Micro-CT

The left tibia was prepared for micro-CT imaging. To avoid an influence of the water content within the bone, soft tissue was removed from the tibia and then tibia was dried at 65 °C. Preliminary tests showed that the procedure did not affect the imaging process. Furthermore, it allowed for a more uniform positioning. The samples were imaged in the custom-designed micro-CT scanner HECTOR of the Ghent University Center for X-ray Tomography (UGCT; www.ugct.ugent.be) [42]. For each micro-CT scan, 2,001 projection images were acquired at an exposure time of 1,000 ms for each projection, covering the full 360° angular range. A tube voltage of 130 kV was employed at 10 W target power. An additional filter of 0.5 mm Al was used to reduce beam hardening effects. Using geometrical magnification, the reconstructed isotropic voxel size was 22³ μm³. Projection data were reconstructed to a stack of 2D slices using the in-house developed reconstruction platform Octopus Reconstruction [43]. Next, the micro-CT images were analyzed using custom-written scripts in Image J (National Institutes of Health, USA). The volume of interested cortical bone was chosen from the tibial mid-diaphysis and extended proximal-distally for a total length of 37 mm. The center of the diaphysis of the tibia was automatically classified into trabeculae, cortex and ‘total cortex’ (including pores), using an algorithm similar to that of Buie et al. [44]. The total volume and the volume of cortical bone and trabecular bone were quantified, and bone volume to total volume ratio (BV/TV), cortical thickness and trabecular thickness were calculated. To measure the trabecular bone in the proximal end of the tibia (metaphysis), a region 4 mm in length was determined, starting 9 mm below the surface of the condyles. To exclude denser cortical regions at the bone surface, the outer 0.5 mm of the bone surface was removed from the region of interest. The BV/TV of trabecular bone and trabecular thickness were calculated. The average thickness of the structures was measured using the thickness plugin from Bone J [45].