Major and minor centroidal axes serve as objective, automatable reference points to test mechanobiological hypotheses using histomorphometry.

Recent studies show that minor and major centroidal axes (CA) of long bone cross sections provide valuable predictions of prevailing loading patterns in age and treatment matched cohorts of animals. Furthermore, using CA, we recently showed that the degree of mineralization and area of woven bone laid down in the first two weeks after creation of a critical sized bone defect relate inversely and correlate significantly to loading patterns. Here, we aim to determine how closely independent measures of the spatial distribution of bone apposition determined using the major and minor CA as reference points correlate to those using anatomically defined axes as reference points. In histological sections from the previous study, we found no statistically significant difference between the anatomical and centroidal axes with respect to the centroid location or axis rotation, but there is a significant albeit small difference in the average distance between centroids. Outcome measures calculated in areas of bone defined by 15°, 30°, 45°, 60°, or 90° sectors when using the CA differ less than 5% from those calculated using anatomical axes as reference points. Hence, the major and minor CA provide objective reference points for comparison of mechanobiological outcome measures between animals in matched cohorts. Calculation of major and minor CA is automated, which reduces the potential for observer bias. A major advantage of using the major and minor CA as reference points is that it allows for direct relation of outcome measures to loading patterns in age and treatment matched cohorts, ultimately providing a tool to test mechanobiological hypotheses on histological cross sections of bone.