Fig. 2.

Reduction in bone formation and osteoblast function in Chip^−/− mice. a, b Representative images of Alcian blue/H&E Orange G stained tibiae sections from 1-month-old WT and Chip^−/− mice showed reduced bone volume in long bone (indicated by yellow arrowhead). b The quantification of bone volume was performed. c, d Calcein double labeling of trabecular bones in WT and Chip^−/− mice that were injected with calcein 4 and 1 day before the mice were sacrificed. Pictures of trabecular bone in tibiae were taken under the fluorescence microscope. Chip^−/− mice showed a decrease in bone formation rates (d). e Results of histomorphometric analysis also showed that osteoblast numbers were also significantly reduced in Chip^−/− mice. f, g TRAP staining was performed using tibial sections of 1-month-old WT and Chip^−/− mice. TRAP-positive cells were indicated by blue arrowheads. The numbers of TRAP-positive osteoclasts were higher in Chip^−/− mice (g). h, i Bone marrow stromal (BMS) cells were isolated from 1-month-old WT and Chip^−/− mice, and were cultured with ascorbic acid and β-glycerophosphate for 3 days for Ki67 expression (h) and ALP staining (i). The BMS cells were also cultured with osteoblast differentiation medium in the presence of BMP-2 (50 ng·mL^-1) for 2 weeks and Alizarin red staining was performed (i). Ki67 expression, ALP activity, and mineralized bone matrix formation were significant decreased in Chip^−/− mice. *P < 0.05, **P < 0.01, unpaired Student’s t-test, n = 6.