μct evaluation program

Femurs harvested from 8-week-old mice and cleaned of surrounding soft tissues were immediately wrapped with HBSS-soaked gauze and stored at -20 °C until proceeding with μCT scanning and mechanical testing. Once samples were brought to room temperature, the femur and its mid-diaphysis were located with a scout scan, and the mid-diaphyseal cortical bone was analyzed by scanning 1 mm of mid-diaphysis on Scanco μCT50 (Scanco Medical, Switzerland) with 55 kVp intensity, 109 μA current, 6 W energy, and 10 μm resolution. To minimize dehydration of the bones prior to mechanical testing, mid-diaphysis, which cannot be determined once the bone has fractured, was scanned prior to 3-point bending while the epiphysis was scanned afterwards. Following mechanical testing, 2 mm of epiphysis below the growth plate separation was scanned using the same scanning parameters. Cortical bone sections with endosteal and periosteal surfaces automatically contoured were analyzed using the cortical bone analysis algorithm provided in μCT Evaluation Program (Scanco Medical, Switzerland). To assess trabecular parameters, including but not limited to BV/TV (bone volume/total volume), Tb. N. (trabecular number), and Tb. Sp. (trabecular spacing), contours were manually segmented on 100 sections distal to the growth plate. Data were collected from n = 8 mice/group for females and 5-11 mice/group for males.