Close dependence of fibroblast proliferation on collagen scaffold matrix stiffness

Human dermal fibroblasts (HDFs) in free-floating collagen matrices show minimal proliferation, although this may increase when the matrix is 'under tension'. We have investigated the detailed mechanics underlying one of the possible controls of this important cell behaviour, in particular the hypothesis that this is a response to substrate stiffness. Hyperhydrated collagen gels were plastic-compressed (PC) to give a predetermined collagen density and stiffness. Mechanical properties were tested using a dynamic mechanical analyser; cell number by Alamar blue assay. In the stiffest PC matrices, cell proliferation was rapid and seeding density-dependent, with a population doubling time of 2 days. In contrast, compliant attached matrices showed a 4 day lag period and a doubling time of 6 days. HDF growth was directly related to matrix stiffness, such that increasing stiffness using a range of compression levels (0-75% fluid removal) supported increasing proliferation rate, doubling times and matrix elastic modulus. HDF quiescence in compliant matrices was reversible, such that increasing stiffness in situ by compression at 1 and 5 days initiated proliferation. We conclude that collagen matrix stiffness regulates proliferation of fibroblasts (a duro-response), with important implications for understanding fibroblast-matrix feedback controls during wound healing and the design and regulation of engineered connective tissues based on collagen and other hydrogel-based scaffolds.