The sample surface was carbon coated prior to qBEI. A digital electron microscope (DSM 962, Zeiss, Oberkochen, Germany) equipped with a four quadrant semiconductor BE detector was used for backscattered electron imaging. The accelerating voltage of the electron beam was adjusted to 20 kV, the probe current to 110 pA, and the working distance

to 15 mm. The digital backscattered (BE) images of trabecular bone areas were acquired by a single frame with a scan speed of 100 s/frame and a pixel resolution of 1 μm. Areas with high backscattered electron intensities – light gray levels – represent mineralized matrix with high Ca contents, whereas areas with low intensities – dark gray levels – indicate low mineral density. For the characterization and quantification of changes learn more in the bone mineralization density distribution (BMDD) curve, four outcomes were used: CaMean (the weight mean calcium content of the bone area obtained from the integrated area under the BMDD curve), CaPeak (the mode of calcium content indicated by the peak position in the BMDD diagram), CaWidth (the heterogeneity of mineralization

caused by the coexistence of BSU of different ages measured at the full width at one-half maximum of the BMDD-peak), CaLow (reflects the fraction low mineralized bone areas (< 17.68 wt Ca)). Details of the analysis method have been published previously [26]. Additionally, these qBEI images were also used later, to evaluate this website the mean gray level/mineral content (CaInd) at the nanoindentation sites similar as described in Ref. [27]. After μCT scanning, the L2 vertebral bodies were rehydrated, mounted in a servo-hydraulic testing system (858 Mini Bionix II, MTS, USA), preconditioned with 10 cycles in the elastic range and tested to failure Quisqualic acid in axial compression

with a rate of 0.033 mm/s. Stiffness, maximal load to failure, and energy to failure were computed from the resulting force–displacement curves. A mean tissue modulus for each vertebral body was then back-calculated from the experimental stiffness using a 12 μm resolution, homogeneous, linear elastic (v = 0.3) finite element model of the same loading scenario. Nanoindentation was performed using a Scanning Nanoindenter (Hysitron Inc., Minneapolis, USA) with a Berkovich diamond indenter tip as described elsewhere [28]. The calibration of the instrument was performed by doing indents of increasing depth in fused quartz with a known reduced modulus of 72 GPa. The material properties of the diamond tip are known such as the Poisson’s ratio is 0.07 and elastic modulus of the tip is 1140 GPa. Automated area scans of indents were performed using moving sample stage, which has a positional resolution of 1 μm. There was a distance of 10 to 11 μm between indents. A thermal drift correction factor was introduced automatically before each indent, by measuring the drift for 20 s.