Study Of Gold-Titanium Dioxide Heterojunction For Enhanced Sonodynamic Mediated Biofilm Scavenging And Peri-implantitis Treatment

Background and Objective:Titanium implants have now become a routine treatment to replace missing teeth.However,peri-implantitis caused by bacterial biofilm accumulation is the main cause of implant repair failure,and the removal of biofilm is essential for the successful treatment of peri-implantitis.However,traditional mechanical debridement therapy and antibiotic therapy cannot completely remove bacterial biofilm,and new antibacterial therapy is urgently needed to explore.Antibacterial sonodynamic therapy（a SDT）is considered a promising new treatment for infectious diseases,which uses low-frequency ultrasound（US）to activate sonosensitizers to produce reactive oxygen species（ROS）to kill bacteria,with the advantages of no drug resistance,large penetration depth,convenience and low cost.In this study,a simple and safe sonodynamic therapy has been proposed by fabrication of gold nanoparticles modified titanium dioxide nanotubes（Au-TNT）on the surface of the implant to endow it an outstanding antibacterial ability.Au-TNT can be activated by US as a sonosensitizer,and we studied its antibacterial effect on oral single-species and multi-species biofilms,as well as its antibacterial effect in a rat peri-implantitis model,aiming to provide inspiration for a SDT treatment of peri-implant infectious diseases.Methods:1.Titanium dioxide nanotubes（TNT）were formed on the surface of titanium implants by anodic oxidation,and then gold nanoparticles（Au NPs）were synthesized by sodium citrate reduction.The surface of TNT was grafted with silane coupling agent（APS）so that the surface showed high positive charge,and the negatively charged Au NPs could be loaded on the nanotubes by electrostatic attraction,labeled as Au-TNT.2.The physical and chemical properties of the Au-TNT were characterized using scanning electron microscope（SEM）,transmission electron microscope（TEM）,X-ray diffraction（XRD）,X-ray photoelectron spectroscopy（XPS）,contact angle measuring instrument,and WS-2005 coating adhesion auto-scratcher.3.The oxygen production（O₂）capacity of the materials was measured using a portable dissolved oxygen meter,and the reactive oxygen species（ROS）production rate of the materials was measured using DPBF probe,SOSG probe,and MB probe.4.The biocompatibility of Au-TNT to MC3T3-E1 was determined by CCK-8assay.5.In vitro antibiofilm experiments:single-species biofilms（P.gingivalis,F.nucleatum,S.sanguinis）were established respectively,and the killing effect of Au-TNT on single-species biofilms and the morphological changes of bacteria under ultrasound irradiation were determined using CFU assay,MTT assay,and SEM assay.Multi-species biofilms were established,and the killing effect of Au-TNT on multi-species biofilm under ultrasonic irradiation was determined by the live/dead staining assay,MTT assay,crystalline violet assay,and extracellular polysaccharide assay.6.Animal experiments:a rat peri-implantitis model was established,and CFU assay and Giemsa staining were performed to assess the in vivo antibacterial effect of Au-TNT under the action of ultrasound;hematoxylin and eosin（H&E）staining,immunohistochemical staining,and Micro-CT were used to evaluate the status of peri-implant tissue inflammation regression and alveolar bone repair.Results:1.SEM images showed Au NPs（15 nm in diameter）loaded on TNT（100 nm in diameter）,XRD and XPS proved that the crystalline phase was anatase phase Ti O₂with elemental composition of Ti,O and Au,which proved the successful synthesis of Au-TNT.The coating adhesion between the nanotubes and the substrate is about 9 N.The contact angles of Au-TNT was 35.9°.2.Au-TNT has excellent sonodynamic efficiency and could produce O₂and ROS.The results of CCK-8 assay showed that Au-TNT has good biocompatibility in vitro.3.Killing efficiency of Au-TNT against single-species biofilm:CFU assay results showed that the antibacterial rates of the Au-TNT+US group against P.gingivalis,F.nucleatum,and S.sanguinis were 96.59%,96.25%,and 95.31%,respectively,and SEM images showed that the three bacteria in the Au-TNT+US group all showed shrinkage and rupture of bacterial walls,and the bacterial contents flowed out.The MTT results of three single species biofilms showed that the biofilm metabolic activity of Ti group>TNT group>Au-TNT group>Ti+US group>TNT+US group>Au-TNT+US group.4.Killing efficiency of Au-TNT against multi-species biofilm:the live/dead staining assay showed that Ti,TNT,Au-TNT and Ti+US groups showed large areas of green fluorescent live bacteria,while Au-TNT+US group had a considerable number of dead bacteria with severely damaged cell membranes（red fluorescence）;the biofilm metabolic activity,thickness,and extracellular polysaccharide yield of Au-TNT+US group were the lowest among the groups as demonstrated by MTT experiment,crystalline violet experiment,and extracellular polysaccharide experiment.5.Au-TNT-mediated a SDT in a rat peri-implantitis model:the least number of bacteria was found in the Au-TNT+US group by CFU assay and Giemsa staining,which was reduced by 3 logs compared with the control group.H&E staining and immunohistochemistry results showed that the number of inflammatory cells and IL-1βand TNF-αpositive markers in the Au-TNT+US group were minimal,which indicated that inflammation was alleviated.In addition,3D reconstruction of the peri-implant tissue after 4 weeks of treatment showed the least amount of alveolar bone loss and the highest BV/TV in the Au-TNT+US group.Conclusion:1.Au-TNT nanocomposites with highly efficient sonodynamic antibacterial ability were successfully prepared on the surface of titanium implants.2.Au-TNT can produce ROS and O₂under ultrasound irradiation and has good biocompatibility.3.In vitro experiments showed that Au-TNT-mediated a SDT could effectively kill oral single-species and multi-species biofilms.4.Au-TNT-mediated a SDT has outstanding antibacterial effect in rat peri-implantitis model,which is conducive to the regression of inflammation and promotes the recovery of bone tissue.