Vx-11e Protects Against Titanium-particle-induced Osteolysis And Osteoclastogenesis By Supressing ERK Activity

Background and aims:Wear particle-induced osteolysis around the prosthesis is the most common long-term complication after total joint replacement surgery which often leads to aseptic loosening of the prosthesis.Despite the complex molecular and cellular interplays of the osteolytic bone destruction,wear particle-induced inflammation,enhanced osteoclast formation and activation,leading to excessive bone resorption remains the most important cause As such,pharmacological interventions with bisphosphonates,estrogens,denosumab,and/or teriparatide have been widely used after arthroplasty to inhibit the wear particle-induced osteoclast-mediated osteolysis.Although there are substantial advances in biomaterials and prosthesis design,loosening of the prosthesis caused by osteolysis remain the most difficult problem to overcome.Currently there is a lack of clinically effective measures to prevent or treat peri-prostheticosteolysis.Osteoclasts play key roles in the osteolytic process,thus identification of new agents that can inhibit the enhanced osteoclastic bone resorption is warranted.The pathophysiology of wear particle-induced osteolysis is complex but can be generalized as the local induction of inflammation near and around the prosthesis leading to release of pro-inflammatory signals that result in the recruitment of immune cells,lymphocytes and osteoclast precursors such as monocytes and macrophages.Subsequently,various pro-inflammatory cytokines are released by these cells into the tissues surrounding the loose prosthesis.These factors such as TNF-?,IL-6 and IL-17 stimulate osteoblasts to release receptor activator of nuclear factor-?B ligand(RANKL)which then promotes the osteoclastogenesis of BMMs and bone resorption leading to peri-prosthetic bone loss.Osteoclasts are therefore key players in mediating the osteolytic bone destruction induced by wear particles,and are prime targets for pharmacological intervention.Vx-11 e is a potent,selective,and orally bioavailable ERK1/2 inhibitor and ERK pathway plays an important role in osteoclast differentiation.In our study,we found that Vx-11 e has a significant effect on inhibiting osteoclastogenesis in vitro.Futhermore,we explored that Vx-11 e can protecte mice against titanium particle-induced calvarial osteolysis.Experimental methods:(1)In vitro experiment: 1)BMMs(bone marrow macrophage)were isolated from femurs and tibias of 4-week-old C57BL/6J mice by marrow flushing.BMMs were treated without or with serial dilutions of Vx-11 e in the stimulation of RANKL until multinucleated ‘pancake' shaped osteoclasts were formed in RANKL-treated only control groups.Cells were stained with TRAcP Staining Kit after fixed with 4% paraformaldehyde for 10 mins.The number of TRAcP-positive osteoclasts with 3 or more nuclei was quantified by ImageJ software.In order to rule out the toxic effect of Vx-11 e on cells,the proliferation experiment of BMMs was detected with CCK8 kit.2)To further show an impairment of precursor cell fusion,we examined the actin cytoskeleton and nuclei number in BMM-derived osteoclasts treated without or with Vx-11 e.For bone resorption assay,small pre-osteoclasts cultured with RANKL for at least 3 to 4 days were dissociated from culture plates and re-seeded onto hydroxyapatite-coated 96-well plates.3)Total RNA was extracted from the cells,Total RNA were extracted from BMM-derived osteoclasts cultured with RANKL in the absence or presence of serial dilutions of Vx-11 e and the expression of osteoclast specific genes were detected by Real-time PCR.Western blot was used to investgate the related signaling pathway of Vx-11 e inhibiting osteoclast differentiation induced by RANKL.(2)In vivo experiments: To evaluate the potential therapeutic efficacy of Vx-11 e in vivo,we generated a mouse calvarial osteolysis model.Result: In our study,we explored that Vx-11 e can inhibit the osteoclastogenesis induced by RANKL,the mRNA expression of osteoclastic-specific genes and bone resorption by attenuating the ERK activity in vitro.Furthermore,we explored that Vx-11 e can protecte mice against titanium particle-induced calvarial osteolysis in vivo.Conclusion: To sum up,Vx-11 e can inhibit osteoclast differentiation and bone resorption by attenuating the ERK activity,thereby protect the osteolysis induced by titanium particles.