Comprehensive Microstructural Bone Analysis

μ-CT of the femur/Tibia (trabecular/cortical) and lumbar vertebrae-V (trabecular) was performed using SkyScan 1076 scanner (SkyScan). Scanning was done at 70 kV, 100 mA using a 0.5-mm aluminum filter and exposure set to 590 ms. In total, 1800 projections were collected at a resolution of 6.93 μm/pixel. For purpose of carrying out reconstruction process NRecon software was used. Bone length was determined from the rendered 3D images in CTAn software. The distance from trochanter to edge of femoral condyles defined total femoral length. Tibial length calculated as distance from medial condyle to medial malleolus. Manual segmentation of 2D slice of sagittal images was done to isolate growth plates from the surrounding bone tissue in micro-CT images followed by reconstruction to render 3D images. It was from these images that growth plate height was measured using data viewer software. For trabecular region analysis, ROI was drawn at a total of 100 slices in secondary spongiosa at a distance of 1.5 mm from distal border of growth plates excluding the parts of cortical bone and primary spongiosa. In vivo measurement of LV5 trabecular was done by encompassing 50 continuous slices which start from the beginning of trabecular bone within spinal body (Li et al., 2016 ; Dempster et al., 2013 (link)). The CTAn software was used to analyze cortical bone by taking into consideration 350 consecutive image slides discarded from growth plate to leave out all trabecular regions, out of these 200 continuous images were selected as such. Various bone histomorphometric trabecular parameters (3D) and cortical parameters (2D) were analyzed by already established protocols (Dempster et al., 2013 (link); Srivastava et al., 2013 ). For determination of BMD of LV5, femur and tibia μ-CT scans were utilized as determined from VOI made for trabecular and cortical region. For BMD calibration, 2-mm-diameter hydroxyapatite phantom rods with known BMD (0.25 and 0.75 g/cm³) were used. For each analysis, the estimated BMD was determined based on linear correlation between the μCT attenuation coefficient and BMD (Srivastava et al., 2013 ).