title: Histomorphometric Evaluation of Cartilage Degradation using Rabbit Articular Chondrocytes Cultured in Alginate Beads - Effects of Hyaluronan.
abstract: OBJECTIVE
  A 3-dimensional alginate bead culturing method using rabbit articular chondrocytes was studied for the screening of the effectiveness of drugs for articular diseases.
  
  
  METHODS
  The beads cultured with IL-1β, TGF-β, and Hyaluronan (HA) were evaluated histochemically with Alecian blue and immunohistochemically with CS-56 antibody. Chondrocytes in alginate beads were arbitrarily classified into four groups: 1) chodrocyte surrounded with cell-associated matrix (CAM) in which proteoglycan (PG) was positively stained (PG-possitive chondrocyte); 2) chondrocyte with PG-negative CAM; 3) PG-positive CAM alone, and 4) PG-negative CAM alone. Total sulfated GAG concentrations in the culture media were quantitated by dimethylmethylene blue (DMMB) assay. ProMMP-3, TIMP-1 and -2 concentrations in the culture media were determined by sandwich enzyme immunoassays.
  
  
  RESULTS
  Significant increase of PG-nagative cells were immunohistochemically found by IL-1β stimulation. The pretreatment with TGF-β almost fully suppressed those increase of PG-negative cells by IL-1β. Both GAG and proMMP-3 concentrations in the culture media were significantly increased after IL-1β stimulation. There were no significant differences in both TIMP-1 and TIMP-2 concentrations in the culture media with or without IL-1β stimulation. 800-kDa HA reduced significantly the number of PG-negative cells and proMMP-3 concentration in the culture media, but showed no effects on the concentrations of both TIMPs.
  
  
  CONCLUSIONS
  Because this 3-dimensional chondrocyte culture in alginate beads is close to in vivo conditions, this method can be used for evaluation of the effectiveness of novel drugs for articular diseases.
authors.affiliation: Department of Oral and Maxillofacial Surgery, School of Dentistry, Aichi-Gakuin Unuverity, Japan;
authors.forename: K
authors.initials: K
authors.lastname: Nakatsuka
authors.forename: K
authors.initials: K
authors.lastname: Kurita
authors.forename: Taro
authors.initials: T
authors.lastname: Hayakawa
authors.forename: Katsuhito
authors.initials: K
authors.lastname: Nakashima
authors.forename: Kyoko
authors.initials: K
authors.lastname: Yamashita
authors.forename: Takeshi
authors.initials: T
authors.lastname: Hoshino
authors.forename: Kyosuke
authors.initials: K
authors.lastname: Miyazaki
keywords: alginate beads; chondrocyte; matrix metalloproteinases; proteoglycan