A Study of a Cell Mechanosensing System Under Mechanical Vibration Considering its Modes of Vibration and Calcium Ion Response

2017 ◽  
Author(s):  
Yuki Nakamura ◽  
Toshihiko Shiraishi
Keyword(s):  
Calcium Ion ◽  
Mechanical Vibration ◽  
Ion Concentration ◽  
Experimental Result ◽  
Osteoblastic Cells ◽  
Confocal Laser ◽  
Fluorescent Intensity ◽  
Modes Of Vibration ◽  
A Cell ◽  
Cell Mechanosensing

It was reported that osteoblastic cells respond to mechanical vibration and generate the bone mass with a peak at a specific frequency like a resonance curve [1]. There seems to be an analogy between its cell response and the resonance of a cell as a mechanical system. This paper describes a novel method to measure the cellular modes of vibration of a cell and its calcium ion response under mechanical vibration, and the evaluation of the obtained results to clarify the mechanism of the cell mechanosensing. Nuclei and calcium ion in osteoblastic cells were visualized with fluorescent labelling. Mechanical vibration was applied to cells in a dish in the horizontal direction under a confocal laser scanning microscope by an exciter. Since the fluorescent intensity was very weak due to high frame rate to capture moving cells under Mechanical vibration, we used a high-speed and high-sensitive camera adjusting various conditions such as exposure time. We realized the spatial resolution of approximately 2 μm in the captured micrographs even under mechanical vibration using the experimental setup. As a result, the modes of vibration of nuclei was not obtained in this resolution. We found that the intracellular calcium ion concentration began to increase in a few seconds after mechanical vibration was applied. This experimental result indicates that applying mechanical vibration to cells can produce calcium signals as a second messenger by causing the entry of the ion.

Investigation of a Cell Mechanosensing System by Measuring Cytoskeletal Deformation and Intracellular Calcium Ion Concentration

2011 ◽  
Author(s):  
Toshihiko Shiraishi ◽  
Kazuhiro Sakata ◽  
Shin Morishita ◽  
Ryohei Takeuchi
Keyword(s):  
Mechanical Stimulation ◽  
Calcium Ion ◽  
Fluorescent Protein ◽  
Gene Transfection ◽  
Local Deformation ◽  
Ion Concentration ◽  
Fluorescent Intensity ◽  
A Cell ◽  
The Relationship ◽  
In Cells

This paper describes a method to investigate the relationship between cytoskeletal deformation by mechanical stimulation and its corresponding intracellular signals for identifying mechanosensors of cells. Gene transfection of green fluorescent protein to osteoblasts enabled visualization of actin in cells. When local deformation was applied to a cell by a micropipette, the distribution of cytoskeletal actin deformation in the whole cell was automatically obtained from the two images of the cell before and after deformation by using KLT method. Calcium ion signaling response to the same mechanical stimulation was measured as the spatial and temporal changes of fluorescent intensity of fluo-4 loaded to osteoblasts. As a result, we obtained the relationship between the deformation and the biochemical signals in cells.

Study on a Cell Mechanosensing System by Measuring Structural Deformation and Biochemical Response

2015 ◽  
Author(s):  
Atsushi Horiguchi ◽  
Toshihiko Shiraishi
Keyword(s):  
Mechanical Stimulation ◽  
Calcium Ion ◽  
Quantitative Relationship ◽  
Ion Concentration ◽  
Structural Deformation ◽  
Biochemical Response ◽  
Intracellular Calcium Ion ◽  
A Cell ◽  
Calcium Ion Concentration ◽  
Cell Mechanosensing

Mechanical stimulation induces new bone formation in vivo and promotes the metabolic activity and the gene expression of osteoblasts in vitro. It was reported that biochemical signals of osteoblasts to sense mechanical stimulation are activated according to their actin cytoskeletal deformation. However, there have been not so many researches on the relationship between cytoskeletal deformation and biochemical response. Here we show an original method to investigate a cell mechanosensing system and the quantitative relationship between the deformation of cytoskeletal structure and the change of intracellular calcium ion concentration as biochemical response in a living cell stimulated by a micropipette. Gene transfection of green fluorescent protein to osteoblastic cells enabled visualization of actin in cells. When local deformation was applied to a single osteoblastic cell by a micropipette, the displacement distribution of cytoskeletal structure in the whole cell was automatically obtained from the two images of the cell before and after deformation by using Kanade-Lucas-Tomasi (KLT) method. Intracellular calcium ion response to mechanical stimulation was measured as the spatial and temporal changes of intensity of Fura Red loaded to a cell. As a result, we obtained the quantitative relationship between structural deformation and biochemical response of a cell and found that the change of calcium ion concentration increases with increasing the displacement of actin cytoskeleton. It indicates that the deformation of actin cytoskeleton is highly related to the cell mechanosensing system.

A Study of Mechanosensing of an Osteoblast at Focal Adhesions Under Cyclic Strain Using Magnetic Micropillars

2019 ◽  
Author(s):  
Toshihiko Shiraishi ◽  
Kota Nagai
Keyword(s):  
Calcium Ion ◽  
Mechanical Vibration ◽  
Focal Adhesions ◽  
Cyclic Strain ◽  
Mechanical Stimuli ◽  
Osteoblast Cells ◽  
Intracellular Calcium Ion ◽  
A Cell ◽  
Sense And Respond ◽  
Biochemical Signals

Abstract It has been reported that cells sense and respond to mechanical stimuli. Mechanical vibration promotes the cell proliferation and the cell differentiation of osteoblast cells at 12.5 Hz and 50 Hz, respectively. It indicates that osteoblast cells have a mechansensing system for mechanical vibration. There may be some mechanosensors and we focus on cellular focal adhesions through which mechanical and biochemical signals may be transmitted from extracellular matrices into a cell. However, it is very difficult to directly apply mechanical stimuli to focal adhesions. We developed a magnetic micropillar substrate on which micron-sized pillars are deflected according to applied magnetic field strength and focal adhesions adhering to the top surface of the pillars are given mechanical stimuli. In this paper, we focus on intracellular calcium ion as a second messenger of cellular mechanosensing and investigate the mechanosensing mechanism of an osteoblast cell at focal adhesions under cyclic strain using a magnetic micropillar substrate. The experimental results indicate that the magnetic micropillars have enough performance to response to an electric current applied to a coil in an electromagnet and to apply the cyclic strain of less than 3% to a cell. In the cyclic strain of less than 3%, the calcium response of a cell was not observed.

Measurement of Modes of Vibration of a Cultured Cell for its Mechanosensing Mechanisms

2020 ◽  
Author(s):  
Katsuya Sato ◽  
Toshihiko Shiraishi
Keyword(s):  
Single Cell ◽  
Cultured Cells ◽  
Mrna Level ◽  
Mechanical Vibration ◽  
Mode Shapes ◽  
Control Group ◽  
Cellular Mechanisms ◽  
Biochemical Responses ◽  
Modes Of Vibration ◽  
A Cell

Abstract Applying mechanical vibration to cultured cells gives intracellular biochemical responses activated, for instance, their gene expression level is increased, so that it can be applied in the medical field. However, the cellular mechanisms of sensing mechanical vibration and transducing into the biochemical responses have not been clarified. One of the previous studies culturing osteoblastic cells under mechanical vibration showed that some of the intracellular biochemical responses reached peak depending on the frequencies like a resonance in the mechanical engineering field; the mRNA level of alkaline phosphatase at the frequency of 50 Hz reaches approximately 4.5 times as high as that at the control group [1]. Considering the analogy between the mechanical and the biochemical responses of a cell, the modes of vibration of a cell are thought to be related to the mechanosensing. In this study, the mode shapes of a single cell were experimentally measured under mechanical vibration up to 100 Hz using an improved experimental system with high natural frequencies designed to be low mass and high rigidity. This present paper will report the obtained experimental results of the mode shapes of a single cell nucleus and may contribute to the elucidation of the mechanosensing mechanisms of a cell for mechanical vibration.

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