基于聚多巴胺辅助表面沉积含银涂层的聚醚醚酮的制备及抗菌性能与生物相容性研究

发布时间：2018-05-02 21:20

本文选题：聚醚醚酮(PEEK) + 多巴胺　；参考：《苏州大学》2016年博士论文

【摘要】：第一部分表面载银的PEEK材料的制备与表征检测目的:在弱碱性条件下自聚合形成的聚多巴胺涂层沉积在聚醚醚酮(Polyetheretherketone,PEEK)表面,利用聚多巴胺自身的弱还原性,可以将银氨离子还原为银纳米颗粒沉积在PEEK材料表面,制备出表面载银的PEEK复合材料,并对其进行检测与表征。方法:将表面打磨光滑的PEEK圆片用等离子体清洗后,在表面负载大量的羟基,将上述材料浸泡在2 mg/ml多巴胺溶液中(10 m M Tris-HCl溶液,p H=8.5)24小时,在PEEK表面制备出聚多巴胺涂层(PEEK-PDA),然后将含有聚多巴胺涂层的PEEK复合材料浸泡在0.02 mol/L的银氨溶液中1小时,制备出表面载银的PEEK复合材料(PEEK-PDA-Ag)。然后对该涂层的形貌特征、化学组成进行检测,材料表面亲水性分析,以及银离子释放等表征。结果:检测显示,经过载银处理后的PEEK材料颜色上发生了很明显的变化,电镜下可以很明显观察到材料表面均匀的沉积了银颗粒,粒径约为90 nm,EDS能谱分析显示载银量为5.83 wt%。接触角测试显示经过载银处理后的PEEK材料表面亲水性发生了很大的改变。当将表面载银的PEEK复合材料浸泡于缓冲溶液中,涂层能向周围溶液中持续释放银离子。结论:利用多巴胺的自聚合以及聚多巴胺的还原性,能够在PEEK材料表面制备出尺寸均匀的银粒子涂层,该涂层能够长期向周围溶液中释放银离子。第二部分表面载银的PEEK材料的抗菌性能检测目的:探讨表面载银的PEEK复合材料(PEEK-PDA-Ag)对金黄色葡萄球菌的抗菌性能。方法:⑴以PEEK-PDA-Ag和PEEK-PDA作为实验组,PEEK作为对照组,以金黄色葡萄球菌菌株ATCC25923为实验菌株。将金黄色葡萄球菌菌株ATCC25923接种与各组材料培养24小时,进行活菌计数,观察抑菌效果,计算其抑菌率。⑵在各组材料表面接种金黄色葡萄球菌菌珠,经过24小时培养后,观察有无抑菌圈生成,并测量抑菌圈直径大小。⑶在各组材料表面接种实验菌株培养24小时后,脱水干燥后,电镜下观测菌落形貌。结果:表面抑菌实验结果提示:培养24小时以后,实验组(PEEK-PDA-Ag)表现出明显的抑菌效应,其表面的金黄色葡萄球菌活菌计数低于接种浓度,抑菌率可达到99.84%,PEEK-PDA组也展现出了一定的抑菌效应,抑菌率为78.6%。实验组与对照组之间抑菌率具有显著统计学差异(P㩳0.05)。将金黄色葡萄球菌接种在各组材料表面,培养24小时后,在PEEK-PDA-Ag组样品周围产生了明显的抑菌圈,抑菌圈直径约为8.8 mm,而在PEEK组和PEEK-PDA组周围未见明显的抑菌圈产生。将金黄色葡萄球菌接种在各组材料表面,培养24小时后,经过脱水干燥处理后,在电镜下可以观察,PEEK-PDA-Ag组样品表面细菌数量明显少于PEEK组和PEEK-PDA组(P㩳0.05)。结论:PEEK-PDA-Ag材料具有明显的抑菌作用,抑菌率可达到99.84%,同时在PEEK-PDA-Ag材料周围可以明显的观测到抑菌圈的产生,抑菌圈直径约为8.8 mm。第三部分表面载银的PEEK材料的生物相容性检测目的:探讨表面载银的PEEK复合材料(PEEK-PDA-Ag)对成骨细胞MC3T3-E1生物活性的影响,并观察细胞在材料表面黏附和增殖的情况。用小鼠巨噬细胞(RAW264.7)为动物细胞模型,利用q RT-PCR技术从细胞分子水平上探讨几种材料对小鼠巨噬细胞分泌的相关炎性因子表达的影响。方法:⑴以PEEK-PDA-Ag材料作为实验组,PEEK材料和PEEK-PDA材料作为对照组,分别按一定的密度接种MC3T3-E1细胞并进行培养。在第1,3,5天,分别用CCK-8试剂检测各组材料表面细胞增殖情况。⑵在每个时间点,每组取出两个样品,经过梯度脱水和临界点干燥后,用扫描电镜观察成骨细胞在各组材料表面的黏附铺展情况。⑶以PEEK-PDA-Ag材料和PEEK-PDA材料作为实验组,空白板作为对照组,用小鼠巨噬细胞(RAW264.7)为动物细胞模型,检测IL-4,IL-10,TGF-β抗炎因子的表达以及IL-1β,IL-6,TNF-α等促炎因子的表达情况。结果:CCK-8检测结果表明:PEEK-PDA-Ag组细胞有明显的增殖趋势,在第1,3,5天时间点时,PEEK-PDA-Ag组细胞都有明显的增殖,对照组PEEK和PEEK-PDA的细胞同样也有明显的增殖(P〉0.05);扫描电镜下观察细胞形貌及黏附:可以看出,实验组PEEK-PDA-Ag表面的细胞在第一天时呈细长形,在第3,5天时,材料表面细胞有明显增殖,材料表面细胞密度明显增多,且细胞立体性较好,对照组PEEK和PEEK-PDA材料在第1天时,表面的细胞黏附很均匀,且向四周铺展,第3天时,材料表面细胞密度明显多于第一天时间点,细胞增殖明显,第5天时,对照组细胞基本长满,且细胞均比较饱满。炎性因子表达的结果显示,RAW264.7细胞在各组材料上培养72h后,在抗炎因子表达方面,与对照组相比,PEEK-PDA-Ag组细胞中IL-4基因表达量显著降低(P㩳0.05),IL-10和TGF-β表达量有降低趋势,但没达到显著水平;在促炎因子表达方面,与对照组相比,PEEK-PDA-Ag组细胞中IL-1β基因表达量显著升高(P㩳0.05),但IL-6与TNF-α表达量无显著差异。结论:通过聚多巴胺修饰还原制备的表面载银的PEEK材料(PEEK-PDA-Ag)与MC3T3-E1共培养观察,显示在PEEK-PDA-Ag复合材料表面,细胞同样可以很好地黏附,增殖分化,无严重的毒性;通过与小鼠巨噬细胞RAW264.7的共培养,检测相关炎性因子表达情况,材料表面载Ag并无抗炎症作用,相反还略有轻微促炎作用。
[Abstract]:The preparation and characterization of the first part of the PEEK material carrying silver on the surface of Polyetheretherketone (PEEK) on the surface of the polyether ether ketone (PEEK) under the weak alkali condition, the silver nanoparticles can be reduced to silver nanoparticles on the surface of the PEEK material by using the weak reducibility of the polyamine itself, and the preparation of the silver nanoparticles can be prepared on the surface of the material. The PEEK composite with silver on the surface was detected and characterized. Method: after cleaning the smooth PEEK wafer on the surface with plasma, a large amount of hydroxyl was loaded on the surface, and the above materials were soaked in 2 mg/ml dopamine solution (10 m M Tris-HCl solution, P H=8.5) for 24 hours, and the polydopamine coating was prepared on the PEEK surface (PEEK-P). DA), then the PEEK composite containing polydopamine coating was soaked in a silver ammonia solution of 0.02 mol/L for 1 hours. The surface silver loaded PEEK composite (PEEK-PDA-Ag) was prepared. Then the morphology, chemical composition of the coating, the hydrophilic analysis of the material surface, and the release of silver ions were characterized. The color of the PEEK material after the silver loading has been obviously changed. Under the electron microscope, it can be observed that the silver particles are evenly deposited on the surface of the material, the particle size is about 90 nm. The EDS spectrum analysis shows that the silver load is 5.83 wt%. contact angle, and the hydrophilicity of the surface of the PEEK material after the silver loading has been greatly changed. When the material is treated, the surface hydrophilicity of the material has been greatly changed. The silver coated PEEK composite is soaked in the buffer solution, and the coating can release silver ions continuously in the surrounding solution. Conclusion: using the self polymerization of dopamine and the reducibility of the poly dopamine, a silver particle coating with uniform size can be prepared on the surface of the PEEK material. The coating can release Silver ions in the surrounding solution for a long time. Second parts. Detection of antibacterial properties of PEEK material carrying silver on the surface: To investigate the antibacterial properties of PEEK composite (PEEK-PDA-Ag) on Staphylococcus aureus. Methods: (1) PEEK-PDA-Ag and PEEK-PDA as the experimental group, PEEK as the control group and Staphylococcus aureus strain ATCC25923 as the experimental strain. Plant ATCC25923 inoculation and each group material culture for 24 hours, carry out the living bacteria count, observe the bacteriostasis effect and calculate its bacteriostasis rate. 2. After inoculation of Staphylococcus aureus beads on the surface of each material, after 24 hours culture, the formation of bacteriostatic ring is observed and the diameter of bacteriostasis circle is measured. 3. (3) inoculation of experimental strains on the surface of each material is 24 small. After dehydration and drying, the morphology of the colony was observed under the electron microscope. The results of the surface bacteriostasis experiment showed that after 24 hours of culture, the experimental group (PEEK-PDA-Ag) showed obvious bacteriostasis effect, the count of Staphylococcus aureus on the surface was lower than the inoculation concentration, the bacteriostasis rate could reach 99.84%, and the PEEK-PDA group also showed a certain bacteriostasis effect. The bacteriostasis rate was significant difference between the 78.6%. experimental group and the control group (P? 0.05). The Staphylococcus aureus was inoculated on the surface of each group. After 24 hours culture, the bacteriostatic circle was found around the PEEK-PDA-Ag group, and the diameter of the bacteriostasis was about 8.8 mm, but there was no obvious inhibition around the PEEK group and the PEEK-PDA group. The micrococcus aureus was inoculated on the surface of each material. After 24 hours culture, after dehydration and drying, the number of bacteria on the surface of PEEK-PDA-Ag group was obviously less than that of group PEEK and PEEK-PDA (P? 0.05). Conclusion: PEEK-PDA-Ag material has obvious bacteriostasis, and the rate of bacteriostasis can reach 99.84%. Meanwhile, the rate of bacteriostasis can reach 99.84%. The production of bacteriostasis circle can be observed around the PEEK-PDA-Ag material. The biocompatibility test of the PEEK material containing 8.8 mm. and third parts of silver containing the bacteriostasis diameter is to investigate the effect of the PEEK composite (PEEK-PDA-Ag) on the bioactivity of the osteoblast MC3T3-E1, and to observe the adhesion of the cells to the surface of the material and the adhesion of the cells on the surface of the material. Using mouse macrophage (RAW264.7) as an animal cell model, the effect of several materials on the expression of related inflammatory factors secreted by mouse macrophages was investigated by Q RT-PCR technology. Methods: (1) the PEEK-PDA-Ag material was used as the experimental group, and the PEEK material and the PEEK-PDA material were used as the control group, respectively. The density of MC3T3-E1 cells was inoculated and cultured. On day 1,3,5, CCK-8 reagents were used to detect the proliferation of the surface cells of each group. 2. At each time point, two samples were taken out of each group. After the gradient dehydration and critical point drying, the adhesion and spreading of the osteoblasts on the surface of each group was observed by scanning electron microscope. 3. (3) PEEK-PD A-Ag and PEEK-PDA materials were used as the experimental group, and the blank plate was used as the control group. The expression of IL-4, IL-10, TGF- beta anti-inflammatory factors and the expression of IL-1 beta, IL-6, TNF- alpha and other proinflammatory factors were detected with mouse macrophage (RAW264.7) as the animal cell model. Results: the results of CCK-8 test showed that the PEEK-PDA-Ag group had obvious proliferation trend. At 1,3,5 day time, the cells of group PEEK-PDA-Ag had obvious proliferation, and the cells of PEEK and PEEK-PDA in the control group also had obvious proliferation (P > 0.05). The morphology and adhesion of the cells were observed under the scanning electron microscope. It can be seen that the cells on the surface of PEEK-PDA-Ag in the experimental group were elongated on the first day, and the surface cells of the material were obvious at day 3,5. On the first day of first days, the cell density of the control group was very much more than the first day point, and the cell proliferation was obvious. At the time of fifth days, the cells of the control group were basically full, and the cells were all cells. The expression of inflammatory factors showed that after the RAW264.7 cells were cultured for 72h in each group, the expression of IL-4 gene in the PEEK-PDA-Ag group was significantly lower than that in the control group (P? 0.05). The expression of IL-10 and TGF- beta was reduced, but it did not reach a significant level. Compared with the control group, the expression of IL-1 beta gene in the cells of the PEEK-PDA-Ag group increased significantly (P? 0.05), but there was no significant difference in the expression of IL-6 and TNF- alpha. Conclusion: the PEEK material (PEEK-PDA-Ag), which is prepared by polydopamine modification and reduction (PEEK-PDA-Ag), is co cultured with MC3T3-E1, showing that the cells can also be well on the surface of the PEEK-PDA-Ag composite material. Adhesion, proliferation and differentiation, no serious toxicity; through co culture with RAW264.7 of mouse macrophages, the expression of related inflammatory factors was detected, and the surface of the material had no anti-inflammatory effect on the surface of Ag. On the contrary, it was slightly proinflammatory.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318.08;R68

【参考文献】