Fig. 8. Hyaline cartilage formation and mineralization. (A) Late removal of TGFß3 and PDGF-BB, and replacement with BMP4 during the in vitro development of cartilage particles. The sorted flk-1^–PDGFR⁺ cells were subjected to the pellet culture in the presence of TGFß3 and 50 ng/ml PDGF-BB. On day 10, the TGFß3and PDGF-BB were removed (d-f) or were substituted with 50 ng/ml BMP4 (g-i) in some cultures. On day 18, particles were fixed with Gendre's fluid, paraffin-embedded, sectioned and stained with Toluidine blue (a,d,g) or immunostained with AB765P (b,e,h) or 2B1.5 (c,f,i). The results are representative of five independent experiments. (B) The sorted flk-1^–PDGFR⁺ cells (a-c) and flk-1⁺PDGFR^– cells (d-f) were pellet-cultured with TGFß3 and 50 ng/ml PDGF-BB for 10 days, with 50 ng/ml BMP4 for 6 days and then in the hypertrophic differentiation medium for 5 days. Particles were formalin-fixed, paraffin-embedded, sectioned and stained individually with von Kossa (a,d), with X53 (b,e) and with 2B1.5 (c,f). The results are representative of three independent experiments. Black staining indicates mineral deposition (Ba,d), and brown areas indicate the accumulation of collagens. Immunostaining with control IgGs showed negative results (data not shown).