Figure 1

Effects of Endothelial Cell Conditioned Medium (EC-CM) on osteoblasts.

Primary endothelial cells isolated from 7 week-old CD1 mouse aorta were subjected to unit gravity (1g) and microgravity (0.008g) on microbeads for the indicated times, then EC-CM were collected and used to treat primary osteoblasts isolated from 7-day old CD1 mouse calvarias for 48 hours. (a) MTT proliferation assay. (b) Representative images of ALP cytochemical staining. (c) Densitometric quantification of ALP staining. The human endothelial cell line (EA.hy926 cells) was subjected for 96 hours to 1g and 0.008g on microbeads, then the EC-CM were collected and used to treat mouse primary osteoblasts for 48 hours. (d) MTT proliferation assay. (e) Representative images of ALP cytochemical staining. (f) Densitometric quantification of ALP staining. Primary endothelial cells isolated from 7-week old CD1 mouse aorta were subjected to 1g, 0.08g and 0.008g on microbeads for 96 hours, then EC-CM were collected and used to treat primary osteoblasts isolated from 7-day old CD1 mouse calvarias for 48 hours. (g) MTT proliferation assay. (h) Representative images of ALP cytochemical staining. (i) Densitometric quantification of ALP staining. (j) RT-PCR analysis of mRNA expression of the proliferation marker, Cyclin D1 and the osteoblast differentiation markers, Osterix, Runx2, Alp and Collagen type 1 chain-α1 (Coll-1), normalized versus Gapdh. Real time RT-PCR of (k) Rankl and (l) Opg mRNA expression in mouse primary osteoblasts treated with 1g-, 0.08g- and 0.008g-EC-CM. Total bone marrow cells were isolated from the long bones of 7-day old CD1 mice and cultured with 1g-, 0.08g- and 0.008 g-EC-CM. (m) Representative images of TRAcP staining and (n) quantification of TRAcP-positive multinucleated cells per field. Images are representative and data are the mean ± SD of 3 independent experiments (one way RM ANOVA). Bar = 100 μm. All gels have been run under the same experimental conditions.