构建种植体表面Ti-GO-Ag多相复合材料抗菌性能及生物活性研究

发布时间：2018-03-11 03:36

  本文选题：种植体　切入点：氧化石墨烯　出处：《第四军医大学》2017年博士论文　论文类型：学位论文

【摘要】：钛及钛合金因其良好的材料学性能和生物学性能被广泛应用于牙科领域,但因其上部结构暴露在口腔环境中,种植体-软组织结合较薄弱部位容易受细菌侵袭;术中淤血及受损种植位点为细菌繁殖提供理想环境;植体下段表面粗糙程度不仅易于形成骨结合,也方便细菌黏附和定植;钛及钛合金的生物惰性性质,这些因素均会诱导种植体周围炎的发生。因此,如何使种植体材料表面修饰具有生物活性和抗菌性能以提高种植成功率是众多学者一直研究的重点和难点。本研究拟采用电镀法和紫外还原法使纯钛表面获得膜状和颗粒状改性,以口腔常见菌为研究对象,就改性后纯钛表面抗菌性能进行研究,并对其生物活性进行初步评价,从抗菌和生物活性角度优化种植体材料表面修饰。第一部分种植体表面Ti-GO-Ag多相复合材料的构建及性能分析[目的]在纯钛(Titanium,Ti)表面构建不同浓度的氧化石墨烯(Graphene oxide,GO)膜和银(Silver,Ag)颗粒,并对其材料学性能进行分析。[方法]通过正交实验设计设计工艺参数;电压为20 V,时间为5 min,浓度分别为20μg/ml、50μg/ml、80μg/ml、100μg/ml条件下通过电镀法对Ti进行修饰;采用紫外还原法在浓度为1 mol/L的硝酸银溶液中对Ti-GO进行加载;利用场发射扫描电子显微镜对表面形貌进行观察;使用拉曼光仪对GO的性状进行分析;通过X射线光电子能谱对Ag进行表征;采用原子力显微镜对材料表面轮廓和粗糙度进行研究;利用接触角测试仪对材料表面亲水性进行研究;使用纳米压痕和纳米划痕对材料表面弹性模量和摩擦力进行探讨;通过电感耦合等离子体质谱仪对Ag离子释放进行研究。[结果]正交实验优化的工艺参数:电压:20 V,时间:5 min;Ti表面经过改性以后表面有类膜物质和类颗粒状物质,经分析可知膜状物质为GO,且浓度不同,GO在Ti表面的形貌和量也不一样;颗粒状物质为Ag单质,与GO的浓度成正比;表面轮廓高低起伏,粗糙度与浓度成正比;Ti-GO与浓度成反比,Ti-GO-Ag与浓度成反比;弹性模量与维氏硬度随GO浓度升高而升高,摩擦力与浓度没有正反比关系;各组在第四天Ag离子释放量出现相似情况。[结论]正交实验设计可以优化实验过程,并使Ti表面获得修饰;改性后的Ti,其表面形貌、轮廓、粗糙度、亲水性能、弹性模量、维氏硬度、摩擦力等均与改性前有所不同,使Ti表面获得生物涂层(如抗菌涂层);四组实验组Ag离子释放总体规律趋向于降低,指导抗菌过程。第二部分种植体表面Ti-GO-Ag多相复合材料的抗菌性能研究[目的]研究不同浓度的GO和Ag涂层表面对三种口腔常见菌的抗菌作用。[方法]以金黄色葡萄球菌、变形链球菌和牙龈卟啉单胞菌为研究对象,评价20μg/ml、50μg/ml、80μg/ml、100μg/ml四种不同浓度Ti-GO-Ag涂层表面形成细菌生物膜的能力,并对细菌表面形貌进行观察;使用激光共聚焦显微镜对细菌胞膜完整性进行检测,并用Image J软件对荧光强度值和涂层表面细菌三维形貌进行研究;利用流式细胞仪对细菌凋亡情况进行评价;采用透射电子显微镜对细菌内部结构进行观察;通过时实定量荧光PCR技术检测细菌相对基因表达情况;对改性后Ti的抗菌机理进行初步探讨。[结果]20μg/ml、50μg/ml、80μg/ml、100μg/ml四种不同浓度Ti-GO-Ag涂层表面对金黄色葡萄球菌、变形链球菌和牙龈卟啉单胞菌的抗菌效果均达到或超过89%,明显优于对照组;三种细菌表面形貌发生明显改变;涂层表面细菌胞膜完整性受损,红色荧光强度值高于绿色荧光强度值,细菌活性均低于对照组,细菌的三维形貌也比对照组量少、薄和松;细菌凋亡情况明显不同于对照组;通过内部结构观察可发现细菌破坏主要是银离子接触并穿过胞膜进入到胞内而使细菌结构破坏;三种细菌的相对基因表达量均低于对照组。[结论]不同浓度的GO和Ag涂层对三种口腔常见菌均有抗菌作用,且对革兰氏阴性菌的效果优于革兰氏阳性菌,其抗菌机制是银离子接触攻击细菌胞膜,使胞膜结构穿孔,胞质外泄,从而使细菌裂解死亡。第三部分种植体表面Ti-GO-Ag多相复合材料的生物活性评价[目的]评价不同浓度的GO和Ag涂层表面生物活性。[方法]通过人体模拟液浸泡实验研究不同浓度的GO和Ag涂层表面钙磷沉积情况;利用CCK-8实验评价不同浓度的GO和Ag涂层表面鼠骨髓间充质干细胞活性情况。[结果]不同浓度的GO和Ag涂层表面均可沉积钙磷等元素,沉积效果较好的是对照组、G20组和G80组,其中对照组和G20组、G20组和G80组之间无统计学差异;四种不同浓度的GO和Ag涂层表面对鼠骨髓间充质干细胞的存活状况较对照组差,G20和G50组优于G80和G100组。[结论]四种不同浓度的GO和Ag涂层表面拥有不同程度的生物矿化能力,且对鼠骨髓间充质干细胞的存活状况均有影响。
[Abstract]:Titanium and titanium alloy due to its good properties and biological properties of materials are widely used in the field of Dentistry, but because of the upper structure is exposed to the oral environment, combined with the weak parts of the implant soft tissue susceptible to bacterial invasion; intraoperative congestion and damaged implant sites provide an ideal environment for bacterial reproduction; implant under a rough surface is not only easy to form osseointegration, also facilitate adhesion and colonization of bacteria; biological inert properties of titanium and titanium alloy, these factors have induced peri implantitis. Therefore, how to make the implant material with surface modification and antibacterial properties of biological activity in order to improve the success rate of planting is the key and difficult point in many scholars have been studied. This research adopts the electroplating method and UV reduction method to the surface of pure titanium film and granular modified to obtain oral common bacteria as the research object, the modified titanium surface antibacterial Study of properties, and to evaluate its biological activity, antibacterial and biological activity from the angle optimization of implant material surface modification. The first part of the implant surface Ti-GO-Ag composite construction and performance analysis [Objective] in pure titanium (Titanium, Ti) the surface of graphene oxide with different concentrations of Graphene (oxide, GO) film and silver (Silver, Ag) on the properties of the particles, and materials are analyzed. Through the orthogonal experiment design method of process parameters; the voltage is 20 V, time 5 min, the concentration were 20 g/ml, 50 g/ml, 80 g/ml, 100 g/ml conditions by electroplating method of Ti was modified by UV; reduction method for loading Ti-GO in the concentration of silver nitrate was 1 mol/L; on the surface morphology were observed by field emission scanning electron microscope; analyzed using Raman spectrometer on the GO characters; by X ray photoelectron spectroscopy The Ag was characterized by atomic force microscope; and the roughness of the surface profile of materials; using the instrument of material surface hydrophilic contact angle measurement; using nanoindentation and nano scratch on the surface of the material of elastic modulus and friction force; optimize the process parameters. The results of orthogonal experiment on the release of Ag ions through. Inductively coupled plasma mass spectrometry: voltage: 20 V, time: 5 min; Ti surface modified after surface class membrane materials and class of granular material. The analysis shows that membrane material was GO, and the different concentration of GO on the morphology and quantity of Ti surface is not the same; granular material as Ag elements, a is proportional to the concentration of GO; surface profile undulating, roughness and is proportional to the concentration of Ti-GO; and is inversely proportional to the concentration, Ti-GO-Ag and concentration is inversely proportional to the elastic modulus and hardness; Vivtorinox with GO concentration increased and increased friction No positive relationship with the ratio of concentration; in each group fourth days Ag ion release appeared similar to the situation. We can optimize the process of experiment conclusion] orthogonal experimental design, and the Ti surface modification; modified Ti, the surface morphology, profile, roughness, hydrophilicity, elastic modulus, hardness and friction force of Vivtorinox. Before modification is different, the Ti surface coating (such as antibacterial coating); four experimental groups, Ag ion release tends to reduce the overall law, guide the antibacterial process. The second part of the implant surface Ti-GO-Ag composite antibacterial antibacterial effect. Methods can study [Objective] to study the effects of different concentrations of GO and Ag coating on the surface of three kinds of common oral bacteria Staphylococcus aureus, Streptococcus mutans and Porphyromonas gingivalis as the research object, the evaluation of 20 g/ml, 50 g/ml, 80 g/ml, 100 g/ml formed four different concentrations of Ti-GO-Ag coating The ability of bacterial biofilm, and the bacterial surface morphology were observed; detection of bacterial cell membrane integrity was performed using laser confocal microscopy, and the fluorescence intensity values of bacteria and coating the surface topography by using Image J software; the evaluation of bacterial apoptosis by flow cytometry was observed by transmission electron microscopy; the internal structure of bacteria; by real-time fluorescence PCR detection of bacterial gene expression relative to preliminary study results.;]20 g/ml of the antibacterial mechanism of Ti after 50 g/ml, 80 g/ml, 100 g/ ml four different concentration of Ti-GO-Ag coating on Staphylococcus aureus, antibacterial effect Streptococcus mutans and Porphyromonas gingivalis were reached or exceeded 89%, significantly better than the control group; three kinds of bacterial surface morphology changed obviously; the integrity of the damaged coating on the surface of the bacterial membrane, strong red fluorescence The degree is higher than the value of green fluorescence intensity values, bacterial activity was lower than the control group, the three-dimensional morphology of bacteria than the control group less, Bo Hesong; bacterial apoptosis is significantly different from the control group; the observation of the internal structure of bacteria found damage is mainly silver ions and contact through the membrane into the intracellular bacteria caused structural damage; the relative gene expression of three kinds of bacteria were lower than the control group. Conclusion: different concentrations of GO and Ag coating of common bacteria had antimicrobial effects on three kinds of oral cavity, and the effect is better than the gram positive bacteria of gram negative bacteria, the antibacterial mechanism of silver ions is to attack the bacterial membrane contact, the membrane perforation, cytoplasm leak, causing bacterial lysis and death. GO biological surface Ag coating. The third part activity method of implant surface Ti-GO-Ag multiphase biological activity evaluation of composites [Objective] evaluation of different concentrations through the body Simulation experiment of Soaking with different concentration of GO and Ag coating on the surface of calcium and phosphorus deposition; evaluation of different concentration using CCK-8 experimental GO and Ag coating surface of rat bone marrow mesenchymal stem cells. The activity of the different concentrations of GO and Ag coating could be deposited calcium phosphate precipitation and other elements, better control group, G20 group and G80 group, the control group and G20 group, there was no significant difference between G20 group and G80 group; four different concentrations of GO and Ag coating on the surface of bone marrow mesenchymal stem cell survival situation worse than that of control group, G20 group and G50 group is better than that of G80 and G100. [Conclusion] four different concentrations of GO and Ag coatings have different degrees of biological mineralization, and the influence of bone marrow mesenchymal stem cell survival in both.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R783.1
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本文编号：1596429