Experimental Investigations On Antimicrobial Mechanisms Of Tantalum Nanofilm

OBJECTIVE: Tantalum(Ta)is widey used in orthopedic surgeries which has demonstrated excellent biocompatibility and osseointegration in clinical practice for decades.However,the antimicrobial effect of Ta is still controversial.Current study aims to investigate the antimicrobial effect and relevant mechanisms of Ta nanofilm in vivo and vitro.METHODS: Megnetron sputtering was used to deposite Ta nanoparticles on the substrate of Ti to form different film thickness,and scanning electron microscope(SEM),X-ray photoelectron spectroscopic analysis(XPS)and X-ray diffraction(XRD)were utilized to investigate the material characterizations of different samples.CCK8 test was used to evaluate the biocompatibility in vitro.Mouse subcutaneous prosthesis model was constructed which was further tested by the analysis of complete blood count and blood chemistries as well as pathological sections of major organs.The immune antimicrobial effect of Ta nanofilms was preliminarily assessed using the coculture of fluroscent mcherry Escherichia coli,HL60-eGFP neutrophil-like cells and biomaterials model.Mouse subcutaneous prosthesis infection model was constructed with bioluminescent Staphylococcus aureus(ST1792-lux),the progression of infection in the mouse model was monitored by IVIS system,pathological sections of surrounding tissues was undertaken to evaluate infection severity and bacterial CFU count was done to quantify the bacterial load on samples and in surrounding tissues.Bacteria and samples were cocultured in vitro.The bacterial load in planktonic bacteria and biofilm were measured by CFU count.Live/dead flurouscent staining and SEM were utilized to qualitatively study the biofilm on the surfaces of different samples.Biomaterials-venous blood-bacteria cocultured model was constructed,blood smear and Wright–Giemsa staining were used to study the phagocytosis rates of neutrophils in different systems.The lysis rate of neutrophils in the presence of bacteria and biomaterials were assessed with the cocultural model of biomaterials-isolated neutrophils-bacteria.LPS-mouse macrophage-biomaterials cocultural model was constructed to deterimine the levels of IL-6,TNF-? and IL-1ra with Elisa kits.Lastly,rat osteomyelitis model was construted with different samples and bacteria,X-ray and microCT were utilized to evaluate the severity of osteolysis and bone destruction.RESULTS: Ta nanofilms on the surface of Ti became thicker and thicker with the increase of deposition time.Both Ta metals and oxides presented which was revealed by XPS and XRD.The proliferation of preosteoblast and macrophage was enhanced in the presence of Ta nanofilm,and Ta nanofilm did no harm to major organs and blood system in mouse subcutaneous prosthesis model.Dual fluroscent coclulture model revealed that the phagocytic efficiency of neutrophil-like cells was enhanced in the presence of Ta nanofilm.Mouse subcutaneous prosthesis infection model showed the infection severity was alleviated in the presence of Ta nanofilm thickness.However,Ta nanofilm showed no intrinsic antimicrobial effect in vitro.Ex vivo models revealed Ta could enhance the phagocytic ability of neutrophils towards bacteria,and inhibit the lysis of neutrophils.Meanwhile,Ta nanofilm could enhance the secretion of proinflammatory cytokines(i.e.,IL-6 and TNF-?),while inhibit the secretion of anti-inflammatory cytokine(i.e.,IL-1ra).Rat osteomyelitis model showed that Ta nanofilm could alleviate osteolysis and bone destruction in infected bones.CONCLUSIONS: Ta nanofilm showed favorable antimicrobial efficiency in vivo,while no intrinsic antimicrobial effect in vitro.The antimicrobial effect is accomplished by the immune activation of neutrophils and macrophages.Besides,Ta nanofilm showed excellent biocompatibility in vivo and in vitro which is promising for clinical translation.