【作者】 陈赟；

【导师】 何福明；

【作者基本信息】 浙江大学 ， 口腔临床医学， 2015， 硕士

【摘要】 在口腔种植领域,Branemark教授提出的骨结合(osseointegration)理论一直指导着世界各国学者对种植体表面改性的研究。如何促进细胞增殖、分化从而诱导骨形成,以期获得早期骨结合一直成为种植体表面改性研究的热点。锶(Sr)元素因其有促进成骨和抑制骨吸收的作用而具有很大的医用潜力。本课题组在一系列水热参数下可控地制备出掺锶二氧化钛涂层。在表征分析的基础上择出最优水热制备参数,并进一步进行体外生物相容性实验以及体内早期对骨髓间充质干细胞行为影响的评估。研究目的：1.通过分析不同参数下使用水热法制备出的掺锶种植体表面的表征,推导掺锶纳米表面形成的机理。2.通过体外细胞实验,研究掺锶种植体和非掺锶种植体对前成骨细胞、骨髓间充质干细胞增殖、分化以及体外成骨的影响。3.通过体内植入实验,研究掺锶和非掺锶种植体周围早期骨组织形态学改变及对骨髓间充质干细胞行为的影响。研究方法：1.根据不同的Sr(OH)2水热液浓度、水热温度、时间制备出一系列掺锶二氧化钛种植体表面。使用扫描电镜、透射电镜、X射线衍射、X射线光电子能谱分析、接触角等方法分析掺锶二氧化钛种植体表面的表征。2.在掺锶种植体和非掺锶种植体表面培养前成骨细胞(MC3T3-E1)及骨髓间充质干细胞(BMSC),一定时间点下检测增殖能力、粘附能力、碱性磷酸酶(ALP)活性、骨钙素(OC)释放、关键成骨基因表达、钙盐沉积量来评价体外细胞在不同表面的增殖和分化能力。3.掺锶种植体和非掺锶种植体植入SD大鼠股骨干骺端,6天后取出,扫描电镜下观察种植体表面形貌,种植体周骨组织进行HE染色及Stro-1免疫组化标记,分析早期骨愈合能力及不同种植体对骨髓间充质干细胞招募的影响。结果：1.水热液浓度、水热温度、时间可显著影响种植体表面形貌和表面物质成分、晶型。2.掺锶种植体可释放锶离子长达一周以上,与非掺锶种植体相比可显著的促进细胞分化,上调成骨相关基因的表达,增加体外钙盐沉积(P<0.05),利于体外成骨。3.体内植入实验证实了种植体植入初期的类炎症性反应,同时扭出的种植体表面布满类骨胶原纤维样物质,而掺锶种植体周围骨组织含有更多的Stro-1标记的骨髓间充质干细胞(P=0.0125)。结论：1.本研究中水热法使用的最佳参数为：0.02M Sr(OH)2,220℃,12h,使用此参数可获得具有良好的亲水性掺锶微、纳米二氧化钛涂层,并且可促进体外细胞分化、成骨。2.最佳参数下制备的掺锶表面可招募Stro-1高表达的骨髓间充质干细胞,因而具有潜在的促进早期骨结合的能力。更多还原

【Abstract】 Last century, the "osseointegration" investigated by professor Branemark inducted the surface modification of implant for researchers all over the world in the field of oral implant. Improving the osteoconduction and osseointegration of biomedical titanium has been the main purpose to pursue. Strontium, a potential ion, was proved to increase bone formation and decrease resorption. Our group optimized the production of nano-structured strontium permeated into titanium oxide surface using hydrothermal technique and evaluated the biological characterization properties and response of bone mesenchymal stem cells (BMSCs) in vivo in early stage.Objective:1. Detect the characterization of Sr containing titanium oxide surface prepared by hydrothermal treatment in the series of parameters and confirme the nanostructure evolution.2. Investigate the proliferation, differentiation and osteogenesis of MC3T3-E1and BMSCs cultured on Sr containing surface and Mod Ti.3. In vivo experiments were used to evaluate the response of peri-implant tissues and BMSCs. Methods:1. A series concentration of Sr(OH)2solution was used for hydrothermal treatment at different temperature and time. Optimization of the solution, temperature, heating time and subsequent characterization of the hydrothermal derived oxide titanium layers were carried out using FESEM, TFXRD, XPS, contact angle measurement and TEM.2. We evaluated the MC3T3-E1and BMSCs response by initial adhesion, proliferation, alkaline phosphatase (ALP), osteocalcin (OC), osteogenesis related genes and calcium mineralization in vitro.3. Implants were implanted into proximal tibia of SD rats. After6days, the implants were unscrewed for SEM observation. H&E staining and immunohistochemistry analysis with Stro-1antibody on tissue adjacent to implants were used to evaluate the ability of bone healing and BMSCs recruiting in vivo.Results1. The concentration, heating temperature and time affected the surface morphology significantly and varied the composition and phase of surfaces.2. The Sr containing surface could release Sr for more than one week, which promoted the differentiation of cells, up regulated osteogenesis related genes, increased extracellular matrix mineralization(P<0.05) and eventually enhanced the osteogenesis in vitro.3. The unscrewed implants were covered with quite organized mesh formed by discrete and small bundles of collagen and transitory hematoma in adjacent bone tissue by H&E staining. The Stro-1labeled MSCs were observed noticeably around Sr containing implants in immunohistochemistry analysis (P=0.0125).Conclusions:1. The hydrophilic micro/nano-structured strontium-containing oxide surface could be obtained under the best parameter (0.02M Sr(OH)2,220℃,12h) and this surface showed improvement on cell differentiation and osteogenesis in vitro.2. This Sr containing surface had a potential ability to enhance early osseointegration as it could also recruit rat Stro-1positive MSCs to peri-implant tissue in vivo.更多还原