Matrix maturation and cytoskeletal tension define strain thresholds for stretch-induced calcium signaling in human tendon cells

The extracellular matrix (ECM) and mechanical loading shape cellular behavior, yet their interaction remains obscure. We developed a dynamic proto-tissue model using human tendon cells and live-cell calcium imaging to study how ECM and cell mechanics regulate mechanotransduction. Stretch-induced calcium signaling served as a functional readout. We discovered that ascorbic acid-dependent ECM deposition is essential for proto-tissue maturation and stretch-induced calcium signaling at physiological strains. Proto-tissue maturation enhanced stretch sensitivity, reducing the strain needed to trigger a calcium response from ∼40% in isolated cells to ∼5% in matured proto-tissues. A strong correlation between tissue rupture and calcium signaling suggests a mechanistic link to ECM damage. Disrupting ECM integrity, cell alignment, or cytoskeletal tension reduced mechanosensitivity, showcasing the influence of ECM and cytoskeletal mechanics on stretch-induced calcium signaling. Fundamentally, our work replicates calcium signaling observed in rodent tendon explants in vitro and bridges the gap between cell-scale and tissue-scale mechanotransduction.