Large Gradient High Magnetic Field Research Articles

Effects of large gradient high magnetic field (LG-HMF) on the long-term culture of aquatic organisms: Planarians example.

Large gradient high magnetic field (LG-HMF) is a powerful tool to study the effects of altered gravity on organisms. In our study, a platform for the long-term culture of aquatic organisms was designed based on a special superconducting magnet with an LG-HMF, which can provide three apparent gravity levels (µ g, 1 g, and 2 g), along with a control condition on the ground. Planarians, Dugesia japonica, were head-amputated and cultured for 5 days in a platform for head reconstruction. After planarian head regeneration, all samples were taken out from the superconducting magnet for a behavioral test under geomagnetic field and normal gravity conditions. To analyze differences among the four groups, four aspects of the planarians were considered, including head regeneration rate, phototaxis response, locomotor velocity, and righting behavior. Data showed that there was no significant difference in the planarian head regeneration rate under simulated altered gravity. According to statistical analysis of the behavioral test, all of the groups had normal functioning of the phototaxis response, while the planarians that underwent head reconstruction under the microgravity environment had significantly slower locomotor velocity and spent more time in righting behavior. Furthermore, histological staining and immunohistochemistry results helped us reveal that the locomotor system of planarians was affected by the simulated microgravity environment. We further demonstrated that the circular muscle of the planarians was weakened (hematoxylin and eosin staining), and the epithelial cilia of the planarians were reduced (anti-acetylated tubulin staining) under the simulated microgravity environment. Bioelectromagnetics. 2018;39:428-440. © 2018 Wiley Periodicals, Inc.

GeneChip expression profiling reveals the alterations of energy metabolism related genes in osteocytes under large gradient high magnetic fields.

The diamagnetic levitation as a novel ground-based model for simulating a reduced gravity environment has recently been applied in life science research. In this study a specially designed superconducting magnet with a large gradient high magnetic field (LG-HMF), which can provide three apparent gravity levels (μ-g, 1-g, and 2-g), was used to simulate a space-like gravity environment. Osteocyte, as the most important mechanosensor in bone, takes a pivotal position in mediating the mechano-induced bone remodeling. In this study, the effects of LG-HMF on gene expression profiling of osteocyte-like cell line MLO-Y4 were investigated by Affymetrix DNA microarray. LG-HMF affected osteocyte gene expression profiling. Differentially expressed genes (DEGs) and data mining were further analyzed by using bioinfomatic tools, such as DAVID, iReport. 12 energy metabolism related genes (PFKL, AK4, ALDOC, COX7A1, STC1, ADM, CA9, CA12, P4HA1, APLN, GPR35 and GPR84) were further confirmed by real-time PCR. An integrated gene interaction network of 12 DEGs was constructed. Bio-data mining showed that genes involved in glucose metabolic process and apoptosis changed notablly. Our results demostrated that LG-HMF affected the expression of energy metabolism related genes in osteocyte. The identification of sensitive genes to special environments may provide some potential targets for preventing and treating bone loss or osteoporosis.

Diamagnetic levitation promotes osteoclast differentiation from RAW264.7 cells.

The superconducting magnet with a high magnetic force field can levitate diamagnetic materials. In this study, a specially designed superconducting magnet with large gradient high magnetic field (LGHMF), which provides three apparent gravity levels (μg, 1 g, and 2 g), was used to study its influence on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation from preosteoclast cell line RAW264.7. The effects of LGHMF on the viability, nitric oxide (NO) production, morphology in RAW264.7 cells were detected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, the Griess method, and the immunofluorescence staining, respectively. The changes induced by LGHMF in osteoclast formation, mRNA expression, and bone resorption were determined by tartrate-resistant acid phosphatase staining, semiquantity PCR, and bone resorption test, respectively. The results showed that: 1) LGHMF had no lethal effect on osteoclast precursors but attenuated NO release in RAW264.7 cells. 2) Diamagnetic levitation (μg) enhanced both the formation and bone resorption capacity of osteoclast. Moreover, diamagnetic levitation up-regulated mRNA expression of RANK, Cathepsin K, MMP-9, and NFATc1, while down-regulated RunX2 in comparison with controls. Furthermore, diamagnetic levitation induced obvious morphological alterations in osteoclast, including active cytoplasmic peripheral pseudopodial expansion, formation of pedosome belt, and aggregation of actin ring. 3) Magnetic field produced by LGHMF attenuated osteoclast resorption activity. Collectively, LGHMF with combined effects has multiple effects on osteoclast, which attenuated osteoclast resorption with magnetic field, whereas promoted osteoclast differentiation with diamagnetic levitation. Therefore, these findings indicate that diamagnetic levitation could be used as a novel ground-based microgravity simulator, which facilitates bone cell research of weightlessness condition.

Gap junction mediated regulation of osteocytes to osteoblastic alkaline phosphatase activity is independent of microgravity

Decreased bone formation is one of the main causes of bone loss under microgravity. As a mechanical perceiver, osteocyte regulates bone formation by sending mechanical signals to osteoblast. However, the regulation of osteoblastic bone formation by osteocytes is less known so far under the microgravity conditions. The aim of this study was to investigate the regulation of bone formation/loss by detection of the osteocytic regulatory effects on the bone-specific alkaline phosphatase (ALP) activity of osteoblasts under altered gravitational environment. The altered gravitational environment was provided by a large gradient high magnetic field that could produce high magneto-gravitational environment (HMGE) and provided three apparent gravity levels (µg, 1g, and 2g). After the MLO-Y4 and 2T3 cells were cocultured in direct physical contact for 24 h and established gap junction intercellular communication under HMGE, a highly significant rapid increase in ALP activity was observed in µg, 1g, 2g, and control group (p < 0.001). Conversely, application of 50-µM beta-glycyrrhetinic acid gap junction inhibitor or remote coculture significantly decreased the ALP activity. There were no obvious differences of osteoblastic 2T3 ALP activity among µg, 1g, 2g, and control groups. Based on these findings, we interpreted that gap junction was probably the main route of osteoblastic ALP activity regulation by osteocyte. Gravity had no significant impact on the osteoblastic ALP activity regulated by osteocyte in a short period of time.

Large Gradient High Magnetic Fields Affect Osteoblast Ultrastructure and Function by Disrupting Collagen I or Fibronectin/αβ1 Integrin

The superconducting magnet generates a field and field gradient product that can levitate diamagnetic materials. In this study a specially designed superconducting magnet with a large gradient high magnetic field (LG-HMF), which can provide three apparent gravity levels (μ-g, 1-g, and 2-g), was used to simulate a space-like gravity environment. The effects of LG-HMF on the ultrastructure and function of osteoblast-like cells (MG-63 and MC3T3-E1) and the underlying mechanism were investigated by transmission electromicroscopy (TEM), MTT, and cell western (ICW) assays. Under LG-HMF significant morphologic changes in osteoblast-like cells occurred, including expansion of endoplasmic reticulum and mitochondria, an increased number of lysosomes, distorted microvilli, and aggregates of actin filaments. Compared to controls, cell viability and alkaline phosphatase (ALP) secretion were significantly increased, and collagen I (col I), fibronectin (FN), vinculin, integrin α3, αv, and β1 expression were changed under LG-HMF conditions. In conclusion, LG-HMF affects osteoblast ultrastructure, cell viability, and ALP secretion, and the changes caused by LG-HMF may be related to disrupting col I or FN/αβ1 integrin.

Dynamic and quantitative phase-contrast imaging of living cells under simulated zero gravity by digital holographic microscopy and superconducting magnet

The experiment of dynamic and quantitative phase-contrast imaging of living cells in simulated zero gravity environment were performed by using digital holographic microscopy (DHM) combined with a superconducting magnet (SM). The SM with large gradient high magnetic field was used to simulate zero gravity by levitating biological living samples. The proposed DHM system provided highly efficient and versatile means for dynamically and quantitatively phase-contrast imaging MC3T3-E1 cells. To our knowledge, the phase images of living cells undergoing modifications and division under simulated zero gravity were firstly obtained by using DHM-SM prototype.

Large gradient high magnetic field affects FLG29.1 cells differentiation to form osteoclast-like cells

Purpose: We aimed to investigate the effects of different apparent gravities (μ g, 1 g and 2 g) produced by large gradient high magnetic field (LGHMF) on human preosteoclast FLG29.1 cells.Materials and methods: FLG29.1 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium. Cells were exposed to LGHMF for 72 h. On culture day 1, 2, 3, cell proliferation was detected by 3-(4,5)-dimethylthiahi-azo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) method. On day 3, cell apoptosis and necrosis were assayed by Hoechst and propidium iodide (PI) staining. After cells were exposed to LGHMF for 72 h with the induction of 12-o-tetradecanoylphorbol 13-acetate (TPA), Tartrate-Resistant Acid Phosphatase (TRAP) positive cells and nitric oxide (NO) release were detected by TRAP staining and Griess method, respectively. Intracellular TRAP activity was measured using nitrophenylphosphate (pNPP) as the substrate.Results: MTT detection revealed that compared to control, FLG 29.1 cell proliferation in the μ g and 2 g groups were promoted. However, there is no obvious difference between the 1 g and control groups. Hoechst-PI staining showed that LGHMF promoted cell apoptosis and necrosis, especially in the 2 g group. Exposure to LGHMF inhibited the NO concentration of supernatant. Both the TRAP activity and the number of TRAP positive cells were higher in cells of μ g group than those in 2 g group. In the 1 g group, they were decreased significantly compared to control.Conclusions: These findings indicate that LGHMF could directly affect human preosteoclast FLG29.1 cells survival and differentiation. High magnetic flux inhibited osteoclasts formation and differentiation while reduced apparent gravity enhanced osteoclastogenesis.

Selection of suitable reference genes from bone cells in large gradient high magnetic field based on GeNorm algorithm

Studies of animals and humans subjected to spaceflight demonstrate that weightlessness negatively affects the mass and mechanical properties of bone tissue. Bone cells could sense and respond to the gravity unloading, and genes sensitive to gravity change were considered to play a critical role in the mechanotransduction of bone cells. To evaluate the fold-change of gene expression, appropriate reference genes should be identified because there is no housekeeping gene having stable expression in all experimental conditions. Consequently, expression stability of ten candidate housekeeping genes were examined in osteoblast-like MC3T3-E1, osteocyte-like MLO-Y4, and preosteoclast-like FLG29.1 cells under different apparent gravities (μg, 1 g, and 2 g) in the high-intensity gradient magnetic field produced by a superconducting magnet. The results showed that the relative expression of these ten candidate housekeeping genes was different in different bone cells; Moreover, the most suitable reference genes of the same cells in altered gravity conditions were also different from that in strong magnetic field. It demonstrated the importance of selecting suitable reference genes in experimental set-ups. Furthermore, it provides an alternative choice to the traditionally accepted housekeeping genes used so far about studies of gravitational biology and magneto biology.

Diamagnetic Levitation Causes Changes in Actin Architecture and Actin-Binding Proteins in Bone Cells

Diamagnetic levitation technology is a novel approach for simulated weightlessness and has only recently been applied in biological research. We have developed a superconducting magnet platform with large gradient high magnetic field (LG-HMF), which can provide three apparent gravity levels, namely μ-g (diamagnetic levitation), 1-g, and 2-g for diamagnetic materials. Here we studied the effects of LG-HMF on the morphology of actin cytoskeleton and the expression of actin-binding protein vinculin, paxillin, talin and MACF1 (microtubule actin crosslinking factor) in bone cells. The results showed that LG-HMF treatment of 48 hours had no acute lethal effects on both osteoblast-like and osteocyte-like cells. Compared to the nature 1-g control, μ-g significantly altered the cell shape, actin cytoskeleton structure (Fig 1), and the distribution and expression of actin-binding proteins in bone cells. In contrast, LG-HMF-produced 1-g and 2-g apparent gravity did not have such effects on bone cells. The results indicate that bone cells are sensitive to microgravity and vinculin, paxillin, talin and MACF1 may be involved in the mechanosensation and adaptation of bone cells. The diamagnetic levitation system provides a novel ground-based microgravity simulator for cell biological studies.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Diamagnetic Levitation Causes Changes in the Morphology, Cytoskeleton, and Focal Adhesion Proteins Expression in Osteocytes

Diamagnetic levitation technology is a novel simulated weightless technique and has recently been applied in life-science research. We have developed a superconducting magnet platform with large gradient high magnetic field (LG-HMF), which can provide three apparent gravity levels, namely, μg (diamagnetic levitation), 1g, and 2g for diamagnetic materials. In this study, the effects of LG-HMF on the activity, morphology, and cytoskeleton (actin filament, microtubules, and vimentin intermediate filaments) in osteocyte - like cell line MLO-Y4 were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) methods, hematoxylin-eosin (HE) staining, and laser scanning confocal microscopy (LSCM), respectively. The changes induced by LG-HMF in distribution and expression of focal adhesion (FA) proteins, including vinculin, paxillin, and talin in MLO-Y4 were determined by LSCM and Western blotting. The results showed that LG-HMF produced by superconducting magnet had no lethal effects on MLO-Y4. Compared to control, diamagnetic levitation (μg) affected MLO-Y4 morphology, nucleus size, cytoskeleton architecture, and FA proteins distribution and expression. The study indicates that osteocytes are sensitive to altered gravity and FA proteins (vinculin, paxillin, and talin) may be involved in osteocyte mechanosensation. The diamagnetic levitation may be a novel ground-based space-gravity simulator and can be used for biological experiment at cellular level.

Application of Magnetic Levitation of Diamagnetic Materials for Space Biology and Biotechnology

失重是特定空间运动条件下的重要环境物理特征之一, 一般以微重力环境来表示. 几十年来人类利用空间失重环境进行了多学科领域的科学研究与探索. 由于真实空间失重环境下科学实验机会稀少, 人类为研究空间失重环境或效应, 开发了多种地基的空间模拟实验技术方法. 然而, 对于空间生物学和空间生物技术研究而言, 已有的各种模拟实验技术手段在原理上和应用上均存在一定的局限性. 本文介绍了抗磁性物质在大梯度强磁场中的悬浮现象, 及将其用于模拟空间失重环境的方法与原理；简述了近年来利用抗磁性物质悬浮方法进行生物大分子晶体生长、分子细胞生物学及整体生物学等方面研究与应用的进展.

Effects of Microgravity Modeled by Large Gradient High Magnetic Field on the Osteogenic Initiation of Human Mesenchymal Stem Cells

Microgravity (MG) leads to a decrease in osteogenic potential of human bone marrow-derived mesenchymal stem cells (hMSCs). In the present study, we used large gradient high magnetic field (LGHMF) produced by a superconducting magnet to model MG (LGHMF-MG) and analyzed the effects of LGHMF-MG on survival, cytoskeleton and osteogenic potential of hMSCs. Results showed that the LGHMF-MG treatment for 6 h disrupted the cytoskeleton of hMSCs, and the LGHMF-MG treatment for 24 h led to cell death. LGHMF-MG treatments for 6 h in early stages of osteogenic induction (the pre-treatment before osteogenic induction, the beginning-treatment in the beginning-stage of osteogenic induction and the middle-treatment in the middle-stage of osteogenic induction) resulted in suppression on osteogenesis of hMSCs. The suppression intensity was reduced gradually as the treatment stage of LGHMF-MG was postponed. The LGHMF-MG treatment for 6 h in the ending-stage of osteogenic induction (the ending-treatment) had no obvious effect on osteogenesis of hMSCs. These results indicated that LGHMF-MG should affect the initiation of osteogenesis. Finally, the possible mechanism for the inhibition effect of LGHMF-MG on osteogenesis of hMSCs is discussed.

Large gradient high magnetic field affects the association of MACF1 with actin and microtubule cytoskeleton

The intense inhomogeneous magnetic fields acting on the diamagnetic materials naturally present in cells can generate strong magnetic forces. We have developed a superconducting magnet platform with large gradient high magnetic field (LG-HMF), which can produce three magnetic force fields of -1360, 0, and 1312 T(2)/m, and three corresponding apparent gravity levels, namely 0, 1, and 2-g for diamagnetic materials. In this study, the effects of different magnetic force fields on osteoblast-like cells (MG-63 and MC3T3-E1) viability, microtubule actin crosslinking factor 1 (MACF1) expression and its association with cytoskeleton were investigated. Results showed that cell viability increased to different degrees after exposure to 0 or 1-g conditions for 24 h, but it decreased by about 30% under 2-g conditions compared with control conditions. An increase in MACF1 expression at the RNA or protein level was observed in osteoblast-like cells under the magnetic force field of -1360 T(2)/m (0-g) relative to 1312 T(2)/m (2-g). Under control conditions, anti-MACF1 staining was scattered in the cytoplasm and partially colocalized with actin filaments (AFs) or microtubules (MTs) in the majority of osteoblast-like cells. Under 0-g conditions, MACF1 labeling was concentrated at perinuclear region and colocalization was not apparent. The patterns of anti-MACF1 labeling on MTs varied with MTs' changing under LG-HMF environment. In conclusion, LG-HMF affects osteoblast-like cell viability, MACF1 distribution, expression, and its association with cytoskeleton to some extent.

Development of a Ground-Based Simulated Experimental Platform for Gravitational Biology

<para xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> The purpose of this study is to develop a ground-based experimental platform which can provide a long-term, persistent, and stable weightless environment to provide technical support for the fundamental study of space life sciences. The platform included a superconducting magnet which can generate a large gradient high magnetic field, temperature control system, object stage, observing system, and gas control system. The platform can accurately measure and control comprehensive parameters, such as temperature, humidity, sample location, gravity level, and magnetic induction intensity. The biological experiment by means of the platform indicated that diamagnetic levitation affected cell morphology but had nonlethal effects on cell growth, which is consistent with the results of spaceflight. It suggests that the platform can meet the need of the simulated experimental conditions for gravitational biology research from molecule, cell, tissue, and organ to model animal. </para>

Gravitational environment produced by a superconducting magnet affects osteoblast morphology and functions

The aims of this study are to investigate the effects of gravitational environment produced by a superconducting magnet on osteoblast morphology, proliferation and adhesion. A superconducting magnet which can produce large gradient high magnetic field (LGHMF) and provide three apparent gravity levels ( 0 g , 1 g and 2 g ) was employed to simulate space gravity environment. The effects of LGHMF on osteoblast morphology, proliferation, adhesion and the gene expression of fibronectin and collagen I were detected by scanning electron microscopy, immunocytochemistry, adhesion assays and real time PCR, respectively, after exposure of osteoblasts to LGHMF for 24 h. Osteoblast morphology was affected by LGHMF ( 0 g , 1 g and 2 g ) and the most evident morphology alteration was observed at 0 g condition. Proliferative abilities of MC3T3 and MG-63 cell were affected under LGHMF ( 0 g , 1 g and 2 g ) conditions compared to control condition. The adhesive abilities of MC3T3 and MG-63 cells to extracellular matrix (ECM) proteins (fibronectin, laminin, collagen IV) were also affected by LGHMF ( 0 g , 1 g and 2 g ) , moreover, the effects of LGHMF on osteoblast adhesion to different ECM proteins were different. Fibronectin gene expression in MG63 cells under zero gravity condition was increased significantly compared to other conditions. Collagen I gene expression in MG-63 and MC3T3 cells was altered by both magnetic field and alerted gravity. The study indicates that the superconducting magnet which can produce LGHMF may be a novel ground-based space gravity simulator and can be used for biological experiment at cellular level.