Research On Establishment Of A Zinc Oxide/Titanium Dioxide Coating To Regulate Osteoimmunomodulation And Antibacterial Activity On The Titanium Implant

ObjectiveDental implant has become one of the main therapies for dentition defects and edentulous patients at present.Promoting osseointegration at early stage is the basis of successful implantation,and antibacterial function is the key to maintain implant long-term stability.Herein,an immune osteogenesis functional surface was constructed,and the effect of osteoimmunomodulation was observed.Furthermore,a photosensitive coating with both immune osteogenesis and antibacterial functions was prepared.The illumination parameters of the antibacterial function were studied to realize the coating immune osteogenesis and photocontrol antibacterial function,and to improve the success rate of implantation.MethodsPartⅠ The effects of zinc oxide/titanium dioxide nanotube composite coating on osteogenic activity of titanium implants by regulating macrophage functions1.Preparation and characterizationAnodic oxidation and hydrothermal methods were used to construct zinc oxide/titanium dioxide nanotube composite coatings.SEM was used to observe the surface morphology,EDS was employed to detect the elementary composition,XRD was introduced to detect the crystal structure,XPS was used to study the element types and valence states and ICP-AES was employed to determine the total load and release amount of zinc oxide.The surface roughness and hydrophilicity were detected by the surface profiler and contact angle tester.The BCA kit was employed to test the sample protein adsorption capacity.2.Regulation of macrophage by coatingsCCK-8 kit and LDH kit were used to detect the proliferation and toxicity of macrophages on the coating surface.SEM and CLSM were employed to observe the morphology and cytoskeleton.RT-q PCR and ELISA were used to reveal the pro-inflammatory,pro-regenerative gene and protein expression of macrophage.3.Promotion of the osteogenic activity by coatings regulating macrophagesThe macrophage conditional medium environment was created.CCK-8 kit and LDH kit were used to detect osteoblast proliferation and toxicity.SEM and CLSM were employed to observe the morphology and cytoskeleton.RT-q PCR and ELISA were used to reveal the osteogenesis related gene and protein expression of osteoblast.The expression of osteogenic markers in the supernatant of osteoblasts in each group was detected by ALP kit.The mineralization of osteoblasts in each group was detected by ARS staining.PartⅡ The study of zinc oxide/black titanium dioxide nanotube photocontrol coating on titanium implant surface for prevention of peri-implant infection and promotion of osteoimmunomodulation1.Preparation,characterization,mechanical properties,photocontrol and ZnO release propertiesThe black titanium dioxide nanotube coating was prepared by anodic oxidation and electrochemical reduction.The ZnO coating（ZnO/B-TNT/Ti）was constructed by ALD.SEM was used to observe the surface morphology and EDS was used to detect the composition.The crystal structure was detected by XRD.XPS was employed to study the element types and valence states.Raman spectroscopy was used to characterize the molecular structure of the coating.The nanoindentation was used to evaluate the strength of each coating.The absorption spectra and band gap widths were measured by UV-Vis-NIR spectrophotometer.The photothermal properties and photothermal stability were evaluated.The total load amount,heat release amount and natural release amount of ZnO were determined by ICP-AES.2.Evaluation of antibacterial propertiesThe antibacterial properties were verified by P.gingivalis,F.nucleatum and S.aureus,respectively.The antibacterial rate was calculated.The effect of each coating on membrane permeability and mitochondrial ATP of each bacterium were detected by ONPG and ATP kits.3.Evaluation of immune osteogenesis propertiesCCK-8 kit was used to evaluate the effects of NIR and material surface on cell proliferation.Flow cytometry was employed to detect the macrophage membrane surface marker.RT-q PCR,Western Blot and ELISA were used to detect the effects of different samples on the expression of inflammation-related genes and proteins in macrophages.CLSM was used to observe the skeleton of osteoblasts on the surface.The immune osteogenesis properties of the samples were evaluated by RT-q PCR,Western Blot and ARS staining4.In vivo detection of antibacterial and immune osteogenesis propertiesThe intraoral implant model of rats was established.The antibacterial properties of the implants were evaluated by plate colony count and turbidity.The local and systemic immunomodulatory properties of the implants were evaluated by IHC,HE staining sections and blood routine.Micro-CT assay,HE and Masson staining were used to evaluate the osseointegration performance of the implants.ResultsPartⅠ1.Results of SEM,EDS,XPS and XRD showed that at the voltages of 5V,15V and25V,the diameters of TiO₂ nanotubes were 30±5 nm,70±7 nm and 100±12 nm,respectively.The nano-sized zinc oxide particles were loaded onto the opening and walls of the nanotubes.ICP-AES results showed that ZnO could release for more than21 days in15VZnand 25VZngroups.Compared with Ti group,the ZnO loaded groups had appropriate roughness,hydrophilicity and protein adsorption capacity.2.15VZnand 25VZngroups exhibited no macrophage toxicity.The results of SEM and CLSM showed that the cells in 15VZnand 25VZngroups were M2-shaped polarized.The RT-q PCR and ELISA results showed that ZnO and TNT had synergistic effects on inhibiting pro-inflammatory genes,proteins expression and promoting pro-regenerative genes,proteins expression.The effects of 15VZngroup were the most obvious.3.Under the conditioned medium,15VZnand 25VZngroups showed good proliferation ability of osteoblasts and effectively reduced cell apoptosis.The results of SEM and CLSM showed that the cells in the 15VZngroup displayed the best spreading and adhesion state.15VZngroup had the greatest ability of the osteogenic related genes expression,the osteogenic related markers secretion and the mineralization ability.Therefore,appropriate diameter ZnO/TiO₂ nanotube composite coating could regulate the immune performance of macrophages,create an immune osteogenic microenvironment,enhance the osteogenic ability of osteoblasts,and promote the early osseointegration of implants.PartⅡ1.SEM result showed that the ZnO/black titanium dioxide nanotubes（ZnO/B-TNT/Ti）owned a regular surface structure.The diameter of the nanotube was about 100±6nm and the length of the nanotube was about 2±0.3μm.Compared with the black titanium dioxide nanotubes（B-TNT/Ti）,the ZnO/B-TNT/Ti group tube wall was significantly thicker,about 15±0.9nm,and the texture was uniform.XRD result showed that ZnO/B-TNT/Ti had the characteristic peaks of anatase and wurtzite.Raman spectroscopy result showed that there were oxygen defects on the surface of B-TNT/Ti group.EDS and XPS further confirmed the existence of oxygen deficiency in ZnO/B-TNT/Ti group and the successful binding of ZnO.Nanoindentation result showed that ZnO/B-TNT/Ti had good hardness and elastic modulus,which could meet the requirements of implant useage.The UV-Vis-NIR spectra showed that ZnO/B-TNT/Ti could absorb the light wavelength in the range of 390-850nm.The band gap width of ZnO/B-TNT/Ti was significantly reduced,about 2.04 e V.The photothermal properties showed that ZnO/B-TNT/Ti had controllable,stable and long-lasting photothermal properties.The ZnO release test showed that ZnO/B-TNT/Ti had a stable sustained ZnO release curve,had more than 28 days release potential.Meanwhile,ZnO/B-TNT/Ti had photothermal controlled release effect.2.ZnO/B-TNT/Ti group had excellent antibacterial ability under the synergistic effect of photothermal and zinc oxide heat release.The antibacterial rates of P.gingivalis,F.nucleatum and S.aureus could reach 99.5%,99.4%and 99.9%,respectively.ZnO could be coordinated with photothermal therapy to destroy bacterial cell membrane and bacterial mitochondria,killing bacteria in the end.3.CCK-8 results showed that photothermal effect had no significant influence on the long-term activity of cells.Flow cytometry,RT-q PCR,Western Blot and ELISA results showed that ZnO/B-TNT/Ti surface could regulate the polarization of macrophages towards M2-shaped,inhibit pro-inflammatory genes,proteins expression and promote pro-regenerative genes,proteins expression.In vitro immune osteogenic experiments showed that ZnO/B-TNT/Ti could promote osteoblast proliferation,osteogenic related genes,proteins expression,and mineralization.4.In vivo experiments further verified the antibacterial and immune osteogenesis properties.ZnO/B-TNT/Ti had stable and controllable photothermal properties with excellent antibacterial effects.HE,IHC staining and blood routine results showed that ZnO/B-TNT/Ti had strong immunomodulatory ability,which could reduce the secretion of inflammatory cytokines and regulate the pro-regenerative immune cells secretion at the early stage of implant implantation.Combined with micro-CT,HE and Masson staining of decalcified bone sections,the results showed that ZnO/B-TNT/Ti group had excellent immune osteogenesis properties.ConclusionEarly osseointegration and long-term antimicrobial treatment are keys to maintain implant stability.In this study,in order to improve the antibacterial performance and osteogenic activity of pure titanium implants,two kinds of titanium dioxide nanocoating structures loaded with zinc oxide were designed and successfully constructed.1.The composite coatings of different diameters ZnO/TiO₂ nanotubes were constructed on the surface of titanium,which had regular anatase nanotube morphology,good hydrophilicity and controllable ZnO release mode.An immune osteogenesis micro-environment model composed of biomaterials,macrophages and osteoblasts was established in vitro.By regulating the immunity of macrophages,the coating could create an immune osteogenic microenvironment and enhance the osteogenic ability of osteoblasts.2.By further modification,the photocontrol coating of zinc oxide/black titanium dioxide nanotubes were constructed,which retained its good immune osteogenesis function and was endowed with stronger antibacterial properties.The coating had long-term repeatable photothermal properties,and ZnO could cooperate with bacteria through temperature-sensitive controlled release.The antibacterial rate was more than99%.Meanwhile,zinc oxide/black titanium dioxide nanotubes photo-controlled coating had better immune osteogenesis properties.In vivo experiments also demonstrated the antimicrobial and immune osteogenesis activity of the coating.In this study,two types of zinc oxide loaded titanium dioxide nano coating structure showed good early immune osteogenesis properties.At the same time,the zinc oxide/black titanium dioxide nanotube photocontrol coating had good photocontrol antimicrobial properties.It had a great potential to maintain long-term stability of implant.This modification method provided a new idea and reference for promoting early implant osseointegration,as well as preventing and treating peri-implantitis.