FIG. 4.

Effects of COL1A1 and/or HtrA1 siRNA on collagen synthesis. (A) The combination of hypoxia, BMP-2, COL1A1, and HtrA1 siRNAs promotes redifferentiation of chondrocyte at the protein level. HACs seeded onto collagen sponges were transfected with an INTERFERin-siRNA complex (5 nM NC siRNA, or 5 nM COL1A1 siRNA and/or 5 nM HtrA1 siRNA) as described in the “Materials and Methods” section. HACs were then cultured in 3% O₂ for 7 days and treated with or without BMP-2 (50 ng/mL). Protein extracts were analyzed by Western blotting for type I, II, and X collagens, and HtrA1 versus GAPDH. Representative blots are shown. (B) Immunostaining of type I collagen and type II collagen reveals effective redifferentiation of HACs with siRNAs. HACs were treated as described earlier but with 50 nM of each siRNA. Immunostaining was carried out to detect type I and type II collagens (red). The nuclei were counterstained with DAPI (blue). Images shown are representative of the experimental results. Magnification ×40. Scale bar: 40 μm. Color images available online at www.liebertpub.com/tec