构建不同杨氏模量且表面粗糙度相近的聚丙烯酰胺水凝胶模型，观察不同基质刚度条件下小鼠骨髓间充质干细胞（mBMSC）的微丝形态、铺展及早期成骨相关基因表达，以评价该模型用于细胞力学响应研究的可行性。

制备低、中、高刚度3组聚丙烯酰胺水凝胶，检测其杨氏模量和表面粗糙度，并经sulfo-SANPAH活化后偶联Ⅰ型胶原。将mBMSC接种于水凝胶表面，通过F-actin/DAPI荧光染色观察细胞微丝形态并分析相对铺展面积；采用细胞松弛素D干预微丝聚合，成骨诱导7 d后检测Alpl和Runx2的相对表达。采用GraphPad Prism 8软件进行统计学分析。多组间比较根据数据分布及方差齐性选择单因素方差分析或Kruskal-Wallis检验，组间两两比较分别采用Tukey或Dunn多重比较检验。以P<0.05为差异有统计学意义。

低、中、高刚度组水凝胶平均杨氏模量分别为1.09、11.37和29.24 kPa，图示统计学标记提示组间差异有统计学意义（F = 766.60，P<0.001）；3组表面粗糙度差异无统计学意义（H = 5.956，P>0.05）。随水凝胶刚度增加，mBMSC微丝束更加明显，相对铺展面积增大（H = 14.73，低刚度组与高刚度组差异有统计学意义，P = 0.000 4）。对照条件下，高刚度组Alpl和Runx2表达高于低刚度组（F_Alpl = 91.54，P_Alpl = 0.003 9；F_Runx2 = 12.93，P_Runx2 = 0.002 5）；细胞松弛素D干预后，低、高刚度组间上述基因表达差异无统计学意义（F_Alpl = 91.54，P_Alpl = 0.401 9；F_Runx2 = 12.93，P_Runx2 = 0.887 3）。

本研究构建的聚丙烯酰胺水凝胶模型可在表面粗糙度相近的条件下形成明确的杨氏模量分级，基质刚度调控干细胞的早期成骨效应一定程度上可受到细胞骨架微丝的影响。

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.