Effect And Mechanism Of AKBA On Osteogenesis Of Osteolysis Induced By Titanium Particles

Part ? acetyl-11-keto-?-boswellic acid enhances bone formation to reduce titanium particle-induced osteolysisObjective:To investigate the therapeutic effect of AKBA in titanium particle-induced osteolysis.Methods:8-week-old male C57BL/6 mice were used in this study and divided into four groups randomly.Control group(Sham group),the surgical operation was the same as that of the model group,and normal saline was injected under the periosteum.Model group,20mg titanium particle solution was injected under the periosteum.Low-concentration AK.BA treatment group(low-AKBA group),mice in the model group were treated with 5mg/kg/day AKBA.High-concentration AKBA treatment group(high-AKBA group),model group mice were treated with 20mg/kg/day AKBA.After two weeks of treatment,the mice were sacrificed,and their skulls were collected for Micro-CT scanning,Hematoxylin and eosin(H&E)staining,TRAP Stainning,Masson's trichrome staining,Calcein double labeling assay and immunohistoche:mical analysis.Results:Micro-CT showed that osteolysis decreased after AKBA treatment,and the bone mineral density(BMD)increased significantly compared with the model group,118.8±4.293 mg/mm3 in the Low-AKBA group and 122.1±5.684 mg/mm3 in the High-AKBA group,and 103.0±4.425mg/mm3 in the model group(p<0.05).After AKBA treatment the bone volume(BV)and bone volume/tissue volume(BV/TV)were significantly increased,and the number of pores in the ROI were decreased compared with the model group(p<0.05).The results of H&E staining showed that Ti particles implanted in the model group had an obvious inflammatory response,with a large number of inflammatory cells infiltration.Inflammatory cells in the periosteum of the Low-AKBA group and the High-AKBA group were reduced,with a mild inflammatory response.After AKBA treatment,bone thickness(BT)increased significantly to 0.258±0.020 mm(Low-AKBA group)and 0.281±0.031 mm(High-AKBA group),respectively compared with the model group(0.173±0.029 mm)(p<0.05).Eroded bone surface to bone surface(EBS/BS)was significantly decreased after AKBA treatment compared with the model group(p<0.05).TRAP staining results showed that a large number of stained positive osteoclasts were observed in the model group,while the number of positive cells decreased in the AKBA treatment group.The Osteoclast surface/bone surface(Oc.s/BS)and average number of osteoclasts to bone surface(N.oc/BS)were significantly reduced in the Low-AKBA group and the High-AKBA group compared with the model group(p<0.05).The Oc.s/BS of the Low-AKBA group and the High-AKBA group were 8.06%±0.37%and 6.49%±0.93%,respectively,showing significant differences compared with the Model group(20.63%±2.74%)(p<0.05).The mineral apposition rate(MAR)was significantly increased in the Low-AKBA group and the High-AKBA group compared with the model group(P<0.05).Masson's trichrome staining showed that no significant blue collagen fiber staining was observed in the Model group,while the formation of blue collagen fiber was observed after AKBA treatment,which showed dispersed or continuous presence and increased bone formation.Immunohistochemical staining results showed that the number of positive alkaline phosphatase(ALP),osteocalcin(OCN)and Osterix cells was relatively a few in the Model group,while the number of positive cells of the three markers increased significantly after AKBA treatment(p<0.05).The expression of bone formation markers increased in the AKBA treatment group,which increased bone formation.At the same time,immunohistochemical staining results showed that the number of positive cells for pSer9-GSK-3? and ?-catenin staining was significantly reduced in the Model group,while a large number of positive cells were observed in the AKBA group.The difference between the treatment group and the model group was significant(p<0.05).This indicates that AKBA can promote the formation of new bone in osteolysis induced by Ti particles by activating GSK-3 ?/?-catenin signaling pathway.Conclusion:AKBA can inhibit titanium particle-induced osteolysis.On the one hand,AKBA can reduce bone absorption by reducing the formation of osteoclasts,and on the other hand,it can also increase the osteogenesis of osteoblasts through the GSK-3?/?-catenin signaling pathway.Therefore,AKBA is expected to be a new drug therapy for the treatment of periprosthetic osteolysis.Part ? Effect of acetyl-11-keto-?-boswellic acid on osteoblast in titanium particle-induced osteolysisObjective:To observe the effect of AKBA on differentiation and function of MC3T3-E1 cells stimulated with titanium(Ti)particles in vitro.Methods:MC3T3-E1 cells was stimulated with Ti particles and then treated with different concentration of AKBA.The effect of Ti particles and AKBA on the viability of MC3T3-E1 cells were determined using a cell counting kit-8(CCK-8)assay.Then four groups were involved in the current study.Control group,MC3T3-E1 cells were cultured in osteogenic induction medium containing;model group(Ti group),MC3T3-E1 cells were in osteogenic induction medium supplied with 5?g/cm2 Ti particles;low-AKBA group:5?g/cm2 Ti particles+100nM AKBA;high-AKBA group:5?g/cm2 Ti particles+1000nM AKBA.Alkaline phosphatase staining was used to detect the number of ALP positive celles,Alizarin red S staining was used to observe the mineralization of osteoblasts.qRT-PCR was used to detect the mRNA expression of Runt-related transcription factor 2(Runx-2),Osterix,OCN,ALP,Osteopontin(OPN)and Axin-2.Western blot analysis was performed to analyze the protein expression levels of Runx-2,OCN,Osterix,ALP,pSer9-GSK-3?,total GSK-3?and Axin-2.Results:Ti(10?g/cm2)and AKBA(10?M)did not affect MC3T3-E1 cell viability.The results of ALP staining showed that no significant positive osteoblasts were observed in the Ti particles group,while more positive osteoblasts were observed in the Low-AKBA group and the High-AKBA group,showing a concentration dependence.Quantitative analysis showed that the number of positive cells in the AKBA treatment group increased significantly compared with that in the Ti particles group,showing a statistical difference(p<0.05).ALP staining showed that AKBA could promote osteoblast differentiation in vitro.The results of Alizarin red S staining showed that there were no obvious orange calcium nodules in the Ti group,while more calcium nodules were formed in the Low-AKBA group and the High-AKBA group,with scattered or patchy distribution.Quantitative analysis showed that the mineralization rates of osteocytes in the Low-AKBA group and the High-AKBA group were significantly higher than those in the Ti granule group,which were about 224.3%and 324.3%higher than those in the Ti particles group,with significant differences(p<0.05).The results of qRT-PCR showed that the expression of osteogenic markers in Ti particles group(Runx-2,Osterix,OCN,ALP,OPN)was significantly decreased,while in the Low-AKBA group and the High-AKBA group,the expression was significantly different from that in the Ti particles group(p<0.05).The results of Western blot and qRT-PCR were basically the same.Western blot and qRT-PCR analysis showed that Ti particles could inhibit the expression of ?-catenin and Axin-2.However,there was a significant increase after AKBA treatment.Western blot analysis of pSer9-GSK-3? and GSK-3? showed that after AKBA treatment,the titanium particle-induced pSer9-GSK-3?/total GSK-3? ratio was increased.These results showed that AKBA could regulate differentiation and maturation of osteoblasts through the GSK-3?/?-catenin signaling pathway under the intervention of Ti particles in vitro.Conclusion:AKBA can promote the differentiation and maturation of osteoblasts and promote bone formation under the intervention of Ti particles in vitro.AKBA's effect on osteoblasts may be achieved by activating the GSK-3?/?-catenin signaling pathway.Part ? Blocking GSK-3?/?-catenin signaling pathway inhibited the effect of AKBAObjective:To determine the effect of AKBA on differentiation and function of MC3T3-E1 cells stimulated with Ti particles in vitro,and to explore the role of GSK-3?/?-catenin signaling pathway in AKBA promoting osteogenesis.Methods:The effect of ICG-001 on the viability of MC3T3-E1 cells were determined using a cell counting kit-8(CCK-8)assay.ICG-001 was detected to inhibit osteoblast differentiation and mineralization by blocking the ?-catenin signaling pathway.Then four groups were involved in the study.Control group,MC3T3-E1 cells were cultured in osteogenic induction medium containing;ICG-001 group,MC3T3-E1 cells were in osteogenic induction medium supplied with 20?M ICG-001;Ti group,MC3T3-E1 cells were in osteogenic induction medium supplied with 5?g/cm2 Ti particles;Ti+ICG-001 group,5?g/cm2 Ti particles+20?M ICG-001.Secondly,the effect of ICG-001 blocking GSK-3?/?-catenin signaling pathway on AKBA promoting osteoblast differentiation was studied.Four groups were involved in the study.Control group,MC3T3-E1 cells were cultured in osteogenic induction medium containing;Ti group,MC3T3-E1 cells were in osteogenic induction medium supplied with 5?g/cm2 Ti particles;AKBA group,5?g/cm2 Ti particles+1000nM AKBA,AKBA+ICG-001 group,5?g/cm2 Ti particles+1000nM AKBA+20?M ICG-001.Alkaline phosphatase staining was used to detect the number of ALP positive celles,Alizarin red S staining was used to observe the mineralization of osteoblasts.Immunofluorescence analysis was used to detection the ?-catenin in cells.qRT-PCR and Western blot analysis were performed to detect the mRNA expression and protein espression levels of Axin-2,?-catenin,Runx-2,Osterix,OCN and ALP,respectively.Results:CCK-8 results showed that the viability of the cells was not affected when ICG-001 concentration was less than 50?M.In the results of ICG-001 blocking experiment,ALP staining showed that a large number of osteoblasts were formed in the control group,while the number of positive cells in the ICG-001 group and the Ti group decreased compared with the control group,and the number of positive cells in the Ti+ICG-001 group was the least,with significant differences(p<0.05).Azidine staining showed that a large number of red calcium nodules were formed in the control group,while fewer calcium nodules were formed in the Ti group and ICG-001 group,and the least in the Ti+ICG-001 group.Western blot and qRT-PCR results showed that the expression of bone markers of ICG-001 group,Ti group and Ti+ICG-001 group significantly decreased compared with the control group,and the expression of Axin-2 and ?-catenin in GSK-3?/?-catenin signaling pathway also significantly decreased compared with the control group,with significant difference(p<0.05).The results confirmed that ICG-001 could inhibit the differentiation,maturation and osteogenic ability of osteoblasts by blocking GSK-3?/?-catenin signaling pathway.Cell immunofluorescence after intervention of Ti particles treated with ICG-001 and AKBA showed that AKBA treatment promoted the activation of ?-catenin and increased nuclear transport,and these effects were suppressed in the presence of ICG-001.Western blot and qRT-PCR were used to detect the mRNA and protein expressions of experimental?-catenin and Axin-2 in AKBA+ICG-001 group,which were significantly lower than those in AKBA group(p<0.05).Meanwhile,the expressions of osteogenic markers Runx-2,Osterix,OCN and ALP were significantly lower than those in AKBA group,with statistical differences(p<0.05).The number of alkaline phosphatase staining positive cells in AKBA+ICG-001 group was consistent with the number of calcium nodules detected by azidine staining,compared with AKBA group were both significantly decreased(p<0.05).All results showed that AKBA could promote the differentiation,maturation and osteogenesis of osteoblasts stimulated with Ti particles in vitro,but these effects could be reversed by ICG-001.Conclusion:AKBA treatment can promote the differentiation,maturation and osteogenesis of osteoblasts stimulated with Ti particles in vitro through activation of GSK-3?/?-catenin signaling pathway,and can be reversed by ICG-001.