Study On The Antibacterial Properties And Biocompatibility Of SLM Copper-cobalt Alloys

Objective:Cobalt base alloy is one of the most widely used medical metal materials in clinic.It has strong corrosion resistance,good biocompatibility and low elastic modulus.Currently,it is mostly used as implant,crown repair material and orthodontic filament.However,the conventional cobalt-based alloy molding process is complex and is a kind of biological inert material,which limits its clinical application to some extent.Studies have confirmed that copper ions have strong broad-spectrum bactericidal ability,and scholars have developed copper-containing stainless steel and copper-nickel-titanium alloy with good antibacterial properties.The copper-rich phase formed by this antibacterial alloy in the physiological environment can effectively kill bacteria on the surface of the alloy,thus playing an antibacterial role.In this study,a new type of copper-cobalt alloy was prepared by adding copper element to cobalt-based alloy and using selective laser melting technology,aiming to optimize the molding mode of cobalt-based alloy and endow cobalt-based alloy with antibacterial ability.Therefore,the antibacterial properties and biocompatibility of the new alloy were studied and analyzed.Materials and Methods:Two alloys were fabricated using SLM and prepared into circle with the size of?10×1mm~3,SLM-CoCrW was used as the control group to verify the antibacterial properties of SLM-CoCrCu.Staphylococcus aureus(ATCC 6538)was selected as the bacteria,and quantitative antibacterial experiments were conducted in vitro to preliminarily detect the antibacterial properties of SLM-CoCrCu.After the bacteria and the material were co-cultured for 24h,the bacterial biofilm formed on the material surface was observed by scanning electron microscopy,and the adhesion and biofilm formation of the bacteria on the material were analyzed.Using the Bacteria dead/live assay dyeing bacteria,material surface using laser confocal further observate live bacteria and dead bacteria distribution and the thickness of the biofilm,combining with the Real-time PCR results of comprehensive evaluation of the new-type alloy antibacterial properties.Selecting MC3T3-E1 as the object for this study.Antimicrobial activity was analyzed by the spread plate analysis;Biocompatibility was analyzed using Cell Counting Kit-8 for cell proliferation and toxicity,using Cytoskeleton staining for cell morphology and adhesion ability,using Flow cytometry for cell apoptosis.The material also was cultured with fresh anticoagulant to evaluate the effect of the material on blood dynamic coagulation time and hemolysis of erythrocytes.Results:1.The result highlighted a clear inhibitory effect of SLM-CoCrCu alloys on bacterial growth after direct contact.The antibacterial property had significant different between two groups(P<0.01).2.SEM images and live/dead staining results further indicated that SLM copper-cobalt based alloy could inhibit bacterial adhesion,thus greatly reducing the formation of biofilms.Real-time PCR results also verified its antibacterial performance.3.Compared with SLM-CoCrW,The cytotoxicity of SLM-CoCrCu was declined gradually with the extension of time;4.There were significant differences in the cell proliferation rate between SLM-CoCrCu and SLM-CoCrW after co-culture with MC3T3-E1 for 1,3,7 days(P=0.01);The group of SLM-CoCrW cell proliferation rate was better than the other one after 1 day of co-culture,and the effect of the SLM-CoCrCu group on cell proliferation was significantly better than that of the SLM-CoCrW group after 3days and 7 days.And two groups had no significant difference on cell adhesion and morphology.5.Two groups had no significant difference on cell apoptosis.At the initial stage of co-culture,the hemolysis amount in the copper-free group was higher than that in the copper-containing group.The hemolysis results showed that the hemolysis rate of SLM-CoCrW alloy was 0%,that of SLM-CoCrCu alloy was 0.25%,and that the hemolysis rate was much lower than 5%.Conclusion:SLM-CoCrCu alloy had excellent antibacterial property to S.aureus.It has a certain inhibitory effect on the growth,survival ability of bacteria and the formation of biofilms on materials.Moreover,SLM-CoCrCu alloy showed good cytocompatibility,appearing as beneficial to cell proliferation,adhesion and apoptosis.SLM-CoCrCu has good blood compatibility,and the coagulation function is still good and the hemolysis rate is low after the addition of Cu.