Micro-computed tomography

Excised femurs from mice were scanned using the Scanco µCT40 desktop cone-beam micro-CT scanner (Scanco Medical AG, Brüttisellen, Switzerland; using µCT Tomography v5.44). The femur was placed inverted in a 12 mm diameter scanning holder and scanned at the following settings: 12 µm resolution, 70 kVp, 114 µA, 500 projections/180 degrees with an integration time of 200 ms. Scans were automatically reconstructed into 2D slices and all slices were analyzed using the µCT Evaluation Program (v.6.5-2, Scanco Medical). 3D images were obtained from the 3D evaluation software (µCT Ray v.3.8, Scanco Medical). For the cortical analysis, the bone was scanned at the midshaft of the bone for a scan of 25 slices. The region of interest (ROI) was drawn on every slice and fitted to the outside of the cortical bone, to include all the bone and marrow. The threshold for cortical bone was set at 1033 mgHA/cm³. The 3D reconstruction (µCT Ray v3.8) was performed using all the outlined slices. Data were obtained on bone volume (BV), total volume (TV), BV/TV, bone density and cortical thickness. For the trabecular bone, the scan was started at the growth plate and consisted of 210 slices. The ROI was outlined starting 20 slices below the growth plate. 100 slices were outlined from this point, on the inside of the cortical bone, enclosing only the trabecular bone and marrow. Trabecular bone was thresholded at 547 mgHA/cm³ and the 3D analysis performed on the 100 slices. Data were obtained on bone volume, density, total volume, trabecular number, thickness and separation.