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The detail of how mechanical forces are transmitted to cells is poorly understood at present and represents a key missing link in Tissue Engineering. As cells attach to the fibrils in fibroblast-seeded 3D collagen scaffolds they generate contractile forces to levels, which depend on cell type, attachment, density, growth factors and matrix stiffness. The aim here was to use external applied strain to increase matrix stiffness in collagen constructs. Embedded resident cells (from three different sites) were then subjected to specific mechanical loading regimes in scaffolds of increasing stiffness and matrix remodelling genes quantified as markers of mechanoregulatory cellular response. A strong co-relation between substrate stiffness, mechanical loading and regulation of key ECM turnover genes was identified. This knowledge is crucial to successful tissue engineering outcomes. The differential lineage dependent response is a key finding and will have to be tailored depending on cell source and specific outcomes desired.