A colony-forming assay was performed to evaluate the proliferation rates of rat FCSCs in vitro, showing that rat FCSCs formed sixfold more colonies than donor-matched cartilage cells (Embree et al., 2010, 2016). Canonical Wnt signaling was closely tied to the proliferative activity of rat FCSCs. By adding sclerostin (SOST), an inhibitor of Wnt signaling, the proliferation rates of FCSCs were significantly reduced (Embree et al., 2016). In H. sapiens, FCSCs show self-renewal ability and are able to maintain their spindle-shaped morphometry, proliferation, apoptosis, and senescence abilities after expansion. They have comparable propagation ability, apoptosis rates, and colony-forming efficiency to orofacial bone marrow-derived mesenchymal stem cells (OMSCs) (Bi et al., 2020).

Fibrocartilage stem cells possess many in vitro features of MSCs, including clonogenicity and multipotential differentiation capacity. Under stimulation, FCSCs can differentiate into osteogenic, chondrogenic, and adipogenic cells. When rat FCSCs were cultured in chemically defined media, over 87% of individual colonies exhibited heterogeneous differentiation potential (22.5% trilineage, 64.5% bilineage) (Embree et al., 2016). Human FCSCs, comparing to human OMSCs, had a comparable adipogenic potential but a reduced osteogenesis potential during multi-lineage differentiation in vitro. Increasing evidence has revealed that multiple signaling pathways control FCSC differentiation in vitro, such as the canonical Wnt and Notch signaling pathways. After transfection of β-catenin, FCSCs exhibited decreased expression of cartilage-related transcription factors, such as sox5, sox6, and sox9 (Embree et al., 2016). Another study found that SOX9 was more highly expressed in human FCSCs than in cartilage cells (Embree et al., 2010, 2016) and other mesenchymal stem cells (Bi et al., 2020; Jiang et al., 2020). When SOX9 expression was interfered with, FCSCs were unable to form well-organized cartilaginous tissue under chondrogenic induction (Bi et al., 2020). Furthermore, Ruscitto et al. (2020) revealed that Notch signaling plays a key role in promoting FCSC differentiation into chondrocytes and osteoblasts but not adipogenic cells in vitro.

Fibrocartilage stem cells also present multipotential capacity in vivo. FCSCs traced by the skeletal stem/progenitor cell marker α-SMA in adult mice showed that the α-SMA⁺ cells in the SZ gradually infiltrated into the mature chondrocyte layer and expressed the chondrocyte marker Col II. This result indicated that FCSCs have the potential to differentiate toward chondrocytes to maintain the homeostasis of condylar cartilage under physiological conditions (Embree et al., 2016). Wnt signaling is also involved in the chondrogenesis of FCSCs. The downstream Wnt mediator β-catenin is expressed in mature chondrocytes but not in the SZ, implicating that Wnt activity is restrained in the SZ. Notably, SOST suppressed FCSC proliferation as previously noted, yet SOST knockout mice showed depletion of the FCSC pool (Embree et al., 2016). It has been speculated that in SOST knockout mice, Wnt signaling was enhanced significantly in the SZ and thus induced the differentiation of FCSCs toward chondrocytes, emphasizing the function of Wnt signaling in directing FCSC fate.

Moreover, recent research has confirmed the strong osteogenic and chondrogenic capability of exogenic FCSCs. Subcutaneously transplanted rat FCSCs with collagen sponges could form cartilaginous-like tissue, which then gradually transformed into transitional tissue (bone, cartilage, and osteoclast-mediated tissue) and resulted in well-organized trabecular bone-like tissue (Embree et al., 2016). Collectively, these results indicate that FCSCs are able to differentiate into multiple cell lineages and spontaneously recapitulate endochondral ossification when transplanted in vivo (Yang et al., 2014). Human FCSCs have different fates than rat FCSCs in xenograft models. Human FCSCs need chondrogenic induction before transplantation; otherwise, they are not able to undergo a similar process to rat FCSCs (Bi et al., 2020).

The regulation of stem cell populations is tightly controlled by the local microenvironment according to the requirements of the host tissue (Fuchs and Segre, 2000; Bianco and Robey, 2001). When injected into mandibular condylar cartilage defects, FCSCs spontaneously formed cartilage, and no bone-forming process was observed during follow-up (Bi et al., 2020). FCSCs undergo a chondrogenic differentiation fate in the microenvironment of the lower joint compartment. However, ectopic xenografts of rat FCSCs begin to form bone-like tissue after 4 weeks of observation, indicating that FCSCs have the tendency to undergo hypertrophy to form bone in a subcutaneous environment (Embree et al., 2016). These distinct differentiation patterns highlight the importance of the microenvironment in FCSC fate decisions. To date, the detailed modulatory mechanisms by which the microenvironment affects FCSC fate have not been fully characterized, but this could be a target for researchers in future studies of FCSCs.

The analysis of FSCS migration is somewhat limited. By performing the scratch wound healing assay, we found that human FCSCs presented a weaker migration capability than OMSCs (Bi et al., 2020). The migratory ability of FCSCs in vivo and whether these endogenous stem cells can be recruited to defect sites remain to be determined.

Increasingly, the mechanisms underlying the therapeutic effects of MSCs are attributed to the secretion of trophic factors, particularly extracellular vesicles (EVs) (Meirelles Lda et al., 2009). EVs are cell-derived membrane-bound nanoparticles that play an important role in the maintenance of biophysiological homeostasis as well as cellular, physiological, and pathological processes (Yáñez-Mó et al., 2015). EVs have significant diagnostic and therapeutic potential. MSCs, as prolific producers of EVs, have recently attracted much attention (Baglio et al., 2012; Liang et al., 2014). Of note, exosomes, one type of EV with a 40–100 nm diameter (Raposo and Stoorvogel, 2013), secreted by MSCs are found to have a great effect on the treatment of OA (Zhu et al., 2017). In TMJOA treatment, Zhang et al. (2016, 2019) administered exosomes isolated from human embryonic stem cell-derived MSCs to treat TMJOA and found that they could promote TMJ repair. However, the trophic function of FCSCs is still not well characterized. FCSCs have the potential to secrete trophic factors, particularly exosomes, which are crucial for therapeutic function. Moreover, a previous report compared exosomes secreted by synovial membrane MSCs and induced pluripotent stem cell-derived MSCs in the treatment of OA in the knee joint. Both exosomes could attenuate OA, but the latter had a better therapeutic effect (Zhu et al., 2017). Because exosomes secreted by different types of MSCs show distinct regenerative capacities, it is crucial to investigate the trophic function of FCSCs as well as their cell-specific properties of trophic factors in TMJOA treatment.

Moreover, increasing evidence indicates that MSCs play an immunomodulatory role primarily through the release of EVs and paracrine factors (Spees et al., 2016; Li and Hua, 2017). Previous reports have found that MSC-derived exosomes and microparticles play an anti-inflammatory role independently to modulate T and B lymphocytes in inflammatory arthritis (Cosenza et al., 2018). Whether FCSCs have immunomodulatory capacity during condylar cartilage regeneration under pathological conditions remains to be determined. Scholars have found that FCSCs can secrete VEGF-A in a paracrine manner in vitro (Nathan et al., 2018). This may help to explain how FCSCs organize the hematopoietic microenvironment in vivo (Embree et al., 2016), highlighting the possibility of interactions of FCSCs and surrounding cells in a paracrine manner. Further study of the trophic and immunomodulatory functions of FCSCs is needed.

Compared with BMMSCs, FCSCs express similar cell surface markers, including CD90, CD44, CD29, CD105, and CD146, but lack leukocyte markers, such as CD45, CD79a, and CD11b (Soleimani and Nadri, 2009; Robey et al., 2021). As noted above, they show heterogeneous differentiation potential similar to that of BMMSCs in vitro (Embree et al., 2016). It is important to note that FCSCs show distinct progress of osteogenesis when transplanted onto the dorsum of athymic nude mice. FCSCs formed cartilaginous-like tissue first and then transformed into bone-like tissue, while BMMSCs directly formed bony tissue without cartilaginous tissue transition (Embree et al., 2016). Notably, chondrogenically precultured BMMSCs could form unstable cartilage with hypertrophy, vascular invasion, and terminal matrix calcification (Pelttari et al., 2006). In general, compared to BMMSCs, FCSCs have innate chondrogenic capacity in the context of transplantation.

While FCSCs are stem cells in the fibrocartilage of the TMJ, there are various stem cells that can be isolated from fibrocartilage in other organs, including meniscus-derived mesenchymal stem cells (MMSCs), annulus fibrosus-derived stem cells (AFSCs), and tendon-derived stem cells (TDSCs). MMSCs are isolated from avascular zone of meniscus, which express MSCs surface markers, such as CD44 and CD90 (Gui et al., 2015; Huang et al., 2016). When compared to BMMSCs, MMSCs showed a stronger chondrogenesis in vitro and a better repair of damaged meniscus in vivo (Ding and Huang, 2015). Similar to FCSCs, MMSCs preferentially differentiate into chondrocytes (Huang et al., 2016). Annulus fibrosus is a fibrocartilaginous tissue in intervertebral disc (Liu et al., 2014). AFSCs express common MSCs surface markers, including CD29, CD44, and CD166 (Liu et al., 2014; Guo et al., 2018). They could form a hierarchical structure approximating native AF tissue (Chu et al., 2018; Zhou et al., 2021). TDSCs express a similar surface marker with MSCs, including CD44 and CD90 (Bi et al., 2007; Liu et al., 2018). Unlike FCSCs and MMSCs, TDSCs preferentially differentiated into tenocyte-like cells but not chondrocytes (Guo et al., 2016), emphasizing the potential of TDSCs in repairing bone-tendon junction, a fibrocartilaginous structure in tendon (Benjamin and Ralphs, 1998; Qin et al., 2020). These data suggest that stem cells originated from certain fibrocartilage tissue may have their unique differentiation signature, possibly reflecting their site of origin.

Angiogenesis is a tightly regulated process involved in the growth and repair of bone tissue. Several studies have verified that human umbilical vein endothelial cells (HUVECs) can indirectly regulate BMMSCs via angiocrine factors (Villars et al., 2000; Zhu et al., 2020). However, Nathan et al. proved that the secreted factors of HUVECs were not sufficient to stimulate FCSCs in vitro. Only when in direct contact with HUVECs were the osteogenic transcription factors of FCSCs markedly upregulated (Nathan et al., 2018). Furthermore, FCSCs in turn support angiogenesis. Vascular endothelial growth factor A (VEGF-A), a cytokine promoting HUVEC proliferation, was highly expressed in FCSCs when cultured in vitro. The number of HUVECs significantly increased when cultured in FCSC-conditioned medium (Nathan et al., 2018). However, some studies reported different results. The fibrinogen gel bead angiogenesis assay (FIBA) suggested that direct interactions between FCSCs and HUVECs impeded angiogenesis (Nathan et al., 2018). Therefore, more research is needed to explore whether other FCSC-derived factors affect HUVECs in addition to their paracrine function through VEGF-A.

Temporomandibular joint OA is one of the most severe subtypes of TMD due to degeneration of various hard and soft tissues, including cartilage degeneration, viscous synovial fluid accumulation, and osteophyte formation (Rando and Waldron, 2012; Bechtold et al., 2016; Ibi, 2019). Existing treatments for TMJ OA mainly focus on pain relief and functional rehabilitation. There is difficulty in recovering the physiological morphology and function of condylar cartilage. Therefore, clinical therapy is urgently needed to restore the TMJ structure and regenerate defects. Residing in the SZ of cartilage, FCSCs harbor multilineage differentiation potential and participate in cartilage formation, implying their potential in repairing defects in TMJ OA. It was discovered that the application of an exogenous Wnt inhibitor could repair and regenerate injured fibrocartilage by maintaining the FCSC pool and regulating FCSC differentiation. Embree et al. arranged SOST injection into a rabbit TMJ OA model and found that the condyles had mild surface irregularities after SOST administration. The contralateral PBS-treated condyles displayed severe surface irregularities and had significantly higher Osteoarthritis Research Society International (OARSI) recommended macroscopic scores. Moreover, SOST treatment led to a significantly greater number of cells surviving in the SZ, indicating that Wnt inhibitors could protect FCSCs from depletion and improve the morphology of condylar cartilage in the progression of TMJ OA (Embree et al., 2016).

Accelerated cartilage-to-bone transformation is one of the main causes of condylar bone reconstruction in TMJ OA (Liu et al., 2015). Ruscitto et al. discovered that Col II/Runx2 double-positive cells located at the cartilage/bone interphase did not express Notch1 in the normal mandibular condyle. However, after local delivery of TNF-α to induce TMJ OA, Col II/Runx2⁺ cells appeared in the SZ and were positive for Notch1, implying that Notch1 mediated FCSCs cartilage-to-bone transformation in the setting of TMJ OA (Ruscitto et al., 2020). Therefore, Notch inhibitors offer promising therapeutic potential in the treatment of TMJ OA by maintaining the morphology of the condyle. The Notch inhibitors γ-secretase inhibitor IX and N-[N-(3,5-difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t-butyl ester (DAPT) significantly reduced the expression of Notch 1, Runx2, and Ocn in FCSCs and suppressed osteogenesis of FCSCs in vitro (Ruscitto et al., 2020). Although the effect of Notch inhibitors on FCSCs has not been evaluated in vivo, increasing evidence suggests the potential role of Notch inhibitors in the treatment of TMJ OA by targeting FCSCs (Luo et al., 2018).

More recently, we transplanted exogenous FCSCs into a TMJ defect rat model to assess their function in cartilage repair (Bi et al., 2020). After 4 weeks, engrafted FCSC lineages could be observed in the SZ, polymorphic zone, and zone of chondrocytes. Under gross observation, the condylar surface of the defect sites was smoother in the FCSC-treated group than in the vehicle-treated group. The International Cartilage Regeneration and Joint Preservation Society (ICRS) score and modified Mankin score were utilized to evaluate the effectiveness of FCSC treatment, showing that FCSC treatment improved the arrangement of cartilage structures. These results indicate that FCSCs are an optimal stem cell source facilitating TMJ cartilage repair in vivo.

Previous studies have demonstrated that the interactions of FCSCs and HUVECs could promote osteogenic differentiation of FCSCs in vitro. Researchers further generated a mouse model with critical-size defects in the calvaria to mimic the vascularized bone niche and found that FCSC transplantation directly formed bone-like tissue in the defect region (Nathan et al., 2018). FCSCs were able to differentiate and form de novo bony tissue that expressed OCN. In addition, the neovasculature localized at the periphery of the FCSC engraftment area was CD31⁺, suggesting that FCSC integration was coupled with endothelial cell recruitment (Embree et al., 2016; Nathan et al., 2018). Notably, dorsum-transplanted FCSCs regenerated cartilage before calcification, which differed from the direct formation of bone-like tissue in calvarial defects. Scholars speculated that the microenvironment may contribute to FCSC fate decisions. In the ectopic xenograft model, the innate chondrogenic capacity of FCSCs dominated the regeneration process, while the osteogenesis of FCSCs observed in the vascularized bone defect may rely on FCSC–HUVEC interactions. At present, the mechanisms modulating FCSC differentiation toward chondrogenesis and osteogenesis in vivo remain uncertain, and further investigation is warranted.