Volumes of interest (VOI) are commonly assigned to image stacks generated from micro-computed tomography to specify areas for bone quantification. However, the size of the VOI can impact the values obtained for trabecular bone parameters. This study aims to investigate the effect of VOI size by applying VOIs of four different diameters (10%, 15%, 20% and 30% of the antero-posterior width). Ten juvenile right distal femora were included, aged from pre-natal to three years. Smaller VOIs were placed within the largest VOI, with multiple locations used for the same VOI size. The observed parameters included bone volume fraction (BV/TV), trabecular bone thickness (Tb.Th), separation (Tb.Sp), number (Tb.N), degree of anisotropy by mean intercept length (DA.MIL) and star volume distribution (DA.SVD). Statistically significant differences between VOI sizes were found for Tb.Sp, Tb.N, DA.MIL and DA.SVD. A possible effect of localized variation was found due to significantly different values for Tb.Th between VOI locations. The effect related to VOI geometry was reflected by DA.MIL and DA.SVD as no significant difference was found between locations. The minimum diametric strut quantity (DSQ, diameter multiplied by Tb.N) of 4 can be implied based on the zero DA.SVD value found in particular VOIs.
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