Figure 1.

Effect of p27 deficiency on osteoblastic bone formation and osteogenesis. (A) Representative micro-CT scans of 3-dimensional longitudinal reconstructions of proximal ends of tibiae and midshaft diaphysis from 4-week-old WT and p27^-/- mice, and (B) Trabecular bone volume relative to tissue volume (BV/TV, %). Representative micrographs of paraffin-embedded sections of tibias from 4-week-old WT and p27^-/- mice showing (C) H&E staining and (D) the number of osteoblasts per mm² tissue area (N.Ob/T.Ar, #/mm²), (E) histochemical staining for ALP and (F) ALP-positive areas as a percent of the tissue area (%), (G) immunostaining for type I collagen (Col-I), and (H) the percentages of Col-I positive areas, (I) histochemical staining for TRAP, (J) the number of osteoclasts/bone perimeter (Oc.N/B.Pm, #/mm) and (K) osteoclast surface/bone surface (Oc.S/BS, %). BM-MSCs from 4-week-old WT and p27^-/- mice were cultured ex vivo in osteogenic differentiation medium for 10 days. Resulting cultures were analyzed as follows: (L) staining with methyl blue for the total CFU-f and (M) total CFU-f-positive areas, (N) cytochemical staining for ALP to show CFU-fap and (O) ALP-positive areas, (P) immunocytochemical staining for Ki67 and (Q) the percentage of Ki67 positive cells. Real-time RT-PCR analyses of BM-MSC extracts for the expression of (R) Runx2, ALP, Col-I and OCN. Messenger RNA expression assessed by RT-PCR is expressed as a ratio relative to GAPDH expression. Values are mean ± s. e. m. of 5 determinations per group. *: P< 0.05; **: P< 0.01, compared with WT mice.