经皮钛植入体的纳米结构化表面改性及其生物学性能
发布时间：2018-07-28 11:52
【摘要】：经皮植入器械穿过皮肤固定于体内并保持部分于皮肤创口或伸出皮肤外,由于部分暴露于体外,就要求植入体与周围皮肤组织形成良好的生物密封以避免发生感染。钛及钛合金以其良好的力学性能和骨整合性能被广泛用作经皮器械,然而钛是生物惰性材料,植入体内后与软组织整合能力差,同时钛植入体本身也无抗菌性能。为了降低术后感染的发生率,实现良好的生物密封,我们对钛植入体进行纳米结构化的表面改性,以期获得既有抑菌性能又有利于皮肤组织细胞黏附生长的功能表面。大量的研究显示钛表面二氧化钛纳米管具有多孔形貌和纳米级粗糙度,有利于细胞粘附,并且能促进细胞增殖分化,这都表明二氧化钛纳米管改性的钛植入体在植入材料领域有着良好的应用前景。本研究通过体外蛋白质、细菌、巨噬细胞、表皮角质细胞,成纤维细胞实验以及动物皮下体内实验,探究纳米结构改性后的钛经皮植入体是否能与周围组织形成良好结合,及其与体内相关组织的整合过程,为其应用提供理论与实验基础。此外,还采用钦结合噬菌体对钛植入体进行纳米结构化表面改性并对其表面理化性能及生物相容性进行初步评价。本论文的主要工作及结论如下:采用阳极氧化技术在钛植入体表面构建的二氧化钛纳米管膜(TNT)具有优良的润湿性和较高的比表面积,表面吸附的蛋白质使材料具有更强的生物活性,可以促进细胞在材料表面的粘附等行为。由于TNT的表面粗糙度属于纳米级,不利于细菌在其表面粘附。在紫外和日光条件下,TNT的光催化效应使其表现出较好的抑菌能力。TNT的抑菌性能有助于减少经皮感染的发生。在TNT表面培养巨噬细胞发现纳米结构抑制了巨噬细胞的增殖迁移及炎性因子分泌。脂多糖(lipopolysaccharide,LPS)修饰的TNT表面能够促进巨噬细胞的迁移并调控其功能转化,在短时间内大量表达促炎性因子及时清除周围的细菌等病原体,并且在炎症消退后诱导巨噬细胞向促进组织修复的方向转化。这表明在植入体诱发的感染环境下TNT不仅具有良好的炎性调控功能,而且还能促进组织修复。在不同生理环境下,研究TNT与人表皮角质细胞(Human epidermal keratinocyte,HaCat)和人真皮成纤维细胞(human skin fibroblast,HSF)之间的作用机制。TNT能促进HSF细胞的增殖迁移及相关因子分泌,但抑制了 HaCat细胞的增殖及相关蛋白的表达。将两种细胞接触共培养时,TNT通过促进成纤维细胞的增殖分化进而促进角质细胞的成熟分化,有利于在植入体表面形成与其紧密结合且完整的表皮真皮及皮下组织结构。动物皮下短期(4h到7d)植入时,感染部位的TNT能有效激活免疫细胞释放炎性因子,在炎症得到控制后调节炎性因子的快速消退,促进皮肤组织细胞粘附于植入体表面。这表明TNT抑制细菌的初始粘附,在短时间内能清除术后感染,有利于随后组织愈合并与材料紧密结合。长期植入(2 w到8 w)实验时,感染部位的TNT能避免炎性因子大量持续释放,促进皮肤组织与其紧密结合和血管生成。这表明TNT植入体与皮肤组织形成良好的生物密封,从而抵御外界细菌侵袭,降低经皮植入体感染风险。此外,通过钛结合噬菌体对钛植入体改性,表明钛结合噬菌体与钛表面具有较强的亲和力及稳定的结合性能,改性后的表面具有多孔结构且有良好的细胞相容性。综合体内外实验,发现TNT改性的钛植入体能够降低感染的发生率,促进皮肤组织与其紧密结合。当TNT改性的钛植入植入后,血液和组织液蛋白迅速吸附到植入体表面,利于组织细胞粘附,而不利于细菌粘附。同时,细菌的存在可以激活巨噬细胞迁移至植入部位参与炎症反应。细菌清除后,炎症减弱,TNT及时调控巨噬细胞极化参与组织修复。随后,真皮成纤维细胞粘附于植入体表面开始增殖分化、沉积基质,表皮细胞也沿着真皮层迁移、锚定于植入体表面开始增殖分化形成表皮组织。TNT促进早期炎症反应的及时消退以及成纤维细胞和表皮角质细胞的相互作用,最终在植入体表面形成完整生物密封。
[Abstract]:The percutaneous instruments are fixed in the body through the skin and remain partially on the skin or out of the skin. Due to partial exposure to the body, the implant is required to form a good biological seal with the surrounding tissue to avoid infection. Titanium and titanium alloys are widely used as a percutaneous instrument for their good mechanical and bone integration properties. However, titanium is a biological inert material, and the ability to integrate with soft tissue after implantation is poor, and titanium implants themselves have no antibacterial properties. In order to reduce the incidence of postoperative infection and achieve good biological seal, we carry out the nano structured surface modification to titanium implants, in order to obtain both bacteriostasis and skin tissue fine. A large number of studies show that titanium dioxide nanotube has porous morphology and nanoscale roughness, which is beneficial to cell adhesion and promotes cell proliferation and differentiation. This shows that titanium dioxide nanotube modified titanium implant has a good application prospect in the field of implant materials. External proteins, bacteria, macrophages, epidermal keratinocytes, fibroblast experiments and animal subcutaneous experiments are used to explore whether the titanium transdermal implant after nanostructure modification can form a good combination with the surrounding tissue, and the integration process with the related tissues in the body, and provides a theoretical and experimental basis for its application. A preliminary evaluation of the surface physical and chemical properties and biocompatibility of titanium implants was carried out with phage. The main work and conclusion of this paper are as follows: the titanium dioxide nanotube membrane (TNT) constructed on the surface of titanium implant with anodic oxidation technology has excellent wettability and high specific surface area. The surface adsorbed protein makes the material more bioactive and can promote the adhesion of the cell to the surface of the material. Because the surface roughness of TNT belongs to the nanoscale, it is not conducive to the adhesion of bacteria on its surface. Under the ultraviolet and sunlight conditions, the photocatalytic effect of TNT makes it show that the Bacteriostasis of.TNT is better. It helps to reduce the incidence of transdermal infection. The macrophages on the TNT surface have found that nanostructures inhibit the proliferation and migration of macrophages and the secretion of inflammatory factors. The TNT surface modified by lipopolysaccharide (lipopolysaccharide, LPS) can promote the migration of macrophages and regulate their functional transformation, and in a short time the expression of proinflammatory factors is timely. Clear the surrounding bacteria and other pathogens, and induce macrophages to convert to the direction of promoting tissue repair after the inflammation subsided. This shows that TNT not only has good inflammatory regulatory function, but also promotes tissue repair under the environment of implant induced infection. In different physiological rings, the study of TNT and human epidermal keratinocyte (Human EP) The mechanism of action between idermal keratinocyte, HaCat) and human dermal fibroblasts (human skin fibroblast, HSF),.TNT can promote the proliferation and migration of HSF cells and the secretion of related factors, but inhibit the proliferation of HaCat cells and the expression of related proteins. When the two cells are exposed to co culture, TNT by promoting the proliferation and differentiation of fibroblasts. To promote the maturation and differentiation of keratinocytes, it is beneficial for the formation of the dermal and subcutaneous tissue of the implant surface, which is closely combined with it. When the animal subcutaneous (4h to 7D) implantation, the TNT of the infected site can effectively activate the immune cells to release inflammatory factors and regulate the rapid decline of inflammatory factors after the inflammation is controlled. The cells that enter the skin tissue adhere to the implant surface. This indicates that TNT inhibits the initial adhesion of the bacteria, clears the postoperative infection in a short time, and is beneficial to the subsequent consolidation of the tissue with the material. In the long term implantation (2 w to 8 W), the TNT of the infected site can avoid the continuous release of the inflammatory cells and promote the skin tissue to close closely with it. This indicates that the TNT implant has a good biological seal with the skin tissue, thus resisting the invasion of the external bacteria and reducing the risk of infection of the transdermal implant. In addition, the titanium binding phage is modified to show that the titanium binding phage has a stronger affinity and stable binding performance on the surface of the titanium. The surface has porous structure and good cellular compatibility. In vitro and in vivo experiments, it is found that the TNT modified titanium implant can reduce the incidence of infection and promote the close combination of skin tissue. When TNT modified titanium implants are implanted, the blood and tissue liquid protein quickly adsorb the implant surface, which is unfavorable to the adhesion of tissue cells. At the same time, the presence of bacteria can activate macrophages to migrate to the implant site to participate in the inflammatory response. After bacteria clearance, the inflammation weakens, and TNT regulates macrophage polarization in time to participate in tissue repair. Subsequently, dermal fibroblasts adhere to the implant surface and begin to proliferate and differentiate, deposition matrix, and epidermal cells along the dermis. Migration, anchored to the implant surface, began to proliferate and differentiate into the epidermal tissue to form the epidermal tissue.TNT to promote the early fading of the early inflammatory response and the interaction between the fibroblasts and the epidermal keratinocytes, and eventually formed a complete biological seal on the implant surface.
【学位授予单位】：西南交通大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R318.08;TB383.1
本文编号：2150051
[Abstract]:The percutaneous instruments are fixed in the body through the skin and remain partially on the skin or out of the skin. Due to partial exposure to the body, the implant is required to form a good biological seal with the surrounding tissue to avoid infection. Titanium and titanium alloys are widely used as a percutaneous instrument for their good mechanical and bone integration properties. However, titanium is a biological inert material, and the ability to integrate with soft tissue after implantation is poor, and titanium implants themselves have no antibacterial properties. In order to reduce the incidence of postoperative infection and achieve good biological seal, we carry out the nano structured surface modification to titanium implants, in order to obtain both bacteriostasis and skin tissue fine. A large number of studies show that titanium dioxide nanotube has porous morphology and nanoscale roughness, which is beneficial to cell adhesion and promotes cell proliferation and differentiation. This shows that titanium dioxide nanotube modified titanium implant has a good application prospect in the field of implant materials. External proteins, bacteria, macrophages, epidermal keratinocytes, fibroblast experiments and animal subcutaneous experiments are used to explore whether the titanium transdermal implant after nanostructure modification can form a good combination with the surrounding tissue, and the integration process with the related tissues in the body, and provides a theoretical and experimental basis for its application. A preliminary evaluation of the surface physical and chemical properties and biocompatibility of titanium implants was carried out with phage. The main work and conclusion of this paper are as follows: the titanium dioxide nanotube membrane (TNT) constructed on the surface of titanium implant with anodic oxidation technology has excellent wettability and high specific surface area. The surface adsorbed protein makes the material more bioactive and can promote the adhesion of the cell to the surface of the material. Because the surface roughness of TNT belongs to the nanoscale, it is not conducive to the adhesion of bacteria on its surface. Under the ultraviolet and sunlight conditions, the photocatalytic effect of TNT makes it show that the Bacteriostasis of.TNT is better. It helps to reduce the incidence of transdermal infection. The macrophages on the TNT surface have found that nanostructures inhibit the proliferation and migration of macrophages and the secretion of inflammatory factors. The TNT surface modified by lipopolysaccharide (lipopolysaccharide, LPS) can promote the migration of macrophages and regulate their functional transformation, and in a short time the expression of proinflammatory factors is timely. Clear the surrounding bacteria and other pathogens, and induce macrophages to convert to the direction of promoting tissue repair after the inflammation subsided. This shows that TNT not only has good inflammatory regulatory function, but also promotes tissue repair under the environment of implant induced infection. In different physiological rings, the study of TNT and human epidermal keratinocyte (Human EP) The mechanism of action between idermal keratinocyte, HaCat) and human dermal fibroblasts (human skin fibroblast, HSF),.TNT can promote the proliferation and migration of HSF cells and the secretion of related factors, but inhibit the proliferation of HaCat cells and the expression of related proteins. When the two cells are exposed to co culture, TNT by promoting the proliferation and differentiation of fibroblasts. To promote the maturation and differentiation of keratinocytes, it is beneficial for the formation of the dermal and subcutaneous tissue of the implant surface, which is closely combined with it. When the animal subcutaneous (4h to 7D) implantation, the TNT of the infected site can effectively activate the immune cells to release inflammatory factors and regulate the rapid decline of inflammatory factors after the inflammation is controlled. The cells that enter the skin tissue adhere to the implant surface. This indicates that TNT inhibits the initial adhesion of the bacteria, clears the postoperative infection in a short time, and is beneficial to the subsequent consolidation of the tissue with the material. In the long term implantation (2 w to 8 W), the TNT of the infected site can avoid the continuous release of the inflammatory cells and promote the skin tissue to close closely with it. This indicates that the TNT implant has a good biological seal with the skin tissue, thus resisting the invasion of the external bacteria and reducing the risk of infection of the transdermal implant. In addition, the titanium binding phage is modified to show that the titanium binding phage has a stronger affinity and stable binding performance on the surface of the titanium. The surface has porous structure and good cellular compatibility. In vitro and in vivo experiments, it is found that the TNT modified titanium implant can reduce the incidence of infection and promote the close combination of skin tissue. When TNT modified titanium implants are implanted, the blood and tissue liquid protein quickly adsorb the implant surface, which is unfavorable to the adhesion of tissue cells. At the same time, the presence of bacteria can activate macrophages to migrate to the implant site to participate in the inflammatory response. After bacteria clearance, the inflammation weakens, and TNT regulates macrophage polarization in time to participate in tissue repair. Subsequently, dermal fibroblasts adhere to the implant surface and begin to proliferate and differentiate, deposition matrix, and epidermal cells along the dermis. Migration, anchored to the implant surface, began to proliferate and differentiate into the epidermal tissue to form the epidermal tissue.TNT to promote the early fading of the early inflammatory response and the interaction between the fibroblasts and the epidermal keratinocytes, and eventually formed a complete biological seal on the implant surface.
【学位授予单位】：西南交通大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R318.08;TB383.1
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