Mechanobiological Modulation of Cytoskeleton and Calcium Influx in Osteoblastic Cells by Short-Term Focused Acoustic Radiation Force

Shu Zhang,Jiqi Cheng,Yi-Xian Qin,Chi Zhang

doi:10.1371/journal.pone.0038343

Abstract

Mechanotransduction has demonstrated potential for regulating tissue adaptation in vivo and cellular activities in vitro. It is well documented that ultrasound can produce a wide variety of biological effects in biological systems. For example, pulsed ultrasound can be used to noninvasively accelerate the rate of bone fracture healing. Although a wide range of studies has been performed, mechanism for this therapeutic effect on bone healing is currently unknown. To elucidate the mechanism of cellular response to mechanical stimuli induced by pulsed ultrasound radiation, we developed a method to apply focused acoustic radiation force (ARF) (duration, one minute) on osteoblastic MC3T3-E1 cells and observed cellular responses to ARF using a spinning disk confocal microscope. This study demonstrates that the focused ARF induced F-actin cytoskeletal rearrangement in MC3T3-E1 cells. In addition, these cells showed an increase in intracellular calcium concentration following the application of focused ARF. Furthermore, passive bending movement was noted in primary cilium that were treated with focused ARF. Cell viability was not affected. Application of pulsed ultrasound radiation generated only a minimal temperature rise of 0.1°C, and induced a streaming resulting fluid shear stress of 0.186 dyne/cm2, suggesting that hyperthermia and acoustic streaming might not be the main causes of the observed cell responses. In conclusion, these data provide more insight in the interactions between acoustic mechanical stress and osteoblastic cells. This experimental system could serve as basis for further exploration of the mechanosensing mechanism of osteoblasts triggered by ultrasound.

Highlights

Bone is in a constant state of dynamic equilibrium in its mechanical loading and adopts changes in function and architecture due to these stimuli
We develop a methodology to allow for in-vitro mechanical manipulation of osteoblastic cells using focused acoustic radiation force (ARF) and observe the morphological and calcium signaling responses
Cell Cultures Cells from the MC3T3-E1 mouse osteoblastic cell line (ATCC, Manassas, VA) were grown on 35 mm plastic cell culture Petri dishes in 95% air–5% CO2 in Dulbecco’s modified Eagle Medium (DMEM; Gibco, Grand Island, NY) which was supplemented with 20 mM HEPES and 10% heat-inactivated FBS, 2 mM glutamine, penicillin (100 U/ml), and streptomycin (100 Ag/ml)

Summary

Introduction

Bone is in a constant state of dynamic equilibrium in its mechanical loading (or reduced loading) and adopts changes in function and architecture due to these stimuli. Osteoblasts, known as boneforming cells, can sense mechanical stimuli such as stress or strain [1]. A wide variety of cell-level mechanical stimuli occur due to loading, including but not limited to substrate strain, direct cellular deformation, compressive loading (increased hydrostatic pressure), intramedullary pressure and interstitial fluid flow [2,3,4]. These mechanical stimuli have been utilized for in vitro experiments to clarify the characteristics of osteoblastic responses. Despite the fact that many types of mechanical stimuli have been studied intensively during the past two decades, very little is known about the cellular and molecular mechanisms triggered by ultrasound in bone mechanobiology

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Conclusion