic differentiation is derived from animal models and in vitro
studies. Nonetheless, these provide pivotal information for both basic understanding
as well as studies of clinical relevance using fundamental knowledge to translate to
clinical practice through the development of novel therapeutics (Bartold et al., 2010,
Cantley et al., 2017).

Role of Growth Factors
Growth factors are a collective group of highly biologically active molecules

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that can stimulate cell proliferation, migration and differentiation by either autocrine or
paracrine mechanisms (Zarei and Soleimaninejad, 2018). While the effects of growth
factors are well recognized the mechanisms of their actions are still under intense
investigation. Growth factors are integral components regulating embryonic tissue
formation, maintaining tissue homeostasis and the co-ordinating tissue healing and
regeneration. The use of growth factors for bone regeneration has been under
intense investigation for several decades and in recent times this has resulted in
many of these being approved for human clinical use or undergoing evaluation in
human clinical trials. A search of clinicaltrials.gov (May 10, 2018) using the search
words “bone and growth factor” identified 391 registered clinical trials. The list of
known biologically active growth factors is voluminous with around 33 families of
structurally and evolutionary related proteins identified to date (Bafico and Aaronson,
2003). An appraisal of each and every known growth factor is beyond the scope of
this review. Accordingly this review will consider only the most widely studied growth
factors in the context of bone biology and regeneration. These include the bone
morphogenetic proteins (BMPs), vascular endothelial cell growth factor (VEGF),
fibroblast growth factors (FGF), platelet derived growth factors (PDGF). Although
blood derived products, such as platelet rich plasma (PRP) and platelet rich fibrin
(PRF), are a mixture of a number of growth and differentiation factors, they are
considered as a biologic agent of considerable interest in the context of bone
regeneration due to their ready availability as a chair-side source of biologic factors.

Inflammatory Phase of Bone Formation
Inflammation is essential for any wound healing and bone is no exception.

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Immediately following bone injury a blood clot forms and this serves to achieve
hemostasis. During this phase of bone healing inflammatory cells are recruited to the
site of injury through the release of a number of pro-inflammatory cytokines released
by platelets. The inflammatory infiltrate is extensive and comprises neutrophils,
lymphocytes, macrophages and eosinophils and if compromised, healing is
significantly affected (Bastian et al., 2011). The initial role of these cells is to “clean”
the wound site prior to formation of new tissue. Additional roles for these cells are to
initiate neovascularization and also to promote recruitment of fibroblasts to the site.
At this time there is a significant increase in a number of additional pro-inflammatory
mediators. These include prostaglandin-E2, bone morphogenetic protein-2,
monocyte chemoattractant proteins (MCP-1 and MCP-3), tumor necrosis factor- ,
transforming growth factor-β and numerous interleukins (IL-1, IL-3, IL-6,IL-10).
These factors have been shown to be crucial to bone repair in gene knock-out and
receptor blocking experiments that have demonstrated diminished granulation tissue
formation post clot formation at the healing site (Walters et al., 2018). The highly
iterative wound healing process leads to the formation of granulation tissue that is
slowly replaced with a more mature connective tissue rich in collagen and fibroblasts.
Cell Recruitment for Bone Formation
Mesenchymal stem cells with the capacity to differentiate into bone forming cells as
well as endothelial progenitor cells need to be attracted to the healing site. The
source of these cells for osseous healing in the maxilla, mandible and healing tooth
sockets is unclear but the periosteum, bone marrow cavity and endosteum are all
likely to be able to provide an ample supply of mesenchymal stem cells and

endothelial progenitor cells for bone formation. A crucial phase in this recruitment of
precursor cells is the release of initiating molecular signals by platelets such as

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platelet-derived growth factor (PDGF) in both its AA and BB isoforms. PDGF
promotes cell migration and proliferation. It acts through receptor tyrosine kinases
which in turn activate a number of intracellular pathways including mitogen-activated
protein kinase (MAPK), phosphoinositide-3 kinase (PI3K), Stat3 and Rho-Rac.
Through these mechanisms PDGF induces mesenchymal stem cell migration and
proliferation as well as, importantly, angiogenesis (Graham et al., 2009). While
PDGF AA has some osteogenic capacity (Li et al., 2014) it is PDGF BB that has
received most attention as a potential therapeutic agent for bone formation and
regeneration (Graham et al., 2009, Gronthos and Simmons, 1995). A number of
reports have demonstrated the clinical efficacy of PDGF for bone regeneration
(Younger et al., 2016, Li et al., 2017). Specifically, PDGF has been investigated for
its osseous regenerative properties in the management of repair and regeneration of
orthopedic and periodontal osseous defects (Hollinger et al., 2008, Friedlaender et
al., 2013, Nevins et al., 2013). The positive results from these studies have have led
to U.S. Food and Drug Administration (FDA) approval of rhPDGF for these
indications.
The fibroblast growth factors are another important group of biological
mediators involved in the recruitment and proliferation of mesenchymal stem cells
and endothelial progenitor cells (Charoenlarp et al., 2017). This family is composed
of 22 related proteins that have the capacity to play a role in bone formation. Within
this family FGF2, FGF9 and FGF 18 have been proposed to have the greatest
potential for use in bone regeneration (Charoenlarp et al., 2017) and of these FGF2
has received the most attention. Osteogenic differentiation of mesenchymal stem
cells derived from FGF2 knockout mice is reduced but this can be restored by the
addition of exogenous FGF2 (Montero et al., 2000). Furthermore, FGF knockout

animals demonstrate a number of developmental osseous abnormalities as well as
adverse bone healing and reduced bone formation (Du et al., 2012). Human trials
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have commenced using FGF to assist repair of bone fractures (Kawaguchi et al.,
2010, Tanaka et al., 2012) and also for promoting regeneration of periodontal
intraosseous defects (Kitamura et al., 2011, Li et al., 2017, Kitamura et al., 2016,
Kitamura et al., 2008) with promising results.
Angiogenesis and Bone Formation
Angiogenesis is a critical part of wound healing and tissue regeneration
(Stegen et al., 2015). For bone formation, angiogenesis is also a critical process to
prevent ischemia at the healing site and permit adequate supply of nutrients and cells
necessary for healing. Indeed ischemia is a significant cause of impaired bone
healing. The newly formed vessels also provide additional signaling molecules
involved in cell differentiation and ossification.
Until complete revascularization of the reparative tissue occurs, the wound is
hypoxic and any cells that are present must be able to adapt to these conditions
through oxygen sensing. In general, this is achieved through hypoxia inducible factor
(HIF) which leads to the release of vascular endothelial cell growth factor (VEGF)
and stimulation of neovascularization (Fan et al., 2014). VEGF is one of the most
widely studied growth factors in bone healing and regeneration because of its critical
role in neovascularization (Hu and Olsen, 2017). VEGF is highly expressed by
endothelial progenitor cells at sites of bone healing and this is thought to be an
important mechanism stimulating angiogenesis and facilitate the commencement of
bone repair (Hu and Olsen, 2017). VEFG has been shown to potentiate the bone
forming capacity of bone morphogenetic proteins (Cui et al., 2013) but these
synergistic effects have not been observed consistently (Li et al., 2016).

Angiopoietin is another important growth factor involved in angiogenesis.
While not directly osteogenic, it can, like VEGF, it can also act synergistically with
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bone morphogenetic protein 2 to upregulate the expression of osteogenic genes in
mesenchymal stem cells (Jeong et al., 2010). Furthermore, animal studies show that
implantation of angiopoietin and BMP2 into bone defects has been shown to induce
regeneration (Choi et al., 2015)
Cell differentiation and Bone Formation
Initial bone formation at sites of bone injury in the craniofacial region is
performed by osteoblasts that have differentiated from the mesenchymal stem cell
infiltrate in a manner consistent with that of intramembranous ossification. The
mesenchymal stem cell differentiation process is controlled not only by soluble
mediators, but also by the cells’ responses to hypoxia and mechanical stresses.
While mechanical forces are of interest to bone healing around teeth and dental
implants this is beyond the scope of this review and only the most significant soluble
biological mediators will be considered here.
Bone Morphogenetic Proteins, TGF- and bone cell differentiation
The discovery that demineralized bone matrix, when implanted into animals
(rabbits, rats, mice and guinea pigs) as well as humans, could induce ectopic bone
formation and new bone formation in osseous defects was a significant advance in
understanding the role of the extracellular matrix in osseoinduction (Urist, 1965).
Since then the active components have been identified as a family of proteins
collectively referred to as bone morphogenetic proteins that are part of the TGF
supergene family (Wozney et al., 1988). To date, 14 BMPs have been identified and

of these BMP-2,4,5,6,7 and 9 demonstrate osteogenic capacity. The osteogenic
activity of BMP-2 and BMP-7 has been the most widely studied both in experimental
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animals and in human trials for use in the repair of osseous defects and deformities
(Wu et al., 2016).
TGF was originally isolated from human platelets (Assoian et al., 1983) and,
like the BMPs belongs to the TGF supergene family. It is more commonly
associated with the differentiation of chondrocytes and endochondral bone formation
rather than intramembranous bone formation. While there is considerable evidence
to suggest that TGF inhibits osteoblast differentiation and intramembranous bone
formation, it may it may also have both stimulatory and inhibitory effects on
osteoblast formation mediated via complex intracellular signaling pathways. (Tang et
al., 2009).
Although the BMPs and TGF bind to related cell surface receptors and
activate similar signaling pathways (SMADS) their endpoint of action on bone
formation is different. The BMPs act through SMAD1, 5 and 8 which are associated
with osteogenic pathways whereas TGFβ acts through SMAD2 and 3 and drives
chondrocyte differentiation (Wu et al., 2016). The BMPs and TGFβ also act through
SMAD-independent pathways (e.g. p38 mitogen-activated protein kinase/p38 MAPK)
to influence mesenchymal stem cell differentiation. Both the SMAD and p38 MAPK
signaling pathways can activate specific transcription factors (e.g. Runx2) leading to
the differentiation of mesenchymal stem cells into chondrocytes and osteoblasts.
However, this is probably a highly simplistic view of the roles of TGFβ, BMPs and
SMADS, as their various interactions with many transcription factors are very
complex and yet to be fully determined (Nickel et al., 2018).

Cell Surface Signaling Pathways for bone cell differentiation
The Wnt signaling pathway is another important process associated with

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osteogenesis and bone regeneration (Leucht et al., 2018). Wnt exerts its action via
cell surface receptors (LRP 4,5 and 6) and β-catenin resulting in gene regulation
necessary for bone formation (Duan and Bonewald, 2016). The expression of Wnt
receptors is upregulated during bone regeneration (Wise et al., 2010). Disruption of
the canonical signalling pathway of Wnt has been demonstrated to influence bone
formation. For example, sclerostin is a soluble inhibitor of Wnt that, if deficient or
blocked, can lead to increased bone mass (Pietrzyk et al., 2017). Conversely,
knockout mice deficient in the Wnt cell surface receptors LRP 5 and LRP 6
demonstrate diminished bone mass (Holmen et al., 2004). Our expanding
understanding of the role of Wnt signalling in bone development, formation and
regeneration has led to translational studies investigating how this pathway can be
manipulated for regenerative purposes (Brommage, 2015, Leucht et al., 2018).
Notch signalling is another pathway that has received considerable attention
for its role in osteoinduction (Majidinia et al., 2018). The Notch signalling pathway is
mediated through four transmembrane Notch receptors (NOTCH1,2,3 & 4). Notch is
a crucial pathway involved in mesenchymal stem cell differentiation and subsequent
bone healing (Wang et al., 2016). It exerts its influence via direct osteoinductive
effects on osteoblasts. Inhibition of Notch in progenitor cells leads to reduced
osteoblastogenesis with resultant bone loss (Tu et al., 2012). Interestingly, activation
of Notch (as well as TGF and Wnt) can lead to enhanced osteoblast differentiation
at modified titanium surfaces (Chakravorty et al., 2014).

Remodeling of newly formed bone
The final stage in bone formation and regeneration is the remodeling phase
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that is mediated by both osteoclasts and osteoblasts to replace the early formed
woven bone with more mature lamellar bone. This is a complex process that can take
months or even years to complete. Bone remodeling in healing tooth sockets has
been extensively studied and provides a very useful background to understanding
how modeling and remodeling take place to result in both qualitative and quantitative
changes in bone healing and repairing bone (Araujo et al., 2015). The most critical
regulators of this process are osteoprotegerin, the signaling pathway of Receptor
Activator of Nuclear Factor B and its ligand (RANK/RANKL) and parathyroid
hormone.
RANKL is a transmembrane ligand expressed on osteoblasts, and other
stromal stromal cells, that binds to RANK, a transmembrane receptor found on
osteoclast precursor cells (Lacey et al., 1998). Interaction between RANKL and
RANK propels a cascade of signalling and gene expression that culminates in the
differentiation and maturation of osteoclast precursor cells into active osteoclasts with
bone resorbing capabilities. Osteoprotegerin is a naturally occurring inhibitor of
osteoclastogenesis (Simonet et al., 1997). It acts as a decoy receptor by binding to
RANKL and blocking its interaction with RANK, thereby preventing osteoclast
development. In addition, many of the hormones and cytokines involved in the
regulation of calcium levels in blood and bone, including prostaglandin E2,
parathyroid hormone, vitamin D3 and interleukin-11, play key roles in stimulating
osteoclastogenesis. These effects are mediated via a combination of inhibiting OPG
production and stimulating RANKL production (Martin and Sims, 2015).

RANK and RANKL knockout mice demonstrate a severe lack of
ostgeoclastogenesis and associated osteopetrosis, defective B & T lymphocytes and

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a number of skeletal defects (Ono and Nakashima, 2018). Therapeutics targeting the
RANK/RANKL axis have been developed to assist in the management of
osteoporosis, metastases to bone and giant cell tumor of bone (Deeks, 2018, Sohn
et al., 2014, Diedhiou et al., 2015, Jamshidi et al., 2018) but to date no significant
trials have been undertaken to determine if these have any effect on bone
remodeling associated with bone repair and regeneration (Vannucci and Brandi,
2016).
Parathyroid hormone is a very important mediator of bone mass which can
lead to either bone resorption or bone formation (Jilka, 2007). Parathyroid hormone
helps to maintain calcium ion levels in the blood and its effects are mediated through
a calcium-sensing receptor in the parathyroid gland. When serum calcium levels are
elevated the receptor mediates a decrease in release of parathyroid hormone.
Depending on the length of time of exposure to parathyroid hormone, its effects on
bone can be either catabolic or anabolic. For example, chronic exposure to
parathyroid hormone results in bone remodeling, whereas intermittent exposure to
the hormone results in bone apposition (Jilka, 2007). Intermittent exposure to
parathyroid hormone leads to bone apposition through three cellular mechanisms
including osteoblast proliferation, inhibition of osteoblast apoptosis and activation of
bone cells to produce new bone matrix (Lindsay et al., 2007). Induction of osteoblast
differentiation (and thereby bone apposition) by parathyroid hormone is assisted by a
number of osteogenic cytokines and growth factors, including insulin-like growth
factor-1 and fibroblast growth factor (Lindsay et al., 2007). Parathyroid hormone
stimulation of osteoclast differentiation is principally mediated through the
osteoprotegerin (OPG) and RANK/RANKL axis (Greenfield, 2012). Overall, the
anabolic and catabolic effects of parathyroid hormone on bone through its cell

surface receptor are mediated by complex regulatory mechanisms controlled by
growth factors, transcription factors, regulatory molecules and signaling pathways

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(Silva and Bilezikian, 2015).
A modified form of parathyroid hormone consisting of its first 34 amino acids
(teriparatide) has been developed and trialled for the management of osteoporosis
(Neer et al., 2001). This medication has been found to increase bone mineral
formation and density in extraction sockets and prevent periodontal bone loss in rats
with induced osteoporosis. Furthermore in a canine model teraparatide assisted new
bone formation around dental implants (Valderrama et al., 2010). Early human trials
have demonstrated the potential for teraparatide to positively influence osseous
regeneration in the oral cavity (Bashutski et al., 2010).
CELLS, BIOLOGICS AND REGENERATION
The cellular and molecular processes of bone formation during
embryogenesis, remodelling and regeneration are considered to be almost identical
(Bruder et al., 1994). It is evident that a principal underlying requirement for these
processes is the presence or supply of mesenchymal stem cells. The preferred
therapeutic strategy moving forward is the use of ex vivo expanded MSC
preparations rather than whole bone marrow aspirate transplantation or autografts
that contain limited MSC numbers diluted within a heterogeneous population of
blood/ immune cells. The move away from autograft-based therapy would also
alleviate the risk of morbidity at secondary bone sites. Originally, for the biological
process of bone formation, it was thought that mesenchymal stem cells arising from
bone were the only cells that could be involved. However, with time it has become
evident that mesenchymal stem cells from a variety of tissues have the potential to
participate in bone formation and regeneration. These include mesenchymal stem

cell-like populations from diverse tissues such as the tooth forming elements and
adipose tissue. While the simple injection of cells into osseous defects has been
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studied it is apparent that a more efficient method for the utilization of mesenchymal
stem cells for osseous regeneration is to combine them with tissue growth factors
and space maintaining and delivery systems. With these concepts in mind the field
of tissue engineering emerged and is defined as the science of reconstructing or
mimicking natural processes through the use of synthetic polymer scaffolds with the
expectation of tissue regeneration. The vision is that suitable stem cells produced in
large enough quantities through cell culture methods and aided by appropriate
growth and differentiation factors, are injected into tissues and organs in appropriate
carrier vehicles. As such, they provide a source of progenitor cells that replace the
lost or damaged resident cells allowing for regeneration of damaged tissues. Thus
stem cell based regenerative technologies rely on the input from a large number of
scientific fields including nanotechnology, three-dimension bioprinting,
bioengineering, cell biology, molecular biology, gene manipulation and good
manufacturing processes (Li et al., 2018, Oryan et al., 2017).
A number of principles have been proposed that must be taken into account
for the use of mesenchymal stem cells for bone formation, repair or regeneration
(Bruder et al., 1994):
1. An adequate supply of mesenchymal stem cells must be available
2. Mesenchymal stem cells require specific molecular cues in order to differentiate
into bone forming cells
3. Without rapid vascularization of the MSC-implanted site, the graft will fail
4. Turnover and remodelling are essential phases leading to mature and fully
regenerated bone

5. Delivery scaffolds and devices must be designed within the context of
biocompatibility and appropriate resorption/replacement rates, consistent with

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new woven bone formation and subsequent remodelling into lamellar bone.
Thus, understanding the interface between biology and engineering is central to
achieving acceptable regenerative outcomes (Li et al., 2018). Accordingly, new bone
formation and regeneration can only be achieved by combining our understanding of
cell biology, growth and differentiation factors and bioengineering principles and not
pursuing these processes in isolation.
CONCLUSION
Bone formation and regeneration involve very complex and highly regulated
cellular and molecular processes. By studying these processes new clinical
opportunities will arise for therapeutic bone regenerative treatments. These
processes can be studied both in vitro and in vivo with in vivo studies including
preclinical animal models and human clinical trials. Where possible we have
discussed human clinical trial outcomes but where this was not possible in vitro and
preclinical animal studies have been presented to highlight the potential of stem cells
and biologic agents to play significant roles in both bone formation and bone
regeneration. This review has highlighted the potential roles of stem cells and
specific growth and biologic agents in bone formation and therefore, as a corollary,
bone regeneration. Future research efforts will need to target both stem cells and
biologics through well-controlled clinical trials based on the in vitro and preclinical
published to date. Not only is there considerable opportunity for stem cell based
therapies but also by combining these with regulatory molecules through the
controlled temporal delivery using tissue engineering approaches, offer many exciting
prospects for bone regeneration. Since it is widely recognized that a thorough

appreciation of the biological basis of clinical therapies is essential, it is not until we
understand the process of formation that regeneration will become an achievable

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clinical endpoint for managing disease and trauma to tissues. This will certainly be
the case for bone regeneration.
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Figure Legends
Figure 1. Bone is composed of periosteal, cortical and cancellous compartments
Figure 2. Structural components of the alveolar bone include the alveolar process of
the maxilla and mandible that contains developing teeth as well as the roots of fully
erupted teeth. The other type of alveolar bone is termed “alveolar bone proper” and
this is the portion of the alveolar bone that houses the tooth roots of erupted teeth
and associated periodontal ligament.
Figure 3. Stem cell lineages. Classification of stem cells is based upon their
differentiation potency and is determined by the site, stage of development or cell
culture induction environment in which the populations are derived. Cells may be
categorised as totipotent, pluripotent, multipotent or induced pluripotent. (Adapted
with permission (Lin et al., 2008).

Figure 4. Cellular and molecular players in bone formation, resorption and
remodelling. The complex interplay between stem cells, growth factors, cell surface
ligands and bone cells is illustrated.
Accepted Article
Abbreviations:
BMPs: bone morphogenetic proteins; Ca2+; Calcium ions CTGF: connective tissue
growth factor; EGF: epidermal growth factor ERBs: a family of receptor tyrosine
kinase receptors related to the epidermal growth factor receptor ET-1: endothelin 1;
IGFs; insulin-like growth factors; IL-1: interleukin-1; IL-11: interleukin-11 IL-6:
interleukin-6; OPG: osteoprotogerin; PDGF: platelet derived growth factor; PTHrP;
parathyroid related protein; RANK: Receptor activator of nuclear factor kappa-
Β; RANKL: Receptor activator of nuclear factor kappa-Β ligand; TGF-
growth factor beta; uPA: urokinase-type plasminogen activator.
Table 1
Different Stem Cell Populations used for Bone and Periodontal Regeneration
Bone Marrow Stromal Cells (BMSC)
Dental Derived Mesenchymal Stem cell-like Cells
Dental Pulp Stem Cells (DPSC)
Periodontal Ligament Stem Cells (PDLSC)
Stem Cells from Human Exfoliated Deciduous Teeth (SHED)
Stem Cells from Apical Papilla
Dental Follicle Cells (DFC)
Gingival Mesenchymal Stem Cells (GMSC)
Adipose Tissue Stem Cells (ASC)

Table 2
Biologic Factors Associated with Bone Formation and Regeneration

Accepted Article
Bone–derived Growth Factors & Differentiation Factors
Bone Morphogenetic Proteins (BMPs)
BMP-2
BMP-7
Platelet Derived Growth Factor (PDGF)
Fibroblast Growth Factors (FGF)
aFGF
bFGF
Transforming Growth Factor (TGF-beta)
Growth Differentiation Factors (GDF)
Insulin-like Growth Factors (IGF)
IGF-1
IGF-2
Vascular Endothelial Cell Growth Factor (VEGF)
Skeletal Growth Factor (SGF)
Parathyroid Hormone-related Peptide (PTHrP)
Bone Growth and Regeneration Signalling Pathways
TGF-β Family Signaling
FGF Signaling
Wnt Signaling
Hh Signaling

Bone Growth and Regeneration Families of Transcription Factors
Homeobox Gene Family of Transcription Factors
Accepted Article
Dlx Homeobox Gene Family
Homeobox Gene Family
Hox Homeobox Gene Family
Paired Box (Pax) Homeobox Gene Family
LIM Homeobox Gene Family (Lhx)
Paired-Like (Pitx) Homeobox Gene Family
Runx Transcription Factors

SRY-Related HMG-Box Family of Transcription Factors
bHLH Family of Transcription Factors
Twist
D proteins
The myogenic regulatory factors (MRFs)

Snail Family of Transcription Factors
Smad Transcription Factors
β-Catenin/LEF/TCF Transcription Factors
Gli Transcription Factors
Forkhead Family of Transcription Factors

Accepted Article

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PROF.

PETER MARK BARTOLD (Orcid ID : 0000-0002-5695-3877)

Mesenchymal Stem Cells and Biologic Factors leading to Bone Formation

PM Bartold1, S Gronthos2, D Haynes3, S Ivanovski4

Adelaide, South Australia, Australia

Running Title: Stem cells and biologics in osteogenesis

Key Words: Mesenchymal stem cells, growth factors, bone regeneration

Address for Correspondence

This article is protected by copyright. All rights reserved.

Adelaide, South Australia

Statement of Sources of Funding:

This review was not funded by any external funding agency

Disclosure of any conflicts of interests:

All authors declare they have conflicts of interest to declare.

Background: Physiological bone formation and bone regeneration occurring during

discussing the clinical relevance of in vitro and in vivo animal studies.

bone regeneration. The results are summarized in a narrative review format.

This article is protected by copyright. All rights reserved.

associated with bone formation in the maxilla and mandible.

regulated cellular and molecular processes. By studying these processes new

clinical opportunities will arise for therapeutic bone regenerative treatments.

Although it is widely recognized that a thorough appreciation of the biological basis of

This article is protected by copyright. All rights reserved.

This review is presented as part of the discussion papers for the XV

conclusions can be drawn concerning clinical outcomes, rather consideration of the

information to date should be helpful in defining future clinical studies.

BONE BIOLOGY, STRUCTURE AND REGENERATION

Bone is a hard connective tissue constituting the various skeletal elements

and macro-structural hierarchical arrangement (Rho et al., 1998). It has a number of

specific roles including providing mechanical support and protection, maintaining

mineral homeostasis, hematopoiesis and endocrine functions (Florencio-Silva et al.,

where structured cross-linked collagen matrices are embedded within a mineralized

mechanical and functional requirements of the tissue.

This article is protected by copyright. All rights reserved.

(Figure 1). Woven bone is a developmentally early form of bone characterized by

resulting in low mineral content (Garcia-Rodriguez and Martinez-Reina, 2017). It is

eventually resorbed and replaced. Secondary, or lamellar bone, is comprised of four

general forms: osteons, trabecular, circumferential and interstitial bone (Buckwalter

attachment of ligaments (including the periodontal ligament) as well as providing a

rich blood supply to the outer bone layers.

This article is protected by copyright. All rights reserved.

Developmentally bone is derived from the mesoderm although there is also

formed via two mechanisms know as endochondral and intramembranous bone

regeneration are almost identical processes (Shapiro, 2008). Intramembranous bone

formation is characterized by the formation of bone matrix without the intermediary

step of chondrogenesis (endochondral ossification). Ossification occurs through the

osteoblasts. All of this is intricately orchestrated by myriad growth, differentiation and

continual remodelling dictated predominantly by mechano-transduction signals

significant ramifications for bone grafts or regenerated bone protected from

mechanical forces through excessive resorption resulting in lost architecture and

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(Araujo et al., 2015).

proliferation and the potential to reproducibly differentiate into functional cells

indicative of several different lineages (Dominici et al., 2006). Stem/progenitor cells

This article is protected by copyright. All rights reserved.

others exhibit more limited differentiation capacity.

regenerative cell-based therapies (Ilic and Ogilvie, 2017).

renewal capacity comparable to embryonic stem cells (Takahashi and Yamanaka,

considered to provide an ethically viable alternative to embryonic stem cells in

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medicine (Kanji and Das, 2017).

Due to their accessibility, high growth capacity and multi-potential,

overcome, including identification and isolation of appropriate precursor cell types,