title: An in vitro model of cartilage degradation by chondrocytes in a three-dimensional culture system.
abstract: 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-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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Oral and Maxillofacial Surgery, School of Dentistry, Aichi-Gakuin University, Nagoya, Japan;
authors.forename: Katsuhito
authors.initials: K
authors.lastname: Nakashima
authors.forename: Kensuke
authors.initials: K
authors.lastname: Nakatsuka
authors.forename: Kyoko
authors.initials: K
authors.lastname: Yamashita
authors.forename: Kurita
authors.initials: K
authors.lastname: Kenichi
authors.forename: Hayakawa
authors.initials: H
authors.lastname: Taro
keywords: TIMPs; cell culture; chondrocytes; matrix