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Objective: To investigate the effect of tissue interface stiffness change on the spreading, proliferation, and osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs), and to find the suitable stiffness range for stem cell differentiation., Methods: Bone marrow of male Sprague Dawley rats (4 weeks old) were selected to isolate and culture BMSCs by whole bone marrow cell adherent method. The third generation BMSCs (1×10 5 cells/mL) were inoculated into the ordinary culture dishes covered with polyacrylamide hydrophilic gel (PA) which elastic modulus was 1, 4, 10, 40, and 80 kPa (cells seeded on PA), and ordinary culture dish (75 MPa extreme high elastic modulus) as control. Spreading of cells in different stiffness of PA was observed under light microscope. The elastic modulus values of 4, 10, and 40 kPa PA were selected as groups A, B, and C respectively; the ordinary culture dish (75 MPa extreme high elastic modulus) was used as control group (group D). Cell counts was used to detect the growth conditions of BMSCs, alkaline phosphatase (ALP) kit to detect the concentration of ALP, alizarin red staining technique to detect calcium deposition status, and real-time quatitative PCR technique to detect the expressions of bone gla protein (BGP), Runx2, and collagen type I mRNA., Results: With increased PA stiffness, BMSCs spreading area gradually increased, especially in 10 kPa and 40 kPa. At 1 and 2 days after culture, the growth rate showed no significant difference between groups ( P >0.05); at 3-5 days, the growth rate of groups B and C was significantly faster than that of groups A and D ( P <0.05), but difference was not statistically significant between groups A and D ( P <0.05); at 5 days, the proliferation of group C was significantly higher than that of group B ( P <0.05). ALP concentrations were (53.69±0.89), (97.30±1.57), (126.60±14.54), and (12.93±0.58) U/gprot in groups A, B, C, and D respectively; groups A, B, and C were significantly higher than group D, and group C was significantly higher than groups A and B ( P <0.05). Alizarin red staining showed that the percentages of calcium nodules was 20.07%±4.24% in group C; group C was significantly higher than groups A, B, and D ( P <0.05). The expression levels of BGP and collagen type I mRNA were significantly higher in groups A, B, and C than group D, and in group C than groups A and B ( P <0.05). The expression level of Runx2 mRNA was significantly higher in groups B and C than group D, and in group C than group B ( P <0.05), but no significant difference was found between groups A and D ( P >0.05)., Conclusions: PA elastic modulus of 10-40 kPa can promote the proliferation and osteogenic differentiation of BMSCs, and the higher the stiffness, the stronger the promoting effect.