An in vitro model of cartilage degradation by chondrocytes in a three-dimensional culture system

摘要

Using the alginate bead three-dimensional culturing method, which is considered to be advantageous for the in vitro study of chondrocytes, we confirmed earlier reports concerning the inhibitory effect of TGF-β on IL-1β-induced cartilage destruction and serially evaluated changes in proteinases and their inhibitors in cartilage destruction. Methods: Chondrocytes were cultured on alginate beads with IL-1β or TGF-β alone or both. The glycosaminoglycan (GAG) concentration in the culture medium was determined by use of the DMMB assay; and the levels of TIMP-1,-2 and proMMP-3 were measured with their respective sandwich EIAs. Sections of the beads were prepared and stained with toluidine blue or anti-TIMP-1-2,-3 antibodies. The numbers of chondrocytes negative for pericellular proteoglycan staining and TIMP-positive chondrocytes were counted, and positive staining for TIMP-3 in the extracellular matrix was examined. RT-PCR was performed to evaluate the gene expression of TIMP-1,-2,-3, and MMP-3. Results: The number of TIMP-1(+)chondrocytes, TIMP-1 concentration in the culture medium, and TIMP-1-gene expression all increased maximally as early as 6 hours after IL-1β stimulation, and then gradually decreased. However, the number of cells immunopositive for TIMP-3 increased somewhat later. GAG and proMMP-3 concentrations in the culture medium increased gradually with time. The number of TIMP-3(+)chondrocytes and positive staining for TIMP-3 in the extracellular matrix significantly increased in the TGF-β group compared with the values for the IL-1β group. The proMMP-3 concentration in the culture medium of TGF-β-treated cells was significantly decreased compared with that for the IL-1β-treated ones at all times examined. Discussion: We suggest that TIMP-1 plays a primary role in the prevention of articular cartilage destruction in its early stage but that TIMP-3 gradually takes over this role. Also, TGF-β was shown to regulate these TIMPs and act as a suppressor of articular cartilage destruction. These results suggest that TIMP-1 and TIMP-3 are closely involved in preventing the progression of joint disorders such as OA.