| Summary: | Many physiological, biomechanical, evolutionary and clinical studies that explore skeletal structure and function require successful separation of trabecular from cortical compartments of a bone that has been imaged by X-ray micro-computed tomography (micro-CT) prior to analysis. Separation often involves manual subdivision of these two similarly radio-opaque compartments, which can be time-consuming and subjective. We have developed an objective, semi-automated protocol which reduces user bias and enables straightforward, user-friendly segmentation of trabecular from the cortical bone without requiring sophisticated programming expertise. This method can conveniently be used as a ‘recipe’ in commercial programmes (Avizo herein) and applied to a variety of datasets. Here, we characterize and share this recipe, and demonstrate its application to a range of murine and human bone types, including normal and osteoarthritic specimens, and bones with distinct embryonic origins and spanning a range of ages. We validate the method by testing inter-user bias during the scan preparation steps and confirm utility in the architecturally challenging analysis of growing murine epiphyses. We also report details of the recipe, so that other groups can readily re-create a similar method in open access programmes. Our aim is that this method will be adopted widely to create a reproducible and time-efficient method of segmenting trabecular and cortical bone.
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