A new open-source tool for measuring 3D osteocyte lacunar geometries from confocal laser scanning microscopy reveals age-related changes to lacunar size and shape in cortical mouse bone

<p class="first" id="P1">Osteocytes can participate in systemic mineral homeostasis through perilacunar maintenance and remodeling, where changes to osteocyte lacunar morphology may affect bone structural integrity, tissue strains, and osteocyte mechanosensitivity. Though aging is associated with both decreased bone quality and altered mineral metabolism, it is not known if osteocyte lacunae undergo age-related changes in geometry. In order to survey lacunar changes with age, we developed an open-source program whereby 3D osteocyte lacunae are automatically segmented and then subsequently reconstructed from confocal laser scanning microscopy (CLSM) depth stacks for quantitative analysis of geometry and orientation. This approach takes advantage of the availability and speed of CLSM while avoiding time-consuming and bias-prone manual segmentation. Unlike conventional approaches used to quantify osteocyte lacunar morphology, CLSM enables facile analysis in three-dimensions with clear identification of osteocyte lacunae. We report that 3D osteocyte lacunae measured by CLSM become smaller, more spherical, more oblate, more spatially disorganized, and more sparsely populated with increased age in C57Bl/6 mouse cortical bone in groups spanning 6 – 24 months old. Critically, these age-related changes are in large part not observed in 2D analyses from the same samples. These results (1) demonstrate proof-of-concept of an efficient method to quantitatively assess osteocyte lacunae in 3D for application to a wide range of studies and (2) motivate further inquiry into how changes to osteocyte lacunar geometries and perilacunar material contribute to diminished bone quality in aging. </p>