Therapeutic Effect And Mechanism Of Strontium Ranelate On Wear Particle-induced Mouse Peri-prosthetic Osteolysis

Part?Strontium ranelate inhibits peri-prosthetic osteolysis induced by wear particles in miceObjective To investigate the therapeutic effect of titanium granules on osteoporosis around the prosthesis in mice,using a new type of drug,Strontium ranelate(SR),a new drug for the treatment of postmenopausal osteoporosis.Methods Forty-two female C57BL/6j mice were selected to establish a titanium nail model and intra-articular injection of titanium metal particles.They were randomly divided into three groups:control group(Control),low dose of strontium ranelate.The treatment group(625SR group)and the high-dose treatment group(1800SR group),14 mice per group.The Control group received only titanium nail injection and titanium granule injection,and 400?l isotonic saline was administered daily after operation.Low-SR and High-SR groups were administered with 625 or 1800mg kg^-1d^-1SR after model establishment.After 12 weeks of administration,the mice in each group were sacrificed in a CO₂ box.After treatment with bone tissue samples,micro-CT was used to detect the osteolysis around the prosthesis,the thickness and number of trabecular bones.The observation of hematoxylin and eosin staining,immunohistochemical staining was conducted to observe the expression levels of RANKL and OPG proteins in bone tissue.Results Scanning electron microscopy showed that the shape of titanium metal particles was irregular and the size was different.The average diameter of Ti particles was3.13?m±1.65?m,93%of the particles were less than 10?m in diameter,50%of the particles were less than 2.6?m in diameter,and 20%of the particles were smaller than 1.0?m in diameter.Micro-CT results showed that the osteoporosis around the titanium nail prosthesis was significantly reduced in the strontium ranelate treatment group.Compared with the control group(0.731±0.057mm³),the bone mass around the prosthesis in the SR625 group was significantly increased(0.808±0.035 mm³,P<0.05)and the bone mass of the SR1800 group(0.936±0.034 mm³,P<0.01)also increased significantly.In terms of bone volume fraction,compared with the control group(33.71%±2.35%),the SR1800 group had a significant increase in BV/TV(43.06%±2.48%,P<0.01)and the SR625 group(37.49%±3.68%)bone volume.There was no statistical difference in score growth compared with the control group.In contrast,compared with the control group(41.12±2.18 1/mm),a significant decrease in bone surface area density was observed in the SR625 group(34.49±1.75 1/mm,P<0.01)and in the SR1800group(31.83±0.77 1/mm,P<0.01)also decreased.In the trabecular bone generation,compared with the control group(0.108±0.014mm),the thickness of the trabecular bone in the SR625group significantly increased(0.190±0.033mm,P<0.01)and The SR1800 group also increased significantly(0.232±0.016mm,P<0.01).In addition,compared with the control group(1.761±0.097 1/mm),the number of trabecular bone in the SR625 group was also significantly increased(2.825±0.444 1/mm,P<0.01)and in the SR1800 group(3.341±0.460 1/mm,P<0.01)also observed a significant increase in the number of trabecular bone.However,there was no significant difference in the trabecular bone separation distance compared with the control group.H&E staining and immunohistochemical staining showed that strontium ranelate could alleviate the inflammatory reaction caused by wear particles around the prosthesis.At the same time,SR can increase the expression of OPG,down-regulate the expression of RANKL,and then increase the ratio of OPG/RANKL,the difference is statistically significant(p<0.05).Conclusion In a dose-dependent manner,strontium ranelate can effectively promote the new bone star city,inhibit bone resorption,reduce the inflammatory reaction caused by wear particles,and inhibit osteolysis around the prosthesis.In addition,strontium ranelate can down-regulate the ratio of RANKL/OPG,revealing that strontium ranelate may be a potential new means of preventing aseptic loosening of artificial joints.Part ? Strontium ranelate promotes the proliferation and differentiation of inflammatory osteoblasts induced by titanium particlesObjective To establish an in vitro aseptic loosing cell model to observe the effect of Ti particles stimulating the release of inflammatory cytokines from mononuclear macrophage cell line on the proliferation and differentiation of osteoblasts and the expression of RANKL and OPG.To explore the regulation of osteogenesis by strontium ranelate on wear particle-induced osteolysis.Methods The mouse mononuclear/macrophage cell line RAW264.7 was first stimulated with titanium metal particles that were autoclaved and endotoxin removed,and conditioned medium containing inflammatory cytokines was collected.The mouse pre-osteoblast cell line MC3T3-E1 was used as the main research object,and treated with 0.1,0.5,1.0m M strontium ranelate and/or conditioned medium containing inflammatory cytokines to establish aseptic loosening at the cellular level.The proliferation rate of MC3T3-E1 cells was detected by CCK-8 method.The activity of alkaline phosphatase was detected by p-nitrophenyl phosphate(p NPP).The differentiation of MC3T3-E1 cells was promoted by osteogenic induction medium,and quantified by alizarin red staining.Osteoblast mineralization ability,RT-PCR detection of m RNA expression levels of Rnux2,OCN,OPG,RANKL,and GAPDH was conducted.Immunofluorescence staining was carried out for double-staining observation of subcellular localization of RANKL and OPG to explore the effect of strontium ranelate and conditioned medium on the ratio of RANKL/OPG.ELISA was used to detect the content of inflammatory cytokines such as TNF-?,IL-1? and IL-6 in conditioned medium.Results The optimal stimulation condition of Ti particles was 1:100,24 hours.The conditioned medium was co-cultured with MC3T3-E1 cells.The ALP activity was detected,and the ALP activity of MC3T3-E1 cells was decreased by 45.8%(p<0.05).After the application of strontium ranelate,strontium ranelate can restore the reduction of ALP activity caused by conditioned medium,with 0.5 m M SR being the most effective(74.5%,p<0.05)followed by 1.0 m M SR(62.9%,p<0.05),and 0.1 m M had no effect on the recovery of ALP activity,and the difference was not statistically significant.The effect of intervention factors was measured in the early treatment of MC3T3-E1 cells with ceric acid and conditioned medium for 2 days.MC3T3-E1 cells were induced by osteogenic induction medium culture,and osteogenic mineralization ability of MC3T3-E1 cells was observed by alizarin red staining on the 14 th day.The results were as follows: the red stained area in the Ti CM group was significantly less,and It was observed that the cell density was lower than that of the Cont CM control group,but in the strontium ranelate treatment group,it was observed that the MC3T3-E1 cells had a large amount of red staining of mineralized nodules,suggesting that strontium ranelate can promote osteoblasts differentiation and mineralization of MC3T3-E1.RT-PCR was used to detect the relative m RNA expression of RANKL and OPG.Cellular immunofluorescence technique was used to localize the expression of these proteins.It can be seen that strontium ranelate can also regulate the expression of RANKL and OPG protein at the cellular level.Conclusion The conditioned medium from RAW264.7 cells stimulated by titanium particles can inhibit the activity and mineralization ability of mouse precursor osteoblast MC3T3-E1.0.5m M and 1.0m M strontium ranelate can relieve the effect of conditioned medium in the short-term and medium-term.The osteoblast activity decreased by the conditioned medium,and strontium ranelate's possible mechanism of action is the regulation of RANKL/OPG in MC3T3-E1.Part ? Strontium ranelate regulates WNT/?-catenin signaling pathway to treat aseptic loosening of artificial jointsObjective To investigate the role of strontium ranelate in the apoptosis of osteoblasts induced by titanium particles in an aseptic loosening cell model,and to explore the role of WNT/?-catenin signaling pathway in the regulation of osteoblast by strontium ranelate.Methods An aseptic loosing cell experimental model was established as in the second part.In this part,the mouse pre-osteoblast cell line MC3T3-E1 was used as a research object.The osteoblast double luciferase reporter gene system was constructed by transfecting a plasmid containing TOPFlash and p-TK Green Renilla gene fragment,and the sputum was observed in strontium ranelate.Transcriptional activity of the WNT/?-catenin signaling pathway under the intervention of conditioned medium.On this basis,recombinant mouse Wnt-3a protein and ICG-001,which binds to CBP protein and specifically antagonize WNT signaling pathway,were used to WNT/?-catenin signaling pathway for specific activation and blockade.The apoptosis of osteoblasts was detected by Annexin V-FITC/PI double staining flow cytometry.Western blotting is used to detect the proteins expression of RANKL,OPG,SOST and ?-actin in osteoblasts.Results Co-culture with conditioned medium and MC3T3-E1 cells can lead to apoptosis of osteoblasts.The number of apoptotic cells increased from 16.2% to 30%,but 0.1m M and 0.5m M strontium ranelate may inhibit this effect.The percentage of death was 25% and 19%,respectively.Furthermore,by constructing the dual fluorescein reporter gene system,we obtained that conditioned medium can reduce the transcriptional activity of WNT/?-catenin signaling pathway in osteoblasts,but the reduced transcriptional activity of WNT signaling pathway can be restored after the application of strontium ranelate.After blocking the WNT signaling pathway,this effect was attenuated.What's more,western blotting analysis showed that the expression of sclerostin in osteoblasts decreased after application of strontium ranelate.But the expression of ?-catenin was increased.Conclusion Strontium ranelate can suppress the conditioned media induced osteoblast apoptosis,and this process is likely to act through down regulate SOST indirectly activates WNT/?-catenin signaling pathway.