Matrix stiffness and serum concentration effects matrix remodelling and ECM regulatory genes of human bone marrow...

The effects of mechanical stimulation of cell-seeded collagen constructs on cell orientation, intracellular signalling and molecular responses have been widely reported. In this study we investigated in vitro the contractile responses of human bone marrow stem cells (HBMSCs) to increasing collagen gel substrate stiffness and their effect on extracellular matrix (ECM) regulatory genes. Human dermal fibroblasts (HDFs) were used as controls. Cells were cultured in 10% and 20% FCS and embedded in collagen constructs at a density of 1 million cells/ml collagen. Matrix stiffness was achieved by subjecting the constructs to three different strain regimes (0%, 5% and 10%), using a computer-driven tensional culture force monitor (t-CFM) capable of uniaxial loading. The contraction forces generated by the cells were quantified over 24 h. Molecular outputs were quantified using RT-PCR. HBMSCs significantly increased force generation to increasing serum concentration (i.e 10% to 20%). 10% FCS concentration significantly reduced contraction as pre-strain stiffness was increased in HBMSCs and HDFs (0% > 5% > 10%). However, at 20% FCS HBMSCs generated similar peak force contraction at 24 h to 5% and 10% pre-strain (0% = 5% = 10%). The ECM regulatory gene for MMP2 showed upregulation at 5% pre-strain, but a 50% downregulation when pre-strain was increased to 10%. MMP9 was upregulated at 5% pre-strain and further upregulated at 10% pre-strain. In designing tissue-engineering solutions, predictable responses of cells, embedded within bio-artificial matrices, to external mechanical forces are critical. To take into account the increasing stiffness of the matrix as increasing ECM is deposited, it would be necessary to take mechanical stimulation into account to determine predictable cellular responses.