Preparation And Characterization Of Antibacterial Compound Film Containing Ca/P/Ag By Micro-arc Oxidation On Titanium Surfaces

Background:With the improvement of living standards, the application of artificial implants become more and more widely and ordinarily, people have higher requirements for safety and biological activity of the biological material. Because of strength, good heat resistance, low temperature resistance, low modulus of elasticity, non-magnetic non-toxic, biocompatible and its alloys have good heat intensity, low temperature toughness and fracture toughness. Metal titanium has been widely used in hard tissue repair and replacement materials, as the preferred metal medical materials. But titanium itself lacks of bone induction properties and a strong mechanical bond with the surrounding tissue and biological activity and in the body environment can be corrosive to the release of metal ions, which affected the stability of the titanium implants and long-term success rate. With the development of materials science in recent years, the titanium surface treatment technology has been the focus and emphasis of the study. How to get a better osseointegration with shorten the bone healing time by surface treatment has become a research goal of many scholars and it has got good results. After surface modification, it formed a micro-rough or rough surfaces on titanium surface, although this is conducive to osseointegration, but also more conducive to bacterial adhesion; while the titanium itself does not have antibacterial function so antibacterial modification of titanium surface is of great significance.Currently, surface modification methods of titanium contains plasma sprayed coating (PSC), ion beam assisted deposition (IBAD), pulsed laser deposition(PLD), biomimetic deposition, sol-gel processing, alkaline and heating treatment, sandblasted and acid-etching, SLA, resorbable blast media (RBM), Ion implantation method, Laser ablation, micro-arc oxidation et al. Micro-arc oxidation is a new technology that can directly grow ceramic coating in situ on the surface of Titanium metals. And the ceramic coating has such characteristics as high corrosion resistance, good wear resistance, et al. It generats a harder micro-arc oxidation film than other methods and the film has a stronger base combined. Through the control of the electrolyte composition and concentration, it can regulate the proper ratio of calcium and phosphorus in the film of the titanium surface. Studies have reported that through this method it can generate rutile and anatase TiO2with good biological activity in the titanium surface, so as to promote osseointegration and accelerate bone healing.Resently Ag is considered as the most promising antibacterial materials for its advantage of bactericidal broad-spectrum, strong sterilization function, talerance, non-toxic to the body, and long-lasting bactericidal effect. In this study, AgNO3was added into the traditional electrolyte of micro-arc oxidation which contained phosphorus salt and calcium salt to produce composite ceramic film with calcium, phosphorus, silver. Cytology testing and anti-bacterial performance testing were use to evaluate the biological performance of the film.Objective:This research is to investigate the preparation of compound film containing Ca/P/Ag on titanium surface by micro-arc oxidation and the feature and cell biological and antibacterial of the film to provide data basis for the antibacterial modification of titanium surface.Methods:1. The experiment was divided into three groups, the blank control group was group M which was polishing mechanically, the experimental control group was group MAO which was modify in the traditional electrolyte with phosphorus salt and calcium salt, and the Experimental group was group MAO-Ag which was modify in the electrolyte phosphorus salt and calcium salt and AgNO3. There were two specifications of titanium plate respectively used in cell experiments and antibacterial tests. The kind of titanium using in the cell experiments was round with a diameter of15mm and a thickness of lmm, the other one using in antibacterial tests was squre with a side length of2.3mm and a thickness of1mm. Prepared titanium plates were dried at room temperature and then detected on physical and chemical properties. Scanning electron microscope was used to observe the surface morphology of it, and surface profiler and surface contact angle tester were used to measure the roughness and static water contact angle of each specimen. X-ray energy dispersive spectroscopy and X-ray diffraction were used to analyze the element components and the form of elements in the film of titanium surface.2. Human osteosarcoma cell line MG63and ATCC fibroblast were respectively cultured in48hours soaking of titanium in each group. After24hours cultured, cell morphology was observed under optical microscope to qualitatively analyze the cytotoxicity of sample and MTS test was performed to quantitatively analyze the cytotoxicity of sample.3. After Human osteosarcoma cell line MG63cells were cultured on disks after2h,4h,8h and24h, scanning electron microscopy (SEM) was used to observe the morphology of cells. And Hoechst33342dye was used to stained cells which were cultured on disks after2h,4h and8h to take photos under fluorescence microscopy which could be used to count to analyze adhesion rate.4. After Human osteosarcoma cell line MG63cells were cultured on disks after Id,3d,5d and7d, MTS measured the proliferation by OD value. ALP activity was measured at time of7d,14d and21d.5. Antibacterial test was performed by film method to quantitatively evaluate the samples antibacterial capacity to E.coli and Staphylococcus aureus and Porphyromonas gingivalis bacteria.6. In cell test each time-period above each group had six samples, each sample was measured for three times. According to national standards, we set three samples in each group in antibacterial tests. Datas were described as x±s, statistical analysis software was SPSS13.0. The between-subjects effects were tested by factorial analysis of variance. The comparisons among goups were analyzed by one-Way ANOVA. After test of Homogeneity of variances multiple comparisons were analyzed by LSD or Dunnentt’s T3after Welch. The hypothesis test was two-sided test and the mean difference was significant at the0.5level.Result:1. It appeared grooved scratches in surface of group M under scanning electron microscopy and uniform fire hills like microporous which was about1-5μm on group MAO and group MAO-Ag. After micro-arc oxidation preparation the titanium surface roughness increased, and there was no difference between group MAO and group MAO-Ag, the result of roughness value Ra turned out to be group MAO=group MAO-Ag>group M. The result of surface contact angle measurement was group MAO=group MAO-Ag<group M. The result of EDS measurement was that the surface of group M only contained element of Ti, meanwhile there were four elements containing Ti, Ca, P and O on the surface of group MAO; and five elements containing Ti, Ca, P, O and Ag on the surface of group MAO-Ag. The XRD measurement analyzed the form of the elements on the titanium surface, it discovered that there were Rutile and Anatase TiO2being generated in the film of group MAO and MAO-Ag and there was Ag appearance on group MAO-Ag. The above physical and chemical properties analysis showed that the micro-arc oxidation process has changed the morphology and roughness contact angle and chemical composition of titanium surface It generated Rutile and Anatase TiO2with excellent biological activity. Adding AgNO3into the electrolyte of micro-arc oxidation process just changed the composition of titanium surface but not change the morphology of it.2. Cytotoxicity test results showed that ATCC fibroblasts and Human osteosarcoma cell line MG63cells which cultured in the three titanium plates soaking appeared a normal cell morphology and good growth. Results of quantitative detection of cytotoxicity by MTS showed that the cytotoxicity grade of the three titanium plates wree0, it implied that titanium surface load trace amounts of silver were not cytotoxic.3. SEM observations showed that the Human osteosarcoma cell line MG63cells of group MAO and group MAO-Ag had a better adhesion and spreading and differentiation at time2,4,8and24hours than group M, but there were no differentce between group MAO and MAO-Ag. The adhesion rates of group MAO and MAO-Ag were higher than in the group M at time2and4hours, but there were no difference among three groups at time8hours. The cell adhesion rates of three groups were time-dependent gradual increase.4. In the MTS test, the OD value was higher in group MAO and MAO-Ag than in group M at time1,3,5and7days, and value in group MAO was equal to it in group MAO-Ag. The OD values of three groups were time-dependent gradual increase too. It suggested that titanium of group MAO and MAO-Ag can promote the proliferation of MG63. ALP test results showed that there was a higher OD value in group MAO and MAO-Ag than in group M at time7,14and21days. And the highest OD value appeared at14days in all groups. It displayed a better biological activity in titanium modified by micro-arc oxidation than puer titanium.5. Film method antibacterial experiment showed an antibacterial rate of99.0%to E.coli and Staphylococcus aureus and Porphyromonas gingivalis bacteria in group MAO-Ag, but no antibacterial ability in group M and group MAO. Group MAO-Ag showed an exellent antibacterial property.Conclusion:1. It can be formed a composite ceramic film which contains calcium, phosphorus and silver on the surface of commercial pure titanium by micro-arc oxidation and the film has a similar surface morphology and physical properties to film generated by traditional micro-arc oxidation. After modified by micro-arc oxidation, it can increase the roughness and contact angle of titanium surface and generate Rutile and Anatase TiO2on the surface of titanium.2. Composite ceramic film with calcium, phosphorus and silver modified by micro-arc oxidation has an excellent bio-security. It is no cell toxicity to Human osteosarcoma cell line MG63cell and ATCC fibroblast. It can promote the adhesion and stretching and differentiation and ALP vitality of MG63cell.3. The composite ceramic film with calcium, phosphorus and silver on titanium surface in this study has an antibacterial while having an excellent bio-security.