Fig. 5.

The absence of CD200 increases bone density. (A) Bone marrow cells from 6- to 8-week-old CD200-deficient and wild-type mice were plated in 24-well plates (5 × 10⁶ cells/well) and cultured for 9–11 days in α-MEM supplemented with ascorbic acid (50 μg/ml) and β-glycerophosphate (10 mM) to acquire the osteoblast phenotype. Osteoblast lysates were analyzed for protein concentration and subjected to Western blot analysis with antibodies directed against mouse CD200, CD200R, and GAPDH. (B) Bone-marrow-derived osteoblasts were examined for ALP activity and stained for calcium with alizarin red S to allow quantitation of the number of nodules per well (SD; n = 6). Cell lysates were analyzed for ALP activity (Left; SD; n = 6). These experiments were repeated three times with similar results. (C) Toluidine blue-stained sections of proximal tibiae from 2-month-old CD200-deficient male and female mice and wild-type mice. (Scale bar, 1 mm.) (D) Microcomputed tomography analysis of distal femurs from 6-month-old male and female CD200-deficient mice. Note the increased density of trabeculae inside the distal femur of CD200-deficient male and female mice as compared with wild types. The widest diameter of the bone sections correspond ≈3 mm.