不同管径钛纳米管对大鼠骨髓间充质干细胞增殖及成骨分化的影响  

Effects of different-diameter TiO_2 nanotubes on proliferation and osteogenic differentiation of rat mesenchymal stem cells

作　　者：王明[1] 杨生[1] 邓锋[1] 王璐[1] 张翼[1] 张赫[1] 

机构地区：[1]重庆医科大学口腔医学院,口腔疾病与生物医学重庆市重点实验室,重庆401147

出　　处：《第三军医大学学报》2014年第12期1273-1278,共6页Journal of Third Military Medical University

基　　金：重庆医科大学口腔医学院博士科研启动基金(2011);重庆市卫生局面上项目(2012-2-121);重庆市科委自然科学基金(CSTC2012jjA0178);高等学校博士学科点专项科研基金新教师类资助课题(20125503120009)~~

摘　　要：目的研究不同管径钛纳米管表面结构对体外培养SD大鼠骨髓间充质干细胞(rat mesenchymal stem cells,rMSCs)粘附、增殖及成骨向分化的影响。方法分离并扩增rMSCs,成骨向诱导分化及流式细胞鉴定;阳极氧化法分别制备管径30、70、100 nm的TiO2纳米管,扫描电镜观察表面形貌、接触角测试材料表面亲疏水性;MTT法检测细胞在材料表面粘附及增殖情况,激光共聚焦显微镜观察细胞在材料表面形貌。评价细胞在纯钛,管径30、70、100 nm的TiO2纳米管表面成骨向分化过程中碱性磷酸酶活性变化特征及矿化情况。Real-time PCR检测诱导14 d后Runx2及OSX基因表达情况。结果分离获得的rMSCs具有成骨向分化的能力,CD29阳性细胞为99.65%;扫描电镜观察各组材料表面管径均达到设计要求;管径为70 nm的TiO2纳米管材料表面亲水性较好(P<0.05);MTT结果显示70 nm组rMSCs的粘附、增殖均较其他组明显增高(P<0.05);30 nm组在成骨诱导7 d后,碱性磷酸酶活性均高于其他组(P<0.05)。30 nm组在成骨诱导21 d后表面矿化高于其他组(P<0.05)。30 nm组在成骨诱导14 d后Runx2及OSX基因表达高于其余组(P<0.05)。结论与光滑面纯钛相比,不同管径的TiO2纳米管对rMSCs的作用各不相同,rMSCs在70nm表面增殖能力和粘附性较好,但在30 nm表面的成骨向分化能力较强。Objective To investigate the effects of different-diameter TiO2 nanotubes prepared by anodization on the adhesion, proliferation and osteogenic differentiation of Sprague-Dawley rat mesenchymal stem cells （rMSCs）. Methods The isolated rMSCs were characterized by osteogenic differentiation and flow cytometry. TiO2 nanotubes having diameter of 30, 70 and 100 nm were fabricated by anodization with different voltages. The topography and hydrophilicity of the nanotubes were accessed with scanning electron microscopy （SEM） and contact angle test, respectively. MTT assay was applied to evaluate the cell adhesion and proliferation on different substrates. Cell morphologies were observed by confocal microscopy. The activity of alkaline phosphatase and mineralization of rMSCs on plane titanium and TiO2 nanotubes （diameter=30, 70 and 100 nm） were also detected during osteogenic differentiation. The expression of Runx2 and OSX was analyzed by real-time PCR in 14 d after osteogenic differentiation.Results The osteogenic differentiation of rMSCs was successfully induced, and CD29 positive cells were 99.65%. TiO2 nanotubes of different diameters were generated. The TiO2 nanotubes of 70 nm diameter were more hydrophilic （P〈0.05） and exhibited better cell adhesion and proliferation compared with other substrates （P〈0.05）. The cells cultured on the TiO2 nanotubes of 30 nm diameter displayed much higher activity of alkaline phosphatase than those on other substrates in 7 d after osteogenic induction （P〈0.05）, higher mineralization in 21 d after osteogenic induction （P〈0.05） and more Runx2 and OSX gene expression in 14 d after osteogenic induction （P〈0.05）. Conclusion TiO2 nanotubes with different diameters have different influences on rMSCs as compared to plane titanium. The TiO2 nanotubes of 70 nm diameter are more advantageous for adhesion and proliferation of rMSCs, while TiO2 nanotubes of 30 nm diameter facilitate osteogenic differentiation.

关 键 词：TIO2纳米管 骨髓间充质干细胞 成骨向分化 

分 类 号：R318.08[医药卫生—生物医学工程] R329.28[医药卫生—基础医学]