Unravelling the Distinct Phenotype and Mechanosensitive Properties of Different Tendon Cell Populations.

Tendinopathy arises from maladaptive cellular responses, though the drivers remain unclear. Here we isolate and characterise an overlooked tendon cell population residing within interfascicular matrix (IFM), demonstrating its importance as a highly mechanosensitive cell in tendon. We describe the first successful isolation and long-term culture of enriched populations of primary IFM and fascicular matrix (FM) cells, enabling direct comparison of their phenotypes and mechanosensitivity. IFM cells exhibited a potent response to stiff substrates, displaying cytoskeletal remodelling, rapid drifting of tenogenic and ECM gene expression, and proliferative decline, while FM cells remained largely unaltered. Crucially, transferring IFM cells to compliant, IFM-like substrates recovered their proliferative capacity, morphology, and partially restored their gene expression. This work defines IFM cells as a highly mechanosensitive tendon cell population. Importantly, it also enables the establishment of a defined culture framework that supports the maintenance of key IFM phenotypic characteristics in vitro and provides a new perspective on tendon cell heterogeneity by identifying distinct compartment-specific responses to the mechanical microenvironment.