, Scottsdale, AZ, USA) and a 25-hydroxyvitamin D RIA kit (Diasorin S.p.A., Saluggia [Vercelli], Italy). Areal BMD (aBMD) of the lumbar spine (L1–L4) and right proximal femur (total hip)

were measured by DXA (QDR 2000 plus; Hologic Inc., Bedford, MA, USA) at baseline and at month 6 (experiment 1) or this website at months 3 and 6 (experiment 2). The coefficient of variation of DXA scanning with repositioning ranged from 0.8% for lumbar spine aBMD to 4.5% for femoral neck aBMD. pQCT (XCT Research SA +; Stratec Medizintechnik GmbH, Germany) was used to measure volumetric bone mineral content (vBMC) and volumetric BMD (vBMD) at metaphyseal and diaphyseal sites of the right tibia at baseline and at month 6 (experiment 1) or at months 3 and 6 (experiment 2). Metaphyseal data were generated as an average from 3 scans separated by 0.5 mm at the tibia/fibula junction (contour mode 2: threshold 0.446 cm− 1; peelmode 2: threshold 0.550 cm− 1). A diaphyseal scan was taken at approximately 12% of the bone length (peelmode 2, cortmode 2: threshold 0.930 cm− 1) toward the center of the tibia from the metaphyseal scans. SB431542 in vitro Nominal voxel size was 0.35 mm. The coefficient of variation of pQCT

scanning with repositioning was 0.2% to 1.1% at the proximal tibia across all variables obtained at metaphyseal and diaphyseal sites. L2 vertebrae and right proximal femurs were collected for bone histomorphometric analysis. Each animal was injected with 8 mg/kg of calcein subcutaneously, 15 days and 5 days prior to termination. All bone samples were fixed in 10% neutral-buffered formalin for 3 days and transferred to 70% ethanol. Bones were then trimmed, Thiamet G dehydrated, and embedded in methyl methacrylate. Trabecular bone sections were prepared in the frontal plane for the femur neck, and in the sagittal plane for the L2 vertebral body. Dynamic histomorphometry was performed on 7 μm-thick unstained sections, while 5 μm-thick sections were stained with Goldner’s trichrome for static parameters and with toluidine blue for wall thickness (W.Th).

Cortical bone histomorphometry was performed on 2 unstained transverse sections at the femur diaphysis, ground to 20–40 μm in thickness. Measurements were collected with a Bioquant image analyzer (Bioquant Image Analysis Corporation, Nashville, TN, USA) linked with an Olympus BX-60 microscope (Olympus Corporation, Tokyo, Japan) equipped with bright and epifluorescence illumination Static and dynamic parameters were measured and reported as outlined by the ASBMR histomorphometry nomenclature committee [18]. Bone biomechanical testing was performed with an MTS 858 Mini Bionix servohydraulic test system (TestResources Inc., Shakopee, MN, USA), and data was collected using Testworks (v3.8A) for Teststar II (v.4.0c) software.