Curr Oral Health Rep (2014) 1:1–8

DOI 10.1007/s40496-013-0010-7

STEM-CELL BIOLOGY FOR TOOTH AND PERIODONTAL REGENERATION (M BARTOLD, SECTION EDITOR)

Mesenchymal Stem Cells and Periodontal Regeneration

Francis J. Hughes

Published online: 3 January 2014

# Springer International Publishing AG 2014

Abstract The use of mesenchymal stem cell (MSC) therapy notably that they are applicable in only very specific clinical
offers the potential to develop a completely novel and im- situations and are generally considered to provide limited,
proved method of periodontal regeneration compared to unpredictable outcomes. Consequently, there continues to be
existing methods. Since the initial demonstration of the pres- considerable research activity in an attempt to improve these
ence of MSCs in the periodontal ligament, many recent stud- therapies and to develop novel treatments to address this issue.
ies have now demonstrated the potential for the transplanta- With the advent of tissue engineering, there is great interest
tion of MSCs from PDL and other sources to enhance peri- in the potential therapeutic application of stem cells for peri-
odontal regenerative outcomes in a variety of animal models. odontal regeneration, and there has been a marked increase in
In addition, the recent demonstration of the possible utility of publications in this area over the past 5 years. However, there
allogeneic MSCs and MSCs derived from inducible pluripo- remain many basic scientific, translational, clinical, and prac-
tent stem cells may offer new methods of delivering such tical issues that must be addressed before this research activity
therapies. Determination of the specific roles that MSCs can be translated into viable therapies. Some of these research
may play in enhancing regenerative outcomes requires further questions are summarized in Table 1. This article reviews
investigation. The principle of MSC-directed periodontal re- recent research into the potential application of mesenchymal
generative therapy is accepted in the field, but extensive stem cells (MSCs) for periodontal regeneration, with particu-
investigation is necessary to establish viable, efficacious, and lar emphasis on how these studies may advance the field by
practically applicable human therapies. addressing some of the questions set forth in Table 1. We
focus particularly (but not exclusively) on the potential use of
Keywords Mesenchymal stem cell . Periodontal . periodontal ligament-derived MSCs, as these have attracted
Regeneration . Tissue engineering . Bone marrow . Dental the most attention in the field. In many respects, this is a
stem cells . Inducible pluripotent stem cells, Allogeneic . relatively recent and novel area of research, and as such, it is
Periodontal ligament . Cementum . Bone . inevitable that much of the recent research addresses some of
Immunomodulation . Scaffolds the more fundamental biological questions listed in Table 1.

Introduction Current Status of Periodontal Regeneration –

The Context for Developing Stem Cell Therapies
Periodontal regeneration, the restoration of the periodontal
tissues destroyed by periodontitis, has been an important Although the normal outcome of healing following treatment
objective of periodontal therapies for many years. Existing of periodontitis is tissue repair, with little or no restoration of
regenerative therapies have considerable limitations, most the previously damaged tissues, it has long been suggested
that the periodontal ligament has high inherent regenerative
potential [1-3] and can give rise to osteoblasts, periodontal
F. J. Hughes (*)
ligament (PDL) fibroblasts, and cementoblasts. More recently,
Dental Institute, Kings College London, Floor 21 Tower Wing, Guys
Hospital, London SE1 9RT, UK this concept is supported by the demonstration of the presence
e-mail: [email protected] of stem cells within the periodontal ligament [4, 5].
2 Curr Oral Health Rep (2014) 1:1–8

Table 1 Some of the basic

scientific, translational, clinical, Basic science
and practical questions to be Fundamental biology of MSCs Phenotypic features of MSCs from different sources;
addressed in studies oninto the Factors regulating MSC differentiation;
application of mesenchymal stem
cells for periodontal regeneration. Role of MSCs Do implanted MSCs contribute to wound healing?
Do implanted MSCs modulate native cells during healing?
Potential for inducible pluripotent stem cells;
Cell delivery systems Role of tissue engineering scaffolds;
Other cell delivery systems;
Other functions of MSCs Immunomodulatory roles of MSCs;
Potentials for vehicles for gene therapies;
Translational
Sources of MSCs Are some MSC sources best suited to this application ?
How are MSCs obtained?
Autologous vs. Allogeneic cells;
In vitro expansion of MSCs Optimizsing isolation and in vitro expansion of MSCs;
Use of appropriate culture media ( without animal products);
Use of appropriate preclinical models. Development and utilizsation of suitable animal models for
investigation of regenerative therapies;
Safety Short- or long- term risks of the therapy (EG genetic stability of cells
during in vitro expansion);
Clinical
Clinical Outcomes Efficacy compared with existing regenerative treatments in humans
Superior outcomes to existing Does the procedure work in new indications;, e.g.,. nNon-contained
treatments bony lesions, suprabony defects, Class III furcations?
Is it superior to alternate treatments ( e.g., dental implant
placement)?
Patient benefits Acceptability to patients;
Patient- centertred outcomes ( e.g., quality of life, risk of morbidities);
Practical
Feasibility of treatment Need for GMP facilities for cell therapies;
Cost- /effectiveness Costs compared with alternative therapies;

Biologically, current regenerative therapies such as guided of interdental papillae typically seen in periodontitis. There is
tissue regeneration (GTR), grafting, and application of bio- little evidence that any of these current techniques show
logical agents can be considered as attempting to ‘unlock’ this consistent significant benefits compared to others, and indeed,
inherent regenerative potential. In the case of GTR, the place- combinations of the techniques do not generally show mark-
ment of a barrier membrane is intended to exclude the gingival edly improved efficacy, with the possible exception of the use
tissues and promote wound repopulation by PDL cells. The of PDGF combined with alloplastic ß-tricalcium phosphate
use of grafts is intended to provide a scaffold for wound graft [9]. Interestingly, in recent studies of treatment of highly
repopulation by PDL cells, and in some cases it may trigger contained narrow infrabony defects, a minimally invasive
osteogenesis. The addition of biological agents such as enamel surgical approach without any additional regenerative thera-
matrix proteins or rhPDGF-BB are intended to direct cellular pies appears to provide equivalent results to those in which
wound repopulation and appropriate differentiation outcomes. treatment with enamel matrix proteins or alloplastic grafting
Although there is evidence supporting the efficacy of these were also used [10, 11].
techniques in promoting periodontal regeneration, their over- The current status of periodontal regenerative therapies
all effects are modest [6-8]. Moreover, they are applicable establishes the base from which any stem cell-based therapy
only in very specific circumstances, specifically in narrow needs to improve. The treatment of non-contained bony de-
contained infrabony defects and class II furcation lesions. In fects, regeneration of supracrestal periodontal, and regenera-
addition, although these techniques address the issue of re- tion of gingival soft tissue appear to be particularly critical
generation of deep periodontal tissues, they do not reverse the long-term goals upon which to judge potential novel regener-
poor aesthetics resulting from the gingival recession and loss ative therapies.
Curr Oral Health Rep (2014) 1:1–8 3

Mesenchymal Stem Cells Phenotype of PDL MSCs

Stem cells are undifferentiated cells that are defined by their Although these various tissue-derived MSCs may share many
properties of self-renewal and differentiation potential – the properties, an important question arises as to whether they are
ability to give rise to a range of mature cell phenotypes. They phenotypically distinct. Within the field of periodontal regener-
may be isolated from embryonic tissue (embryonic stem cells) ation, in particular, a number of studies have aimed to investigate
or from adult tissues (somatic stem cells). Embryonic stem (ES) the specific phenotypic characteristics of periodontal ligament-
cells can be maintained indefinitely and are totipotent, meaning derived MSCs. This may be the result of observations that PDL
they have the capacity to give rise to all cell phenotypes. MSCs can give rise to tissues resembling bone, cementum, and
Somatic stem cells have been isolated from a wide range of periodontal ligament when implanted in nude mouse models.
adult tissues and, in general, have a more restricted repertoire of Methodologically, these observations are hampered by the ab-
differentiation potential (i.e., are multipotent). They exhibit the sence of truly specific markers of PDL and cementum. Howev-
capacity for extensive self-renewal but have a finite lifespan. er, it is known that certain genes such as periostin, periodontal
In addition, a third type of stem cell, the induced pluripo- ligament-associated protein (PLAP), and scleraxis are highly
tent stem cell (iPS), has now been reported. iPS cells were first expressed in the PDL, and that cementum expresses bone matrix
described following the stable transfection of embryonic and proteins, including bone sialoprotein, and is high in cementum
adult mouse fibroblasts with genes associated with ES cells, attachment protein and cementum protein 1 (CEMP1), which
including Oct3/4, Sox-2, c-Myc and Klf-4 [12, 13]. Since that may also be found in bone [26, 27]. Given this caveat, there is a
time, iPS cells have been produced from many human cell risk that some of these observations may be subject to inadver-
types, including gingival and periodontal fibroblasts [14-16]. tent bias, particularly when based on histological appearance,
As they demonstrate many of the same properties as ES cells, whereby observation of mineralized tissue is described as “ce-
including a very wide range of differentiation potential, iPS mentum-like” based on the finding of mineralized tissue with
cells have garnered much interest within the stem cell associated connective tissue fibers.
research community for a number of reasons, not the Nevertheless, a number of studies do suggest significant
least of which is the absence of ethical issues surround- phenotypic differences between PDL MSC and MSCs derived
ing the generation of ES cells. from other sources in vitro, despite the fact that they carry the
Mesenchymal stem cells are somatic stem cells that were same immunological surface markers. Studies have reported
originally described by Friedenstien and colleagues as fibro- increased proliferative potential and osteoblastic differentia-
blastic cells derived from bone marrow stroma with the ability tion from PDL and other dental stem cells when compared
to form colonies in vitro and with the potential to differentiate with bone marrow stromal cells (BMSC) in vitro, and have
to form bone, cartilage, and adipose tissue [17]. In 2006, the suggested their increased potential for neurogenic differentia-
International Society for Cellular Therapy proposed minimum tion, possibly reflecting their neural crest origin [28, 29, 30•,
criteria to define MSCs, the first of which was that MSCs must 31]. In addition, preliminary studies using microarray analysis
be plastic-adherent in certain cultures. Secondly, they must of gene expression have described marked quantitative differ-
express the surface markers CD105, CD73 and CD90, and ences in gene expression patterns between BMSC, dental pulp
must not express CD45, CD34, CD14, CD19, or HLA-DR. stem cells, and PDL MSC [32].
Thirdly, they must be able to differentiate into osteoblasts, Two interesting recent studies investigated differences in
adipocytes, and chondroblasts in vitro [18]. In addition to the proteomes of MSCs of distinct sources. Using mass spec-
these criteria, MSCs exhibit self-renewal and are clonogenic. trometric analysis, Mrozik and colleagues investigated prote-
MSCs exhibiting these properties have now been isolated omic differences between BMSC, DPSC, and PDL MSC
from a wide range of tissues, including bone marrow, adipose derived from the same (ovine) donor and reported 58 proteins
tissue, fetal liver and blood, Wharton’s Jelly of the umbilical that were differentially expressed among the different MSC
cord, and dental tissues, including periodontal ligament (PDL sources [33]. Similarly, Eleuterio and colleagues carried out
MSC), dental pulp (DPSC), exfoliated deciduous teeth mass spectrometric analysis of proteomic differences between
(SHED cells), and dental follicle [4, 19-21]. Recent studies PDL MSC and matched DPSC and from BMSC (derived
have also described the isolation and characterization of from a difference source) [34•]. This study reported a number
MSCs from healthy and inflamed gingival tissue by isolation of marked quantitative differences in protein expression
of clonogenic cells or magnet immunosorting, and this has among MSC populations, although the expression profiles
been proposed as a potentially more abundant source of tissue were qualitatively quite similar.
for deriving autologous MSCs when compared to PDL Collectively, these studies support the hypothesis that PDL
[22-24]. A recent study also suggested that gingiva-derived MSCs are phenotypically distinct when tested in vitro. There
MSCs include both neural crest-derived and mesodermally are a number of possible explanations for these observations.
derived cells [25•]. Firstly, the cells may have inherently different properties, such
4 Curr Oral Health Rep (2014) 1:1–8

as differentiation potentials, and this may be because of their the potential suitability of allogeneic PDL cells for use in
embryological (neural crest) origins [35-38]. Interestingly, therapeutic development of regenerative procedures [45].
however, in a study using GFP-labeled BMSCs for lineage The broader subject of mechanisms of immunomodulatory
tracing, Zhou and colleagues reported that BMSCs contribute properties of MSCs, including those derived from dental
to the total MSC population within the PDL and can differen- tissues, was the subject of a recent excellent review, but this
tiate into dental-specific tissues, suggesting regulation by local is beyond the scope of this article [46].
environmental conditions. Secondly, despite the persistence of
the characteristic cell surface markers of MSCs, MSC cultures
are heterogeneous, containing cells of more restricted lineage Preclinical Models – Animal Studies
potential, and may show reduced stemness and proliferative
capacities with aging both in terms of donor age and time in Studies of MSCs demonstrate the potential to isolate multipotent
culture [39-42]. Consequently, studies of phenotypic differ- cells which can differentiate into the tissues of the PDL both
ences between cells from different donors should be in vitro and in ectopic implantation animal models, typically
interpreted with caution. Studies using matched cultures de- using nude mice. There are, of course, a number of important
rived simultaneously from the same donor may therefore be challenges that need to be addressed to translate these basic
particularly valuable models in which to investigate true phe- findings into successful tissue engineering strategies to achieve
notypic differences between cells [33, 43]. periodontal regeneration in the clinic. These issues include
With respect to the goal of utilizing MSC for therapeutic optimization of cell sources, how cells are expanded ex vivo
application in periodontal regeneration, phenotypic differ- prior to implantation, choice of delivery system/scaffold for
ences in vitro between MSCs of different origin may not implantation, and ultimately the efficacy and magnitude of effect
necessarily translate to superior clinical outcomes when using of the proposed treatments. These questions pose difficult prac-
PDL MSC compared to other cell types. These questions will tical problems, involving the use of animal models, for example,
require specific studies, including animal models of regener- to compare different cell sources and delivery systems.
ation, as further considered below. In terms of the practicality of ex vivo expansion, it is
known that expansion can be greatly enhanced by appropriate
Immunomodulatory Properties of PDL MSCs medium supplements [47], suggesting the feasibility of utiliz-
ing chemically defined serum-free medium [48•]. A recent
It has been known for a number of years that MSCs derived study with human BMSC has shown that use of FGF2, PDGF,
from bone marrow exert powerful immunosuppressive effects or ascorbate can increase by over 1000-fold the total number
[44]. These properties have 2 potentially important clinical of cells generated while retaining their stem cell properties
implications – firstly, that transplanted MSCs might be uti- [42]. In addition, it has been demonstrated that the age of the
lized as specific immunosuppressive therapeutic agents for the donor may have a marked effect on the capacity for ex vivo
management of autoimmune diseases, and secondly, that im- expansion of MSCs [49].
plantation of allogeneic MSCs might be used in regenerative
procedures rather than requiring the use of autologous cells Use of MSC for Periodontal Regeneration in Animal Models
isolated from the patient and expanded ex vivo prior to the
procedure. It has now become clear that dental MSCs, includ- The choice of animal model for preclinical PDL regenerative
ing those derived from PDL, exhibit similar immunomodula- studies – albeit essentially for “proof of principle” studies –
tory properties to those of BMSCs. Immunomodulation by may significantly influence findings. Of course, findings in
dental MSCs was first described by Wada and colleagues, who animal models may be difficult at times to translate to human
showed that these cells produced soluble factors, including studies. However, a wide range of animal models of periodon-
TGF-β1, hepatocyte growth factor (HGF), and indoleamine tal regeneration have been used, including rodent, canine,
2,3-dioxygenase (IDO), which resulted in inhibition of ovine, and porcine models, that have demonstrated beneficial
mitogen-induced T cell proliferation. Ding and colleagues effects on PDL and mineralized tissue formation [4, 24, 50,
demonstrated that porcine PDL MSCs failed to elicit a T cell 51•, 52, 53, 54••, 55]. The majority of these studies utilized
proliferative response and did not express human leucocyte surgically created infrabony defects. Liu and colleagues also
antigen (HLA)-II DR, CD80, and CD 86. They further dem- described the use of a periodontitis model in minipigs by first
onstrated that PDL MSCs inhibited mitogen-stimulated T cell surgically creating a defect in the interdental region and then
proliferative responses via a PGE2-mediated mechanism [45]. placing a silk ligature to induce a periodontal lesion prior to
This study, along with a recently published study using an placement of the stem cell treatment [45, 56].
ovine model, have now demonstrated successful transplanta- A range of different cell sources, including PDL MSC
tion and survival in animal studies of periodontal regeneration [54••], DPSC [55], adipose tissue [52], BMSC [57], and gingi-
without induction of an inflammatory response, suggesting val MSC, have demonstrated the ability to increase
Curr Oral Health Rep (2014) 1:1–8 5

regeneration of periodontal tissues [24]. Although these studies tissue, suggesting that the ES cells contributed directly to the
have not demonstrated superior results of any particular cell type tissue formation process. In addition, some transplanted cells
over another, PDL cells were found to have superior outcomes became incorporated into distant organs and tissues, suggesting
in a study by Park and colleagues that tested the effects of systemic cell transmission. Aside from ethical issues of ES cell
autologous PDL, DPSC, and follicular stem cells on regenera- use, the totipotent nature of these cells does raise important
tion of defects extending to the root apex in a canine model [58]. safety issues and the possibility of teratoma formation in such
Most of these studies have employed autologous cells that models, although this was not observed here.
have been isolated, expanded ex vivo, and reimplanted. How-
ever, given the immunosuppressive effects of MSCs as previ- Contribution of Transplanted Cells to Tissue Regeneration
ously described, the possibility of utilizing allogeneic cells for
implantation is an attractive alternative. Ding and colleagues In most of the above-described studies, the precise contribution
compared the regenerative capacity of autologous and alloge- of stem cells implanted into wounds is an important consider-
neic PDL MSCs in the minipig periodontitis model and found ation, particularly as it relates to transplanted MSCs. This is
equivalent regenerative outcomes [45]. In recent studies, allo- especially the case given the increasing evidence that intrave-
geneic SHED cells were also shown to promote regeneration nous administration of MSCs can enhance clinical regenerative
in the minipig model [59], and allogeneic PDL cells were able outcomes in a number of conditions despite the apparent lack of
to enhance regeneration in a rat fenestration defect model persistence of these cells or their incorporation into newly
[51•]. Mrozik and colleagues utilized an ovine model of formed tissues [62, 63]. In these cases, it is postulated that
regeneration using surgically created uncontained buccal bony transplanted MSCs are acting principally through mechanisms
defects to test the effect of allogeneic PDL cells in a Gelfoam involving the paracrine regulation of immune responses and by
carrier [50•]. Although the cells were well tolerated and release of trophic cytokines that direct endogenous cell regener-
showed trends towards more periodontal regeneration, this ation. Additionally, the possibility of crosstalk between im-
was not significantly greater than the use of the Gelfoam planted MSCs and local cells has been described in vitro [64].
carrier alone (which demonstrated a statistically significant Clearly, there are potentially important differences be-
increase of regeneration compared to control defects) [50•]. tween the local implantation of MSCs in a periodontal
wound and the systemic administration of MSCs, and this
Inducible PS Cells and ES Cells remains an important issue in stem cell-mediated PDL
regeneration. In an attempt to investigate this difference,
In addition to the MSCs, embryonic stem (ES) cells and iPS Yu and colleagues recently reported their study of trans-
cells have also been investigated in a small number of periodon- plantation of GFP-labeled PDL MSC in gelatin sponges in
tal regeneration studies. Duan and colleagues described the a rat infrabony defect model using GFP-labeled gingival
implantation of iPS cells combined with enamel matrix deriva- fibroblasts as controls. PDL MSC-treated defects showed
tive (EMD) into rat periodontal fenestration defects, which increased bone and PDL regeneration. Rather strikingly,
demonstrated increase bone, cementum, and PDL formation PDL-labeled MSCs were specifically densely located ad-
with both EMD and EMD+iPS cells, with slightly greater jacent to the regenerated tissue, suggestive of cell homing
effects seen with iPS treatment [60]. In a recent study, Hynes to the defect, whereas gingival cells were sparsely distrib-
and colleagues reported the induction of iPS into MSC and uted throughout the tissue. However, there were few la-
subsequent implantation of iPS-derived MSCs into rat fenestra- beled cells incorporated into the new tissue, and the
tion defects, resulting in enhancement of bone, cementum, and authors suggest that the improved outcomes may be large-
periodontal regeneration, with particular enhancement of the ly the result of paracrine regulation of endogenous cells.
complete “bridging” of the bony fenestration defects [61•].
The induction of iPS into MSC as a precursor for use in
regenerative studies may be an important step in order to avoid Delivery Systems for Implantation of Cells
the potential risks of implantation of totipotent cells.
Yang and colleagues recently reported the use of ES cell In the studies described above, a wide range of different
transplantation in a minipig periodontitis model that demon- carrier systems and scaffolds have been utilized for the deliv-
strated improved clinical periodontal parameters (approximate- ery of stem cells. These range from the very simple cell sheets
ly 1.5 mm extra pocket depth reduction and approximately to rather complex 3D structures. Many studies have success-
2 mm increased CAL gain compared to controls) and improved fully utilized collagen, gelatin, or fibrin sponges [54••, 65-67].
cementum formation, although this was not quantified Other studies have variously reported the use of
histomorphometrically [53]. Furthermore, by using GFP label- hydroxyapatite/tricalcium phosphate [56], alginate hydrogels
ing of cells prior to transplantation, they demonstrated persis- [68], silk scaffolds [60], amniotic membrane [69], and increas-
tence of ES cells and incorporation of cells into regenerated ingly, the use of cell sheets utilizing the matrix produced
6 Curr Oral Health Rep (2014) 1:1–8

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