Microcomputed tomography (μCT) of cortical and trabecular bone in femurs.

Femurs were dissected from mice at day 14 postinfection, fixed in 10% neutral buffered formalin for 48 h, and then moved to 70% EtOH before storage at 4°C. Fixed femurs were scanned using a μCT50 (Scanco Medical, Switzerland) instrument and analyzed with μCT Tomography V6.3-4 software (Scanco USA, Inc.). The scans were acquired at a 10.0 μm voxel size at 70 kV, 200 μA, and an integration time of 350 ms in a 10.24 mm view to result in 1,088 image slices. To accommodate the analysis of trabecular and cortical bone, a region of interest was selected on each femur to encompass the trabecular bone of the distal epiphysis, as well as the entire diaphysis, so that the cortical defect into which bacteria were inoculated could be visualized. The proximal epiphysis was excluded. Cortical bone destruction, callus formation, and trabecular bone volume were determined by contouring the indicated regions of interest as previously described (43, 44, 72). Briefly, to analyze cortical bone loss and callus formation, 818 slices were contoured, centering around the midpoint of the defect. For trabecular bone analysis, measurements to the distal trabecular bone were standardized to begin 30 slices above the growth plate. 101 slices were analyzed in total.