Lumbar 5 vertebrae were scanned at a resolution of 5 μm. The X-ray tube was operated at 41 kV and 240 μA. A lower grey threshold value of 81 and upper grey threshold value of 252 was used as thresholding values in each trabecular bone sample. A cylindrical region of interest (150 slices or 0.774 mm) was selected from the centre of each vertebral body. Calibration

of the standard unit of X-ray CT density from Hounsfield units to volumetric bone mineral density (vBMD) was conducted. ROIs were analysed for the following parameters: PD98059 price trabecular thickness, trabecular separation, trabecular bone volume, trabecular porosity, as well as degree of anisotropy (DA) and structure model index (SMI). Right tibial and femoral shafts from each comparison group were subjected click here to mechanical testing (three point bending and microindentation tests respectively) after the μCT. The mechanical tests were designed to test the cortical part of bone. The tests were performed using a Zwick/Roell z2.0 testing machine (Leominster, UK) with a 100 N load cell [32]. Tibias were placed on the lower supports, at 8 mm separation, with the posterior surface of the tibia facing down. Load was applied with a loading rate of 0.1 mm s− 1 on the shaft of the tibia using the Zwick/Roell testing machine until the fracture occurred (Fig. 3A). Data were

analysed to determine values of stiffness, ultimate load and Young’s modulus using the following formula: equation(1) Young’smodulus=stiffτ⋅Ls348⋅Iwhere stiffτ is the stiffness. Ls is the separation of the supports and I is the second moment of area of the tibias. The stiffness was calculated by measuring the slope of the force-displacement graph and the ultimate load by measuring the maximum force that the bone was able to resist. The second moment of area was calculated using the microCT data and ImageJ software v1.47 and the plug-in Bone J. The micro indentation

hardness test was performed unless on equivalent transverse distal mid-shaft sections of right femur for each mouse/genotype. Bone sections were air dried and embedded in metallurgical mounting resin (EPO Set Resin, Meta Prep, UK) and the moulds allowed to solidify at room temperature for 24 h. The bone cross-section surface was subsequently polished using silicon carbide papers with decreasing grain size (240, 400, 600, 800, 1200) and diamond paste (15, 6 and 1 μm) to produce a smooth surface. After the sample preparation, micro hardness testing was performed using a Wilson Wolport Micro-Vickers 401MVA machine (UK), with an applied load of 25 g for 100 sec. The bone was tested at seven points for each specimen (Fig. 4A). The Vickers pyramid hardness number (HV) was calculated using Eq. 2 where the load (L) is in grammes force and the average length of the two diagonals (D) is in millimetres: equation(2) HV=1.854LD2. The femoral neck fracture test was used to test the mechanical properties of the femoral neck.