Tendon Cells Root Into (Instead of Attach to) Humeral Bone Head via Fibrocartilage-Enthesis

Large joints are composed of two closely linked cartilages: articular cartilage (AC; rich in type II collagen, a well-studied tissue) and fibrocartilaginous enthesis (FE; rich in type I collagen, common disorder sites of enthesopathy and sporting injuries, although receiving little attention). For many years, both cartilages were thought to be formed by chondrocytes, whereas tendon, which attaches to the humeral bone head, is primarily considered as a completely different connective tissue. In this study, we raised an unconventional hypothesis: tendon cells directly form FE via cell transdifferentiation. To test this hypothesis, we first qualitatively and quantitatively demonstrated distinct differences between AC and FE in cell morphology and cell distribution, mineralization status, extracellular matrix (ECM) contents, and critical ECM protein expression profiles using comprehensive approaches. Next, we traced the cell fate of tendon cells using Scx^Lin (a tendon specific Cre Scx^CreERT2; R26R-tdTomato line) with one-time tamoxifen induction at early (P3) or young adult (P28) stages and harvested mice at different development ages, respectively. Our early tracing data revealed different growth events in tendon and FE: an initial increase but gradual decrease in the Scx^Lin tendon cells and a continuous expansion in the Scx^Lin FE cells. The young adult tracing data demonstrated continuous recruitment of Scx^Lin cells into FE expansion during P28 and P56. A separate tracing line, 3.2 Col 1^Lin (a so-called "bone-specific" line), further confirmed the direct contribution of tendon cells for FE cell formation, which occurred in days but FE ECM maturation (including high levels of SOST, a potent Wnt signaling inhibitor) took weeks. Finally, loss of function data using diphtheria toxin fragment A (DTA) in Scx^Lin cells demonstrated a significant reduction of Scx^Lin cells in both tendons and FE cells, whereas the gain of function study (by stabilizing β-catenin in Scx^Lin tendon cells via one-time injection of tamoxifen at P3 and harvesting at P60) displayed great expansion of both Scx^Lin tendon and FE mass. Together, our studies demonstrated that fibrocartilage is an invaded enthesis likely originating from the tendon via a quick cell transdifferentiation mechanism with a lengthy ECM maturation process. The postnatally formed fibrocartilage roots into existing cartilage and firmly connects tendon and bone instead of acting as a simple attachment site as widely believed. We believe that this study will stimulate more intense exploring in this understudied area, especially for patients with enthesopathy and sporting injuries.