Fig. 2

Volume expansion regulates osteogenesis of MSCs in fast-relaxing hydrogels. a Representative 3D renderings of single MSCs from confocal imaging of a cell membrane dye within fast-relaxing hydrogels for 7 days under the indicated osmotic pressure. Scale bar, 10 µm. b, c Quantification of cell volumes (b) and sphericity (c) of MSCs cultured in fast-relaxing gels with a RGD concentration of 1500 µM under varied osmotic pressures for 7 days (n ≥ 30 single cells from three biological replications per each condition). d Representative images of alkaline phosphatase staining for MSC cultured in fast-relaxing gels under indicated osmotic pressure for 7 days. Scale bar, 25 µm. e Quantification of the percentage of cells positively stained for alkaline phosphatase produced by MSCs cultured for 7 days under indicated conditions (n ≥ 10 images from three biological replications per each condition). f Scatter plot of cell volume with the percentage of cells positively stained for alkaline phosphatase produced by MSCs when volume of single cells was modulated by varying stress relaxation, RGD density, or osmotic pressure. A non-linear regression analysis showed a global trend of osteogenic differentiation with volume of single cells. g Schematic of protocol for application of hypoosmotic pressure to enhance volume expansion. h, i Quantification of cell volume (h) and sphericity (i) for MSCs cultured under hypoosmotic pressure or control conditions at days 3 and 7 (n ≥ 30 single cells from three replications per each condition). The box plots show 25/50/75th percentiles and whiskers show minimum/maximum. j Representative images of ALP staining of MSCs cultured in hypoosmotic media or control media at days 3, 5, and 7 (n ≥ 10 images from three replications per each condition). k Quantification of ALP-positive cells for MSCs in hypoosmotic or control medium at days 3, 5, and 7. (n ≥ 8 images from three replications per each condition). All data are shown as mean ± s.e.m. For one-way ANOVA comparisons; *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 and for Spearman’s rank correlation; ^####p<0.0001