Keywords
3D-bioprinting - exosomes - immune phenotype - periodontal regeneration - periodontal
stem cells - regenerative medicine - stem cells - tissue engineering
Introduction
Periodontal disease is a chronic inflammatory disease characterized by a dysregulation
of balance between the native oral commensals and the pathogenic microorganisms, leading
to activation of the inflammatory cascade thereby causing host mediated destruction
of the periodontal soft and hard tissues.[1] Nonsurgical periodontal therapy (NSPT) primarily aims at regulating the immune-inflammatory
profile by mechanical debridement. Despite this, in approximately 67% of instances,
disease persists even after NSPT owing to areas of persistent pockets that do not
allow complete resolution of inflammation, thereby warranting surgical treatment.[2]
Traditional surgical periodontal therapies rely on synthetic materials and biological
agents for regeneration, although their effectiveness is debatable due to lack of
histological evidence of regeneration.[3] Tissue engineering with a triad of cells cultivated on a scaffold with suitable
biophysical and chemical cues to finally rebuild the lost tissues has been proposed
for attaining optimal regeneration.[4]
Stem cells have been considered a promising approach for regeneration as they have
unique properties of stemness, migration, differentiation, and immune modulation.[5] Traditionally, stem cells have been harvested from the dental pulp and the exfoliated
deciduous teeth; however, recently the use of stem cells sourced from the periodontium
has been advocated as they are a reservoir of highly undifferentiated cells that can
migrate to regenerate the lost periodontium. Mesenchymal stem cells from periodontal
tissues such as periodontal ligament stem cells (PDLSCs), gingival mesenchymal stem
cells (GMSCs), oral periosteal stem cells (OPSCs), and dental follicle stem cells
(DFSCs) have been tested with varying results in vitro and in vivo for regeneration. The best possible stem cell needs to be assessed and compared to
aid in the selection of the candidate cell to achieve complete regeneration. This
review summarizes the properties of each unique stem cell population harvested within
the periodontium and their regenerative potential.
Origin and Distinct Phenotypes of Stem Cells within the Periodontium
Origin and Distinct Phenotypes of Stem Cells within the Periodontium
The periodontal complex's postnatal root development that parallels the tooth growth
process heightens the possibility of a bountiful supply of dental stem cells that
are more embryonic in nature than other dental stem cell sources. The nomenclature
of periodontal MSCs is strongly linked to their tissue origins ([Fig. 1]). PDLSCs are produced from ectomesenchymal cells originating from the neural crest
and are principally extracted from the mid-third of the root surface post extraction
of permanent teeth.[6] The stem cells isolated from root surface are termed root surface derived PDLSCs
(r-PDLSCs) and the stem cells isolated from the tissue collected from the bone surface
are called alveolar socket derived PDLSCs (a-PDLSCs). It has been observed that a-PDLSCs
retain more proliferative capacity, high osteogenic, and adipogenic potential compared
to r-PDLSCs.[7] Seo et al, 2004, found that PDLSCs have the ability to differentiate into periodontal
ligament, alveolar bone, cementum, peripheral nerves, and even blood vessels. PDLSCs
are found in the periodontal ligament and the developing follicle of permanent teeth.[8]
Fig. 1 Origin and development of stem cells within the periodontium. (A) The neural crest ectomesenchyme and the neural crest cells—The thickening of ectomesenchyme
with migration of neural epithelial cells form a tooth bud. (B) Condensation of mesenchyme to form dental papilla and follicle giving rise to dental
follicle stem cells. Inner layer of follicle (dental follicle proper) gives rise to
periodontal ligament cells, the denotgingival fiber system harboring periodontal ligament
stem cells (PDLSCs) and alveolar bone harboring oral periosteal stem cells (OPSCs),
outer layer of follicle (perifollicular mesenchyme) forms gingival lamina propria
that harbors gingival mesenchymal stem cells (GMSCs). (C) Mature tooth showing location of PDLSCs, GMSCs, and OPSCs.
Gingiva, the most important periodontal organ, is the next source of stem cells that
originate from the neural crest and even from the bone marrow. GMSCs are extracted
from gingival tissue samples acquired during gingivectomy procedures and de-epithelialized
to leave only connective tissue.[9] Neural-crest-derived GMSCs (N-GMSCs) and mesoderm-derived GMSCs (M-GMSCs) are two
subpopulations of gingival mesenchymal stem/progenitor cells, with N-GMSCs having
a stronger ability to develop into neural cells than M-GMSCs.[10]
Alternatively, a loose ectomesenchyme-derived connective tissue called dental follicle
surrounds the enamel organ and dental papilla of the growing tooth germ. The DFSCs
are comprised of a series of pluripotent stem cells formed from neural crest cells
originating from the ectoderm.[11] Morsczeck was the first to discover that periodontal tissue progenitors were present
in dental follicular cells, known to regulate osteoclastogenesis and osteogenesis
that is vital for tooth eruption coordination, in addition to its role in periodontal
development.[12] OPSCs are derived from the periosteum, a complex structure that includes undifferentiated
mesenchymal cells and envelopes the bone.[13] The periosteal stem cells regulate chondrogenesis and osteogenesis that can be exploited
for the maintenance of bone mass in both physiological remodeling and in periodontal
surgical healing process.
Dental tissues in the craniofacial complex have become a readily available source
of MSCs with multilineage differentiation capabilities comparable to bone marrow MSCs.[14] Despite the fact that there are numerous dental stem cell populations, periodontal
stem cells have recently attracted a lot of attention because they are native to the
periodontal complex and can be induced to accomplish regeneration.[15] MSCs from the periodontal complex have morphology and marker expression that are
very similar to fibroblasts, despite the fact that they only make up around 1% of
the cell culture population. There are two main methods for isolating SCs from the
periodontium, and both have been successfully employed to isolate MSCs from oral tissues,
including gingiva for cell culture.
The first method entails growing cells from a tissue sample in plastic-adherent fibroblast:
explant culture method or by enzymatic release method.[16] The second method includes MSCs sorting from parental fibroblast cultures or cell
population enzymatically liberated directly from connective tissue biopsy based on
a panel of preselected cell surface markers utilized to isolate MSCs from oral and
other tissues. This method of prospective separation is based on the ability of the
chosen markers to accurately identify MSCs.[17] Based on these above techniques, MSCs can be identified within the periodontium
and probably the biggest colonies may be identified as putative MSCs with highest
potential for proliferation and self-renewal.
Distinct Phenotypes of Stem Cells within the Periodontium:
The periodontal stem cells originate from closely related tissues; however, there
is wide difference in the type of markers they express, which determines the unique
phenotype and differentiation capacity of each of these stem cells ([Table 1]).
Table 1
Phenotypic expression and regenerative potential of periodontal stem cells
|
Stem cell type
|
Markers expressed
|
In vivo tissue formation
|
In vitro tissue
formation
|
Reference
|
|
Neural crest
|
Embryonic
|
CD antigen (+) / (-)
|
|
PDLSC
|
Sox 10, P75NTvnR, Snail, Twist,
Sox-9, CD49d
|
Nanog, Sox 2, SSEA4
Oct 4,
Klf4
|
CD-9 + , CD-10 + , CD-13 + , CD-29 + ,
CD-44 + , CD-59 + , CD-73 + , CD-90 + , CD-105 + , CD-106 + , CD-146 + , CD-166+ CD14-,
CD31-, CD34-,CD45-
|
Cementum, PDL, adipose, dentin, bone
|
Osteo, adipo, chondro, myo, neuro, cardiomyo, HLC, melanocyte
|
[18]
|
|
GMSC
|
Snail1, Twist 1, Sox 9, NES, FoxD3, PAX3
|
Nanog,
Sox-2
SSEA3
|
CD-29 + , CD-44 + , CD-73 + , CD-90 + , CD-105 + , CD-106 + , CD-146 + , CD-166+
CD34-, CD45-,CD117-
|
Cartilage, bone, muscle
|
Adiopo, chondro,
osteo, neuro, endothelial cells
|
.[19]
|
|
DFSC
|
HNK1, NES, P75NTR, Nestin, βIII-Tubulin
|
Oct 4,
Sox 2,
Nanog
|
CD-13 + ,CD-29 + , CD-9 + , CD10 + ,
CD-44 + , CD-59 + , D-53 + ,CD-73 + ,CD-90 + ,CD-105 + , CD106 + ,CD146 + , CD166 + ,CD34-,
CD45-,CD31-, CD117-, CD14-
|
PDL like, cementum like, alveolar bone
|
Osteo, adipo, chondro, myo, neuro, cemento, odonto, HLCs
|
.[12]
|
|
OPSC
|
Nestin, NG2
|
Hox-11, Nanog
|
CD-13, CD-29, CD-44, CD-71, CD-73, CD-146, CD34-, CD45-, CD105-,CD166-, D117-, CD90-
|
Bone
|
Osteogenic, neurogenic chondrogenic
|
[20]
|
Abbreviations: adipo, adipocyte; cardiomyo, cardiomyocyte; CD, cluster of differentiation;
cemento, cementoblast; chondro, chondrocyte; FOXD3, forkhead box D3; DFSC, dental
follicle stem cell; GMSC, gingival mesenchymal stem cell; Klf4, Kruppel-like factor
4; HLC, hepatocyte like cells, myo, myoblast; HNK-1, human natural killer 1; Hox,
homeobox; Nanog, nanog homeobox; NES, neuro epithelial stem cell protein; neuro, neuronal
cell; NG2, neuron glial antigen 2; Nestin, neuroepithelial stem cell protein; odonto,
odontoblast; OPSC, oral periosteal stem cell; osteo, osteoblast; Oct 4, octamer 4,
PAX3, paired box 3; PDLSC, periodontal ligament stem cell; PDL, periodontal ligament;
P75NTR, p75-neurotrophin receptor; Snail-1, Slug- Zinc finger protein; Sox-SRY-related
HMG-box genes; SSEA4, stage specific embryonic antigen; Twist 1, twist related protein
1.
+ - positive expression, - negative expression; elevated expression, decreased expression.
The PDLSCs, for instance, are found to be more of adult mesenchymal-like as they express
the entire range of MSC markers and less of neural crest markers apart from sharing
similar phenotype to pericytes as seen by positive expression of CD146, neural/glial
antigen-2, and CD140B.[21]
The majority of putative stem cell markers have been found to be expressed by GMSCs,
and it has been observed that GMSCs express high levels of the embryonic stem cell
markers Oct 4, Nanog, and SSEA3, which are essential for maintaining progenitor status,
when cultured in three-dimensional (3D) scaffolds primed with ascorbic acid.[22] The significant expression of pluripotent markers in GMSCs indicates that gingival
stem cells have the propensity for regeneration and incorporate stem cells that constitute
pluripotent properties. It is discovered that the DFSCs are more embryonic in origin
and express Oct 4, but only infrequently Nanog and CD90 that demonstrate the increased
heterogeneity and neural crest genesis of DFSCs.[23]
The periosteum offers mechanical support while also acting as a major source of progenitor
cells and growth factors for bone regeneration.[24] Further, OPSCs showed high expression of CD-73, CD-90, CD-105, and CD-29, whereas
hematological markers CD-45 and CD-34 were not expressed.[25] OPSCs have a low level of CD-117 surface marker positivity, which is a stem antigen
expressed on MSCs that are not yet committed to bone phenotype.[26] OPSCs are distinguished from other types of stem cells by the selective expression
of cathepsin K in the periosteum as early as embryonic day 14.[27]
Overall, the stem cells of periodontal origin exhibit a wide spectrum of characteristics
that can be harnessed for optimal regeneration based on a case-to-case scenario. While
the GMSCs exhibit a wide range of embryonic markers, selecting a PDLSC or an OPSC
for extensive periodontal regeneration would be more preferred as they have unique
properties such as expression of greater osteogenic and chondrogenic differentiation
markers that can achieve cementum and periodontal ligament like structures when transplanted
in animal models, thereby making them a superior choice when compared to the application
of GMSCs.[28] Further, the data for PDLSCs and OPSCs to form bone suggest that they can be adequately
osteogenic only when they are used in a combination with suitable grafting materials
until which their regenerative potentials cannot be tapped appropriately. On the other
hand, the DFSCs have been shown to provide a suitable microenvironment for enhanced
regeneration of PDLSCs in vivo, thereby highlighting the adjunctive role of DFSCs by acting as a scaffold in facilitating
the differentiation of other types of stem cells.[29]
Inflammatory Environment and Periodontal Stem Cells—The Bidirectional Link
Inflammatory Environment and Periodontal Stem Cells—The Bidirectional Link
The behavior of stems cells in an inflammatory environment differs markedly from the
healthy state as inflammation affects the stem cells and in turn the stem cells exert
immune-modulatory properties in an inflammatory microenvironment.[19]
[30] Understanding this bidirectional link of how periodontal inflammation can influence
stem cells and how they interact in an exacerbated immune inflammatory environment
can help devise future regenerative strategies ([Fig. 2]).
Fig. 2 Immunomodulation and regeneration potential of periodontal stem cells. (A) Inflammation and stem cells “The Bidirectional Effect” affecting their differentiation.
(B) Immunomodulatory dentistry—Regeneration of periodontal tissues like bone, cementum,
and periodontal ligament. (C) Immunomodulatory medicine—regeneration into various tissues of the body.
Effect of Periodontal inflammation on Stem Cells
In periodontal disease, gram-negative bacteria predominantly Porphyromonas gingivalis,
Treponema denticola, and Tannerella forsythia are key microbial regulators of the
disease, causing increased expression of inflammatory mediators and adhesion molecules
triggering an inflammatory cascade there by altering the immune phenotype of the stem
cells in the periodontally destructed sites. This inflammatory response acts as a
regulator of tissue stemness either by directly affecting periodontal tissue stem
cells or by shifting differentiated cells toward a stem cell like characteristic.
The balance in this inflammatory response and mediated stemness is a critical driver
of either maintaining tissue integrity or promoting aberrant homeostasis and disease.
PDSLCs have been studied to have scope to regenerate the supporting structures as
long-term stimulation of PDLSCs by P. gingivalis lipopolysaccharide (LPS) resulted in increase in cellular cytokine release compared
to GMSCs. It is also suggested that LPS restrains the osteoblast differentiation by
impairment of alkaline phosphatase activity and mineral deposition in DFSCs and PDLSCs.[31] Activation of TLR2 also caused increased proliferation of OPSCs.[32] On the contrary, studies also reported that P. gingivalis LPS treatment did not alter the cell viability of both OPSCs and DFSCs. This data
suggest that the activation of inflammatory cascade has a role in the stimulation
of various periodontal stem cells; however, the availability of contradictory evidences
questions the phenomenon of this effect thereby warranting the need of future research.
Immunomodulatory Effect of Periodontal Stem Cells on Inflammatory Environment
The effect of periodontal inflammation could sensitize the stem cells; however, recent
data suggest that these stem cells also exert an immunomodulatory effect that explains
the opposing link between SCs of periodontal origin and local inflammation. Immunomodulation
is characterized by an induction, amplification, attenuation, or prevention of the
functioning of the immune system by the activity of an immunomodulator such as the
periodontal stem cells.
PDLSCs induce the secretion of tumor growth factor-β, indoleamine-2,3 di-oxygenase-1
and hepatocyte growth factor that have immunomodulatory effect on periodontal regeneration.[33] PDLSCs also alter the innate immune response by elevating the proliferation and
diminishing the apoptotic potential of neutrophils. In addition to the inhibition
of T cell proliferation by the PDLSCs, the anti-inflammatory M2 macrophage phenotype
polarization is also enhanced as the PDLSCs stimulate CD-136, interleukin-10 (IL-10)
and arginase 1.[34]
Similarly, GMSCs facilitate macrophage M2 polarization and also inhibit the M1 macrophages
by producing prostaglandin E2 (PGE2), IL-6, and IL-10. Furthermore, they also reduce
the maturation of dendritic cells that further suppresses its capability to present
the antigens in a PGE2-related phenomenon, thereby dampening the inflammatory cascade.[35] The DFSCs in a similar fashion suppress bone resorption by diminishing the phagocytic
activities and neutrophil extracellular trap formation and also cause M2 macrophage
polarisation.[36] DFSCs also elevate the expression of anti-inflammatory cytokines such as IL-10 and
suppress the concentration of the proinflammatory cytokines, thereby preventing bacterial
internalisation.[37] A recent study by He et al proved that OPSCs effectively inhibited M1 polarisation.[38] The overall picture suggests that periodontal stem cells attenuate inflammation
by various mechanisms as motioned above; however, the immunomodulatory capacities
of these cells, which are essential participants in the modulation of immune responses
to accomplish regeneration in periodontitis models, have not yet been fully explained.
Boundless Regenerative Potential of Periodontal Stem Cells
Boundless Regenerative Potential of Periodontal Stem Cells
The predictability of current treatment protocols to limit the spread of periodontal
disease and facilitate regeneration is questionable. However, the principles of tissue
engineering can be adapted to expedite regeneration of oral and extraoral tissues
tat diversifies the application of these intimately related stem cells—the periodontal
stem cells ([Table 2]).[39]
Table 2
Periodontal stem cells in regenerative periodontics and regenerative medicine
|
Stem cell
|
Growth factors
|
Carrier/scaffold used
|
Cell numbers achieved In vitro in Vivo
|
Model used
|
Regenerative outcome
|
Reference
|
|
PDLSCs
|
FGF2
RhFGF-2
|
Chitosan conjugated Nano HA coating
|
5× 104
|
1× 107
|
Mice-calvarial defect
|
• Osteogenic potential of PDLCs are enhanced
• Superior hard tissue regeneration
• Increased mineralization by Notch signaling
|
[40]
|
|
TGF-β3
|
RGD Modified alginate microspheres
|
1× 106
alginate solution
|
1× 106
|
Mice
Subcutaneous
|
• Enhanced tendon regeneration capacity
• Higher chondrogenic and adipogenic differentiation
|
[41]
|
|
rAd- BMP-2
|
Hydroxyapatite and bone grafts
|
2× 106 cells/mL
|
2× 106 cells/mL
|
Mice and canine
|
• BMP-2 enhances new bone formation and promotes osteogenesis
|
[42]
|
|
BMP-2
BMP-9
|
1% collagen hydrogel
|
2× 106
|
2× 106
|
Canine
|
• Higher osteogenic differentiation
|
[43]
|
|
IGF
|
Absorbable gelatine sponge
Gelfoam
|
8× 103 cells / cm2
|
1× 106
|
Mice
|
• Promotes osteogenic differentiation via osteogenesis of PDL progenitor cells
|
[42]
|
|
1% PRP
|
PDLSC sheets
|
8× 103 cells / cm2
|
1× 106
|
Mice
|
• Increases extra-cellular matrix
|
[44]
|
|
FGF2
|
Amnion
|
3× 105 cells
|
3× 105 cells
|
Human
|
• Increased osteo, adipo differentiation
|
[45]
|
|
GMSCs
|
TGF-β3
|
Alginate microspheres
|
1× 106
alginate solution
|
1× 106
|
Mice
Subcutaneous
|
• Enhanced regeneration capacity
• Greater chondrogenic and adipogenic differentiation
|
[46]
|
|
BMP-2
|
Collagen Scaffold
|
2mm × 3mm
|
2× 106
|
Rats
|
• Higher osteogenic differentiation
|
[47]
|
|
TNF-α
|
Exosomes
|
200 µg
|
1× 106
|
Mice
|
• Higher chondrogenic and osteogenic differentiation
|
[48]
|
|
BMP-9
|
Hyaluronic acid synthetic ECM
|
250µl
|
5× 106
|
Dog
Mini
Pig
|
• Enhanced adipogenic and chondrogenic lineage
|
[19]
|
|
BMP-2
|
Collagen membrane covering a scaffold with β-TCP
|
2 × 105
|
8× 106 cells / cm3
|
Human
|
• Periodontal defects, enhanced osteogenic, adipogenic differentiation
|
[49]
|
|
TGF-β
|
Alginate based adhesive and cross-linked hydrogel
|
4× 106
|
4× 106
|
Rat
|
• Higher osteogenic potential in repairing peri implantitis model
|
[50]
|
|
Hydorgel scaffold(PuraMatrix)
|
1 × 106
|
1 × 106
|
Rat
|
• Maxillary alveolar defects-higher osteogenic bone formation
|
[51]
|
|
FGF2
|
(PLA) 3D bioengineered scaffold
Enriched with GMSCs
|
2× 106 cells
|
2× 106 cells
|
Rat
|
• Calvarial defects enhanced regeneration into osteocytes and adipocytes
|
[52]
|
|
IGF-1
BMP-4
|
Axo guard Nerve Conduits
|
0.5× 106
|
0.5 × 106
|
Rat
|
• Facial nerve - Enhanced neuronal and glial differentiation
|
[53]
|
|
DFSCs
|
BMP-2
BMP-9
|
HA powder
|
2 × 105
|
2× 105
|
Mice (Subcutaneous)
|
• Fibrous tissue formation and cementum matrix
|
[54]
|
|
BMP-9
|
HA coated dental implant
|
5× 104
|
1× 107
|
Murine
|
• Osteogenic differentiation and periodontal ligament like tissues
|
[55]
|
|
BMP-2
|
HA/ Collagen gel
|
2× 106
|
2× 106
|
Mice
|
• Higher differentiation into cementum like tissues – Acellular cementum
|
[56]
|
|
IGF
|
Collagen nano HA/ phosphoserine biocomposite cryogel
|
1 × 106
|
1 × 106
|
Mice
|
• Enhanced osteogenic differentiation to bone like tissues
|
[57]
|
|
FGF-2
|
Treated dentin matrix (TDM)
|
5× 104
|
1× 107
|
Canine
|
• Enhanced osteogenic, cementogenic, periodontal ligament tissue formation in bony
defects
|
[58]
|
|
FGF-2
|
TDM
|
5× 104
|
1× 107
|
Mice
|
• Increased formation of periodontal tissues like cementum and alveolar bone
|
[59]
|
|
TGF- β1
|
Ceramic bovine bone
|
2× 106
|
2× 106
|
Mice
|
• Enhanced cementogenic, osteogenic and fibroblastic potential (forms cementum-PDL
complex)
|
[60]
|
|
BMP-2
|
Extra cellular matrix (ECM)
|
250µl
|
5 × 106
|
Rat
|
• Enhanced bone regeneration and higher osteogenic differentiation
|
[61]
|
|
OPSCs
|
TGF-β
BMP-2
|
Periosteal cell sheets
|
1× 106
|
2× 106
|
Human bone defects
|
• Increased bone formation by osteogenic differentiation
|
[62]
|
|
BMP-2
|
OPSC cell sheets
|
1× 106
|
2× 106
|
Human
|
• Sinus elevation procedures- enhanced bone formation
|
[63]
|
|
FGF-2
VEGF
|
Cell sheets OPSC
|
1× 106
|
2× 106
|
Mice
|
• Enhanced osteogenic and chondrogenic differentiation
|
[64]
|
|
BMP-2
|
HA powder
|
2 × 105
|
2× 105
|
Rat
|
• Higher osteogenic differentiation
|
[65]
|
|
BMP-9
|
HA bone graft
|
2× 106
|
2× 106
|
Mice
|
• Enhanced bone formation
|
[66]
|
Abbreviations: BMP, bone morphogenetic protein; DFSCs, dental follicle stem cells;
FGF, fibroblast growth factor; GMSCs, gingival mesenchymal stem cells; HA, hydroxy
apatite; IGF, insulin-like growth factor; OPSCs, oral periosteal stem cells; PDLSCs,
periodontal ligament stem cells; PDL, periodontal ligament; PLA, polylactic acid;
PRP, platelet-rich plasma; RGD, arginylglycylaspartic acid; RhFGF, recombinant human
fibroblast growth factor; rAdBMP-2, recombinant adenovirus (rAd) encodingBMP-2; TCP,
tricalcium phosphate; TGF, transforming growth factor; TNF, tumor necrosis factor;
VEGF, vascular endothelial growth factor.
The potential of PDLSCs in tissue regeneration as explained by Seo et al suggested
that PDLSCs could generate tissue similar to the cementum and periodontal ligament.[8] Coextensive research on PDLSC sheets exhibited the property of giving rise to structures
similar to the periodontal tissues in a tricalcium phosphate matrix. The amalgamation
of PDLSCs with chitosan-based scaffolds aided in bone regeneration in calvarial defect
models.[67] Human DFSC sheets transplant was able to achieve optimal periodontal regeneration,
including new periodontal ligament attachments, new alveolar bone development, and
even a periodontal ligament–cementum complex structure. Further, DFSC sheets were
more conducive to periodontal regeneration than PDLSC sheets, as observed in canine
model, which might be attributable to DFSC's greater ability to adapt to the chronic
inflammatory milieu of periodontitis.
Wang et al first demonstrated that transplanted GMSCs formed new bone in mandibular
wounds and calvarial defects of nude mice and rats, which suggested that GMSCs can
repair bone.[68] GMSCs exhibited a moderate osteogenic capability, similar to PDLSCs; however, GMSCs
were stronger at forming mineralized nodules and differentiated into osteogenic, chondrogenic,
and adipogenic lineages.[69] While some studies suggest that inflamed GMSCs have a reduced capability for osteogenic
and adipogenic differentiation than healthy subjects, epigenetic variables linked
to chronic periodontitis could impact the cell line's different orientations. The
“real mesenchymal stem cells”, the OPSCs have the characteristics to differentiate
into osteoblasts and chondroblasts.[25] Cacceralli et al also suggested OPSCs to be a valuable and precious alternative
compared to other mesenchymal stem cells from bone marrow for tissue engineering applications
in oral cavity. The extraoral applications of OPSCs need to be studied further; however,
the other periodontal stem cells have widely studied in regenerative medicine for
their varied differentiation capabilities.
The therapeutic potential of PDLSCs in medicine has been used for differentiation
of corneal stromal keratinocytes as both PDLSCs and corneal cells are derived from
the neural crest. In the personalized treatment of multiple sclerosis (MS), a comparison
of PDLSCs obtained from systemically healthy patients and MS patients showed identical
proliferative and differentiative potential of PDLSCs, thereby validating the use
of PDLSCs in such auto-immune conditions.[70] Lee and Park demonstrated the transdifferentiation of PDLSCs into pancreatic islet
cells thereby providing an alternative treatment strategy for diabetes.[71] Periodontal mesenchymal stem cells can also differentiate into cardiac muscles,
skeletal muscles, endothelial and neuronal cells suggesting their therapeutic application
in medical regenerative procedures.
DFSCs have a potential for neuronal differentiation as they differentiate into mature
neurons and oligodendrocytes but not astrocytes. The application of DFSCs in myasthenia
gravis was pioneered by Ulusoy et al, whereas the therapeutic effect of DFSCs in asthma
was researched by coculturing DFSCs with the blood mononucleocytes of asthmatic patients
in vitro.[72]
GMSCs, on the other hand, have been shown to demonstrate antiageing potentials and
this property can be harnessed to develop cell free treatment strategies for ageing-related
and vascular disorders.[73] GMSCs have also been known to differentiate into neuronal and glial cells and this
property has been harnessed in facial nerve regeneration and the management of spinal
cord injuries.[74] Ansari et al reported that GMSCs encapsulated in alginate underwent osteogenic differentiation
and also have chondrogenic potential without the need of additional growth factors.[75] It has been suggested that GMSCs and OPSCs exhibit similar degree of bone regeneration
in defects created in rabbit models suggesting that these stem cells maybe an useful
alternative in regenerative strategies.[76]
Stem Cell Derivatives in Regeneration
Stem Cell Derivatives in Regeneration
E1. 3D Bioprinted Scaffolds—Self-Scaffolded Models
In scaffold-free tissue engineering, cells produce and arrange their own endogenous
ECM to create a 3D structure. Scaffold-free tissue engineering, in contrast to conventional
tissue engineering techniques, forgoes the use of an external scaffold material to
create a 3D tissue. In 2007, a 3D bioengineered tooth—“organoid”—was made by combining
dental epithelium and mesenchyme to form a complete tooth germ and it was proposed
that combining in with biomaterials such as collagen can enhance the possibility of
forming a bioengineered tooth in animal models. 3D spheroids can be used in a variety
of culture methods to optimize the property and function of MSCs as they allow close
cell–cell and cell–matrix interactions that closely resemble the microenvironment.[77] The various methods of culturing in 3D are using patterned microwells, floating
culture for neurosphere formation, chitosan, ultra-low culture dishes, and poly-L-ornithine.
These can be enhanced by adding growth factors like fibroblast like growth factors,
lovastatins, spheroids, and mesenspheres.
PDLSCs have been successfully cultured to form 3D structures mimicking cementum and
periodontal ligament using this technique. After in vitro cultivation, the periodontal tissue organization was evident, and it was preserved
in vivo as well as after subcutaneous implantation in mice. These results show that PDLCs
can self-assemble into an ordered cementum-PDL-like complex through scaffold-free
tissue engineering.[78]
Self-Assembly of a 3D spheroid culture of GMSCs can enhance the differentiation and
neural stem cell properties as shown by Hsu et al, where the GMSCs were found to spontaneously
aggregate into 3D spheroids with enhanced stemness and increases trilineage differentiation.[79] The GMSCs cultured in patterned microwells aggregate into 3D spheroids and have
higher osteogenic potential that is augmented by addition of growth factors, while
floating culture technique allows for aggregation into neurospheres and elevated neural
crest markers and neuronal differentiation. The possibility of reprogramming GMSCs
into neural crest like cells to differentiate into nerve cells is also possible by
3D culturing.[53]
DFSCs have been seeded on 3D porous scaffolds laden with collagen-nanohydroxyapatite/phosphoserine
biocompatible cryogel with osteogenic factors in the culture medium and the resultant
3D spheroids showed dynamic growth and osteogenic differentiation when implanted in
mice models.[57]
E2. Use of Dental Stem Cell-Derived Exosomes in Regenerative Medicine Inverting the
Disease Paradigm:
Extracellular vesicles (EVs) including ectosomes and exosomes are essential for intracellular
communication as they can carry bioactive molecules such as lipids, nucleic acids,
proteins, and metabolites. EVs are released as membrane bound agents from all types
of cells and even found to be released from periodontal stem cells and used for treating
diseases. EVs can be categorized as apoptotic vesicles, microvesicles, and exosomes.
EVs are successful in treating a variety of disorders by encapsulating and conveying
essential bioactive components (e.g., proteins and nucleic acids) to affect the phenotype
of target cells.
PDLSCs produce exosomes that harbor the potential of repair and regeneration, which
can induce angiogenesis, alleviate neurological diseases, and reduce the inflammatory
microenvironment.[80] In periodontitis, they can be used to induce osteogenesis and enhance bone regeneration
as seen by Pizzicannella et al, in lesions of the rat skull, where addition of PDLSC-derived
exosomes to a 3D collagen membrane and polyethyleneimine scaffold, showed bone regeneration.[81] Further, it has been observed that human PDLSC-derived exosomes promote osteogenesis
by the expression of their exosomal miRNAs in vitro.[82] Similarly, GMSC-EVs in periodontitis models in rats show periodontal regeneration
by delivery of mIR-120b to inhibit osteoclastogenic activity of PDL cells by targeting
the Wnt5a-mediated RANKL pathway.[48]
DFSCs-sEVs were found to greatly increase PDLSC migration, proliferation, and osteogenic
differentiation and regeneration of periodontal tissue by stimulation of the p38-MAPK
signaling pathway. Small EVs from DFSCs provide biochemical cues for periodontal tissue
regeneration.[83] There is potential to use OPSC-derived EVs in future for bone regeneration as they
are more committed to an osteogenic lineage. However, to achieve an optimal periodontal
regeneration of the intricate structures in the periodontium, more research is still
required to identify ideal and standardized sources of EVs, their effective concentration,
frequency of treatment, and suitable scaffolds or delivery routes. Better insight
into the therapeutic potential of periodontal stem cells derived-EVs would provide
more reasonable options for the future treatment of periodontal diseases.
Clinical Application of Stem Cells in Human Periodontal Regeneration
Clinical Application of Stem Cells in Human Periodontal Regeneration
The use of stem cells cultured in scaffolds has shown promising results in vitro and animal model studies. The subsequent application of these stem cells needs to
be explored whether it can bring about regeneration as previous clinical trials have
been performed in this regard to achieve periodontal regeneration using autologous
transplanted stem cells from the periodontal complex. PDLSC sheet transplants in humans
have shown no adverse effects and have generated a reduction in probing depth; however,
this was not significant. Further, a clinical trial with autologous PDLSC transplants
in periodontitis patients had shown improvement in periodontal parameters as seen
by reduction in pocket depths and bone regeneration. Transplants of PDLSC sheets mixed
with granules of β-tricalcium phosphate in bone defects have also shown no adverse
effects till 6 months.[84] PDLSCs implanted in animal models show superior periodontal regeneration in the
form of greater cementum, bone, and PDL formation. A recently concluded clinical trial
on animal model showed that when the PDLSC was combined with collagen membrane on
fenestration defects, it showed a greater cementum formation but no difference in
bone formation when applied without the membrane.[85] Similarly, in furcation defects when PDLSCs were combined with hyaluronic acid sheets,
it resulted in greater cementum, bone, and PDL formation than controls.[86] Further, PDLSC sheets combined with β-tricalcium phosphate in infrabony defects
resulted in nearly complete regeneration of periodontal tissues.[87] Over all, it can be deciphered that the application of PDLSCs in animal models is
predictable for cementum formation; however, the results are conflicting when it comes
to bone regeneration.
Use of GMSC in future clinical trial seems to be a promising approach as systemically
administered GMSCs have the ability to home to the injury site and differentiate into
osteoblasts, cementoblasts, and periodontal ligament fibroblasts as tested in animal
models. In class III furcation defect animal models, GMSC sheets significantly have
enhanced the regeneration of periodontal tissues.[51] Combination of the GMSC with HA gel has also showed a significant regeneration in
porcine model by exhibiting formation of newly formed bone and PDL fibres.[88] GMSC human clinical trials have also been employed in treating periodontal defects
while embedding these cells in collagen scaffolds mixed with β-tricalcium phosphate,
thereby reducing probing depth, attachment gains, and alveolar bone gain as seen in
6 months follow-up.[49]
DFSC sheets implanted in animal models have shown to regenerate whole periodontal
tissue as observed by formation of complex–periodontal ligament like structure within
a month. Implanting into periodontal irregularities in vivo, DFSCs show a better capacity for cementum and periodontal attachment healing than
PDLSCs due to higher involvement of extracellular matrix.[29]
[89] The DFSCs work by providing an ideal microenvironment for the growth of PDLSCs and
act as a scaffold.
Human OPSCs, on the other hand, have shown promising results in vivo when transplanted in human intrabony periodontal defects and in sinus elevation procedures
for forming bone.[63] There are enhanced osteogenic properties of OPSCs when transplanted with growth
factors and enriched with collagen scaffolds.[62]
Conclusion
Multiple stem cell populations including PDLSCs, GMSCs, DFSCs, and OPSCs coexist in
close proximity and still stay in function in spite of continual remodeling and inflammation
in the periodontal complex. Periodontal stem cells have a strong interaction with
the inflammatory milieu, as well as the ability to modulate the immune system, making
them lucrative candidates for cell treatment in periodontitis and inflammatory disorders.
The periodontal stem cells are one-of-a-kind, with a variety of morphologies and multipotency,
both in vitro and in vivo. Each stem cell population's differentiation capacity is diverse, and it can repair
bone, neurons, and tendons in addition to mesenchymal tissues in the oral cavity.
The PDLSC are thought to be harboring diverse regenerative potential within the oral
cavity; however, they are more differentiated cells. The DFSCs in contrast exhibit
greater propensity for extraoral tissue differentiation as shown by higher expression
of embryonic markers like Oct4 and Nanog. GMSCs are touted to be the stem cells with
greater accessibility and higher differentiation capacity as seen by various clinical
models and 3D bioprinting studies. The use of GMSCs for nerve regeneration is promising
in future. The OPSCs, on the other hand, need to be studied further to understand
their behavior both in vitro and in clinical human models. Although it would not be appropriate to state the superiority
of one stem cell type over another, it is plausible that therapeutic application of
these stem cells to regenerate hard and soft tissues and alleviate degenerative diseases
may become a reality in the future. To this regard, additional prospective and long-term
trials are needed to determine the true characteristics of each population and how
they might be used for exogenous MSC grafting, 3D bioprinting, specific exosomal derived
vesicles, and cell homing in periodontal tissue engineering.
