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Magnetic Therapy Research: Cellular Activity

General

 

Proliferation / Differentiation / Apoptosis (Life / Growth / Death)

 

Orientation / Behavior

 

Calcium incorporation in cultured chondroblasts perturbed by an electromagnetic field.

Norton LA, Rovetti LA.

Department of Orthodontics, University of Connecticut Health Center, School of Dental Medicine, Farmington 06032.

We tested the hypothesis that electric perturbation influences 45Ca incorporation in extracellular matrix (ECM) of cartilage in vitro. Hypertrophic chondroblasts of tibial epiphyses (HC), sternum (SC), and skin fibroblasts (F) were cultured from chick embryos. HC, SC, and F cells were micromass seeded three times per week and maintained at 37.5 degrees C with 5% CO2 for two weeks. Cultures were randomly designated control (C) or exposed (E) to a pulsed electromagnetic field (PEMF). A time course experiment of calcium incorporation for all cultured groups showed that 24 h of exposure produced the largest biological response in chondroblasts. Calcium incorporation required supplemental phosphate. Autoradiography data indicated that the calcium incorporation into macromolecules largely occurred in the ECM. 45Ca steady-state perturbation was enhanced by Streptomyces hyaluronidase (SH) but not by testicular hyaluronidase (TH). 45Ca incorporation experiments tested the effects of phosphate, SH, TH, and PEMF alone and in various combinations on these cultures. Only PEMF or SH plus PEMF with phosphate enhanced 45Ca incorporation. Other experiments examined the effect of rotenone or freeze-thawing on cells exposed to PEMF. PEMF plus freeze-thaw enhanced calcium incorporation in HC only. PEMF appeared to cause disruption of the ECM, enhancing the probability of matrix calcification.

J Orthop Res. 1988;6(4):559-66.

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Effect of pulsed electromagnetic fields (PEMF) on osteoblast-like cells. Alterations of intracellular Ca2+.

Satake T.

Department of Oral Biochemistry, Kanagawa Dental College.

Low-energy electromagnetic fields pulsed at frequencies of 60-90 Hz significantly increase healing of chronic fracture nonunions in man. These fields are effective at tissue current levels as low as several orders of magnitude lower than those required for transmembrane depolarization of normal cells. In this study, the effects of PEMF on culture of rat osteoblast-like cells have been examined. The PEMF promoted the growth of these cells, were also found to increase the basal level of [Ca2+]i, and to decrease the responses towards epidermal growth factor (EGF) and serum, when the degree of response was based on the intracellular Ca2+ transient. These effects of PEMF were mimicked by 12-O-tetradecanoyl phorbol 13-acetate (TPA), a potent activator of protein kinase C. Pretreatment of TPA enhanced the cell growth and suppressed the intracellular Ca2+ transient induced with EGF and then serum to about 170% of the control. Then, present study investigated how the PEMF and TPA modulate EGF receptors of these cells. Both PEMF and TPA decreased the level of EGF binding to these cells down to about 65% and 75%, respectively. Scatchard analysis revealed the decrease of EGF receptor without a significant change in the affinity for EGF by both. In conclusion, it was indicated that PEMF acts at cell membrane and modulates the receptors which is essential for cell growth and DNA synthesis.

Kanagawa Shigaku. 1990 Mar;24(4):692-701.

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Membrane response to static magnetic fields: effect of exposure duration.

Rosen AD.

Department of Neurology, School of Medicine, State University of New York, Stony Brook 11794-8121.

The time-course for the reversible alteration in presynaptic membrane function associated with exposure to a 123 mT static magnetic field was examined in an attempt to help define the mechanism whereby these fields influence biomembranes. Miniature endplate potentials (MEPPs) were recorded in the isolated murine neuromuscular junction preparation, maintained at a temperature of 35.5 degrees C. A minimum field duration of 50 s was found to be necessary for MEPP inhibition, with the efficacy of the field in inducing further inhibition being a function of its duration, but only for periods up to 150 s. Longer durations were not associated with additional inhibition. The time required for MEPP frequency to return to baseline, following deactivation of the field, was found to be linear for field durations up to 150 s. At and above this limit, recovery time remained constant at 135 s. These findings are consistent with the slow reorientation of diamagnetic molecular domains within the membrane and suggest tight coupling to the mechanism responsible for neurotransmitter release. The limits on this effect are compatible with the mechanical constraints imposed by the membrane's cytoskeleton.

Biochim Biophys Acta. 1993 Jun 5;1148(2):317-20.

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Resting potential of excitable neuroblastoma cells in weak magnetic fields.

Sonnier H, Kolomytkin OV, Marino AA.

Department of Orthopedic Surgery, LSU Medical Center in Shreveport, Louisiana 71130-3932, USA.

The mechanism by which static and low-frequency magnetic fields are transduced into biological signals responsible for reported effects on brain electrical activity is not yet ascertained. To test the hypothesis that fields can cause a subthreshold change in the resting membrane potential of excitable cells, we measured changes in transmembrane current under voltage clamp produced in SH-SY5Y neuroblastoma cells, using the patch-clamp method in the whole-cell configuration. In separate experiments, cells were exposed to static fields of 1, 5, and 75 G, to time-varying fields of 1 and 5 G, and to combined static and time-varying fields tuned for resonance of Na+, K+, Ca2+, or H+. To increase sensitivity, measurements were made on cells connected by gap junctions. For each cell, the effect of the field was evaluated on the basis of 100 trials consisting of a 5-s exposure immediately followed by a 5-s control period. In each experiment, the field had no discernible effect on the transmembrane current in the vicinity of zero current (- 50 mV voltage clamp). The sensitivity of the measuring system was such that we would have detected a current corresponding to a change in membrane potential as small as 38 microV. Consequently, if sensitivity of mammalian cells to magnetic fields is mediated by subthreshold changes in membrane potential, as in sensory transduction of sound, light, and other stimuli, then the ion channels responsible for the putative changes are probably present only in specialized sensory neurons or neuroepithelial cells. A change in transmembrane potential in response to magnetic fields is not a general property of excitable cells in culture.

Cell Mol Life Sci. 2000 Mar;57(3):514-20.

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Pulsed electromagnetic fields affect the intracellular calcium concentrations in human astrocytoma cells.

Pessina GP, Aldinucci C, Palmi M, Sgaragli G, Benocci A, Meini A, Pessina F.

Institute of General Physiology and Nutritional Science, University of Siena, Italy. pessina@unisi.it

Experiments assessed whether long term exposure to 50 Hz pulsed electromagnetic fields with a peak magnetic field of 3 mT can alter the dynamics of intracellular calcium in human astrocytoma U-373 MG cells. Pretreatment of cells with 1.2 microM substance P significantly increased the [Ca(2+)](i). The same effect was also observed when [Ca(2+)](i) was evaluated in the presence of 20 mM caffeine. After exposure to electromagnetic fields the basal [Ca(2+)](i) levels increased significantly from 143 +/- 46 nM to 278 +/- 125 nM. The increase was also evident after caffeine addition, but in cells treated with substance P and substance P + caffeine we observed a [Ca(2+)](i) decrease after exposure. When we substituted calcium-free medium for normal medium immediately before the [Ca(2+)](i) measurements, the [Ca(2+)](i) was similar to that measured in the presence of Ca(2+). In this case, after EMFs exposure of cells treated with substance P, the [Ca(2+)](i), measured without and with addition of caffeine, declined from 824 +/- 425 to 38 +/- 13 nM and from 1369 +/- 700 to 11 +/- 4 nM, respectively, indicating that electromagnetic fields act either on intracellular Ca(2+) stores or on the plasma membrane. Moreover the electromagnetic fields that affected [Ca(2+)](i) did not cause cell proliferation or cell death and the proliferation indexes remained unchanged after exposure. Copyright 2001 Wiley-Liss, Inc.

Bioelectromagnetics. 2001 Oct;22(7):503-10.

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The effect of strong static magnetic field on lymphocytes.

Aldinucci C, Garcia JB, Palmi M, Sgaragli G, Benocci A, Meini A, Pessina F, Rossi C, Bonechi C, Pessina GP.

Department of Physiology, University of Siena, Siena, Italy.

We investigated whether static electromagnetic fields (EMFs) at a flux density of 4.75 T, generated by an NMR apparatus (NMRF), could promote movements of Ca2+, cell proliferation, and the eventual production of proinflammatory cytokines in human peripheral blood mononuclear cells (PBMC) as well as in Jurkat cells, after exposure to the field for 1 h. The same study was also performed after activation of cells with 5 mg/ml phytohaemagglutinin (PHA). Our results clearly demonstrate that static NMRF exposure has neither proliferative, nor activating, nor proinflammatory effects on both normal and PHA activated PBMC. Moreover, the concentration of interleukin-1beta, interleukin-2, interleukin-6, interferon, and tumour necrosis factor alpha (TNFalpha) remained unvaried in exposed cells. Exposure of Jurkat cells statistically decreased the proliferation and the proliferation indexes, which 24 and 48 h after exposure were 0.7 +/- 0.29 and 0.87 +/- 0.12, respectively. Moreover, in Jurkat cells the [Ca2+]i was higher than in PBMC and was reduced significantly to about one half after exposure. This is consistent with the decrease of proliferation and with the low levels of IL-2 measured. On the whole, our data suggest that NMRF exposure failed to affect the physiologic behaviour of normal lymphomonocytes. Instead in Jurkat cells, by changing the properties of cell membranes, NMRF can influence Ca2+ transport processes, and hence Ca2+ homeostasis with improvement of proliferation. Copyright 2003 Wiley-Liss, Inc.

Bioelectromagnetics. 2003 Feb;24(2):109-17.

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Interaction between weak low frequency magnetic fields and cell membranes.

Baureus Koch CL, Sommarin M, Persson BR, Salford LG, Eberhardt JL.

Department of Radiation Physics, Lund University Hospital, Lund, Sweden.

The question of whether very weak low frequency magnetic fields can affect biological systems, has attracted attention by many research groups for quite some time. Still, today, the theoretical possibility of such an interaction is often questioned and the site of interaction in the cell is unknown. In the present study, the influence of extremely low frequency (ELF) magnetic fields on the transport of Ca(2+) was studied in a biological system consisting of highly purified plasma membrane vesicles. We tested two quantum mechanical theoretical models that assume that biologically active ions can be bound to a channel protein and influence the opening state of the channel. Vesicles were exposed for 30 min at 32 degrees C and the calcium efflux was studied using radioactive (45)Ca as a tracer. Static magnetic fields ranging from 27 to 37 micro T and time varying magnetic fields with frequencies between 7 and 72 Hz and amplitudes between 13 and 114 micro T (peak) were used. We show that suitable combinations of static and time varying magnetic fields directly interact with the Ca(2+) channel protein in the cell membrane, and we could quantitatively confirm the model proposed by Blanchard. Copyright 2003 Wiley-Liss, Inc.

Bioelectromagnetics. 2003 Sep;24(6):395-402.

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Effect of a 125 mT static magnetic field on the kinetics of voltage activated Na+ channels in GH3 cells.

Rosen AD.

Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA. arosen@bilbo.bio.purdue.edu

Voltage activated Na(+) channels were examined in GH3 cells, using the whole cell patch clamp method. Channel currents were recorded before, during, and after a 150 s exposure to a 125 mT static magnetic field. There was a slight shift in the current-voltage relationship and a less than 5% reduction in peak current during magnetic field exposure. More pronounced, however, was an increase in the activation time constant, tau(m), during and for at least 100 s following exposure to the field. This change in tau(m) was seen primarily at lower activation voltages. No change was noted in the inactivation time constant, tau(h). Changes were clearly temperature dependent, being evident only at and above 35 degrees C. These findings are consistent with the hypothesis that reorientation of diamagnetic anisotropic molecules in the cell membrane are capable of distorting imbedded ion channels sufficiently to alter their function. The temperature dependence of this phenomenon is probably due to the greater ease with which a liquid crystal membrane can be deformed. Copyright 2003 Wiley-Liss, Inc.

Bioelectromagnetics. 2003 Oct;24(7):517-23.

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Effect of a static magnetic field on ion transport in a cellulose membrane.

Ohata R, Tomita N, Ikada Y.

Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, 606-8507 Kyoto, Japan. rohata@mech.kyoto-u.ac.jp

A cellulose membrane was exposed to the static magnetic field (SMF) in the presence of KCl solution and ion transport through the membrane was measured before and after the SMF exposure. SMF at 0.24 T significantly enhanced the rate of ion transport, especially after the first exposure (p<0.05), while the increased ion transport rate did not return to the initial basal level after exchange of the aqueous medium. These results suggest that an irreversible, temporal conformation change took place on the cellulose membrane or on the water bound to the cellulose surface. The accelerating effect of SMF on the ion transport seems to have occurred as a result of stabilized hydration layer on the cellulose surface.

J Colloid Interface Sci. 2004 Feb 15;270(2):413-6.

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Effects of pulsed electromagnetic fields on human chondrocytes: an in vitro study.

Pezzetti F, De Mattei M, Caruso A, Cadossi R, Zucchini P, Carinci F, Traina GC, Sollazzo V.

Dipartimento di Morfologia ed Embriologia, Universita di Ferrara, via Fossato di Mortara 64, 44100 Ferrara, Italy.

(3)H-thymidine incorporation was studied in cultured human nasal and articular chondrocytes exposed to low-energy, low-frequency pulsed electromagnetic fields (PEMFs) (75 Hz, 2.3 mT). The reverse transcriptase polymerase chain reaction (RT-PCR) analysis shows that human secondary chondrocytes derived from both nasal and articular cartilage express collagen type II mRNA, which is a specific marker of the chondrocyte phenotype. In a preliminary series of experiments, cells were exposed to PEMF for different time periods ranging from 6 to 30 hours (time-course), in medium supplemented with 10% or 0.5% fetal calf serum (FCS) and in serum-free medium. The ratios between the (3)H-thymidine incorporation in PEMFs and control cultures show an increase of the cell proliferation in cultures exposed to PEMFs when serum is present in the culture medium, whereas no effect was observed in serum-free conditions. The increase in DNA synthesis, induced by PEMFs, was then evaluated only at the times of maximum induction and the results were analyzed by the three-factor analysis of variance (ANOVA). The data presented in this study show that even if (3)H-thymidine incorporation is higher in nasal than in articular chondrocytes, PEMF induce an increase in the proliferation of both cell types. Moreover, the concentration of FCS in the culture medium greatly influences the proliferative response of human chondrocytes to the PEMF exposure. Though normal human osteoblast cells increase their proliferation when exposed to PEMFs if only 10% FCS is present in the medium, human chondrocytes are able to increase their cell proliferation when exposed to PEMFs in the presence of both 0.5% and 10% of FCS in the medium. The results obtained may help to explain the basic mechanisms of PEMF stimulation of fracture healing.

Calcif Tissue Int. 1999 Nov;65(5):396-401.

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Effects of low-frequency pulsed electromagnetic fields on the proliferation of chondrocytes.

Indouraine A, Petersen JP, Pforringer W.

Symbion Science Park (Symbion-Wissenschaftspark), Kopenhagen, Danemark.

Chondrocytes isolated from the human cartilage of 5 patients between the ages 23 and 56 were exposed to low frequency pulsed electromagnetic fields (9 mT; 3 Hz) for a daily period of 60 minutes on 5 consecutive days and then every 48 hours for the next 6 days (11 days in total). Cell viability was estimated using trypan blue exclusion and proliferation was estimated by counting the cells in a haemacytometer. Cell morphology was compared for control purposes by directly observing the cells under a light microscope after staining cells in a haematoxylin and eosin solution. The results were statistically analysed and compared to a control sample. Data revealed that exposing cells isolated from human cartilage to pulsed electromagnetic fields (9 mT; 3 Hz) led to a significantly higher number of cells in comparison to the control sample. Among the cells from the 5 patients, growth varied between 1.1 to 3.0 folds compared to the control sample. The difference in cell viability between the exposed cells and the control sample was, however, not significant. Some morphological variations were revealed when the cells were observed under a light microscope. The exposed cells were thinner and longer than the control cells which were large and flat. The exposed cells tended to grow in a more uniform direction while the control cells grew in all directions. These differences in morphology and growth may be related to the higher density of the exposed cells.

Sportverletz Sportschaden. 2001 Mar;15(1):22-7.

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Pulsed electromagnetic fields increase growth factor release by nonunion cells.

Guerkov HH, Lohmann CH, Liu Y, Dean DD, Simon BJ, Heckman JD, Schwartz Z, Boyan BD.

Department of Orthopaedics, University of Texas Health Science Center at San Antonio, 78229-3900, USA.

The mechanisms involved in pulsed electromagnetic field stimulation of nonunions are not known. Animal and cell culture models suggest endochondral ossification is stimulated by increasing cartilage mass and production of transforming growth factor-beta 1. For the current study, the effect of pulsed electromagnetic field stimulation on cells from human hypertrophic (n = 3) and atrophic (n = 4) nonunion tissues was examined. Cultures were placed between Helmholtz coils, and an electromagnetic field (4.5-ms bursts of 20 pulses repeating at 15 Hz) was applied to 1/2 of them 8 hours per day for 1, 2, or 4 days. There was a time-dependent increase in transforming growth factor-beta 1 in the conditioned media of treated hypertrophic nonunion cells by Day 2 and of atrophic nonunion cells by Day 4. There was no effect on cell number, [3H]-thymidine incorporation, alkaline phosphatase activity, collagen synthesis, or prostaglandin E2 and osteocalcin production. This indicates that human nonunion cells respond to pulsed electromagnetic fields in culture and that transforming growth factor-beta 1 production is an early event. The delayed response of hypertrophic and atrophic nonunion cells (> 24 hours) suggests that a cascade of regulatory events is stimulated, culminating in growth factor synthesis and release.

Clin Orthop. 2001 Mar;(384):265-79.

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Cell proliferation/cell death balance in renal cell cultures after exposure to a static magnetic field.

Buemi M, Marino D, Di Pasquale G, Floccari F, Senatore M, Aloisi C, Grasso F, Mondio G, Perillo P, Frisina N, Corica F.

Dipartimento di Medicina Interna, Facolta di Medicina e Chirurgia, Universita di Messina, Italy. Buemim@unime.it

The effect of a static magnetic field (MF) of 0.5 mT of intensity on the cell proliferation/cell death balance was investigated in renal cells (VERO) and cortical astrocyte cultures from rats. Magnetic stimulation was delivered by magnetic disks at known intensities. The percentage of apoptotic and necrotic cells was evaluated using flow cytometry and morphological analysis following Hoechst chromatin staining. An index of cell proliferation was determined using sulfonated tetrazolium (WST-1). Control cultures were prepared without exposure to MFs. After 2, 4 and 6 days of exposure to a MF, we observed a gradual decrease in apoptosis and proliferation and a gradual increase in cells with a necrotic morphology with respect to the control group. In astrocyte cultures, over a 6-day exposure period, a gradual increase was observed in apoptotic, proliferating, and necrotic cells. Our findings suggest that the effect of exposure to MFs varies, depending on the cell type; MFs may also have a nephropathogenic effect. Copyright 2001 S. Karger AG, Basel

Nephron. 2001 Mar;87(3):269-73.

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Effects of pulsed electromagnetic fields on human articular chondrocyte proliferation.

De Mattei M, Caruso A, Pezzetti F, Pellati A, Stabellini G, Sollazzo V, Traina GC.

Dipartimento di Morfologia ed Embriologia, Universita di Ferrara, Italy.

Low-energy, low-frequency pulsed electromagnetic fields (PEMFs) can induce cell proliferation in several cell culture models. In this work we analysed the proliferative response of human articular chondrocytes, cultured in medium containing 10% FBS, following prolonged exposure to PEMFs (75 Hz, 2.3 mT), currently used in the treatment of some orthopaedic pathologies. In particular, we investigated the dependence of the proliferative effects on the cell density, the availability of growth factors and the exposure lengths. We observed that PEMFs can induce cell proliferation of low density chondrocyte cultures for a long time (6 days), when fresh serum is added again in the culture medium. In the same conditions, in high density cultures, the PEMF-induced increase in cell proliferation was observed only in the first three days of exposure. The data presented in this study show that the availability of growth factors and the environmental constrictions strongly condition the cellular proliferative response to PEMFs.

Connect Tissue Res. 2001;42(4):269-79.

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Low frequency EMF regulates chondrocyte differentiation and expression of matrix proteins.

Ciombor DM, Lester G, Aaron RK, Neame P, Caterson B.

Department of Orthopaedics, School of Medicine, Brown University, Providence, RI, USA. deborah_ciombor@brown.edu

This study describes the enhancement of chondrogenic differentiation in endochondral ossification by extremely low frequency pulsed electric/magnetic fields (EMFs). The demineralized bone matrix (DBM)-induced endochondral ossification model was used to examine the effects of EMF stimulation. [35S]-Sulfate and [3H]-thymidine incorporation and glycosaminoglycan (GAG) content were determined by standard methods. Proteoglycan (PG) and GAG molecular size and composition were determined by gel chromatography and sequential enzyme digestion. Immunohistochemical and Western blot analysis of PGs were done with antibodies 2B6, 3B3, 2D3 and 5D4. Northern analysis of total RNA extracts was performed for aggrecan, and type II collagen. All data was compared for significance by Student's t- or analysis of variance (ANOVA)-tests. The EMF field accelerated chondrogenesis as evidenced by an increase in: (1) 35SO4 incorporation and GAG content, (2) the number of chondrocytes at day 8 of development, (3) the volumetric density of cartilage and (4) the extent of immunostaining for 3B3 and 5D4. No differences in DNA content or [3H]-thymidine incorporation were observed between control and stimulated ossicles, suggesting the absence of enhanced cell proliferation or recruitment as a mechanism for the acceleration. PG and GAG molecular sizes and GAG chemical composition were similar in stimulated and control ossicles, indicating that stimulation resulted in an accelerated synthesis of normal cartilage molecules. The increased expression of PG and type II collagen mRNA as well as a greater immunoreactivity of 3B3 and 5D4 suggest an increase in the rate of differentiation of chondrocytes and enhanced phenotypic maturation.

J Orthop Res. 2002 Jan;20(1):40-50.

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Effects of pulsed electromagnetic field (PEMF) stimulation on bone tissue like formation are dependent on the maturation stages of the osteoblasts.

Diniz P, Shomura K, Soejima K, Ito G.

Department of Orthodontics, Kagoshima University Dental School, Kagoshima, Japan.

The effects of pulsed electromagnetic field (PEMF, 15 Hz pulse burst, 7 mT peak) stimulation on bone tissue-like formation on osteoblasts (MC3T3-E1 cell line) in different stages of maturation were assessed to determine whether the PEMF stimulatory effect on bone tissue-like formation was associated with the increase in the number of cells and/or with the enhancement of the cellular differentiation. The cellular proliferation (DNA content), differentiation (alkaline phosphatase activity), and bone tissue-like formation (area of mineralized matrix) were determined at different time points. PEMF treatment of osteoblasts in the active proliferation stage accelerated cellular proliferation, enhanced cellular differentiation, and increased bone tissue-like formation. PEMF treatment of osteoblasts in the differentiation stage enhanced cellular differentiation and increased bone tissue-like formation. PEMF treatment of osteoblasts in the mineralization stage decreased bone tissue-like formation. In conclusion, PEMF had a stimulatory effect on the osteoblasts in the early stages of culture, which increased bone tissue-like formation. This stimulatory effect was most likely associated with enhancement of the cellular differentiation, but not with the increase in the number of cells. Copyright 2002 Wiley-Liss, Inc.

Bioelectromagnetics. 2002 Jul;23(5):398-405.

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Nitric oxide mediates the effects of pulsed electromagnetic field stimulation on the osteoblast proliferation and differentiation.

Diniz P, Soejima K, Ito G.

Department of Orthodontics, Kagoshima University Dental School, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan.

The purpose of this research was to investigate whether the effects of pulsed electromagnetic field (PEMF) stimulation on the osteoblast proliferation and differentiation are mediated by the increase in the nitric oxide (NO, nitrogen monoxide) synthesis. The osteoblasts (MC3T3-E1 cell line) were cultured in the absence (-NMMA group) or in the presence (+NMMA group) of the NO synthase inhibitor L-NMMA. First, osteoblasts were subjected to PEMF stimulation (15 Hz and 0.6 mT) up to 15 days. The DNA content and the NO concentration in the conditioned medium were determined on the 3rd, 7th, and 15th days of culture. Following, osteoblasts were stimulated in the proliferation (P-NMMA and P+NMMA groups) or in the differentiation (D-NMMA and D+NMMA groups) stages of maturation, and the alkaline phosphatase (AlPase) activity was determined on the 15th day of culture for all groups. PEMF stimulation increased significantly the nitrite concentration in the -NMMA group on the 3rd, 7th, and 15th days of culture. However, this effect was partially blocked in the +NMMA group. The DNA content in the -NMMA group, but not in the +NMMA group, increased significantly on the 3rd and 7th days of culture. The AlPase activity in the P-NMMA and D-NMMA groups, but not in the P+NMMA and D+NMMA groups, also increased significantly. In conclusion, the PEMF stimulatory effects on the osteoblasts proliferation and differentiation were mediated by the increase in the NO synthesis. Copyright 2002 Elsevier Science (USA)

Nitric Oxide. 2002 Aug;7(1):18-23.

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Exposure of cells to static magnetic field accelerates loss of integrity of plasma membrane during apoptosis.

Teodori L, Grabarek J, Smolewski P, Ghibelli L, Bergamaschi A, De Nicola M, Darzynkiewicz Z.

Department of Biomedicine and Toxicology, UTS BIOTEC, ENEA-Casaccia, Rome, Italy.

BACKGROUND: Much attention is being paid to the biologic effects of magnetic fields (MFs). Although MFs enhance tumorigenesis, they are neither mutagenic nor tumorigenic. The mechanism of their tumorigenic effect has not been elucidated.

METHODS: To investigate the effect of MFs on apoptosis in HL-60 cells, we exposed the cells to static MFs of 6 mT generated by a magnetic disk of known intensity. Apoptosis was triggered by the DNA topoisomerase I inhibitor, camptothecin (CPT). Activation of caspases in situ using the fluorochrome-labeled inhibitor (FLICA) method and determination of plasma membrane integrity by excluding propidium iodide (PI) were measured by both laser scanning cytometry (LSC) and flow cytometry (FC). LSC and FC identified cells at three sequential stages of their demise: early apoptosis (cells with activated caspases and PI negative); late apoptosis (cells with activated caspases but unable to exclude PI); secondary necrosis (cells with apoptotic morphology no longer stained with FLICA, not excluding PI).

RESULTS: MF alone did not induce any apoptogenic or necrogenic effect. CPT exposure led to the sequential appearance of apoptotic cells. In the presence of CPT and MF, the overall proportion of cells undergoing apoptosis was not significantly changed. However, we consistently observed a significant increase in the frequency of late apoptotic/necrotic cells when compared with samples treated with CPT alone (P < 0.001), as well as a decrease in the percentage of early apoptotic cells (P = 0.013). The data obtained by FC and LSC were consistent with each other, showing a similar phenomenon.

CONCLUSION: Whereas MF alone or with CPT did not affect overall cell viability, it accelerated the rate of cell transition from apoptosis to secondary necrosis after induction of apoptosis by the DNA-damaging agent, CPT. Modulation of the kinetics of the transition from apoptosis to secondary necrosis by MF in vivo may play a role in inflammation and tumorigenesis. Copyright 2002 Wiley-Liss, Inc.

Cytometry. 2002 Nov 1;49(3):113-8.

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Evaluation of the morphology of the human placental cotyledon following dual in vitro perfusion in variable magnetic field.

Lopucki M, Lancut M, Rogowska W, Czerny K, Jedrych B, Pietruszewski S, Kornarzynski K, Kotarski J.

I Katedry i Kliniki Ginekologii AM w Lublinie.

Objectives and the aim of the study was electron-microscopy morphological estimation of the human placental cotyledon after 180 minutes of dual closed perfusion in vitro.

MATERIALS AND METHODS: In the experimental group the cotyledons were exposed to variable magnetic field of 2 mT magnetic induction and 50 Hz frequency. The control group K (10 perfusions) was not subjected to magnetic field while the experimental group B (10 perfusions) was influenced by magnetic field.

RESULTS: It was found that homogeneous variable magnetic field disturbs the ultrastructure of the nuclei and cytoplasma and it increases the density of the vascular-epithelial membrane of villi cells of human placenta in vitro.

CONCLUSION: Variable sinusoildal, magnetic field of 2 mT magnetic induction and 50 Hz frequency disturbs the ultrastructure of the nuclei and cytoplasma and it increases the density of the vascular-epithelial membrane of villi cells of human placenta in vitro after 180 minutes of dual closed perfusion in vitro.

Ginekol Pol. 2003 Oct;74(10):1187-93.

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50-Hz magnetic field exposure influences DNA repair and mitochondrial DNA synthesis of distinct cell types in brain and kidney of adult mice.

Schmitz C, Keller E, Freuding T, Silny J, Korr H.

Department of Anatomy and Cell Biology, RWTH Aachen University, Pauwelsstrasse/Wendlingweg 2, 52057, Aachen, Germany.

Despite several recent investigations, the impact of whole-body magnetic field exposure on cell-type-specific alterations due to DNA damage and DNA repair remains unclear. In this pilot study adult mice were exposed to 50-Hz magnetic field (mean value 1.5 mT) for 8 weeks or left unexposed. Five minutes after ending exposure, the mice received [(3)H]thymidine and were killed 2 h later. Autoradiographs were prepared from paraffin sections of brains and kidneys for measuring unscheduled DNA synthesis and mitochondrial DNA synthesis, or in situ nick translation with DNA polymerase-I and [(3)H]dTTP. A significant ( P<0.05) increase in both unscheduled DNA synthesis and in situ nick translation was only found for epithelial cells of the choroid plexus. Thus, these two independent methods indicate that nuclear DNA damage is produced by long-lasting and strong magnetic field exposure. The fact that only plexus epithelial cells were affected might point to possible effects of magnetic fields on iron transport across the blood-cerebrospinal fluid barrier, but the mechanisms are currently not understood. Mitochondrial DNA synthesis was exclusively increased in renal epithelial cells of distal convoluted tubules and collecting ducts, i.e., cells with a very high content of mitochondria, possibly indicating increased metabolic activity of these cells.

Acta Neuropathol (Berl). 2003 Dec 19. [Epub ahead of print]

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Effects of 0.2 T static magnetic field on human skin fibroblasts.

Pacini S, Gulisano M, Peruzzi B, Sgambati E, Gheri G, Gheri Bryk S, Vannucchi S, Polli G, Ruggiero M.

Department of Human Anatomy, Histology and Forensic Medicine, University of Firenze, viale Morgagni 85, 50134, Firenze, Italy.

Human skin fibroblasts were exposed to 0.2 T static magnetic field generated by a magnetic resonance tomograph. After 1h exposure, cell morphology was modified in association with a concomitant decrease in the expression of some sugar residues of glycoconjugates. Study of cell proliferation and mitogenic signal transduction showed a decrease of thymidine incorporation and of second messenger formation. However, cell viability, assessed by colony forming assay, was unaffected. These results demonstrate that the static magnetic field generated by routinely used magnetic resonance tomograph induces alterations on human skin fibroblasts.

Cancer Detect Prev. 2003;27(5):327-32.

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Exposure to 900 MHz electromagnetic field induces an unbalance between pro-apoptotic and pro-survival signals in T-lymphoblastoid leukemia CCRF-CEM cells.

Marinelli F, La Sala D, Cicciotti G, Cattini L, Trimarchi C, Putti S, Zamparelli A, Giuliani L, Tomassetti G, Cinti C.

Institute for Organ Transplantation and Immunocytology, ITOI-CNR, Bologna unit, c/o IOR, Bologna, Italy.

It has been recently established that low-frequency electromagnetic field (EMFs) exposure induces biological changes and could be associated with increased incidence of cancer, while the issue remains unresolved as to whether high-frequency EMFs can have hazardous effect on health. Epidemiological studies on association between childhood cancers, particularly leukemia and brain cancer, and exposure to low- and high-frequency EMF suggested an etiological role of EMFs in inducing adverse health effects. To investigate whether exposure to high-frequency EMFs could affect in vitro cell survival, we cultured acute T-lymphoblastoid leukemia cells (CCRF-CEM) in the presence of unmodulated 900 MHz EMF, generated by a transverse electromagnetic (TEM) cell, at various exposure times. We evaluated the effects of high-frequency EMF on cell growth rate and apoptosis induction, by cell viability (MTT) test, FACS analysis and DNA ladder, and we investigated pro-apoptotic and pro-survival signaling pathways possibly involved as a function of exposure time by Western blot analysis. At short exposure times (2-12 h), unmodulated 900 MHz EMF induced DNA breaks and early activation of both p53-dependent and -independent apoptotic pathways while longer continuous exposure (24-48 h) determined silencing of pro-apoptotic signals and activation of genes involved in both intracellular (Bcl-2) and extracellular (Ras and Akt1) pro-survival signaling. Overall our results indicate that exposure to 900 MHz continuous wave, after inducing an early self-defense response triggered by DNA damage, could confer to the survivor CCRF-CEM cells a further advantage to survive and proliferate. Copyright 2003 Wiley-Liss, Inc.

J Cell Physiol. 2004 Feb;198(2):324-32.

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Low electromagnetic field (50 Hz) induces differentiation on primary human oral keratinocytes (HOK).

Manni V, Lisi A, Rieti S, Serafino A, Ledda M, Giuliani L, Sacco D, D'Emilia E, Grimaldi S.

Istituto di Neurobiologia e Medicina Molecolare (INeMM), CNR, Rome, Italy.

This work concerns the effect of low frequency electromagnetic fields (ELF) on biochemical properties of human oral keratinocytes (HOK). Cells exposed to a 2 mT, 50 Hz, magnetic field, showed by scanning electron microscopy (SEM) modification in shape and morphology; these modifications were also associated with different actin distribution, revealed by phalloidin fluorescence analysis. Moreover, exposed cells had a smaller clonogenic capacity, and decreased cellular growth. Indirect immunofluorescence with fluorescent antibodies against involucrin and beta-catenin, both differentiation and adhesion markers, revealed an increase in involucrin and beta-catenin expression. The advance in differentiation was confirmed by a decrease of expression of epidermal growth factor (EGF) receptor in exposed cells, supporting the idea that exposure to electromagnetic field carries keratinocytes to higher differentiation level. These observations support the hypothesis that 50 Hz electromagnetic fields may modify cell morphology and interfere in differentiation and cellular adhesion of normal keratinocytes. Copyright 2004 Wiley-Liss, Inc.

Bioelectromagnetics. 2004 Feb;25(2):118-26.

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Pulsing electromagnetic fields induce cellular transcription.

Goodman R, Bassett CA, Henderson AS.

Weak, pulsing electromagnetic fields can modify biological processes. The hypothesis that responses to such induced currents depend on pulse characteristics was evaluated by using transcription as the target process. Two pulses in clinical use, the repetitive single pulse and the repetitive pulse train, were tested. These pulses produced different results from each other and from controls when transcription in dipteran salivary gland cells was monitored with tritiated uridine in transcription autoradiography, cytological nick translation, and analysis of isolated RNA fractions. The single pulse increased the specific activity of messenger RNA after 15 and 45 minutes of exposure. The pulse train increased specific activity only after 45 minutes of exposure.

Science. 1983 Jun 17;220(4603):1283-5.

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Pulsed electromagnetic fields alter phenotypic expression in chondroblasts in tissue culture.

Norton LA, Witt DW, Rovetti LA.

Department of Orthodontics, School of Dental Medicine, University of Connecticut Health Center, Farmington 06032.

We hypothesize that pulsed electromagnetic fields (PEMF) alter phenotypic expression of chondroblasts by promoting the production of alkaline phosphatase (AP) and altering the structure of proteoglycans. Chondroblasts from the hypertrophic zone of tibial epiphyses (HC), sternum (SC), and skin fibroblasts (F) were cultured from 16 day chick embryos. Cultures were randomly designated control (C) or experimental (E). E received PEMF for 24 h in a 6 h on, 6 h rest sequence. The controls were in the same incubator shielded by Mu metal. Assays for AP activity were performed and normalized to protein content. Proteoglycan synthesis assay involved labeling with 35S fractionating in a 5% to 20% surcrose gradient determining total protein and chondroitin sulfate content. PEMF showed no change of AP activity on F. A high AP basal activity was found in HC, but was not increased above the control. PEMF increased AP in the SC samples (E/C ratio). The sucrose gradient data showed a shift in peaks for SC only altering the ratio of carbohydrate to protein for the SC. Analysis of carbohydrate and protein indicated that the effect was decreased synthesis or degradation of protein. We conclude that PEMF alters the phenotypic expression of sternal chondroblasts in our in vitro system.

J Orthop Res. 1988;6(5):685-9.

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Magnetic behavior of human erythrocytes at different hemoglobin states.

Sakhnini L, Khuzaie R.

Physics Department, College of Science, University of Bahrain, State of Bahrain. l_sakhnini@yahoo.com

The effect of a static magnetic field on human erythrocytes at different hemoglobin states (normal, oxidized and reduced hemoglobin) was investigated. Three different blood samples, normal, iron deficiency anemic and beta thalassemia minor, were studied. Measurements of the magnetization curves of the erythrocytes for all blood samples in all states showed diamagnetic behavior; however, oxidation was found to enhance this behavior. These measurements have also shown that the normal and iron deficiency samples in the reduced states exhibit a less diamagnetic response in comparison with the normal state. This result indicates that the reduction process gave rise to a paramagnetic component of the magnetization. Analysis of the measured paramagnetic behavior, using a Brillouin function, gave an effective magnetic moment of 8 muB per reduced hemoglobin molecule for both normal and anemic samples. This result shows that both anemic and normal blood have similar magnetic behavior and the only difference is the number of hemoglobin molecules per erythrocyte. For the beta thalassemia minor blood sample, magnetic measurements showed that both the normal and reduced states have almost the same diamagnetic behavior. However, this diamagnetic response is less than that for the normal state of the iron deficiency anemic sample. This result may indicate a low oxygen intake for the blood in the normal state for the beta thalassemia minor blood. All magnetic measurements were made using a vibrating sample magnetometer using field steps of 0.001 T from 1 T to -1 T.

Eur Biophys J. 2001 Oct;30(6):467-70.

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Effects of static magnetic field on human leukemic cell line HL-60.

Sabo J, Mirossay L, Horovcak L, Sarissky M, Mirossay A, Mojzis J.

Department of Medical Biophysics, Medical Faculty, P.J. Safarik University, Trieda SNP 1, 04011 Kosice, Slovak Republic. grosiar@central.medic.upjs.sk

A number of structures with magnetic moments exists in living organisms that may be oriented by magnetic field. While most experimental efforts belong to the area of effects induced by weak and extremely low-frequency electromagnetic fields, we attempt to give an attention to the biological effects of strong static magnetic fields. The influence of static magnetic field (SMF) on metabolic activity of cells was examined. The metabolic activity retardation is observed in human leukemic cell line HL-60 exposed to 1-T SMF for 72 h. The retardation effect was observed as well as in the presence of the mixture of the antineoplastic drugs 5 fluorouracil, cisplatin, doxorubicin and vincristine.

Bioelectrochemistry. 2002 May 15;56(1-2):227-31.

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The effect of extremely low frequency magnetic fields on cytochrome oxidase subunit 1 mRNA transcription.

Zhong T, Chen Q, Wu R, Yao G, Lu D, Chiang H.

Bioelectromagnetic Lab., Zhejiang University School of Medicine, Hangzhou 310031, China.

OBJECTIVE: To clone and identify MF-1 gene which responded to extremely low frequency magnetic fields(ELF MF) in Daudi cells, and explore the response universality of MF-1 gene in several MF-sensitive cell lines, so as to provide experimental basis for revealing the mechanism of biological effects induced by magnetic field.

METHODS: The DNA fragment of MF-1 was cloned and sequenced; the mRNA level of MF-1 gene were analysed in MF-sensitive cell lines(HL-60, L1210 and CHL) by Northern blot after these cells being treated with 0.1 mT and 0.8 mT MF for 20 minutes and 24 hours, respectively.

RESULTS: The MF-1 cDNA sequence had 100% homology with cytochrome oxidase subunit 1 gene(CO1) by searching Gene Bank database; the transcription of CO1 in HL-60, L1210 and CHL cell lines which exposed to 0.1 mT and 0.8 mT MF for 20 minutes were significantly lower(0.38 +/- 0.12 and 0.37 +/- 0.04) than that of control(0.58 +/- 0.12) and so did for 24 hours exposure(0.46 +/- 0.09 and 0.45 +/- 0.09 vs 0.65 +/- 0.06) (P < 0.05).

CONCLUSION: CO1 is a MF-responsive gene. Cytochrome oxidase activity may be affected through low level of CO1 transcription by magnetic fields, thus induce bioeffects in organisms.

Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2002 Aug; 20(4): 249-51.

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Orientation of human glioblastoma cells embedded in type I collagen, caused by exposure to a 10 T static magnetic field.

Hirose H, Nakahara T, Miyakoshi J.

Laboratory of Radiation Biology, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo, Kyoto 606-8502, Japan.

We investigated the preferred orientation of human glioblastoma cells (A172) following exposure to static magnetic fields (SMF) at 10 Tesla in the presence or absence of collagen. A172 cells embedded in collagen gel were oriented perpendicular to the direction of the SMF. A172 cells cultured in the absence of collagen did not exhibit any specific orientation pattern after 7 days of exposure to the SMF. Thus we succeeded in evoking the magnetic orientation of human glioblastoma cells by exposure to the SMF. Our results suggest that the orientation of glioblastoma cell processes may be due to the arrangement of microtubules under the influence of magnetically oriented collagen fiber. Copyright 2002 Elsevier Science Ireland Ltd.

Neurosci Lett. 2003 Feb 20;338(1):88-90.

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Magnetic field effects on assembly pattern of smooth muscle cells.

Iwasaka M, Miyakoshi J, Ueno S.

Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan (M. I., S. U.) and Department of Radiological Technology, School of Health Science, Faculty of Medicine, Hirosaki University, 66-1 Hon-cho, Hirosaki 036-8564, Japan (J. M.).

Under a strong magnetic field, the diamagnetic properties of biological cells modulate the behavior of the cells themselves, under conditions of both floating and adherence. The morphological effects of strong static magnetic fields on adherent cells are less well understood than the effects of magnetic fields on red blood cells. In the present study, a high-intensity magnetic field of 14 T affected the morphology of smooth muscle cell assemblies, and the shapes of the cell colonies extended along the direction of the magnetic flux. The phenomenon was most notable under magnetic fields of more than 10 T, where an ellipsoidal pattern of smooth muscle cell colonies was clearly observed. The ellipticity of the cell colony pattern with a 14-T magnetic field was 1.3, whereas that with a field of 0-8 T was close to a circle at about 1.0. The evidence that smooth muscle cells detect high-density magnetic flux and thus change their cell orientation was shown as a visible pattern of cellular colonies. The speculated mechanism is a diamagnetic torque force acting on cytoskeleton fibers, which are dynamically polymerizing-depolymerizing during cell division and cell migration.

In Vitro Cell Dev Biol Anim. 2003 Mar;39(3):120-123.

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Control of orientation of rat Schwann cells using an 8-T static magnetic field.

Eguchi Y, Ogiue-Ikeda M, Ueno S.

Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

Schwann cells aid in neuronal regeneration in the peripheral nervous system via guiding the regrowth of axons. In this study, we investigated the magnetic orientation of Schwann cells, and of a mixture of Schwann cells and collagen, after an 8-tesla magnetic field exposure. We obtained cultured Schwann cells from dissected sciatic nerves of neonatal rats. After 60 h of magnetic field exposure, Schwann cells oriented parallel to the magnetic fields. In contrast, the mixture of Schwann cells and collagen, Schwann cells oriented in the direction perpendicular to the magnetic field after 2 h of magnetic field exposure. In this case, Schwann cells aligned along the collagen fiber oriented by magnetic fields. The magnetic control of Schwann cell alignment is useful in medical engineering applications such as nerve regeneration.

Neurosci Lett. 2003 Nov 13;351(2):130-2.

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Detection of intracellular macromolecule behavior under strong magnetic fields by linearly polarized light.

Iwasaka M, Ueno S.

Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo, Tokyo, Japan. iwasaka@medes.m.u-tokyo.ac.jp

Strong static magnetic fields on the order of 10 T have a diamagnetic force on cell components and generate a clear alignment of a smooth muscle cell assembly, parallel to the direction of the magnetic fields within an exposure period of 3 days. This work shows the effects of diamagnetic torque forces on cell component motion. Linearly polarized light was utilized to detect the displacement of intracellular macromolecules. The polarized light passed through a mass of cells in a magnetic field, and transmission of the light increased and reached a plateau 2 h after the beginning of magnetic field exposure at 14 T. However, no distinct change was observed in transmission of the light under zero magnetic field exposure. The change in polarized light intensity through the lamellar cell assembly under magnetic fields corresponds to behavioral changes in cell components. It was speculated that intracellular macromolecules rotated and showed a displacement due to diamagnetic torque forces during 2-3 h of magnetic field exposure at 14 T. Copyright 2003 Wiley-Liss, Inc.

Bioelectromagnetics. 2003 Dec;24(8):564-70.

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