The Effect Of Titanium Dioxide Particles On Biological Behaviour And Inflammatory Response Of Human Gingival Fibroblast

Background:Peri-implantitis was an important factor that affected the survival rate of implant restoration.The pathogenesis and influence factors of the disease had been wildly studied.Various risk indicators including systematic diseases and local factors such as smoking habits,plaque microbes,exess cement had been reported.Although titanium material have a well biocomatibility,there could be corrosion wear under low dissolved oxygen conditions as in the oral cavity because of the mechanical abrasion chemical corrosion and so on.Titanium wera debris have been detected in the hard and soft tissues around the implant.Titanium wear particle was one type of titanium wear debris and was ususally detected in the tissues from peri-implantitis.Many studies had reported that the titanium wear particlles could enhance the expression of inflammatory cytokines in types of cells,which could promote tissue destruction.However,the influence of titanium wear debris on the soft tissues around the implant were rarely reported both in China and abroad.Peri-implantitis always begin with peri-implant mucositis.The soft tissues around implant were different from that around nature teeth.At the interface between the peri-implant mucosa and dental implants,there was a biological seal formed by soft tissues which played an important role in the stability of the peri-implant tissues.Once the biological seal were destroyed,the osseointegration under the soft tissues would be easy to disturbed.Human gingival fibroblast?HGF?cells were the main cell of the soft tissues.It not only provide mechanical barrier to protect tissue structure around implant from invading,but also participate in immune repsonse to stimulation.It played a vital role in soft tissue health.These important biological function was based on the adhesion,migration,proliferation of HGF cells on the surface of titanium implant.Therefore,it is necessary to clarify the influence of titanium wear particles on the HGF biological behaviour.Moreover,in oral cavity,lipopolysaccharides always presence around the dental implant.It was reported that titanium wear particles promote endotoxin tolerance in macrophages.However,titanium nanoparticles enchance the inflammatory response in many cells induced by LPS.Therefore,the possible immune response in HGF cells induced by titanium wear particles with or without LPS was worthy to study.As titanium dioxide particles were used to modified the titanium implant surface,we used titanium dioxide nanoparticles?TiO₂ NPs?and titanium dioxide microparticles?TiO₂ MPs?in our research.Demonstrating the influence of TiO₂ NPs and TiO₂ MPs on inflammatory response in HGF cells induced by LPS in vitro,could reveal the role of TiO₂ NPs and TiO₂ MPs in the pathogenesis or progress of pei-implantitis and peri-implant mucositis.Furthermore,we were interested in the anti-inflammatory effect of caffeic acid phenethyl ester?CAPE?in LPS induced HGF cells.As a NF-?B inhibitor,the mechanism of its inhibition was still unclear.In our study CAPE was to used to inhibite the inflammatory reponse in HGF cells induced by TiO₂ NPs and TiO₂ MPs.CAPE was demonstraed to have enormous potential in the novel therapeutic strategies for periodontal and peri-implant diseases.Objective:The aim of this stuy was to clarify the effect of TiO₂ NPs and TiO₂ MPs on the adhension,migration,proliferation of HGF cells with or without LPS and demonstrate the possible mechanism.The interaction between titanium dioxide particles and LPS was discussed.Furthermore,the anti-inflammatory effect and mechanism of CAPE on HGF cell induced by LPS or titanium dioxide particles was detected to find a possible treatment.Part I The influence of TiO₂ NPs and TiO₂ MPs on HGF cell cytoskeletal organization,adhesion,migration and proliferation1.Materials and methods.?1?HGF cells were isolated and cultured with the adherent method.Cell morphology was observed under inverted microscope and anti-vimentin protein expression was detected by immumofluorescence method.?2?TiO₂ NPs and TiO₂ MPs were purchased from Sigma corporation.The particles was reiterative washed with acid to remove adherent endotoxins.Dopamine was used to modify the particles surface and rhodamine B was used to labelled the dopamine.RhB-labelled TiO₂ NPs and TiO₂ MPs were achieved.?3?HGF cells were co-cultured with 0.1mg/ml TiO₂ NPs or Ti02 MPs for 24h.Laser scanning confocal microscope?LSCM?was used to detected the internalization of TiO₂ NPs or TiO₂ MPs in HGF cells as well as F-actin,vinculin,vimentin expression.?4?HGF cells were co-cultured with different concentration of TiO₂ NPs or TiO₂ MPs for 24h.CCK-8 assay was employed to detect the cell viability at 1 day,3day,7day.?5?HGF cells were plated into 6-well plates.Cells were co-cultured with TiO₂ NPs or TiO₂ MPs with or without LPS until confluence.A scratch?wound?was created on each confluent monolayer to detect the migration and proliferation of HGF cells.Wound area was observed at 12h and 24h under inverted microscope and images were captured.?6?HGF cells were co-cultured with TiO₂ NPs or TiO₂ MPs with or without LPS for 8h.Then cells were seeded in the upper chamber.Cells were cultured in gradient FBS concentration DMEM for 24h.The number of HGF cell migrated through the mebrane was detected under inverted microscope and photographed.?7?FAK,p-FAK,paxillin,p-paxillin protein expression in HGF cells were measured with western blot.2.Results?1?Primary HGF cells were adherent cells with spindle or star shape.They grew into a radiate or gyrate pattern.And anti-vimentin expression was postive.?2?RhB-labelled TiO₂ NPs and RhB-labelled TiO₂ MPs were observed in red under LSCM.?3?The internalization of TiO₂ NPs and TiO₂ MPs in HGF cells was observed under LSCM.F-actin,vinculin and vimentin were differently disrupted and rearranged after exposured to TiO₂ NPs and TiO₂ MPs.Furtermore these negative effect was enhanced by P.g-LPS.?4?TiO₂ NPs at higher concentration than 0.5mg/ml and TiO₂ MPs at higher than 0.25mg/ml concentration inhibited cell viability.TiO₂ MPs,LPS,TiO₂NPs+ LPS and TiO₂ MPs+LPS groups at 3day and 7day significantly inhibited HGF cell proliferation.?5?TiO₂ NPs did not affect the migration ability of HGF cells.However TiO₂ MPs inhibited the migration ability of HGF cells.And LPS enhanced this inhibitation.?6?The number of HGF cells migrate through the membrane in TiO₂ MPs group was lower than in TiO₂NPs group.inhibited significantly.The number of cells migrated in LPS exposure groups was lower than that in non-LPS groups.?7?The expression of p-FAK and p-paxillin of HGF cells in TiO₂ MPs group and LPS exposure groups was significantly lower.It indicated that the adhension ability of HGF cells was remarkably inhibited.3.Conclusions?1?TiO₂ NPs and TiO₂ MPs at high concentration remarkably inhibited the HGF cells viabitily and proliferation.?2?TiO₂ NPs and TiO₂ MPs can be uptaken by HGF cells and affected the cytoskeletal organization.Subsquently inhibited HGF cells adhension and migration.However,the effect of TiO₂ NPs was less than that of TiO₂ MPs.?3?P.g-LPS significantly enhanced the inhibitory effect of TiO₂ NPs and TiO₂ MPs on HGF cells biologcial behaviour.It indicated that P.g-LPS reduced the resistance of HGF cells to titanium wear particles.Part ? The expression of inflammatory cytokines in HGF cells induced by TiO₂ NPs and TiO₂ MPs with or without LPS1.Materials and methods.?1?HGF cells were pretreated with LPS for 8h,and then were co-cultured with TiO₂ NPs or TiO₂ MPs for another 24h.Cells were collected.ICP-MS was employed to measure the titanium content.?1?HGF cells were pretreated with 1mg/ml TiO₂ NPs or TiO₂ MPs for 12h,then were co-cultured with P.g-LPS for 24h.Supernatant was collected to measure the secretion of TNF-a,IL-6 and IL-1? by ELISA.Total cell mRNA was extracted and RT-PCR was used to measure relative mRNA expression.?3?HGF cells were pretreated with 1mg/ml 1 TiO₂ NPs or TiO₂ MPs for 12h,then cells were co-cultured with P.g-LPS for 30min.Total cell protein was extracted and western blot was used to detected TLR4 and p65 protein expression.2.Results?1?P.g-LPS remarkably increased the titanium content in HGF cells.?2?TiO₂ NPs significantly enhanced the secretion level of TNF-a in HGF cells,and the secretion level of TNF-? and IL-1? was remarkably upregulated by TiO₂ MPs in HGF cells.Furthermore,TiO₂ NPs increased the expression level of TNF-??IL-6?IL-1? in HGF cells induced by P.g-LPS and TiO₂ MPs decreased the expression level of TNF-??IL-6?IL-1? in HGF cells induced by P.g-LPS.?3?The protein expression of TLR4 and p-p65 were increased in HGF cells stimulated by TiO₂ NPs only but significantly less than LPS group.However,after pretreated with TiO₂ NPs,TLR4 and p-p65 expression remarkably enhanced in HGF cells induced by P.g-LPS.?4?The protein expression of TLR4 and p-p65 were significantly increased in HGF cells stimulated by TiO₂ MPs only.However,after pretreated with TiO₂ MPs,TLR4 and p-p65 expression remarkably decreaded in HGF cells induced by P.g-LPS compared with LPS group.3.Conclusions?1?P.g-LPS increased the internalization of TiO₂ NPs and TiO₂ MPs in HGF cells.?2?TiO₂ NPs increased secretion level of TNF-a in HGF cells induced by LPS via TLR4 mediated NF-?B signaling pathway.And significantly enhance the expression of TNF-a,IL-6 and IL-1? stimulated by P.g-LPS.?3?TiO₂ MPs increased secretion level of TNF-a and IL-1? in HGF cells via TLR4 mediated NF-?B signaling pathway.However,Ti02 MPs promoted LPS tolerance in HGF cell.TiO₂ MPs decreased TNF-a,IL-6 and IL-1? expression in HGF cells induced by P.g-LPS.Part III the inhibitory effect and mechanism of CAPE on inflammatory cytokines secretion in HGF cell1.Materials and methods.?1?CCK-8 assay was performed to evalutated different concentration of CAPE on HGF cell viability.?2?ELISA test was used to measure the secretion levels of IL-6 and IL-8 in HGF cells induced by P.g-LPS.Western blot was employed to detected COX-2 and iNOS protein expression.?3?The activation of TLR4,NF-?B,PI3K/AKT and MAPK pathway as well as thenuclear translocation of p65 in HGF cell induced by P.g-LPS was detected by western blot.Immunofluorescence was used to detected the expression of p65 in nuclearposition.DNA-binding activity of NF-?B in nuclear extracts was assessed by usingthe enzyme-linked immunosorbent assay-based NF-?B p65 transcription factor assaykits.?4?HGF cells were pretreated with CAPAE for 1h,then co-cultured with TiO₂ NPs and TiO₂ MPs for 24h.Subsequently,ICP-MS was used to measure the titanium concetration in HGF cells.?5?The effect of CAPE on the TiO₂ NPs and TiO₂ MPs induced TNF-?,IL-6 and IL-1? expression in HGF cells was measured by ELISA.And the mRNA were detected by RT-PCR.?6?TLR4 and p-p65 expression in HGF cells induced by TiO₂ NPs and TiO₂ MPs was measured by western blot.2.Results?1?CAPE could not affect HGF cells viability at the concentration lower than 40?M.?2?CAPE significantly inhibited the upregulation of IL-6,IL-8,COX-2 and iNOS on LPS-stimulated HGF cells in a dose-dependent manner.?3?CAPE significantly inhibited the activation of TLR4,NF-?B and PI3K/AKT signaling pathway in HGF cells induced by P.g-LPS but not MAPK pathway.the nuclear translocation of p65 was blocked by CAPE,and p65 DNA bingding was also reduced by CAPE.?4?CAPE didnot affect the internalization of TiO₂ NPs and TiO₂ MPs in HGF cells.?5?CAPE significantly inhibited the expression of TNF-a,IL-6 and IL-1? in HGF cells induced by TiO₂ NPs and TiO₂ MPs.?6?CAPE significantly inhibited the activation of TLR4,NF-?B in HGF cells induced by TiO₂ NPs and TiO₂ MPs.3.Conclusions?1?CAPE attenuated the production of IL-6,IL-8,iNOS,and COX-2 in LPS-induced HGFs in a dose-dependent manner.?2?The anti-inflammatory mechanisms of CAPE may be based on the inhibited activation of the TLR4-mediated NF-?B and PI3K/AKT pathways,except in the MAPK pathway.?3?CAPE inhibited the expression of inflammatory cytokines in HGF cells induced by TiO₂ NPs and TiO₂ MPs via TLR4/NF-?B signaling pathway.However,CAPE did not affect the internalization of TiO₂ NPs and TiO₂ MPs in HGF cells.?4?The results indicated that CAPE have enormous potential in the novel therapeutic strategies for periodontal and peri-implant diseases.