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Human mesenchymal stem cells (hMSCs) have been demonstrated to exhibit characteristic cytosolic Ca2+ ([Ca2+]i) oscillations in response to physicochemical factors, including soluble factors, matrix proteins and microenvironment. It has been postulated that the calcium oscillation at the early stages of hMSC differentiation may serve as a biophysical predictor. The goal of the present study is therefore aimed at elucidating the matrix proteinmediated [Ca2+]i oscillation that occurs in the early stages of hMSC osteogenic differentiation. On the collagen substrates of different stiffness and concentrations, intracellular [Ca2+]i oscillations of hMSCs exhibited rapid spikes on the hard substrates and the opposite on the soft substrate. Cells seeded on a hard substrate also expressed signi ficantly high levels of osteogenic gene markers, suggesting a key role of intracellular calcium in the transduction of ECM signaling to the hMSC differentiation. Our findings revealed that the characteristics of [Ca2+]i oscillation are regulated by the biochemical and mechanical properties of the collagen substrate. Furthermore, β1-integrin and associated signaling proteins found in focal adhesions (FAs) were involved in the signaling pathways. Because the [Ca2+]i signaling and stemcell differentiation appear closely correlated, the regulation of [Ca2+]i signaling through the modification of engineeredECMmay provide a controllable exogenous technique to direct the fate of hMSCs.
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