To construct polyacrylamide hydrogel models with graded Young's moduli and comparable surface roughness, and to evaluate their feasibility for studying stiffness-related changes in the actin cytoskeleton, spreading, and early osteogenic gene expression of mouse bone marrow mesenchymal stem cells (mBMSCs) .

Three groups of polyacrylamide hydrogels with low, medium, and high stiffness were prepared, characterized for Young's modulus and surface roughness, activated with sulfo-SANPAH, and functionalized with type I collagen. The morphology of F-actin and the relative spreading area of mBMSCs were assessed by fluorescence imaging. Actin polymerization was perturbed with cytochalasin D, followed by 7 days of osteogenic induction and measurement of Alpl and Runx2 expression. Statistical analyses were performed using GraphPad Prism 8. Comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA) or the Kruskal-Wallis test, as appropriate, according to the data distribution and homogeneity of variances. Post hoc pairwise comparisons were performed using Tukey's multiple comparisons test or Dunn's multiple comparisons test, respectively. A two-sided P value of <0.05 was considered statistically significant.

The average Young's moduli of the low-, medium-, and high-stiffness hydrogels were 1.09, 11.37, and 29.24 kPa, respectively, with statistically significant differences (F = 766.60, P<0.001 for all pairwise comparisons), whereas surface roughness did not differ significantly among groups (H = 5.956, P>0.05). Increasing stiffness was associated with more prominent actin bundles and greater cell spreading (H = 14.73, low stiffness group-high stiffness group: P = 0.000 4). Under control conditions, Alpl and Runx2 expression were higher on the high-stiffness group than in the low-stiffness group (F_Alpl = 91.54, P_Alpl = 0.003 9; F_Runx2 = 12.93, P_Runx2 = 0.002 5). After cytochalasin D treatment, the differences between the low- and high-stiffness groups were not significant (F_Alpl = 91.54, P_Alpl = 0.401 9; F_Runx2 = 12.93, P_Runx2 = 0.887 3) .

The polyacrylamide hydrogel model developed in this study exhibited distinct levels of Young's modulus while maintaining comparable surface roughness. The effects of matrix stiffness on the early osteogenic responses of stem cells may depend, at least in part, on cytoskeletal actin filaments.