Fig. 6. Involvement of a BMP activity during cartilage particle development in the presence of TGFß or TGFß and PDGF-BB. (A) The sorted flk-1^–PDGFR⁺ cells were subjected to the pellet culture in the presence of TGFß3+1 µg/ml IgG-Fc (a) and TGFß3+1 µg/ml noggin-Fc (b). The sorted flk-1⁺PDGFR^– cells were also subjected to the pellet culture in the presence of TGFß3+1 µg/ml IgG-Fc (c), TGFß3+0.1 µg/ml noggin-Fc (d) and TGFß3+1 µg/ml noggin-Fc (e). Particles formed in 15 day (a,b) or 18 day (c-e) cultures were formalin-fixed, paraffin-embedded, sectioned and stained with Toluidine blue. These results are representative of three (c-e) to five (a,b) independent experiments. Note that the addition of 1 µg/ml IgG-Fc did not have any effect on the growth and maturation of the cartilage-containing particle (a,c). The addition of noggin-Fc resulted in a small aggregate and sometimes left part of the initial cell pellet unincorporated into the round cell aggregate, which occasionally attached to the particle throughout the culture (top and bottom cell debris in e). (B) The sorted flk-1^–PDGFR⁺ cells were subjected to the pellet culture with TGFß3 and 50 ng/ml PDGF-BB in the presence of 1 µg/ml IgG-Fc (a,c) and 1 µg/ml noggin-Fc (b,d). On day 18, particles were formalin-fixed, paraffin-embedded, sectioned and stained with Toluidine blue. A contiguous section of each particle was also immunostained with 2B1.5 (c,d). Brown areas indicate the accumulation of COL2. The immunostaining with mouse IgG showed negative results (data not shown). Note that the addition of 1 µg/ml noggin-Fc resulted in a small aggregate (b,d). These results are representative of two independent experiments.

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