Tendon Cells Directly Form Bone Ridges via Multiple Cell Transdifferentiation: Tendon‐Fibroblast‐Bone Cells beyond Simply Connecting Bone And Muscles

Recent studies suggest that tendons play a critical role in the initiation of bone ridges by transducing SCX/BMP4 signaling to osteochondroprogenitors, although tendons are mainly considered to simply control skeletal movement and joint stabilization. Currently, intramembranous ossification and endochondral ossification are thought to be the only osteogenic mechanisms. However, the rough bone ridges appear different from the surrounding smooth bone structure. The aim of this study is to test a hypothesis: tendons directly form bone ridges (a third type of bone) by multiple cell transdifferentiation steps: tendon‐fibroblast‐bone cells. To test this hypothesis, we used the humerus bone ridge (deltoid tuberosity) as a model. We injected Alizarin Red to 2.3kb Col 1‐GFP (reflecting bone cells) mice 4 hours prior to harvesting, followed by imaging of the non‐decalcified bone to analyze the mineralization status of the humerus bone (including the deltoid tuberosity). In contrast to a monolayer of GFP+ bone cells in the periosteum‐formed bone (PFB), there were multilayers of GFP+ fibroblast‐like bone cells between the tendon and deltoid tuberosity beneath, indicating a cell transdifferentiation from tendon to bone (Fig a). The H&E staining showed a gradual transdifferentiation from tendon cells to fibroblast‐like cells to bone cells with a different cell distribution from PFB (Fig b). The quantitative uCT‐analysis displayed a low bone mineral density (BMD, Fig c, left). The cell lineage tracing data (using a tendon specific ScxCreERT2‐R26‐tdtomato line induced at P3 by one‐time injection of tamoxifen) showed a rapid expansion of tendon‐formed bone (TFB) cells from few at P9 to many TFB cells at P33, and maturation at P60 (Fig c). These TFB cells expressed high levels of periostin (tendon marker), aggrecan (cartilage marker), RUNX2 and SOST (two bone markers; Fig d, upper panels), which were sharply reduced when DTA (diphtheria toxin subunit A) was activated in the Scx‐Cre+ cells at P3 and harvested at p30 (Fig d, lower panel). Finally, we stabilized β‐catenin in Scx expressing cells (β‐cateninfx(exon3)/fx(exon3)) at P3 and harvested at P60. These mice displayed an expanded deltoid tuberosity mass and sharply increased Scx+ bone cells (Fig e, lower panels). Based on the data above, we proposed that tendon cells directly form bone ridges by a continuous cell transdifferentiation from tendon cells to fibroblast‐like cells, and then to bone cells. These TFB cells display a distinct feature from conventional bone, including a low BMD, a mixture profile of extracellular matrix proteins that are normally expressed in tendon, cartilage and bone (Fig f). This novel discovery on the broader roles of tendon explains why bone ridges embrace different features from conventional bone and makes the tendon‐bone interface a mineralized continuum rather than a simple attachment.