Objective: To investigate the effect of polydimethylsiloxane (PDMS) matrix elasticity on osteogenic differentiation of rat marrow stromal cells (rBMSC). Methods: A series of PDMS composite substrates with different elastic modulus were constructed by adjusting the relative concentrations of cross-linking agent. The Young's modulus was used to describe the elasticity of PDMS after measurement by atomic force microscope (AFM). After surface modification, rBMSC was seeded on PDMS matrix, and 7 days after rBMSC was cultured on the five different Young's moduli matrix, the differences of osteogenic differentiation of rBMSC were observed by the method of real-time PCR, Western blotting, and alkaline phosphatase assay. Results: The PDMS was suitable for cell culture after surface modification, and by altering the concentration of cross-linking agent, PDMS could mimic the majority of the tissues' elasticity in vivo. The related osteogenic differentiation markers expression showed significant difference between the five matrixes (P<0.05), including type Ⅰ collagen (Col-Ⅰ), osteocalcin (OCN), osteopontin (OPN) and bone morphogenetic protein 2 (BMP2). The expression of osteogenic markers was up-regulated in the group that the Young's modulus was (354.1±40.9) kPa (P<0.05). Conclusions: PDMS is a tunable elasticity matrix which could be used in the investigation of inducing rBMSCs into osteoblastic lineages. PDMS substrate stiffness has an obvious influence on rBMSC osteogenic differentiation.
目的： 模拟体内组织弹性微环境，构建聚二甲基硅氧烷（polydimethylsiloxane，PDMS）基底，探究该基底的弹性模量对大鼠骨髓间充质干细胞（rat bone marrow stromal cells，rBMSC）成骨分化的影响。 方法： 通过调节PDMS基底固化剂及预聚物的比例（质量比1∶10、1∶20、1∶35、1∶60、1∶80）制备5组弹性基底（分别为1∶10、1∶20、1∶35、1∶60及1∶80组）；采用原子力显微镜观察并测量基底表面的弹性模量；rBMSC在不同弹性模量基底上诱导培养7 d后，采用实时荧光定量PCR、蛋白质免疫印迹法、免疫荧光染色检测及碱性磷酸酶（alkaline phosphatase assay，ALP）活性测定等方法检测成骨标志物骨桥蛋白、骨形态发生蛋白（bone morphogenetic protein 2，BMP2）、Ⅰ型胶原、骨钙蛋白的蛋白及mRNA表达。 结果： PDMS能模拟生物体内不同组织的弹性，经过表面修饰后，rBMSC在其上贴壁良好，形态规则。培养7 d后，不同弹性基底上rBMSC的4种成骨标志物的表达不同，差异有统计学意义（P<0.05）。1∶20组[弹性模量为（354.1±40.9）kPa]，其rBMSC表达的ALP活性显著高于其他4组（P<0.05），其骨桥蛋白、BMP2、Ⅰ型胶原的蛋白表达量（分别为1.80±0.37、1.79±0.28及4.12±0.94），BMP2、骨桥蛋白、Ⅰ型胶原及骨钙蛋白的mRNA表达量（分别为1.30±0.11、1.93±0.29、1.55±0.13及2.56±0.37）均有显著增高的趋势（P<0.05）。免疫荧光染色结果显示，Ⅰ型胶原、骨桥蛋白及骨钙蛋白3种成骨标志物在细胞内部呈阳性表达。 结论： PDMS可满足弹性基底诱导rBMSC骨向分化实验的要求，可作为良好的可观察的实验基底材料，其基底的弹性模量对rBMSC的骨向分化有明显影响。当PDMS弹性模量在特定范围内时，rBMSC向成骨系方向分化最理想。.
Keywords: Bone Mesenchymal stem cells; Matrix elasticity; Osteogenic differentiation; Polydimethylsiloxane.