μct40

μCT has been used extensively to quantify the 3-dimensional microarchitecture of trabecular bone.^{36 (link)–38} The hemi-mandibles in 70% alcohol were placed in the μCT specimen holder and sealed using styrofoam blocks to stabilize the bones. Bone mineral volume and microarchitecture of mandibular body and condyle were evaluated using the μCT (μCT 40, Scanco Medical, Basserdorf) with a resolution of 20 μm. One hundred and fifty μCT slices were imaged over the areas of interest using 55kVp energy and 145 μA current. The integration time utilized was 200 ms and total scanning time of 21.1 minutes. The μCT slices were images which were filtered using a constrained three-dimensional Gaussian filter and binarized using a fixed threshold of 240. 3-D construction of the condylar cross sections were made with a reference point set approximately 100 slices from condyle tip and body of mandible, 25 slices mesial to the first molar. The 3D structural parameters (BV/TV and porosity) were obtained using Sigma 1.2, Support 1.0, software provided by the manufacturer.

Femurs were harvested from transplanted female oim mice and frozen in PBS soaked gauze at −20°C (for later mechanical testing), or fixed in 10% formalin for subsequent histology. Femurs were imaged by microcomputed tomography (μCT40, Scanco Medical AG) at 55 kV at a 16 μm voxel size. Cortical morphometry near the mid-diaphysis was measured in a 600 μm span referenced 5.1 mm from the growth plate. Segmentation of bone was performed in conjunction with a constrained Gaussian filter to reduce noise, applying a calibrated hydroxyapatite-equivalent density threshold of 740 mg/cm³ for cortical bone. Femurs with fractures were excluded from the microCT analysis (n=9 sham, n=10 transplanted intact femurs were analyzed). One sample was excluded due to poor injection noted during transplantation.

Quantity and quality of bone across the distal femoral, proximal tibial metaphysis, and L5 vertebrae were measured ex vivo using X-ray micro-computed tomography (μCT40; Scanco Medical; Wayne, PA, USA). X-ray source voltage was set to 55kVp, current set to 145μA, integration time was 300ms, and voxel size was 10μm. Parameters measured were as follows: trabecular bone volume fraction (Tb.BV/TV), cortical bone volume fraction (Ct.BV/TV), and the number of transcortical perforations. Starting 700μm distal to the epiphyseal growth plate in the tibia and 700μm proximal to the growth plate of the femur, 1 000μm of metaphyseal bone was evaluated. A 500μm uniform core of trabecular bone, taking care not to include primary spongiosa, was evaluated within the vertebral body of the L5 vertebrae. Thresholds for each slice were set exclusively to separate cortical and trabecular bone using an automated script (55 (link)). Reconstructed solid 3D images were then used to quantify bone microarchitecture and the number of perforations that extended from the periosteum to the endosteal surface. Digitally slicing the 3D reconstructed image along the sagittal and transverse axes revealed perforations, and these were included if they breached the periosteal surface.