Poster 100. Hedgehog Signaling Upregulation in Heterotopic Ossification Associated with Insertional Achilles Tendinopathy and Tendon Degeneration

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Objective: Heterotopic ossification (HO) involves ectopic bone formation in soft tissues including muscles and tendons. This occurs to patients with insertional Achilles tendinopathy (IAT) and triggers pain, swelling, and limited range of motion. The exact mechanism driving HO is complex, involving the dysregulation of multiple molecular and biological process. Therefore, identification of critical factors that primarily regulate ectopic tissue formation in insertional Achilles tendinopathy could motivate the development of new therapeutic strategies. Interestingly, our preliminary data showed that only activation of hedgehog (Hh) signaling, without injury, in Achilles tendons of mouse models caused HO development. Considering these findings, our study aim to determine whether unexpected upregulation of Hh signaling in mature tendons drives tenocyte phenotype shift to chondrogenic and osteogenic lineages, contributing to ectopic ossification. Methods: First, a new in vivo animal model was created using 9-week-old C57BL/6J mice. Mice's right Achilles tendons were injected with an Hh agonist (Hh-Ag 1.5, 7.5 mM), while left tendons received 0.9% sodium chloride twice weekly for three weeks. Tendons were harvested three weeks post-injection. To assess HO formation, tendons were stained with 0.2% Fast Green (FCF; Sigma-Aldrich, F7252) and 1% Safranin O (Sigma-Aldrich, S2255) and analyzed via microCT. Similarly to quantify bone volume and tissue mineral density, human AT samples from three patients undergoing treatment for insertional AT tendinopathy, including tendon debridement, bony resection and reattachment of the tendon, were collected. Samples were fixed in 10% Neutral Buffered Formalin for 1-2 days, embedded in OCT, and cryosectioned. Stains included Hematoxylin & Eosin and Safranin O & Fast Green. Bone volume and tissue mineral density were quantified. Results: Hh pathway activation induced increased cartilaginous tissue formation in treated mouse Achilles tendons. Safranin O-Fast Green staining revealed cartilaginous tissue in 75% of Hh-agonist-treated right tendons, with microCT confirming mineralized tissue (Figure 1). Similarly, human AT samples showed proximal cartilaginous tissue. Sox9, a chondrogenic marker tied to Hh signaling, was detected in cartilaginous tissue in both mouse HO and human AT samples (Figure 2). Additionally, Hh-agonist-treated mouse tendons exhibited significantly higher bone volume (0.2536 mm³) and tissue mineral density (603.05 mgHA/cm³) compared to controls (Figure 3). These findings demonstrate activation of Hh signaling as the major driver for HO formation. Conclusions: This study establishes an in vivo model demonstrating the critical role of Hh signaling in HO progression. The presence of Hh-related markers in cartilaginous tissue from patients with IAT suggests a possible HO and Hh signaling pathway involvement in tendon degeneration. Future studies will use RNA sequencing to confirm Hh pathway activation in human samples compared to cadaveric controls and related change of cell transcriptional profiles in diseased tissues. Long-term, targeting Hh signaling with small-molecule inhibitors may provide novel treatments for AT-associated HO, meriting further clinical exploration.

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