Identification of a Tenochondral Ossification Sequence Producing Heterotopic Ossification in Human Achilles...

Achilles tendinopathy is a common musculoskeletal disorder. A frequent late-stage manifestation is tendon heterotopic ossification (HO). Achilles HO forms predominantly via endochondral ossification, yet the cellular and molecular mechanisms underlying this pathologic condition remain elusive. Here, we analyzed surgically excised biopsy samples with symptomatic human Achilles tendons alongside non-diseased Achilles tendon controls primarily from lower-limb amputation donors to characterize the morphologic and mineralization patterns of the disease. Based on these analyses, we propose a stepwise endochondral model of Achilles HO that we term "tenochondral ossification" to emphasize the chondrogenic intermediate stage and its tendon origin. This process includes tendon-to-cartilage metaplasia, characterized by increased glycosaminoglycan content (Alcian blue-positive matrix) and the appearance of chondrocyte-like cells aligning the collagen fibers; some cells have substantial RUNX2 or SOX9 expression; subsequent mineralization of the metaplastic tissue, indicated by alkaline phosphatase immunoreactivity and neovessel ingrowth; osteoclast differentiation and resorption of the calcified metaplastic tissue; and new bone deposition on metaplastic tendon remnants accompanied by robust RUNX2 immunoactivity. Furthermore, TNC expression remains high in the metaplastic cartilage but diminishes at the mineralization front. Taken together, these data support a tendon-origin, endochondral pathway for Achilles tendon HO and highlight stage-specific therapeutic opportunities for treating or preventing Achilles tendinopathy and HO development and progression.