Ex vivo assessment of osteoblast and osteoclast activity.
Bone marrow was flushed from the tibia and femora of juvenile 4-week-old Sam68+/+ and Sam68−/− mice to obtain stromal cells that were maintained in differentiation medium for 6 or 18 days as described [63]. Cultures were stained in situ for ALP activity and with von Kossa stain to detect mineralized nodules. Total RNA was harvested from parallel cultures for RT-PCR analysis of osteoblast-related gene expression over time as described previously [63].
Osteoclasts were isolated from the crushed long bones of juvenile Sam68+/+ and Sam68−/− mice and used for quantitative studies as described [64]. Briefly, cells were plated on glass coverslips or on dentin slices in 24-well cluster plates for assessment of cell number and pit number, respectively. Cover slips were immersed in 4% paraformaldehyde and stained for TRAP activity after 2 h. Dentin slices were left for 28 h before removing the cells with 1 M ammonium hydroxide and air drying before coating with sputter Au-Pd and examination with scanning electron microscopy (McGill Electron Microscopy Centre). Light microscope images were captured using a Leica DMR microscope equipped with a Retiga 1300 camera. Quantitative analyses were performed using Adobe Photoshop and the data presented as the mean ± SD of two or three independent experiments. Statistical comparisons were made using the Student's t test.