Analysis and Imaging of Osteocytes

Summary

This study outlines the method to visualize and develop three-dimensional (3D) models of osteocytes within the lacunar-canalicular network (LCN) for computational fluid dynamics (CFD) analysis. The generated models using this method help to understand osteocyte mechanosensation in healthy or diseased bones.

Abstract

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.

Introduction

Osteocytes are postulated to regulate bone mass in response to physical exercise¹. Membrane deformation of osteocytes and their dendritic processes due to mechanical loading, subjects them to FFSS, which is detected by the osteocytes and triggers intracellular signaling^2,3,4. Bone microstructure undergoes through deterioration or alterations in its lacunar-canalicular morphology due to aging or bone diseases such as osteoporosis and diabetes and in conditions such as perlecan deficiency that causes impaired mechano-responsiveness of osteocytes

Protocol

Animal experiments were carried out with the approval of the Institutional Animal Care and Use Committee at the University of Missouri, Kansas City (UMKC), and conformed to relevant federal guidelines.

1. Bone preparation process

1. Collect femurs from 4-month-old and 22-month-old female C57BL6 mice and fix them in cold 4% paraformaldehyde in PBS for 24 h at 4 °C with gentle rocking, then rinse them in PBS and store them in 70% ethanol before embedding.
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Discussion

This protocol outlines a confocal imaging technique for visualization and computational modeling of the osteocytes. Before confocal imaging, the bone preparation process for sectioning and staining bone samples is performed. Confocal images of 100x magnification are imported into various software to develop computer models of osteocytes and the lacunar-canalicular space. A CFD analysis is conducted lastly on the confocal image-based models to model FFSS surrounding the osteocytes and dendritic membranes due to physical a.......

Materials

Name	Company	Catalog Number	Comments
1,200 Grit sandpaper	Buehler	30-5170-012-100
3-Matic software	Materialise	https://www.materialise.com/en/industrial/software/3-matic	3D data optimization software
600 grit sandpaper	Buehler	30-5118-600-100
800 Grit sandpaper	Buehler	30-5170-800-100
ANSYS software	ANSYS	https://www.ansys.com/	simulation software
Fluorescein Isothiocyanate (FITC)	Sigma-Aldrich	F7250
ImageJ software		https://imagej.net/ij/
Immersion Oil for Microscopes	Leica Microsystems	195371-10-9
Leica TCS Sp5 II confocal microscope	Leica Microsystems	TCS Sp5 II
Leitz 1600 inner hole diamond saw	Leica
MIMICS Innovation Suite software	Materialise	https://www.materialise.com/en/healthcare/mimics-innovation-suite	3D image-based processing software
Permount mount medium	Fisher scientific	SP15-500
Sampl-Kwick Fast Cure Acrylic Kit	Buehler	20-3560
Single Platform Laboratory Shaker	Reliable scientific INC	Model 55S