| Periimplantitis is one of the common complications of implant denture.Currently,the main therapies of periimplantitis include mechanical debridement,local medication,laser therapy,surgical treatment and photodynamic therapy(PDT).Recently,PDT emerged as the therapies of periimplantitis for triggering bacterial inactivation by employing the intrinsic photocatalytical activity of photosensitizer,with low damage,wide antibacterial spectrum and anti-drug resistance.However,photosensitive reaction caused by photosensitizer induced irritation blister,swallowwing photosensitizer and other complications.How to prevent periimplantitis without introducing exogenous media has become a hot research topic.TiO2 nanotube(TNT)has strong photocatalytic oxidation capacity which claims an excellent application in detecting organic pollutants.The surface activity of TNT treated by ultraviolet radiation was enhanced the hydroxylions(OH-),H2O2 and oxygen free radical groups with strong oxidation capacity were formed.Considering that TNT arrays are potential drug carriers,antibacterialagentscan be loaded.Whereas,due to the rapid release of antibiotics from TNT,it's hard to maintain the long-term antibacterial ability,and meanwhile the use of antibiotics could increase the resistance to antibiotics.Another effective and facial strategy to trigger bacterial inactivation is to employ the intrinsic photocatalytical activity of these coating on Ti implant material.However,the drawback of TNT was its wide band gap(~ 3.2 e V for anatase crystalline),therefore electron–hole pair generation can only be achieved triggered under UV-light irradiation(? < 388 nm).Many biomolecules could suffer from denaturation or decomposition when exposed to UV irradiation for long time.Even more importantly,exposure in UV light is harmful to eyes and increases the risk of suffering skin cancer.To date,lots of approaches have been reported to expand the light harvest efficiency of TiO2 in visible-light region.For example,improved photocatalytic activities in visible light can be achieved by doping with N,C ortransition metal elements.Another solution is the decoration of noble metal onto TiO2 surface.The surface plasmon resonance(SPR)feature of noble metal nanostructures,i.e.,Au nanostructure has been attracted increasing attention in the scientific community as it can harvest and convert light into chemical energy via plasmonic excitation.The generated “hot spots” can subsequently transfer to nearby substrates.Furthermore,as co-catalyst,these metal nanoparticles can facilitate electron–hole(e––h+)separation and to promote interfacial electron transfer process that in turn increases the surface reactivity,which has been confirmed as a promising antibacterial therapeutic method.Objective:TiO2 nanotubes were prepared in the system of fluorinated glycerine and loaded with Au nanoparticles by the method of UV photocatalytic.To optimize the ideal preparation process,the physical and chemical properties were explored.To confirm the compatibility of TNT-Au,the osteogenic activity of MC3T3-E1 cells and the proliferation,secretion and migration performance of the Human gingival fibroblasts(HGFs)were studied.To verify the antibiaterial activity of TNT-Au,the effects of TNT-Au on the main pathogens of periimplantitis and animal model under visible light catalysis were observed.Method:1.TiO2 nanotubes were prepared in the system of fluorinated glycerine and loaded with Au nanoparticles by the method of UV photocatalytic.The surface morphology of TiO2 nanotubes were observed by SEM,the crystal structure was observed by X-ray diffractometer(XRD),the composition of element and and relative content were analyzed by X-ray photoelectron spectroscopy(XPS),the contact angle was measured by contact angle tester and the absorption spectrum was measured by UV-Visible Spectroscopy.2.Human gingival fibroblasts(HGFs)were inoculated to the surface of the samples.Cell proliferation was detected by flow cytometry and CCK-8.DAPI staining was used to detect cell adhesion.Cells migration was detected by scratch test.Confocal laser microscopy was used to observe the expression of integrin ?1 and Vinculin.Real-time quantitative polymerase chain reaction(RT-q PCR)was used to detect the expression of the genes(FAK,integrin ?2,integrin ?1,FN1,VCL and Col-1)involved in HGFs proliferation and differentiation.Western blot was used to detect the expression of protein(FN1,P-FAK,FAK)involved in HGFs proliferation and differentiation.3.The MC3T3-E1 cells were inoculated to the surface of five groups of materials.CCK-8 was used to detect the proliferation of cells.Alkaline phosphatase(ALP)activity was evaluated by thymolphthalein release from thymolphthalein monophosphate.The collagen secretion of cells was detected by staining with Sirius red,the matrix mineralization was observed by staining with alizarin red,RT-q PCR was used to detect the expression of osteogenic related genes and Western blot was used to detect the expression of the protein involved in Wnt pathway.4.P.gingival(ATCC33277)and F.nucleate(ATCC25586)were inoculated to the surface of samples.LED light illuminated samples for 30 s and 60 s.Then,the samples were dehydrated progressively and sprayed gold to observe the morphology of bacterias by SEM.The antimicrobial rates were calculated.The samples were implanted in the anterior maxillary region of the rabbit.The experimental group was set periimplanttits model by wrapping silk thread for 30 days and LED light was applied for 30 and 60 s respectively.Then the silk thread was removed and the animals were sacrificed 7 days later.The specimens were fixed with 4% paraformaldehyde solution,stained with HE,and observed by microscope.Results:1.TNT array was orderly and uniform,without impurities,collapse,fragmentation,and stripping.The average diameter of TNT was about 100 nm.The characteristic diffraction peaks of anatase and Au were observed by XRD.XPS showed that TNT-Au contained Ti,O and Au elements.The content of Au in each group was 2.75%(TNT-Au1),5.52%(TNT-Au2)and 10.83%(TNT-Au3),respectively.The contact Angle of each group was TNT-Au1 < TNT-Au2 < TNT < TNT-Au3.After 1,2 and 3 modification cycles,Uv-vis spectra showed that the peak absorption of TNT-Au was 530-600 nm.2.TiO2 nanotubes loading with Au promoted the early adhesion,proliferation and migration of HGFs(P<0.05).In this process,integrin?1 and Volcolin distributed more widely in TNT-Au group than Ti and TNT group.The results of RT-PCR showed that the expression of gene FAK,integrin?2,integrins?1,Col-I,FN1 and VCL in TNT–Au group were higher than Ti and TNT group.The results of Wester-blot showed that the level p-FAK in TNT-Au2 and TNT-Au3 group was significantly higher than other groups(P<0.05).3.The results of CCK8 and ALP showed that the proliferation and activity of ALP were time dependent and the results in TNT-Au were higher than that in Ti and TNT groups(P<0.05).Meanwhile,the secretion collagen-I and of the extracellular matrix were higher in the TNT-Au groups than other groups(P<0.05).The results showed that the secretion of collagen-I in TNT-Au3 group was the highest(P <0.05).The expression of genes and proteins related to osteogenesis,Dishevelled and ?-catein,were significantly higher in the TNT-Au groups than the other two(P<0.05).4.SEM showed that the bodies of bacterias were smooth and intact on the surface of Ti and TNT samples photocatalyzed.While,the surfaces of bacterias were showed pits,shrinkage,collapse,transparent in some parts or traces of broken bacteria only in TNT-Au group.Statistical results of antibacterial rates showed that the antibacterial rates of TNT-Au for was bove 90% Statistical results of antibacterial rates showed that the antibacterial rates of TNT-Au for P.gingivalis and F.nucleatum in TNT-Au groups were above 90% and 85%,respectively.5.In vivo experiments,the results showed that the periimplantitis model was successfully constructed after 30 days.In the blank group,the gingiva was red,swollen and bleeding.Some sites showed pyorrhea and large number of lymphocyte infiltration was observed in HE staining.The control group was rinsed wirh H2O2,the gingiva redness,swelling lymphocyte infiltration were reduced.In the experimental group 1(illumination 30s),the gingival redness and swelling was slightly reduced.There was a large amount of lymphocyte infiltration in the Ti and TNT groups.The gingival redness and swelling was significantly reduced in TNT-Au groups,and only a small amount of lymphocyte infiltration was observed.In the experimental group 2(illumination 60s),the gingival redness and swelling in Ti and TNT group were decreased slightly while lymphocyte infiltration was observed.In TNT-Au groups,the gingival color was basically normal without swelling and the tissue structure tended to be normal.In conclusion,TNT-Au groups showed excellent biocompatibility for promoting the biological behavior expression of HGFs and no adverse effect on osteoblasts,and showed.TNT-Au can effectively kill the pathogenic bacteria of periimplantitis and ease the inflammatory response of periimplantitis in vivo with illumination. |
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