Research Of Antibacterial Properties And Biocompatibility Of Nano-Silver-Zirconium Phosphate Antibacterial Polyurethane

BackgroundBiological material is medical objects of non-living materials work with biological systems,also known as biomedical materials. The inflammatory response is one of the major side effect of the biological materials implanted in the body.Biological material implantation is free of bacterial adhesion sites, making infection more likely to occur. With the bacterial adhesion, gradually formed a layer of biofilm by the polysaccharides. In this biofilm, bacterial resistance is much greater than the free bacteria.Traditional antimicrobial treatment effect is bad, so the use of implants is often complicated by biomaterial centered infection. BCI and other tissue reaction is the major problems of clinical implantation,often the main reason for reoperation and medical malpractice, and bring significant economic burden to patients. Therefore, the research on biomedical materials with anti-bacterial ability has become a hot issue in the field of biological materials.Polyurethane is a general term of polymer with the structure containing repeated urethane groups (-NHCO-), its physical and chemical properties.can be carried adjustment through the design of hard and soft segment ratio.The polyurethane material has excellent mechanical strength, high elasticity,abrasion resistance, lubricity, fatigue resistance, biocompatibility, workability, so widely used in the biomedical field, as well as good biocompatibility and anticoagulant properties, known as the "ideal biological material,has been widely applied in many medical devices and artificial organs..Bacteria can easily grow and multiply on the surface of the medical products,which become serious threat to human health, also bring a medical malpractice and huge economic losses.Development of antimicrobial polyurethane material are of great significance at protection of people’s health and prevention of the spread of bacteria, even prevention of local or worldwide epidemic disease outbreaks.ObjectivePrepare Nano-Silver-Zirconium phosphate antibacterial polyurethane.Observe the antibacterial properties and biocompatibility in vitro and in vivo in order to create a superior physical and chemical properties, high-biocompatibility of artificial materials,which provided for the best choice of materials applied to human implantation materials in further.Methods1、Prepare the antimicrobial polyurethane by the current widespread use of masterbatch.First, put nano-silver-zirconium phosphate, the polyurethane (EG100A-B20) and the dispersant by a certain percentage in the high-speed mixer, in order to improve the dispersion and adhesion of antimicrobial agent in the polyurethane;then mixed materials are made in antibacterial masterbatch by the twin-screw extruderand pelletizer; at last mix antimicrobial masterbatch with blank polyurethane by a certain percentage, antimicrobial agent quality scores of0%(blank),0.5%,1%,1.5%,2%,2.5%,5%of the antimicrobial polyurethane are prepared.Then mold into5×5×0.5cm standard model by injection molding machine, soak with distilled water for24h, and sterilize.2、Study the antibacterial properties of nano-silver-zirconium phosphate antimicrobial polyurethane material in vitro.Antimicrobial properties of nano-silver zirconium phosphate antimicrobial polyurethane material against Staphylococcus aureus and Escherichia coli are detected by the foil method.Culture Standard strains of Staphylococcus aureus and E. coli, suspend in TSB culture medium, configure to a concentration of1.5×108CFU/mL of bacteria, and dilute in turn10-fold increments.Pipette100μL of bacteria to inoculated on the surface of the specimen, covere with a sterile PE film and culcure for24hours.Then place the specimen in the sterilized test tube with9.99mL of TSB, vortex for1min. Taken1mL of bacteria liquid to inoculate on TS A plates, incubate for24h, count the number of colonies.The experiment was repeated five times and the results are averaged.3、According to the ISO10993.12-2009and national GB/T16886-2008,we detect the biocompatibility of nano-silver-zirconium phosphate antimicrobial polyurethane with cell toxicity test, acute systemic toxicity test, haemolysis test, pyrogen test, rabbit muscle implantation experiment.Cell Toxicity Test:The experimental group (2%antibacterial polyurethane extracts), the negative control group (polyethylene extract),blank control group(0%antibacterial polyurethane extracts)and positive control group (6.4%phenol) each3ml, were placed in35mm cell culture dishes, join3ml of6x104/ml L929cells suspended solution, observe cell morphology and growth24h,48h and72h later. Then100ul6×104/mL cell suspension were seeded in96-well culture plates, abandon liquid cells after adherent to culture plates,added100ul of the experimental group (antibacterial polyurethane extract), negative control group (polyethylene extract) and positive control group (6.4%phenol), detected the cytotoxicity of the material by MTT assay of L929cells with relative growth rate (RGR) after cultured for24h,48h,72h.Acute Systemic Toxicity Test:In accordance with the standards of50ml/kg dose, the extract of2%nano-silver zirconium phosphate polyurethane material was injected into the abdominal cavity of test mice under sterile conditions, the negative control group injected with saline, the positive control group were injected with phenol. After administration for7days in mice, the mice breathing, eating, exercise, etc. In general, toxicity, weight change, or death. After administration for7days, Observed in the basic situation of mice such as breathing,eating,exercise, and toxicity, weight change,or death.Haemolysis Test:Take10ml extracts of the tested samples (experimental group),10ml saline (negative control group),10ml distilled water (positive control), added into0.2ml fresh anticoagulant dilution rabbit blood. Observe whether there significant hemolysis with naked eye. Detect the absorbance of supernatant of each sample with microplate reader, and evaluate blood compatibility of the test materials in vitroPyrogen Test:In accordance with the5ml/kg dose, slowly injected into the test material extract from the rabbit ear vein, after administration measure the rabbit body temperature continuously,and compared to the preflight temperature before the experiment, evaluate the heating effect of the test material.Rabbit Muscle Implantation Experiment:Implanted2%nano-silver-zirconium phosphate antimicrobial polyurethane material in the back muscles of New Zealand white rabbits. Respectively, remove the specimen after1w,4w,12w.Conduct by gross observation and histopathology. Observe the degree of tissue inflammation and fibrous capsule formation after implantion of the material. Evaluate tissue reaction of materials implanted in muscle.Results1、Evaluation of antibacterial properties of nano-silver-zirconium phosphate antimicrobial polyurethane material in vitro.The experimental groups added with antibacterial agent had good inhibitory effect on Staphylococcus aureus and Escherichia coli. The inhibition percents for Staphylococcus aureus groups (0.5%-5%) were80.23%,91.32%,95.23%,99.19%,99.87%and99.93%, respectively; and were76.70%,86.96%,92.92%,99.14%,99.34%and99.87%, respectively, for Escherichia coli group.The antimicrobial properties of antibacterial polyurethane were strengthened with the increasing doses of antibacterial agent.2、Evaluation of biocompatibility of nano-silver zirconium phosphate antimicrobial polyurethane materialCell Toxicity Test:The cells of experimental group are adherent to culture plates, fusiform, strong refraction,and fully extended. After cultured for24h,48h and72h,the RGR of L929cells in experimental group were97.3%、96.5%and96.3%respectively.there is no significant difference with blank group (P>0.05); and significant difference compared with negative control group at the same observed period (P<0.05).The cytotoxicity of the antibacterial polyurethanes was graded Level Acute Systemic Toxicity Test:After injection,the mice of experimental group showed abnormal symptoms, activiting, eating, excreting normally. Unsteady gait, convulsions, paralysis and respiratory depression and other toxic reactions are not observed, weight tended to increase, with no deaths within7days.Haemolysis Test:The test material extract does not cause a hemolytic reaction in vitro, hemolysis rate is1.56%.Pyrogen Test:After injection of test material extract,the temperature of rabbits are creasing lower than0.60℃,the total temperature is lower than0.5℃.The test material has no adverse heating effect according to National Pharmacopoeia Standards.Rabbit Muscle Implantation Experiment:After the test materials are implanted.The incision are no bleeding, exudation, infection obviously, and the fibrous capsule is progressively thinner over time. Histopathological examinations showed that: the local tissue is infiltration of inflammatory cells, mainly neutrophils and scattered lymphocytes, with no tissue necrosis. With increasing implantation time, the inflammation almost disappeared.The test materials are wrapped.with a thin, transparent fiber membrane.Conclusions1、We prepared Nano-silver-zirconium phosphate antimicrobial polyurethane by melt-blending process for the first time at this topic,and confirmed that it has good antibacterial effects and excellent biocompatibility.2、The antimicrobial properties of antibacterial polyurethane were strengthened with the increasing doses of antibacterial agent. When the proportion is added up to1.5%, inhibition rate of Staphylococcus aureus reach up to95.23%, E. coli reache up to92.92%. In accordance with the QB/T2591-2003,materials can be referred to as antimicrobial materials when its microbial inhibition rate is>90%.So, sarting antibacterial angle, recommended ratio of nano-silver base inorganic antibacterial agent in the polyurethane should be not less than1.5%.3、According to ISO10993-2009and GB/T16886-2008series standards for compatibility testing methods and requirements of biological materials, the experimental results show that the prepared nano-silver-zirconium phosphate antimicrobial polyurethane material has no cytotoxicity,no acute systemic toxicity, no hemolytic reaction, no pyrogenic effect.Biocompatibility was no significant difference compared with blank medical polyurethane.The nano-silver-zirconium phosphate antimicrobial polyurethane has good biocompatibility, and provide an experimental basis for further applied to the biological material of the artificial anal sphincter.