Micro-CT Analysis of Decalcified Tibias

Formalin‐fixed, decalcified tibias were washed in Sorensen's phosphate buffer (81 mM KH2PO4, 19 mM Na2HPO4 · 7H2O, pH 7.4) and stained with 1% osmium tetroxide solution (2% w/v; Agar Scientific, Stansted, UK; diluted 1:1 in Sorensen's phosphate buffer) for 48 hours at room temperature, washed, and stored in Sorensen's phosphate buffer at 4°C. Stained tibias were arranged in parallel in 1% agarose in a 30‐mL universal tube and mounted in a SkyScan 1172 desktop micro‐CT (Bruker, Kontich, Belgium). The samples were then scanned through 360° using a step of 0.40° between exposures. A voxel resolution of 12.05 μm was obtained in the scans using the following control settings: 54 kV source voltage, 185 μA source current with an exposure time of 885 ms. A 0.5‐mm aluminium filter and two‐frame averaging were used to optimize the scan. After scanning, the data were reconstructed using SkyScan software NRecon v1.6.9.4 (Bruker). The reconstruction thresholding window was optimized to encapsulate the target image. Volumetric analysis was performed using CT Analyzer v1.13.5.1 (Bruker).24 (link)