本文選題：納米載銀無機抗菌劑 + 聚氨酯��； 參考：《南方醫(yī)科大學(xué)》2012年碩士論文

【摘要】：研究背景 生物材料是與生物系統(tǒng)起作用的醫(yī)療物件的非生命材料,也稱作生物醫(yī)學(xué)材料。炎癥反應(yīng)是生物材料植入人體后主要副作用之一。生物材料的植入為游離細菌提供粘附的位點,使感染更容易發(fā)生。隨著細菌的感染粘附,逐漸形成一層水合多聚糖性質(zhì)的生物膜。在這種生物膜內(nèi),細菌的抗藥性要遠遠大于游離細菌,傳統(tǒng)的抗菌治療作用不大,因此植入物的使用常并發(fā)以生物材料為中心的感染(BCI)。BCI及其它組織反應(yīng)是臨床植入手術(shù)的重大問題,經(jīng)常是再次手術(shù)和醫(yī)療事故的主要原因,并給病人帶來經(jīng)濟上的重大負擔(dān)。因此,研究本身具有抗細菌感染能力的生物醫(yī)用材料已經(jīng)成為生物材料領(lǐng)域的一個熱點問題。 聚氨酯是結(jié)構(gòu)中含有重復(fù)的氨基甲酸酯基(-NHCO-)的聚合物的總稱,可以通過設(shè)計軟硬段的比例對其物理、化學(xué)性質(zhì)進行比較大的調(diào)整。聚氨酯材料具有優(yōu)異的力學(xué)強度、高彈性、耐磨性、潤滑性、耐疲勞性、生物相容性、可加工性等而被廣泛應(yīng)用于生物醫(yī)學(xué)領(lǐng)域,以及良好的生物相容性和抗凝血性能,被稱為“理想的生物材料”。在許多醫(yī)療裝置和人工器官中得到了廣泛應(yīng)用,是一種很重要的生物醫(yī)用材料。但在適宜的溫度和濕度條件下,醫(yī)用制品表面極易生長和繁殖細菌,嚴重威脅人類的健康,同時也會帶來醫(yī)療事故和巨大的經(jīng)濟損失。 開發(fā)抗菌聚氨酯材料,保護人民的身體健康,防止細菌的傳播,乃至預(yù)防局部或世界流行疾病的爆發(fā)都具有十分重要的意義。 目的 研制納米載銀磷酸鋯抗菌聚氨酯材料,并觀察其在體外及體內(nèi)的生物相容性,以期制作出一種理化性質(zhì)優(yōu)越,生物相容性高的人工生物材料,為其進一步應(yīng)用于人體植入材料提供最佳材料的選擇。 方法 1、采取目前普遍使用的母粒法制備抗菌聚氨酯。 首先將納米載銀磷酸鋯、聚氨酯(EG100A-B20)和分散劑按一定比例在高速混合機中混合均勻,以提高抗菌劑在聚氨酯中的分散和附著能力,然后將混合均勻的物料經(jīng)雙螺桿擠出機、切粒機制成抗菌母粒,最后將抗菌母粒同空白聚氨酯按一定比例混合,制備出抗菌劑質(zhì)量分數(shù)分別為0%(空白)、0.5%、1%、1.5%、2%、2.5%、5%的抗菌聚氨酯,經(jīng)注塑機注塑成型為5×5×0.5cm標準樣板,蒸餾水中浸泡24,消毒備用。 2、納米載銀磷酸鋯抗菌聚氨酯材料的體外抗菌性能研究。 采用貼膜法檢測納米載銀磷酸鋯抗菌聚氨酯材料對金黃色葡萄球菌和大腸桿菌的抗菌性能。將標準菌株金黃色葡萄球菌、大腸桿菌傳代培養(yǎng),混懸于TSB培養(yǎng)液中,采用麥氏比濁法配置成濃度為1.5×108CFU/mL的菌液,并依次做4次10倍遞增稀釋。分別吸取100μL菌液接種在各組抗菌試件表面,蓋上消毒PE膜,培養(yǎng)24h。然后將試件放置在含9.99mL TSB培養(yǎng)液滅菌試管中,旋渦振蕩1min。分別取1mL接種于TSA平板上,培養(yǎng)24h后,計數(shù)菌落數(shù)CFU。實驗重復(fù)5次,結(jié)果取平均值。 3、按ISO10993.12-2009和國家GB/T16886-2008中的系列標準要求,對制備的納米載銀磷酸鋯抗菌聚氨酯材料進行細胞毒性實驗、急性全身毒性實驗、溶血實驗、熱原實驗、兔肌肉植入實驗,以綜合評價其生物相容性。 細胞毒性實驗：將實驗組(抗菌聚氨酯浸提液)、陰性對照組(聚乙烯浸提液)與陽性對照組(6.4%苯酚溶液)各3ml,分別置于35mm細胞培養(yǎng)皿內(nèi),加入6×104/ml L929細胞懸液3ml,培養(yǎng)箱培養(yǎng)24h、48h及72h后,觀察細胞形態(tài)及生長情況。將100ul濃度為6×104/mL的細胞懸液接種于96孔培養(yǎng)板,細胞貼壁后棄原液,分別加入實驗組(2%抗菌聚氨酯浸提液)、空白對照組(0%抗菌聚氨酯)、陰性對照組(聚乙烯浸提液)與陽性對照組(6.4%苯酚溶液)各100ul,采用MTT比色法測定L929細胞培養(yǎng)24h、48h、72h的相對增殖率(RGR),評價材料的細胞毒性。 急性全身毒性實驗：按50ml/kg劑量標準,無菌條件下將2%納米載銀磷酸鋯聚氨酯材料的浸提液注入試驗組小鼠腹腔內(nèi),陰性對照組注入生理鹽水,陽性對照組注入苯酚。給藥后連續(xù)7天觀察小鼠,記錄小鼠呼吸、進食、運動等一般情況、毒性反應(yīng)、體重變化或死亡 溶血實驗：取10ml受試樣品浸提液(實驗組)、10ml生理鹽水(陰性對照組)、10ml蒸餾水(陽性對照組),分別加入0.2ml的新鮮抗凝稀釋兔血,肉眼觀察是否出現(xiàn)明顯的溶血現(xiàn)象；酶標儀檢測各樣本上清液的吸光度,評價受試材料的體外血液相容性。 熱原實驗：以5ml/kg劑量將受試材料浸提液自兔耳緣靜脈緩慢注入,給藥后連續(xù)測量兔體溫并與實驗前預(yù)檢體溫相比較,評價受試材料的制熱作用。 兔肌肉植入實驗：將2%納米載銀磷酸鋯抗菌聚氨酯材料植入新西蘭大白兔背部肌肉中,分別于術(shù)后1w、4w、12w后取出進行大體觀察與組織病理學(xué)檢查,觀察材料植入后的組織炎癥反應(yīng)程度與纖維囊形成情況,評價材料植入肌肉后的組織反應(yīng)。 結(jié)果 1、抗菌聚氨酯的抗菌性能評價 添加納米載銀磷酸鋯的熱塑性聚氨酯能有效抑制大腸桿菌和金黃色葡萄球菌的生長�？咕鷦┨砑颖壤�0.5%-5%組對金黃色葡萄球菌的抑菌率分別為80.23%、91.32%、95.23%、99.19%、99.87%、99.93%,對大腸桿菌的抑菌率分別為76.70%、86.96%、92.92%、99.14%、99.34%、99.87%,與0%比例組(對照組)比較均有顯著差異(P0.05),且抗菌效能隨抗菌劑添加比例的上升而顯著提高。 2、抗菌聚氨酯材料的生物相容性評價 細胞毒性實驗：2%納米載銀磷酸鋯抗菌聚氨酯浸提液細胞貼壁生長,細胞呈梭形,折光性強,細胞突充分伸展；浸提液作用小鼠成纖維細胞24h,48h和72h后RGR分別為97.3%、96.5%、96.3%,組細胞毒性毒性反應(yīng)均為Ⅰ級,無細胞毒性。提示2%納米載銀磷酸鋯抗菌抗菌聚酯與普通抗菌聚氨酯(0%)均有良好的細胞相容性。 急性全身毒性實驗：實驗組小鼠注射后沒有出現(xiàn)異常癥狀,活動、進食、排泄正常,未見步態(tài)不穩(wěn)、驚厥、癱瘓以及呼吸抑制等毒性反應(yīng),在7天觀察期內(nèi),體重呈增加趨勢,無死亡。 溶血實驗：受試材料浸提液不引起體外溶血反應(yīng),溶血率為1.56%。 熱原實驗：注射受試材料浸提液后體溫升高均低于0.60℃,三只兔體溫升高總數(shù)為0.5℃,按國家藥典的標準受試材料無不良的的制熱作用。 兔肌肉植入實驗：大體觀察受試材料植入后切口無明顯滲血、滲液,無感染發(fā)生,纖維囊隨時間逐漸變薄。組織病理學(xué)檢查提示,材料植入后局部有少量炎細胞浸潤,以中性粒細胞和散在的淋巴細胞為主,無組織壞死。隨著植入時間增加,炎癥基本消失,且形成微薄且透明的纖維膜包裹。 結(jié)論 1、本課題首次將納米載銀磷酸鋯通過熔融共混法加入聚氨酯制備出納米載銀磷酸鋯抗菌聚氨酯,并證實其具有良好的抗菌效果和優(yōu)異的生物相容性。 2、隨著納米載銀無機抗菌劑添加比例的上升,聚氨酯的抗菌性能明顯提高。當添加比例達到1.5%時,納米抗菌聚氨酯對金黃色葡萄球菌抑菌率達到95.23%,對大腸桿菌的抑菌率達到92.92%。由于QB/T2591-2003規(guī)定材料對微生物的抑菌率90%才能被稱為抗菌材料,所以從抗菌角度出發(fā),推薦納米載銀無機抗菌劑在聚氨酯中的添加比例應(yīng)不低于1.5%。 3、據(jù)IS010993-2009和GB/T16886-2008系列標準對于生物材料相容性的檢測方法與要求,實驗結(jié)果表明本課題組制備的納米載銀磷酸鋯抗菌聚氨酯材料無細胞毒性、無急性全身毒性、不引起溶血反應(yīng)、無致熱作用,與空白的醫(yī)用聚氨酯比較,生物相容性無明顯差異。納米載銀磷酸鋯抗菌聚氨酯具有良好生物相容性,為下一步應(yīng)用于人工肛門括約肌的生物材料提供了實驗依據(jù)。
[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.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R318.08

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