Preparation Of Copper-loaded Antibacterial Functional Film On Titanium Surface And Investigation On Its Properties

Objective: Titanium implants with antibacterial properties were designed by loading copper ions on the surface.Simultaneously,the cytotoxicity and biocompatibility was evaluated.We hope that our study can provide the experimental basis for improving infection of titanium implants clinically.Methods: Firstly,the polylactic acid polymer material with certain viscosity and non-toxic and degradable was dissolved in dichloromethane,and different concentrations of copper ions were added to prepare the polylactic acid solution containing copper.Poly(lactic acid)(PLA)films with different concentrations of copper ions were prepared on the surface of titanium by dip method.Poly lactic acid films with the different concentration of copper ions(0.1 mg/mL?0.5 mg/mL?1.0 mg/mL)was performed by sol-gcl dip-coating(SGDC)method.Then,the loading condition between the titanium implant and film(Group A: control group;Group B: no copper-loading film group;Group C: 0.1 mg/mL copper-loading film group;Group D: 0.5 mg/mL copper-loading film group;Group E: 1.0 mg/mL copper-loading film group)were observed by scanning electron microscope(SEM).Further,the antibacterial activity test in vitro was detected with bacterial staining and counting method.Biocompatibility was also performed by cell proliferation assay with the CCK-8 kit,detecting the expression of OSX?OPN?ALP?OCN with the relevant kit and quantitative polymerase chain reaction(qPCR).Results: The results of scanning electron microscopy showed that the surface of group A was rough and no particles attached.B.In group C,D and E,there were thin films attached to the films,and in group C,D and E,copper particles with a diameter of about 100 nm were uniformly distributed in the films,and the density of copper particles on the surface of titanium increased with the increase of the concentration of impregnated copper ions;the release of copper ions on the surface of titanium was the highest in group E,followed by that in group D and C,indicating that there was a positive correlation between the release of copper ions and the content of impregnated copper ions.In vitro bacteriostatic experiment showed that there was no significant difference in bacterial colony growth between group A and group B without copper,indicating that PLA had no antibacterial activity.However,compared with the groups(C,D,E)loaded with different concentrations of copper ions,the copper group had a significant killing effect on bacteria,and the number of colonies attached to the surface of materials from C to E decreased in turn,indicating that with the increase of copper ion content in the functional film on the surface of titanium plate,the killing effect on bacteria gradually strengthened.The results of living and dead bacteria staining and counting under microscope showed that the survival rate of E.coli and staphylococcus aureus decreased with the increase of copper ion content,the number of living bacteria in group A and group B was the most,and then decreased in turn according to group C,D and E.CCK-8 test results showed that the results of co culture of rabbit bone marrow mesenchymal stem cells with each group of copper loaded polylactic acid membrane titanium samples were as follows: group C had the strongest cell proliferation ability,followed by B,A,D,E group;after co culture with each group of copper loaded polylactic acid membrane titanium samples soaked in DMEM for 6 hours,the results were: group C was still the best,followed by D,B,E,A group(The results showed that rBMSCs survived most when the content of copper ion in the solution was 1mg.ALP detection results showed that the highest ALP activity was found in group C(P < 0.05)after 7 and 14 days of co culture with rBMSCs.In group C,ALP expression was in the order of B,D,A and E(P < 0.05)from high to low the results showed that the activity of alkaline phosphatase was the highest when the content of copper ions in titanium samples of each group was 1.0 mg/ml,and the activity of alkaline phosphatase was the highest in D,E,B,A group(P < 0.05),OSX,OPN,ALP,OCN and other osteogenic related genes are all expressed in different degrees.The expression of each gene in group A,B,C,D,E shows a trend,that is,the highest expression in group C(P There was significant difference in gene expression between the two groups(P < 0.05).Conclusion: Through the preparation of copper loaded functional films on titanium surface,titanium implants with antibacterial function were successfully prepared.With the increase of copper ion concentration(0.1 mg / mL ~ 1.0 mg /mL),the antibacterial ability of titanium surface to Escherichia coli and Staphylococcus aureus increased.When the concentration of copper ion is 0.1 mg/mL,its ability of promoting cell proliferation and osteogenic differentiation is the strongest,but its bactericidal ability is relatively weak;when the concentration of copper ion is 1.0 mg/ml,its bactericidal ability is the strongest,DMEM soaking for 6 hours has no cytotoxicity,and it can promote cell osteogenic differentiation.Therefore,when the concentration of copper ion is 1.0 mg/mL,it can not only endow the implant with antibacterial properties,but also facilitate the cell differentiation.