Antibacterial Property Of Sr-Ag Dopped Porous TiO₂ Coating With Different Components Of Ag Prepared By Micro-arc Oxidation

Objectives Micro-arc oxidation（MAO）,a one-step method,was used to construct Sr/Ag-dopped porous TiO₂ coatings with different silver contents on the surface of pure titanium.The physical and chemical properties,cell compatibility,short-term and long-term antibacterial properties of the coatings were tested,and the optimum Ag content within the Sr/Ag-dopped porous TiO₂ coatings was determined,which provided experimental basis for development of a new implant coating with both good biological comparability and antibacterial property.Methods Micro-arc oxidation（MAO）was used to prepare Sr/Ag-dopped porous TiO₂coatings with different silver contents on pure titanium surface.The coating samples were divided into five groups,group Ag0,Ag0.04,Ag0.08,Ag0.17 and Ag0.34.Lanthanum acetate solution with a concentration of 90g/L was used as work solution and 0 g/L,0.04g/L,0.08 g/L,0.17 g/L and 0.34 g/L silver nitrate was added into the solution,respectively.The different coatings were detected as follows.1.Scanning electron microscope（SEM）observation,energy dispersive spectrometry（EDS）,X-ray diffractometer analysis（XRD）,surface roughness testing,contact angle measuring and current thickness gauge measurement were used to determine physical and chemical properties of these coatings:2.The adherence,growth and proliferation of mouse pre-osteoblast MC3T3-E1 cells on different coatings in five groups were also examined to evaluate the biological comparability of these coatings.3.Bacteria inhibiting zone,plate bacteria flora counting well as SEM observation were used to determine short-and long-term antibacteria properties of different coatings.Ag⁺release from coatings at different time points were also detected by inductively coupled plasma spectrometry.Results 1 Preparation and determination of physical and chemical properties of different Sr/Ag-dopped porous TiO₂ coatings.Sr/Ag-dopped porous TiO₂ coatings with different contents of Ag were successfully prepared by micro-arc oxidation（MAO）technique.SEM observation showed that microholes with a diameter from 0.5μm to 5.0μm were evenly distributed on the surfaces of different coatings and Ag within the coatings led no significant changes on surface micromorphology among these coatings.EDS analysis demonstrated that Ti,O,Sr and Ag elements were evenly distributed on surface of different coatings and weight ratios of Ag in group Ag0,Ag0.04,Ag0.08,Ag0.17 and Ag0.34were0.00±0.00wt%,0.07±0.02wt%,0.26±0.04wt%,0.58±0.19wt%and1.29±0.05wt%,respectively.XRD showed that diffraction peaks of TiO₂ in both anatase phase and rutile phase,as well as SrTiO₂,were detected in all five coatings,whereas no Ag phase was observed.Roughness measurement showed that the surface roughness of coatings in the five groups was 0.442±0.033 mm,0.430±0.021 mm,0.442±0.012 mm,0.446±0.009 mm and 0.452±0.026 mm,respectively.There was no significant difference among the five groups（P>0.05）.Contact angle of five coatings were 78.77±4.42°,v77.20±3.48°,76.93±4.12°,77.01±4.73°and 74.47±3.93°,respectively,and no significant difference was observed among five groups（P>0.05）,which suggested that the five groups had similar surface hydrophilicity.Eddy current thickness gauge measurement showed that the thinnest coating was observed in group Ag0.34（34.70±2.72μm）,and followed by group Ag0.17（36.22±1.16μm）,which were both significantly lower than that in group Ag0（38.85±1.46μm）,Ag0.04（38.72±1.05μm）and Ag0.08（38.68±1.87μm）（P<0.05）,and no significant difference was observed among the last three groups（P>0.05）.Above results suggested that increase of Ag content in the coatings slightly decrease the thickness of TiO₂ coating.2 Biological comparability analysis of different coatings.Cell adhesion analysis showed that the number of adherent cells decreased with the increase of Ag incorporated in the coatings and cell number were significantly different among the five groups at both 1h and 2h（P<0.01）,except the cell number between group 0.17 and Ag0.34at 1h,which showed no significant difference（P>0.05）.The result indicated that increase of Ag content within the coatings had slight negative influence on initial adhesion of preosteoblasts.SEM and fluorescent microscope observation demonstrated that much more cells were observed on coatings from group Ag0 to group Ag0.17 and the cells were polygonal and grew well,whereas the cells in group Ag0.34 were much less,small sized and became shrunk,which suggested that the coating in group Ag0.34 was poisonous to preosteoblasts.Cell proliferation measurement showed that no significant difference for cell proliferation were observed among group Ag0,Ag0.04,Ag0.08 and Ag0.17 at 1d,4d and 7d（P>0.05）,except at 1d,the cell proliferation in group Ag0.17 was lower than that in other three groups（P<0.05）,whereas the cell proliferation in group Ag0.34 was significantly lower than that in all former four groups in all three time points（P<0.01）.Above results suggested that the coatings from group Ag0 to Ag0.17（with Ag content from 0wt%to0.58wt%）had good biological comparability and had no significant influence on preosteoblasts proliferation,whereas coating of group Ag0.34（with Ag content of1.29wt%）was poisonous and could inhibit preosteoblast proliferation.3 Antibacteria property of different TiO₂ caotings.Bacteria inhibiting zone analysis showed that the diameter of bacteria inhibiting zone in group Ag0,Ag0.04,Ag0.08,Ag0.17 and Ag0.34was 0.0±0.0 mm,2.8±0.5 mm,4.2±0.6 mm,8.4±0.8 mm and 12.9±0.2 mm,respectively.The diameter of bacteria inhibiting zone increased significantly with the increase of Ag content in coatings of five groups（P<0.05）.Plate bacteria flora counting demonstrated that compared with group Ag0,the bacteria inhibiting rate of coating in group Ag0.04 was about 85%before PBS soaking and that in group Ag0.08,Ag0.17 and Ag0.34 was up to 100%.Thirty days after PBS soaking,bacteria inhibiting rate in group Ag0.04,Ag0.08,Ag0.17 and Ag0.34 was 29.47±4.13%,44.13±2.34%,77.60±3.39%and87.04±1.77%,respectively（P<0.05）.Above results indicated that the short-term bacteria inhibiting rate could reach up to 100%when Ag content in coatings was 0.25wt%（group Ag0.08）or more,and the long-term bacteria inhibiting rate could remain as high as 77.6%or more when Ag content was or more than 0.58wt%（group Ag0.17）.SEM observation showed that After soaking in PBS for 30 days,the number of bacteria on coating surface decreased obviously with the increase of Ag content in coatings.Furthermore,the bacteria morphology was complete in group Ag0,Ag0.04 and Ag0.08,whereas some bacteria in group Ag0.17 was broken and most of bacteria in group Ag0.34 showed similar phenomenon.the result indicated that the coating with a 0.58wt%or more Ag content had good long-term antibacteria property.Above results suggested that TiO₂ coatings acquired antibacteria property with incorporation of Ag and the coatings with a 0.58wt%or more Ag content had both good short-and long-term antibacteria property.Ag⁺release measurement showed that at each time point,average Ag⁺release concentration increased significantly with the increase of Ag content incorporated in coatings in five groups（P<0.05）,while with the increase of soaking time,Ag⁺release decreased sharply in all of five groups.After 30 and 120 days PBS soaking,the coating in group Ag0.17 remained Ag⁺release at a very low level from 0.0142 to 0.0031ppm/d.Conclusions 1 Sr/Ag-doped porous TiO₂ coatings with different Ag contents were prepared by micro-arc oxidation（MAO）,a one-step technique,on the surface of pure titanium,and incorporation of Ag showed no significant influence on micromorphology,element distribution,physical phase form,hydrophilicity and roughness of the coating surface,except coating thickness,which was slightly decreased with the increase of Ag content in coatings.2 Sr/Ag-dopped porous TiO₂ coatings with a 0.58wt%Ag content showed good biological comparability and both strong short-and long-term antibacteria property of S.aureus.Figure 22;Table 3;Reference 123...