Fig. 7

Ionomycin treatment rescued osteoporosis in BMSC-specific conditional alpl knockout mice. a MicroCT analysis showed that Prrx1-alpl^−/− mice (n = 12) had significantly decreased bone mineral density (BMD), Bone volume relative to tissue volume (BV/TV), and trabecular number (Tb.N) in trabecular bone (TB) of the distal femur compared with the values of their alpl^f/f littermates and Prrx1-alpl^−/− mice treated with ionomycin (n = 10). b H&E staining showed a decreased TB volume (yellow circled area) in the distal femurs of Prrx1-alpl^−/− mice compared with that of the alpl^f/f control group and Prrx1-alpl^−/− mice treated with ionomycin. Scale bar, 1 mm. c Representative histological images of distal femurs show a significantly increased number of adipocytes in Prrx1-alpl^−/− mouse bone marrow, as assessed by oil red O staining. Scale bar, 25 μm. d Calcein double labeling assay showed a significantly decreased bone formation rate in Prrx1-alpl^−/− mice compared with that of the alpl^f/f controls and Prrx1-alpl^−/− mice treated with ionomycin. Scale bar, 25 μm. e The intracellular level of Ca²⁺ in alpl^−/− BMSCs was decreased compared with that of the WT BMSCs, and ionomycin treatment elevated the intracellular level of Ca²⁺ in alpl^−/− BMSCs. f Alizarin red staining showed that alpl^−/− BMSCs had a decreased capacity to form mineralized nodules when compared with the capacity of WT BMSCs cultured under osteoinductive conditions. BMSCs from Prrx1-alpl^−/− mice treated with ionomycin showed an elevated capacity to form mineralized nodules. g Western blot analysis showed that alp^−/− BMSCs had decreased expression of the osteogenic-related proteins RUNX2 and Sp7 compared with what was observed in WT BMSCs and alpl^−/− BMSCs treated with ionomycin. β-actin was used as a protein loading control. alpl^−/− BMSCs showed an increased number of oil red O-positive cells when cultured under adipogenic conditions (h), and western blotting showed an upregulation of the adipogenic-related proteins PPARγ2 and LPL (i) compared with what was observed in WT BMSCs and alpl^−/− BMSCs treated with ionomycin. The representative results from three independent experiments are shown. Error bars represent the s.d. from the mean values. *P < 0.05; **P < 0.01; ***P < 0.001