Changes in osteoblast differentiation and function of 4 week-old Lmna^−/− mice.

(A and B) Formation of colony forming units-osteoblasts (CFU-OB) in ex-vivo cultures of bone marrow cells from 3-week-old Lmna^−/− mice (lower panels) and WT controls (upper panels). The number of colony forming units-osteoblast (CFU-OB) per femur was significantly higher after 3 wks of differentiation in WT mice compared to Lmna^−/− mice (B). (C) Sections of plastic embedded proximal tibiae (secondary spongiosa) from Lmna^−/− mice and WT controls (n = 10 per group) were stained sequentially with toluidine blue (upper panels, Magnification ×40) for osteoblasts (black arrows) and osteocytes (red arrows) and ALP (lower panels, Magnification ×20) for osteoblasts (arrows). (D) A significant decrease (−47%) in the number of ALP expressing osteoblasts (N.Ob) and a significant decrease in osteocyte number (N.Ot) (−50%) were seen in Lmna^−/− mice compared with WT^+/+ mice. Micrographs are representative of those from eight different mice of each genotype. *P<0.001. Changes in osteoblast differentiation and function correlated with changes in a serum biochemical marker of bone formation (P1NP) in Lmna^−/− mice compared with WT^+/+ mice. *P<0.001. (E) The reduction in osteoblast differentiation in Lmna^−/− mice was associated with lower expression of osteocalcin (OCN) and osteopontin (OPN) at the mRNA level. There were no differences in Runx2 expression between Lmna^−/− mice and their WT controls. For PCR, data analysis is expressed as the ratio of the gene of interest vs. GAPDH as control. Data represent the mean±SD of triplicate determinations. *P<0.001.