Exercise-primed Trpv4 ⁺ progenitors sense mechanical to regulate graded transition in enthesis regeneration

Aims To identify the molecular mechanisms of mechanotransduction that drive functional regeneration rather than scar formation at the tendon-bone interface, addressing the high clinical failure rates of enthesis injuries. Methods Single-cell RNA sequencing was performed on enthesis tissues at multiple post-injury timepoints. A mouse tendon-bone insertion injury model was established with treadmill exercise versus sedentary controls. Transient receptor potential vanilloid 4 (TRPV4)+ cells were isolated by fluorescence-activated cell sorting for functional assays. Mechanistic studies included bulk RNA sequencing, immunofluorescence, and in vitro mechanical stimulation. Functional outcomes were assessed by histomorphometry, micro-CT, and biomechanical testing. Results Single-cell transcriptomics identified Trpv4 + cells specifically enriched in chondrogenic lineages during enthesis repair. Mechanical stimulation significantly increased Trpv4 + chondrocyte density and enhanced chondrogenic marker expression. Lineage tracing confirmed that Trpv4 + progenitors possess clonogenicity and multipotent differentiation capacity, with mechanical stimulation enhancing their chondrogenic potential. Mechanistically, Trpv4 activation facilitated calcium influx, leading to calcineurin-NFATc1 signalling cascade activation. Transplantation of exercise-primed Trpv4 + progenitor cells significantly improved cartilage matrix deposition and biomechanical properties compared to controls. Conclusion Trpv4 + progenitors constitute a mechanosensitive population critical for enthesis regeneration. Exercise enhances Trpv4 -dependent chondrogenic differentiation through the calcium-calcineurin-NFATc1 axis, providing a scientific basis for optimizing postoperative rehabilitation. Cite this article: Bone Joint Res 2026;15(7):836–849.