Analysis and Imaging of Osteocytes.

Osteocytes are the bone cells that are thought to respond to mechanical strains and fluid flow shear stress (FFSS) by activating various biological pathways in a process known as mechanotransduction. Confocal image-derived models of osteocyte networks are a valuable tool for conducting Computational Fluid Dynamics (CFD) analysis to evaluate shear stresses on the osteocyte membrane, which cannot be determined by direct measurement. Computational modeling using these high-resolution images of the microstructural architecture of bone was used to numerically simulate the mechanical loading exerted on bone and understand the load-induced stimulation of osteocytes. This study elaborates on the methods to develop 3D single osteocyte models using confocal microscope images of the Lacunar-Canalicular Network (LCN) to perform CFD analysis utilizing various computational modeling software. Prior to confocal microscopy, the mouse bones are sectioned and stained with Fluorescein isothiocyanate (FITC) dye to label the LCN. At 100x resolution, Z-stack images are collected using a confocal microscope and imported into MIMICS software (3D image-based processing software) to construct a surface model of the LCN and osteocyte-dendritic processes. These surfaces are then subtracted using a Boolean operation in 3-Matic software (3D data optimization software) to model the lacunar fluidic space around the osteocyte cell body and canalicular space around the dendrites containing lacunocanalicular fluid. 3D volumetric fluid geometry is imported into ANSYS software (simulation software) for CFD analysis. ANSYS CFX (CFD software) is used to apply physiological loading on the bone as fluid pressure, and the wall shear stresses on the osteocytes and dendritic processes are determined. The morphology of the LCN affects the shear stress values sensed by the osteocyte cell membrane and cell processes. Therefore, the details of how confocal image-based models are developed can be valuable in understanding osteocyte mechanosensation and can lay the groundwork for future studies in this area.