Skyscan 1172 x ray microtomograph

To evaluate trabecular microarchitecture and cortical bone geometry of the tibia from control and DSS-treated mice, we used microCT (Skyscan 1172 X-ray microtomograph, Bruker, Kontich, Belgium) as described previously [43 (link)]. In brief, high-resolution scans with an isotropic voxel size of 5 µm were acquired (60 kV, 0.5 mm aluminium filter, 0.6° rotation angle). Two images were averaged at each rotation angle. Scan reconstruction was conducted using NRecon software (Bruker) and each bone was analysed using CtAN (Bruker). For the trabecular analysis, the base of the growth plate was used as a standard reference point. A 1.25 mm trabecular bone region, located at 5% of the total length beneath this reference point, was analysed. To investigate the changes in the cortical bone geometry, two 0.5 mm sections were analysed at 37% and 50% of the total bone length from the reference starting slice (first appearance of the medial tibial condyles). To assess BMD, phantoms were used to calibrate the CTAn software. BMD phantoms of known calcium hydroxyapatite mineral densities of 0.25 and 0.75 g/cm³ were scanned and reconstructed using the same parameters as used for bone samples.

Tibiae were scanned using a μCT system (Skyscan 1172 X-Ray microtomograph, Bruker Corporation, Kontich, Belgium) to evaluate trabecular architecture and cortical bone geometry. For trabecular scanning, high-resolution scans with an isotropic voxel size of 5 μm were acquired (60 kV, 0.5 mm aluminium filter, 0.6° rotation angle). The isotropic voxel size was changed to 10 μm for cortical analysis. Two images were averaged at each rotation angle. The scans were reconstructed using NRecon software (Bruker). For analysis, a 1 mm section of the metaphysis was taken for the analysis of trabecular bone, using the base of the growth plate as a standard reference point. A 500 μm section of the mid-shaft was taken for the analysis of cortical bone, using the articulation with the fibula as a standard reference point. CTAn software (Bruker) was used to analyse the appropriate parameters previously outlined (Bouxsein et al. 2010 (link)).

Prior to scanning, the aortae were immersed for a minimum period of time (10 min) in a macro-molecular iopamidol-based contrast agent (Niopam 300; Brako UK Ltd., High Wycombe, Buckinghamshire, UK) diluted 1:4 in water as previously described (44 (link)). To allow tissue differentiation, aortic luminae were filled with corn oil and the aortae were submersed in oil for the duration of the scan. Tissues were imaged using a Skyscan 1172 X-Ray Microtomograph (Bruker Daltonics, Brussels, Belgium). Sequential high-resolution scans were acquired using a rotation step of 0.3° with the averaging of 3 frames at each step, applying a 0.5-mm aluminium filter, with an X-ray source set at 60 kV and 167 µA, and with an isotropic voxel size of 7 µm. The scans were reconstructed using NRecon (Bruker Daltonics). Noise in the reconstructed images was reduced by applying a median filter (radius = 1). The region of interest was selected to be the aortic arch, 200 lices (1.4 mm) under the subclavian artery. Soft and calcified tissue was identified by thresholding using CTAn software (Bruker Daltonics).