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The crack regions in the DVC sub-regions were determined for each sample from the final scan (after global yield) of each sample (Fig. 2f) using two consecutive steps: (1) a custom-made automated crack identification and segmentation algorithm followed by (2) a manual inspection (both in MatLab). A crack was defined as a visible discontinuity in the structure in the last scan, which was not present in earlier scans. The final segmentation was based solely on visual inspection. Although the automated crack identification and segmentation algorithm was used only as a tool to aid the final visual segmentation, it is introduced here briefly.
As the automated procedure also captured canals within the trabeculae and did not reveal cracks that had low contrast compared to the surrounding bone or cracks that were too small, the automated segmentation was corrected manually. The manual correction included removal of falsely detected cracks (e.g. canals) and addition of undetected cracks by comparing visually the corrected image stack with cracks with an image stack at an earlier time-point to ensure that the cracks were not present before. Finally, the corrected BSc was overlaid on the original stack to mark the crack regions (Fig. 2f).
The DVC strain analysis was performed only on images from load-steps before reaching global yield, thus when no cracks were visually observed. Only strains from nodes with a correlation coefficient greater than 0.95 were included in the analysis. The DVC strain maps were linearly interpolated to fit the voxel size of the TMD and trabecular thickness maps to enable comparison of the same regions. Cumulative volumetric strains were calculated for each voxel by summing strains from every loading step voxel-by-voxel (Fig. 4). Each voxel at each step had a negative (compressive strain) or positive (tensile strain) volumetric strain value (Supplementary Fig. 2). Strain values after global yield or in regions where a crack was visible were not included in the analysis. After cumulative summing of each load step, absolute cumulative strain maps for each load step were generated. The absolute cumulative strain curves were normalized to extend from the first DVC step until global yield point by interpolation. Since the DVC was performed between two image stacks, the local strains obtained at each loading step represent the total accumulated local strains at the latter acquisition. Average absolute volumetric strain magnitudes, TMDs, and trabecular thicknesses from regions surrounding cracks and from non-crack regions were calculated, analyzed and plotted using custom made MatLab scripts. 2b1af7f3a8