一种新型含铜不锈钢的生物学性能研究

发布时间：2018-06-15 19:31

本文选题：317L-Cu不锈钢 + 抗菌性能　；参考：《重庆大学》2016年博士论文

【摘要】：众所周知,不锈钢材料具有较低的成本、理想的生物相容性、综合力学性能、机械加工性能以及耐腐蚀性能,目前已经被广泛应用于生物医用领域,如口腔、骨科、外科手术器械和生物医疗设备等。然而,传统的医用不锈钢材料仅仅发挥了其机械支撑属性,无法避免植入生物体内后导致微生物感染而出现炎症反应的问题。此外,该类感染的出现将持续反复,难以彻底杜绝。因此,为了解决植入物术后感染问题,在不牺牲其力学性能、机械加工性能以及耐腐蚀性能的先决条件下,赋予新型的317L不锈钢以优秀、广谱和耐久的抗菌属性,与此同时兼顾良好的生物相容性,是本论文研究的主旨所在。因此依据该研究理念,本研究在317L不锈钢基体内掺杂一定比例的具有抗菌作用的铜元素,并进一步借助特殊的热处理工艺对该含铜不锈钢进行加工,从而设计和开发出一类新型的317L-Cu不锈钢抗菌材料。在本研究工作中,设计并制备了一种新型的317L-Cu不锈钢。首先通过系统的材料表征来观察其材料和表面性能的变化情况;随后,结合多种方法评价其抗菌性能;此外,联合利用多种生物学手段对其生物相容性进行系统地评价;最后,阐述317L-Cu不锈钢的抗菌作用机理,评估其在临床医学和日常生活等领域的潜在应用前景。具体而言,主要的研究内容和结论如下:(1)根据课题组前期研究结果,综合考虑商用成本因素,设计并制备出一种铜元素含量为4.5wt.%的新型的317L-Cu不锈钢,并借助扫描电镜、表面元素能谱分析、硬度检测、接触角及表面能计算、金相显微组织观察和耐腐蚀性能测试等实验对材料进行表征。测试结果表明:在铜元素添加前后,317L-Cu不锈钢材料的力学及表面元素参数变化不大,保持了较好的力学及表面特性,为材料的生物学研究及应用提供了可靠的基础。(2)目前广泛采用的JIS Z2801:2000标准无法检测到细菌VBNC状态,而此状态下的细菌在复苏后仍然会对机体造成威胁。本研究针对该标准的不足,创新地对材料抗菌性能的评价进行了扩展,借助平板覆膜法、活死染色法和q PCR法分别在细胞活力、细胞膜和基因水平表征了317L-Cu不锈钢的抗菌性能。实验结果显示:317L-Cu不锈钢在细菌共培养1天的抑菌性能高达98.3%,并在共培养3天时长内持续展现出良好的杀菌性能。在S.aureus与317L-Cu不锈钢共培养3天后,扫描电镜观测到不锈钢表面的细菌生物膜出现了质膜内陷,内含物外泄等现象,展现了317L-Cu不锈钢优异的抗菌效用。与此同时,为今后医用材料尤其是植入物材料的抗菌性能评价提供了丰富的科学数据和进一步的优化建议。(3)为了探究317L-Cu不锈钢材料溶出的Cu2+的潜在生物学毒性,本研究借助血液相容性、细胞相容性以及斑马鱼模型综合评价了317L-Cu不锈钢在体外和体内的毒性。其中MTT标记法和RTCA实时无标记细胞分析技术的结果相符合,均表明:两种不锈钢对MC3T3-E1细胞的增殖没有显著的毒副作用,甚至317L-Cu不锈钢还在一定程度上表现出了促进MC3T3-E1细胞增殖的现象。急性溶血实验和血小板粘附观察结果显示:两种不锈钢几乎都不存在溶血性(0.61±0.07%和0.72±0.13%)。与此同时,斑马鱼模型已经在药物筛选领域取得瞩目的成果,我们首次将其作为整体动物模型应用到317L-Cu不锈钢体内毒性的全面评价中,结果显示两种不锈钢对斑马鱼胚胎和幼鱼的发育前期不具有显著的毒副作用。(4)通过对细菌在两种不锈钢材料表面形成生物膜内胞外聚合物(EPS)的傅立叶红外光谱(FTIR)分析及细胞膜表面铜离子情况的X射线光电子能谱学(XPS)检测探索了317L-Cu不锈钢对S.aureus生物膜的影响。此外,进一步通过活死(Live/Dead)荧光染色法及透射电子显微镜(TEM)表征317L-Cu不锈钢溶出的铜离子对于细菌细胞膜及内含物的损伤过程,并探究其抗菌机制:Cu2+对细菌的细胞膜和胞外分泌物均造成了损伤,致使细菌内含物泄露,生物膜形成受到抑制;317L-Cu不锈钢并未能对细菌的基因组DNA完整性造成显著影响,即317L-Cu不锈钢的抗菌性能并非通过破坏细菌基因组的完整性而体现。于此同时,我们推测了317L-Cu不锈钢同时具有良好杀菌能力及低细胞毒副作用的初步解释原因:细菌和细胞的膜结构、组成成分以及代谢活动存在差异造成其对外界相同刺激的反应也不一致,耐受能力也存在差异。低浓度的Cu2+本身是有助于细胞增殖,而在该浓度区间内,对细菌而言则是致命的。综上所述,317L-Cu不锈钢具有理想的耐腐蚀性和生物安全性,并且在不显著降低317L不锈钢的优秀材料属性的前提下,兼具有良好的抗菌性能。因此,本论文研究的317L-Cu不锈钢是一种极具潜力可用于日常生活及临床医学上代替原有不锈钢材料的新型生物功能化不锈钢改进材料。
[Abstract]:It is well known that stainless steel has low cost, ideal biocompatibility, comprehensive mechanical properties, mechanical properties and corrosion resistance. It has been widely used in biomedical fields, such as oral cavity, Department of orthopedics, surgical instruments and biological medical equipment. However, traditional medical stainless steel materials have only been used. Mechanical support properties can not avoid the problem of inflammatory response to microbial infection after implantation. In addition, the occurrence of this type of infection will continue to be repeated and difficult to eradicate. Therefore, in order to solve the problem of postoperative infection of the implant, without sacrificing its mechanical properties, mechanical properties and corrosion resistance The purpose of this paper is to give a new type of 317L stainless steel with excellent, broad-spectrum and durable antibacterial properties and good biocompatibility at the same time. Therefore, based on this research concept, this study doped a certain proportion of copper elements with antibacterial activity in the 317L stainless steel matrix and further assisted with special heat treatment. A new type of 317L-Cu stainless steel antibacterial material was designed and developed by processing the copper containing stainless steel. In this study, a new type of 317L-Cu stainless steel was designed and prepared. First, the material and surface properties of the stainless steel were observed by the material characterization of the system. Then, the resistance of the material was evaluated by a variety of methods. In addition, a variety of biological methods are used to systematically evaluate their biocompatibility. Finally, the antibacterial mechanism of 317L-Cu stainless steel is described and its potential applications in clinical medicine and daily life are evaluated. In particular, the main research contents and conclusions are as follows: (1) according to the previous research group of the project group In view of the commercial cost factors, a new type of 317L-Cu stainless steel with copper element content of 4.5wt.% was designed and prepared. The materials were characterized by scanning electron microscopy, surface element energy spectrum analysis, hardness testing, contact angle and surface energy calculation, metallographic microstructure observation and corrosion resistance test. Before and after the addition of copper elements, the mechanical and surface element parameters of 317L-Cu stainless steel have little change, maintaining good mechanical and surface properties, providing a reliable basis for the biological research and application of materials. (2) the JIS Z2801:2000 standard which is widely used at present can not detect the state of bacterial VBNC, and the bacteria under this state are complex. This study expanded the evaluation of antibacterial properties of the material creatively in view of the shortage of the standard. The antibacterial properties of 317L-Cu stainless steel were characterized by the live death staining method and Q PCR method, respectively, with the help of flat sheet mulching, and the cell membrane and gene level. The experimental results showed that the 317L-Cu stainless steel was in the test. The bacteriostasis performance of bacteria co culture for 1 days was up to 98.3%, and it showed good bactericidal performance during the long period of co culture for 3 days. After 3 days co culture of S.aureus and 317L-Cu stainless steel, the bacterial biofilm on the surface of stainless steel appeared in the membrane of the surface of the stainless steel and the leakage of the inclusions, which showed the excellent antibacterial activity of the 317L-Cu stainless steel. At the same time, there are abundant scientific data and further optimization suggestions for evaluating the antibacterial properties of medical materials, especially implants. (3) in order to explore the potential biological toxicity of the dissolved Cu2+ of 317L-Cu stainless steel materials, this study is based on the comprehensive evaluation of blood compatibility, cytocompatibility and zebrafish model. The toxicity of 317L-Cu stainless steel in vitro and in vivo, in which the MTT labeling method and the RTCA real-time unmarked cell analysis technique were in accordance with the results, all showed that the two kinds of stainless steel had no significant toxic and side effects on the proliferation of MC3T3-E1 cells, and even 317L-Cu stainless steel showed the phenomenon of promoting the proliferation of MC3T3-E1 cells to a certain extent. The results of sexual hemolysis and platelet adhesion observation showed that there was almost no hemolysis in the two kinds of stainless steel (0.61 + 0.07% and 0.72 + 0.13%). At the same time, the zebrafish model has achieved remarkable results in the field of drug screening. We should use it as a whole animal model for the first time in the comprehensive assessment of the toxicity of 317L-Cu stainless steel. The results showed that two kinds of stainless steel had no significant toxic and side effects on the early development of zebrafish embryos and young fish. (4) detection of X ray photoelectron spectroscopy (XPS) of the cell membrane surface by Fu Liye infrared spectroscopy (FTIR) analysis of the bacteria on the surface of two kinds of stainless steel materials on the surface of two kinds of stainless steel. The effect of 317L-Cu stainless steel on S.aureus biofilm was investigated. In addition, the damage process of copper ions dissolved in 317L-Cu stainless steel was characterized by Live/Dead fluorescence staining and transmission electron microscopy (TEM), and its antibacterial mechanism was explored: Cu2+ was made to the cell membrane and extracellular secretions of the bacteria. Damage has resulted in leakage of bacterial inclusions and inhibition of biofilm formation; 317L-Cu stainless steel has not significantly affected the integrity of bacterial genome DNA, that is, the antibacterial properties of 317L-Cu stainless steel are not reflected by destroying the integrity of the bacterial genome. At the same time, we speculate that the 317L-Cu stainless steel is good at the same time. The preliminary explanation of bactericidal and low cytotoxic side effects: the membrane structure, composition and metabolic activity of bacteria and cells cause differences in the response to the same stimulus to the outside world, and there are differences in tolerance. Low concentrations of Cu2+ itself contribute to cell proliferation, and in this concentration range, for bacteria, It is fatal. To sum up, 317L-Cu stainless steel has ideal corrosion resistance and biological safety, and has good antibacterial properties without significantly reducing the excellent material properties of 317L stainless steel. Therefore, the study of 317L-Cu stainless steel in this paper is a great potential for daily life and clinical medicine. The new type of stainless steel is a new type of bio functional stainless steel modified material.
【学位授予单位】：重庆大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318.08

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