%0 Journal Article
%T An In Vitro Model of Cartilage Degradation by Chondrocytes in a Three-Dimensional Culture System
%A Katsuhito Nakashima1
%A Kensuke Nakatsuka
%A Kenichi Kurita
%A Kyoko Yamashita
%J International Journal of Biomedical Science
%D 2012
%I 
%X Objective: 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-¦Â on IL-1¦Â-induced cartilage destruction and serially evaluated changes in proteinases and their inhibitors in cartilage destruction. Methods: Chondrocytes were cultured on alginate beads with IL-1¦Â or TGF-¦Â 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-1¦Â 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-¦Â group compared with the values for the IL-1¦Â group. The proMMP-3 concentration in the culture medium of TGF-¦Â-treated cells was significantly decreased compared with that for the IL-1¦Â-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-¦Â 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.
%K chondrocytes
%K matrix
%K TIMPs
%K cell culture
%U http://www.ijbs.org/User/ContentFullTextFrame.aspx?VolumeNO=8&StartPage=249