Study On The Role And Mechanism Of The Local Bone Renin-angiotensin System In Wear-particle-induced Periprosthetic Osteolysis

Objective: To explore the role and mechanism of the local bone renin-angiotensin system(RAS)in wear-particle-induced periprosthetic osteolysis(PPOL).A mouse calvarial osteolysis model was established to investigate the role and mechanism of local bone RAS in titanium(Ti)-particle-induced osteolysis,so as to further contribute to understanding the molecular mechanism of the pathogenic process of wear-particle-induced PPOL,and such understanding may shed light on the potential preventive or therapeutic treatment for PPOL.Methods: In the present study,the expressions of RAS components in the interface membrane tissue of patients with PPOL undergoing revised total hip arthoplasty and the synovial tissue of patients with femoral neck fracture undergoing primary total hip arthroplasty were detected.The histological morphology and RAS components expressions in the interface membrane and synovium tissue were detected by HE staining and immunohistochemistry.The expressions of RAS components in the interface membrane and synovium were determined by western blot and RT-q PCR.60 healthy8-week-old male C57/BL6 mice were randomly assigned to three groups(n=20): sham PBS control(sham group),Ti particles with PBS(Ti group),and Ti particles with perindopril(Ti+Peri group).Ti-particle-induced mouse calvarial osteolysis model was established.During the next 2 weeks,mice in the Ti + Peri group were injected intraperitoneally with perindopril(20 mg/kg/day),while those in the sham and Ti groups were injected with the same dose of PBS.Three mice from each group were selected randomly and injected intraperitoneally with calcein(20 mg/kg)and alizarin red(40mg/kg)10 and 3 days before sacrifice,respectively,to identify newly formed bone.The expressions of RAS components in the mouse calvarial tissue were examined by immunohistochemistry,western blot and RT-q PCR.The bone mass parameters and total porosity of each sample were obtained using Micro-CT.HE staining was used to detect bone destruction in mouse calvarial tissue.The number of osteoclasts in mouse calvarial tissue was determined by tartrate-resistant acid phosphatase(TRAP)staining.The m RNA levels of osteoclast and osteoblast-related genes were detected using RT-q PCR.The expressions of m RNA and proteins of RANKL/RANK/TRAF6 signaling pathway were measured by RT-q PCR and western blot.Results: HE staining revealed that the inflammatory response in the interface membrane was significantly higher than that in the synovial tissue.Immunohistochemistry,western blot and RT-q PCR showed that RAS components,including AT1R,AT2R and ACE,were expressed in the interface membrane and synovial tissue,and the expressions of AT1R,AT2R and ACE were up-regulated in the interface membrane(p<0.05).In animal experiments,the expressions of AT1R,AT2R and ACE in the Ti group were significantly increased compared with these in the sham group(p<0.05).Micro-CT results showed that the bone destruction was significantly increased in the Ti group as compared with in the sham group,which was ameliorated by perindopril(p<0.05).The results of quantitative analysis indicated significantly reduced bone matrix density(BMD)and bone volume to tissue volume ratio(BV/TV)and increased percentage of total porosity in the Ti group as compared with in the sham group,and such effects were markedly mitigated by the treatment with perindopril(p<0.05).H&E staining revealed extensive erosion of the calvarial surface in the Ti group,which was remarkably reduced by the treatment with perindopril.TRAP staining revealed an increased area of bone erosion with increased number of osteoclasts on the calvarial surface in the Ti group as compared with in the other two groups.Quantitative analysis showed that the number of TRAP-positive cells and the values of osteoclast surface to bone surface(OC.s/BS)and eroded surface to bone surface(ES/BS)in the Ti group were significantly greater than those in the other two groups(p<0.05).Calcein/alizarin red fluorescent signals were obviously observed in the three groups.And quantitative analysis exhibited that the mineral apposition rate(MAR),bone formation rate per bone surface(BFR/BS)and mineralizing surface to bone surface(MS/BS)values in the Ti group were significantly lower than those in the sham group,which were significantly elevated in the Ti+Peri group(p<0.05).RT-q PCR results showed that the levels of osteoclast-related genes were significantly increased and the osteoblast-related genes were significantly reduced in the Ti group as compared with in the other two groups(p<0.05).Western blot and RT-q PCR results indicated that the expressions of RANKL signaling pathway related proteins and m RNA in the Ti group were significantly higher than these in the other two groups(p<0.05).Conclusion: The RAS components AT1R,AT2R and ACE are expressed in synovial and interface membrane tissues,and are up-expressed in the interface membrane tissues of PPOL patients.Local bone RAS promotes the Ti-particle-induced mouse calvarial osteolysis via RANKL signaling pathway,which suggests that the local bone RAS plays an important role in wear-particle-induced PPOL.