MSC osteogenic differentiation on different ECM + BMP2 modified substrates. MSCs were cultured on different substrates for 28 days, followed by OPN and OCN staining, as well as von Kossa staining. FN + VN + BMP2 substates could significantly support MSC osteogenic differentiation, in terms of OPN and OCN expression as well as mineralization deposition via increased activation of the pSMAD 1/5/9 pathway. (A), Representative images of OPN staining from MSCs on FN/FN + VN + BMP2 substates. Scale bar 100 μm. (B), Representative images of OCN staining from MSCs on FN/FN + VN + BMP2 substates. The scale bar is 100 μm. (C), Representative images of von Kossa staining from MSCs on FN/FN + VN + BMP2 substates. Scale bar 100 μm. (D) and (E), Quantification of immunofluorescence images of OPN and OCN staining from MSCs on FN/FN + VN + BMP2 substates. MSCs cultured on TCP surfaces were used as a control. Fluorescence intensity fold change to the TCP group was analyzed. MSCs were isolated from 3 donors. (F), Quantification of mineralized area based on von Kossa staining from MSCs on FN/FN + VN + BMP2 substates. Mineralized area fold change to the FN BMP2 group was analyzed. MSCs were isolated from 3 donors. (G), Quantification of pSMAD ICW results. MSCs treated with soluble BMP2 were set up as a control. Fluorescence fold change to the soluble BMP2 group was analyzed. MSCs were isolated from 2 donors. Each shape represents a different donor, each small shape represents each cell measured or each technical replicate, and each large shape represents the mean from each donor. The scatter plot and the bar plot show mean values and standard deviation. Paired T-test was applied for two-group comparisons. Ordinary two-way ANOVA with Tukey's test was applied for multi-group comparison. ∗p < 0.05, ∗∗p < 0.01.