Fig. 6

Long term culturing of primary neonatal calvarial osteoblasts (OBs) using a combination of frequent subculture and bFGF. a Long term growth curves of primary isolated OBs cultured by traditional method and two independent OBs-FS cell lines cultured by our new protocol in the presence of bFGF. b Phase contrast representative images (20x) of OBs (p5) and two independent OBs-FS cell lines (at 50 PDL, p30). c Osteoblast differentiation of OBs-FS, p30 measured by quantitative ALP activity at different time points during differentiation course of 15 days. Representative images of ALP staining are shown. d In vivo ectopic bone formation by mOBs versus 2 independent OBs-FS cell lines (at 50 PDL, p30). Cells were mixed with HA/TCP powder and implanted subcutaneously in immune-deficient mice for 2 months (n = 6 implants for each cell line). Images of H&E sections of implants show the formation of ectopic bone by implanted cells. Bone histomorphometric analysis of the percentage of total bone area per total area of the implants. Data are mean ± SD of three independent experiments, (*p < 0.05, **p < 0.005, compared to mBOs control). For panel c, columns with different letters at each time point indicate significant differences according to Duncan’s multiple range tests at p < 0.05