Shear Stress Modulates Osteoblast Cell and Nucleus Morphology and Volume.

Jianfeng Jin,Gang Wu,Richard T Jaspers,Jenneke Klein-Nulend,Astrid D Bakker,Joannes A.M Korfage

doi:10.3390/ijms21218361

Abstract

Mechanical loading preserves bone mass and function—yet, little is known about the cell biological basis behind this preservation. For example, cell and nucleus morphology are critically important for cell function, but how these morphological characteristics are affected by the physiological mechanical loading of bone cells is under-investigated. This study aims to determine the effects of fluid shear stress on cell and nucleus morphology and volume of osteoblasts, and how these effects relate to changes in actin cytoskeleton and focal adhesion formation. Mouse calvaria 3T3-E1 (MC3T3-E1) osteoblasts were treated with or without 1 h pulsating fluid flow (PFF). Live-cell imaging was performed every 10 min during PFF and immediately after PFF. Cytoskeletal organization and focal adhesions were visualized, and gene and protein expression quantified. Two-dimensional (2D) and three-dimensional (3D) morphometric analyses were made using MeasureStack and medical imaging interaction toolkit (MITK) software. 2D-images revealed that 1 h PFF changed cell morphology from polygonal to triangular, and nucleus morphology from round to ellipsoid. PFF also reduced cell surface area (0.3-fold), cell volume (0.3-fold), and nucleus volume (0.2-fold). During PFF, the live-cell volume gradually decreased from 6000 to 3000 µm3. After PFF, α-tubulin orientation was more disorganized, but F-actin fluorescence intensity was enhanced, particularly around the nucleus. 3D-images obtained from Z-stacks indicated that PFF increased F-actin fluorescence signal distribution around the nucleus in the XZ and YZ direction (2.3-fold). PFF increased protein expression of phospho-paxillin (2.0-fold) and integrin-α5 (2.8-fold), but did not increase mRNA expression of paxillin-a (PXNA), paxillin-b (PXNB), integrin-α5 (ITGA51), or α-tubulin protein expression. In conclusion, PFF induced substantial changes in osteoblast cytoskeleton, as well as cell and nucleus morphology and volume, which was accompanied by elevated gene and protein expression of adhesion and structural proteins. More insights into the mechanisms whereby mechanical cues drive morphological changes in bone cells, and thereby, possibly in bone cell behavior, will aid the guidance of clinical treatment, particularly in the field of orthodontics, (oral) implantology, and orthopedics.

Highlights

In orthodontics, implantology, and orthopedics, mechanical stimuli are known to modulate bone mass, strength, and microstructure [1]
Since osteoblasts respond to fluid flow-induced shear stress in vitro, osteoblasts provide a practical model for studying bone cell responses to shear stress
We focused on the morphology (F-actin, paxillin, integrin-α5, and α-tubulin) and volume of cell and nucleus, since these can change by pulsating fluid flow (PFF)-treatment

Summary

Introduction

In orthodontics, (oral) implantology, and orthopedics, mechanical stimuli are known to modulate bone mass, strength, and microstructure [1]. Direct sensing of mechanical loads, or lack thereof, by osteocytes and osteoblasts plays an important role in the shaping of bone tissue [3,4,5]. Both osteocytes and osteoblasts are responsive to the loading-induced flow of fluid, albeit osteocytes are most responsive [6]. Since osteoblasts (which terminally differentiate into osteocytes) respond to fluid flow-induced shear stress in vitro, osteoblasts provide a practical model for studying bone cell responses to shear stress. Osteocytes or osteoblasts translate the mechanical signal into a biological response, through a process known as mechanotransduction

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