钛系材料微纳结构表面成骨细胞及胶原蛋白吸附机理研究

发布时间：2018-05-05 07:15

本文选题：表面拓扑结构 + 成骨细胞　；参考：《哈尔滨工业大学》2017年博士论文

【摘要】：随着材料制备、表面检测及表征技术的迅猛发展,生物材料逐步向功能化和智能化方向发展,其在分子水平上对细胞行为的调控能力也备受关注。由于骨置换术需求的不断增加,在骨组织替换领域,对植入体的生物学性能,如骨整合性的要求越来越高。因此,合理设计和制备生物材料并使其具备分子识别能力,诱导细胞产生特定的响应或形成新的组织,已成为当前组织工程领域迫切需要解决的关键问题。钛合金是常用的医用金属材料,在硬组织替换领域有非常广泛的应用,对钛合金医用材料实施合理的表面改性有助于提高其生物相容性。在众多影响组织/细胞-材料界面相互作用的表面特征因素中,表面拓扑形貌能够独立影响细胞行为,阐明表面微纳拓扑形貌诱导和调控细胞行为的作用机理是指导和设计生物材料的重要前提。基于细胞外蛋白质在细胞黏附过程中所起的重要调节作用,研究蛋白质与材料之间的相互作用对揭示细胞黏附机理具有重要意义。本文将围绕生物材料与细胞之间的界面相互作用机理,采用细胞实验和分子模拟相结合的方法,探讨表面拓扑结构对成骨细胞黏附的影响规律,分析表面纳结构对细胞外黏附蛋白的吸附过程可能产生的影响,为建立材料表面拓扑结构的生物相容性评价体系提供实验和理论指导。鉴于生物材料表面拓扑结构具有独立影响细胞行为的能力,本文首先从细胞实验角度分析了Ti6Al7Nb表面粗糙度对MC3T3-E1成骨细胞黏附和增殖特性的影响,探讨了PDMS表面沟槽结构的几何参数对细胞形成“接触引导”现象的影响规律。实验结果表明,成骨细胞的铺展能力和增殖能力随钛合金表面粗糙度增加而减小。随着PDMS表面沟槽宽度增加,成骨细胞的“接触引导”现象愈加凸显。生物材料表面拓扑结构能够约束细胞伪足的生长,从而诱导细胞产生“接触引导”等特定的细胞行为。钛合金表面纳结构在水环境下可能发生羟基化,继而影响细胞黏附和蛋白质的吸附特性,本文基于第一原理计算方法,建立了金红石型TiO2(110)表面台阶结构的羟基化模型。研究结果表明TiO2(110)表面沿11—1晶向分布的台阶结构具有较高的表面能密度,是水分子分解的活性位点,故由此利用第一原理能量最小化方法获得了稳定羟基化模型的原子结构及分子力场。本文开展的水分子在表面台阶结构附近分解的动力学模拟结果显示,水分子分解后产生的自由质子能被台阶附近不饱和氧原子夺走;在较高的水分子覆盖率条件下,自由质子又能以水合氢离子的形式逐渐扩散到水环境中。在羟基化模型基础上,借助经典分子动力学方法研究了表面羟基化对近表面水层分布的影响,发现表面羟基化能够弱化水分子在TiO2表面分层分布的现象。为了研究细胞外黏附蛋白在纳结构TiO2表面的吸附特性,本文采用经典分子动力学方法,建立了胶原蛋白和胶原三肽(脯氨酸-羟脯氨酸-甘氨酸,Gly-HypPro)分子在TiO2表面的吸附模型,分析了表面台阶和沟槽结构对胶原蛋白吸附特性的影响规律。模拟结果表明未羟基化表面近表层水分子能够间接为胶原蛋白吸附提供位点,然而沟槽内致密的水分子则会阻碍胶原蛋白的吸附。通过对胶原三肽在电中性和电负性完全羟基化TiO2表面吸附过程的比较分析,确定了胶原三肽在吸附过程中的自由能变化及其与TiO2表面的结合强度。研究对比了羟基化前后的表面沟槽结构对三肽吸附过程的影响效果,发现羟基化后沟槽内水分子分层现象减弱且扩散能力增强,导致胶原三肽扩散吸附至沟槽内时所受的阻力减小。为了分析溶液条件下钙/磷酸根离子团对胶原三肽在纳结构表面吸附特性的影响,分别建立了磷酸根离子HPO42-和H2PO4-的分子模型,分析了钙/磷酸根离子在TiO2表面的团聚过程,研究发现羟基化的TiO2表面有利于钙/磷酸根离子的形核,表面纳结构在限制钙/磷酸根离子扩散的同时,亦能为离子吸附提供大量活性吸附位点,从而能够有效促进钙磷酸盐的矿化过程。研究了钙离子在TiO2表面吸附的动态特性及自由能变化,确定了钙离子和磷酸根离子在TiO2表面的吸附构象,分析了钙离子协助胶原三肽吸附到羟基化TiO2表面的动态过程。鉴于溶液中钙/磷酸根离子的扩散能力大于胶原三肽,故其能够首先扩散抵达TiO2表面,并成为胶原三肽吸附的活性位点。当表面存在沟槽结构时,大量钙/磷酸根离子能够快速地在沟槽中富集,促进胶原三肽在沟槽中形成稳定的吸附。通过分析基底表面特征(拓扑结构、羟基化等)和溶液离子对胶原蛋白及胶原三肽吸附特性的影响,本文能为合理设计和制备生物材料表面拓扑结构提供理论依据。
[Abstract]:With the rapid development of material preparation, surface detection and characterization technology, biomaterials are gradually developing towards functional and intelligent direction, and their ability to regulate cell behavior at the molecular level is also concerned. The increasing demand for bone replacement, the replacement of bone tissue in the bone tissue, and the biological properties of the implant, such as bone integration Therefore, it has become the key problem in the field of tissue engineering to rationally design and prepare biomaterials and make them have the ability to identify the molecules, to induce specific responses or to form new tissues. Titanium alloys are commonly used as medical metal materials and are very widely used in the field of hard tissue replacement. The rational surface modification of titanium alloy materials can help to improve their biocompatibility. In many surface characteristic factors that affect the interaction of tissue / cell material interface, surface topologies can affect cell behavior independently, and the mechanism of surface micronano topological morphologies to induce and regulate cell behavior is pointed out. Based on the important precondition of conducting and designing biological materials. Based on the important regulatory role of extracellular proteins in cell adhesion, the study of the interaction between proteins and materials is of great significance to reveal the mechanism of cell adhesion. The effect of surface topology on the adhesion of osteoblasts is discussed by the method of molecular simulation, and the effects of surface nanostructure on the adsorption process of extracellular matrix adhesion proteins are analyzed. The experimental and theoretical guidance for establishing the biocompatibility evaluation system for the surface topology of materials is provided. The effect of Ti6Al7Nb surface roughness on the adhesion and proliferation of MC3T3-E1 osteoblasts was analyzed from the angle of cell experiment. The effect of geometric parameters on the "contact guidance" of cell formation on the surface of the PDMS surface was discussed. The spreading ability and proliferation ability decreases with the increase of the surface roughness of the titanium alloy. With the increase of the groove width of the PDMS surface, the "contact guidance" phenomenon of osteoblasts becomes more prominent. The topologic structure of the biomaterial surface can restrain the growth of the cell pseudo foot, thus inducing the cell to produce the specific cell behavior such as "contact guidance". The surface nanostructures may be hydroxylation in the water environment, and then influence the adhesion of cells and the adsorption characteristics of protein. Based on the first principle calculation method, the hydroxylation model of the surface step structure of the rutile TiO2 (110) surface is established. The results show that the surface of the TiO2 (110) surface has a higher surface along the step structure along the 11 to 1 crystal direction. The energy density is the active site of the decomposition of water molecules. Therefore, the atomic structure and molecular force field of the stable hydroxylation model are obtained by the first principle energy minimization method. The kinetic simulation of the decomposition of water molecules near the surface step structure shows that the free protons produced by the decomposition of the water subsurface can be near the steps. Under the condition of high water molecular coverage, free protons can gradually spread to the water environment in the form of hydrated hydrogen ions. On the basis of the hydroxylation model, the effect of surface hydroxylation on the distribution of near surface water is studied with the help of the classical molecular dynamics. It is found that the surface hydroxylation can weaken the water molecules. In order to study the adsorption characteristics of the TiO2 surface on the surface of the TiO2 surface, the classical molecular dynamics method was used to establish the adsorption model of collagen and collagen three peptide (proline hydroxyproline glycine, Gly-HypPro) on the surface of TiO2 surface, and the surface steps and groove structure were analyzed. The simulation results show that the near surface water molecules on the surface of the surface of the non hydroxylation can indirectly provide the site for collagen adsorption, but the dense water molecules in the grooves will impede the adsorption of collagen. The ratio of the adsorption process of the collagen three peptide to the surface of the electrically neutral and electronegative completely hydroxylated TiO2 surface The free energy change of collagen three peptide during the adsorption process and the binding strength to the TiO2 surface were determined. The effect of the surface groove structure on the adsorption of three peptide was compared before and after the hydroxylation. It was found that the stratification of water molecules in the grooves was weakened and the diffusion capacity was enhanced after hydroxylation, which resulted in the diffusion and adsorption of collagen three peptide. In order to analyze the effect of calcium / phosphate group on the adsorption properties of collagen three peptide on the nano structure surface under the condition of solution, the molecular model of HPO42- and H2PO4- was established, and the agglomeration process of calcium / phosphate ion on the surface of TiO2 was analyzed. It was found that the hydroxylated TiO2 surface was beneficial to the surface. The nucleation of calcium / phosphate ion, the surface nanostructure can also limit the diffusion of calcium / phosphate ions, and can also provide a large number of active adsorption sites for ion adsorption, which can effectively promote the mineralization process of calcium phosphate. The dynamic characteristics of calcium ion adsorption on the TiO2 surface and the change of free energy are studied, and calcium ions and phosphate ions are determined. On the surface of TiO2, the dynamic process of calcium ion assisted collagen three peptide adsorbed on the surface of hydroxylated TiO2 was analyzed. In view of the diffusion capacity of calcium / phosphate radical ion in the solution is greater than that of collagen three peptide, it can first spread to the TiO2 surface and become the active site for the adsorption of collagen three peptide. Calcium / phosphate ions can be enriched in the grooves rapidly, promoting the stable adsorption of collagen three peptide in the grooves. By analyzing the characteristics of the surface of the substrate (topological structure, hydroxylation, etc.) and the effect of solution ions on the adsorption properties of collagen and collagen three peptide, this paper can provide a rational design and preparation of the topologic structure of the biomaterial surface. Theoretical basis.

【学位授予单位】：哈尔滨工业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R318.08;O647.3

【相似文献】