Study On The Corrosion Resistance Antibacterial Property And Biocompatibility Of Ti-Cu Alloy Surface Modified By Micro-arc Oxidation

Objective:Biomedical materials are a kind of multifunctional materials that can be used for diagnosis,treatment and repair of human tissues and organs,whether biomedical materials can be widely applied in clinic depends on whether they can meet the conditions of strong biomechanics,good corrosion resistance and good biocompatibility.Titanium and titanium alloys have become the most widely used medical metal materials because of their advantages such as low density,high strength,non-toxic,good corrosion resistance and biocompatibility.Pure titanium metal has no anti-bacterial ability,and bacteria are easy to adhere to its surface and form biofilm,which leads to inflammation around the implant,while in clinical application,implant-related infection is sometimes difficult to control.Therefore,how to alloying pure titanium to make it antibacterial has become a hot issue.Cu is an essential trace element for human body,which constitutes the active components of a variety of enzymes,participates in cell metabolism in the body,can promote the growth of vascular endothelial cells and the formation of early bone,and has antibacterial effect.Cu can be added in titanium alloy as alloying element,which can play a long-term antibacterial property.Previous studies have found that ti-10Cu alloy with 10%mass fraction of copper has good antibacterial property,but its corrosion resistance and bone-forming property are insufficient.The decrease of corrosion resistance of Ti-Copper alloy can lead to the increase of Cu²⁺dissolution,which will cause cytotoxicity when accumulated to a certain amount.Osteogenesis is the key in the repair of bone injury.Whether the implant material is conducive to osteogenesis is one of the indicators of its biocompatibility.Lack of osteogenesis will affect the ability of bone integration and decrease the mechanical properties,which may cause premature loosening,fracture and failure of metal implants.Therefore,how to improve the corrosion resistance and osteogenesis of titanium copper alloy is one of the research hotspots of biomaterials.Micro-arc oxidation（MAO）is a surface modification process,which can form a firm oxidized ceramic layer on the metal surface and increase the corrosion resistance of the material.By adding calcium and phosphorus elements into the electrolytic solution,the oxide layer containing calcium and phosphorus is obtained,which has biological activity.It is still unknown whether Ti-10Cu alloy can improve its corrosion resistance and promote bone properties,as well as its antibacterial properties and biocompatibility after surface modification by micro-arc oxidation.In this study,the sintered Ti-10Cu alloy material was treated by micro-arc oxidation for the first time.Its corrosion resistance,antibacterial property,biocompatibility and bone promoting properties were evaluated through material performance test,in vivo and in vitro antibacterial and biocompatibility assay,histological analysis and molecular biology assay to provide a good new titanium alloy material for clinical application in orthopedics.Methods:Ti-10Cu alloy prepared by sintering method was cut into thin wafers and rods.MAO was carried out under the conditions of voltage 400 V,frequency 400 Hz,pulse width 100μs and oxidation time 3 min to produce micro-arc oxidation Ti-10Cu,namely Ti-10Cu（MAO）.Adding electrolytes containing calcium and phosphorus into micro-arc oxidation electrolyte can improve the biocompatibility of materials and the ability to induce osteoblast differentiation.In the first part,the composition of alloy phase was identified by X-ray diffraction（XRD）.The surface morphology of the samples was detected by scanning electron microscope（SEM）.The hydrophilicity of sample surface was evaluated by water contact Angle measurement.The release amount of Cu²⁺in each metal sample was determined by copper ion stripping test.Electrochemical experiments were conducted to evaluate the corrosion resistance of the alloy materials in the simulated environment of human body fluids.The antibacterial ability of Ti-10Cu（MAO）samples was investigated by implanting metal materials in vivo into a rabbit femur infection model and counting bacterial culture plate on the surface of the materials in vitro.The antibacterial rate was calculated and the antibacterial ability was evaluated.C-reactive protein and IL-6 were detected by ELISA,IL-1,IL-6,IL-8,IFN-γand TNF-αwere detected by fluorescence quantitative PCR,and the antibacterial enhancement effect of micro-arc oxidation on Ti-10Cu alloy was evaluated.In the second part,in vivo and in vitro experiments were used to evaluate the effect of micro-arc oxidation on the biocompatibility and osteogenesis of Ti-10Cu alloy.The bone integration and biocompatibility of Ti-10Cu（MAO）were observed by X-ray image and HE staining.The effects of Ti-10Cu（MAO）on induced differentiation and bone formation of MG63 cells were evaluated by ALP activity detection combined with ALP（alkaline phosphatase）staining and ARS（alizarin red）staining.Cell proliferation was detected by MTT assay and cell cloning and proliferation assay.Immunofluorescence and Western-blot were used to detect the expression of OPN,Runx2,cyclin D1,P21 and P27,TRAIL,DR3,DR4,Caspase3 and VEGFR.The effects of micro-arc oxidation on cell proliferation,cytotoxicity,apoptosis,formation of vascular endothelial cells and bone repair of Ti-10Cu alloy were evaluated.Results:1.Material performance test results:X-ray diffraction（XRD）observations show that Ti-10Cu（MAO）has three different phases,namelyα-Ti,Ti₂Cu and Ti O₂phases.Scanning electron microscopy（SEM）shows that the surface coating of the alloy is composed of a large number of molten aggregates,forming micron discharge holes with uniform distribution,which is similar to the"volcanic cone"grid structure.The measurement of water contact Angle showed that Ti-10Cu（MAO）group was more hydrophilic than Ti-10Cu group,which was conducive to protein adsorption and initial cell adhesion.The copper ion dissolution experiment showed that the copper ion release of Ti-10Cu（MAO）in normal saline solution was significantly higher than that of Ti-10Cu（P<0.001）.Electrochemical experiments show that Ti-10Cu（MAO）material has higher open circuit potential and self-corrosion potential than Ti-10Cu,and lower self-corrosion current density,indicating that Ti-10Cu（MAO）material has better corrosion resistance than Ti-10Cu.2.Antibacterial test results:Results of antibacterial experiments in vitro:The colony numbers of S.aureus and E.coli cultured on Cu,Ti-10Cu and Ti-10Cu（MAO）materials were significantly lower than those cultured on Ti materials.The bacteriostasis rate of TI-10Cu（MAO）against S.aureus and E.coli was 99.34±0.04%and 99.42±0.09%,respectively.Results of antibacterial experiments in vivo:The implantation of Ti-10Cu and Ti-10Cu（MAO）metal rods can inhibit infection and prevent local bone destruction,and the inhibition effect of Ti-10Cu（MAO）is stronger than that of Ti-10Cu.In vivo experimental results of local tissue HE staining of Ti-10Cu（MAO）antibacterial activity showed that a large number of inflammatory cell infiltration was observed in the tissues around the implant site of femoral rod in the Ti-Cu group,while only a small number of inflammatory cell infiltration was observed in the Ti-10Cu and TI-10Cu（MAO）groups.The expressions of CRP and IL-6 in rabbit infection model blood in ti-10cu（MAO）group and ti-10cu group were significantly lower than those in Ti group（P<0.05）,and the expressions of two factors in Ti-10Cu（MAO）group were also lower than those in Ti-10Cu group.Fluorescence quantitative PCR showed that compared with Ti group,m RNA expressions of IL-1,IL-6,IL-8,IFN-γand TNF-αin the soft tissue surrounding bone rod implantation site in Cu,Ti-10Cu and TI-10Cu（MAO）groups were significantly decreased（P<0.001）.3.Cell compatibility test results:Contributes to the results of the osteogenic test showed that the 90 d after implantation of each group of materials into the rabbit femur,X-ray slides show good bonding on the implant/bone interface in the Ti-10Cu（MAO）group,There is no loosening of the implant,The surrounding bone is dense,It shows that the Ti-10Cu（MAO）group has stronger osseointegration ability.ALP detection results showed that the expression of ALP in Ti-10Cu（MAO）group was significantly higher than that in Ti-10Cu（P<0.01）and Ti（P<0.001）groups when the extract was applied to MG63 cells.ALP staining results showed that the expression of ALP in cytoplasm of MG63 cells in TI-10Cu（MAO）group was significantly stronger than that in Ti-10Cu group.ARS staining results showed that the number of mineralized nodules in Ti-10Cu（MAO）group was significantly higher than that in Ti-10Cu group.The material was implanted into the rabbit femur 90 d later,and X-ray showed that the Ti-10Cu（MAO）group had good bonding at the implant/bone interface,no loosening of the implant and dense surrounding bone.These results indicated that Ti-10Cu（MAO）group had stronger bone binding ability.MTT assay showed that the effect of Ti-10Cu（MAO）material on cell proliferation was not significantly different from that of the blank control group.Compared with Ctrl group,the expression of F-actin and Ki67 protein was significantly decreased in Cu group.The expression of F-actin and Ki67 protein in Ti group,Ti-10Cu group and Ti-10Cu（MAO）group had no significant difference compared with control group.The expression of VEGFR protein in Ti-10Cu（MAO）group was significantly higher than that in Ti-10Cu group.The results of cell clone formation ability assay showed that Compared with control group,the number of cell clone formation in Ti group,Ti-10Cu group and Ti-10Cu（MAO）group was not significantly different,while the number of cell clone formation in Cu group was significantly reduced.Flow cytometry apoptosis detection results demonstrated that,the apoptosis number of MG63cells in Ti-10Cu（MAO）group,Ti-10Cu group and Ti group was not significantly different compared with control group,while the apoptosis of MG63 cells in Cu group was significantly increased.The cell scratch assay showed that there was no significant difference in the percentage of wound healing distance between the Ti group,Ti-10Cu group and Ti-10Cu（MAO）group and the normal control group.These results indicated that Ti,Ti-10Cu and Ti-10Cu（MAO）materials had no effect on the migration ability of MG63 cells.The western-blot showed that the expression of OPN and Runx2 in Ti-10Cu（MAO）group was significantly higher than that in Ti-10cu group.Compared with control group,the expression levels of P21,P27 and cyclin D1 proteins in Ti-10Cu（MAO）group were not significantly different.The detection results of apoptosis-related proteins TRAIL,DR3,DR4 and Caspase proteins showed no significant difference between the Ti-10Cu（MAO）group and the control group.The expression of VEGFR in Ti-10Cu（MAO）group was significantly higher than that in Ti-10Cu groupConclusion:1.Ti-10Cu alloy modified by micro-arc oxidation improves its corrosion resistance,and its three-dimensional porous rough surface morphology is more conducive to the growth of osteoblasts and increases its osteogenesis.2.Surface modification by micro-arc oxidation can improve the antibacterial ability of Ti-10Cu alloy against E.coli and S.aureus in vitro and in vivo.3.Ti-10Cu（MAO）has no cytotoxicity,no effect on cell proliferation,cell cycle and apoptosis after surface modification by micro-arc oxidation,and has good biocompatibility.