Alumina Oxide And Bortezomib Prevented Inflammation And Osteolysis Activity Induced By Titanium Oxide Via Autophagy And NF-κB Signaling

Objective: As a result of rapid advances of the biomaterials science,rel-iable implants were considered to be most widely accepted operative pr-ocedure for treating damaged skeleton and diseased organs,such as vari-ous skeletal defects,tooth deficiency,joints replacements.Due to the ro-utine use of implants,wear particles or debris can be observed from su-rfaces in relative friction between implants and physiological environme-nt,which will result in peri-implant osteolysis and implant failure after the surgical.The pathophysiology is still unclear although researchers a-re attempting to study its potential mechanism to prevent osteolysis ind-uced by wear particles.Therefore,in order to search for more perfect biomaterial,more and more researches have been conducted on compos-ite materials.However,the biological interaction between different typ e of particles was little studied.In this study,we investigated the potent ial mechanism of osteolysis induced by titanium oxide particles(Ti)an d the biological interaction between titanium oxide and alumina oxide p articles(Al)in MG-63 cells and mouse calvarial osteolysis model.Methods: To mimic the main process of particles-induced osteogenic inhibition and inflammation in vivo,MG-63 cells(Human osteosarcoma cell line,purchased from Obio Technology)were exposed to particles.Different concentrations of titanium oxide particles were used.MTT assay and apoptosis assay were used to detecte cell viability and apoptosis,and then appropriate concentration of particles was used in cell experiment.The experiment was divided into 5 groups: control group,titanium oxide particles stimulation group,alumina oxide particles stimulation group,titanium oxide and alumina oxide particles co-stimulation group,titanium oxide and alumina oxide particles mixed bortezome(BTZ)group.Western Blot was used to detect the expression of NF-κB signaling related molecules at the protein level,for example IκBα,p-IκBα,p65,autophagy marker LC3 and inflammatory factor IL-6.At the mRNA level,expression levels of inflammatory factors IL-6、TNF-α、IL-1 and apoptosis gene casepase-3 were detect using real-time PCR.The expression of autophagy marker LC3 was also examined by cell immunofluorescence staining techniques.To confirm the results of previous experiments: Twenty-five C57BL/6J male mice,aged 6-8 weeks were used to established the calvarial osteolysis model.Mice were randomly assigned to five groups: sham operation group,titanium oxide particles group,alumina oxide particles group,titanium oxide and alumina oxide particles mixed group,titanium oxide and alumina oxide particles mixed group + BTZ.After 2 weeks,mice were sacrifice.The skulls were fixed and decalcified.Micro-CT reconstruction and data analysis、 HE staining and immunocytochemistry(IL-1、 IL-6、 TNF-α、 RANKL、 OPG and casepase-3)were performed.Results: 1.Apoptosis and necrosis assay showed that titanium oxide particles had markedly pro-apoptotic effect.However,alumina oxide particles had no distinct pro-apoptotic effect on MG-63 cells,which can effectively alleviate the apoptosis of MG-63 cells induced by titanium oxide particles.Based on these results,in order to minimize the effect of concentration and figure out the effect of titanium oxide particles on the toxicity of titanium oxide particles,the low concentration of materials(10 μg/mL titanium oxide,10μg/mL alumina oxide,5μg/ mL titanium oxide +5 μg/ mL alumina oxide)were used in the following experiments.2.Bortezomib(BTZ),which is one of the reversible proteasome inhibitors,has been shown to be a potent inhibitor of NF-κB.The concentration above 10 nM markedly suppressed cells proliferation(P<0.05).While the concentration up to 5nM induced apoptosis of the cells obviously.Based on results of above and previous experimental results,the non-cytotoxic concentration(BTZ 0.5nM)was used in subsequent experiments.3.Western blot and real-time PCR analysis showed a lower expression of IκBα and enhanced expression P-IκBα/IκBα in Ti treated group.Titanium oxide particles activated NF-κB signaling pathway,then overwhelming released pro-inflammatory cytokines and casepase-3.Meanwhile,the inflammation response(IL-1、TNF-α)and expression of apoptotic factor caspase-3 were attenuated the by co-cultured titanium oxide particles with alumina oxide or treated with BTZ 4.In calvarial osteolysis model,titanium oxide particles could obviously reduce the BV/TV(Bone Volume/ Total Volume)and induce osteolysis compared to the sham group.Alumina oxide and BTZ could reduce inflammation and osteolysis induced by titanium oxide particles.Conclusion: Collectively,alumina oxide could evoke autophagy and reduce the apoptosis,inflammation and osteolysis induced by titanium oxide particles,offering a basic data for implant design.What’s more,proteasome inhibitor BTZ could be a potential therapy for wear particle-induced inflammation and osteogenic activity via regulating the activity of NF-κB signaling pathway.