Fig 2. Pellet cultures of expanded chondrocytes.

Expanded chondrocytes from P1 to P6 were subjected to pellet cultures without or with TGF-β1. (A) Pellet cultures were analyzed histologically for the presence of glycosaminoglycans (Safranin-O) and COL2 and COL1 proteins (immunohistochemistry [IHC]). Cells from P1, and to a lesser extent from P2, formed cartilage-like tissue autonomously. Addition of TGF-β1 (10 ng/ml) induced formation of cartilage-like tissue in cells from P1 to P6, although a decrease in the staining intensities for glycosaminoglycans and COL2 was observed in cells at P4 and later time points. Cultures treated with TGF-β1 stained more intense for COL1 compared to cultures without TGF-β1. (B) The decreased expression of transcripts coding for cartilage-specific matrix proteins (ACAN and COL2) in pellet cultures from late passages in the absence of TGF-β1 was compensated for by the addition of TGF-β1. TGF-β1 treatment of pellet cultures resulted in an up-regulation of COL10A1 in P1–P4 and increased COL1A1 expression in all passages. Data are shown as mean with the 95% confidence interval from one experiment with three separately analyzed cultures per group. The experiment was repeated with P1–P4 cells and P1/P3 cells from two other animals, respectively, yielding similar results. Levels of transcripts of cultures without TGF-β1 were compared to the levels in P1 without exogenous TGF-β1 (ANOVA). TGF-β1 treated cultures from each passage were compared to the corresponding culture from the same passage without exogenous TGF-β1 (Student’s t-test). Exact P values are indicated by numbers or * P < .001. n.d.: not detectable.