Figure 3

CL82198 prevents and decelerates MLI-induced osteoarthritis progression. (A and B) CL82198 inhibits matrix metalloproteinase (MMP13) activity in vitro. (A) Addition of 5 ng of MMP13, 5 ng of MMP13 and 10 μg/mL of CL82198, or substrate control into a 96-well plate. MMP13 enzyme activity was determined using the SensoLyte 520 MMP13 Assay Kit, and showed that CL82198 could block > 90% MMP13 activity in vitro. (B) Primary sternal chondrocytes were isolated from three-day-old wild type pups. Cells were plated in 12-well plates and treated with 100 ng bone morphogenetic protein 2 (BMP-2), 100 ng of BMP-2 and 1 μM of CL82198, 100 ng of BMP-2 and 5 μM of CL82198, 100 ng of BMP-2 and 10 μM of CL82198, or vehicle control for 60 hours. Cell culture media was collected and MMP13 activity was determined and showed that CL82198 could inhibit > 90% MMP13 activity produced by BMP-2-treated primary chondrocytes. (C) Inhibition of MMP13 by CL82198 protects against meniscal-ligamentous injury (MLI)-induced osteoarthritis (OA). MLI and sham surgeries were performed on 10-week-old wild type mice. CL82198 was administered to wild type mice one day after surgery by intraperitoneal injection every other day for 12 weeks at doses of 1, 5, or 10 mg/kg body weight. Normal saline was used as a control. Knee joints were collected, and sectioned 12 weeks post-surgery, and Alcian blue/Hematoxylin/Organge G staining was performed. (D) Histological grading by two blinded observers confirmed decreased articular cartilage degradation in mice treated with 1, 5, and 10 mg/kg of CL82198 compared to saline control mice (*P < 0.001). (E) The articular cartilage area at proximal tibiae was quantified by tracing the Alcian blue-positive area in the proximal tibia. Compared to saline MLI, tibial cartilage area was increased 9, 15, and 43% after treatment with 1, 5, and 10 mg/kg of CL82198, respectively (*P < 0.05). (F) Total articular cartilage area was quantified by tracing the Alcian blue-positive area in both the proximal tibia and the distal femur. Total cartilage area was increased 21%, 19%, and 38% with treatment of 1, 5, and 10 mg/kg of CL82198, respectively (*P < 0.05). (G) Tibia cartilage thickness was quantified by tracing the Alcian blue-positive thickness in the center of the tibial plateau. Compared to saline treatment, tibia cartilage thickness was increased 11%, 37%, and 70% with treatment of 1, 5, and 10 mg/kg of CL82198, respectively (*P < 0.05). (H) Total thickness was quantified by tracing the Alcian blue-positive thickness in both the proximal tibia and the distal femur. Total cartilage thickness was increased 23%, 27%, and 50% after injection of 1, 5, or 10 mg/kg CL82198, respectively (*P < 0.05).