【摘要】：生物材料植入宿主體內(nèi)后，其與生物系統(tǒng)的相互作用（蛋白吸附、細(xì)胞粘附/增殖等）發(fā)生在材料表面。細(xì)胞的生物學(xué)行為主要由材料表面化學(xué)成分和宏觀、介觀與微觀多尺度的拓?fù)浣Y(jié)構(gòu)構(gòu)成的局部微環(huán)境決定。因此，如何構(gòu)建材料表面適宜的細(xì)胞外微環(huán)境，進(jìn)而調(diào)控細(xì)胞的生理功能，已成為相關(guān)領(lǐng)域的研究熱點(diǎn)之一。 鈦及鈦合金由于具有良好的物理性能已被作為植入體材料廣泛應(yīng)用于骨科臨床領(lǐng)域。不足之處是，鈦及鈦合金材料表面有生物惰性，缺乏誘導(dǎo)骨生成潛能，導(dǎo)致鈦基植入體與周邊自然骨組織的整合性差，使用壽命短，是其臨床應(yīng)用面臨的普遍挑戰(zhàn)。鑒于此，為了實(shí)現(xiàn)鈦材表面原位調(diào)控細(xì)胞生物學(xué)行為，進(jìn)而誘導(dǎo)骨組織形成，亟需研發(fā)新的鈦材表面改性技術(shù)。從模擬類骨納米結(jié)構(gòu)及細(xì)胞外基質(zhì)組分角度出發(fā)，本論文利用機(jī)械研磨技術(shù)、陽極氧化等方法制備了表面納米拓?fù)浣Y(jié)構(gòu)的鈦基材，進(jìn)而沉積磷灰石/明膠，構(gòu)建適宜的細(xì)胞外微環(huán)境，以期提高鈦基植入體的骨整合性。主要研究?jī)?nèi)容和結(jié)論如下： 1.表面納米結(jié)構(gòu)化鈦材對(duì)骨髓間充質(zhì)干細(xì)胞行為的影響 為探究表面納米結(jié)構(gòu)化鈦材對(duì)骨髓間充質(zhì)干細(xì)胞生理行為的影響，本章利用機(jī)械研磨技術(shù)制備了表面納米結(jié)構(gòu)化鈦材。通過掃描電子顯微鏡（SEM），透射電鏡（TEM），原子力顯微鏡（AFM），X射線衍射儀（XRD）和接觸角測(cè)試對(duì)表面特性進(jìn)行表征。結(jié)果表明，經(jīng)機(jī)械研磨技術(shù)處理后，在鈦材表面上形成了納米結(jié)構(gòu)化薄層。通過纖連蛋白（Fn）與牛血清白蛋白（BSA）進(jìn)行蛋白質(zhì)吸附實(shí)驗(yàn)，結(jié)果表明，與未處理的純鈦相比，表面納米結(jié)構(gòu)化鈦材對(duì)蛋白吸附量無顯著的差異性。此外，在細(xì)胞與分子水平上，通過紐蛋白（vinculin）染色，四唑鹽比色（MTT）檢測(cè)，堿性磷酸酶（ALP）活性檢測(cè)，骨鈣素（OCN）與骨橋蛋白（OPN）染色，茜紅素定量檢測(cè)及骨鈣素（OCN）、骨橋蛋白（OPN）、I型膠原蛋白（collagen I）與轉(zhuǎn)錄因子Runx2在mRNA水平表達(dá)等實(shí)驗(yàn)，分別研究了表面納米結(jié)構(gòu)化鈦材對(duì)骨髓間充質(zhì)干細(xì)胞粘附、鋪展、增殖及分化的影響。結(jié)果表明，表面納米結(jié)構(gòu)化鈦材促進(jìn)了骨髓間充質(zhì)干細(xì)胞粘附、增殖及骨發(fā)生相關(guān)的蛋白及基因在蛋白質(zhì)與mRNA水平上的表達(dá)。本研究提供了一種制備表面納米結(jié)構(gòu)化鈦材的新方法。 2. BMP2功能化TiO_2納米管對(duì)骨髓間充質(zhì)干細(xì)胞行為的協(xié)同效應(yīng) 為探究BMP2功能化TiO_2納米管對(duì)骨髓間充質(zhì)干細(xì)胞行為的影響，本章利用聚多巴胺中間層將BMP2接枝到不同尺寸直徑（30nm、60nm與100nm）的TiO_2納米管上，并利用掃描電子顯微鏡（SEM），X光電子譜（XPS）及接觸角對(duì)材料進(jìn)行了表征。結(jié)果表明，BMP2已成功地接枝到TiO_2納米管上。此外，進(jìn)一步研究了BMP2修飾的TiO_2納米管對(duì)骨髓間充質(zhì)干細(xì)胞行為的影響。紐蛋白熒光染色結(jié)果表明，BMP2功能化的TiO_2納米管促進(jìn)了細(xì)胞粘附與生長(zhǎng)，且經(jīng)過7天與14天培養(yǎng)后，在BMP2功能化的TiO_2納米管上生長(zhǎng)的骨髓間充質(zhì)干細(xì)胞表現(xiàn)更高的堿性磷酸酶（ALP）活性與礦化量（p0.05或p0.01），其中，BMP2功能化的管徑為30nm的TiO_2納米管上生長(zhǎng)的細(xì)胞表現(xiàn)最高。該結(jié)果表明，BMP2功能化的TiO_2納米管拓?fù)浣Y(jié)構(gòu)協(xié)同增效地促進(jìn)了骨髓間充質(zhì)干細(xì)胞的增殖與分化。本研究為研發(fā)高骨整合性鈦植入體提供了新方法。 3.鈦材表面微環(huán)境的構(gòu)建及其對(duì)體外骨髓間充質(zhì)干細(xì)胞成骨分化與體內(nèi)成骨的影響 為模擬自然骨的細(xì)胞外基質(zhì)，，本章利用共沉淀方法將磷灰石/明膠沉積在納米結(jié)構(gòu)化鈦材表面，這種納米結(jié)構(gòu)化鈦材是通過氫氧化鉀與加熱處理后，在表面形成的抗腐蝕納米結(jié)構(gòu)化層。利用紅外（FTIR），場(chǎng)發(fā)射掃描電子顯微鏡（FE-SEM），原子力顯微鏡（AFM）與薄膜X線衍射（TE-XRD）對(duì)材料進(jìn)行了表征。檢測(cè)結(jié)果表明，磷灰石/明膠成功地沉積在納米結(jié)構(gòu)化鈦材表面。紐蛋白的熒光染色結(jié)果表明，磷灰石/明膠納米成分促進(jìn)了細(xì)胞粘附，更重要的是，在第7，14與21天時(shí)，在磷灰石/明膠納米成分鈦材上生長(zhǎng)的骨髓間充質(zhì)干細(xì)胞表現(xiàn)了更高的增殖與堿性磷酸酶（ALP）活性。并且，骨鈣素（OCN）、骨橋蛋白（OPN）和I型膠原蛋白（p0.05或p0.01）的表達(dá)得到了更大地提高。通過OCN與OPN的免疫熒光染色也得到了同樣的結(jié)果。通過組織切片，X光片與micro-CT（micro computedtomography，微計(jì)算機(jī)斷層掃描技術(shù)）分析得知磷灰石/明膠納米成分提高了骨密度和骨-植入體的接觸率（p0.05或p0.01），誘導(dǎo)植入體與骨界面間新骨的生長(zhǎng)。以上結(jié)果表明，磷灰石/明膠納米成分促進(jìn)了體內(nèi)外成骨。本研究為制備高性能鈦材植入體提供了新技術(shù)。
[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
【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R318.08

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

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