钛材表面纳米结构化及其对骨髓间充质干细胞的影响

发布时间：2019-06-02 23:01

【摘要】：生物材料植入宿主体内后，其与生物系统的相互作用（蛋白吸附、细胞粘附/增殖等）发生在材料表面。细胞的生物学行为主要由材料表面化学成分和宏观、介观与微观多尺度的拓扑结构构成的局部微环境决定。因此，如何构建材料表面适宜的细胞外微环境，进而调控细胞的生理功能，已成为相关领域的研究热点之一。钛及钛合金由于具有良好的物理性能已被作为植入体材料广泛应用于骨科临床领域。不足之处是，钛及钛合金材料表面有生物惰性，缺乏诱导骨生成潜能，导致钛基植入体与周边自然骨组织的整合性差，使用寿命短，是其临床应用面临的普遍挑战。鉴于此，为了实现钛材表面原位调控细胞生物学行为，进而诱导骨组织形成，亟需研发新的钛材表面改性技术。从模拟类骨纳米结构及细胞外基质组分角度出发，本论文利用机械研磨技术、阳极氧化等方法制备了表面纳米拓扑结构的钛基材，进而沉积磷灰石/明胶，构建适宜的细胞外微环境，以期提高钛基植入体的骨整合性。主要研究内容和结论如下： 1.表面纳米结构化钛材对骨髓间充质干细胞行为的影响为探究表面纳米结构化钛材对骨髓间充质干细胞生理行为的影响，本章利用机械研磨技术制备了表面纳米结构化钛材。通过扫描电子显微镜（SEM），透射电镜（TEM），原子力显微镜（AFM），X射线衍射仪（XRD）和接触角测试对表面特性进行表征。结果表明，经机械研磨技术处理后，在钛材表面上形成了纳米结构化薄层。通过纤连蛋白（Fn）与牛血清白蛋白（BSA）进行蛋白质吸附实验，结果表明，与未处理的纯钛相比，表面纳米结构化钛材对蛋白吸附量无显著的差异性。此外，在细胞与分子水平上，通过纽蛋白（vinculin）染色，四唑盐比色（MTT）检测，碱性磷酸酶（ALP）活性检测，骨钙素（OCN）与骨桥蛋白（OPN）染色，茜红素定量检测及骨钙素（OCN）、骨桥蛋白（OPN）、I型胶原蛋白（collagen I）与转录因子Runx2在mRNA水平表达等实验，分别研究了表面纳米结构化钛材对骨髓间充质干细胞粘附、铺展、增殖及分化的影响。结果表明，表面纳米结构化钛材促进了骨髓间充质干细胞粘附、增殖及骨发生相关的蛋白及基因在蛋白质与mRNA水平上的表达。本研究提供了一种制备表面纳米结构化钛材的新方法。 2. BMP2功能化TiO_2纳米管对骨髓间充质干细胞行为的协同效应为探究BMP2功能化TiO_2纳米管对骨髓间充质干细胞行为的影响，本章利用聚多巴胺中间层将BMP2接枝到不同尺寸直径（30nm、60nm与100nm）的TiO_2纳米管上，并利用扫描电子显微镜（SEM），X光电子谱（XPS）及接触角对材料进行了表征。结果表明，BMP2已成功地接枝到TiO_2纳米管上。此外，进一步研究了BMP2修饰的TiO_2纳米管对骨髓间充质干细胞行为的影响。纽蛋白荧光染色结果表明，BMP2功能化的TiO_2纳米管促进了细胞粘附与生长，且经过7天与14天培养后，在BMP2功能化的TiO_2纳米管上生长的骨髓间充质干细胞表现更高的碱性磷酸酶（ALP）活性与矿化量（p0.05或p0.01），其中，BMP2功能化的管径为30nm的TiO_2纳米管上生长的细胞表现最高。该结果表明，BMP2功能化的TiO_2纳米管拓扑结构协同增效地促进了骨髓间充质干细胞的增殖与分化。本研究为研发高骨整合性钛植入体提供了新方法。 3.钛材表面微环境的构建及其对体外骨髓间充质干细胞成骨分化与体内成骨的影响为模拟自然骨的细胞外基质，，本章利用共沉淀方法将磷灰石/明胶沉积在纳米结构化钛材表面，这种纳米结构化钛材是通过氢氧化钾与加热处理后，在表面形成的抗腐蚀纳米结构化层。利用红外（FTIR），场发射扫描电子显微镜（FE-SEM），原子力显微镜（AFM）与薄膜X线衍射（TE-XRD）对材料进行了表征。检测结果表明，磷灰石/明胶成功地沉积在纳米结构化钛材表面。纽蛋白的荧光染色结果表明，磷灰石/明胶纳米成分促进了细胞粘附，更重要的是，在第7，14与21天时，在磷灰石/明胶纳米成分钛材上生长的骨髓间充质干细胞表现了更高的增殖与碱性磷酸酶（ALP）活性。并且，骨钙素（OCN）、骨桥蛋白（OPN）和I型胶原蛋白（p0.05或p0.01）的表达得到了更大地提高。通过OCN与OPN的免疫荧光染色也得到了同样的结果。通过组织切片，X光片与micro-CT（micro computedtomography，微计算机断层扫描技术）分析得知磷灰石/明胶纳米成分提高了骨密度和骨-植入体的接触率（p0.05或p0.01），诱导植入体与骨界面间新骨的生长。以上结果表明，磷灰石/明胶纳米成分促进了体内外成骨。本研究为制备高性能钛材植入体提供了新技术。
[Abstract]:After the biological material is implanted in the host, its interaction with the biological system (protein adsorption, cell adhesion/ proliferation, etc.) occurs on the surface of the material. The biological behavior of the cells is mainly composed of the chemical composition of the surface of the material and the local micro-environment which is composed of macroscopic, mesoscopic and micro-scale topological structures. Therefore, how to construct a suitable extracellular microenvironment for the surface of a material, and to control the physiological function of the cells, has become one of the hot spots in the relevant fields. Titanium and titanium alloys have been widely used as implant materials in the clinical neck of orthopedics due to their good physical properties The deficiency is that the surface of the titanium and titanium alloy material is biologically inert and lacks the potential of inducing the bone formation, which leads to the loss of heterozygosity and short service life of the titanium-based implant and the surrounding natural bone tissue, and is a common choice for the clinical application of the titanium-based implant. In view of this, in order to achieve in-situ control of the biological behavior of the cells in the surface of the titanium material, the formation of bone tissue is induced, and a new surface modification technique of the titanium material is urgently needed to be developed. On the basis of the structure of the simulated bone and the component of the extracellular matrix, the titanium substrate of the surface nano-topological structure was prepared by mechanical polishing, anodic oxidation and the like, and then the apatite/ gelatin was deposited to construct the appropriate external microring of the cell. To improve the bone integrity of the titanium-based implant Sex. Main research content and conclusions such as Lower:1. The surface nanostructured titanium material is used for bone marrow mesenchymal stem cell line. In order to study the effect of surface nano-structured titanium material on the physiological behavior of bone marrow-derived mesenchymal stem cells, the surface nano-structure was prepared by mechanical grinding technology. The surface is measured by a scanning electron microscope (SEM), a transmission electron microscope (TEM), an atomic force microscope (AFM), an X-ray diffractometer (XRD), and a contact angle test. The results show that, after the mechanical polishing, the surface of the titanium material is nano-sized. The results of protein adsorption of fibronectin (Fn) and bovine serum albumin (BSA) show that the surface nanostructured titanium material has no significant effect on protein adsorption compared with untreated pure titanium. In addition, on the cellular and molecular level, the detection of the activity of alkaline phosphatase (ALP), the detection of the activity of alkaline phosphatase (ALP), the detection of the activity of alkaline phosphatase (OCN) and the osteopontin (OPN), the quantitative detection of the prodigiosin and the osteopontin (OCN), osteopontin The adhesion, spreading and proliferation of bone marrow-derived mesenchymal stem cells (MSCs), such as bone marrow-derived mesenchymal stem cells (MSCs), were studied by the experiments of the expression of mRNA and the expression of collagen (collagen I) and transcription factor Runx2. The results showed that the surface nano-structured titanium material promoted the adhesion, proliferation and bone-related proteins and genes of bone marrow-derived mesenchymal stem cells in protein and mRNA The present study provides a method for preparing the surface nanostructured titanium A new method of material preparation.2. BMP2 functionalized TiO _ 2 nanotubes can be used to fine bone marrow mesenchymal stem cells The synergistic effect of cellular behavior was to investigate the effect of BMP2-functionalized TiO _ 2 nanotubes on the behavior of bone marrow-derived mesenchymal stem cells. In this chapter, BMP2 was grafted to different sizes (30 nm,60 nm and 100 nm) by using the poly-dopamine intermediate layer. TiO _ 2 nanotubes, and scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and contact The results show that BMP2 has been successfully grafted to In addition, the effect of BMP2-modified TiO _ 2 nanotubes on the bone marrow was further studied. The results of the fluorescent staining of the new protein show that the BMP2 functionalized TiO _ 2 nanotubes promote the adhesion and growth of the cells and After 7 and 14 days, the bone marrow mesenchymal stem cells grown on the BMP2 functionalized TiO _ 2 nanotubes exhibited higher alkaline phosphatase (ALP) activity and mineralization (p0.05 or p0.01), in which the BMP2 functionalized TiO _ 2 nanotubes with a diameter of 30 nm The results showed that the topological structure of the BMP2 functionalized TiO _ 2 nanotube promoted the bone marrow-derived mesenchymal stem cells. The proliferation and differentiation of stem cells. the invention provides a novel method,3. the construction of a titanium material surface micro-environment and the on-vitro bone marrow mesenchymal stem cells The effect of osteogenic differentiation and in vivo osteogenesis is the extracellular matrix of simulated natural bone. the nano-structured titanium material is deposited on the surface of the nano-structured titanium material, Surface-formed corrosion-resistant nanostructured layers. Infrared (FTIR), field emission scanning electron microscopy (FE-SEM), atomic force microscopy (AFM), and thin film X-ray diffraction (TE -XRD) characterization of the material. The results showed that the apatite/ gelatin was successful The fluorescence staining of the new protein in the surface of the nanostructured titanium material shows that the apatite/ gelatin nano-component promotes cell adhesion, and more importantly, the bone marrow mesenchymal stem cells grown on the apatite/ gelatin nano-component titanium material exhibit higher proliferation on days 7,14 and 21. and alkaline phosphatase (ALP) activity. Also, osteocalcin (OCN), osteopontin (OPN) and type I collagen (p0.05 or p0.05) 1) The expression of OCN and OPN is improved. The same results were also obtained by fluorescence staining. The bone mineral density and the bone-implant contact rate (p0.05 or p0.01) were improved by the analysis of the tissue sections, X-ray and micro-CT (micro-computed tomography). The growth of the new bone between the implant and the bone interface. The above results indicate that the apatite/ min The glue nanometer component promotes the external bone formation in the body. The research is high in preparation
【学位授予单位】：重庆大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.08

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