發(fā)布時間：2018-06-15 19:31

  本文選題：317L-Cu不銹鋼 + 抗菌性能�。� 參考：《重慶大學(xué)》2016年博士論文

【摘要】：眾所周知,不銹鋼材料具有較低的成本、理想的生物相容性、綜合力學(xué)性能、機(jī)械加工性能以及耐腐蝕性能,目前已經(jīng)被廣泛應(yīng)用于生物醫(yī)用領(lǐng)域,如口腔、骨科、外科手術(shù)器械和生物醫(yī)療設(shè)備等。然而,傳統(tǒng)的醫(yī)用不銹鋼材料僅僅發(fā)揮了其機(jī)械支撐屬性,無法避免植入生物體內(nèi)后導(dǎo)致微生物感染而出現(xiàn)炎癥反應(yīng)的問題。此外,該類感染的出現(xiàn)將持續(xù)反復(fù),難以徹底杜絕。因此,為了解決植入物術(shù)后感染問題,在不犧牲其力學(xué)性能、機(jī)械加工性能以及耐腐蝕性能的先決條件下,賦予新型的317L不銹鋼以優(yōu)秀、廣譜和耐久的抗菌屬性,與此同時兼顧良好的生物相容性,是本論文研究的主旨所在。因此依據(jù)該研究理念,本研究在317L不銹鋼基體內(nèi)摻雜一定比例的具有抗菌作用的銅元素,并進(jìn)一步借助特殊的熱處理工藝對該含銅不銹鋼進(jìn)行加工,從而設(shè)計和開發(fā)出一類新型的317L-Cu不銹鋼抗菌材料。在本研究工作中,設(shè)計并制備了一種新型的317L-Cu不銹鋼。首先通過系統(tǒng)的材料表征來觀察其材料和表面性能的變化情況;隨后,結(jié)合多種方法評價其抗菌性能;此外,聯(lián)合利用多種生物學(xué)手段對其生物相容性進(jìn)行系統(tǒng)地評價;最后,闡述317L-Cu不銹鋼的抗菌作用機(jī)理,評估其在臨床醫(yī)學(xué)和日常生活等領(lǐng)域的潛在應(yīng)用前景。具體而言,主要的研究內(nèi)容和結(jié)論如下:(1)根據(jù)課題組前期研究結(jié)果,綜合考慮商用成本因素,設(shè)計并制備出一種銅元素含量為4.5wt.%的新型的317L-Cu不銹鋼,并借助掃描電鏡、表面元素能譜分析、硬度檢測、接觸角及表面能計算、金相顯微組織觀察和耐腐蝕性能測試等實驗對材料進(jìn)行表征。測試結(jié)果表明:在銅元素添加前后,317L-Cu不銹鋼材料的力學(xué)及表面元素參數(shù)變化不大,保持了較好的力學(xué)及表面特性,為材料的生物學(xué)研究及應(yīng)用提供了可靠的基礎(chǔ)。(2)目前廣泛采用的JIS Z2801:2000標(biāo)準(zhǔn)無法檢測到細(xì)菌VBNC狀態(tài),而此狀態(tài)下的細(xì)菌在復(fù)蘇后仍然會對機(jī)體造成威脅。本研究針對該標(biāo)準(zhǔn)的不足,創(chuàng)新地對材料抗菌性能的評價進(jìn)行了擴(kuò)展,借助平板覆膜法、活死染色法和q PCR法分別在細(xì)胞活力、細(xì)胞膜和基因水平表征了317L-Cu不銹鋼的抗菌性能。實驗結(jié)果顯示:317L-Cu不銹鋼在細(xì)菌共培養(yǎng)1天的抑菌性能高達(dá)98.3%,并在共培養(yǎng)3天時長內(nèi)持續(xù)展現(xiàn)出良好的殺菌性能。在S.aureus與317L-Cu不銹鋼共培養(yǎng)3天后,掃描電鏡觀測到不銹鋼表面的細(xì)菌生物膜出現(xiàn)了質(zhì)膜內(nèi)陷,內(nèi)含物外泄等現(xiàn)象,展現(xiàn)了317L-Cu不銹鋼優(yōu)異的抗菌效用。與此同時,為今后醫(yī)用材料尤其是植入物材料的抗菌性能評價提供了豐富的科學(xué)數(shù)據(jù)和進(jìn)一步的優(yōu)化建議。(3)為了探究317L-Cu不銹鋼材料溶出的Cu2+的潛在生物學(xué)毒性,本研究借助血液相容性、細(xì)胞相容性以及斑馬魚模型綜合評價了317L-Cu不銹鋼在體外和體內(nèi)的毒性。其中MTT標(biāo)記法和RTCA實時無標(biāo)記細(xì)胞分析技術(shù)的結(jié)果相符合,均表明:兩種不銹鋼對MC3T3-E1細(xì)胞的增殖沒有顯著的毒副作用,甚至317L-Cu不銹鋼還在一定程度上表現(xiàn)出了促進(jìn)MC3T3-E1細(xì)胞增殖的現(xiàn)象。急性溶血實驗和血小板粘附觀察結(jié)果顯示:兩種不銹鋼幾乎都不存在溶血性(0.61±0.07%和0.72±0.13%)。與此同時,斑馬魚模型已經(jīng)在藥物篩選領(lǐng)域取得矚目的成果,我們首次將其作為整體動物模型應(yīng)用到317L-Cu不銹鋼體內(nèi)毒性的全面評價中,結(jié)果顯示兩種不銹鋼對斑馬魚胚胎和幼魚的發(fā)育前期不具有顯著的毒副作用。(4)通過對細(xì)菌在兩種不銹鋼材料表面形成生物膜內(nèi)胞外聚合物(EPS)的傅立葉紅外光譜(FTIR)分析及細(xì)胞膜表面銅離子情況的X射線光電子能譜學(xué)(XPS)檢測探索了317L-Cu不銹鋼對S.aureus生物膜的影響。此外,進(jìn)一步通過活死(Live/Dead)熒光染色法及透射電子顯微鏡(TEM)表征317L-Cu不銹鋼溶出的銅離子對于細(xì)菌細(xì)胞膜及內(nèi)含物的損傷過程,并探究其抗菌機(jī)制:Cu2+對細(xì)菌的細(xì)胞膜和胞外分泌物均造成了損傷,致使細(xì)菌內(nèi)含物泄露,生物膜形成受到抑制;317L-Cu不銹鋼并未能對細(xì)菌的基因組DNA完整性造成顯著影響,即317L-Cu不銹鋼的抗菌性能并非通過破壞細(xì)菌基因組的完整性而體現(xiàn)。于此同時,我們推測了317L-Cu不銹鋼同時具有良好殺菌能力及低細(xì)胞毒副作用的初步解釋原因:細(xì)菌和細(xì)胞的膜結(jié)構(gòu)、組成成分以及代謝活動存在差異造成其對外界相同刺激的反應(yīng)也不一致,耐受能力也存在差異。低濃度的Cu2+本身是有助于細(xì)胞增殖,而在該濃度區(qū)間內(nèi),對細(xì)菌而言則是致命的。綜上所述,317L-Cu不銹鋼具有理想的耐腐蝕性和生物安全性,并且在不顯著降低317L不銹鋼的優(yōu)秀材料屬性的前提下,兼具有良好的抗菌性能。因此,本論文研究的317L-Cu不銹鋼是一種極具潛力可用于日常生活及臨床醫(yī)學(xué)上代替原有不銹鋼材料的新型生物功能化不銹鋼改進(jìn)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.
【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R318.08

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