| AbstractObjective: To access whether calcitonin exerts a positive action on the ACLT rabbit articular cartilage and subchondral bone of rabbit's knees in vivo;To study protective effects of calcitonin on chondrocytes in vitro. Methods: Thirty 3 months Japanese white rabbits were divided randomly into three groups: Sham group, ACLT+CT group, ACLT+NS group. Rabbits in ACLT+CT and ACLT+NS groups received ACLT on the right knee. Every rabbit was placed in a cage (60cm×60cm×80cm) without any further immobilization. ACLT+CT group received a daily subcutaneous injection of salmon calcitonin 5IU/ kg for 8 weeks. In contrast, ACLT+NS group received NS at the same dose. All rabbits were sacrificed at 8 weeks post surgery. Samples of cartilage were harvested from right femoral condyles. The samples were observed for the macro-pathologic changes and stained with HE and graded by Mankin's scale. Cartilage sections from right femoral condyle were immunostained for type II collagen and MMP-13. The right femora were removed and the subchondral of right tibial plateau was cutted and stained for the bone histomorphometric measuring. The chondrocytes obtained from articular surface of five 3 months normal Japanese white rabbits after being sacrificed were cultured in Dulbeccos Modified Eagles Medium (DMEM) containing 10% fetal calf serum and were subcultured in two 24-well microplates at passage 3. Every 24-well microplate was divided into 3 groups, IL-1βgroup: cells were cultured in 10% DMEM containing 10ng/ml IL-1βfor 5 days, IL-1β+CT group: cells were cultured in 10% DMEM containing 10ng/ml IL-1βfor 2 days, and then were exposed to medium containing 50ng/ml salmon calcitonin for another 3 days. normal group: cells were cultured in 10% DMEM for 5 days. The cells in one 24-well microplate were stained immunohistochemically for collagen type II and MMP-13, and mRNA expressions of collagen type II and MMP-13 were examined by realtime RT-PCR in another one.Results: (1) The sham group, ACLT+CT group and ACLT+NS group exhibited that OA were mainly developing in the femoral condyles cartilage. Surfaces of knee joints of the sham group were all smoothy and glossy, displayed blue and transparent. Knee joints of ACLT +NS group were all swelled, their surfaces were uneven or anabrotic with displaying dim and bloomy and there was light yellow and cloudy synovial in knee joints . Their tibial plateaus appeared hypertrophic, osteophyte emerged on the margin. These appearance was accorded with OA. In ACLT+CT group, articular surfaces were uneven and dim, but tibial plateau hypertrophy had not been seen, the cartilage degradation were less than that of ACLT +NS group. So the Mankin score of the ACLT+ NS group were higher than that of the sham group and the ACLT+CT group.(2) Compared with the ACLT+NS group, the rabbits in sham group and ACLT treated with calcitonin markedly increased the trabecular bone relative volume (BV/TV),trabecular bone thickness (Tb.Th)and markedly decreased trabecular bone separation (Tb.Sp).(3) Immunohistochemistry staining showed expression of type II collagen in ACLT+ NS group was lower than that of ACLT+ CT group and sham group. The expression of MMP-13 in ACLT+ CT group was lower than that in ACLT+NS group and sham group.(4) From realtime RT-PCR, salmon calcitonin caused a significant increase of levels of mRNA of type II collagen compared to IL-1βgroup. And the normal group expressed higher type II collagen mRNA than that in the IL-1βgroup. The normal group and the IL-1β+CT group expressed lower MMP-13 mRNA than that in IL-1βgroup.Conclusion: (1)ACLT rabbits showed articular cartilage degeneration, as well as bone loss and microarchitechture deterioration of subchondral bone. (2) Calcitonin probably protects articular cartilage by increasing the expression levels of type II collagen and decreasing the expression levels of MMP-13.(3) Calcitonin could inhibit the development of osteoarthritis from modulating subchondral bone metablism and thereby preventing bone loss and improving microarchitechture of subchondral bone.
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