A Coating Strategy On Titanium Implants With Enhanced Photodynamic Therapy And Gas Therapy For Microenvironmental Regulation Of Peri-implantitis And Mechanism

Dental implant is the preferred method of dental restoration for missing teeth.With the continuous advancement of national collection policy,dental implant will become the mainstream repair method for dentition defects.However,the thread structure on the titanium surface of the implant is easy to be adhered to and clustered by bacteria to form biofilms,and then secondary inflammatory reactions can cause related infections such as peri-implant inflammation,and eventually lead to implant loss.Therefore,how to control the occurrence and development of peri-implantitis has become the key factor of affecting the life of implant teeth.（Antimicrobial photodynamic therapy,aPDT）is considered to be an effective way to treat bacterial infections non-invasively.In particular,aPDT excited by Near Infrared（NIR）has a deeper penetration in human tissues,which further expands the prospect of biological application of aPDT.However,in the clinical treatment of oral diseases（especially peri-implant inflammation）,we still need to solve the bottleneck problems such as elevated local oxidative stress and hypoxia caused by infection and aPDT treatment according to their particularity,thereby reducing and improving inflammation.To solve the above problems,we propose a strategy integrating aPDT and gas therapy,and based on this strategy,we design a multifunctional gas generator integrating aPDT with oxygen（O₂）and carbon monoxide（CO）gas release function.The generator can achieve the purpose of removing pathogenic plaque biofilm by stimulating aPDT with NIR,and the therapeutic gases O₂and CO molecules generated have long life and long transmission distance,which can effectively solve the problem of oxygen deficiency in aPDT under NIR irradiation and the problem of inflammation regulation in the distal part of the implant.In this study,we first constructed a multifunctional nanomaterial system consisting of UCNPs（Upconversion Nanoparticles,UCNPs,U）,SnS₂,and ICG（Indocyanine Green,ICG,I）.Then,the composite nanomaterial was used to modify the surface of titanium（Ti）,and a multifunctional coating was constructed to modify the surface of Ti Ti-U@SnS₂/I.The ICG in this system releases Reactive oxygen species（ROS）under the excitation of NIR and exerts aPDT performance.At the same time,the system can use UCNPs to efficiently convert NIR into visible light.Subsequently,the partially oxidized SnS₂releases the therapeutic gases O₂ and CO under the excitation of visible light,further enhancing the performance of aPDT and regulating the inflammatory microenvironment.The nanosystem can release ROS,CO and O₂ at the same time under the excitation of a single NIR 808 nm,thus exerting multiple therapeutic effects.Finally,the titanium surface was modified by physical spin coating,and the modified titanium surface multifunctional coating was constructed.The successful construction of the nanosystem and the release of ROS,CO and O₂ were confirmed by characterization experiments.The antibacterial and anti-inflammatory properties of the nano-system were evaluated in vitro and in vivo.Finally,the potential mechanism of antibacterial and anti-inflammatory was further discussed through bioinformatics.The constructed multifunctional composite nanomaterial system provides theoretical and experimental basis for the development of new implant surfaces.The following are the experimental contents and results of this study:Experiment 1:Preparation,characterization and biosafety evaluation of a coating strategy on titanium implants with enhanced photodynamic therapy and gas therapyFirstly,prepare U@SnS₂/I nanomaterials,and the surface of pure titanium was modified by spin coating to obtain Ti-U@SnS₂/I.The physical and chemical properties of U@SnS₂/I and Ti-U@SnS₂/I were evaluated by transmission electron microscope,scanning electron microscope,X-ray diffractometer,atomic force microscope,UV-VIS absorption spectrum and Fourier transform infrared spectrum.The gas release performance of the nanosystem was evaluated using CO,O₂ and ROS probes.Finally,the biosafety of Ti-U@SnS₂/I was evaluated.The results show that U@SnS₂/I nanomaterials are successfully prepared and the surface of titanium is successfully modified.Under NIR irradiation,there is an efficient energy transfer between UCNPs and SnS₂ in U@SnS₂/I,and CO and O₂are released with high efficiency.In addition,U@SnS₂/I release ROS more efficiently than SnS₂/I due to the generation of O₂.At the same time Ti-U@SnS₂/I has good stability and biocompatibility.Experiment 2:Study on antibacterial properties and mechanism of a coating strategy on titanium implants with enhanced photodynamic therapy and gas therapyThe antibacterial performance of Ti-U@SnS₂/I was evaluated by 3D live/dead bacterial staining,bacterial metabolic activity assay,colony forming unit,and histological staining.The results of in vitro experiments showed that Ti-U@SnS₂/I could effectively inhibit the adhesion of early biofilm colonizing bacteria and the formation of mature biofilm in the middle and late stages.In vivo experiment results show that Ti-U@SnS₂/I can effectively kill microorganisms in deep tissues and reduce the damage of microorganisms to tissues.The antibacterial mechanism of Ti-U@SnS₂/I was investigated by transcriptomic GO enrichment analysis,KEGG enrichment analysis and PPI network analysis.The results showed that after treatment with Ti-U@SnS₂/I,ion transmembrane transport and sensing of two-component system on bacterial cell membrane were interfered,and ribosom-associated protein transcription was inhibited.Experiment 3:Study on the anti-inflammatory activity and mechanism of a coating strategy on titanium implants with enhanced photodynamic therapy and gas therapyThe anti-inflammatory activity of Ti-U@SnS₂/I was evaluated by RT-PCR,M1/M2 phenotype immunofluorescence,histological staining and immunohistochemical staining.The results of in vitro experiments showed that Ti-U@SnS₂/I has a good inflammatory regulation effect,can regulate the expression of pro-inflammatory/anti-inflammatory cytokines,and regulate the polarization of macrophages from M1 type to M2 type.In vivo experiment results show that Ti-U@SnS₂/I can regulate the changes of inflammatory microenvironment,promote tissue healing and reconstruction,and solve the problem of insufficient regulation of inflammation by aPDT.The anti-inflammatory mechanism of Ti-U@SnS₂/I was investigated by NF-κB/p65 immunofluorescence assay,transcriptomic GO enrichment analysis,KEGG enrichment analysis and PPI network analysis.The results showed that Ti-U@SnS₂/I could up-regulate the expression of AMPK and PPAR pathways and down-regulate the expression of NF-κB signaling pathway,thereby regulating the inflammatory microenvironment and playing an anti-inflammatory role.Through the above three part of the experiments,the following conclusions are obtained:（1）In this study,a coating strategy on titanium implants with enhanced photodynamic therapy and gas therapy（Ti-U@SnS₂/I）was successfully constructed,which has good ROS,O₂and CO release performance.Meanwhile,this gas production property can further increase ROS production in aPDT process by increasing the oxygen partial pressure.The nano-coating has good biosafety and has certain application prospect in clinic.（2）A coating strategy on titanium implants with enhanced photodynamic therapy and gas therapy（Ti-U@SnS₂/I）can interfere with the ion pump and two-component system of bacterial cell wall,inhibit the transcription of ribosome related proteins,and effectively inhibit the adhesion of early colonizing bacteria and the formation of mature biofilms in the middle and late stages.Compared with traditional aPDT,it has better therapeutic effect on deep infection,and can effectively kill microorganisms in deep tissues in the body and reduce the damage of microorganisms to tissues.（3）A coating strategy on titanium implants with enhanced photodynamic therapy and gas therapy（Ti-U@SnS₂/I）can inhibit the NF-κB signaling pathway,balance the polarization of M1/M2 macrophages,improve the inflammatory microenvironment,relieve excessive inflammation induced by aPDT,and promote tissue healing and regeneration.