Effects Of OPN On MMP13 Expression Of Human Osteoarthritic Chondrocyte In Vitro

Objective:Application OPN to intervent human osteoarthritis chondrocytes cultured in vitor to observe the characteristics of cell biology and detect the level of MMP13 in cell culture supernatan,further to discuss the possible mechanism that OPN involvement in the pathogenesis of human osteoarthritis.Methods:.1. A large number of high purity chondrocytes were harvested from specimens of human articular cartilage with type II collagenase digestion and identification the cells by the method of type II collagen immunohistochemistry, calcation cartilage cell viability by Trypan blue staining.2.Normal articular cartilage cells (A group) and OA articular chondrocytes (B group) were respectively cultured under the intervention of blank and three different concentrations of OPN:A1 group (normal cartilage cells as the control group), A2 group (normal Chondrocytes 2ng/ml OPN intervention group), A3 group (normal chondrocytes 4ng/ml OPN intervention group), A4 group (normal chondrocytes 6ng/ml OPN intervention group), B1 group (OA chondrocytes control group), B2 Group (OA chondrocytes 2ng/ml OPN intervention group), B3 group (OA chondrocytes 4ng/ml OPN intervention group), B4 group (OA chondrocytes 6ng/ml OPN intervention group), MTT assay for measuring the Situation of cell growth and survival;3. Application ELISA to detected the level of MMP13 in supernatant after cultured for 48 hours.Results:1.Human normal chondrocytes and OA chondrocytes were successfully obtained by the method of typeⅡcollagenase digestion. The survival rates of primary cultured normal human chondrocytes (A group) and OA chondrocytes (B group) A group survival rates respectively were: A group (95±1.0)%, B group (95±1.0)%, and no significant difference between the two groups(P> 0.05).2.The situation of Cells to stick wall observed under the microscope was no significant differences between groups after adding OPN. Cell survival rates respectively were:A1 group (98±1.0)%, A2 group (97±1.0)%, A3 Group (98±1.0)%, A4 group (97.5±1)%,B 1 group (98.7±0.6)%,B2 group(97.7±0.6)%,B3 group (98±1.0)%,B4 group (97.7±1.5)%,and there was no significant difference (P> 0.05);3.The results about levels of MMP13 in supernatant of cultured chondrocytes:levels of MMP13 in supernatant of normal chondrocytes was no significant difference among groups(P> 0.05). After the Intervention of OPN to OA chondrocytes, the expression of MMP13 there were significant difference between B2 group and B1 group, the same as B4 group and B1 group(P<0.05).Conclusion:1. There was no significant difference in survival rate between OA chondrocytes and normal chondrocytes cultured in vitro.2.OPN has no effect on survival rate of chondrocyte and normal chondrocytes cultured in vitro.3.OPN has no effect on MMP13 expression in normal articular chondrocytes,2ng/ml group concentration and 6ng/ml group concentration of OPN can enhance the expression of MMP13 in chondrocyte.