Microcomputed tomography (microCT) analysis

MicroCT scanning was used to measure cortical and trabecular architecture of the femur and the trabecular architecture of the fourth lumbar vertebra (L4). Bones were dissected, cleaned of soft tissues, fixed in 10% Millonig’s formalin overnight, and transferred gradually from 70 to 100% ethanol. Dehydrated bones were scanned using a model μCT40 scanner (Scanco Biomedical, Bruttisellen, Switzerland) to generate three-dimensional voxel images (1024 × 1024 pixels) of bone samples. A Gaussian filter (sigma  =  0.8, support  =  1) was used to reduce signal noise and a threshold of 220 was applied to all scans, at medium resolution (E  =  55 kVp, I  =  145 μA, integration time  =  200 ms). Femurs were scanned beginning immediately distal to the third trochanter to the beginning of the distal growth plate. Cortical dimensions were determined at the diaphysis. Trabecular bone measurements at the distal femur were made on 151 slices beginning 8–10 slices away from the growth plate in order to avoid the primary spongiosa, and proceeded proximally. The fourth lumbar vertebra (L4) was scanned from the rostral growth plate to the caudal growth plate. Trabecular bone analyses were performed on contours of cross-sectional images, drawn to exclude cortical bone, as described for femoral trabecular bone. All trabecular measurements were made by drawing contours every 10–20 slices and using voxel counting for bone volume per tissue volume and sphere-filling distance transformation indices. Calibration and quality control were performed weekly using five density standards, and spatial resolution was verified monthly using a tungsten wire rod. Beam hardening correction was based on the calibration records. All nomenclature, symbols, and units adhered to guidelines in the literature [18].