Study On The Role Of "Dual Antioxidant-antimicrobial" Coating In Remodeling The Inflammatory Immune Microenvironment And Repairing The Connective Tissue Barrier

Objective:Peri-implantitis is the most common complication of dental implants.The"microgap"between the implant and the surrounding connective tissue is the initial factor contributing to pathogen invasion,and persistent bacterial stimulation exacerbates the macrophage-mediated inflammatory response,leading to implant failure.Unfortunately,most of the existing studies have focused on the improvement of the implant-osteosynthesis surface and have neglected the importance of surface improvement in the transmucosal region of the implant.The abutment,as the transmucosal region of the implant,needs to be modified by biofunctionalization to repair the mucosal barrier and regulate the immune microenvironment.Here,the aim of the present study was to investigate a metallophenolic network structure formed by tannic acid（TA）and cerium（Ce）,on the basis of which a multifunctional coating（TA-Ce-Mino）incorporating minocycline（Mino）was developed and evaluated in ex vivo and in vivo experiments to modulate the immune microenvironment and promote The ability of the coating to modulate the immune microenvironment and promote tissue cell adhesion to enhance the connective tissue barrier,as well as its p H-responsive drug delivery for smart antimicrobial function,were evaluated in ex vivo experiments.Methods:1.Material construction and physicochemical property study:TA-Ce-Mino coating was synthesized;SEM（Scanning electron microscope）and AFM（Atomic Force Microscope）tests were performed to observe the changes of micro-morphology at the surface interface of the material;contact angle test was performed to detect the hydrophilic and hydrophobic properties of the material;PEEK was used to test the hydrophilic and hydrophobic properties of the material;and PEEK was used to test the hydrophilic properties of the material.EDS（Energy Dispersive Spectrometer,energy spectrometer）,FTIR（Fourier transform infrared spectroscopy）,XPS（X-ray photoelectron spectroscopy）,XRD（X-ray Diffractometer）,etc.,to detect the elemental composition and valence of metals,coating stability and coordination bonds.ratio,coating stability,and coordination bonding,whether it is crystalline or amorphous,in order to determine whether the material system is successfully constructed or not;through DPPH（2,2-Diphenyl-1-picrylhydrazyl）,NBT（Nitroblue tetrazolium）and TMB（3,.3’,5,5’-Tetramethylbenzidine）were used to evaluate the oxidation resistance of the coatings;and the oxidation resistance of the coatings was evaluated by ICP-OES（Inductively coupled plasma optical emission spectrometry）and absorbance spectroscopy.Inductively coupled plasma optical emission spectrometry（ICP-OES）and absorbance detection to evaluate the p H responsive release ability of the coatings.2.After co-culturing with Aureobasidium,the free antimicrobial properties of the materials were detected by plate counting,OD600（Optical Density）and Alma blue;after co-culturing with S.aureus（Staphylococcus aureus）and E.coli（Escherichia coli）,the materials were stained live and dead,and the contact antimicrobial properties of the materials with S.aureus and E.coli were detected by confocal microscopy and SEM.3.The biocompatibility of the materials was verified by CCK-8（Cell counting kit-8）and live-dead staining after co-culture of macrophages with the materials,and the effect of the materials on macrophage polarization was verified by immunofluorescence reaction in the inflammatory environment stimulated by LPS（Lipopolysaccharide）,and the effect of the materials on macrophage polarization was verified by q RT-PCR（Quantitative reverse transcription Polymerase chain reaction,real-time fluorescence quantitative nucleic acid amplification assay）and DCFH-DA（2’,7’-dichlorodihydrofluoresceindiacetate）,Ru（dpp）₃Cl₂（Tris（4,7-diphenyl-1,10-phenanthroline）ruthenium（II）dichloride）fluorescent staining to observe the material antioxidant effect,and the repairing effect of TA-Ce-Mino on mitochondrial function was observed by staining with JC-1（mitochondrial membrane potential fluorescent probe）.4.The effect of the material on the angiogenesis of human umbilical vein endothelial cells was investigated by human umbilical vein endothelial cell angiogenesis experiments,and after transcriptome sequencing,the mechanism of the material in promoting angiogenesis was preliminarily investigated by utilizing the KEGG（Kyoto encyclopedia of genes and genomes）pathway enrichment.5.The biocompatibility of the material was verified by CCK-8 and live-dead staining of human gingival fibroblasts co-cultured with the material,laying the foundation of biosafety for the subsequent in vitro experiments and animal in vivo experiments;and the biocompatibility of the material was verified by the F-actin（Fibros actin）/Dapi（4,6-diamino-2-phenyl indole）/Vinculin（adhesive plaque protein）immunofluorescence staining to study the spreading of fibroblasts on the surface of the material,as well as the adhesion;the effect of the coating on the migration of human gingival fibroblasts was verified by scratching experiments.6.Through the establishment of rat subcutaneous implantation infection model,through the observation of vascularization,H&E（Immunohistochemical）staining,CD31 immunohistochemical staining to study the surface of the material vasculogenesis;plate counting to verify the antimicrobial situation;Masson staining,Sirius red staining to study the collagen around the material generation;CD206 immunofluorescence and i NOS immunohistochemical staining to study the improvement of inflammatory microenvironment around the material.Results:The results showed that we successfully synthesized a multifunctional coating（TA-Ce-Mino）based on a metal phenolic network combined with the antibiotic minocycline.It promotes tissue adhesion to enhance the connective tissue barrier and achieves smart antimicrobial function through p H-responsive administration,creating a regenerative microenvironment for macrophage-mediated healing process.In addition,it has dual antioxidant functions against local oxidative stress:（i）exogenous antioxidant,Ce⁴⁺/Ce³⁺valence exchange and ROS（Reactive oxygen species,reactive oxygen species）scavenging ability of TA to convert ROS into harmless H₂O and O₂;（ii）endogenous antioxidant,repairing the mitochondrial homeostasis and maintaining macrophage mitochondrial homeostasis,thereby promoting the expression of antioxidant enzymes and related cytokines,and ultimately promoting macrophage M2 polarization.After observing that TA-Ce-Mino ideally promotes angiogenesis and collagen production,we revealed the underlying mechanism by which the coating stimulates the integrin-mediated Rho A/ROCK,PI3K/Akt,and NOTCH signaling pathways to promote cell migration,adhesion,and differentiation.Conclusion:In conclusion,the research-designed TA-Ce-Mino is a multifunctional coating that promotes soft tissue healing and possesses antimicrobial activity,is able to respond to the bacterial infectious inflammatory microenvironment,and achieves p H-responsive drug release kinetics to match the macrophage immune cascade,thereby promoting connective tissue barrier repair.