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目的探讨基底硬度与形貌协同对大鼠骨髓间充质干细胞(rat bone mesenchymal stem cells,r BMSCs)形态、增殖以及成骨分化的影响。方法分别在硬度为3.5 MPa、槽、脊宽为0.3μm的聚二甲基硅氧烷(polydimethylsiloxane,PDMS)基底;硬度为3.5 MPa、槽、脊宽为1.8μm的PDMS基底;硬度为3.5 MPa的平面PDMS基底;硬度为0.27 MPa、槽、脊宽为0.3μm的PDMS基底;硬度为0.27 MPa、槽、脊宽为1.8μm的PDMS基底;硬度为0.27 MPa的平面PDMS基底上培养r BMSCs,利用倒置荧光显微镜观察r BMSCs的形态,CCK-8试剂盒检测r BMSCs的增殖情况,碱性磷酸酶(alkalinephosphatase,ALP)试剂盒检测r BMSCs的ALP活性,免疫荧光技术检测骨钙蛋白(osteocalcin,OCN)及I型胶原(collagen type I,COL I)的表达,qRT-PCR检测Runx2 mRNA的表达。结果在硬度为3.5 MPa以及槽、脊宽为0.3μm的PDMS上r BMSCs铺展更好、增殖更快,ALP活性更高,OCN、COL I及Runx2mRNA表达量明显多于其他各组。结论基底硬度对r BMSCs的增殖有明显影响,而硬度与形貌能协同促进r BMSCs的增殖及成骨分化。研究结果有助于了解生物物理因素在某些疾病(如骨质疏松)发病机制过程中的作用,并可为骨组织工程新材料的研发提供理论基础。
Objective To investigate the effects of substrate hardness and topography on the morphology, proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs). Methods The surface of PDMS substrate with a hardness of 3.5 MPa, a groove width of 0.3 μm and a polydimethylsiloxane (PDMS) substrate with a width of 0.3 μm, a PDMS substrate with a hardness of 3.5 MPa, a groove width of 1.8 μm and a hardness of 3.5 MPa PDMS substrate with a hardness of 0.27 MPa, a groove width of 0.3 μm, a PDMS substrate with a hardness of 0.27 MPa and a groove width of 1.8 μm, and rMSCs cultured on a flat PDMS substrate with a hardness of 0.27 MPa, The morphological changes of rBMSCs were observed by inverted fluorescence microscope. The proliferation of rBMSCs was detected by CCK-8 kit. ALP activity was detected by alkaline phosphatase (ALP) kit. The expression of osteocalcin was detected by immunofluorescence. OCN) and collagen type I (COL I) expression. Runx2 mRNA expression was detected by qRT-PCR. Results rMSCs proliferated faster and had higher ALP activity on PDMS with a hardness of 3.5 MPa and a groove width of 0.3 μm. The expression of OCN, COL I and Runx2 mRNA was significantly higher than that of other groups. Conclusion The substrate hardness has a significant effect on the proliferation of r BMSCs, while the hardness and morphology can promote the proliferation and osteogenic differentiation of r BMSCs. The findings help to understand the role of biophysical factors in the pathogenesis of certain diseases such as osteoporosis and provide a theoretical basis for the development of new materials for bone tissue engineering.