纳米载银磷酸锆抗菌聚氨酯抗菌性能及生物相容性研究

发布时间：2018-04-28 01:03

本文选题：纳米载银无机抗菌剂 + 聚氨酯　；参考：《南方医科大学》2012年硕士论文

【摘要】：研究背景生物材料是与生物系统起作用的医疗物件的非生命材料,也称作生物医学材料。炎症反应是生物材料植入人体后主要副作用之一。生物材料的植入为游离细菌提供粘附的位点,使感染更容易发生。随着细菌的感染粘附,逐渐形成一层水合多聚糖性质的生物膜。在这种生物膜内,细菌的抗药性要远远大于游离细菌,传统的抗菌治疗作用不大,因此植入物的使用常并发以生物材料为中心的感染(BCI)。BCI及其它组织反应是临床植入手术的重大问题,经常是再次手术和医疗事故的主要原因,并给病人带来经济上的重大负担。因此,研究本身具有抗细菌感染能力的生物医用材料已经成为生物材料领域的一个热点问题。聚氨酯是结构中含有重复的氨基甲酸酯基(-NHCO-)的聚合物的总称,可以通过设计软硬段的比例对其物理、化学性质进行比较大的调整。聚氨酯材料具有优异的力学强度、高弹性、耐磨性、润滑性、耐疲劳性、生物相容性、可加工性等而被广泛应用于生物医学领域,以及良好的生物相容性和抗凝血性能,被称为“理想的生物材料”。在许多医疗装置和人工器官中得到了广泛应用,是一种很重要的生物医用材料。但在适宜的温度和湿度条件下,医用制品表面极易生长和繁殖细菌,严重威胁人类的健康,同时也会带来医疗事故和巨大的经济损失。开发抗菌聚氨酯材料,保护人民的身体健康,防止细菌的传播,乃至预防局部或世界流行疾病的爆发都具有十分重要的意义。目的研制纳米载银磷酸锆抗菌聚氨酯材料,并观察其在体外及体内的生物相容性,以期制作出一种理化性质优越,生物相容性高的人工生物材料,为其进一步应用于人体植入材料提供最佳材料的选择。方法 1、采取目前普遍使用的母粒法制备抗菌聚氨酯。首先将纳米载银磷酸锆、聚氨酯(EG100A-B20)和分散剂按一定比例在高速混合机中混合均匀,以提高抗菌剂在聚氨酯中的分散和附着能力,然后将混合均匀的物料经双螺杆挤出机、切粒机制成抗菌母粒,最后将抗菌母粒同空白聚氨酯按一定比例混合,制备出抗菌剂质量分数分别为0%(空白)、0.5%、1%、1.5%、2%、2.5%、5%的抗菌聚氨酯,经注塑机注塑成型为5×5×0.5cm标准样板,蒸馏水中浸泡24,消毒备用。 2、纳米载银磷酸锆抗菌聚氨酯材料的体外抗菌性能研究。采用贴膜法检测纳米载银磷酸锆抗菌聚氨酯材料对金黄色葡萄球菌和大肠杆菌的抗菌性能。将标准菌株金黄色葡萄球菌、大肠杆菌传代培养,混悬于TSB培养液中,采用麦氏比浊法配置成浓度为1.5×108CFU/mL的菌液,并依次做4次10倍递增稀释。分别吸取100μL菌液接种在各组抗菌试件表面,盖上消毒PE膜,培养24h。然后将试件放置在含9.99mL TSB培养液灭菌试管中,旋涡振荡1min。分别取1mL接种于TSA平板上,培养24h后,计数菌落数CFU。实验重复5次,结果取平均值。 3、按ISO10993.12-2009和国家GB/T16886-2008中的系列标准要求,对制备的纳米载银磷酸锆抗菌聚氨酯材料进行细胞毒性实验、急性全身毒性实验、溶血实验、热原实验、兔肌肉植入实验,以综合评价其生物相容性。细胞毒性实验：将实验组(抗菌聚氨酯浸提液)、阴性对照组(聚乙烯浸提液)与阳性对照组(6.4%苯酚溶液)各3ml,分别置于35mm细胞培养皿内,加入6×104/ml L929细胞悬液3ml,培养箱培养24h、48h及72h后,观察细胞形态及生长情况。将100ul浓度为6×104/mL的细胞悬液接种于96孔培养板,细胞贴壁后弃原液,分别加入实验组(2%抗菌聚氨酯浸提液)、空白对照组(0%抗菌聚氨酯)、阴性对照组(聚乙烯浸提液)与阳性对照组(6.4%苯酚溶液)各100ul,采用MTT比色法测定L929细胞培养24h、48h、72h的相对增殖率(RGR),评价材料的细胞毒性。急性全身毒性实验：按50ml/kg剂量标准,无菌条件下将2%纳米载银磷酸锆聚氨酯材料的浸提液注入试验组小鼠腹腔内,阴性对照组注入生理盐水,阳性对照组注入苯酚。给药后连续7天观察小鼠,记录小鼠呼吸、进食、运动等一般情况、毒性反应、体重变化或死亡溶血实验：取10ml受试样品浸提液(实验组)、10ml生理盐水(阴性对照组)、10ml蒸馏水(阳性对照组),分别加入0.2ml的新鲜抗凝稀释兔血,肉眼观察是否出现明显的溶血现象；酶标仪检测各样本上清液的吸光度,评价受试材料的体外血液相容性。热原实验：以5ml/kg剂量将受试材料浸提液自兔耳缘静脉缓慢注入,给药后连续测量兔体温并与实验前预检体温相比较,评价受试材料的制热作用。兔肌肉植入实验：将2%纳米载银磷酸锆抗菌聚氨酯材料植入新西兰大白兔背部肌肉中,分别于术后1w、4w、12w后取出进行大体观察与组织病理学检查,观察材料植入后的组织炎症反应程度与纤维囊形成情况,评价材料植入肌肉后的组织反应。结果 1、抗菌聚氨酯的抗菌性能评价添加纳米载银磷酸锆的热塑性聚氨酯能有效抑制大肠杆菌和金黄色葡萄球菌的生长。抗菌剂添加比例0.5%-5%组对金黄色葡萄球菌的抑菌率分别为80.23%、91.32%、95.23%、99.19%、99.87%、99.93%,对大肠杆菌的抑菌率分别为76.70%、86.96%、92.92%、99.14%、99.34%、99.87%,与0%比例组(对照组)比较均有显著差异(P0.05),且抗菌效能随抗菌剂添加比例的上升而显著提高。 2、抗菌聚氨酯材料的生物相容性评价细胞毒性实验：2%纳米载银磷酸锆抗菌聚氨酯浸提液细胞贴壁生长,细胞呈梭形,折光性强,细胞突充分伸展；浸提液作用小鼠成纤维细胞24h,48h和72h后RGR分别为97.3%、96.5%、96.3%,组细胞毒性毒性反应均为Ⅰ级,无细胞毒性。提示2%纳米载银磷酸锆抗菌抗菌聚酯与普通抗菌聚氨酯(0%)均有良好的细胞相容性。急性全身毒性实验：实验组小鼠注射后没有出现异常症状,活动、进食、排泄正常,未见步态不稳、惊厥、瘫痪以及呼吸抑制等毒性反应,在7天观察期内,体重呈增加趋势,无死亡。溶血实验：受试材料浸提液不引起体外溶血反应,溶血率为1.56%。热原实验：注射受试材料浸提液后体温升高均低于0.60℃,三只兔体温升高总数为0.5℃,按国家药典的标准受试材料无不良的的制热作用。兔肌肉植入实验：大体观察受试材料植入后切口无明显渗血、渗液,无感染发生,纤维囊随时间逐渐变薄。组织病理学检查提示,材料植入后局部有少量炎细胞浸润,以中性粒细胞和散在的淋巴细胞为主,无组织坏死。随着植入时间增加,炎症基本消失,且形成微薄且透明的纤维膜包裹。结论 1、本课题首次将纳米载银磷酸锆通过熔融共混法加入聚氨酯制备出纳米载银磷酸锆抗菌聚氨酯,并证实其具有良好的抗菌效果和优异的生物相容性。 2、随着纳米载银无机抗菌剂添加比例的上升,聚氨酯的抗菌性能明显提高。当添加比例达到1.5%时,纳米抗菌聚氨酯对金黄色葡萄球菌抑菌率达到95.23%,对大肠杆菌的抑菌率达到92.92%。由于QB/T2591-2003规定材料对微生物的抑菌率90%才能被称为抗菌材料,所以从抗菌角度出发,推荐纳米载银无机抗菌剂在聚氨酯中的添加比例应不低于1.5%。 3、据IS010993-2009和GB/T16886-2008系列标准对于生物材料相容性的检测方法与要求,实验结果表明本课题组制备的纳米载银磷酸锆抗菌聚氨酯材料无细胞毒性、无急性全身毒性、不引起溶血反应、无致热作用,与空白的医用聚氨酯比较,生物相容性无明显差异。纳米载银磷酸锆抗菌聚氨酯具有良好生物相容性,为下一步应用于人工肛门括约肌的生物材料提供了实验依据。
[Abstract]:Research background
Biomaterials are nonliving materials, also known as biomedical materials that play a role in biological systems. Inflammatory reactions are one of the main side effects of biomaterials implanted in the human body. Biomaterials are implanted to provide adherent sites for free bacteria to make infection more easy to occur. A layer of water is gradually formed as the bacterial infection adhered. In this biofilm, the antimicrobial resistance of bacteria is much greater than that of free bacteria, and the traditional antibacterial treatment is not significant. Therefore, the use of BCI.BCI and other tissue reacts with biomaterial based infection is a major problem in clinical implantation, often reoperation and medical treatment. Therefore, the study of biological materials that have the ability to resist bacterial infection has become a hot issue in the field of biomaterials.
Polyurethane is the general name of the polymer containing repeated carbamate group (-NHCO-) in the structure. The physical and chemical properties of the polyurethane can be greatly adjusted by the proportion of the hard and soft segments. The polyurethane material has excellent mechanical strength, high elasticity, wear resistance, lubricity, fatigue resistance, biocompatibility, and processability. In the field of biomedicine, as well as good biocompatibility and anticoagulant properties, it is called "ideal biomaterial". It has been widely used in many medical devices and artificial organs. It is a very important biological medical material. But the surface of medical products is very easy to grow and reproduce under suitable temperature and humidity. Bacteria, which seriously threaten human health, will also bring about medical accidents and huge economic losses.
It is of great significance to develop antibacterial polyurethane materials, protect the health of the people, prevent the spread of bacteria, and even prevent the outbreak of local or world epidemic diseases.
objective
In order to produce a kind of artificial biological material with superior physical and chemical properties and high biocompatibility, the biocompatibility of nano silver phosphate zirconium phosphate antibacterial polyurethane material was developed and its biocompatibility in vitro and in vivo was observed, which could provide the best material for the further application of the material to human implant materials.
Method
1, the antibacterial polyurethane is prepared by the commonly used masterbatch method.
First, nano silver zirconium phosphate, polyurethane (EG100A-B20) and dispersant are mixed uniformly in a high speed mixer to improve the dispersing and adhesion ability of the antibacterial agent in the polyurethane. Then the mixed material is made by the twin screw extruder and the grain cutting mechanism is the anti bacterial masterbatch. Finally, the antibacterial masterbatch is based on the blank polyurethane. The proportion of antibacterial agents was 0% (blank), 0.5%, 1%, 1.5%, 2%, 2.5%, 5%, respectively. The injection molding machine was injected into 5 x 5 x 0.5cm standard sample, and 24 was soaked in distilled water.
2, in vitro antibacterial properties of nano silver loaded zirconium phosphate antibacterial polyurethane materials.
The antibacterial properties of nanoscale silver phosphate zirconium phosphate antibacterial polyurethane material to Staphylococcus aureus and Escherichia coli were detected by membrane method. The standard strains of Staphylococcus aureus and Escherichia coli were cultured and suspended in TSB medium. The bacterial solution with a concentration of 1.5 x 108CFU/mL was prepared by maimei turbidimetry, and 4 times 10 times increased progressively. The 100 mu L bacteria were inoculated on the surface of each group of antibacterial specimens, the PE membrane was sterilized on the cover, the 24h. was cultured and the specimens were placed in the sterilization test tube containing 9.99mL TSB culture solution. The vortex oscillation 1min. was inoculated on the TSA plate respectively, and the 24h was cultured, and the count of the count colony number CFU. was repeated for 5 times, and the average value was obtained.
3, the cytotoxicity test, acute systemic toxicity test, hemolysis experiment, pyrogen test and rabbit muscle implantation were carried out to evaluate the biocompatibility of the prepared nano silver zirconium phosphate antibacterial polyurethane material according to the standard of ISO10993.12-2009 and national GB/T16886-2008.
Cytotoxicity test: the experimental group (Antibacterial Polyurethane extract), negative control group (polythene extract) and positive control group (6.4% phenol solution) each 3ml, respectively placed in 35mm cell culture dish, adding 6 x 104/ml L929 cell suspension 3ml, culture box culture 24h, 48h and 72h, observe the cell morphology and growth condition. 100ul concentration is 6 x 104/. The cell suspension of mL was inoculated in 96 hole culture plate, and after the cell was adhered to the original solution, the cell suspension was added to the experimental group (2% Antibacterial Polyurethane extract), the blank control group (0% Antibacterial Polyurethane), the negative control group (polyethylene extract) and the positive control group (6.4% phenol solution) each were 100ul, and the relative proliferation of 24h, 48h, 72h in L929 cells was measured by MTT colorimetry. The rate (RGR) was used to evaluate the cytotoxicity of the material.
Acute systemic toxicity test: under the 50ml/kg dose standard, under the aseptic condition, the extract of 2% nano silver zirconium phosphate polyurethane material was injected into the abdominal cavity of the experimental group, and the negative control group was injected with saline, and the positive control group was injected with phenol. The mice were observed for 7 days after the drug was given, and the mice were recorded the general condition of respiration, eating, exercise and so on. Reaction, body weight change, or death
Hemolytic experiment: Taking the 10ml samples (experimental group), 10ml physiological saline (negative control group), 10ml distilled water (positive control group), fresh anticoagulant rabbit blood was added to 0.2ml, and the obvious hemolysis was observed in the naked eye; the enzyme labelled instrument detected the absorbance of all the supernatants, and evaluated the blood compatibility of the material in vitro. Sex.
The test of thermometer: the extract of the tested material was injected slowly from the rabbit ear vein with the dose of 5ml/kg. The rabbit body temperature was measured continuously after the drug was given and compared with the pre test temperature before the test. The heat effect of the tested material was evaluated.
The experiment of rabbit muscle implantation: the 2% nano silver phosphate zirconium phosphate antibacterial polyurethane material was implanted into the back muscles of New Zealand white rabbits. After 1W, 4W, and 12W after operation, the gross observation and histopathological examination were taken respectively. The degree of inflammation and the formation of fibrous sac after the implant were observed, and the tissue reaction after the material was implanted into the muscle was evaluated. It should.
Result
1, evaluation of antibacterial properties of Antibacterial Polyurethane
The growth of Escherichia coli and Staphylococcus aureus was effectively inhibited by the addition of nano silver phosphate zirconium phosphate. The antibacterial rates of 0.5%-5% group to Staphylococcus aureus were 80.23%, 91.32%, 95.23%, 99.19%, 99.87%, 99.93%, respectively, and 76.70%, 86.96%, 92.92%, 99.14%, 99.34%, 9, respectively. 9.87%, there was a significant difference between the 0% groups and the control group (P0.05), and the antibacterial efficacy increased significantly with the increase of the proportion of antibacterial agents.
2, evaluation of biocompatibility of Antibacterial Polyurethane Materials
Cytotoxicity test: 2% nanoscale silver phosphate zirconium phosphate antibacterial polyurethane extract cells adhered to wall growth, cells showed spindle shape, strong refraction, and full extension of cell process. The extract acted on mice fibroblasts 24h, 48h and 72h, RGR was 97.3%, 96.5%, 96.3% respectively, and the cytotoxic reaction of the group was I, no cytotoxicity. It suggested 2% nano silver. Zirconium phosphate antibacterial antibacterial polyester has good cytocompatibility with ordinary Antibacterial Polyurethane (0%).
Acute systemic toxicity test: the mice in the experimental group did not have abnormal symptoms, activities, eating, excreting normal, no gait instability, convulsion, paralysis and respiratory inhibition. During the observation period of 7 days, the body weight showed an increasing trend and no death.
Hemolysis test: the extract of the tested material did not cause hemolysis in vitro, and the hemolysis rate was 1.56%.
The thermometer test: the body temperature rises below 0.60 degrees centigrade after injection of the tested material, and the total body temperature of three rabbits is 0.5 degrees centigrade, and there is no bad heat making effect according to the standard of the national pharmacopoeia.
The experiment of rabbit muscle implantation: there was no obvious bleeding, infiltration and no infection in the incision after the implant was implanted. The fibrous capsule gradually thinned with time. Histopathological examination suggested that a small amount of inflammatory cells infiltrated after the implant was implanted, with neutrophils and scattered lymphoblastic cells and no tissue necrosis. With the implantation time increasing, Inflammation disappeared and formed a thin and transparent fibrous membrane.
conclusion
1, the nanoscale silver phosphate zirconium phosphate was first prepared by melt blending to prepare teller silver zirconium phosphate antibacterial polyurethane, and proved that it had good antibacterial effect and excellent biocompatibility.
2, with the increase in the proportion of nano silver carrying inorganic antibacterial agents, the antibacterial properties of the polyurethane improved obviously. When the addition ratio reached 1.5%, the antibacterial rate of nanoscale Antibacterial Polyurethane to Staphylococcus aureus was 95.23%. The bacteriostasis rate of Escherichia coli reached 92.92%. due to the bacteriostasis rate of 90% to microorganism by QB/ T2591-2003. It is called antibacterial material. Therefore, from the perspective of antibacterial, it is recommended that the proportion of nano silver inorganic antibacterial agent in polyurethane should be no less than 1.5%.
3, according to the detection methods and requirements of IS010993-2009 and GB/T16886-2008 series standards for biocompatibility of biomaterials, the experimental results show that the nano silver phosphate zirconium phosphate antibacterial polyurethane material prepared by this group has no cytotoxicity, no acute systemic toxicity, no hemolytic reaction and no heat effect, compared with the blank medical polyurethane, the biological phase is compared. There is no obvious difference in capacity. The nano silver phosphate zirconium phosphate antibacterial polyurethane has good biocompatibility, which provides experimental basis for the next step of artificial anal sphincter.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08

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