Study On The Antibacterial Activity And Cytocompatibility Of TiN Coated Ti-6Al-4V Implanted With Cu²⁺

The surface of Ti-6Al-4V material does not have effective antibacterial properties,resulting in bacterial adhesion to form bacterial biofilm.The biofilm is difficult to cure with antibiotics.Our previous experiments proved that Ag⁺as a material surface bacteriostatic agent can effectively inhibit bacterial biofilm formation.However,the biosafety of Ag is getting more and more attention,and Cu is one of the essential trace elements of the human body and has good antibacterial function as well as osteogenic activity.In this paper,the antibacterial function and cytocompatibility of Cu²⁺as a colonizing bacteriostatic agent were studied preliminarily in comparison with Ag⁺.Objective:To study and compare the antibacterial activity and cytocompatibility of Ti-6Al-4V carrying TiN implanted with Cu²⁺and Ag⁺respectively.Methods:TiN film was deposited by magnetron sputtering on the Ti-6Al-4V sheet and Cu²⁺or Ag⁺was respectively implanted by MEVVA ion implantation.Then the Ti-6Al-4V carrying TiN implanted with Cu²⁺and Ti-6Al-4V carrying TiN implanted with Ag⁺were constructed.L929 cells were inoculated in the blank control group-Ti-6Al-4V?the titanium alloy group?,negative control group-TiN coating titanium alloy?the TiN group?,positive control group-the titanium alloy carrying TiN implanted with Ag+,experimental group-the titanium alloy carrying TiN implanted with Cu²⁺?the Cu group?surface.After 24 hours of inoculation,the cells were stained with rhodamine phalloidin and Heochest 33342,and then the morphology,growth status and adhesion of the cells were observed by Confocal Laser Scanning Method.After 24 hours of inoculation,,the cells were dehydrated using gradient ethanol,lyophilized,and sprayed with gold.Then adhesion and growth status were observed through Scanning Elctron Microscope.After 1day,3days and 5 days,cell proliferation was detected by CCK-8 Kit.The Straphylococcus aureus solution was dropwise added to the titanium alloy group,the TiN group,the Ag group and the Cu group.After 24 hours of inoculation,the number of viable bacteria colonies on the surface of the material was observed by the viable plate count method;the number of bacteria was observed by Confocal Laser Scanning Method;SEM was used to observe the adhesion,morphology and number of bacteria.The contact angle reflects the hydrophilicity of the material.XPS reflects the surface elements composition of the material and ICP-MS can reflect the amount of ion precipitation of the target element of the material.Results:1.CLSM:The cells in the titanium alloy group and the TiN group distributed evenly and densely.A large number of filopodia could be observed,which were intertwined and reticulated,and the cells spread adequately.The cells in the Ag group and the Cu group distributed evenly and densely relatively.Most of the cells spread well,and their filopodia interwove.2.SEM:The adhesion and spreading of the cells in the titanium alloy group and the TiN group were sufficient,and a large number of plate-like pseudopods and filopodia were observed.The adhesion of the cells in the Ag group and the Cu group was sufficient relatively to show the slab pseudopods and filopodia.3.CCK-8:After 1 day,3 days,and 5 days of inoculation,the cells in the Ag group and the Cu group proliferated obviously,which can prove that the two groups had no apparent toxicity.4.Plate count method:There were colonies which distributed densely in the titanium alloy group and the TiN group;while there were few colonies in the Ag group and the Cu group.5.CLSM:The cells in the titanium alloy group and the TiN group distributed evenly and densely.The Cu group and the Ag group had less bacterial adhesion.6.SEM:The bacteria in the titanium alloy group and the TiN group were dense,laminated and interlaced,with a complete shape.The bacteria in the TiN group were dense,and the appearance of the bacteria remained unchanged.The cell wall was intact.The Ag and the Cu group:few bacteria adhesion,the integrity of bacterial wall was damaged,and some bacteria were broken.Conclusion:TiN coated Ti-6Al-4V implanted with Cu²⁺has high cell compatibility and antibacterial properties.