Bioactivity And Antibacterial Property Of Porous Copper -incorporated TiO₂ Coating Prepared By Micro-arc Oxidation

Part I Preparation of porous copper-incorporated TiO2 coating by micro-arc oxidationObjective:To prepare porous and copper-incorporated TiO2 coating(Cu-TiO2) by micro-arc oxidation (MAO) technique, and to detect the characterization of the novel coacting. Method: Porous and copper-incorporated TiO2 coating was prepared on titanium by MAO technique in a Cu, Ca and P containing electrolyte. The surface morphologies and microstructure of the coatings were observed by scanning electron microscopy (SEM). Phase compositions of the coatings were characterized using X-ray diffraction (XRD). The surface roughness of the coatings was measured by a surface profiler. The elemental composition and distribution of the coatings were measured by energy-dispersive X-ray spectrometry (EDS) attached to electron probe X-ray microanalysis system (EPMA). The cross-sectional structure of the coatings was was observed by EPMA in back-scattering mode. Tris-HCl buffer solution immersion tests were conducted to evaluate the apatite forming ability and bioactivity of coatings. Result:Both the TiO2 and Cu-TiO2 coatings mainly consist of anatase phases, their surfaces exhibit grains of about 30-50 nm in size and pores about 3 μm in diameter. No significant difference of surface roughness (Ra) was found between TiO2 and Cu-TiO2 coatings. Ti,O, Ca and P elements in both coatings as well as the Cu element in the Cu-TiO2 coatings distributed homogeneously in the whole coatings. Ca, P and Cu begin to release from the coatings once the coatings were soaked in the buffer solution. Conclusion The Cu-TiO2 coating is worth further consideration for orthopedic implant applications.Part Ⅱ Antibacterial property of porous copper-incorporated TiO2 coating prepared by micro-arc oxidationObjective:To explore the antibacterial property of porous copper-incorporated TiO2 coating against Staphylococcus aureus. Method:Staphylococcus aureus was inoculated with the material culture for 2,4,6,8 days, and inhibitory effect were observed by colony counts; The Staphylococcus aureus were inoculated with ion extractables of each material for 2 days, and bacterial proliferation was evaluated by light microscopy; Staphylococcus aureus were inoculated on each experiment the surface to observed bacterial proliferation by the application of immunofluorescence.Result:These results from inhibition of surface suggested that:from the first two days after culture.the Cu-TiO2 showed significant inhibitory effect. Staphylococcus aureus inoculum count of Cu-TiO2 is below, and gradually decreased with time, and was significant differences between with the other two groups. The TiO2 and titanium groups did not show antibacterial effect, and the surface of Staphylococcus aureus count on the two groups was higher, and increased gradually with time. The numbers of Staphylococcus aureus in ion extractables of TiO2 and Ti were higher on day 2. The number of Staphylococcus aureus co-cultured with Cu-TiO2 was significantly decreased on day 2 as shown by light microscope. After inoculated on day 8, green fluorescent showed there were a large number of bacterial on TiO2 and Ti group surface and a significantly decreased number of bacterial on Cu-TiO2, suggesting less growth of bacterial adhesion on Cu-TiO2. Conclusion:The Cu-TiO2 coating show satisfactory antibacterial property against Staphylococcus aureus.Part III The influence of porous copper-incorporated TiO2 coating prepared by micro-arc oxidation on osteoblastic bioactivityObjective:To explore the influences of porous copper-incorporated TiO2 coating on adhesion of MC3T3-E1 bioactivitv. Method:Osteoblast-like cells were cultured on Cu-TiO2. TiO2 andTi respcetively. Cell spreading was detected by scanning electron microscopy (SEM) after 24 h of culture. After 4 and 8 days of cultivation respectively, samples were collected, and PI were used to detect cell proliferation. After 2,4,6,8 days of cultivation respectively, MTT assay was used to detect cell proliferation. Cell cycles were analyzed by flow cytometry. Markers of osteoblastic differentiation, e.g type I collagen, osteocalcin were investigated by RT-PCR. Result:The SEM indicated that cell spreaded well on Cu-TiO2. The result of PI showed that at 4 and 8 days, cell number on Cu-TiO2 was much more than that on TiO2 and Ti. MTT value of Cu-TiO2 group was significantly higher than that of TiO2 group and Ti group on day 4 and day 6. Flowcytometric analysis showed that the percentage of cells in S phase of the Cu-TiO2 group was obviously higher than that of the TiO2 group and Ti group on day 4 and day 6. At the same time, cells in GO/Gl phase of the Cu-TiO2 group was significantly lower than that of TiO2 and Ti group, while cells in G2/M phase of the Cu-TiO2 group was higher than that of the TiO2 group and Ti group. With the progression of culture time, ALP、Col I and OC activity increased in all groups. ALP activity on day 4 and 6, Col I activity on day 6 and 8, and OC activity on day 8 in the Cu-TiO2 group was statistically significantly higher than that of the TiO2 group and Ti group.Conclusion:Cu-TiO2 coating was able to promote adhesion, proliferation as well as differentiation of MC3T3-E1 osteoblast-like cells, which indicated that Cu-TiO2 coating may be also a favorable bioactive biomaterial.