Microcomputed tomography.

To assess trabecular and cortical bone morphology, mouse femurs were placed in a 19-mm-diameter specimen holder and scanned using microcomputed tomography (microCT; μCT 100; SCANCO Medical, Bassersdorf, Switzerland). Scan parameters were 70 kVp, 114 μA, 0.5-mm aluminum filter, and 500-ms integration time. The metaphysis of the distal femur and the midshaft femur were scanned using an 18-µm isotropic voxel size. Analyses were performed using SCANCO evaluation software with fixed global thresholds of 18% (180 on a grayscale of 0–1,000) for trabecular bone and 30% (300 on a grayscale of 0–1,000) for cortical bone to segment bone from nonbone.

For trabecular bone, we assessed the secondary spongiosa in a 1.08-mm region of interest beginning 180 µm proximal to the distal femur growth plate. Outcomes were bone volume fraction, trabecular thickness, trabecular separation, trabecular number, and connectivity density.

For cortical bone, we obtained transverse computed tomography slices in a 540-µm region of interest at the femoral midshaft to obtain total cross-sectional area, cortical bone area, and medullary area, cortical thickness, and bone area fraction. All methods followed American Society for Bone and Mineral Research guidelines (6). Authors performing the microCT analysis were blinded to treatment group.