Fig. 7. Effects of BMP4 on cartilage particle development. (A) The sorted flk-1^–PDGFR⁺ cells were subjected to the pellet culture in the presence of TGFß3 (a) or 50 ng/ml BMP4 (b). (B) In a separate experiment, the same cells were cultured with TGFß3 (a), TGFß3+5 ng/ml BMP4 (b), TGFß3+20 ng/ml BMP4 (c) or TGFß3+50 ng/ml BMP4 (d). The sorted flk-1⁺PDGFR^– cells were also subjected to the pellet culture in the presence of TGFß3 (e) or TGFß3+50 ng/ml BMP4 (f). Particles formed during 16 days (Aa-b, Be-f) or 17 days (Ba-d) of culture were formalin-fixed, paraffin-embedded, sectioned and stained with Toluidine blue. These results are representative of three independent experiments. (C) Late removal of TGFß3 and replacement with BMP4 during the in vitro development of cartilage particles. The sorted flk-1^–PDGFR⁺ cells were individually subjected to the pellet culture in the presence of TGFß3. On day 10, the TGFß3 was removed (c), or was substituted with 1 µg/ml noggin-Fc (b) or 50 ng/ml BMP4 (d) in some cultures. On day 18, particles were formalin-fixed, paraffin-embedded, sectioned and stained with Toluidine blue. These results are representative of five independent experiments.

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