疏水疏油纳米银修饰不锈钢材料的制备及生物相容性研究

发布时间：2018-07-08 18:17

本文选题：电化学沉积 + 界面修饰　；参考：《吉林大学》2016年博士论文

【摘要】：冠心病介入治疗领域最突出的成就就是经皮冠脉介入治疗及衍生相关技术,成功的降低了患者的死亡率。过去的十年期间,中国的介入治疗在患者例数方面取得了长足的进步,同时技术上日趋成熟,表现为介入相关总死亡人数下降,死亡率逐年下降。尽管如此,经皮冠脉介入治疗本身也不是完美无缺,自身仍然有尚未解决的缺憾,支架内再狭窄及支架内血栓形成仍是目前发生率较高的PCI的并发症,尽管发生机理较为复杂,但总体上其发生与支架平台及涂层基质及生物活性药物的相容性及术中相关操作等因素有密切关系,支架材料以及涂层基质的疏水性和疏油性能对支架平台的相容性有显著影响。从目前的研究可知,与316L不锈钢纳米修饰材料相关的研究数量不足,尤其是与组织相容性和血液相容性相关的研究数量较少,更是缺乏采用改变材料疏水疏油性或者改变316不锈钢性能对动物实验相关研究的报道。故在316L不锈钢材料表面沉积银纳米修饰层,对不锈钢材质的修饰层对血液和细胞的相容性进行研究和分析,并且制备了316L不锈钢冠状动脉支架进行动物实验,检测其在动物体内是否诱导血栓形成及支架内再狭窄,为冠状动脉支架表面修饰研究提供依据。本研究在316L不锈钢材料表面沉积银纳米修饰层,并经全氟硅烷溶液浸泡。扫描电镜及红外光谱检测均证实修饰成功,对颗粒直径在90-130nm之间的不同电镀时间沉积的银颗粒接触角进行测试,使之达到至少130°,疏油角从316L不锈钢的0°提高到62-72°。按照中华人民共和国对医疗器械的国家标准GB/T 16886.5-2003要求用蛋白吸附实验完成生物材料与血浆蛋白的相互作用的评估;血细胞与生物材料间的相互作用评估则通过溶血性能试验、动态凝血实验、血小板粘附性能实验、抗凝血时间测定等完成;组织细胞与生物材料的相容性实验常常用细胞毒性试验来间接评估。本研究按照要求的相容性指标进行系列测试修饰后的316L不锈钢的血液相容性及细胞相容性。测试中发现白蛋白吸附能力最强者为1600s组,且各组间差异显著,而各组在吸附纤维蛋白能力上无显著差异。而且800s(B)、1600s(C)两组在抗凝血实验的各个时间节点总是优于裸钢板组,800s和1600s组的细胞相容性较好。课题组设计的全氟硅烷浸泡的银镀膜修饰316L不锈钢钢板的最大溶血率仅4.1%,最小溶血率0.4%,均小于5%,符合国家标准的要求,对于其大多数凝血指标如凝血酶原时间,部分凝血活酶时间、国际标准化比值、凝血酶原活动度,各项指标同修饰前并无太大区别。电镜检测表明,与裸钢板相比,各组电沉积银镀膜后全氟硅烷浸泡316L不锈钢板其表面粘附的血小板数量明显较少,而且其表面粘附的血小板激活程度也较低。在实验组各个沉积时间不同的钢板中,沉积时间为1600秒(B组)的钢板表面血小板粘附最少,其激活程度也较低,大部分I型。沉积时间为4000秒(D组)钢板表面的血小板粘附最多,而且激活程度较高为V型。沉积时间为800秒(A组)和2000秒(C组)的两组钢板表面血小板粘附数量及激活程度居中。四甲基偶氮唑蓝法(MTT法)检测细胞活性的实验数据表明,随着培养时间的增加,各实验组与对照组细胞的数量增加均显著,并且两者的曲线存在很大的相似性,这也充分说明了使用全氟硅烷对实验组进行修饰之后,并未引发细胞增殖状况的显著变化,并且银修饰的全氟硅烷钢板的细胞相容性更佳。800s和1600s组的银修饰全氟硅烷钢板的细胞相容性较好,但是两组相比无明显的统计学意义。流式细胞仪(Flow Cytometry,FCM)检测细胞周期分布的实验数据表明,处于G期的细胞,无论裸钢板组还是纳米银修饰组均增多,而处于S期和M期的减少,表明实验组细胞的DNA合成和有丝分裂被轻度抑制;而各纳米银修饰组的处于G期、S期及M期的细胞比例与裸钢板组的相比无明显差异(P0.05)。在体试验中,将修饰后的不锈钢制成冠脉支架,置入靶血管,用血管内超声在宏观上判断内皮化及再狭窄,对比病理切片从微观上评判试验材料的相关性能,在体试验显示,修饰后钢板制成的支架置入1月后,未增加血栓发生。组织学未见明显的炎症反应,各试验组只有800s、2000s、4000s在内膜厚度较裸支架薄、内膜面积较裸支架小,具有统计学意义(p0.05),而1600s组的内膜面积与裸支架组无明显差异;在中膜厚度、中膜面积指标上800s、1600s、2000s组均优于裸支架组,具有统计学意义(P0.05);在管腔面积方面,800s、1600s、2000s组较裸支架组增大,各组之间无统计学意义。结论:在316L不锈钢表面进行纳米银进行修饰并经氟化硅烷浸润后,形成的表面结构改善了接触角的同时还具有较好的相容性,并且材料并未触发亚急性血栓形成,减少了支架内再狭窄的出现。
[Abstract]:The most prominent achievement in the interventional therapy of coronary heart disease is percutaneous coronary intervention and derived related techniques, which have successfully reduced the mortality of the patients. During the past ten years, Chinese interventional therapy has made considerable progress in the number of patients, and the technology is becoming more and more mature, showing a decline in the total number of deaths associated with intervention. The mortality rate is declining year by year. However, percutaneous coronary intervention is not perfect and still has unsolved defects. Stent restenosis and stent thrombosis are still the complications of high incidence of PCI. Although the mechanism is complex, it is generally associated with the scaffolding platform and coating matrix and birth. There is a close relationship between the compatibility of active drugs and the related operations in the operation. The hydrophobicity and oil repellency of the scaffold and the coated matrix have a significant influence on the compatibility of the scaffold. From the present study, the number of studies related to the 316L stainless steel nano modified materials is not enough, especially in the tissue compatibility and blood phase. The number of capacitive related studies is less, and it is lack of reports on the study of animal experiments on changing the hydrophobicity of the materials or changing the properties of 316 stainless steel. Therefore, the silver nano modification layer on the surface of 316L stainless steel is deposited on the surface of stainless steel, and the compatibility of the modified layer of stainless steel to the blood and cell is studied and analyzed. 316L stainless steel coronary stent was used in animal experiments to detect whether the thrombus was induced in the animal and the restenosis in the stent, which provided a basis for the study of the surface modification of the coronary stent. The silver nanoparticles were deposited on the surface of 316L stainless steel and soaked through the perfluorosilane solution. It was proved that the modification was successful. The contact angle of silver particles deposited at different electroplating time between 90-130nm particles was tested to reach at least 130 degrees. The oil repellent angle was raised from 0 degrees of 316L stainless steel to 62-72 degrees. According to the national standard GB/T 16886.5-2003 of People's Republic of China medical equipment, the protein adsorption experiment was required to complete the life. Evaluation of the interaction between material and plasma protein; evaluation of the interaction between blood cells and biomaterials by hemolysis test, dynamic coagulation experiment, platelet adhesion test, anticoagulant time determination, and so on. The compatibility of tissue and biomaterials is often used for indirect evaluation of cytotoxicity test. The blood compatibility and cell compatibility of 316L stainless steel after a series of tests were carried out according to the required compatibility index. The highest albumin adsorption capacity was found in group 1600s, and there were significant differences in each group, but there was no significant difference in the ability to adsorb fibrin. Moreover, 800s (B), 1600s (C) two groups were in anticoagulant experiments. Each time node is always better than the bare plate group. The cell compatibility of the 800s and 1600s groups is better. The maximum hemolysis rate of the 316L stainless steel plate modified by the perfluorsilane immersion coating is only 4.1%, the minimum hemolysis rate is 0.4%, which is less than 5%, which meets the requirements of the national standard, and is suitable for most of the coagulation indexes such as the prothrombin time. There was no significant difference between the thromboplastin time, the international standardization ratio, the prothrombin activity and the prothrombin activity before the modification. The electron microscopic examination showed that the number of platelets adhered to the surface of the 316L stainless steel plate after electrodeposition of silver coated with perfluorosilane was less than that of the bare plate, and the adhesion of the surface of the platelets adhered to the surface of the platelets. In the steel plate with different deposition time in the experimental group, the platelets on the plate surface of 1600 seconds (group B) had the least adherence to the platelets, and the activation degree was lower. Most of the I type. The deposition time was 4000 seconds (group D) on the surface of the platelets, and the degree of activation was higher than that of the V type. The time of deposition was 800 seconds (A group) and 2000 seconds (C). The number and activation of platelets on the two groups of the two groups of plates were in the middle. The experimental data of four methyl azazolium blue (MTT) method to detect cell activity showed that the number of cells increased significantly with the increase of culture time, and there was a great similarity in the curve lines between the two groups. After the modification of fluorosilane, there was no significant change in cell proliferation, and the cytocompatibility of the silver modified perfluorosilane steel plate was better than that of the silver modified perfluorosilane steel plate in.800s and 1600s groups, but there was no significant statistical significance in the two groups. Flow cytometry (Flow Cytometry, FCM) The experimental data of cell cycle distribution showed that the cells in the G stage increased in both the bare plate group and the nano silver modifier, and the decrease in the S phase and the M phase showed that the DNA synthesis and mitosis were slightly inhibited in the experimental group, while the nano silver modifier was in the G phase, the proportion of the cells in the S phase and the M phase with the bare steel plate group. There was no significant difference (P0.05). In the body test, the modified stainless steel was made into a coronary stent, inserted into the target vessel, and judged endothelialization and restenosis with intravascular ultrasound on the macro level. The related properties of the experimental material were judged by the pathological sections. In the body test, the stent made of the modified steel plate was placed in January, without increasing blood. There was no obvious inflammatory reaction in histology. Only 800s, 2000s, 4000s were thinner than bare stents in the endometrium, and the area of the intima was smaller than that of bare scaffold (P0.05), but there was no significant difference between the intima area and the bare stents in the 1600s group. The thickness of the middle membrane and the area index of the middle membrane were superior to those of the naked branch in the group of 800s, 1600s and 2000s. There is statistical significance (P0.05) in the frame group, and in the area of the lumen, the group of 800s, 1600s, and 2000s is larger than the bare scaffold group, and there is no statistical significance between the groups. Conclusion: the surface structure of the 316L stainless steel surface is modified and the surface structure improves the contact angle and has better compatibility, and the material has better compatibility. The material did not trigger subacute thrombosis and reduced the occurrence of in stent restenosis.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R541.4;R318.08

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