Imaging and Assessment Methods of Phenotyping Osteocyte Networks

Osteocytes, the master orchestrators of bone remodeling, form an extensive network of F-actin rich dendrites that enable them to sense external mechanical signals and communicate with other cells, which are essential to maintain bone homeostasis and bone mass. Loss of the osteocyte dendrite number and connectivity and similar changes in the lacunar-canalicular pore system (LCS) surrounding the osteocyte dendrites have been reported in aged animals and humans. Increasing evidence demonstrates the close association of osteocyte dendrites/LCS alterations with many bone disorders and diseases that affect the formation, maturation, maintenance, or degeneration of osteocyte dendrites. Given their intrinsic temporal and spatial variations, phenotyping the osteocyte dendrite/LCS requires rigorous evaluation of a large number of cells with reliable methods. The goal of this manuscript is to describe several easy-to-adopt strategies of phenotyping osteocyte dendrites and high-throughput analysis methods developed in previous literature and our laboratories. Protocols will be detailed for (1) preparing various murine and human bone samples; (2) fluorescence staining of the dendrite cytoskeleton; (3) silver nitrate staining of the LCS; and (4) sample imaging and analysis including macro plugins using the free software ImageJ. The protocols provide reliable methods to phenotyping osteocytes in the context of aging, diseases, and treatments.