Primary cilia regulate mechanical adaptation of tendon and enthesis via hedgehog signaling

Background Physical loading mediates postnatal growth, homeostasis, and healing of the tendon and its attachment to bone, which is critical for rotator cuff functional integrity. Our prior studies have highlighted the mechano-sensing role of primary cilia; However, the mechanisms through which cilia convert mechanical stimuli into structural functional adaptation under altered loading conditions remain unanswered. Methods Publicly available scRNA-seq datasets of mechanically loaded human patellar tendon cells were re-analyzed to identify cilia-related transcriptional changes. Tendon-specific cilia knockout mice (ScxCre;Ift88 fl/fl ) and wild-type controls (Ift88 fl/fl ) underwent mechanical unloading induced by botulinum toxin A injection, followed by micro–computed tomography, biomechanical testing, histology, qPCR, and immunohistochemistry to evaluate structural, mechanical, and Hedgehog (Hh) signaling responses. Primary tendon fibroblasts from wild-type and cilia-deletion mice were treated with Hh agonist or antagonist to assess Hh signaling responsiveness in vitro. Student’s t-test for two groups and two-way ANOVA for two groups with two treatments were performed for our statistical analysis. Results Here, we find that mechanical force causes changes in cilia- and hedgehog (Hh)-related gene expression in human tendon fibroblasts. Cilia ablation in the enthesis blunts force-driven remodeling of tissue structure and mechanical strength. Cilia deletion also leads to impaired Hh signaling in tendon cells and decreased responsiveness to activation and inactivation of hedgehog signaling. Conclusions Our results demonstrate loading-regulated ciliary Hh signaling during postnatal growth of the tendon and enthesis and provide proof-of-concept for developing new cilia-targeted mechanical and biological therapies for enthesis repair.