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 identify and characterise a previously undescribed tendon cell population residing within interfascicular matrix (IFM), demonstrating its importance as the primary mechanosensitive cell in tendon. We describe the first successful isolation and long-term culture 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, gene expression. This work defines IFM cells as the primary mechanosensitive tendon cell population, with implications for tendon ageing, injury, and regeneration. Importantly, it also enables identification of cell surface markers to isolate this population from other tendons, opening new avenues to explore mechanobiology-guided tendon therapeutics.