Fig. 5: Osteodifferentiation of encapsulated cells driven by consistent mechanical cues of rigid shells.

a MC3T3-E1 cells in different hydrogels identified by immunostaining of molecular markers (OCN or Runx2, shown in red) and F-actin (phalloidin, shown in green) after 5 and 10 days of culture. MC3T3-E1 cells on the cell culture wells served as the control group. OCN and Runx2, specific markers for osteogenic differentiation, were upregulated in SP-low, NP-high and SP-high hydrogels. Cell nuclei were stained with DAPI (blue). A space of 318 μm × 318 μm × 100 μm in each sample was scanned layer by layer, and the images were projected onto the z-axis to show immunostaining. b Heatmap illustrating mRNA expression levels corresponding to osteogenesis genes (Runx2, OCN, Col I and ALP) for cells in different hydrogels or cell culture wells (Control) after 5 and 10 days of culture. The intensity represents the expression relative to the control group. c Summary of relative mRNA expression levels of osteogenesis-related genes (Runx2, OCN, Col I, and ALP) in cells cultured in different hydrogels or cell culture wells (Control) after 10 days. The expression levels of these genes were normalized to those of the control group. Values represent the mean ± standard deviation (n = 6 independent experiments). The p-values for the comparisons between the control group and the NP-low, SP-low, NP-high, and SP-high groups are as follows: for Runx2, 0.2938, 0.0048, 0.0012, and 0.0098, respectively; for OCN, 0.0068, 0.0025, 0.0019, and 0.0007, respectively; for Col I, 0.5670, 0.0130, 0.0003, and 0.0020, respectively; and for ALP, 0.0127, 0.0028, 0.00001, and 0.0003, respectively. Statistical significance between different groups and the control group was assessed using two-tailed Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001; NS: not significant.