| Objective To observe the changes of subchondral bone remodeling in SD rats with knee osteoarthritis induced by DMM,and to explore the mechanism of the crosstalk between BMSCs and osteoclasts in regulating subchondral bone remodeling in rats with knee OA.MethodsPart 1:Changes of subchondral bone remodeling in SD rats with knee osteoarthritis induced by DMMForty-eight 10-week-old SD rats were randomly divided into the Sham and DMM groups,DMM method was performed to establish OA model.At 2,4,8 and 12 weeks postoperatively,cardiac blood was collected from 6 rats under anesthesia,and the knee joints were taken after euthanasia.Elisa assay was performed to detect levels of PINP and CTX-I in serum,micro-CT was performed to analyze the changes of micro structure in subchondral bone,HE staining and Safranin O-fast green staining were performed to evaluate the cartilage degeneration,TRAP staining was performed to count osteoclasts in subchondral bone.Another 12 10-week-old SD rats were randomly divided into the Sham and DMM groups,RT-qPCR was performed to detect the expression levels of Wnt genes related to subchondral bone remodeling.Part 2:Expression of Wnt5a in subchondral bone and differentiation of osteoclasts in early osteoarthritisFour weeks postoperatively,immunohistochemistry,Western blot and RT-qPCR were performed to detect the expression of Wnt5a and osteoclast related proteins and mRNA in subchondral bone,including RANKL,CXCL12,NFATc1 and TRAP.Primary BMMs and BMSCs were isolated,cultured and purified by whole bone marrow mechanical separation combined with differential adhesion method,and the TRAP staining was used to detect the osteoclast differentiation,TRITC-conjugated phalloidin staining was used to detect the F-actin rings in osteoclasts,the migration and adhesion of BMMs were detected by crystal violet staining,the purity of BMSCs was identified by immunofluorescence,flow cytometry,alizarin red staining and oil red O staining,Western blot was used to detect the expression level of Wnt5a from BMMs and BMSCs in each group.Part 3:Mechanism of BMSCs and Osteoclasts crosstalk through Wnt5a/NFκB/MAPK signaling and regulate subchondral bone remodeling in rats with knee osteoarthritisBMSCs expressing different levels of Wnt5a were constructed by lentivirus transfection technique and divided into NV,EV,Ad-Wnt5a and siRNA-Wnt5a groups,CCk-8 assay was performed to detect the effect of lentivirus transfection on BMSCs proliferation and determine the appropriate MOI,the transfection efficiency of lentivirus and the expression of osteoclast differentiation related proteins were detected by immunofluorescence,Western blot and RT-qPCR,the co-culture system of BMSCs and RAW264.7 was constructed by Transwell plate,and the crystal violet,TRAP,TRITC-conjugated phalloidin,toluidine blue staining,bone slices and SEM methods were performed to detect the migration,differentiation and bone resorption functions.ResultsPart 1:Changes of subchondral bone remodeling in SD rats with knee osteoarthritis induced by DMMHE staining showed that CC/TAC of Sham and DMM group were(32.15±1.84)%and(34.11±2.46)%2 weeks postoperatively,with no statistically significance(P=0.3569),while CC/TAC of Sham and DMM group were(32.87±2.15)%and(85.79±1.67)%respectively at 8 weeks postoperatively(P<0.001).Safranin O-fast green staining showed that The OARSI scores of the DMM group at 2,4,8 and 12 weeks postoperatively were 1.26±0.06,2.37±0.32,5.04±0.38 and 8.00±0.77,respectively,which were statistically significance from those of the Sham group(all P<0.01).Results of micro-CT demonstrated that compared with Sham group,subchondral bone mass in DMM group decreased gradually within 4 weeks postoperatively,BV/TV,Tb.Th,Tb.N and CD in DMM group were lower than those in Sham group,while Tb.Sp was higher than that in Sham group,with statistically significance(all P<0.01).At 8 weeks postoperatively,subchondral bone sclerosis occurred,BV/TV and Tb.Th increased gradually and were higher than Sham group(all P<0.01),Tb.N and CD continued to decrease(all P<0.05).The results of Elisa demonstrated that the CTX-I level in DMM group increased continuously from 2 weeks to 4 weeks postoperatively,and decreased gradually from 4 weeks to 12 weeks postoperatively,but was always higher than that in Sham group(all P<0.01).The PINP level in DMM group increased from 2 weeks to 4 weeks postoperatively,with no statistically significance compared with Sham group(all P>0.05),increased continuously from 4 weeks to 12 weeks postoperatively,and the PINP level at 12 weeks postoperatively was significantly higher than that in Sham group(P<0.001).TRAP staining showed that the number of osteoclasts in DMM group was significantly higher than that in Sham group at 2 and 4 weeks postoperatively(all P<0.01),while there was no statistically significance between Sham and DMM group at 8 and 12 weeks postoperatively(all P>0.05).The result of RT-qPCR demonstrated that the mRNA expression level of Wnt5a in subchondral bone of DMM group was significantly higher than that of Sham group 4 weeks postoperatively(P<0.001).Part 2:Expression of Wnt5a in subchondral bone and differentiation of osteoclasts in early osteoarthritisAt 4 weeks postoperatively,compared with Sham group,immunohistochemical staining showed that the proportion of Wnt5a+cells in subchondral bone of DMM group increased to(60±4.53)%,and the proportions of RANKL+,CXCL12+and NFATc1+cells were(72±2.26)%,(83.8±2.28)%and(77.6±2.07)%,respectively,which were significantly higher than those in Sham group(all P<0.001).Western blot and RTqPCR demonstrated that the expression level of Wnt5a in subchondral bone of DMM group was 3 times higher than that of Sham group,and the expression levels of osteoclast related protein and mRNA were significantly increased(all P<0.001),Wnt5a derived from BMSCs was significantly higher than BMMs(P<0.001).BMMs obtained via whole bone marrow mechanical separation combined with differential adhesion could be induced to differentiate into TRAP+and F-actin ring rich multi-nuclear osteoclasts.The primary BMSCs owned the highest proliferation ability at P3 generation,and the purity was above 90%.DMM promoted differentiation,migration and adhesion of BMMs at 4 weeks postoperatively without affecting BMMs proliferation(all P<0.001).Part 3:Mechanism of BMSCs and Osteoclasts crosstalk through Wnt5a/NFκB/MAPK signaling and regulate subchondral bone remodeling in rats with knee osteoarthritisThe results of CCK-8,immunofluorescence and flow cytometry demonstrated that the proliferation of BMSCs was not affected by lentivirus transfection when MOI was 100,and the percentage of successfully transfected cells was(91.53±3.72)%.Western blot demonstrated that there was no statistically significance in the expression level of Wnt5a derived from BMSCs in EV group compared with NC group(P=0.99).The expression level of Wnt5a in AD-Wnt5a group was significantly increased to 4 times of that in NC group,while that in siRNA-Wnt5a group was significantly decreased to 1/4 of that in NC group(all P<0.001),RT-qPCR results demonstrated that the mRNA expression level of Wnt5a in BMSCs of each group was consistent with the above results.Compared with NC group,Wnt5a derived from BMSCs in AD-Wnt5a group significantly increased the migration,differentiation and bone resorption capacity of RAW264.7 cells(all P<0.001),while adding AMD3100 or OPG to AD-Wnt5a group could reduce the migration,differentiation and bone resorption capacity of RAW264.7 cells to the level of siRNA-Wnt5a group(all P>0.05).Western blot results demonstrated that compared with BMSCs in EV group,the expression level of RANKL in AD-Wnt5a group was significantly inhibited by inhibitors of NF-κB,ERK and JNK,and the expression level of CXCL12 was significantly inhibited by inhibitors of NFκB,P38 and JNK inhibitors(all P<0.001).ConclusionIn the early stage of osteoarthritis,osteoclast mediated bone resorption in subchondral bone is enhanced,bone remodeling is unbalanced,and bone mass is reduced.BMSCs and osteoclasts crosstalk each other,and up-regulate the expression of CXCL12 and RANKL through Wnt5a/NF-κB/MAPK signal,promote the migration and differentiation of osteoclast precursor cells,and regulate subchondral bone remodeling in the early stage of osteoarthritis. |
