The mice’s body weights were recorded during the 5 weeks of vibration treatment. The mice were injected intraperitoneally with a calcein solution (20 mg/kg) at 10 and 3 days before sacrifice in order to assess bone apposition [48]. Mouse sacrifice was performed by CO2 asphyxia and the mouse tibiae and femora were dissected and cleaned of soft tissues. The right bones were stored in gauze soaked with phosphate buffered

solution (PBS) and frozen at − 18 °C. The left bones were fixed in 4% formalin-phosphate buffered solution overnight, rinsed with PBS and stored in 70% ethanol at 4 °C. Right tibiae and femora were scanned using a micro-computer tomography scanner (Metris X-Tek HMX ST 225 CT System) with a 10 μm voxel resolution (80 to 120 kV, 140 μA, 500 μs integration time). NU7441 purchase Trabecular and cortical bone morphology was analysed in the femur and the tibia using the open source ImageJ software and BoneJ plugin [49]. The cortical bone morphology was analysed (every 10 slices) between 20% and 80% of the femur total length (%TL distal to proximal) and 20% to 90%TL of the tibia after segmenting out the trabecular bone (see Fig. 1). Cortical parameters analysed were as follows: cross section area (CSA, mm2), minimum and selleck inhibitor maximum moment of inertia (Imin, Imax, mm4) and mean cortex thickness (CtTh, mm). Trabecular bone was

analysed (every slice) between 15 and 25%TL in the femur distal metaphysis and between 83 and 93%TL in the tibia proximal metaphysis (see Fig. 1). The trabecular bone was separated from the cortical bone by manually drawing a contour

in the proximal tibia while, in the distal femur, an elliptical region of interest (length/width ratio of 1.5) was drawn and replicated every slice. Trabecular bone parameters analysed were as follows: trabecular bone surface (BS, mm2), trabecular bone volume on total volume (BVTV), mean trabeculae thickness (TbTh, mm) and mean trabeculae space (TbSp, mm). After CT scanning, right femurs were tested until fracture Progesterone by three-point bending using a standard materials testing machine (5866 Instron, Instron, Norwood, MA, USA). Femurs were placed on their posterior side on two supports separated by 9 mm and were loaded in the anterior-posterior direction at the mid-diaphysis with a deflection rate of 50 μm/s. Force–deflection curves were analysed with a custom program (Matlab, MathWorks Inc, MA, USA) to measure the bending stiffness (S: slope of the linear elastic deformation), the yield force (Fyield, limit between the elastic and plastic deformation) and ultimate force (Fult, maximum force sustained) and the total work to fracture (mJ). The bone elastic modulus E (MPa), ultimate stress σult (MPa) and yield stress σyield (MPa) were calculated using the standard beam theory [50] and the mid femur cross-section dimensions (anteror posterior diameter and medial lateral moment of inertia) measured from the μCT scanner data.