Intrasynovial DDFT injury is a common source of debilitating forelimb lameness in horses. Chronic inflammation during healing and poor intrinsic healing potential are important factors responsible for dysregulated tissue repair. Tendon degeneration and adhesion formation within the synovial sheath are common sequelae that significantly reduce prognosis for return to function (3, 9, 13). Intrasynovial DDFT begins just proximal to the metacarpal(-tarsal) phalangeal (MCP/MTP) joint and extends to the distal second phalanx. Distally, the DDFT is compressed against the distal sesamoid bone within the podotrochlear bursa and surrounded by an annular pulley proximal to its insertion at the third phalanx (25). The dorsal fibrocartilage is robust at the level of MCP/MTP joint and proximal second phalanx and becomes thinner at the distal second phalanx, similar to rabbit (26), mouse (12) and canine (15, 27) DDFT. The large size, grossly distinct fibrocartilaginous-tendinous zones of the equine DDFT and ready access to healthy tissue facilitate cellular analyses described in this study. Intrasynovial TDC evaluated in this study were isolated using low-density plating method commonly used to isolate stem/progenitor cells from extrasynovial equine (18, 19, 28, 29), human (16), and mouse (16, 30) tendon tissues. We have designated these cells as TDC since further characterization is needed to determine if they possess stem/progenitor cell properties or if they are differentiated cells.
The clonogenic and monolayer passage characteristics of fTDC and tTDC were similar and consistent with existing extrasynovial tendon studies (16, 18, 19, 28). Freshly isolated primary cells following enzyme digestion of fibrocartilaginous and tendinous zones were heterogenous, with cells from the fibrocartilaginous zone exhibiting polygonal morphology and cells from the tendinous zone appearing elongate and spindle-shaped. However, after monolayer passage fTDC and tTDC became homogenous, fibroblast-like cells. The tenogenic and chondrogenic markers in fTDC and tTDC significantly decreased relative to their respective terminally differentiated cells and were not significantly different between them. This plasticity property is seen in other tissue-derived stem/progenitor cells and highlights the key role of the ECM on their in-situ phenotype and bioactivities (16, 31, 32).
Cell surface marker profiles of equine intrasynovial TDC are comparable with extrasynovial tendon stem/progenitor cells (16, 33) and bone marrow-derived mesenchymal stem cells (18, 19, 28, 34). From the time of enzyme digestion to third passage, the percentage of CD90+ cells increased from ~ 70% to 85–95%. While there were no differences in the cell surface markers of fTDC and tTDC, less than 5% of the cells were positive for CD44, a mesenchymal stromal cell marker. Further assessment of CD44 gene expression is warranted to determine if this is true to intrasynovial TDC, since an equine-specific antibody was utilized. Overall, it should be noted that our assessments of basal fTDC and tTDC phenotypes were restricted in this study, and a more robust analyses of cell surface markers and gene expression representing stem/progenitor cells such as CD73, CD105, Oct-4 are warranted.
Reflective of the in-situ chondrocyte-like morphology of tendon fibrocartilage cells, fTDC were largely restricted to chondrogenesis in vitro, whereas tTDC underwent osteogenic and chondrogenic differentiation. Although the basal (non-induced) osteogenic and chondrogenic mRNA profiles of fTDC and tTDC were not significantly different, basal fTDC SOX9 mRNA was 3-fold higher than tTDC and approached significance (P = 0.06; Fig. 4b). Subsequently, with chondrogenic stimulation, fTDC SOX-9 and ACAN mRNA expressions were 5- (P = 0.07) and 3.5- (P = 0.06) fold higher than tTDC. Toluidine blue stain uptake reflecting the sGAG content was also higher in fTDC pellets and was uniformly distributed throughout the pellet, whereas the stain uptake was just localized to the periphery of tTDC pellets (Fig. 4a). These findings suggest that fTDC may be ‘committed’ to a chondrogenic phenotype. However, additional analyses are required as it also possible that the low-density plating method selects for a chondrogenic fTDC subpopulation.
The common clonogenic, proliferative, and immunophenotype characteristics, and varying trilineage differentiation potentials of TDC isolated from morphologically distinct tendon zones are consistent in meniscal (35) and intervertebral disc (IVD) (36) tissues. In vitro chondrogenic restriction of stem/progenitor cells isolated from the inner zone of meniscus and IVD nucleus pulposus is partially implicated in the limited intrinsic repair capacity of these tissue zones. To this end, in-depth evaluation of cellular and healing responses of the fibrocartilage zone specifically during intrasynovial tendon injury is minimal. Nessler et al., evaluated healing in intrasynovial fibrocartilaginous and extrasynovial tendinous regions of canine DDFT after experimental longitudinal, partial thickness lacerations (15). The fibrocartilaginous region was stiffer and contained larger diameter collagen fibrils compared to tendinous regions at early timepoints during healing (3- and 6-weeks). This is reflective of local differential cellular synthetic activities and differences in mechanical environment during healing. Accepting that clinical tendon injuries require a prolonged time frame for recovery, longer-term in vivo experimental studies are warranted to delineate the mechanisms related to zonal differences in poor healing and re-rupture following intrasynovial injuries. Identifying these intrinsic cellular responses specific to intrasynovial tendon homeostasis, injury, and healing are critical to develop regenerative therapies aimed at restoring intrasynovial tendon gliding function.
The following factors pertaining to this study are to be taken into consideration. Intrasynovial TDC were isolated from healthy equine donors of a wide age range, albeit representative of age group susceptible to clinical disease (3–5, 9, 13, 37). Secondly, although low-density plating method is routinely used to isolate tendon stem/progenitor cells, alternate approaches such as differential substrate adherence or cell surface epitope FACS-based separation that mitigates monolayer passage and accelerates generation of relatively pure cell populations need to be evaluated. However, feasibility of these technologies when working with equine cell stocks is limited. Lastly, trilineage differentiation mRNA values in fTDC and tTDC were expressed as fold-change from respective (non-induced) terminally differentiated cells. Maintaining terminally differentiated subpopulations in basal medium for the same duration as induction media would have facilitated spontaneous differentiation assessments.
The collective outcomes of our data suggest presence of stem/progenitor cells in the fibrocartilaginous and tendinous zones of equine intrasynovial DDFT, and shares similarities with stem/progenitor cells characterized from equine extrasynovial superficial digital flexor tendon (18, 28). Consistent with Cadby et al., and Williamson et al., fTDC and tTDC expressed MSC properties such as clonogenicity and plasticity. These cells were also negative for hematopoietic stem cells markers (CD45, CD34), owing to poor tissue vascularity and supports that they were isolated from the tendon proper. In regard to trilineage differentiation, our results are similar to Williamson et al.; in that, adipogenic capacity was minimal (18). This is in contrast to Cadby et al., where cells underwent adipogenesis based on upregulated PPARg and FABP4 mRNA expression, as well as positive Oil-red O staining (28). tTDC exhibited osteogenic and chondrogenic capacities similar to stem/progenitor cells isolated from equine superficial digital flexor tendon as reported by Williamson et al. On the other hand, fTDC were largely restricted to chondrogenic differentiation, represents a unique TDC subpopulation and emphasizes the need for multi-assay panels for rigorous assessments of lineage commitment. While we have described fTDC characteristics in relation to their terminally differentiated cell counter parts and tTDC, comparative analyses with ‘gold standard’ bone marrow-derived mesenchymal stem cells could be more informative regarding their stem/progenitor cell characteristics.
The diminished healing capacity of intrasynovial tendons is attributed in part due to limited intrinsic healing mechanisms and inherent low tissue cellularity. The limitations in healing are also reflective of the mechanical environment within the tissue and persistent inflammation, consequently resulting in net tissue catabolism. The results of this study provide a foundation for studies evaluating cell-based therapies for intrasynovial tendon repair as these TDC are potential targets to enhance intrinsic repair. Promoting chondrogenic properties in cells administered exogenously into the intrasynovial space and in cells that are used to revitalize decellularized autografts may be beneficial for intrasynovial tendon regeneration. Future in vivo studies to evaluate the healing effects of intrasynovial TDC in experimental animal models are warranted.