Figure 4.

(A) Comparison of cartilage-specific gene expression in Day 42 static and dynamic CNC cultures analyzed by RT-PCR. Both cultures were supplemented with TGF-β1 and IGF-I. Total RNA was analyzed for collagen types II (Col II), IX (Col IX), and X (Col X), cartilage oligomeric matrix protein (COMP), and aggrecan (AGN), with GAPDH as a control. Collagen types IX and X and aggrecan gene expressions are up-regulated in dynamic CNC cultures, whereas expression of other genes are similar in both culture conditions. S, static culture; D, dynamic culture. (B-G) Histological comparison of CNC based engineered cartilage grown in static and dynamic cultures after 42 days. (B and C) H&E staining; (D-G) alcian blue staining. Spindle or elongated cells are found in static cultures (B), whereas more cells with round or oval, chondrocytic morphology are seen in dynamic cultures (C). Less intense alcian blue staining, indicating a lower content of proteoglycan, is seen in static cultures (D) than dynamic cultures (E). Low magnification view of alcian blue stained CNCs showed less proteoglycan accumulation at the periphery of statically grown CNCs (F) than in similar regions of dynamically grown CNCs (G). (H-O) Immunolocalization of cartilage-specific matrix proteins in static and dynamic CNCs cultured for 42 days. Both types of CNCs showed positive staining for collagen types II (H and I) and IX (J and K), aggrecan (AGN) (L and M), and cartilage proteoglycan link protein (LP) (N and O), but more intense staining was shown in dynamically grown CNCs. Bar = 25 μm (B-E, and H-O) or 3 mm (F, G).