Chondrocytes From Osteoarthritis Up-regulate The OPG Expression In Osteoblasts Via The Indian Hedgehog Signaling Pathway

Objective: Osteoarthritis is a complex,chronic,degenerative disease that affects multiple joints.At present,the existing treatment programs mainly try to delay the progression of patients with osteoarthritis,lack of effective treatment programs to stop the progression of the disease.In addition,the specific pathogenesis of Osteoarthritis is still in the stage of exploration and research,especially in the bone-cartilage interface,there are complex physiological and pathological changes.Studying the changes of bone-cartilage junction is helpful to deepen the understanding of cartilage degeneration and osteophyte formation during osteoarthritis.For this,we collected tissue samples from patients with osteoarthritis(OA),and osteoblasts and chondrocytes were extracted from the bone-cartilage junction to research the effect of human osteoarthritis(OA)chondrocytes with OA osteoblasts.Methods: Specimens of the abandoned tibial plateau from osteoarthritis patients who had total knee arthroplasty were collected.Then,the chondrocytes were extracted by enzymatic hydrolysis.The subchondral bone was isolated by ophthalmic scissors from the same position outside the tibial plateau,and osteoblasts were extracted by enzymatic pre-digestion + bone fragment method.The chondrocytes were identified by toluidine blue staining and immunofluorescence of Col-2.In the meanwhile,the osteoblasts were identified by ALP staining and immunofluorescence of Col-1.Before co-culture of the two cells,chondrocytes were embedded in sodium alginate beads to maintain their phenotype.Inhibitors of the IHH signaling pathway(cyclopamine,10 n M)and activators(Purmorphamine,10 n M)were used in the co-culture system.After each treatment,osteoblasts in each group were cultured together for 48 h,and the m RNA expressions of Gli1,OPG,Runx2,RANKL,PTHr P,ALPL,and OCN in osteoblasts were detected by q RT-PCR.Also,the protein expressions of Gli1,OPG and RANKL in osteoblasts of each group was detected by Western blot.Results: After collecting samples,human OA chondrocytes and human OA osteoblasts were successfully extracted and identified,and the cells grew well in the process of culture without contamination.In addition,when cultured with human OA chondrocytes embedded in sodium alginate beads,the m RNA expression levels of Gli1,OPG and Runx2 in osteoblasts were increased significantly,and the RNA expression levels of PTHRP were decreased(P<0.05).After adding IHH signaling pathens inhibitors,the expression of the IHH signaling path line target gene Gli1 decreased significantly at m RNA and protein levels(P<0.05).Moreover,the expression of Gli1 increased significantly at m RNA and protein levels(P<0.05)after IHH signal pathentic activators were added(P <0.05).The expression of Gli1 was significantly increased at both m RNA and protein levels(P <0.05)with the addition of IHH signaling pathway activato.In the meantime,OPG showed the same tendency as GLI1 in the experiment.OPG expression on m RNA and protein levels were significantly decreased(P <0.05)with the addition of IHH signaling pathway inhibitor,and after adding IHH signaling pathway activator,OPG were significantly increased(P <0.05).In order to evaluate the effect of IHH signaling pathway on the OPG/RANKL ratio,we further measured the expression of RANKL in each treatment group.Although the expression of RANKL did not show a completely opposite trend with OPG,the trend of OPG/RANKL was the same as that of OPG.Conclusion: These results suggest that human OA chondrocytes can promote the expression of osteoblast-related genes such as OPG and Runx2.And OA chondrocytes regulate the expression of OPG in osteoblasts through the IHH signaling pathway,thereby affecting the OPG/RANKL ratio.