Primary Cilia Regulate the Mechanical Adaptation of the Tendon and Enthesis via Hedgehog Signaling

Abstract

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 single-cell RNA sequencing (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 microcomputed tomography, biomechanical testing, histology, quantitative polymerase chain reaction (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 analysis of variance (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 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 Hh 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.