【作者】 干锦波；

【导师】 陈红； 常江；

【作者基本信息】 武汉理工大学 ， 生物材料学， 2011， 硕士

【摘要】 蛋白质在表／界面的吸附以及细胞在材料表面的响应行为对材料的生物相容性起着至关重要的作用。当生物材料和机体环境接触后,首先发生的是体液中的蛋白质在表／界面的吸附,吸附的蛋白质层还可进一步诱导后续的细胞黏附、铺展、增殖等行为。由于材料表面拓扑结构是影响蛋白质和细胞行为的一个重要因素,因此,研究表面拓扑结构对蛋白质吸附和细胞响应行为的影响并阐明三者之间的内在联系,这对设计合理的生物材料表面、提高材料的生物相容性具有重要的理论意义和应用价值。基于此,本研究以聚二甲基硅氧烷和生物陶瓷为基材,利用微／纳加工技术在基材表面构筑特定的拓扑结构,分别研究化学组分均质和异质的表面拓扑结构以及表面拓扑结构和化学组分的协同作用对蛋白质吸附行为和细胞响应行为的影响。具体内容如下：1.化学组分均质的表面拓扑结构对蛋白质、细胞行为的影响采用水热法在45S5生物活性玻璃陶瓷表面构筑化学组分均质(羟基磷灰石(HA))的有序纳米棒结构(Ceramic 1)、无规纳米棒结构(Ceramic 2)和纳米片结构(Ceramic 3)。通过蛋白质吸附测试(单一蛋白质吸附体系和血浆蛋白质吸附体系)、蛋白质电泳(SDS-PAGE)和蛋白质印迹(Western-Blot)分析,研究单纯的表面拓扑结构对蛋白质吸附以及细胞黏附行为的影响。结果表明,不论是在单一蛋白质吸附环境还是在人血浆吸附环境下,具有微／纳拓扑结构的表面均有利于纤维蛋白原(Fg)和人血清白蛋白(HSA)的吸附,且样品比表面积越大(Ceramic 1>Ceramic 2>Ceramic 3)蛋白质吸附量也越高。从人骨髓原代细胞培养结果来看,表面纳米棒结构比纳米片结构(Ceramic 3)更有利于细胞的贴壁和铺展,而有序的纳米棒结构(Ceramic 1)又比无规纳米棒结构(Ceramic 2)的效果要好。由上述结果可知,对化学组分均质的表面拓扑结构而言,拓扑结构是影响蛋白质吸附和细胞响应行为的主要因素。2.化学组分异质的图案化表面拓扑结构对蛋白质、细胞行为的影响以聚二甲基硅氧烷(PDMS)为基材,通过硅氢加成反应在PDMS表面接枝烯丙基聚乙二醇(PDMS-PEG)。以透射电镜用铜网为光掩膜,采用紫外光刻蚀技术(Xe2 excimer:172 nm)在PDMS-PEG膜片上制备出化学组分异质的图案化表面拓扑结构。而后在此图案化表面上进行荧光素标记蛋白质(Fg-FITC)吸附以及L929细胞培养实验,结果表明,由于曝光区域的PEG被刻蚀除去并暴露出底层的PDMS基材从而促使Fg在该区域吸附,而未曝光部分的PEG则得以保留并能有效的排斥蛋白质的吸附。因此,Fg限定性地吸附于曝光区域并呈现图案化分布。当进行细胞培养时,细胞培养液中的蛋白质(包括与细胞黏附相关的蛋白质如纤粘蛋白(Fn)、玻粘蛋白(Vn)等)也限定性的吸附于曝光区域,而后L929细胞在该蛋白质吸附层的引导下黏附、铺展于曝光区域,从而实现了细胞图案化。从以上结果可知,对化学组分异质的表面拓扑结构而言,化学组分对蛋白质吸附行为和细胞响应行为的影响占主导地位。3.表面拓扑结构和化学组分的协同作用对蛋白质、细胞行为的影响以镁黄长石(Akermanite)为基材,将其浸入模拟体液环境中从而在样品表面生成一层类骨磷灰石层(Akermanite-HAp),扫描电子显微镜(SEM)和X射线衍射仪(XRD)测试结果表明新生层的化学组分为羟基磷灰石并具有一定的微／纳米级棒状拓扑结构。而后以未修饰的镁黄长石作为对照组,考察Fg、HSA、Fn、Vn在样品表面的吸附行为以及骨髓间充质干细胞(BMSC)的增殖生长状况。结果表明：由于Akermanite-HAp样品表面生成的类骨磷灰石层具有一定的微／纳拓扑结构,有利于蛋白质的吸附(包括与细胞黏附相关的蛋白质如Fn、Vn等)以及细胞的黏附和附着,同时微／纳米棒之间的缝隙也有利于溶液的流动、营养物质的交换以及氧气的输送等。另外,这种新生成的类骨磷灰石层具有一定的生物活性,自身也能促进成骨细胞的黏附和增殖。在这些因素的共同作用下使得BMSC在Akermanite-HAp样品表面的增殖水平高于对照组。以上结果说明,表面拓扑结构和化学组分是紧密联系、相辅相成的,两者共同决定着蛋白质和细胞在材料表面的行为。更多还原

【Abstract】 Protein adsorption on interface and cell responsive behaviors such as adhesion, spreading and proliferationon on biomaterial’s surface plays a vital role in determining biomaterial’s biocompatibility. As is known to us, proteins (from body fluid) adsorption on surface is the first response after biomaterials contacting with biological environment and this preadsorbed protein layer could further mediate subsequent cell behaviors. Since surface topography has evident effects on protein adsorption and cell responsive behaviors; investigating and elucidating the relationship among surface topography, protein and cell is significant for the designing of rational surface of biomaterials and the improvement of their biocompatibility.In this research, different surface topographical features were prepared on the substrates of poly(dimethylsiloxane) and bioceramic by micro/nano fabrication techniques. Further experiments were conducted to investigate the single effect of either chemical homogeneous or chemical heterogeneous surface topography as well as the synergic effect of surface topography and chemical composition on protein adsorption and cell responsive behaviors. Detail works were concluded as follows:1. Effect of chemical homogeneous surface topography on protein and cellDifferent surface topographical features (ordered hydroxyapatite (HA) nanorod arrays (Ceramic 1), disordered HA nanorod arrays (Ceramic 2) and HA nanosheet arrays (Ceramic 3)) were prepared on 45S5 bioactive glass-ceramic substrate through hydrothermal process. Protein adsorption (single protein adsorption and human plasma adsorption environment), protein electrophoresis (SDS-PAGE), Western-Blot and cell culture experiments were conducted to investigate the effect of surface topography on protein adsorption and cell adhesion. Results indicated that surfaces with micro/nano topography could enhance protein adsorption in both adsorption environment and higher specific surface area (Ceramic 1>Ceramic 2>Ceramic 3) meant a higher protein adsorption amount. Primary human bone marrow cells adhesion and spreading status on the surface of nanorod array samples were better than that on nanosheet array surface, and cell behaviors on ordered nanorod array sample were the best of all. It is concluded that surface topographical feature is the chief factor in mediating protein adsorption and cell responsive behaviors for chemical homogeneous surface topography.2. Effect of chemical heterogeneous patterned surface topography on proteinadsorption and cell behaviorAllyl-polyethylene glycol (PEG) was grafted onto poly(dimethylsiloxane) surface (PDMS-PEG) using hydrosilylation reaction. Then chemical heterogeneous patterned PDMS-PEG surface topography was fabricated by ultraviolet lithography (Xe2 excimer:172nm) with copper mesh as a photomask. Fluorescence labelled protein (Fg-FITC) adsorption results indicated that, as PEG in UV exposed domains was etched and PDMS substrate was revealed which could promote protein adsorption while reserved PEG in unexposed regions effectively resisted protein adsorption, fibrinogen (Fg) restrictively adsorbed on UV exposed domains and presented a patterned distribution in accord with the patterned substrate. Likewise, proteins in cell culture medium (including cell adhesive proteins, such as fibronectin (Fn) and vitronectin (Vn)) were confined in UV exposed domains, which promoted L929 cell adhesion and spreading, forming a regular cell pattern. Our results informed that chemical composition is the dominant issue in mediating protein adsorption and cell responsive behaviors for chemical heterogeneous surface topography.3. The synergic effect of surface topography and chemistry on protein and cellAkermanite bioceramics were immersed in simulated body fluid to obtain bone-like apatite layer coated samples (Akermanite-HAp). Scanning electron microscope (SEM) and X-ray diffraction (XRD) characterizations demonstrated that this apatite layer exhibited specific micro/nano topography with a chemical composition of hydroxyapatite. Investigation of protein adsorption and bone marrow mesenchymal stem cells (BMSC) proliferation were conducted with unmodified bioceramics (Akermanite) as control samples. Results showed that micro/nano-scale rods topography in apatite layer could enhance protein adsorption, which resulted in a higher protein adsorption amount on Akermanite-HAp than that on Akermanite. This preadsorbed protein layer (including cell adhesive proteins, such as Fn and Vn) could favor BMSC adhesion and differentiation on Akermanite-HAp. Besides, microstructure of bone-like apatite layer may increase the capacity of biological liquid adsorption into ceramic substrates, enhance ionic exchanging with body fluids and facilitate the transporting of oxygen and nutrients, which could permit the close contact of cells on surface and provide convenient sites for bone cell colonization. Furthermore, the newly-formed apatite layer could promote the adhesion and proliferation of osteoblast cells due to its bioactivity. This synergic effect of surface topography and chemical composition made Akermanite-HAp more favorable for cell adhesion and proliferation than that of Akermanite. These results suggested that surface topography and chemical composition were closely interconnected and complementary to each other, affecting protein adsorption and cell responsive behaviors on surface together.更多还原