Figure 6
Echistatin improved the quality of matrix synthesized by pellet-cultured chondrocytes. Human articular chondrocytes were cultured in pellets in the media containing echistatin or CP4715, or in those containing none of them (Control). Pellets were cultured for 5 weeks in the presence or absence of echistatin or CP-4715. (a) Gross appearance (top row), photomicrographs of Alcian blue (second row) or Safranin O/fast green-stained sections of the pellets (third row) are shown together with those immunostained for type I collagen (bottom row). Scale bar = 1 mm (top row) or 200 μm (the other rows). (b) Expression of type II procollagen (COL2A1), aggrecan (ACAN), type I procollagen (COL1A1) and type III procollagen (COL3A1) in the pellets were evaluated by quantitative PCR. Results are shown by relative ratios against control pellets (Control; open bars). (c) DNA contents in the pellets. (d) Incorporation of [35S]sulfate into the pellets. Radioactivity was normalized by DNA content. For these experiments, echistatin and CP4715 were used at 1 μM and 100 nM, respectively. (b), (c), (d) Results are mean ± standard error of the mean of three to five pellets of four independent experiments. *P <0.05, **P <0.01 against control GADPH, glyceraldehyde 3-phosphate dehydrogenase.