Effects Of Calcitonin On OA And Normal Rat Tissue-derived Chondrocyte-Osteoblast Interaction And Wnt Pathway Activity

Objectives To observe the expression levels of key molecules in Wnt signaling pathway and the expressions of relevant factors in chondrocytes and osteoblasts of each group by chondrocyte-osteoblast co-culture system and we explore the effects of calcitonin(CT)on chondrocyte-osteoblast interaction and activity of Wnt signaling pathway in addtion.Methods Twenty SPF female SD rats with 14 weeks old,10 were established with osteoarthritis(OA)model through anterior cruciate ligament excision(ACLT),10 were sham-operated,and SD was sacrificed by cervical dislocation 6 weeks after surgery.In rats,the cartilage and subchondral bone of both knee joints were isolated under sterile conditions,normal primary chondrocytes(NC),OA chondrocytes(OAC)and normal osteoblasts(NOB)were cultured by enzyme digestion.We identified chondrocytes phenotypes with alcian blue staining and immunofluorescence staining of collagen type ?(COLII);Osteoblasts phenotypes were identified by Alizarin Red S staining,alkaline phosphatase(ALP)staining and immunofluorescence staining of collagen type(COLI).?We select the second-generation cells with good growth status into 5 groups: NC + NOB group,NC + OAOB group,OAC + NOB group,NC + OAOB + CT group and OAC + NOB + CT group,3 days with Transwell co-culture system.The protein levels of COL?,MMP-13,Wnt3 a and ?-catenin expressions in chondrocytes and expressions of COL ?and osteocalcin(OCN)in osteoblasts were evaluated by Western blot analysis in each group.The m RNA expressions of COL2A1,MMP-13,Wnt3 a and ?-catenin in each group of chondrocytes and the m RNA expressions of COL1A1 and OCN in each group of osteoblasts were evaluated by Real-time PCR.Immunofluorescence staining was used to detect OCN expression of osteoblasts and ?-catenin expression of chondrocytes.Results 1 The extracted chondrocytes were stained with Alisin blue and COL ?immunofluorescence staining.2 Alizarin Red Staining,alkaline phosphatase staining and COLI immunofluorescence staining of osteoblasts were all positive.3 Western blot: 1)The expression of chondrocyte COL ?in NC + OAOB group and OAC + NOB group was significantly lower than that in NC + NOB group(P <0.05);MMP-13 was significantly higher in NC + OAOB group and OAC + NOB group than that in NC + NOB group(P <0.05),NC + OAOB + CT group was significantly lower than NC + OAOB group(P <0.05);Wnt3a and ?-catenin expression in NC + OAOB group and OAC + NOB group was significantly higher than NC + NOB group(P <0.05).2)The expression of osteoblast COL in NC + OAOB group was significantly lower than that in NC + NOB group(? P <0.05);the expression of OCN in OAC + NOB group was significantly higher than that in NC + NOB group(P <0.05).4 Real-time: PCR detection: 1)The expression of chondrocyte COL2A1 m RNA in NC + OAOB group and OAC + NOB group was significantly lower than that in NC + NOB group(P <0.05);MMP-13 m RNA in NC + OAOB group and OAC + NOB group Group expression was significantly higher than NC + NOB group(P <0.05),NC + OAOB + CT group was significantly lower than NC + OAOB group(P <0.05);The expression of chondrocyte Wnt3 a,?-catenin m RNA in NC + OAOB group and OAC + NOB group was significantly higher than that in the NC + NOB group(P <0.05).2)The expression of COL1A1 m RNA of osteoblasts in NC + OAOB group was significantly lower than that in NC + NOB group(P <0.05);the expression of OCN m RNA in OAC + NOB group was significantly higher than that in NC + NOB group(P <0.05).5 Immunofluorescence staining: the average fluorescence intensity of chondrocyte ?-catenin fluorescence in NC + OAOB group and OAC + NOB group was significantly higher than that in NC + NOB group(P <0.05),and the average fluorescence intensity of osteoblast OCN fluorescence in OAC + NOB group was significantly higher than that in NC + NOB group(P <0.05).Conclusions OA chondrocytes can promote the expression of OCN in normal osteoblasts;OA osteoblasts can reduce the expression of COL?,increase the expression of MMP-13 and activate canonical Wnt signaling pathway in normal chondrocytes,CT can protect matrix metabolism of cartilage by inhibiting the promotion of expressing of MMP-13 in normal chondrocyte by OA osteoblasts.Figure 17;Table 11;Reference 55...