Micro-CT Imaging

At the end of the study period, the rats were euthanized with an overdose of CO₂ and the knee joints were removed for high-resolution micro-CT imaging. The micro-CT images were acquired using a desktop ex vivo micro-CT system (μCT-40; Scanco Medical, Brüttisellen, Switzerland) as described previously.²⁰ Briefly, images spanning 1,000 angles were collected over 180° with each projection sampled for 200 ms. The projections were then reconstructed with a matrix size of 2,048 × 2,048 and 16.4-mm FOV, resulting in an isotropic nominal resolution of 8 μm. Axial reconstructions were produced starting 9 mm proximal from the proximal extent of the distal femoral metaphyseal compartment and extending 9 mm distal to the distal extent of the proximal tibial metaphyseal compartment. The images were reconstructed into sagittal plan, where semiautomatic contours were generated in the subchondral bone area (epiphysis) to define the volume of interest that included only subchondral trabecular bone. Specifically, these contours identified the endosteal boundary to compartmentalize cancellous and cortical bone regions. An adaptive iterative threshold algorithm was applied to segment the mineralized bone and marrow phases.²³ In the subchondral bone compartment, bone volume fraction (BV/TV) was determined by simple voxel counting of the bone and background phases. Mean trabecular dimensions including trabecular thickness (Tb.Th), number (Tb.N) and spacing (Tb.Sp) were calculated directly using a model-independent sphere filling technique.^24–26