Chondrogenic Potential of Dental-Derived Mesenchymal Stromal Cells

The field of tissue engineering has revolutionized the world in organ and tissue regeneration. With the robust research among regenerative medicine experts and researchers, the plausibility of regenerating cartilage has come into the limelight. For cartilage tissue engineering, orthopedic surgeons and orthobiologists use the mesenchymal stromal cells (MSCs) of various origins along with the cytokines, growth factors, and scaffolds. The least utilized MSCs are of dental origin, which are the richest sources of stromal and progenitor cells. There is a paradigm shift towards the utilization of dental source MSCs in chondrogenesis and cartilage regeneration. Dental-derived MSCs possess similar phenotypes and genotypes like other sources of MSCs along with specific markers such as dentin matrix acidic phosphoprotein (DMP) -1, dentin sialophosphoprotein (DSPP), alkaline phosphatase (ALP), osteopontin (OPN), bone sialoprotein (BSP), and STRO-1. Concerning chondrogenicity, there is literature with marginal use of dental-derived MSCs. Various studies provide evidence for in-vitro and in-vivo chondrogenesis by dental-derived MSCs. With such evidence, clinical trials must be taken up to support or refute the evidence for regenerating cartilage tissues by dental-derived MSCs. This article highlights the significance of dental-derived MSCs for cartilage tissue regeneration.


Impact Statement
Dental tissues are the richest sources of mesenchymal stromal cells, which are not much used in clinical practice. Researchers and regenerative medicine clinicians are more interested in exploring the regenerative potential of dental-derived MSCs, which can be translated from bench to bedside clinical applications. The evidence stated that dentalderived MSCs can be used for cartilage regeneration. To explore the chondrogenicity of dental-derived MSCs, clinical trials have to be taken up to support or refute the evidence for cartilage regeneration.

Introduction
Cartilage is an avascular and aneural structure with poorly cellularized connective tissue [1,2]. Cartilage tissue facilitates mechanical load transmission with a low frictional coefficient resulting in cartilage injury that has an inherent limited healing potential [3]. The recent idea of "Orthobiologics" led to the exploration of stem cells and regenerative medicine in treating musculoskeletal disorders [4]. Orthobiologics provide administration of osteoinductive and osteoconductive micromolecules to enhance regeneration of degenerated tissues, tendons, bones, and cartilages [4].

Dental Pulp MSCs (DP-MSCs)
Embryologically, dental pulp arises from ectomesenchyme where the periphery of the neural tube gives rise to ectodermal cells and migrates to the oromaxillary region, and differentiates into mesenchymal phenotype. The dental pulp remains an unmineralized connective tissue that comprises a heterogeneous population including stromal cells, progenitors, odontoblasts, ameloblasts, fibroblasts, granulocytes, macrophages, vascular fragments, capillaries, and extracellular matrices. Dental pulp tissue is the richest source of MSCs, which are extracted from the teeth recovered during routine dental procedures [32]. DP-MSCs are isolated from impacted third molars and supernumerary teeth, which possess extreme clonigenicty, plasticity, regenerative and reparative potential [43,44]. These cells reside within the dental crown called "niche" or "pulp chamber" [45]. The usage of DP-MSCs was documented with diabetes mellitus [46], neurological disorders [47], maxillofacial and dental disorders [48], disorders of bone and cartilage [49], hepatic disorders [50], and immunological diseases [51]. The extraction of MSCs from dental structures remains non-invasive and can be cryopreserved for future usage.

Dental Pulp MSCs (DP-MSCs)
Embryologically, dental pulp arises from ectomesenchyme where the periphery of the neural tube gives rise to ectodermal cells and migrates to the oromaxillary region, and differentiates into mesenchymal phenotype. The dental pulp remains an unmineralized connective tissue that comprises a heterogeneous population including stromal cells, progenitors, odontoblasts, ameloblasts, fibroblasts, granulocytes, macrophages, vascular fragments, capillaries, and extracellular matrices. Dental pulp tissue is the richest source of MSCs, which are extracted from the teeth recovered during routine dental procedures [32]. DP-MSCs are isolated from impacted third molars and supernumerary teeth, which possess extreme clonigenicty, plasticity, regenerative and reparative potential [43,44]. These cells reside within the dental crown called "niche" or "pulp chamber" [45]. The usage of DP-MSCs was documented with diabetes mellitus [46], neurological disorders [47], maxillofacial and dental disorders [48], disorders of bone and cartilage [49], hepatic disorders [50], and immunological diseases [51]. The extraction of MSCs from dental structures remains non-invasive and can be cryopreserved for future usage.
During dentinogenesis, the interplay between dental pulp epithelial cells leads to the differentiation of odontoblasts and ameloblasts, which deposit dentin and enamel, respectively. The inner lining of dental pulp contains progenitor cells with high regenerative potential throughout the lifetime [52]. DP-MSCs possess a similar regenerative potential to BM-MSCs, but DP-MSCs act as a non-invasive source for extraction of MSCs for therapeutic usage in various diseases [51,53].
Upon addition of appropriate growth factors, micromolecules, transcriptional factors, and ECM proteins, DP-MSCs differentiate into multilineages, namely, adipogenesis, chondrogenesis, osteogenesis, neurogenesis, and dentinogenesis both in in-vitro and invivo studies [54,55]. During any insult to dental structures, due to the higher proliferative capacity, the quiescent DP-MSCs activate and immediately differentiate into ameloblasts, odontoblasts, osteoblasts, adipocytes, and chondrocytes to produce dentin, bone, fat, and cartilage for their repair process, respectively [27,56]. Yu et al. demonstrated the reduced odontoblastogenesis after the ninth passage and differentiation of osteoblast precursors [57]. Since DP-MSCs are derived from the neural crest, they possess neural crest stem cell During dentinogenesis, the interplay between dental pulp epithelial cells leads to the differentiation of odontoblasts and ameloblasts, which deposit dentin and enamel, respectively. The inner lining of dental pulp contains progenitor cells with high regenerative potential throughout the lifetime [52]. DP-MSCs possess a similar regenerative potential to BM-MSCs, but DP-MSCs act as a non-invasive source for extraction of MSCs for therapeutic usage in various diseases [51,53].
Upon addition of appropriate growth factors, micromolecules, transcriptional factors, and ECM proteins, DP-MSCs differentiate into multilineages, namely, adipogenesis, chondrogenesis, osteogenesis, neurogenesis, and dentinogenesis both in in-vitro and in-vivo studies [54,55]. During any insult to dental structures, due to the higher proliferative capacity, the quiescent DP-MSCs activate and immediately differentiate into ameloblasts, odontoblasts, osteoblasts, adipocytes, and chondrocytes to produce dentin, bone, fat, and cartilage for their repair process, respectively [27,56]. Yu et al. demonstrated the reduced odontoblastogenesis after the ninth passage and differentiation of osteoblast precursors [57]. Since DP-MSCs are derived from the neural crest, they possess neural crest stem cell markers and hence they differentiate into neuron-like cells [58][59][60]. Superior chondrogenesis was exhibited by DP-MSCs both in-vitro and in-vivo when compared with BM-MSCs [61][62][63][64].

Stem Cells from Human Exfoliated Deciduous Teeth (SHEDs)
SHEDs are stromal cells extracted from exfoliated deciduous teeth, which behave in the line of embryonic stem cells [65]. They possess MSC markers, epidermal stem cell markers, and tumor recognition markers, but do not exhibit hematopoietic stem cell markers [66]. SHEDs demonstrate higher proliferation and differentiation ability and form cell cluster formation like a sphere when compared with DP-MSCs [67,68]. They enhance osteogenic differentiation due to the presence of higher levels of osteocalcin, and alkaline phosphatase. SHEDs when mixed with platelet-rich plasma, the osteogenic response accelerated in mandibular defects in canines [69] SHEDs play a major role in treating diabetes mellitus [70,71] and neurological disorders such as spinal cord injury [72], focal cerebral ischemia [73,74], and Alzheimer's disease [75,76]. Muhammad et al. observed the upregulation of MMP-13, NF-kB, aggrecan, ECM proteins, and COL-2 in the regenerated chondrocytes with SHEDs, which underscored the basis for OA knee management [77].

Dental Follicle Precursor Cells (DFPCs)
The dental follicle (DF) is a loose connective tissue derived from ectomesenchyme that surrounds the tooth germ [85]. Compared to other stem cells of dental origin, DFPCs exhibit robust immunomodulation, proliferative and diverse differentiative potential [37,86]. Evidence proved that DFPCs differentiate into the alveolar bone, PDL, cementum, adipocyte, osteoblast, cementoblast/chondrocyte, neuron-like cell cardiomyocyte, and dentin-like tissues [45,85]. Researchers have extensively utilized DFPCs for regenerating bone defects, cartilage engineering, tooth root, and periodontal tissue regeneration, and neural tissue regeneration. DFPCs express NOTCH-1 transmembrane protein, which decides the fate of the cell to be differentiated and HLA-ABC, which is acquired during culture [87]. When co-cultured with lymphocytes, DFPCs exhibit decreased INF-γ and IL-4 and increased IL-6, and -10 and TGF-β. Transplanted DFPCs regenerate bone in the calvarial defect when loaded with polycaprolactone (PCL) scaffold along with hyaluronate and β-tricalcium phosphate [88]. Periodontal osteogenesis of DFPCs is enhanced with nanosilicates and fluoride additives [89].
Biodentine, a bioactive dentine substitute, is capable of inducing DP-MSCs differentiation of odontoblasts. Luo et al. demonstrated odontoblast differentiation of DP-MSCs by increased expression of ALP, OCN, DSPP, DMP1, and BSP [126]. Optimal mechanical compression increased the expression of DSPP, BMP-7, and Wnt10a genes for odontoblast differentiation by DP-MSCs [127]. BBX gene expression induces the differentiation of odontoblasts by DP-MSCs [128]. DNA methylation and PTEN expression were increased in DP-MSCs, which are responsible for lineage differentiation and reduced oncogenesis when compared with BM-MSCs [129]. The differential characteristics of individual MSCs of dental origin are given in Table 1.

Harvesting and Delivery Methods of D-MSCs
Various regenerative medicine experts followed different methods to extract and harvest stromal cells from dental pulp.
Raoof et al. used three different methods to isolate DP-MSCs, namely, (a) digestion of dental pulp tissue with collagenase and placement of isolated trypsinized cells in petri dishes, (b) explantation of undigested dental pulp pieces to culture plates, and (c) explantation of trypsinized dental pulp tissues to petri dishes for outgrowth [130]. These tissues are plated to MEM medium supplemented with 20% fetal bovine serum at 37 • C with a 5% CO 2 incubator. A total of 60% cellular confluency was achieved within days of culture and checked for pluripotency markers by RT/PCR analysis [130].
Naz et al. expanded DP-MSCs and SHEDs via the explant culture method after extirpation of dental pulp tissues from deciduous teeth [131]. As a result of culture expansion, MSCs exhibit fibroblast-like cells with long cytoplasmic processes. DP-MSCs and SHEDs characterization was done and cryopreserved for future use as shown in Figure 2.

Chondrogenicity of Dental-Derived MSCs
Though dental-derived stem cells possess higher osteogenic potential, they are being explored very marginally for chondrogenicity. DP-MSCs act as a promising source for cartilage tissue engineering and regeneration. DP-MSCs possess a strong potential to differentiate into hyaline and fibrocartilage [138]. Sophia et al. demonstrated that hyaline cartilage contains few chondrocytes in their extracellular matrix rich in GAGs and type 2 collagen [2], whereas Allen et al. stated that fibrocartilage contains fibroblastic cells with small amounts of GAGs and type 1 collagen [139].
Longoni et al. expanded DP-MSCs from seven molar teeth and induced chondrogenesis in a 3D pellet culture system [63]. These culture-expanded DP-MSCs display GAGs, No significant change was observed in the differentiating capabilities and immunophenotypic properties of cryopreserved and non-cryopreserved DP-MSCs isolated from dental pulp, but there were significant differences in the morphology and proliferative potential of cryopreserved DP-MSCs than non-cryopreserved DP-MSCs [132].
The survival rates of DP-MSCs in DMSO free medium by static magnetic cryopreservation increased by 2 to 2.5 fold when the cells were exposed to 0.4 or 0.8-T static magnetic fields [133]. Gioventù et al. demonstrated that cryopreserved teeth by laser piercing have maintained cellular viability [134].
To isolate a pure population of DP-MSCs, the identification of cell surface markers like LNGFR and THY-1 are significant [87,88]. The assessment of the number of colonies showed that LNGFR Low+ THY-1 High+ cells in the dental pulp have a significantly higher colony-forming potential than LNGFR + THY-1 + cells in the bone marrow [135][136][137].

Chondrogenicity of Dental-Derived MSCs
Though dental-derived stem cells possess higher osteogenic potential, they are being explored very marginally for chondrogenicity. DP-MSCs act as a promising source for cartilage tissue engineering and regeneration. DP-MSCs possess a strong potential to differentiate into hyaline and fibrocartilage [138]. Sophia et al. demonstrated that hyaline cartilage contains few chondrocytes in their extracellular matrix rich in GAGs and type 2 collagen [2], whereas Allen et al. stated that fibrocartilage contains fibroblastic cells with small amounts of GAGs and type 1 collagen [139].
Longoni et al. expanded DP-MSCs from seven molar teeth and induced chondrogenesis in a 3D pellet culture system [63]. These culture-expanded DP-MSCs display GAGs, aggrecan, and type 2 collagen after three weeks. The assessment of culture-expanded cells revealed fibroblastic cells with long cytoplasmic processes with a predominance of type 1 collagen to state the formation of fibrocartilage. They concluded that DP-MSCs regenerate fibrocartilage in joints, rather than hyaline cartilage. DP-MSCs provide a rapid ex-vivo expansion and chondrogenic differentiation potential and hence provide a favorable cell type for treating cartilage disorders. Khajeh et al. demonstrated a significant role of hypoxia mimicking agent and cobalt chloride on chondrogenesis with DP-MSCs [140]. Cobalt chloride exposure to DP-MSCs increases the cellular pellet mass in culture, cellular morphology and integrity, ECM deposition, and cellular organizations. There were elevated levels of GAGs and type 2 collagen expression [141]. Cobalt chloride enhances the stemness of DP-MSCs where flow cytometry reveals the increased expression of STRO-1 + cells [142].
Hsu et al. cultured human gingival fibroblast (HGF) cells on chitosan membrane to observe in-vitro chondrogenesis. On culture, increased spheroid formation resulted, which indicates the stemness of HGF. Spheroid formation by HGF was supported by Rho/Rho-associated kinases and the connexion 43 pathway. Hence, they concluded that culturing HGF on chitosan membrane induces spheroid formation, which further induces chondrogenesis by the ROCK pathway [154].
Ferre et al. demonstrated osteogenesis and chondrogenesis by human gingival stem cells in vitro in 3D floating micromass pellet cultures in a specified medium. Osteogenic cells exhibited the increased expression of Runx-2, ALP, presence of osteoid-like mass, and osterix expression whereas chondrogenic cells exhibited increased expression of type 2 collagen, GAGs, and SOX-9 transcription gene [155].
Ferre et al. demonstrated the differentiation of type 10 collagen secreting hypertrophic chondrocytes and fibroblast-like synoviocytes by human gingival stem cells. Under hypoxia and hypoxic mimicking environment, G-MSCs express high levels of VEGF-α, which promote vasculogenesis for regenerative therapies [155].
NOTCH ligand signaling plays a major role in the chondrogenic differentiation of cells [162]. NOTCH-2 modulates the activities of NOTCH-3 and -1, hence influence the growth and development, and homeostasis of chondrocytes and articular cartilage [163][164][165]. NOTCH-3 represses the proliferation of terminally differentiated chondrocytes within the cartilaginous tissues [166]. In a 3D-cultured chondrogenesis, there is a downregulation of NOTCH ligands and receptors [167,168]. While MSC undergoes terminal chondrocyte differentiation, NOTCH-3 receptors were upregulated and were highly expressed [162,169]. Various studies on the in vitro and in vivo chondrogenic potential of MSCs of dental origin are given in Tables 2 and 3, respectively.

Engineered Chondrogenesis by D-MSCs
Due to the intrinsic limited potential of cartilage tissue to heal, cartilage tissue engineering gave a robust breakthrough in the field of regenerative and translational medicine. The field of tissue engineering provides the biological substitute of limited available tissues to restore and maintain the naïve homeostasis and to improve the biomechanical strength and function of the tissues. The integral components of tissue engineering are stem/stromal cells, scaffolds, and bio-micromolecules [178][179][180]. The successfully engineered tissue relies on tissue ergonomics that include harvest and expansion of appropriate cells, the addition of optimum levels of growth factors and cytokines, and provision of 3D scaffold and extracellular matrices until the healing gets completed.
Bioactive molecules: Kartogenin (KGN) enhances MSC chondrogenesis by upregulation of CBFβ-RUNX1 transcription [192]. Evidence states that kartogenin promotes tendon and meniscus regeneration [193,194]. KGN inhibits pain stimulus, attenuates chondral degeneration and inflammation, supplements the biomechanical strength of repaired bones and tendons in-vivo animals, and robust chondrogenic differentiation of DP-MSCs [195]. Simvastatin, a hypolipidaemic molecule, enhances positive effects on synovium and cartilage tissues, thereby reducing inflammation, degeneration and halts arthritis progression [196]. A higher concentration of statins decreased the production of nitric oxide in chondrocytes and cartilage explants [197].
Bioactive scaffolds: Scaffolds, an integral part of tissue engineering, are of natural [collagen, fibrin, hyaluronan, alginate, agarose, and chitosan] and synthetic [polylactic acid (PLA), polyglycolic acid (PGA), and copolymer polylactic-co-glycolic acid (PLGA)] polymers. The ideal scaffold should be biocompatible, optimum porosity, biodegradable, elastic natured, mechanical strength, easy fabrication, non-toxic, long-term effectiveness, and support cell attachment and proliferation [198,199]. Platelet-rich plasma seeded with agarose enhance cartilage and tendon regeneration [200]. MSCs cocultured with collagen or agarose enhance chondrocyte differentiation along with increased production of ECM and GAGs [201]. Alginate, an injectable scaffold, is used in regenerating focal chondral defects and in autologous chondrocyte implantation [202,203]. DP-MSCs accelerate chondrogenesis when cultured with growth factors and alginate beads [204]. The synergistic effects of chitosan and hyaluronic acid hydrogel enhance the healing of cartilage defects in rabbits [205]. Synthetic polymer scaffolds are used in the repair of osteochondral defects in rabbits [206] and meniscal lesions in dogs [207].
Chondrocytes release factor XIIIA, whose upregulation leads to hypertrophic chondrocyte differentiation in OA chondrocytes. In the murine OA cartilage model, there is an interplay between FXIIIA and α1 subunit of α1β1 integrin and tissue glutaminase 2 (TG2) mobilization, which leads to remodeling of the cartilage matrix. In absence of TG2, FXIIIA fails to undergo chondrocyte hypertrophy [208]. The conjunction of plasma membranebound TG2 and FXIIIA with a raised expression of FXIIIA upregulates the p38 MAP kinase signaling pathway in chondrocytes of OA cartilage in situ [209]. In turn, p38 signaling sig-nificantly increases SOX-9, which inhibits both in vitro and in vivo chondrocyte maturation to hypertrophy by DP-MSC-induced chondrogenesis [210].
Expanded chondrocytes from MSCs co-cultured with PGA-fibrin scaffolds revealed considerable expression of type 1 and 2 collagen and further resulted in the formation of hyaline cartilage. Upon optimal addition of platelet-rich plasma to cartilage tissue, the formation of hyaline cartilage was robust with higher expression of collagen type 2 [214].
Loading of human dedifferentiated chondrocytes into collagen sponge, in the presence of hypoxia and BMP-2, resulted in chondrogenesis, which is transfected onto siRNAs targeting collagen type 1 and HtrA1 serine protease, which are raised in OA cartilage. Such a mechanism led to the improvement of chondrocyte phenotype differentiation. Transplantation of in vitro cultured cells into nude mouse model in vivo resulted in neochondrogenesis with hyaline matrix formation [215]. The in vitro and in vivo studies on cartilage tissue engineering using stem cells of dental origin are tabulated in Tables 4 and 5. Table 4. In vitro studies on cartilage tissue engineering using stem cells of dental origin.

Methods Main Results
Fernandes et al. [170]. (2018) Transmission electron microscopy MSCs derived from dental pulp had intact membranes and scattered microvilli-like structures on their surfaces, showing good attachment to the biomaterial scaffold.
Westin et al. [171]. (2017) Indirect toxicity of scaffolds to DP-MSCs Biomaterial produced is not capable of affecting the growth of DP-MSCs

Histology
The formation of collagen fibers by DP-MSCs in culture can be observed, confirming that DP-MSCs were able to effectively differentiate into chondrocytes within the cultured matrix Mata et al. [172]. (2017

Methods Main Results
Fernandes et al. [170]. (2018) Transmission electron microscopy MSCs derived from dental pulp had intact membranes and scattered microvilli-like structures on their surfaces, showing good attachment to the biomaterial scaffold.
In vitro analysis Cultured chondrocytes enhance the expression of SOX-9, ACAN, and COL2 and downregulate the expression of the early hypertrophic marker COL10 Markers in DP-MSCs in response to FGF9 or CM.
Intense FGFR3 staining and strong phosphorylation of ERK1/2 staining in monolayer DP-MSCs cultured in CM with FGF9.
Yu et al. [125]. (2010) In vitro analysis DP-MSCs differentiation markers showed significant upregulation in the 9th passage. Table 5. In vivo studies on cartilage tissue engineering using stem cells of dental origin.

Methods Main Results
Fernandes et al. [170]. (2019) Macroscopic and histological evaluation Scaffolds plus DP-MSCs showed coverage of the defect and new tissue growth over the cartilage. Scaffold alone showed a regular defect border and shallow tissue coverage over the defect.
Macroscopic evaluation The loss of cartilage was diminished in animals implanted with alginate containing either chondrocytes or hDP-MSCs.

Immunochemistry
Higher expression of type II collagen with no significant expression of type I collagen in groups with chondrocytes and hDP-MSCs.

Methods Main Results
Chen et al. [174]. (2014) Histological evaluation SHED produces rich ECM on day 7 with β-tricalcium phosphate Immunohistochemistry Cartilage-like tissue stained positive for type II collagen Rizk et al. [175]. (2013) Western blot analysis Expression of collagen II, Sox9, and aggrecan in constructs seeded with TGF-β3-transduced cells RT-PCR TGF-β3-transduced DP-MSCs exhibited an increased expression of collagen II Dai et al. [177]. (2012) Histological evaluation of cellular morphology and GAG quantification Co-cultured DP-MSCs with cultured chondrocyte transplants were larger and had increased cartilaginous matrix deposition and higher GAG quantification Yu et al. [125]. (2010) Histology evaluation of STRO1+DP-MSCs in transplanted P1 and P9 pellets Eat DP-MSC-P1 cell pellets developed into dentin, bone, and cartilage structures, while DP-MSC-P9 cells could only generate bone tissues.

Future Perspectives
The U.S. Army has invested over $250 million towards stem cell research to aid in the management of their injured soldiers in the field through a project called Armed Forces Institute for Regenerative Medicine. In recognition of the regenerative potential of dental stem cells, a trend towards establishing tooth banks is on the rise in developed and developing countries [216][217][218][219]. More research is directed towards their application in cardiac ailments [220], regenerating brain tissue [221], muscular dystrophy [222], and bone regeneration [223]. With the pursuit towards their regenerative potential, future clinical studies are needed to validate the results of various animal model and in vitro studies to prove the concept of chondrogenic differentiation of the implanted MSCs of dental origin to be of use in various common clinical conditions such as osteoarthritis that leads to cartilage degeneration.

Conclusions
MSC-based therapies are the upcoming biological modality of management of various ailments in the current decade. Having discussed the potential of the various dentalderived stem cells towards various regenerative domains including dental pulp stem cells towards chondrogenesis, their potential for future utility in mitigating various pathomechanisms and restoring the normal homeostasis needs further exploration. Future clinical studies are needed to evaluate their effectiveness in varied clinical scenarios to enable practical utility. With the evolution of the understanding of these cell-based regenerative therapies, dental tissue-derived cells would prove to be a more promising tool in the management of various diseases and help in the further advancement of medicine in the future.

Conflicts of Interest:
The authors declared no potential conflicts of interest concerning the research, authorship, and/or publication of this article.