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

Abstract

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.

YAP and TAZ Mediate Osteocyte Perilacunar/Canalicular Remodeling

“…Tibias from P84 mice were harvested and removed of all nonosseous tissue. Additionally, custom python code based on previous work (39,42,43) was implemented to analyze osteocyte lacunar shape measures, oblateness, and stretch. Tibias were originally scanned with a 4× objective to determine regions of interest.…”

“…Osteocyte volume, surface area, and aspect ratio were measured for each osteocyte. Additionally, custom python code based on previous work (39,42,43) was implemented to analyze osteocyte lacunar shape measures, oblateness, and stretch. Oblateness and stretch are calculated from the eigenvalues of the shape tensor for each individual osteocyte lacuna.…”

Aging and Mechanoadaptive Responsiveness of Bone

“…These observations are however open to alternative interpretations: Are they due to inherent age-related changes in bone formation and resorption activities or drifting bone cell behavior, an impairment in the ability of bone cells to transduce mechanical stimuli into biochemical signals or due to modified local microenvironmental cues? Might this failure be underpinned by micro-structural changes such as modified osteocyte lacunar organization or directional orientation, or changes in the number/length of inter-canniculular connecting dendrites [76,77]? Irrespective of its basis, many have proposed that this failure in functional adaptation is responsible for the osteoporosis which develops in the aged skeleton [78, 16, 79-81, 34, 82, 83, 63, 84].…”

No Role of Osteocytic Osteolysis in the Development and Recovery of the Bone Phenotype Induced by Severe Secondary Hyperparathyroidism in Vitamin D Receptor Deficient Mice

“…This suggests that we were able to observe dietary effects only, and we can generally rule out any significant influence of age on BMDD in the present work. This was likely also the case for OLS characteristics where no age-differences were observed in mice between 6 and 18 months [23].…”