Ex Vivo Activity of Sam68+/+ and Sam68−/− Osteoblasts and Osteoclasts
Marrow stromal cells were isolated from the long bones of juvenile mice and maintained under conditions that promote osteoblast differentiation.
(A) Cultures were fixed in 4% paraformaldehyde after 6 or 18 days and stained in situ for ALP activity and with silver nitrate (von Kossa) to detect mineralized nodules. Sam68−/− cultures stained more intensely for ALP at early and late time points and produced significantly more mineralized nodules after 18 days. Asterisks represent p < 0.01.
(B) Primary osteoclasts were isolated from the crushed long bones of the same mice and plated on glass coverslips or on dentin slices to quantify numbers and activity, respectively. Osteoclasts were identified as cells with three or more nuclei that stained positive for TRAP activity (upper) and excavated pits in dentin slices, as demonstrated by SEM (lower, bar = 20 μm). No statistical differences were observed either in the number of TRAP-positive cells or in their resorptive activity.