FIG. 3.

System B: Coculture of chondrocytes with MSCs predifferentiated for n days after treatment with mitomycin C. Alizarin red staining of MSCs harvested before coculture period demonstrates that even after treatment with mitomycin C, MSCs are capable of producing matrix characteristic of osteogenic differentiation in as little as 4 days. MSCs are shown after mitomycin C treatment and 4 days of growth in MSC medium (A) or OM (B). (C) GAG accumulation by chondrocytes grown for 1 week of coculture in COMBO medium with MSCs predifferentiated for n days in OM after inactivation with mitomycin C. GAG accumulation was significantly increased (P < 0.05) when chondrocytes were cocultured with MSCs predifferentiated for up to 8 days; however, an increase was not observed when MSCs were predifferentiated for 10 or more days. (D–I) Alizarin red staining of MSCs treated with mitomycin C and predifferentiated for n days in OM before culture for 1 week in COMBO medium alone (D, E, F) or in cell culture insert coculture with chondrocytes (G, H, I). n = 0 (D, G), 4 (E, H), or 14 days (F, I). Alizarin red staining demonstrates that undifferentiated MSCs produce increased calcified matrix in coculture, but after longer periods of predifferentiation, this increase is no longer observed. Color images available online at www.liebertonline.com/scd.