Fig. 6. Bone matrix formation by MMCs. MMCs were cultured for 6 days in pmDM supplemented with either BMP2 alone (A) or both BMP2 and 10 mM ß-glycerophosphate (B-H). Transparent nodules were formed by the cells cultured with BMP2 alone (A). By contrast, many opaque nodules were detected in the presence of ß-glycerophosphate (B). Nodules formed by multiclonal (C) and monoclonal (D) MMCs stained with Alizarin Red S. Incubation of fixed cells with 200 mM EGTA for 5 minutes resulted in the removal of the opaque material; the same field of a culture before and after EGTA treatment is shown in E and F, respectively. (G,H) Osteocalcin was detected in the opaque matrix by immunofluorescence analysis with antibodies to osteocalcin and FITC-conjugated secondary antibodies. (H) The same field is shown in G. Scale bars: 100 µm. Images in A and B were obtained by phase-contrast microscopy, whereas those in C-G were obtained by bright-field microscopy.