Influence Of Ag/HA Nanoparticles Composite Coating On Biofilm Formation And Its Mechanism

BackgroundInfection is the most severe complication after artificial joint replacement, and the outcome is disastrous. Once the infection happens, the surgery will totally fail. The infection after artificial joint replacement has its own pathogenesis and features. The root cause of the incurable infection is related with the biomembrane formation on the surface of prothesis. Biomembrane is an existence form of bacteria corresponding to that of planktonic cells, and it is formed by bacteria for adapting to the living environment in the growth process. Biomembrane consists of bacterial and extracellular polymeric substance (EPS), which can attach to the surface of the implant and form a highly organized multicellular structure, which is the protective growth pattern of bacterial. The adhesion and attachment are beneficial for bacterial to attach to the surface of the support. What's more, compared with single or suspended bacterial cell, it is easier to resist the attack of host immune cells, immune molecules and antibiotics. At the same time, it can increase the transfer of toxicology gene and drug resistant gene. These structures and functional features can explain why the biomembrane is hard to eliminate.At present, the major therapeutic methods for biomembrane are mechanical methods (sonication, eddy and ultrasonic wave), biochemical methods (enzyme treatment) and electrical stimulation. However, the clinical efficacy is unsatisfactory. There are no exact and reliable measures for preventing or inhibiting biomembrane formation at home and abroad. The antibiotic-loaded bone cement widely applied in revision surgery also cannot prevent biomembrane formation, and even cannot kill the planktonic drug resistant strains, so cause the infection of drug resistant strains.A feasible way is to apply antibacterial materials on the surface of prothesis to prepare a sterile coating. Nano-silver is an ideal antibacterial coating material, because it has wide antibacterial spectrum and strong antibacterial activityPart OneObjective:To prepare antibacterial coating by loading nano-silver on Ti-based surfaceMethods:silver powder with average particle diameter of 5μm and original hydroxylapatite powder with average particle diameter of 10μm were proportioned at the mass ratio of 1:20. The mixed original powder was placed in the blender to blend and mix for 30min in order to make the two kinds of powder fully and uniformly dispersed. The powder was ball-milled by QM-3SP2 planetary ball mill and was electrophoretically deposited on Ti-base in HH-S 112 thermostatic waterbath oven. The composition and characterization of the coating were detected by D/MAX 2550 VB/PC X-ray Diffractometer, Magna-IR 550 Fourier Transform Infrared Spectroscopy and X-650 Scanning Electron Microscope.Results:The average particle diameter of nano-silver particles was about 100nm. Nano-silver particles were uniformly dispersed in hydroxylapatite powder. Conclusion:Nano-silver particles were successfully loaded in hydroxylapatite coating by mechanical ball milling and chemical coprecipitation method. Part TwoObjective:to check the influences of nano-silver on biofilm formation after nano-silver loading on the surface of the implantMaterial and method:Staphylococcus epidermidis was clinically isolated and cultured and was taken as the test strain. The sterile Ag/HA coating sample was implanted in 1.Ox 107CFU/ml 10ml bacterial liquid for culture at 37℃. The biofilm on the surface of the sample was tested at 6h,24h,48h and 72h. Morphological observation for the biofilm was carried out by using laser scanning confocal microscope. Biofilm bacterial on the surface of the separately cultured sample were counted at 24h and 28h. HA coating was used as the control group.Result:Biofilm appeared on the surface of Ag/HA coating after 48h. Mature biofilm appeared on HA coating after 12h. The difference in adherence of staphylococcus epidermidis between the two coatings at 24h and 48h had statistical significance (p<0.01).Conclusion:Ag/HA composite coating obviously inhibited biofilm formation. The coating loaded with silver obviously changed the surface activity of the coating, and thus bacterial adhesion decreased.Part ThreeObjective:to discuss the mechanismof silver-loaded coating inhibiting biofilm formation Material and method:The nano-silver release rate of the coating in fetal calf serum was detected by using ICP-Ms (Agilent Company from America). The prepared coating was placed in air for 3 months, and, then, was retested. At the same time, the expression of staphylococcus epidermidis initial adhesion factor at1E was checked by using quantitative PCR method.Result:Nano-silver continuously released within 3 days and the concentration gradually increased, wherein, the highest concentrations could reach 266μg/L and 261μg/L. After being placed for 3 months, nano-silver release obviously reduced (p<0.01).30min after implanting bacterial liquid in the sample, the expression of staphylococcus epidermidis initial adhesion factor at1E began to decrease (p<0.01).Conclusion:The antibacterial action of Ag/HA composite coating was mainly realized by nano-silver release. At the same time, the biofilm inhibition by Ag/HA composite coating also influenced the molecular modulation mechanism of biofilm formation.