Objective: To investigate the role of osteopontin (OPN) on the expressions of matrix metalloproteinase 13 (MMP-13) mRNA and protein in human knee osteoarthritic chondrocytes and to find the optimal time and concentration for OPN treatment.
Methods: Chondrocytes were isolated from articular cartilage tissues of patients with primary osteoarthritis (OA) and cultured using one step digestive treatment with collagenase type II. The chondrocytes were then identified using immunohistochemistry of collagen type II. The first generation of chondrocytes were stimulated with OPN at a concentration of 1 μg/mL for 0, 24, 48, and 72 hours, and with OPN at the concentrations of 0, 0.5, 1, 2, and 4 μg/mL for 48 hours. The levels of MMP-13 mRNA and protein expressions were measured with real-time fluorescent quantitative PCR and Western blot.
Results: The immunohistochemical staining showed that first generation of chondrocytes expressed collagen type II. Both MMP-13 mRNA and protein expression levels in OA chondrocytes increased significantly in the presence of OPN (1 μg/mL) and peaked at 48 hours after incubation, showing significant difference between different time points (P < 0.05). The MMP-13 mRNA expression level in OA chondrocytes at the OPN concentration of 1 μg/mL was significantly higher than those at the other concentrations (P < 0.05), and the MMP-13 protein expression level at the OPN concentration of 1 μg/mL was significantly higher than that at 0 μg/mL (P < 0.05). MMP-13 protein expression level at the OPN concentrations of 0.5, 2, and 4 μg/mL were significantly higher than that at 0 μg/mL (P < 0.05).
Conclusion: OPN induces up-regulation of MMP-13 mRNA and protein expressions in human knee osteoarthritic chondrocytes in time- and dose-dependent manners. The optimal time and concentration for OPN treatment are 48 hours and 1 μg/mL, respectively.