Tendon Cells Root into Rather Than Attach to the Humerus Bone Head via Enthesis Fibrocartilage.

Abstract

Large joints are composed of two closely linked cartilages: articular cartilage (AC) and enthesis fibrocartilage (EF). For many years, both cartilages were thought formed by chondrocytes, whereas tendon, which attaches to the humerus head, is largely considered as a completely different connective tissue. In this study, we raised an unconventional hypothesis: tendon cells directly form EF via cell transdifferentiation. To test this hypothesis, we first qualitatively and quantitatively demonstrated distinctions between AC and EF using comprehensive approaches. Next, we traced the fate of tendon cells using ScxLin (a tendon specific Cre ScleraxisCreERT2; 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 EF: an initial increase but gradual decrease in the ScxLin tendon cells and a continuous expansion in the ScxLin EF cells. The young adult tracing data demonstrated continuous recruitment of ScxLin cells into EF expansion during P28 and P56. A separate tracing line, 3.2 Col1Lin (a so-called "bone-specific" line), further confirmed the direct contribution of tendon cells for EF cell formation, which occurred in days but EF 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 ScxLin cells demonstrated a significant reduction of ScxLin cells in both tendons and EF cells, whereas the gain of function study (by stabilizing β-catenin in ScxLin tendon cells via one-time injection of tamoxifen at P3 and harvesting at P60) displayed great expansion of both ScxLin tendon and EF mass. Together, the studies demonstrated that fibrocartilage is an invaded enthesis likely originating from 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 anticipate this study to stimulate more intense exploring in this understudied area, especially for patients with enthesopathy and sporting injuries.