Oxid Med Cell Longev. 2017; 2017: 2181942.
Published online 2017 Sep 12. doi: 10.1155/2017/2181942
Benign Effect of Extremely Low-Frequency
Electromagnetic Field on Brain Plasticity Assessed by Nitric Oxide
Metabolism during Poststroke Rehabilitation
1 Piotr Czarny, 2 Micha? Bijak, 1 El?bieta Miller, 3 , 4 Tomasz liwiski, 5 Janusz Szemraj, 2 and Joanna Saluk-Bijak 11Department of General
Biochemistry, Faculty of Biology and Environmental Protection,
University of Lodz, Pomorska 141/143, Lodz, Poland
2Department of Medical Biochemistry, Medical University of Lodz, Mazowiecka 6/8, Lodz, Poland
3Department of Physical Medicine, Medical University of Lodz, Pl. Hallera 1, Lodz, Poland
4Neurorehabilitation Ward, III General Hospital in Lodz, Milionowa 14, Lodz, Poland
5Department of Molecular
Genetics, Laboratory of Medical Genetics, Faculty of Biology and
Environmental Protection, University of Lodz, Lodz, Pomorska 141/143,
Nitric oxide (NO) is one of the most
important signal molecules, involved in both physiological and
pathological processes. As a neurotransmitter in the central nervous
system, NO regulates cerebral blood flow, neurogenesis, and synaptic
plasticity. The aim of our study was to investigate the effect of the
extremely low-frequency electromagnetic field (ELF-EMF) on generation
and metabolism of NO, as a neurotransmitter, in the rehabilitation of
poststroke patients. Forty-eight patients were divided into two groups:
ELF-EMF and non-ELF-EMF. Both groups underwent the same 4-week
rehabilitation program. Additionally, the ELF-EMF group was exposed to
an extremely low-frequency electromagnetic field of 40Hz, 7mT, for
15min/day. Levels of 3-nitrotyrosine, nitrate/nitrite, and TNF? in
plasma samples were measured, and NOS2 expression was determined in
whole blood samples. Functional status was evaluated before and after a
series of treatments, using the Activity Daily Living, Geriatric
Depression Scale, and Mini-Mental State Examination. We observed that
application of ELF-EMF significantly increased 3-nitrotyrosine and
nitrate/nitrite levels, while expression of NOS2 was insignificantly
decreased in both groups. The results also show that ELF-EMF treatments
improved functional and mental status. We conclude that ELF-EMF therapy
is capable of promoting recovery in poststroke patients.
Cardiovascular diseases, including
ischemic stroke (IS), are a serious problem of the modern age, killing 4
million people each year in Europe .
Stroke is caused by ischemia of brain tissue. Brain structure damage
occurring during ischemia/reperfusion is due to the generation of
significant amounts of reactive oxygen species and inflammatory
Damage to brain tissue as a result of a stroke cannot be undone.
However, the most important part of poststroke therapy is immediate and
long-term rehabilitation, considering the enormous plasticity of the
Although extremely low-frequency electromagnetic field (ELF-EMF)
therapy is not a standard treatment in the poststroke rehabilitation,
some authors suggest its increased positive effect on patients .
ELF-EMF treatment is based on regeneration, osteogenesis, analgesics,
and anti-inflammatory action. Its biological effect is related to
processes of ion transport, cell proliferation, apoptosis, protein
synthesis, and changes in the transmission of cellular signals .
The regenerative and cytoprotective effect of ELF-EMF is based on
mechanism associated with nitric oxide induction, collateral blood flow,
opioids, and heat shock proteins .
Nitric oxide (NO) is an unstable, colourless, water-soluble
gas with a short half-life (3–6?sec). The compound has one unpaired
electron, which makes it a highly reactive free radical. It is
characterized by the multiplicity of action in the body, in both
physiological and pathological conditions .
Synthesis of NO in the organism is catalysed by nitric oxide synthase
(NOS), occurring in three isoforms: neuronal (nNOS), inducible (iNOS),
and endothelial (eNOS), encoded by different genes whose expression is
subject to varying regulation. The constituent isoforms of NOS are eNOS
and nNOS, whose activity is associated with concentration of calcium
ions and the level of calmodulin in a cell, as well as with hypoxia,
physical activity, and the level of certain hormones, that is,
In contrast, because it is closely related with the calmodulin, iNOS
does not require a high concentration of calcium ions but is regulated
by various endogenous and exogenous proinflammatory factors .
The two-stage synthesis of NO consists of the oxidation of
L-arginine to N-hydroxy-L-arginine and, under the influence of NOS and
oxygen, formation of L-citrulline and release of NO. All isoforms of NOS
require the same cofactors: nicotinamide adenine dinucleotide phosphate
(NADPH), flavin mononucleotide (FMN), flavin adenine dinucleotide
(FAD), tetrahydrobiopterin (BH4), iron protoporphyrin IX (heme), and O2.
Nitric oxide is one of the most important signal molecules,
involved in both physiological and pathological processes. One of the
major functions of NO is as a potent vasodilation, increasing the blood
flow and regulation of blood pressure, which has been used in clinical
practice for many years. Deficiency of this compound is observed in
various disorders of many systems: cardiovascular, gastrointestinal,
respiratory, and genitourinary .
The beneficial effects of NO lie in its platelet inhibition, macrophage
cytotoxicity (antibacterial, antiviral, and antiparasitic), and
protection of the mucosal lining of the digestive system. On the other
hand, excessive expression of iNOS can be disadvantageous, for example,
during sepsis. The adverse action of NO is associated with the
production of superoxide anions and subsequent generation of
peroxynitrite and hydroxyl radicals, which are highly toxic .
In the central nervous system, NO as a neurotransmitter
regulates cerebral blood flow, as well as neurogenesis and synaptic
plasticity. Furthermore, neuronal death is caused by high concentrations
of NO by caspase-dependent apoptosis process and promotion of
inflammation. Elevated levels of nitric oxide promote necrosis by energy
depletion. On the basis of these mechanisms, NO is involved in the
etiology of many neurological diseases, such as major depression,
schizophrenia, epilepsy, anxiety, and drug addiction .
Our study was designed to investigate the
effect of ELF-EMF on the metabolism of NO, as a signal molecule in the
central nervous system, in the rehabilitation of acute poststroke
2. Materials and Methods
2.1. Blood Sample Collection
Blood samples were collected into CPDA1-containing
tubes (Sarstedt, Nümbrecht, Germany). Immediately upon doing so, a
portion of the sample was frozen at ?80°C and the rest of the samples
centrifuged to isolate the plasma (15?min, 1500g) at 25°C.
Blood samples were collected twice, at an interval of 14 days before and
after a standard 10 sessions of therapy. For additional analysis of
3-nitrotyrosine levels, the blood samples were collected three times, at
an interval of 28 days: before treatment, after 10 treatments, and
after 20 treatments. All blood samples were taken in the morning
(between 7am and 9am) under patient fasting condition and stored using
the same protocol.
2.2. Subject Presentation
Forty-eight poststroke patients were enrolled in the study. Participants were randomly divided into two groups: ELF-EMF (n = 25) and non-ELF-EMF (n =
23). Patients with metal and/or electronic implants (pacemakers, etc.)
were excluded from the ELF-EMF group, for safety reasons. The ELF-EMF
group had already undergone ELF-EMF therapy with specific parameters
(40Hz frequency, magnetic induction of 5mT (B), rectangular and bipolar waveforms) (Figure 1),
which was conducted using a Magnetronic MF10 generator (EiE Elektronika
i Elektromedycyna, Otwock, Poland). The parameters were selected on the
basis of the fact that low-intensity stimuli improve the vital
functions of the body. In addition, rectangular pulses are more intense
than sinusoidal and trapezoid, while bipolar pulses show more range of
changes than unipolar pulses .
The ELF-EMF and non-ELF-EMF groups were treated for the same amount of
time (15minutes). The non-ELF-EMF subjects were given only sham
exposure. The pelvic girdle of the patients was exposed to the
electromagnetic field, because exposure of the head to ELF-EMF can
affect the activation of the epilepsy focus in the brain. The same
therapeutic program was used for both subject groups. This consisted of
aerobic exercise (30min), neurophysiological routines (60?min), and
psychological therapy (15?min). Poststroke patients with moderate stroke
severity according to NIHSS scores of 4.9 ±3.1 in the ELF-EMF group
(aged 48.8 ±7.7) and 5.4 ±2.9 (aged 44.8 ±8.0) in the non-ELF-EMF group
were enrolled in the study. Table 1 shows
the clinical and demographic characteristics. Participants with
haemorrhagic stroke, dementia, chronic or significant acute inflammatory
factors, decreased consciousness, and/or neurological illness other
than stroke in their medical prestroke history were excluded. The
subjects had undergone neurorehabilitation for 4 weeks in
Neurorehabilitation Ward III of the General Hospital in Lodz, Poland, as
well as internal and neurological examinations. The Bioethics Committee
of the Faculty of Biology and Environmental Protection of The
University of Lodz, Poland, approved the protocol with resolution
numbers 28/KBBN-U/II/2015 and 13/KBBN-U/II/2016. All participants
provided written informed consent prior to participation. Depression was
screened in both groups using the Geriatric Depression Scale (GDS).
Cognitive status was estimated in a Mini-Mental State Examination
(MMSE), and functional status using the Barthel Index of Activities of
Daily Living (ADL). The GDS, ADL, and MMSE were administered either on
the same day as the blood sampling or on the afternoon before.
ELF-EMF therapy was performed by a
Magnetronic MF10 generator as per accepted guidelines. This device is
able to produce pulses in rectangular, trapezoid, and sinusoidal shapes.
The pulses were applied using an AS-550 applicator (EiE, Otwock,
Poland), which has the following properties: 550 mm in diameter, 270mm
in length, and 5 layers of 187 turns of 1.45mm twin-parallel wires.
Magnetic induction was set at 5mT. The electromagnetic field intensity
was not uniformed; its distribution is vertical, while the induction
coils are set horizontally. Induction of the electromagnetic field of
5mT is present at the geometric center of the applicator, and the value
increases in the proximity to the surface about 7mT. Other factors that
could affect EMF were eliminated (electronic measuring instruments
occurring in rehabilitation room and other electronic equipment).
2.4. Immunodetection of 3-Nitrotyrosine by c-ELISA
Levels of 3-NT-containing proteins in plasma were determined using a modified c-ELISA method, as described by Khan et al. .
96-well microtiter plates were coated with nitro-fibrinogen (nitro-Fg)
(1mg/mL) and kept overnight at 4°C. Concentrations of nitrated proteins
inhibiting the binding of anti-nitrotyrosine antibodies were assessed
from the standard curve (10–100nM nitro-Fg equivalents) and expressed as
nitro-Fg equivalents .
2.5. Nitrate/Nitrite Estimation
Plasma samples were diluted twice
before the measurement of nitrate/nitrite concentration using a
Nitrate/Nitrite Colorimetric Assay Kit (Cayman Chemical Company, USA),
based on the two-step Griess method. In the first step, the nitrate is
converted to nitrite with nitrate reductase, while in the second step,
after addition of the Griess reagent, the nitrite is converted to a deep
purple azo compound. The absorbance measurement was performed at 540nm
in a 96-well microplate reader (SPECTROstarNano, BMG Labtech, Ortenberg,
2.6. Determination of NOS2 Expression in Whole Blood Samples
RNA was isolated from the frozen
whole blood samples (?80°C), in accordance with the manufacturer’s
protocol using TRI Reagent® (Sigma-Aldrich, USA). The aqueous phase was
purified in accordance with the manufacturer’s protocol using an
InviTrap Spin Universal RNA Mini Kit (Stratec Biomedical Systems,
Germany). The purity and quantity of isolated RNA were assessed using a
Synergy HTX Multi-Mode Microplate Reader equipped with a Take3
Micro-Volume Plate and connected to a PC running Gen5 Software (BioTek
Instruments Inc., Winooski, VT, USA). Isolated RNA (20ng/L) was
transcribed onto cDNA with a High-Capacity cDNA Reverse Transcription
Kit (Applied Biosystems™, Waltham, MA, USA). Quantitative assays were
executed using a TaqMan Hs01075529_m1 probe for human NOS2 genes and an Hs02786624_g1 for endogenous control, which was GAPDH (Life
Technologies). Reactions were carried out using a TaqMan Universal
Master Mix II, without UNG (Life Technologies) in a BioRad CFX96
real-time PCR system (BioRad Laboratories, Hercules, CA, USA), all in
accordance with the manufacturers’ protocols. Relative expression of NOS2 was obtained using the equation 2Ct, where Ct is the threshold cycle (Ct) value for the target gene minus Ct values obtained for the housekeeping gene GAPDH .
2.7. Determination of TNF?
Measurements of human tumour
necrosis factor alpha (TNF?) in plasma samples were made with a Human
TNF? ELISA development kit (MABTECH, Cincinnati, OH, USA), in accordance
with the manufacturer’s protocol. The combination of two coating
antibodies (TNF3 and TNF4) were used for the analysis. The absorbance
was measured at 450nm, and TNF? concentration was expressed as pg/mL .
2.8. Data Analysis
Biochemical and clinical data were
expressed as mean ±SD. All measurements were executed in duplicate.
Output value (100%) was determined for each measured parameter of each
patient before treatment. Data from tests performed on the same study
subjects after therapy constituted a percentage of the output value.
Percentage values were presented as mean ± SD. Statistical analyses were
performed using the Statistica 12 statistical software (StaftSoft
Inc.). A Shapiro-Wilk test was used to analyse for normality. A paired
Student t-test was used to the calculate differences between
the values obtained for subjects before and after therapy, whereas
unpaired Student t-test or Mann–Whitney U tests were used to determine differences between the ELF-EMF and non-ELF-EMF groups. p values of 0.05 were accepted as statistically significant for all analyses.
Our comparative analysis demonstrated an increased level of 3-nitrotyrosine (3-NT) (p< 0.05) (Figure 2) and an elevated nitrate/nitrite concentration (p < 0.01) (Figure 3)
in the plasma of patients from the ELF-EMF group. The gain in the 3-NT
level was significantly higher with an increased amount of sessions (Figure 2).
In the non-ELF-EMF group, we saw that the effect of rehabilitation on
nitrative stress was largely weaker and not statistically significant (p > 0.05) (Figures (Figures22 and and3).3). The 3-NT level increased more in the ELF-EMF group than in the non-ELF-EMF after 10 treatments (68% versus 17%, p < 0.05) (Figure 2).
The level of nitrate/nitrite in the non-ELF-EMF group even decreased
after 10 treatments (although not statistically significantly) (Figure 3).
The comparison of 3-NT levels in plasma proteins obtained from the
ELF-EMF group versus those from the non-ELF-EMF group. Statistical
significance between the ELF-EMF and non-ELF-EMF groups: B versus D (p < 0.05).
The comparison of nitrate/nitrite levels in plasma proteins
obtained from the ELF-EMF group versus those from the non-ELF-EMF group.
Statistical significance between ELF-EMF and non-ELF-EMF groups: B
versus D (p < 0.05).
In the next set of experiments, we determined the effect of magnetotherapy on gene expression in the whole blood samples of NOS2 mRNA.
Its expression was unmeasurable in 35% of subjects from both the
ELF-EMF and non-ELF-EMF groups. We observed a statistically
insignificant decrease in the level of NOS2 mRNA expression after treatment in both the ELF-EMF and non-ELF-EMF groups (Figure 4).
The comparison of NOS2 mRNA expression obtained from the ELF-EMF group versus that from the non-ELF-EMF group.
Subsequently, we determined the concentration of proinflammatory cytokine TNF?.
We found that the concentration of TNF? was comparable before treatment
in both the ELF-EMF and non-ELF-EMF-groups. The cytokine level did not
change in either groups after rehabilitation (Figure 5).
The comparison of TNF? levels in plasma proteins obtained from the ELF-EMF group versus those from the non-ELF-EMF group.
The ADL, MMSE, and GDS were used to evaluate the
functional and mental status of poststroke patients undergoing
rehabilitation. We demonstrated that treatment using ELF-EMF improves
their clinical parameters, particularly in cognitive and psychosomatic
Motor abilities estimated by ADL score changed at similar
levels in both groups, with the observed improvement being statistically
significant in all rehabilitated patients (p < 0.001) (Table 2).
Clinical parameters: ADL, MMSE, and GDS measured in the ELF-EMF and
non-ELF-EMF groups. Data presented as the delta of a clinimetric scale
before and after the standard series of treatments ADL=the increase of
ADL; MMSE= the …
The baseline MMSE values before treatment in both groups were comparable, but statistically different (p < 0.05) after rehabilitation. After 2 weeks of rehabilitation, MMSE parameters improved markedly in the ELF-EMF group (p = 0.002), while a small increase in the non-ELF-EMF group was not statistically significant (p = 0.2) (Table 2).
Depression syndrome expressed by GDS
improved significantly in both groups after rehabilitation. However, the
GDS value reached about a 60% lower result in the ELF-EMF group than in
the non-ELF-EMF group (p = 0.018), starting from a similar base level in both groups (p > 0.05) (Table 2).
In this study, we provide the evidence
that application of extremely low-frequency electromagnetic field
increases nitric oxide generation and its metabolism, as well as
improving the effectiveness of poststroke ischemic patients’ treatments.
Ischemic stroke is one of the major causes of morbidity
and mortality in the world’s population and is one of the main causes of
long-term disability. The mechanisms of neurological function recovery
after brain injury associated with neuroplasticity (cortical
reorganization) are still insufficiently understood. Poststroke
neurorehabilitation is designed to provide external stimuli, improving
the effectiveness of compensatory plasticity .
In the central nervous system, NO is both a pre- and
postsynaptic signal molecule. The activity of NO is associated with a
cGMP-mediated signalling cascade. The presynaptic excitatory action of
NO is related to the phosphorylation of synaptophysin by the
cGMP-dependent protein kinase G (PKG) pathway and the subsequent
potentates of glutamatergic neurotransmission .
On the other hand, NO causes a neurotransmission inhibition through
gamma-aminobutyric acid- (GABA-) ergic synaptic communication. It is
associated with ion exchange and regulation of membrane excitation [21, 22].
Moreover, NO as an important vasodilation factor mediates neurovascular
coupling. The enlargement of vessel diameter is caused by increasing
metabolic consumption as a result of neuronal activity. Neurovascular
coupling maintains functional and structural brain integrity .
This study was designed to investigate the impact of
ELF-EMF on the metabolism of nitric oxide in the rehabilitation of acute
In our study, we demonstrate that poststroke
rehabilitation increases the level of 3-NT and nitrate/nitrite
concentrations. Due to its vasodilating and proangiogenic effects, NO
serves as a protective function during cerebral ischemia. Su et al.
investigated the role of simvastatin-regulated TRPV1 receptors
(transient receptor potential vanilloid type 1) in NO bioavailability,
activation of eNOS, and angiogenesis in mice. They demonstrated that
simvastatin causes an influx of calcium ions through the TRPV1-TRPA1
(transient receptor potential ankyrin 1) pathway, which then causes
activation of CaMKII (Ca2+/calmodulin-dependent protein
kinase II). This then enhances the formation of the TRPV1-eNOS complex,
which also includes CaMKII, AMPK (5AMP-activated protein kinase), and
Akt (protein kinase B), which leads to activation of eNOS, production of
NO, and thus the promotion of endothelial angiogenesis . There have been numerous reports of the protective effects of NO against inflammation and oxidative stress .
Transgenic eNOS-deficient mice demonstrated a more extensive infarct of
the middle cerebral artery (MCA), compared to controls .
NO effects on the regulation of endothelial integrity,
anti-inflammatory and anti-apoptotic effects, as well as maintenance of
cerebral blood flow, inhibition of platelet aggregation, and reduction
of leukocyte adhesion [25, 27].
Khan et al. studied structurally different NO donors as agents of
cerebrovascular protection in experimentally induced stroke in rats.
They showed that NO donors promote cerebral blood flow through
S-nitrosylation and may be an effective drug for acute stroke [28, 29].
Furthermore, Greco et al. proved the protective effect of
nitroglycerin (donors of NO) on cerebral damage induced by MCA occlusion
in Wistar rats. They observed a significant reduction in stroke volume
in preinjected rats compared to their control group, which confirms the
protective effect of nitroglycerin in vivo. They speculated
that the mechanism of action is associated with the generation of a
complex chain of phenomena, triggering activation of apoptosis and
subsequent activation of antiapoptotic responses .
The biological action of ELF-EMF is still being
investigated. It is suggested that ELF-EMF has an impact on the
physicochemical properties of water, the liquid crystal structure
generated by cholesterol, and its derivatives [31, 32].
Changes in ion balance caused by ELF-EMF appeal to the structure of
tissue with piezoelectric and magnetostrictive properties, free
radicals, diamagnetic molecules, and uncompensated magnetic spins of
paramagnetic elements . Therefore, ELF-EMF causes depolarization of cells having the ability to spontaneously depolarize, predominantly through Ca2+ influx .
In our previous study, we investigated the effect of ELF-EMF on
oxidative stress in patients after ischemic stroke. We demonstrated that
ELF-EMF causes activation of antioxidant enzymes , which leads to reduction of the oxidative modification of plasma protein (this is detailed in an article published in Advances in Clinical and Experimental Medicine).
As a highly reactive molecule, NO can also regulate the level of
oxidative stress. Through the covalent interaction, NO influences the
activity of various enzymes. Mechanisms of this modulation can be
varied: NO reacts with coenzymes and active centers containing metal
ions and interacts with cysteine residues of proteins .
In the current study, we observed that in the ELF-EMF group, the level of plasma 3-NT was increased (Figure 2). The formation of 3-NT in protein molecules occurs in vivo by
the action of nitrating agents on the polypeptide chain. The formation
of 3-NT is mainly attributed to NO and superoxide anions (O2??), which react rapidly to form peroxynitrite (ONOO?). This is one of the major oxidizing and nitrating agents produced in vivo in
acute and chronic inflammation, as well as in ischemia/reperfusion.
Endothelial cells, macrophages, and neutrophils release large amounts of
NO and O2?. Thus, increased amounts of NO contribute to the creation of 3-NT .
To investigate the effect of ELF-EMF on NO metabolism, we
determined nitrate/nitrite concentrations in plasma. We showed that in
the ELF-EMF group, the level of nitrate/nitrite compounds in plasma
increased after treatment (Figure 3), and these results correspond with the data presented by Chung et al. .
The authors investigated the effects of ELF-EMF (60Hz, 2mT) on the
level of NO, biogenic amines, and amino acid neurotransmitters in the
hippocampus, cortex, thalamus, cerebellum, and striatum in rats. They
found a significant increase in NO concentration in the hippocampus,
thalamus, and striatum. Moreover, ELF-EMF also caused a change in the
level of biogenic amines and amino acid neurotransmitters in the brain.
However, the observed effect and range were different, depending on the
brain area. Balind et al. determined the effect of ELF-EMF (50Hz, 0.5mT)
on oxidative stress in gerbils with induced cerebral ischemia. They
measured the level of NO using the Griess reagent and showed an
increased level of NO, provoked by electromagnetic fields. Moreover,
ELF-EMF reduces oxidative stress generated during cerebral ischemia,
thus leading to a decrease in the damaged brain tissue .
NO is produced from L-arginine with the involvement of
nitric oxide synthase. Three NOS isoforms are expressed in different
tissues. Although, in the blood, only NOS2 is expressed, in 35% of the
subjects in both the ELF-EMF and non-ELF-EMF groups, mRNA expression of NOS2 was under detection. In the remaining patients, the expression of NOS2 had
not significantly changed after treatment. The NOS2 gene in fact
encodes for iNOS, which is primarily activated during inflammation. In
order to exclude deeper inflammation, we measured the concentration of
TNF?, one of the main proinflammatory cytokines. TNF? is a
pleiotropic cytokine that is involved in nearly all phenomena of
inflammatory responses: initiating chemokine synthesis, promoting the
expression of adhesion molecules, promoting the maturation of dendritic
cells, and inducing the production of inflammatory mediators and other
proinflammatory cytokines .
TNF? stimulates collagenase synthesis in synovial fibroblasts and
synovial cartilage chondrocytes and activates osteoclasts, leading to
joint cartilage damage, hypertrophy, bone resorption and erosion, and
angiogenesis. It also activates monocytes and macrophages, enhancing
their cytotoxicity and stimulating cytokine production. Chemokines and
growth factors are responsible for T cell proliferation, proliferation
and differentiation of B lymphocytes, and the release of inflammatory
cytokines by the lymphocytes. Moreover, in the hypothalamus, TNF?
stimulates prostaglandin E and IL-1 synthesis .
Pena-Philippides et al. investigated the effect of pulsed
electromagnetic fields on injury size and neuroinflammation in mice
after middle cerebral artery occlusion (MCAO). They found, using
magnetic resonance imaging (MRI), that EMF reduced infarct size, as well
as changed expression of genes encoding pro- and anti-inflammatory
cytokines in the hemisphere with ischemic injury. After EMF exposure,
genes encoding IL-1 and TNF superfamily were downregulated, while IL-10 expression
was upregulated. Thus, the authors suggested that application of EMF to
poststroke patients could have been beneficial through
anti-inflammatory effect and reduction of injury size .
On the basis of our results, we suggest that the observed
increase in NO level is associated with nNOS and/or eNOS activities, but
not with iNOS expression. Our research is consistent with evidence
shown by Cho et al., who established that ELF-EMF (60Hz, 2mT) increased
the expression and activation of nNOS in rat brains .
The activities of nNOS and eNOS depend on calcium ions.
There are many reports that the biological effect of ELF-EMF is related
to the control of calcium channels [44–48].
In view of these findings, the observed mechanism of increased NO
generation and metabolism may be associated with calcium-ion flux.
Additionally, we noticed that ELF-EMF treatment enhances the effectiveness of poststroke rehabilitation (Table 2).
Some researchers suggest that electromagnetic fields have a beneficial
effect on ischemic/reperfusion injury, and in some places, therapeutic
programs using ELF-EMF are considered to be standard therapy for
poststroke patients [49, 50].
The beneficial effects of ELF-EMF include the following: improvement in
the transport of cellular and mitochondrial membranes; normalization of
blood rheological values; counteraction of tissue oxidation;
intensification of regenerative processes; stimulation of axon growth in
undamaged neurons; intensification of neuronal dissociation and
differentiation; reduction of stress-induced emotional reactions and
free radicals; acceleration of the return of fibre function in
functional disorders; reduction of periapical scarring; and increase of
the level of energetic substances in the brain tissue and erythrocytes [48–53].
Grant et al. estimated the impact of low-frequency pulsed
electromagnetic field on cerebral ischemia in rabbit. They observed
using MRI that exposure to electromagnetic field caused extenuation of
cortical ischemia oedema and reduction of neuronal injury in cortical
In conclusion, ELF-EMF therapy increases
the metabolism and generation of NO, which has both neuroprotective and
cytotoxic properties. An increase in NO level is probably associated
with nNOS and/or eNOS activities, but not with iNOS expression, which
increases mainly during inflammation. We suggested that in poststroke
patients, NO demonstrated a protective effect due to significant
improvement in patient functional status. Thus, our studies promote the
validity of this method in poststroke rehabilitation therapy.
This study was supported by the Department of General
Biochemistry, Faculty of Biology and Environmental Protection,
University of Lodz (no. 506/1136), and Laboratory of Medical Genetics,
Faculty of Biology and Environmental Protection, University of Lodz (no.
B161100000004601), and Grants for Young Scientists and PhD Students,
Faculty of Biology and Environmental Protection, University of Lodz
Conflicts of Interest
The authors declare that there is no conflict of interest regarding the publication of this article.
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Neurosci Behav Physiol. 2010 Oct;40(8):920-5.
The potential of transcranial magnetotherapy in color and rhythm therapy in the rehabilitation of ischemic stroke.
Sholomov II, Cherevashchenko LA, Suprunov OV, Raigorodskii YM.
Saratov State Medical University, Saratov, Russia.
A total of 116 patients with ischemic stroke were studied during the
early recovery period. The patients were divided into four groups –
three experimental groups and one control group. Of these, 87 patients
in the first three groups received transcranial magneto- and/or color
and rhythm therapy (TcMT, CRT) along with traditional treatment, while
the 29 patients of the control group received basal treatment only. TcMT
was performed using a bitemporal method, with a running field regime
with a modulation frequency of 1-10 Hz. CRT consisted of an alternating
scheme of stimulation of the left and right eyes with green and/or blue
light with a period of 2-4 sec and an on time of 1 sec. Each of the
three experimental groups (group 1 received TcMT, group 2 received CRT,
and group 3 received TcMT + CRT) received two courses of treatment
separated by 1.5 months. After treatment, all experimental groups,
particularly group 3, showed more marked improvements than the control
group. Regression of neurological symptomatology on the Lindmark scale
in group 3 was 9.5% greater than that in controls; improvements in
impairments to activity and self-care ability on the Barthel scale were
greater by 8.8%; memory and intellectual changes were also seen on the
MMSE and the Luriya and Schulte tests. Rheography and
electroencephalography demonstrated significant improvements in
hemodynamics and alpha-rhythm differentiation and a 14.6% reduction in
the proportion of patients with dysrhythmia in group 3 compared with the
control group. The best result on all measures were obtained in
patients given the combination of TcMT and CRT; TcMT had the greater
influence on hemodynamics, while CRT had the greater effect on
psychoemotional status. Both treatments were well tolerated and produced
no side effects.
Clin Neurophysiol. 2010 Mar;121(3):441-7. Epub 2010 Jan 13.
Reduced motor cortex plasticity following inhibitory rTMS in older adults.
Todd G, Kimber TE, Ridding MC, Semmler JG.
Discipline of Physiology, School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, SA, Australia. email@example.com
OBJECTIVE: Ageing is accompanied by diminished practice-dependent
plasticity. We investigated the effect of age on another plasticity
inducing paradigm, repetitive transcranial magnetic stimulation (rTMS).
METHODS: Healthy young (n=15; 25+/-4 years) and old (n=15; 67+/-5
years) adults participated in two experiments. Motor evoked potentials
(MEPs) were measured in the target muscle (first dorsal interosseus,
FDI) and a remote muscle (abductor digiti minimi) during a set of single
stimuli. Subjects then received real or sham inhibitory rTMS
(intermittent subthreshold trains of 6Hz stimulation for 10min). MEPs
were measured for 30min after rTMS.
RESULTS: In young adults, MEPs in the target FDI muscle were
approximately 15% smaller in the real rTMS experiment than in the sham
rTMS experiment (P<0.026). In old adults, FDI MEP size did not differ
CONCLUSIONS: Advancing age is associated with reduced efficacy of inhibitory rTMS.
SIGNIFICANCE: This work has important implications for the potential therapeutic use of rTMS in stroke and neurological disease.
Zh Nevrol Psikhiatr Im S S Korsakova. 2009;109(7):23-8.
Possibilities of transcranial magnetic therapy and color and rhythm therapy in rehabilitation of ischemic stroke.
[Article in Russian]
Sholomov II, Cherevashchenko LA, Suprunov OV, Ra?gorondski? IuM.
One hundred and sixteen post-stroke patients were studied in the
early rehabilitation period. All patients were divided into 4 groups: 3
main and 1 control groups. Three main groups (87 patients) received
transcranial magnetic therapy (TMT) and/or color and rhythm therapy
(CRT) along with traditional treatment and the control group (29
patients) received only basic therapy. TMT was conducted using
bitemporal technique, running regime with modulation frequency 1-10 Hz.
In CRT, the alternating stimulation of the right and left eye with green
and/or blue color with a period of 2-4 s and duration of luminescence
1s was applied. Each of 3 main groups received 2 treatment sessions with
an interval of 1,5 month (1st – TMT, 2nd – CRT, 3rd – TMT + CRT). After
the treatment, the marked positive changes were seen in all main
groups, in particular in group 3. The improvement of neurologic symptoms
on the B. Lindmark scale was higher by 9,5% in group 3 compared to the
control one, on the Barthel index – by 8,8%, on MMSE and A. Luria and
Schulte test – by 5,4 and 14,3%, respectively. Rheographic and
encephalographic study revealed the significant improvement of
hemodynamics and alpha-rhythm differentiation, decrease of patients with
dysrhythmia by 14,6% in group 3 as compared to the control group. The
best results were seen in the combination of TMT and CRT, TMT exerted a
higher effect on the hemodynamics and CRT – on the psychoemotional
state. Both therapies were well tolerated and had no side-effects.
Stroke. 2009 Jan;40(1):309-12. Epub 2008 Oct 9.
Safety and behavioral effects of high-frequency repetitive transcranial magnetic stimulation in stroke.
Yozbatiran N, Alonso-Alonso M, See J, Demirtas-Tatlidede A, Luu D, Motiwala RR, Pascual-Leone A, Cramer SC.
BACKGROUND AND PURPOSE: Electromagnetic brain stimulation might have
value to reduce motor deficits after stroke. Safety and behavioral
effects of higher frequencies of repetitive transcranial magnetic
stimulation (rTMS) require detailed assessment.
METHODS: Using an active treatment-only, unblinded, 2-center study
design, patients with chronic stroke received 20 minutes of 20 Hz rTMS
to the ipsilesional primary motor cortex hand area. Patients were
assessed before, during the hour after, and 1 week after rTMS.
RESULTS: The 12 patients were 4.7+/-4.9 years poststroke (mean+/-SD)
with moderate-severe arm motor deficits. In terms of safety, rTMS was
well tolerated and did not cause new symptoms; systolic blood pressure
increased from pre- to immediately post-rTMS by 7 mm Hg (P=0.043); and
none of the behavioral measures showed a decrement. In terms of
behavioral effects, modest improvements were seen, for example, in grip
strength, range of motion, and pegboard performance, up to 1 week after
rTMS. The strongest predictor of these motor gains was lower patient
CONCLUSIONS: A single session of high-frequency rTMS to the motor
cortex was safe. These results require verification with addition of a
placebo group and thus blinded assessments across a wide spectrum of
poststroke deficits and with larger doses of 20 Hz rTMS.
Lancet Neurol. 2008 Jun;7(6):507-13. Epub 2008 May 1.
Contralesional repetitive transcranial magnetic stimulation for
chronic hemiparesis in subcortical paediatric stroke: a randomised
Kirton A, Chen R, Friefeld S, Gunraj C, Pontigon AM, Deveber G.
BACKGROUND: Arterial ischaemic stroke (AIS) can cause disabling
hemiparesis in children. We aimed to test whether contralesional,
inhibitory repetitive transcranial magnetic stimulation (rTMS) could
affect interhemispheric inhibition to improve hand function in chronic
subcortical paediatric AIS.
METHODS: Patients were eligible for this parallel, randomised trial
if they were in the SickKids Children’s Stroke Program and had
subcortical AIS more than 2 years previously, had transcallosal sparing,
were more than 7 years of age, had hand motor impairment, had no
seizures or dyskinesia, and were taking no drugs that alter cortical
excitability. Patients were paired for age and weakness and were
randomised within each pair to sham treatment or inhibitory,
low-frequency rTMS over contralesional motor cortex (20 min, 1200
stimuli) once per day for 8 days. An occupational therapist did
standardised tests of hand function at days 1 (baseline), 5, 10, and 17
(1 week post-treatment), and the primary outcomes were changes in grip
strength and the Melbourne assessment of upper extremity function
(MAUEF) between baseline and day 10. Patients, parents, and occupational
therapists were blinded to treatment allocation. Analysis was per
FINDINGS: Ten patients with paediatric stroke were enrolled (median
age 13.25 [IQR 10.08-16.78] years, mean time post-stroke 6.33 [SD 3.56]
years): four with mild weakness, two with moderate weakness, and four
with severe weakness. A repeated-measures ANOVA showed a significant
interaction between time and the effect of treatment on grip strength
(p=0.03). At day 10, grip strength was 2.28 (SD 1.01) kg greater than
baseline in the rTMS group and 2.92 (1.20) kg less than baseline in the
sham group (p=0.009). Benefits in mean grip strength persisted at day 17
(2.63 [0.56] kg greater than baseline with rTMS and 1.00 [0.70] kg less
than baseline with sham treatment; p=0.01). Day 10 MAUEF score improved
by more in the rTMS group than in the sham group (7.25 [3.8] vs 0.79
[1.3] points greater than baseline; p=0.002), but this benefit did not
persist to day 17. Function of the unaffected hand remained stable. rTMS
was well tolerated with no serious adverse events.
INTERPRETATION: Contralesional inhibitory rTMS was safe and feasible
for patients with paediatric subcortical AIS, and seemed to improve hand
function in patients with hemiparesis. Further studies are required to
confirm the potential role of rTMS in paediatric neurorehabilitation.
FUNDING: Canadian Stroke Consortium; Canadian Institutes of Health
Research; American Academy of Neurology Foundation; Alberta Heritage
Foundation for Medical Research.
Arch Neurol. 2008 Jun;65(6):741-7.
Effects of low-frequency repetitive transcranial magnetic
stimulation of the contralesional primary motor cortex on movement
kinematics and neural activity in subcortical stroke.
Nowak DA, Grefkes C, Dafotakis M, Eickhoff S, Küst J, Karbe H, Fink GR.
Department of Neurology, University Hospital Cologne, University of Cologne, Kerpener Strasse 62, D-50924 Cologne, Germany. firstname.lastname@example.org
BACKGROUND: Following the concept of interhemispheric competition,
downregulation of the contralesional primary motor cortex (M1) may
improve the dexterity of the affected hand after stroke.
OBJECTIVE: To determine the effects of 1-Hz repetitive transcranial
magnetic stimulation (rTMS) of the contralesional M1 on movement
kinematics and neural activation within the motor system in the subacute
phase after subcortical stroke.
DESIGN: Crossover investigation.
SETTING: A university hospital.
METHODS: Fifteen right-handed patients with impaired dexterity due to
subcortical middle cerebral artery stroke received 1-Hz rTMS for 10
minutes applied to the vertex (control stimulation) and contralesional
M1. For behavioral testing, patients performed finger and grasp
movements with both hands at 2 baseline conditions, separated by 1 week,
and following each rTMS application. For functional magnetic resonance
imaging, patients performed hand grip movements with their affected or
unaffected hand before and after each rTMS application.
RESULTS: Application of rTMS to the contralesional M1 improved the
kinematics of finger and grasp movements in the affected hand. At the
neural level, rTMS applied to the contralesional M1 reduced overactivity
in the contralesional primary and nonprimary motor areas. There was no
significant correlation between the rTMS-induced reduction in blood
oxygen level-dependent responses within the contralesional M1 and the
degree of behavioral improvement of the affected hand. Overactivity of
the contralesional dorsal premotor cortex, contralesional parietal
operculum, and ipsilesional mesial frontal cortex at baseline predicted
improvement of movement kinematics with the affected hand after rTMS of
the contralesional M1.
CONCLUSION: The functional magnetic resonance imaging data suggest
that rTMS of the contralesional M1 may normalize neural activation
within the cortical motor network after subcortical stroke. Identifying
patients suitable for rTMS intervention based on individual patterns of
cortical activation may help to implement rTMS in motor rehabilitation
Przegl Lek. 2007;64(2):74-7.
Effect of low frequency magnetic fields used in magnetotherapy and
magnetostimulation on the rehabilitation results of patients after
New methods of rehabilitation should be introduced in order to reduce
disability resulting from stroke. During the twelve months of
follow-up, effect of low frequency magnetic field (If mf) on the course
of patient rehabilitation following ischemic stroke was evaluated on
in-patient (acute and subacute period of the stroke) and outpatient
(chronic period) basis with the use of Mathew et al’s and Barthel’s
scales. Lf mf (20 procedures of 20-min. duration) of magnetotherapy (I
group–placebo, II–group 5.6 mT induction, 10 Hz frequency and sinusoidal
shape, III group–2.8 mT induction, 10 Hz frequency and sinusoidal
shape) and magnetostimulation (IV group–M1P1 program of Viofor JPS
system, 16 min a day) was applied as early as in the subacute period of
the stroke (1-8 weeks). The data obtained were presented in the form of
percentage changes in the pain levels as well as in the form of the
arithmetical mean and standard deviation (X +/- SD). The ANOVA test was
used for a statistical evaluation of the data obtained in the tests. The
results obtained indicate beneficial effects of If mf in the III and IV
group of patients in the Barthel’s scale and Mathew scale, which were
observed during the examination 12 months after the stroke. The
recommended doses of If mf seem to be adequate to obtain therapeutic
effects and may be used in the early period of rehabilitation. The
neurological and functional improvement persisted for a long-period of
the out-patient treatment, which was confirmed during the control
examination 12 months after the ischemic stroke. As no adverse effects
(which could be attributed to If mf), were observed, this method of
physical therapy can be recognized as a safe one and worth making
popular in clinical practice.
Restor Neurol Neurosci. 2007;25(5-6):461-5.
Improvement of dexterity by single session low-frequency repetitive
transcranial magnetic stimulation over the contralesional motor cortex
in acute stroke: a double-blind placebo-controlled crossover trial.
PURPOSE: Increasing evidence suggests that the contralesional motor
cortex (M1) inhibits the ipsilesional M1 in stroke patients. This
inhibition could impair motor function of the affected hand. We
investigated if inhibitory 1~Hz repetitive transcranial magnetic
stimulation (rTMS) over the contralesional M1 improved motor performance
of the affected hand in acute stroke.
METHODS: A double-blind study of real versus placebo rTMS was
conducted. Twelve patients early after subcortical stroke (mean: 7 days)
received 1200 stimuli of real and placebo rTMS in a crossover design.
The sequence of stimulations was counterbalanced across subjects.
Stimulus intensity was subthreshold (90% of motor threshold at rest).
Motor function was tested by grip strength recordings and Nine Hole Peg
Test (NHPT) executions before and after each rTMS session.
RESULTS: Compared to sham stimulation, real rTMS improved NHPT
results but not grip strength in the affected hand. No change of
performance was observed for the unaffected hand. NHPT baseline
repetitions in a subgroup of patients indicated stable motor performance
prior to the rTMS sessions.
CONCLUSIONS: The study suggests that therapeutic rTMS applications
over the contralesional hemisphere are feasible in acute stroke patients
and can transiently improve dexterity of the affected hand. RTMS may
become an additional tool for early neurorehabilitation.
Prog Brain Res. 2005;150:527-35.
Neural plasticity and recovery of function.
Wellcome Department of Imaging Neuroscience, Institute of Neurology,
University College London, 12 Queen Square, London WC1N 3BG, UK.
Recovery of the function after stroke is a consequence of many
factors including resolution of oedema and survival of the ischaemic
penumbra. In addition there is a growing interest in the role of central
nervous system (CNS) reorganization. Much of the evidence supporting
this comes from animal models of focal brain injury, but non-invasive
techniques such as functional magnetic resonance imaging, transcranial
magnetic stimulation, electroencephalography and magnetoencephalography
now allow the study of the working human brain. Using these techniques
it is apparent that the motor system of the brain adapts to damage in a
way that attempts to preserve motor function. This has been demonstrated
after stroke, as part of the ageing process, and even after disruption
of normal motor cortex with repetitive transcranial magnetic
stimulation. The result of this reorganization is a new functional
architecture, one which will vary from patient to patient depending on
the anatomy of the damage, the biological age of the patient and lastly
the chronicity of the lesion. The success of any given therapeutic
intervention will depend on how well it interacts with this new
functional architecture. Thus it is crucial that the study of novel
therapeutic strategies for treating motor impairment after stroke take
account of this. This review maps out the attempts to describe
functionally relevant adaptive changes in the human brain following
focal damage. A greater understanding of how these changes are related
to the recovery process will allow not only the development of novel
therapeutic techniques that are based on neurobiological principles and
designed to minimize impairment in patients suffering from stroke, but
also to target these therapies at the appropriate patients.
Neurology. 2005 Aug 9;65(3):466-8.
Therapeutic trial of repetitive transcranial magnetic stimulation after acute ischemic stroke.
Repetitive transcranial magnetic stimulation (rTMS) or sham
stimulation was given over the motor cortex daily for 10 days to two
randomly assigned groups of 26 patients with acute ischemic stroke.
Patients otherwise continued their normal treatment. Disability scales
measured before rTMS, at the end of the last rTMS session, and 10 days
later showed that real rTMS improved patients’ scores more than sham.
Stroke. 2005 Oct 27; [Epub ahead of print]
Repetitive Transcranial Magnetic Stimulation of Contralesional Primary Motor Cortex Improves Hand Function After Stroke.
Takeuchi N, Chuma T, Matsuo Y, Watanabe I, Ikoma K.
From the Department of Rehabilitation Medicine, Hokkaido University Graduate School of Medicine, Sapporo 060-0814, Japan.
BACKGROUND AND PURPOSE: A recent report has demonstrated that the
contralesional primary motor cortex (M1) inhibited the ipsilesional M1
via an abnormal transcallosal inhibition (TCI) in stroke patients. We
studied whether a decreased excitability of the contralesional M1
induced by 1 Hz repetitive transcranial magnetic stimulation (rTMS)
caused an improved motor performance of the affected hand in stroke
patients by releasing the TCI.
METHODS: We conducted a double-blind study of real versus sham rTMS
in stroke patients. After patients had well- performed motor training to
minimize the possibility of motor training during the motor
measurement, they were randomly assigned to receive a subthreshold rTMS
at the contralesional M1 (1 Hz, 25 minutes) or sham stimulation.
RESULTS: When compared with sham stimulation, rTMS reduced the
amplitude of motor-evoked potentials in contralesional M1 and the TCI
duration, and rTMS immediately induced an improvement in pinch
acceleration of the affected hand, although a plateau in motor
performance had been reached by the previous motor training. This
improvement in motor function after rTMS was significantly correlated
with a reduced TCI duration.
CONCLUSIONS: We have demonstrated that a disruption of the TCI by the
contralesional M1 virtual lesion caused a paradoxical functional
facilitation of the affected hand in stroke patients; this suggests a
new neurorehabilitative strategy for stroke patients.
Stroke. 2005 Nov 3; [Epub ahead of print]
Motor Strokes. The Lesion Location Determines Motor Excitability Changes.
Liepert J, Restemeyer C, Kucinski T, Zittel S, Weiller C.
From the Departments of Neurology and Neuroradiology, University
Medical Center Eppendorf, Hamburg, and Department of Neurology,
University Hospital, Freiburg, Germany.
BACKGROUND AND PURPOSE: The purpose of this research was to
investigate the impact of lesion location on motor excitability and
METHODS: We studied patients with pure motor strokes in 4 different
brain areas: motor cortex lesions (n=7), striatocapsular lesions (n=13),
lacunar lesions of the internal capsule (n=13), and paramedian pontine
lesions (n=10). Motor performance tests included the 9-hole-peg test and
grip strength recordings. Motor excitability was determined by
transcranial magnetic stimulation. Motor thresholds, stimulus-response
curves, silent periods, motor cortical inhibition, and facilitation were
RESULTS: The 4 groups were clinically similar but showed major
differences in motor excitability. Only motor cortex lesions had a loss
of intracortical inhibition in the affected hemisphere. In the internal
capsule lesion group and the pontine lesion group, stimulus-response
curves were depressed on the affected side. All of the subcortical
lesions showed a prolongation of the silent period in the paretic side.
Motor thresholds were predominantly elevated in the lesioned hemisphere
of patients with internal capsule or pontine lesions. Motor performance
was correlated with silent period duration in internal capsule lesions
and with motor thresholds in internal capsule and pontine lesions.
CONCLUSIONS: Motor cortex lesions exhibited deficient inhibitory
properties. In contrast, subcortical lesions displayed an enhancement of
inhibition. Internal capsule and pontine lesions affecting the
corticospinal tract on different levels particularly impaired neuronal
recruitment. Our results suggest that the lesion location determines a
specific pattern of motor excitability changes.
i Yi Jun Yi Da Xue Xue Bao. 2004 Aug;24(8):946-9, 952.
Effect of power-frequency electromagnetic fields on stroke during rehabilitation.
[Article in Chinese]
Deng AW, Yuang XG, Wei D, Zhang JH, Ran CF, Wang M.
Department of Rehabilitation, Longgang center Hospital of Shenzhen City, Shenzhen 518116, China. email@example.com
OBJECTIVE: To explore the effects and mechanism of power-frequency
electromagnetic fields on lipoprotein metabolism and homodynamic during
METHODS: One hundred fifteen patients with stroke were divided into 2
groups, 55 cases of them were treated by exposure to power-frequency
electromagnetic fields, 60 cases were treated as control group. Barthel
index and Functional Independence Measure (FIM) were used to evaluate
rehabilitation outcome. The lipoprotein and its subclasses, homodynamic
parameters were compared at pre- and post-rehabilitation.
RESULTS: The treatment group showed a statistically significant
better prognosis compared with the control group(P<0.01). The score
of Barthel index also increased after treatment(P<0.001). Total
cholesterol(Tc), triglycerides(TG), low-density lipoprotein
cholesterol(LDL-c) levels dropped and high-density lipoprotein
cholesterol(HDL-c) increased significantly. The Tc/HDL-c, LDL-c/HDL-c
ratio along with the blood and plasma viscosity decreased significantly
compared with the controls (P<0.01). The ratio of stroke recurrence
decreased significantly after the treatment compared with the control
CONCLUSION: The power-frequency electromagnetic fields can improve
lipoprotein metabolism and homodynamic parameters. It can improve the
ADL and FIM of stroke patients, which may have significant implications
for stroke patients.
Magnetic and laser therapy of acute ischemic stroke.
[Article in Russian]
The paper presents the technique of frequency-modulated magnetolaser
therapy (FMMLT) used in combined treatment of 121 patients with ischemic
stroke in acute period. The results were compared with those in the
control group of 30 patients who received conventional drug treatment.
The results of the comparison allowed the author to recommend FMMLT in
ischemic stroke especially in the period of “therapeutic window”.
Magnet therappy in rehabilitation of patients with cerabral ischemia.
[Article in Russian]
Provotorov VM, Putilina MV.
Basing on the results of clinical examination and treatment of 420
patients with aftereffects of acute circulatory disturbance, the authors
propose a pathogenetically grounded approach to correction of the
residual phenomena following stroke. The method proposed includes three
courses of impulse electromagnetotherapy used in combination with either
standard chemotherapy or massage and therapeutic exercise
The use of extremely high frequency electromagnetic fields during acute period of ischemic stroke.
[Article in Russian]
Podoliako VA, Makarchik AV.
The rheological, coagulatory and clinical examination of 70 patients
with acute ischemic stroke has demonstrated that EHF therapy (53.53 GHz)
improves rheological and coagulatory parameters in such patients, has a
positive effect on hemostasis and clinical course of acute ischemic
stroke. The technique and doses are presented.
Stroke. 2000 Jun;31(6):1210-6.
Treatment-induced cortical reorganization after stroke in humans.
Liepert J, Bauder H, Wolfgang HR, Miltner WH, Taub E, Weiller C.
BACKGROUND AND PURPOSE: Injury-induced cortical reorganization is a
widely recognized phenomenon. In contrast, there is almost no
information on treatment-induced plastic changes in the human brain. The
aim of the present study was to evaluate reorganization in the motor
cortex of stroke patients that was induced with an efficacious
METHODS: We used focal transcranial magnetic stimulation to map the
cortical motor output area of a hand muscle on both sides in 13 stroke
patients in the chronic stage of their illness before and after a
12-day-period of constraint-induced movement therapy.
RESULTS: Before treatment, the cortical representation area of the
affected hand muscle was significantly smaller than the contralateral
side. After treatment, the muscle output area size in the affected
hemisphere was significantly enlarged, corresponding to a greatly
improved motor performance of the paretic limb. Shifts of the center of
the output map in the affected hemisphere suggested the recruitment of
adjacent brain areas. In follow-up examinations up to 6 months after
treatment, motor performance remained at a high level, whereas the
cortical area sizes in the 2 hemispheres became almost identical,
representing a return of the balance of excitability between the 2
hemispheres toward a normal condition.
CONCLUSIONS: This is the first demonstration in humans of a long-term
alteration in brain function associated with a therapy-induced
improvement in the rehabilitation of movement after neurological injury.
Magnetotherapy and laser therapy were used in complex and
complex-combined regimens in 75 patients after cerebral ischemic or
hemorrhagic stroke starting on the poststroke week 4-5.
Clinico-neurologic, neurophysiological and cerebrohemodynamic findings
evidence for the highest effectiveness of neurorehabilitation including
complex magneto-laser therapy in hemispheric ischemic and hemorrhagic
stroke of subcortical location in the absence of marked
clinico-tomographic signs of dyscirculatory encephalopathy.
Complex-combined magneto-laser therapy is more effective for correction
of spastic dystonia. Mutual potentiation of magnetotherapy and laser
therapy results in maximal development of collateral circulation and
cerebral hemodynamic reserve (84% of the patients). Complex effects
manifest in arteriodilating and venotonic effects. Complex magneto-laser
therapy is accompanied by reduction of hyperthrombocythemia and
Motor hand recovery after stroke. Prognostic yield of early transcranial magnetic stimulation.
Cruz Martínez A, Tejada J, Díez Tejedor E.
Unidad de Electromiografía, Hospital La Luz, Madrid, Spain.
Transcranial magnetic stimulation (TMS) was performed in 20 patients
within the first days after stroke. Motor evoked potentials (MEPs) were
bilaterally recorded over thenar eminence muscles, and central motor
conduction time (CMCT), amplitude of the MEPs (A%M) and threshold
intensity compared between both sides. Six months later the patients
were reexamined. Within the first days after stroke the obtention of
MEPs at rest or during voluntary muscle activation have a favorable
prognostic value. All patients with early response by TMS reached a good
motor function in the following months. The follow-up showed that the
electrophysiological improvement was closely related to clinical
recovery of the hand function. However, even in cases with a good
recovery, the CMCT and, mainly, the A%M, may be significantly different
related to those in normal hand. TMS may be an early and valuable
prognostic indicator of hand function recovery after stroke, and their
prognostic yield is higher than clinical evaluation and CT study. TMS is
a quantifiable method of motor disability and may have practical
application in the management and rehabilitation therapy in stroke
The effect of combined transcerebral magnetic and electric impulse
therapy on the cerebral and central hemodynamic status of stroke
patients in the early rehabilitation period.
[Article in Russian]
Gorbunov FE, Orekhova EM, Isaev SV, Bugaev SA.
75 clinical cases of acute impairment of cerebral circulation treated
with sinusoidal modulated currents in combination with transcerebral
magnetic field have been analysed. Functional and clinical findings
indicate that the above combination is much more effective than magnetic
field and sinusoidal modulated currents alone: 62% against 35% and 33%,
Zh Nevrol Psikhiatr Im S S Korsakova. 1997;97(9):41-3.
Magnetic and electrical stimulation in the rehabilitative treatment of patients with organic nervous system lesions.
[Article in Russian]
Tyshkevich TG, Nikitina VV.
89 patients with organic damages of nervous system with paralyses and
pareses as the main symptoms in clinical pattern were treated. Their
treatment was complex with application of impulse magnetic field and
electrostimulation which permitted to achieve multilevel
electrostimulation. The control group was formed by 49 patients with
analogous diseases which were treated by sinusoidal current
electrostimulation only. Combined application of magnetic stimulation
and electrostimulation was more effective. That was confirmed by data of
roentgenographic and electromyographic studies.
Protection against focal cerebral ischemia following exposure to a pulsed electromagnetic field.
Grant G, Cadossi R, Steinberg G.
Department of Neurosurgery, Stanford University, California 94305.
There is evidence that electromagnetic stimulation may accelerate the
healing of tissue damage following ischemia. We undertook this study to
investigate the effects of low frequency pulsed electromagnetic field
(PEMF) exposure on cerebral injury in a rabbit model of transient focal
ischemia (2 h occlusion followed by 4 h of reperfusion). PEMF exposure
(280 V, 75 Hz, IGEA Stimulator) was initiated 10 min after the onset of
ischemia and continued throughout reperfusion (six exposed, six
controls). Magnetic resonance imaging (MRI) and histology were used to
measure the degree of ischemic injury. Exposure to pulsed
electromagnetic field attenuated cortical ischemia edema on MRI at the
most anterior coronal level by 65% (P < 0.001). On histologic
examination, PEMF exposure reduced ischemic neuronal damage in this same
cortical area by 69% (P < 0.01) and by 43% (P < 0.05) in the
striatum. Preliminary data suggest that exposure to a PEMF of short
duration may have implications for the treatment of acute stroke.
Zh Nevropatol Psikhiatr Im S S Korsakova. 1992;92(1):63-7.
Magnetotherapy of initial manifestations of cerebrovascular disorders in hypertension.
[Article in Russian]
The paper is concerned with the data on 147 subjects who underwent
magnetotherapy with the unit “Magniter-AMT-01” applied to the cervical
area. The main group included 102 subjects, 45 person served as control.
The purpose of the work was to base the application of MT under
inpatient and home conditions with the use of the above-indicated unit.
In view of this fact, a study was made of cerebral hemo- and
thermodynamics with the aid of rheoencephalography and
encephaloradiothermography under the action of different modes of the
functioning of the unit “Magniter-AMT-01” (pulse and variable magnet
induction fields 12-15 mTl and 30-35 mTl). A method of measuring
magnetosensitivity of patients depending on the temperature reaction of
the brain to a single MT session was elaborated. The greatest clinical
effect was attained with the use of pulse magnetic field 15 mTl.
Magnetotherapy with the use of the unit “Magniter-AMT-01” provided good
results under inpatient and home conditions. The magnetosensitive
patients demonstrated the highest effect.
J Cell Biochem. 1993 Apr;51(4):387-93.
Beneficial effects of electromagnetic fields.
Bioelectric Research Center, Columbia University, Riverdale, New York 10463.
Selective control of cell function by applying specifically
configured, weak, time-varying magnetic fields has added a new, exciting
dimension to biology and medicine. Field parameters for therapeutic,
pulsed electromagnetic field (PEMFs) were designed to induce voltages
similar to those produced, normally, during dynamic mechanical
deformation of connective tissues. As a result, a wide variety of
challenging musculoskeletal disorders have been treated successfully
over the past two decades. More than a quarter million patients with
chronically ununited fractures have benefitted, worldwide, from this
surgically non-invasive method, without risk, discomfort, or the high
costs of operative repair. Many of the athermal bioresponses, at the
cellular and subcellular levels, have been identified and found
appropriate to correct or modify the pathologic processes for which
PEMFs have been used. Not only is efficacy supported by these basic
studies but by a number of double-blind trials. As understanding of
mechanisms expands, specific requirements for field energetics are being
defined and the range of treatable ills broadened. These include nerve
regeneration, wound healing, graft behavior, diabetes, and myocardial
and cerebral ischemia (heart attack and stroke), among other conditions.
Preliminary data even suggest possible benefits in controlling
Stress-related psycho-physiological disorders: randomized single
blind placebo controlled naturalistic study of psychometric evaluation
using a radio electric asymmetric treatment.
Rinaldi S, Fontani V, Aravagli L, Mannu P, Castagna A, Margotti ML, Rosettani B.
Department of Neuro-Psycho-Physio Pathology and Neuro Psycho Physical
Optimization, Rinaldi Fontani Institute, Viale Belfiore 43, Florence
50144, Italy. firstname.lastname@example.org
The aim of this study is to investigate the effects of a radio
electric asymmetric treatment on psycho-physiological disorders (PPD).
PPD are often stress related and are under the unconscious control of
the patient and cannot be traced back to any serious physical disease.
The brain stimulation treatment protocol used is called Neuro Psycho
Physical Optimization (NPPO) with a Radio Electric Asymmetric Conveyer
Psychological stress and PPD were measured for a group of 888
subjects using the Psychological Stress Measure (PSM) test, a
self-administered questionnaire. Data were collected immediately before
and after the 4-weeks of REAC treatment cycle.
This study showed a significant reduction in scores measuring
subjective perceptions of stress for subjects treated with a cycle of
NPPO REAC treatment. At the end-point the number of subjects reporting
symptoms of stress-related PPD on the PSM test was significantly
reduced, whereas in the placebo group the difference was not
A cycle of NPPO treatment with REAC was shown to reduce subjective
perceptions of stress measured by the PSM test and in particular on PPD.
This trial has been registered in the Australian New Zealand Clinical
Trials Registry (ANZCTR) with the number: ACTRN12607000463471.
Stem Cell Res Ther. 2018 May 21;9(1):143. doi: 10.1186/s13287-018-0883-4.
Extremely low frequency electromagnetic fields promote mesenchymal stem cell migration by increasing intracellular Ca2+ and activating the FAK/Rho GTPases signaling pathways in vitro.
Zhang Y1, Yan J1, Xu H1, Yang Y1, Li W1, Wu H2, Liu C3.
Department of Orthopedics, Tongji Hospital, Tongji Medical College,
Huazhong University of Science and Technology, Jiefang Avenue 1095,
Wuhan, 430030, China.
Department of Orthopedics, Tongji Hospital, Tongji Medical College,
Huazhong University of Science and Technology, Jiefang Avenue 1095,
Wuhan, 430030, China. email@example.com.
Department of Orthopedics, Tongji Hospital, Tongji Medical College,
Huazhong University of Science and Technology, Jiefang Avenue 1095,
Wuhan, 430030, China. firstname.lastname@example.org.
The ability of mesenchymal stem cells (MSCs) to migrate to the
desired tissues or lesions is crucial for stem cell-based regenerative
medicine and tissue engineering. Optimal therapeutics for promoting MSC
migration are expected to become an effective means for tissue
regeneration. Electromagnetic fields (EMF), as a noninvasive therapy,
can cause a lot of biological changes in MSCs. However, whether EMF can
promote MSC migration has not yet been reported.
We evaluated the effects of EMF on cell migration in human bone
marrow-derived MSCs. With the use of Helmholtz coils and an EMF
stimulator, 7.5, 15, 30, 50, and 70 Hz/1 mT EMF was generated.
Additionally, we employed the L-type calcium channel blocker verapamil
and the focal adhesion kinase (FAK) inhibitor PF-573228 to investigate
the role of intracellular calcium content, cell adhesion proteins, and
the Rho GTPase protein family (RhoA, Rac1, and Cdc42) in EMF-mediated
MSC migration. Cell adhesion proteins (FAK, talin, and vinculin) were
detected by Western blot analysis. The Rho GTPase protein family
activities were assessed by G-LISA, and F-actin levels, which reflect
actin cytoskeletal organization, were detected using immunofluorescence.
All the 7.5, 15, 30, 50, and 70 Hz/1 mT EMF promoted MSC migration.
EMF increased MSC migration in an intracellular calcium-dependent
manner. Notably, EMF-enhanced migration was mediated by FAK activation,
which was critical for the formation of focal contacts, as evidenced by
increased talin and vinculin expression. Moreover, RhoA, Rac1, and Cdc42
were activated by FAK to increase cytoskeletal organization, thus
promoting cell contraction.
EMF promoted MSC migration by increasing intracellular calcium and
activating the FAK/Rho GTPase signaling pathways. This study provides
insights into the mechanisms of MSC migration and will enable the
rational design of targeted therapies to improve MSC engraftment.
Cell migration; Electromagnetic fields; Focal adhesion kinase; Intracellular Ca2+?; Rho GTPase protein famil
Int J Mol Sci. 2018 Mar 27;19(4). pii: E994. doi: 10.3390/ijms19040994.
Co-Culture with Human Osteoblasts and Exposure to Extremely Low
Frequency Pulsed Electromagnetic Fields Improve Osteogenic
Differentiation of Human Adipose-Derived Mesenchymal Stem Cells.
Siegfried Weller Institute for Trauma Research,
Eberhard-Karls-Universität Tübingen, 72076 Tübingen, Germany.
Experimental Trauma Surgery, Klinikum rechts der Isar, Technical
University of Munich, 81675 München, Germany.
Experimental Trauma Surgery, Klinikum rechts der Isar, Technical
University of Munich, 81675 München, Germany. email@example.com.
Siegfried Weller Institute for Trauma Research,
Eberhard-Karls-Universität Tübingen, 72076 Tübingen, Germany.
Sachtleben GmbH, 20251 Hamburg, Germany. firstname.lastname@example.org.
Sachtleben GmbH, 20251 Hamburg, Germany. email@example.com.
Experimental Trauma Surgery, Klinikum rechts der Isar, Technical
University of Munich, 81675 München, Germany. firstname.lastname@example.org.
Siegfried Weller Institute for Trauma Research,
Eberhard-Karls-Universität Tübingen, 72076 Tübingen, Germany.
Siegfried Weller Institute for Trauma Research,
Eberhard-Karls-Universität Tübingen, 72076 Tübingen, Germany.
Experimental Trauma Surgery, Klinikum rechts der Isar, Technical
University of Munich, 81675 München, Germany.
Human adipose-derived mesenchymal stem cells (Ad-MSCs)
have been proposed as suitable option for cell-based therapies to
support bone regeneration. In the bone environment, Ad-MSCs will receive
stimuli from resident cells that may
favor their osteogenic differentiation. There is recent evidence that
this process can be further improved by extremely low frequency pulsed
electromagnetic fields (ELF-PEMFs). Thus, the project aimed at (i)
investigating whether co-culture conditions of human osteoblasts (OBs)
and Ad-MSCs have an impact on their proliferation and osteogenic
differentiation; (ii) whether this effect can be further improved by
repetitive exposure to two specific ELF-PEMFs (16 and 26 Hz); (iii) and
the effect of these ELF-PEMFs on human osteoclasts (OCs). Osteogenic
differentiation was improved by co-culturing OBs and Ad-MSCs when
compared to the individual mono-cultures. An OB to Ad-MSC ratio of 3:1
had best effects on total protein content, alkaline phosphatase (AP)
activity, and matrix mineralization. Osteogenic differentiation was
further improved by both ELF-PEMFs investigated. Interestingly, only
repetitive exposure to 26 Hz ELF-PEMF increased
Trap5B activity in OCs. Considering this result, a treatment with
gradually increasing frequency might be of interest, as the lower
frequency (16 Hz) could enhance bone formation, while the higher
frequency (26 Hz) could enhance bone remodeling.
extremely low frequency pulsed electromagnetic fields (ELF-PEMF); primary human adipose-derived mesenchymal stem cells (Ad-MSCs); primary human osteoblasts (OBs); primary human osteoclasts (OCs)
Sci Rep. 2018; 8: 5108.
Published online 2018 Mar 23. doi: 10.1038/s41598-018-23499-9
Pulsed electromagnetic fields increase
osteogenetic commitment of MSCs via the mTOR pathway in TNF-? mediated
inflammatory conditions: an in-vitro study
Letizia Ferroni,1 Chiara Gardin,1 Oleg Dolkart,
2 Moshe Salai,2 Shlomo Barak,3 Adriano Piattelli,4 Hadar Amir-Barak,5and Barbara Zavan11Department of Biomedical Sciences, University of Padova, Via G. Colombo 3, 35100 Padova, Italy
Orthopaedic Surgery, Tel Aviv Sourasky Medical Center, Tel Aviv
University Sackler Faculty of Medicine, Tel Aviv, Israel
3Private Practice, Tel Aviv, Israel
4Department of Medical, Oral, and Biotechnological Sciences, University of Chieti-Pescara, Chieti, Italy
Internal Medicine E, Tel Aviv Sourasky Medical Center, Tel Aviv
University Sackler Faculty of Medicine, Tel Aviv, Israel
Oleg Dolkart, Email: moc.liamg@otraklod.
Pulsed electromagnetic fields
(PEMFs) have been considered a potential treatment modality for fracture
healing, however, the mechanism of their action remains unclear.
Mammalian target of rapamycin (mTOR) signaling may affect osteoblast
proliferation and differentiation. This study aimed to assess the
osteogenic differentiation of mesenchymal stem cells (MSCs) under PEMF
stimulation and the potential involvement of mTOR signaling pathway in
this process. PEMFs were generated by a novel miniaturized
electromagnetic device. Potential changes in the expression of mTOR
pathway components, including receptors, ligands and nuclear target
genes, and their correlation with osteogenic markers and transcription
factors were analyzed. Involvement of the mTOR pathway in osteogenesis
was also studied in the presence of proinflammatory mediators. PEMF
exposure increased cell proliferation and adhesion and the osteogenic
commitment of MSCs even in inflammatory conditions. Osteogenic-related
genes were over-expressed following PEMF treatment. Our results confirm
that PEMFs contribute to activation of the mTOR pathway via upregulation
of the proteins AKT, MAPP kinase, and RRAGA, suggesting that activation
of the mTOR pathway is required for PEMF-stimulated osteogenic
differentiation. Our findings provide insights into how PEMFs influence
osteogenic differentiation in normal and inflammatory environments.
Pulsed electromagnetic fields (PEMFs) have long been known to accelerate fracture repair1.
Exposure to PEMFs has been shown to affect cell proliferation and
differentiation by influencing multiple metabolic pathways, depending
upon lineage and maturation stage. In the osteoblast lineage, PEMFs
contribute to bone formation induced by a demineralized bone matrix and
stimulate fracture healing, probably through the action of progenitors
that are already committed towards bone2.
Data on the mechanism of action of PEMFs and the potential involvement
of specific signal transduction pathways are, however, scarce. It has
been reported that PEMFs increase the activity of certain kinases
belonging to known intracellular signaling pathways, such as the protein
kinase A (PKA) and the MAPK ERK1/23,4, and that they modulate anti-inflammatory effects by increasing the quantity of the adenosine receptors A2A5.
PEMFs stimulation also upregulates BMP2 expression in association with
increased differentiation in mesenchymal stem cells (MSCs)6,7.
Dental implants and total joint replacements are surgical
procedures that involve the implantation of permanent biomaterials. An
increasing number of these procedures has been extended to younger and
middle-age patients, making long-standing biocompatibility, robustness
and functionality crucial requirements for these implants. Despite many
recent advances, revision surgeries of the implants continue to be a
major concern due to the tissue response induced by implanted
biomaterials, as well as the potential for loosening and periprosthetic
osteolysis which remain significant challenges8.
The basis of recent insights into osseointegration range
from the pure bone healing that takes place around the implant to an
immune-mediated foreign body reaction9–11.
That reaction involves a sequence of events, including protein
adsorption on the surface of the implant, activation of complement and
the coagulation system, recruitment of monocyte/macrophages and MSCs,
activation and differentiation of these cells into functional
macrophages, osteoclasts, and osteoblasts, respectively, and the
formation of biological attachments between implant and new bone11.
The continued release of wear debris from the implants and the
potential evolving infection during the lifespan of the implant might
induce peri-implant inflammation, resulting in peri-implant osteolysis,
aseptic loosening and subsequent implant failure necessitating further
Serine/threonine kinase mammalian target of rapamycin
(mTOR) has been shown to play an important role in osteoclast
differentiation. It is activated by macrophage colony-stimulating
factor, and its inhibition leads to decreased osteoclastogenesis13,14.
Furthermore, mTOR expression levels are higher at the earlier stages of
osteoclastogenesis and decrease at the later stages of osteoclast
mTOR exists in cells as part of two complexes: complex 1 (mTORC1) and
complex 2 (mTORC2). mTORC1 is activated by amino acids, growth factors,
oxygen, inflammation, and Wnt signaling15.
mTORC1 is also a negative regulator of autophagy, a lysosomal
degradation process responsible for the removal of long-lived proteins
and damaged organelles16,17.
It has also been confirmed that the mTOR signaling pathway was involved
in the regulation of apoptosis and autophagy in MSCs, and that its
inhibition is able to attenuate age-related changes in MSCs18.
This study aimed to assess the potential
involvement of the mTOR signaling pathway in the osteogenic
differentiation of MSCs, the cells naturally involved in bone repair
processes, under stimulation with PEMFs. To this end, we analyzed
potential changes in the expression of mTOR signaling pathway
components, including receptors, ligands and nuclear target genes, and
their correlation with osteogenic markers and transcription factors.
PEMFs were generated using a miniaturized electromagnetic device (MED)
(Magdent Ltd., Tel Aviv, Israel) that is used successfully to stimulate
implant osseointegration in the clinical setting and in vivo to19. The involvement of mTOR pathway in osteogenesis was also studied in the presence of proinflammatory mediators.
The biocompatibility of the surface
was evaluated by MTT testing for measuring mitochondria activity as well
as by evaluating cell numbers. Figure 1A displays
the results of MTT testing conducted in normal conditions and in the
presence of proinflammatory cytokines. Mitochondrial activity increased
over time in both the control and PEMF groups. The presence of
inflammatory cytokines caused a well-defined decrease in MTT values. The
same pattern of increased cell proliferation was demonstrated by
monitoring the cell numbers (Fig. 1B).
Specifically, fewer cells were found in inflammatory conditions.
Moreover, PEMF treatment was able to increase cell proliferation in both
conditions. The proliferation rate was significantly higher in the PEMF
group compared to the controls, even in an inflammatory environment.
MSCs subjected to PEMF irradiation in the presence of proinflammatory cytokines for 30 days. (A) MTT proliferation assay. Results are expressed as mean?±?SD of at least 3 independent experiments, *p?<?0.05. (B) DNA content quantification. Results are expressed as mean?±?SD of at least 3 independent experiments, *p?<?0.05.
Morphology and cell adhesion properties
Morphologic analyses of MSCs were
performed. Phalloidin-labeled F-actin (red), DAPI nuclear staining
(blue) and overlaid fluorescent image of immunostained cellular
components (merged) for the MSCs of the control and PEMF-treated groups
are seen in Fig. 2. As shown in Fig. 2,
the cells were able to attach to the implant surface in both the PEMF
and control groups. The number of cells present on the implant surface
with PEMF was clearly higher compared to the number of cells in the
Morphologic analyses of MSCs. Phalloidin-labeled F-actin
(red), DAPI nuclear staining (blue) and overlaid fluorescent image of
immunostained cellular components (merged) for the MSCs of the control
and PEMF-treated groups. After 7 days of culture, the cells were
well-colonized throughout the implant surface, demonstrating a star-like
shape associated with osteoblastic features. The cells were also able
to spread after 7 days. PEMF irradiation resulted in a greater number of
cells that were attached to the surfaces.
Cell adhesion properties were assessed by
the analyses of gene expression of molecules involved on hyaluronan
synthesis (HAS1), including receptor for extracellular hyaluronic acid
molecules (CD44), integrin (ITGA1, 2, 3, 4), and cell adhesion molecules
of the cadherine family, such as NCAM, VCAM, and PCAM (Fig. 3).
The results are reported in all the graphs as an increase of gene
expression value in samples of cells cultured in control conditions
compared to cells exposed to PEMFs. PEMFs generated by MED were able to
induce an increase in the expression of all these molecules, thereby
confirming that they may enhance the adhesion properties of the cells.
The presence of inflammatory stimuli (Fig. 3B)
resulted in a reduction of cell adhesion, however, the presence of a
PEMF significantly increased the expression of the integrin and cadherin
receptors, thus potentially improving the ability of the cell to attach
to the surface.
Analyses of cell adhesion properties in normal conditions (A) and in the presence of inflammation (B)
were conducted by searching for the expression of molecules involved in
hyaluronian synthesis (HAS1), i.e., extracellular receptor for
hyaluronic acid (CD44), integrin (ITGA1, 2, 3, 4), and cadherin family
cell adhesion molecules (NCAM; VCAM; PCAM). The results are reported as
an increase in the gene expression value in samples of cells cultured on
implants with MED device compared to the same gene expression obtained
in normal conditions.
Real-time PCR for principal
osteogenic markers, such as Runx, osteopontin, osteonectin, osteocalcin,
collagen type I, wnt, foxO, ALP, BMP2, and BMP7 was performed in order
to evaluate the commitment of MSCs onto osteoblastic phenotypes. The
cells were cultured in the presence (Fig. 4A) and in the absence of inflammatory conditions (Fig. 4B)
in order to compare the variations obtained in the control group with
those obtained in the PEMF group. As illustrated in Fig. 4,
in all the conditions an increase in expression of all osteogenic
markers was noticed, confirming that the presence of PEMF exerts a
positive effect on this process even in the presence of inflammatory
cytokines. This commitment was confirmed by quantified ALP activity when
MSCs were cultured in both the control and PEMF groups in the presence
and absence of inflammatory stimuli (Fig. 5).
Additionally, PEMFs were also able to induce a positive effect on the
osteogenic process. It was clear that MSCs were also able to produce
higher values of ALP in the presence of inflammatory cytokines as well.
There was a significant, time-dependent ALP activity for cells grown
under PEMF treatment, demonstrating the promotion of the crystallization
of hydroxyapatites, a typical feature of pre-osteoblastic cells.
Real-time PCR for principal osteogenic markers, such as
Runx, osteopontin, osteonectin, osteocalcin, collagen type I, wnt, foxO,
ALP, BMP2, and BMP7 was performed in order to evaluate the commitment
of stem cells onto an osteoblastic phenotype. The cells were cultured in
the (A) presence and (B) absence of inflammatory conditions, and the variations obtained in normal implants versus implants?+?MED were compared.
Quantification of intracellular ALP activity (expressed as
U/mL) in MSC exposed to PEMFs and in non-exposed MSC in the presence and
absence of an inflammatory environment at 15 and 30 days. Results are
expressed as mean?±?SD of at least 3 independent experiments,
*p?<?0.05. *; **p?=?0.01; ***p?=?0.001.
In order to test if PEMF is able to
excerpt its osteogenic properties thought mTOR pathway we used rapamicin
to verify following hypothesis:
rapamicin is able to reduce the osteogenic properties in absence of PEMF(control);
The exposure to PEMF in presence of rapamicin could restore the osteogenic commitment of MSCs.
The osteogenic properties of MSCs seeded in the osteogenic
medium have been evaluated as their ability to produce a mineralized
extracellular matrix by means the ARS test. Figure 6 reports
the staining on implant (A); on the medium (B) and the quantification
of ARS staining (C). The osteogenic potential is related to the ability
to produce a mineralized matrix. Higher values of mineralization are
represented by a greater values of the red staining (Fig. 6A,B). Spectroscopy was used to assess these parameters. The quantification of the osteogenic potential is reported in Fig. 6C.
It is well evident that both in normal condition (passive implant) and
in presence of PEMF (active implant) a decent quantity of ARS is
detectable at the time frame of 7 to 14?days. When Rapamicin was added, a
well-defined decline was noticed, predominantly at 14 days in passive
condition. On the contrary, in presence of PEMF, Rapamicin was not able
to inhibit the process and mineralization of the extracellular
environment was demonstrated.
The osteogenic properties of MSCs seeded in the osteogenic
medium have been evaluated as their ability to produce a mineralized
extracellular matrix by means the ARS test. staining on implant (A); on the medium (B) and the quantification of ARS staining (C). Results are expressed as mean?±?SD of at least 3 independent experiments, **p?=?0.01.
Similar phenomenon was observed at gene expression level as well. In Figs 7 and ?and88 we
report the gene expression of markers related to mTOR pathway evaluated
at 14 day on MSCs cultures seeded in osteogenic medium with rapamicin,
with or without PEMF treatment (passive VS active implant). Results have
been grouped in correlation to their involvement in mTOR pathway: mTOR1
Complexes; mTOR2 Complexes; mTOR Upstream Regulators – negative
regulation; mTOR Upstream Regulators – positive regulation; mTOR
Downstream Regulators – negative regulation; mTOR Downstream Regulators –
positive regulation. The results were analyzed and are presented as the
ratio between: active implant in osteogenic medium?+?rapamicin with the
active implant in osteogenic medium without rapamicin; passive implant
in osteogenic medium?+?rapamicin with the passive implant in osteogenic
medium without rapamicin. Value comprises from ?2 to +2 are related to
no significant variation. All genes related to the ratio in presence of a
PEMF (active implant) are from ?2 to +2, indicating that no difference
occurs in co-presence of rapamicine and PEMF. On the contrary in absence
of PEMF defined up or dowregulation to gene related to mTOR1 involved
mostly in Adipogenic commitment rather than osteogenic commitment of
MSCs were demonstrated.
Real-time PCR analysis of mTOR pathway markers. Gene
expression levels of the selected markers are reported as ration of MSC
coltured on active implants in presence of osteogenic medium and
Rapamicin implants with passive implants in presence of osteogenic
medium and Rapamicin. Results are expressed as mean?±?SD of at least 3
independent experiments, **p?=?0.01.
In order to highlight genes responsive to the PEMF
stimulus we analyzed also the results related to the ratio of gene
expression of active implant in presence of osteogenic medium plus
rapamicin with the passive implant in osteogenic medium?+?rapamicine
As reported in Fig. 8, a significant difference was found in presence of PEMF and is related to:
•Decrease in RICTOR (receptor for mTOR2)
•Decrease in Protein phosphatase 2, regulatory subunit B, beta (PPP2R2B) involved on mTOR2 pathway
•Decrease in PKC protein (involved on Adipogenesis)
•Increase on VEGF (involved on angiogenesis)
•Decrease in Upstream regulator of negative mTOR regulator:
Protein kinase, AMP-activated, beta 1 non-catalytic subunit (PRKAB1)
Protein kinase, AMP-activated, beta 2 non-catalytic subunit (PRKAB2)
Protein kinase, AMP-activated, gamma 3 non-catalytic subunit (PRKAG3)
•Decrease in downstream stream regulator of negative mTOR regulator:
Calcium binding protein 39-like (CAB39L)
DNA-damage-inducible transcript 4 (DDIT4)
DNA-damage-inducible transcript 4-like (DDIT4L)
STE20-related kinase adaptor beta (STRADB)
The results indicated that PEMFs enhance
mTOR signaling by inducing an increase in the value of its related
proteins, such as AKT, MAPP kinase, and RRAGA. Additionally, a
significant increase in Rho family of GTPases was detected. Rho family
members play crucial roles in mechanical signal transduction and promote
the differentiation of MSCs into osteoblasts.
MSCs were treated with PEMF in the
presence of inflammatory cytokines as well as in the presence of PEMF.
The results of their effect on inflammatory/anti-inflammatory activities
of a PEMF are shown in Fig. 9, and they indicate that the presence of a PEMF induced a significant increase of in vitro expression
of IL-10 (that exerts anti-inflammatory activity). Conversely, there
was a reduction of expression of pro-inflammatory cytokines, such as
IL-1, following PEMF treatment. There was no significant difference in
expression of the other selected cytokines.
MSC were treated with inflammatory cytokines in the
presence and absence of PEMFs. The results of the effect on
inflammatory/anti-inflammatory activities of the active implants on MSC
indicate a significant increase of in vitro expression of IL-10
(that exerts anti-inflammatory activity) in the presence of PEMFs
generated by the MED device. Conversely, there is a reduction of
expression of inflammatory cytokines, such as IL-1, in the presence of
PEMFs. No significant difference in the expression of the other tested
cytokines is evident.
The principal results of the present
study revealed several novel findings regarding the events involved in
the stimulation of the osteogenic differentiation of MSCs induced by
PEMFs. They identified a significant role of mTOR signaling during the
differentiation driven by PEMF stimulation in an osteogenic
microenvironment. Additionally, PEMFs were able to preserve the
proliferation rate of MSCs in inflammatory conditions equal to that in a
normal environment. MED-induced PEMF treatment resulted in an
immunomodulatory effect in MSCs as expressed by increased IL-10
secretion. We found that PEMF stimulation of MSC proliferation mainly
affected cell cycle regulation, cell structure, extracellular matrix,
and some growth receptors involved in kinase pathways.
The osteointegration process begins with an inflammatory
stage followed by the migration of MSCs. One of the major goals of
dental, orthopedic and maxillofacial surgery is to achieve good and
rapid osteointegration between implants and bone. The main research
strategies to reduce implant failure aim at improving biomaterial
characteristics, or stimulating bone endogenous repair, through a
careful assessment of both processes by means of in vitro and in vivo experimental
models before any application in humans. It had been reported that
MED-generated PEMFs stimulated early bone formation around dental
implants, already resulting in higher peri-implant bone-implant contact
and bone mass after only 2 weeks, which suggests an acceleration of the
osseointegration process by more than 3-fold19.
However, the exact biologic mechanism of the influence of PEMFs on bone
regeneration remains to be elucidated. A recent study by Ferroni et al.
concluded that PEMFs affect the osteogenic differentiation of MSCs only
if they are pre-committed, and that this therapy can be an appropriate
candidate for the treatment of conditions requiring an acceleration of
the repair process20.
We raised two major questions concerning the PEMF-related
mechanism in the current study. First, we looked into the effects of
PEMFs on MSCs in an inflammatory environment with regard to the ability
of the cells to proliferate and adapt to the immunomodulatory changes.
Understanding the mechanism of the implant’s integration, particularly
the inflammatory response, is relevant for finding new treatment
modalities to optimize the osteointegration and subsequent stability of
the implants, which have implications in dentistry and orthopedic
surgery. In this study, we added pro- and anti-inflammatory cytokines,
which simulate the kinetics of their expression during early stages of
implant integration in vivo, and investigated their effects on
the proliferation and osteogenic differentiation of MSCs under PEMF
irradiation. The proliferative capacity of MSCs is highly relevant for
tissue repair21. Cytokines are known to affect proliferation of different cell types21.
Therefore, we first analyzed the effect of selected cytokines on MSC
proliferation. To the best of our knowledge, no previous study had
assessed the influence of PEMF irradiation on the production of
cytokines in MSC cultures. There are published data on the
post-irradiation release of cytokines in mature osteoblasts22 and in osteoclast-like cells23.
In both of those studies, ELISA was used for quantification and
demonstrated an increase of TNF-a, IL-1b and PG-E2 in relation both to
the recruitment of the osteoclast-like cells and to the intensity of the
electrical field. The current study demonstrated the ability of
MED-generated PEMFs to alter the immuno-modulative activity properties
of MSCs. A significant elevation in anti-inflammatory cytokines, such as
IL-10, was clearly present when MSCs were seeded on implants. IL-10
acted as an anti-inflammatory substance by inhibiting the synthesis of
proinflammatory cytokines, and its up-regulation in MSCs may counteract
the detrimental proinflammatory effects.
Second, we examined the effects of PEMFs on the mTOR
signaling pathway, and the results confirmed that PEMFs in the presence
of an inflammatory environment positively affected MSC commitment into
an osteoblastic phenotype through the mTOR pathway. In in vivo model
demonstrated that the IGF-1 released from the bone matrix during bone
remodeling stimulated osteoblastic differentiation of recruited MSCs by
activation of Akt/mTOR24.
It had been reported that the presence of a good bone-like
extracellular matrix was able to maintain bone mass by activation of
mTOR in mesenchymal stem cells24.
We now demonstrated that PEMF irradiation positively stimulated mTOR
signaling, thus increasing the osteoblastic commitment of MSCs in the
presence of inflammatory stimuli as well. This commitment could also be
induced by increased integrin expression, such as ?(4)?(1) integrin that
has a high affinity for bone and improves the homing of MSCs to bone,
thus promoting osteoblast differentiation and bone formation. mTOR is a
central molecule in the regulation of cell growth in a wide variety of
cells including osteoblasts, adipocytes, and myocytes. mTOR interacts
with several proteins to form two distinct complexes named mTOR complex 1
(mTORC1) and 2 (mTORC2) which differ in their unique components, Raptor
and Rictor. Upstream regulation and downstream products of mTORC1 are
much more investigated than that of mTORC2. Though it is widely believed
that the inhibition of mTOR signaling can promote osteoblastic
differentiation, this issue is still controversial. While rapamycin
primarily inhibits mTORC1, prolonged exposure can also disrupt mTORC2
function25. This fact makes difficult the data interpretation regarding the role played by mTORC1 and mTORC2 in osteogenesis. Martin SK et al. demonstrated that using Cre-mediated gene deletion in well established in-vitro differentiation assays, have shown that mTORC1 and mTORC2 have distinct roles in MSCs fate determination26. In agreement with previous studies26,27,
blockade of Raptor in MSCs resultedin reduced adipogenic potential.
Under osteoinductive conditions however, Raptor blockade promoted
osteogenic differentiation. In current study we demonstrated that in
osteogenic medium rapamicin is able to significantely reduce the
mineralization of extracellular matrix. However, PEMF treatment is able
to abolish this event, ensuring a good mineralization of extracellular
environment. In light of these findings, we can assume that in presence
of PEMF, the effect of rapamicin on osteoblasts behavior could be the
opposit. Gene expression of 84 markers associated with mTOR pathway
confirmed that no notable change in gene expression ocurred following
rapamicin treatment coadministered with PEMF. While comparing gene
expression under rapamicin treatment with PEMF to passive implant,
reduction in mTOR2 pathway related genes was found. Namely, we found a
reduction in Rictor expression that is associated to an adipogenic
commitment of MSCs; and a decrease in several markers associated to a
negative regulation of mTOR in both downstream and upstream levels. The
most important changes are related to PKC? that, as we have previously
demonstrated is strongly related to the adipogenic commitment of MSCs28–31.
We showed that PKC? recruits the 66-kD proapoptotic isoform of Shc
(p66Shc) to act as oxidoreductase within mitochondria and in triggering a
feed-forward cycle of ROS production, eventually leading to cell death.
The same players may come together in a radically different context,
i.e., the production of cellular signals linking hyperglicemia to the
regulation of a transdifferentiation scheme of stem cells residing in
adipose tissues. Moreover a downregulation of genes related to
adipofunction such as PRKAG3 involved on insulin signalling is well
evident. Finally, a significant increase in VEGF gene was demonstrated.
These data confirm the ability of PEMF to promote angiogenesis, that is
cruicial during tissue regeneration as we have previously demonstrated
in wound healing processes32,33.
The differentiations of MSCs into the osteoblastic or
adipogenic lineages are inter-dependent process: molecular components
promoting one cell fate inhibit the mechanisms leading the
differentiation of the alternative lineage. Interestingly, inducers of
differentiation along one lineage often inhibit differentiation along
Our results suggest that in presence of osteogenic medium, PEMFs are
able to induce osteogenic commitment of MSCs blocking the pathway of
adipogenesis via mTOR related proteins.
This study reaults are in a line and comparable with a several previusly published papers. Ardeshirylajimi et al.35 investigated
the the influence of prolonged pulsed extremely low frequency
electromagnetic field on the osteogenic potential of cultured induced
pluripotent stem cells. They concluded that combination of osteogenic
medium and pulsed extremely low frequency electromagnetic field can be a
great enhancement for bone differentiation of stem cells and
appropriate candidate the management of bone defects and patients
suffering from osteoporosis. A recently published paper by Arjmand et al.36 investigated
the osteoinductive potential of PEMF in combination with
Poly(caprolactone) (PCL) nanofibrous scaffold. Their results confirmed
that the effects of PEMF on the osteogenic differentiation of ADSCs are
very similar to these of osteogenic medium. They concluded that due to
the immunological concerns regarding the application of bioactive
molecules for tissue engineering, PEMF could be a good alternative for
osteogenic medium. Additional recent article by Ardeshirylajimi et al.37 demonstrated
that PEMF alone can induce osteogenic differentiation, but this
capability was significantly increased when used in combination with
electrospun polycaprolactone nanofibers. In addition, simultaneous use
of osteogenic medium, PEMF and electrospun nanofibers resulted in
increased osteogenic differentiation potential of induced pluripotent
This study has several limitations, including its in vitro nature. Furthermore, the cells were grown in a monolayer, which does not accurately reflect in vivo conditions.
The primary human cell cultures, however, can serve as a relevant model
for examining the effects of PEMFs on bone cell physiology. The
modulation of bone cell proliferation markers observed in this study
have implications with regard to the immediate effects of PEMFs on bone
formation and healing, as well as possible long-term implications for
In summary, the findings of the present
study revealed that MED-generated PEMFs stimulate osteogenic
differentiation and the maturation of the adipose tissue-derived MSCs
via activation of the mTOR pathways. We also demonstrated that PEMF
exposure increased cell proliferation, adhesion and the osteogenic
commitment of MSCs, even in inflammatory conditions. We showed that
PEMFs increased the expression of anti-inflammatory cytokines, such as
IL-10, and reduced the expression of the pro-inflammatory cytokine IL-1.
MSCs provided not only cell sources for connective tissues, but also
had a significant influence on the immune response. Further studies are
required to investigate the precise mechanisms by which mTOR signaling
pathways are influenced and to discover other potential pathways
involved in the PEMF-induced osteogenic effects.
The miniaturized electromagnetic
device (MED) (Magdent Ltd., Tel Aviv, Israel) was the generator used to
stimulate the cells. In the clinical setting, MED technology is used to
actively stimulate osteogenesis and osseointegration. The MED was used
with a Classix Dental Implant (3.3?mm 10?mm?L Non Touch Prime, Cortex
Ltd., Shlomi, Israel). The cells were irradiated continuously for 30
days with the MED inside the incubator and under the same conditions of
temperature, humidity and CO2 concentration as non PEMF irradiated cells which served as the controls.
MSCs were extracted from human
adipose tissues of 5 healthy women and 5 healthy men (age 21–36 years,
body mass index 30–38) who were undergoing cosmetic surgery procedures,
following the guidelines of the University of Padova’s Plastic Surgery
Clinic. The adipose tissues were digested with 0.075% collagenase (type
1?A; Sigma Aldrich, Italia) in a modified Krebs-Ringer buffer [125?mM
NaCl, 5?mM KCl, 1?mM Na3PO4, 1?mM MgSO4,
5.5?mM glucose, and 20?mM HEPES (pH 7.4)] for 60?min at 37?°C, followed
by 10?min with 0.25% trypsin. Floating adipocytes were discarded, and
cells from the stromal-vascular fraction were pelleted, rinsed with
media, and centrifuged, after which a red cell lysis step in NH4Cl was
run for 10?min at room temperature. The resulting viable cells were
counted using the trypan blue exclusion assay and seeded at a density of
106 cells per cm² for in vitro expansion in
Dulbecco’s modified Eagle’s medium (DMEM, SIGMA Aldrich Italia)
supplemented with 10% fetal calf serum and 1% penicillin/streptomycin.
For treatment in inflammatory conditions, the cells were treated for
24?h with 0.1?mg/mL?1 of tumor necrosis factor-alpha
(Celbio). TNF-? concentration used in the study is higher than in
physiologic conditions. However, the aforementioned concentration was
chosen based on the previously published papers in order to achieve
effects in in-vitro studies38,39.
DNA content was determined using a
DNeasy kit (Qiagen, Hilden, Germany) to isolate total DNA from cell
cultures following the manufacturer’s protocol for tissue isolation,
using overnight incubation in proteinase K (Qiagen). DNA concentration
was detected by measuring the absorbance at 260?nm in a
spectrophotometer. The cell number was then determined from a standard
curve (microgram DNA vs. cell number) generated by DNA extraction from
the counted cells. The standard curve was linear over the tested range
of 5–80?µg DNA (r?=?0.99).
To determine the proliferation rate
of cell growth on titanium disks with or without treatment, a methyl
thiazolyl-tetrazolium (MTT)-based cytotoxicity assay was performed
according to the method of Denizot and Lang with minor modifications40.
The test is based on mitochondria viability, i.e., only functional
mitochondria can oxidize an MTT solution, giving a typical blue-violet
endproduct. After harvesting the culture medium, the cells were
incubated for 3?h at 37?°C in 1?mL 0.5?mg/mL MTT solution prepared in
phosphate buffered saline (PBS) solution. After removal of the MTT
solution by pipette, 0.5?mL 10% dimethyl sulfoxide in isopropanol
(iDMSO) was added for 30?min at 37?°C. For each sample, absorbance
values at 570?nm were recorded in duplicate on 200??L aliquots deposited
in 96-well plates using a multilabel plate reader (Victor 3 Perkin
Elmer, Milano, Italy). All samples were examined after 15 and 30 days of
RNA extraction and first-strand cDNA synthesis
RNase-Free DNase Set (Qiagen) from
implants were cultured with adipose tissue derived mesenchymal stem
cells for 15 and 25 days. The RNA quality and concentration of the
samples were measured using a NanoDropTM ND-1000
Spectrophotometer (Thermo Scientific). For the first-strand cDNA
synthesis, 200?ng of total RNA of each sample was reverse transcribed
with M-MLV Reverse Transcriptase (Invitrogen), following the
Human primers were selected for each target gene with Primer 3 software (Table 1).
Real-time PCRs were carried out using the designed primers at a
concentration of 300?nM and FastStart SYBR Green Master (Roche) on a
Rotor-Gene 3000 (Corbett Research, Sydney, Australia). Real-time PCR was
performed also according to the user’s manual for the Human mTOR
signaling Profiler PCR Array (SABiosciences, Frederick, MD, USA) and
using RT2 SYBR Green ROX FAST Master Mix (Qiagen). The data were
analyzed using Excel-based PCR Array Data Analysis Templates
(SABiosciences). The thermal cycling conditions were as follows: 15?min
denaturation at 95?°C, followed by 40 cycles of 15?s denaturation at
95?°C, annealing for 30?s at 60?°C, and 20?s elongation at 72?°C.
Differences in gene expression were evaluated by the 2??Ct method, using
MSCs cultured in the presence and absence of inflammatory cytokines and
in the presence and absence of PEMFs. Values were normalized to the
expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) internal
reference whose abundance did not change under our experimental
conditions. Experiments were performed with 3 different cell
preparations and repeated at least 3 times.
List of gene related to mTOR pathway analized by RT PCR.
MTOR associated protein, LST8 homolog (S. cerevisiae)
Mechanistic target of rapamycin (serine/threonine kinase)
Regulatory associated protein of MTOR, complex 1
Mitogen-activated protein kinase associated protein 1
RPTOR independent companion of MTOR, complex 2
mTOR Upstream Regulators negative regulation:
Eukaryotic translation initiation factor 4E binding protein 1
Eukaryotic translation initiation factor 4E binding protein 2
Protein phosphatase 2, catalytic subunit, alpha isozyme
Protein phosphatase 2, regulatory subunit B, beta
Protein phosphatase 2?A activator, regulatory subunit 4
Total RNA was extracted using an
RNeasy Lipid Tissue kit (Qiagen), including DNase digestion with the
RNase-Free DNase. Set (Qiagen), from the mTOR signalling RT2 profiler
PCR Array (gene analized are reported on Table 1).
In total, 800?ng of RNA was reverse-transcribed using an RT2 First
Strand kit (Qiagen). Real-time PCR was performed according to the user’s
manual for the Human mTOR signalling RT2 profiler PCR Array
(SABiosciences, Frederick, MD, USA) and using RT2 SYBR Green ROX FAST
Master Mix (Qiagen). Thermal cycling and fluorescence detection were
performed using a Rotor-Gene Q 100 (Qiagen). The data were analyzed
using Excel-based PCR Array Data Analysis Templates (SABiosciences).
Alizarin Red S staining
The extracellular mineral deposits
were detected by Alizarin Red S staining. Cells were fixed in 4%
paraformaldehyde (Sigma-Aldrich) in PBS for 10?min at room temperature.
Cells were stained adding 40?mM freshly Alizarin Red S Solution (pH 4.2)
for 10?min at room temperature with gentle shaking. Cells were washed
with ddH2O, then photographed by an optical microscope. Alizarin Red S
stained area were quantified from microscope images of three independent
experiments using ImageJ software (NIH, Bethesda, MD, USA).
ALP activity measurements
Alkaline phosphatase (ALP) activity
was measured for up to 20 days of cell culture in order to evaluate the
initial differentiation of Adipose Tissue Derived Mesenchymal Stem
cells into preosteoblasts. Abcam’s alkaline phosphates kit
(colorimetric) was used to detect the intracellular and extracellular
ALP activities. The kit uses p-nitrophenyl phosphate (pNPP) as a
phosphatase substrate, which is adsorbed at 405?nm when dephosphorylated
by ALP. In accordance with the manufacturer’s protocol, the culture
medium from each sample group was collected and pooled. At the same
time, the cells were washed with PBS and then homogenized with ALP assay
buffer (a total of 300??L for each group) and centrifuged at 13,000?rpm
for 3?min to remove insoluble material. Different volumes of samples
(medium and cells) were then added into 96-well plates, bringing the
total volume in each well up to 80??L with assay buffer. In addition,
80??L fresh medium was utilized as sample background control.
Thereafter, 50??L 5mMpNPP solution was added to each well containing
test samples and background control and incubated for 60?min at 25?°C
while shielding the plate from light. A standard curve of 0, 4, 6, 12,
16, and 20?nmol/well was generated from 1?mM pNPP standard solution,
bringing the final volume to 120??L with assay buffer. All reactions
were then stopped by adding 20??L of stop solution into each standard
and sample reaction, except the sample background control reaction.
Optical density was read at 405?nm in a microplate reader (Victor). The
results were normalized by subtracting the value derived from the zero
standards from all standards, samples and sample background control. The
pNP standard curve was plotted to identify the pNP concentration in
each sample. ALP activity of the test samples was calculated as follows:
ALP activity (U/ml) = A/V/T
where: A is the amount of pNP generated by samples (in ?mol), V is
the amount of sample added in the assay well (in mL), and T is the
reaction time (in minutes).
Cells were fixed in 4%
paraformaldehyde in PBS for 10?min and then incubated in 2% bovine serum
albumin (BSA, Sigma-Aldrich) in PBS for 30?min at room temperature.
They were then incubated with primary antibodies in 2% BSA solution in a
humidified chamber for 12?h at 4?°C. The rabbit polyclonal antihuman
phalloidine antibody (Millipore Corporation, MA, USA) was the primary
antibody. Immunofluorescence staining was performed using the secondary
antibody DyLight 549-labeled anti-rabbit IgG (H?+?L) (KPL, Gaithersburg,
MD, USA) diluted 1/1000 in 2% BSA for 1?h at room temperature. Nuclear
staining was performed with 2??g/mL Hoechst H33342 (Sigma-Aldrich) solution for 2?min.
One-way analysis of variance
(ANOVA) was used for data analyses. Levene’s test was used to
demonstrate the equal variances of the variables. Repeated measures
ANOVA with a post-hoc analysis using Bonferroni’s multiple comparison
was performed. T-tests were used to determine significant differences
(p?<?0.05). Repeatability was calculated as the standard deviation of
the difference between measurements. All testing was performed using
SPSS 16.0 software (SPSS Inc., Chicago, IL, USA) (license of the
University of Padua, Italy).
Research support (including PEMF generting devices and partial support in lab supplies) was provided by Mgdent Ltd.
B.Z., A.P. and H.A.B. conceived and designed the
experiments; L.F., O.D. and C.G. performed the experiments; B.Z., S.B.
and C.G. analyzed the data; B.Z. and H.A.B. contributed
reagents/materials/analysis tools; B.Z., L.F., M.S. and O.D. wrote the
O.D.- payed consultant of the Magdent ltd. Company. S.B.- Co-founder of Magdent ltd.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Neural Regen Res. 2018 Jan;13(1):145-153. doi: 10.4103/1673-5374.224383.
Low-frequency pulsed electromagnetic field pretreated bone
marrow-derived mesenchymal stem cells promote the regeneration of
crush-injured rat mental nerve.
Seo N1, Lee SH2, Ju KW2, Woo J3, Kim B4, Kim S5, Jahng JW6, Lee JH7.
Department of Oral and Maxillofacial Surgery, Graduate School of
Dentistry, Seoul National University; Dental Research Institute, Seoul
National University, Seoul, South Korea.
Department of Oral and Maxillofacial Surgery, Seoul National
University Dental Hospital; Dental Research Institute, Seoul National
University, Seoul, South Korea.
Department of Oral and Maxillofacial Surgery, Seoul National University Dental Hospital, Seoul, South Korea.
Clinical Translational Research Center for Dental Science (CTRC), Seoul National University Dental Hospital, Seoul, South Korea.
Department of Oral and Maxillofacial Surgery, Graduate School of
Dentistry, Seoul National University; Department of Oral and
Maxillofacial Surgery, Seoul National University Dental Hospital, Seoul,
Dental Research Institute, Seoul National University, Seoul, South Korea.
Department of Oral and Maxillofacial Surgery, Graduate School of
Dentistry, Seoul National University; Department of Oral and
Maxillofacial Surgery, Seoul National University Dental Hospital; Dental
Research Institute, Seoul National University; Clinical Translational
Research Center for Dental Science (CTRC), Seoul National University
Dental Hospital, Seoul, South Korea.
Bone marrow-derived mesenchymal stem cells (BMSCs) have been shown to
promote the regeneration of injured peripheral nerves. Pulsed
electromagnetic field (PEMF) reportedly promotes the proliferation and
neuronal differentiation of BMSCs. Low-frequency PEMF can induce the
neuronal differentiation of BMSCs in the absence of nerve growth
factors. This study was designed to investigate the effects of
low-frequency PEMF pretreatment on the proliferation and function of
BMSCs and the effects of low-frequency PEMF pre-treated BMSCs on the
regeneration of injured peripheral nerve using in vitro and in vivo
experiments. In in vitro experiments, quantitative DNA analysis was
performed to determine the proliferation of BMSCs, and reverse
transcription-polymerase chain reaction was performed to detect S100
(Schwann cell marker), glial fibrillary acidic protein (astrocyte
marker), and brain-derived neurotrophic factor and nerve growth factor
(neurotrophic factors) mRNA expression. In the in vivo experiments, rat
models of crush-injured mental nerve established using clamp method were
randomly injected with low-frequency PEMF pretreated BMSCs,
unpretreated BMSCs or PBS at the injury site (1 × 106 cells).
DiI-labeled BMSCs injected at the injury site were counted under the
fluorescence microscope to determine cell survival. One or two weeks
after cell injection, functional recovery of the injured nerve was
assessed using the sensory test with von Frey filaments. Two weeks after
cell injection, axonal regeneration was evaluated using
histomorphometric analysis and retrograde labeling of trigeminal
ganglion neurons. In vitro experiment results revealed that
low-frequency PEMF pretreated BMSCs proliferated faster and had greater
mRNA expression of growth factors than unpretreated BMSCs. In vivo
experiment results revealed that compared with injection of unpretreated
BMSCs, injection of low-frequency PEMF pretreated BMSCs led to higher
myelinated axon count and axon density and more DiI-labeled neurons in
the trigeminal ganglia, contributing to rapider functional recovery of
injured mental nerve. These findings suggest that low-frequency PEMF
pretreatment is a promising approach to enhance the efficacy of cell
therapy for peripheral nerve injury repair.
Sci Rep. 2017; 7: 9421.
Published online 2017 Aug 25. doi: 10.1038/s41598-017-09892-w
Enhancement of mesenchymal stem cell chondrogenesis with short-term low intensity pulsed electromagnetic fields
Dinesh Parate,1 Alfredo Franco-Obregón,
2,3 Jürg Fröhlich,2,4 Christian Beyer,4 Azlina A. Abbas,5 Tunku Kamarul,5James H. P. Hui,
1,6 and Zheng Yang
Orthopaedic Surgery, Yong Loo Lin School of Medicine, National
University of Singapore, NUHS Tower Block, Level 11, 1E Kent Ridge Road,
Singapore, 119288 Singapore
Surgery, Yong Loo Lin School of Medicine, National University of
Singapore, NUHS Tower Block, Level 8, IE Kent Ridge Road, Singapore,
Electromagnetic Pulsing Systems Laboratory, BICEPS, National University
of Singapore, MD6, 14 medical Drive, #14-01, Singapore, 117599 Singapore
4Institute for Electromagnetic Fields, Swiss Federal Institute of Technology (ETH), Rämistrasse 101, 8092 Zurich, Switzerland
Group (TEG), National Orthopaedic Centre of Excellence for Research and
Learning (NOCERAL), Department of Orthopaedic Surgery, Faculty of
Medicine, University of Malaya, Pantai Valley, Kuala Lumpur, 50603
Program, Life Sciences Institute, National University of Singapore, DSO
(Kent Ridge) Building, #04-01, 27 Medical Drive, Singapore, 117510
Alfredo Franco-Obregón, Email: gs.ude.sun@farus.
Pulse electromagnetic fields (PEMFs)
have been shown to recruit calcium-signaling cascades common to
chondrogenesis. Here we document the effects of specified PEMF
parameters over mesenchymal stem cells (MSC) chondrogenic
differentiation. MSCs undergoing chondrogenesis are preferentially
responsive to an electromagnetic efficacy window defined by field
amplitude, duration and frequency of exposure. Contrary to conventional
practice of administering prolonged and repetitive exposures to PEMFs,
optimal chondrogenic outcome is achieved in response to brief
(10?minutes), low intensity (2?mT) exposure to 6?ms bursts of magnetic
pulses, at 15?Hz, administered only once at the onset of chondrogenic
induction. By contrast, repeated exposures diminished chondrogenic
outcome and could be attributed to calcium entry after the initial
induction. Transient receptor potential (TRP) channels appear to mediate
these aspects of PEMF stimulation, serving as a conduit for
extracellular calcium. Preventing calcium entry during the repeated PEMF
exposure with the co-administration of EGTA or TRP channel antagonists
precluded the inhibition of differentiation. This study highlights the
intricacies of calcium homeostasis during early chondrogenesis and the
constraints that are placed on PEMF-based therapeutic strategies aimed
at promoting MSC chondrogenesis. The demonstrated efficacy of our
optimized PEMF regimens has clear clinical implications for future
regenerative strategies for cartilage.
Articular cartilage is an avascular
tissue with low potential for self-repair. When left untreated, lesions
of the articular cartilage can lead to osteoarthritis1–3.
The success of any technology aimed at repairing chondral defects will
thus be based on its ability to produce tissues that most closely
recapitulate the mechanical and biochemical properties of native
cartilage. To this end many technologies have been advanced yet, none
are without drawbacks. The ‘microfracture’ technique is commonly plagued
by the formation of fibro-cartilaginous tissue of low dexterity4.
Autologous chondrocytes implantation and osteochondral autograft
transplantation are limited by scarce cartilage production, low
proliferative capacity of chondrocytes, chondrocyte de-differentiation
and complications due to donor site morbidity5.
Stem cell-based approaches are also being actively pursued in hopes of
improved outcome. Mesenchymal stem cells (MSCs) support chondrogenic
differentiation and are an attractive cell source for cartilage tissue
engineering. However, the neocartilage formed by conventional MSC-based
repair methodologies commonly contain a mixture of fibro- and hyaline
cartilage6–8 that do not achieve the biochemical, mechanical or functional properties of native cartilage.
MSCs can be differentiated along different cell lineages of
mesodermal origin including osteoblasts, chondrocytes, skeletal
myocytes or visceral stromal cells9. Chondrogenic induction of MSCs entails proliferation, condensation, differentiation and maturation10,
necessitating endogenous transcriptional and developmental regulators,
cell-cell and cell-matrix interactions that, in turn, are modulated by
environmental stimuli including mechanical forces, temperature and
oxygen levels10–13. A common objective is to recreate as closely as possible the in vivo environmental conditions in vitro so
that the rate and quality of chondrogenic development is enhanced and
the functionality of the repaired tissue improved. To this end, various
environmental stimuli such as hypoxia, mechanical, electric and
electromagnetic stimulation are currently being explored14–16.
Mechanical stimulation can be applied in a semi-controlled
manner with the use of bioreactors designed to impart shear,
compression, tension, or pressure on developing tissues. Appropriately
applied mechanical stimulation positively influences MSC-induced
chondrogenic differentiation, ECM deposition and the mechanical
properties of the generated cartilage17–20.
At the cellular level the transduction of mechanical signals
(mechanotransduction) involves their conversion into biochemical
responses, often with the assistance of mechanosensitive
Electromagnetic field (EMF)-stimulation has been shown to promote cell
differentiation via the modulation of extracellular calcium entry via
plasma membrane-embedded cation channels25–27, raising the intriguing possibility that EMFs may be recruiting related pathways.
Studies examining time-variant or pulsing
electromagnetic fields (PEMFs) have alluded to a benefit over articular
chondrocytes or cartilaginous tissue in vitro, particularly
with reference to chondrocyte proliferation, extracellular matrix (ECM)
deposition, secretory activity and inflammatory status28–35.
Studies have also examined the effects of PEMF-treatment over the
chondrogenic differentiation of stem cells derived from bone marrow36–38, adipose35, umbilical cord Wharton jelly39, synovial fluid40 or peripheral blood sources40.
The reported consequences of PEMF-stimulation over chondrogenesis,
however, are largely inconsistent. Some studies report modest
enhancements in the gene expression of Sox9, aggrecan, type II collagen
(Col 2) as well as deposition of sulfated glycosaminoglycan (sGAG),
typically on the order of 2-folds35, 36, whereas other studies show little to no effect38–41. On the extreme end of the spectrum, Wang et al.37 reported
inhibition of both Sox9 and Col 2 expression concomitant with induction
of hypertrophy and mineralization in response to exposures of 3?h per
day at an amplitude of 1?mT. Obvious differences in stimulation
protocols likely underlie reported discrepancies. Existing EMF studies
have typically employed exposure durations between 30?minutes to 8?h per
day and more consistently in the low milli Tesla amplitude range
(3–5?mT). Empirical determination of the appropriate exposure and signal
parameters for a specific biological response and given tissue are
essential as there are indications that cell responses to magnetic
fields obey an electromagnetic efficacy window defined by a specific
combination of frequency, amplitude and time of exposure that gives rise
to optimum cell response42, 43.
Here, we systematically characterized the effects of PEMF exposure over
MSC chondrogenic differentiation by varying the field amplitude,
exposure duration and dosage with an emphasis on determining the
briefest and lowest amplitude electromagnetic exposure to render a
developmental outcome. Given that both mechanical stimuli and calcium
entry21, 22 influences
chondrogenic differentiation, we investigated the ability of PEMF
exposure to influence calcium homeostasis during early induction of MSCs
into the chondrogenic lineage, in particular that attributed to the
Transient Receptor Potential (TRP) family of cation-permeable channels,
which has been broadly implicated in cellular mechanotransduction23, 44.
We show that brief and single exposures to low amplitude PEMFs were
most effective at stimulating MSC chondrogenesis. Our results also
implicate the involvement of calcium influx and the mechanosensitive TRP
channels, TRPC1 and TRPV4, in the chondrogenic development stimulated
by targeted PEMF exposure.
Effect of PEMF intensities and exposure durations on MSC chondrogenesis
We first sought to determine the
magnetic field amplitude and duration of exposure at which MSCs
undergoing chondrogenic induction are most responsive using starting
conditions preliminarily tested in MSCs for chondrogenic regeneration39.
MSC pellets in chondrogenic differentiation medium were subjected to
single exposures to PEMFs of 10?min duration at intensities ranging in
amplitudes between 0–4?mT (Fig. 1A), then subjected to exposure durations between 5 and 60?min at 2?mT intensity (Fig. 1B),
applied on the first day of chondrogenic induction. RNA analysis
monitoring MSC chondrogenic progression at 7 days post-induction showed
greatest increases in response to 10?min exposures applied at an
amplitude of 2?mT as evidenced by enhancements in Sox9, aggrecan and Col
2 mRNA expression. By contrast, lower (1?mT) or higher (>3?mT)
amplitude of PEMFs (Fig. 1A), or briefer (5?min) or longer (>20?min) durations of exposure (Fig. 1B),
resulted in overall smaller effect sizes. The same EMF efficacy window
translated to the expression of cartilaginous ECM macromolecular
proteins (Fig. 1C).
In response to 2?mT amplitude pulsing, a 3-fold increase in Col 2
protein was detected 21 days after chondrogenic induction, whereas no
increase was detected with exposure to 3?mT. Moreover, a 2-fold increase
in sGAG was detected in response to exposure to 2?mT PEMFs, whereas
3?mT PEMFs produced a significantly smaller increase. The relative
ineffectiveness of prolonged exposure to PEMFs was also corroborated at
the protein level. Sixty min exposures to 2?mT PEMFs did not elicit
significant increases in Col 2 formation than 10?min exposures (Fig. 1C).
With reference to sGAG production, PEMF amplitudes greater than 2?mT,
or exposure durations of one hour, produced inferior results to 10?min
exposures at 2?mT.
Effects of PEMF amplitude (A) and exposure duration (B)
on MSC chondrogenesis. Real-time PCR analysis of cartilaginous markers
expression after 7 days of differentiation was normalized to GAPDH and
presented as fold-changes relative to levels in undifferentiated MSC. (C)
Quantification of cartilaginous extracellular matrix macromolecules
(Col 2 and sGAG) generated after 21 days of chondrogenic differentiation
of MSC subjected to distinct PEMF parameters. All data shown are
mean?±?SD, n?=?6 from 2 independent experiments. *Denotes significant
increase, or decrease, compared to non-PEMF control. #Denotes significant decrease compared to 2?mT (A), or 10?min (B) PEMF exposure.
Dosage effects of PEMFs over MSC chondrogenesis
We next investigated the effect of
repetitive exposures to PEMFs. MSCs were exposed to PEMFs at 2?mT for
10?min/day once, twice or thrice on days 1, 2 and 4 following
chondrogenic induction (Fig. 2A).
RNA analysis after 7 days of differentiation showed that a single
exposure produced the greatest and most consistent increase in the
expression of chondrogenic markers (Fig. 2A).
In another series of experiments, MSCs pellets were exposed once on the
first day of chondrogenic induction, or weekly for 3 consecutive weeks
RNA analysis after 21 days of chondrogenic differentiation showed that a
single exposure to 2?mT PEMFs for 10?min given on day 1 of induction
gave the greatest and most consistent increase in expression of
chondrogenic markers relative to no exposure (0?mT) (Fig. 2B).
Three weekly exposures either rendered no additional benefit (Col 2) or
gave similar results to control (0?mT) (Sox9 and aggrecan). Moreover,
the amount of ECM produced was inversely related to the total number of
exposures. Single exposures produced >2-folds and ~1-fold increases
in Col 2 and sGAG, respectively (Fig. 2C),
whereas triple weekly exposures for three weeks (9 total exposures)
completely precluded an increase Col 2 and sGAG formation. The change in
the amount of DNA across samples varied less than 0.2-fold, although
reaching significances at 2?mT, indicating that cell proliferation was
only modestly affected within our pellet culture system (Fig. 2C).
Dosage effects of PEMFs over MSC chondrogenesis. (A) MSCs were exposed once (1x), twice (2x) or thrice (3x) per week. (B)
MSCs were subjected to either a single exposure on day 1 of
chondrogenic induction (1x) or once per week for 3 weeks (3x). Real-time
PCR analysis of cartilaginous markers expression at 7 (A) or 21 days (B)
after the induction of differentiation was normalized to GAPDH and
presented as fold-changes relative to level in undifferentiated MSCs. (C)
Quantification of cartilaginous ECM macromolecules generated during
chondrogenic differentiation of MSCs in response to distinct PEMF dosing
as indicated. MSC pellets were subjected to either a single PEMF
exposure given on day 1 of chondrogenic induction (1x) once per week for
3 weeks (3x), or thrice weekly for 3 weeks (9x). Data represents the
mean?±?SD, n?=?6 from 2 independent experiments. *Denotes significant
increase compare to non-PEMF (0?mT) control. #Denotes significant decrease compared to single PEMF (1x) exposure.
Effect of PEMF treatment to deposition of ECM
ECM deposition in response to
PEMF-exposure was also analyzed using Safranin O staining for
proteoglycan and immunohistochemical staining for type II collagen
Stained images of day 21 samples showed an enhanced deposition of
proteoglycan and type II collagen in samples exposed only once to 2?mT
for 10?min as compared to control (0?mT). By contrast, MSC samples
exposed for longer (60?min), to greater amplitude (3?mT) or repeatedly
(3x, 9x) yielded comparable, or inferior, ECM deposition to control.
Histological analysis of pellets exposed to PEMFs of
distinct amplitude, duration and dosage. Pellets were harvested at day
21, sectioned and subjected to Safranin O or type II collagen
immunohistochemistry staining. Images presented were represenation of
n?=?3, taken at 100× magnification.
Ca2+ entry pathways implicated in transducing the effects of PEMFs over MSC differentiation
To investigate whether PEMF-stimulated
MSC chondrogenesis was depended on calcium influx, EGTA (2?mM) was
co-administered to the culture medium during PEMF exposure and summarily
replaced afterwards with age-matched chondrogenic control media. RNA
analysis at day 7 showed that the inclusion of EGTA significantly
decreased the mRNA expression of Sox9, Col 2 and aggrecan in
PEMF-treated samples (Fig. 4A),
indicating that PEMF-exposure stimulates calcium influx. Conversely,
transiently supplementing the differentiation medium with elevated
extracellular Ca2+ (5?mM CaCl2) enhanced the mRNA
expression of Sox9, Col 2 and aggrecan in otherwise non-exposed samples,
and moreover, accentuated chondrogenic gene expression in PEMF-treated
samples. These results corroborate that calcium influx is part of the
upstream signalling cascade recruited by PEMFs contributing to
Investigation of calcium entry pathways implicated in the PEMF-effect. (A) Involvement of Ca2+ influx
in mediating the effects of PEMF-induced MSC chondrogenic
differentiation. MSCs were exposed for 10?min at 2?mT alone (control,
white bars), or in the presence of 2?mM EGTA (dark grey bars) or 5?mM
CaCl2 (hatched bars) transiently added to the culture media. EGTA and CaCl2 were
included to the bathing media 10?min before exposure and replaced with
age-matched media control cultures 10?min after exposure. (B)
Involvement of candidate calcium channels in mediating the effect of
PEMs over MSC chondrogenic differentiation. Control MSC chondrogenic
differentiation medium (white bars) was supplemented with Nifedipine
(1?µM, light grey bars), Ruthenium Red (RR, 10?µM, black bars), or 2-APB
(100?µM, dark grey bars) 10?min before exposure and replaced with
age-matched media control cultures 10?min after exposure. Real-time PCR
analysis was performed on day 7 of differentiation. Data represent the
means?±?SD, n?=?6 from 2 independent experiments. *Denotes significant
increase, or decrease, compared to non-PEMF (0?mT) control. #Denotes significant decrease relativeto 2?mT PEMF treatment.
To reveal the Ca2+ influx pathway recruited by
PEMFs, we pharmacologically dissected the contribution of candidate
channels utilizing 2-APB (100?µM) or Ruthenium Red (RR, 10?µM) as TRPC
or TRPV cation channel antagonists, respectively, or Nifedipine
(1?µM), as a dihydropyridine-sensitive, L-type voltage-gated calcium
channel (VGCC) antagonist. Calcium channel antagonists were included
into the differentiation medium 10?min before exposure to PEMFs and
removed immediately afterwards with age-matched control chondrogenic
media. Both 2-APB and Ruthenium Red completely inhibited the
PEMF-triggered up-regulation of chondrogenic genes, whereas Nifedipine
had no significant inhibitory effect (Fig. 4B).
Chondrogenic inhibition by 2-APB and Ruthenium Red was also observed in
non-exposed samples, indicating that TRPC- and TRPV-mediated calcium
entry are similarly involved in constitutive chondrogenesis upon
induction. By contrast, VGCC-mediated Ca2+ entry does not appear to play a predominant role in the early induction of chondrogenesis.
We next investigated the expression
profiles of TRP channels (TRPC1, TRPC6, TRPV1, TRPV4, TRPV6) previously
implicated in chondrogenesis and correlated these to our PEMF-induced
chondrogenic responses. Amongst the panel of candidate TRP channels, the
expression of TRPC1 and TRPV4 most closely correlated with our
delineated magnetic efficacy window governing chondrogenesis with
reference to PEMF amplitude, duration and dosage (Fig. 5 and Suppl. Figures 1 and 2).
These results corroborate an involvement of TRPC1 and TRPV4 in the
PEMF-induced enhancement of chondrogenic differentiation of MSC we
Expression profiles of TRPC1 and TRPV4 in response to
determined PEMF efficacy window regulating MSC chondrogenesis. Real-time
PCR analysis of TRPC1 and V4 exposed to different (A) intensities, (B) durations, and (C)
dosages of PEMFs. Data represent the means?±?SD, n?=?6 from 2
independent experiments. *Denotes significant increase, or decrease,
compared to non-PEMF (0?mT) control. #Denotes significant decrease relative to 2 mT (A), 10?min (B), or single (1x, C) PEMF treatment.
Effect of recurring calcium influx on MSCchondrogenesis
We next investigated whether calcium
entry, particularly that via TRPchannels underlies the inhibitory effect
observed with repeated PEMF exposures. MSCs were exposed once, twice or
thrice to 2?mT PEMFs for 10?min or, alternatively, exposed for 10?min
to aged-matched control differentiation media containing elevated
extracellular calcium (5?mM CaCl2) in lieu of PEMF exposure.
RNA analysis at day 7 showed that MSCs treated once with PEMFs, or
transiently administered elevated calcium, on day 1 exhibited enhanced
chondrogenesis to comparable levels. By contrast, subsequent exposures
to elevated calcium, on days 2 and 4 suppressed chondrogenesis mirrored
the effect of multiple exposures to PEMFs (Fig. 6A and Suppl. Fig. 3A).
(A) MSC chondrogenic differentiation in
response to multiple exposures to PEMFs or exogenously elevated calcium.
MSCs were subjected to either PEMF stimulation alone (white bars) or
with transient supplementation of CaCl2 alone (5?mM; hatched
bars), once (1x), twice (2x) or thrice (3x) in a week. Dotted lines
refers to expression level of non-treated controls. Real-time PCR
analysis was performed on day 7 of chondrogenic differentiation.
*Denotes significant increase relative to non-PEMF (0?mT) control. # and +denote
significant differences relative to respective single (1x) exposure
(white and hatched bars, respectively). P?=?PEMF treatment, Ca?=?CaCl2 supplementation. (B)
MSC chondrogenic differentiation in response to multiple exposures to
PEMFs alone (white bars) or in combination with calcium chelator (EGTA)
or TRP channel antagonists. EGTA (2?mM; dark grey bars; “E”), Ruthenium
Red (10?µM; RR, black bars; “R”) or 2-APB (100?µM; light grey bars; “C”)
was added to the MSC differentiation medium during PEMF expoure applied
once (1x), twice (2) or thrice (3x) per week. EGTA, RR and 2-APB were
included 10?min before exposure and replaced with media harvested from
age-matched chondrogenic control cultures 10?min after exposure.
*Denotes significant increase compare to non-PEMF (0?mT) control. #Denotes significant decrease compared to single PEMF exposure (1x). +Denotes
significant difference compared to respective PEMF control (white bar).
P?=?PEMF treatment, E?=?EGTA, R?=?Ruthenium Red (RR), C?=?2-APB. Data
shown are means?±?SD, n?=?6 from 2 independent experiments.
Analogously, precluding calcium entry
(with EGTA) also exhibited dichotomous effects if applied during the
first versus the second or third exposition to PEMFs, although in
opposite direction to that observed with calcium administration or
PEMFs. Whereas EGTA added during the initial exposure to PEMFs (1x)
prevented PEMF-induced chondrogenesis, EGTA applied during the second or
third exposure partially counteracted the inhibition of differentiation
exerted by serial PEMF exposure (Fig. 6B and Suppl. Fig. 3B).
Notably, impeding calcium entry with transient application of EGTA
during both the second and third PEMF exposure was capable of almost
completely reversing the inhibition of chondrogenesis observed with
repeated PEMF exposures, suggesting that PEMFs are activating
disparately functioning calcium mechanisms at early (day 1) and later
stages (>day 2) of chondrogenic-induction that confer opposite
effects over chondrogenesis. In contrast to the beneficial effect of
calcium influx induced by PEMF at the initial stage of chondrogenesis,
subsequent induction of calcium influx by repeated pulsing at later
stages of chondrogenesis was suppressive of MSC chondrogenesis. The
contribution of TRPC- and TRPV-mediated calcium entry to the
chondrogenic-inhibition observed with repeated PEMF exposures was
investigated by co-administering 2-APB (100?µM) or Ruthenium Red (RR,
10?µM), respectively, during PEMF exposure. As observed with transient
EGTA application, antagonism of TRPC1/V4-mediated calcium entry during
the first exposition to PEMFs was strongly inhibitory of PEMF-induced
chondrogenesis, whereasTRPC1/V4 antagonism during subsequent PEMF
expositions was somewhat less protective than EGTA over differentiation
implicating other yet to be determined calcium pathways in the later
calcium-dependent inhibitory phase of chondrogenic progression. Notably,
Ruthenium Red (TRPV4 antagonist) was capable of reverting the
inhibition of differentiation and expression of TRPC1 expression in
response to repeated PEMFing, whereas 2-APB (TRPC antagonist) was unable
to revert the inhibition of differentiation and TRPV4 expression in
response to repeated PEMFing, suggesting that TRPV4-mediated calcium
entry antagonizes TRPC1 expression leading up to differentiation
suppression. The dichotomous effects of precluding calcium entry by
EGTA, Ruthenium Red or 2-APB when applied during the first, or the
second and third, exposition to PEMFs was corroborated at the protein
level. Preventing calcium entry during the initial exposure to PEMFs
(1x) prevented PEMF-induced cartilaginous Col 2 and sGAG formation,
while blocking calcium entry at later exposures counteracted the
inhibition of differentiation exerted by serial PEMF exposure (Fig. 7).
Voltage-gated L-type calcium channels, on the other hand, do not appear
to be strongly implicated in the response as the expression of its
subunits (CACNA1C and CACNA2D1) was not perturbed by PEMF or calcium
treatment (Suppl. Fig. 3).
Quantification of cartilaginous ECM macromolecules
generated by chondrogenically differentiated MSC in response to single
or three weekly PEMF exposures alone (white bars) or in combination with
calcium chelator (EGTA) or TRP channel antagonists as indicated. EGTA
(2?mM; dark grey bars; “E”), Ruthenium Red (10?µM; RR, black bars; “R”)
and 2-APB (100?µM; light grey bars; “C”) were included once during
single PEMF exposures, or twice during the second and third PEMF
exposure. EGTA, RR and 2-APB were added 10?min before exposure and
replaced with media harvested from age-matched chondrogenic control
cultures 10?min after exposure. *Denotes significant increase compare to
non-PEMF (0?mT) control. #Denotes significant decrease compared to single PEMF exposure (1x). +Denotes
significant difference compared to respective PEMF control (white bar).
P?=?PEMF treatment, E?=?EGTA, R?=?Ruthenium Red (RR), C?=?2-APB. Data
represent means?±?SD, n?=?3.
Pulsed electromagnetic fields (PEMFs)
have been demonstrated to be influential in numerous biological
functions including progenitor cell fate determination and
differentiation. PEMF-based therapies have been previously shown to
enhance chondrocyte and cartilage explant anabolism while also limiting
the catabolic consequences of inflammatory cytokines29–31, 33–35, 40. PEMF exposure has been also reported to enhance the chondrogenic induction of stem cells34–37, 45.
Nevertheless, inconsistent and conflicting results plague the
scientific literature in this area of study, with PEMF exposures
typically being applied on the order of hours per day for several days
or weeks at a time. Here we report a high-efficacy of unprecedentedly
brief (10?min applied once) PEMF exposure at inducing MSC
chondrogenesis. We consistently detected increases in Sox9, Col 2 and
aggrecan mRNA (>2-folds) in response to lone exposure to 2?mT PEMFs
applied at the commencement of induction for only 10?min (Fig. 1).
These increments in mRNA later translated into increased chondrogenic
ECM protein formation (>2-fold) after 21 days of differentiation. By
contrast, stimulation with greater amplitudes (>3?mT), longer
exposures (>20?min) or more frequently (>2x/week) rendered no
additional benefit, or was even less effective at promoting
chondrogenesis at both the gene and protein levels. Although higher PEMF
amplitudes and longer duration exposures were capable of augmenting
aggrecan mRNA expression and macromolecular sGAG formation, the levels
achieved were no better than those from samples treated only once with
2?mT PEMFs for 10?min. Col 2 expression was especially susceptible to
overstimulation, being negatively impacted by exposures >2?mT or
longer than 10?minutes. To the best of our knowledge, all published EMF
studies examining chondrogenesis have employed exposure durations
between 30?min to 8?h28, 35, 37, 38. For instance, Mayer-Wagner et al.38 using
PEMF of 15?Hz, 5?mT, exposed MSCs undergoing chondrogenesis for 45?min
every 8?h for a total of 21 days and observed less than a 2-fold
increase in type II collagen expression, with no detected effect on Sox9
or aggrecan expression. Wang et al.37 using
1, 2, and 5?mT PEMFs at the frequency of 75?Hz exposed MSCs for 3?h per
day for 4 weeks and instead observed a loss of cartilaginous phenotype
associated with increased cartilage-specific extracellular matrix
degradation in the later stage of chondrogenic differentiation.
Given that most conventional PEMF exposure paradigms
employ a multiple exposure strategy and have reported positive
chondrogenic outcome35, we sought to determine the minimal number of exposures necessary to promote chondrogenesis (Fig. 2).
We found that exposing MSCs once at the commencement of chondrogenic
differentiation (1x) was necessary and sufficient to induce chondrogenic
gene expression (Fig. 2A), which was sustainable for up to 21 days post chondrogenic-induction (Fig. 2B). The superior effect of a single pulse was also confirmed at the level of sGAG and Col 2 protein deposition (Figs 2C and ?and3).3).
Indeed, in response to 3 exposures per week (10?min pulsing) for 3
consecutive weeks (9x treatments), ECM deposition was unchanged, or even
inhibited, relative to unexposed samples. Ours is likely the first
report to demonstrate an effectiveness of lone, 10?min, low amplitude
PEMF exposures over MSC chondrogenesis, while concomitantly
demonstrating the counter productivity of prolonged or repeated
exposures. The possibility that prolonged or repeated PEMF exposures
were merely cytotoxic, rather than truly inhibitory to chondrogenesis,
was ruled out by our finding that total DNA content across all
treatments was largely unchanged, despite lower Col 2 yield. In
addition, the amount of sGAG was either unchanged or higher than that in
control non-pulsed samples, further indicating that prolonged/repeated
PEMF exposure did not adversely influence cell viability. Finally, the
PEMF paradigm demonstrated here to best promote chondrogenesis (2?mT
applied once for 10?min) did not alter the expression of osteogenic
genes, Runx2 and ALP (Suppl. Fig. 4).
Provocatively, osteogenic markers did increase following 20?min
exposure to 2?mT PEMFs, thereby substantiating our assertion that
reduced chondrogenic expression is not a reflection of cell death, but
likely deferred chondrogenesis towards osteogenesis. Our demonstration
of the high efficacy of brief and early PEMF exposure might thus help
explain the existing inconsistencies and the relatively weaker responses
previously reported28, 30, 32, 34–38.
Chondrogenesis is known to be modulated by calcium signaling cascades of specific temporal sensitivity24,46. The dependence of chondrogenesis on extracellular Ca2+ was first alluded to with the demonstration that elevated extracellular Ca2+ promoted chondrogenic differentiation in chick limb bud-derived cultures47, 48. Moreover, Sox9, the master transcription factor of chondrogenesis, is subject to Ca2+-calmodulin regulation49.
Elevation in cytoplasmic calcium downstream of calcium influx has been
demonstrated in response to electric field (EF) or EMF stimulation
during MSC-derived osteogenesis or chondrogenesis27, 50, 51. We show that MSC chondrogenesis depends on the presence of extracellular Ca2+, whereby a transient (10?min) elevation of extracellular Ca2+ or brief (10?min) exposure to PEMFs (Figs 4Aand ?and6A)6A)
enhanced MSC chondrogenic differentiation in an additive manner.
Previous studies have also revealed that chondrogenesis is positively
responsive to intracellular Ca2+ within a tightly controlled concentration window24, 46. A 1.25-fold increase in cytosolic Ca2+ concentration was shown to promote differentiation, whereas a moderately greater increase (1.5-fold) negatively influenced in vitrochondrogenesis48.
It is thus feasible that high amplitude or prolonged PEMF exposures
elevate cytoplasmic calcium levels beyond the beneficial threshold for
MSC chondrogenic differentiation46. It is also well documented that the spatial and temporal patterns of intracellular free Ca2+ concentration play important roles in the regulation of various cellular processes, governed not only by absolute Ca2+ level, but also by periodic oscillatory changes of cytosolic Ca2+ concentration48, 52. MSCs undergoing chondrogenesis increase their frequency of Ca2+oscillations (waves) in the early stages of differentiation48, coinciding with the initial period of cellular condensation during the first 2–4 days10. Conversely, sustained elevations of extracellular calcium inhibit chondrogenesis48,
demonstrating a temporal requirement for calcium. Here we show that
transient pulsing with elevated calcium recapitulates the temporal
characteristic of the inhibitory actions of repeated PEMF exposures
(Fig. 6A). Moreover, preventing calcium entry (with EGTA) during repeated PEMF exposure precludes the inhibition (Figs 6B and ?and7),7),
defining a developmental change in calcium-sensitivity following
calcium-dependent initiation of chondrogenesis. In this respect, single
brief exposition to PEMFs defined by a specified electromagnetic window
applied during the early stages of MSC chondrogenesis may be sufficient
to provide the correct catalytic rise in intracellular Ca2+ to
optimally promote the initiation of chondrogenesis. Conversely, higher
exposure intensities or multiple exposures could result in excessive or
sustained calcium influx that may instead disrupts or interrupts
MSC-induced chondrogenesis, respectively.
Ca2+ influx via membrane-associated cation
channels is a key event in initiating chondrogenesis, that can be
potentially mediated by either TRP channels and/or voltage-gated calcium
channels (VGCC)24, 44, 46.
The transient receptor potential (TRP) channels are a diverse and
widely distributed family of cation channel broadly implicated in
cellular mechanotransduction23, 53–57. The TRPC and TRPV subfamilies have been broadly implicated in calcium homeostasis, ascribed mechanically-mediated gating44, 57–61, as well as implicated in the developmental programs of diverse mechanosensitive tissues62, 63. Previous studies have shown that blocking TRPV4 during the initial stages of induction inhibited chondrogenesis60,64. TRPV4-mediated Ca2+ signaling is also a positive regulator of Sox9 and as such, has been shown to promote chondrogenesis65 and in transducing the mechanical signals that support cartilage extracellular matrix maintenance and joint health44, 59. TRPC1 is expressed during early chondrocyte expansion66, as well as being involved in the proliferation of mesenchymal stem cells67.
We detected time, intensity, and PEMF dosage-dependent up-regulations
of both TRPV4 and TRPC1 that closely correlated with the PEMF-induced
expression pattern of chondrogenic markers (Fig. 5). Blocking TRPC1 and TRPV4 channels with 2-APB68 and Ruthenium Red64,
respectively, in the early stage of differentiation effectively
inhibited chondrogenesis, implicating these TRP channels in the
initiation of chondrogenesis, and indicating that PEMFs recruit the
activity of these channels to enhance chondrogenesis. Notably, blocking
calcium-permeation through TRPV4 channels reverses the inhibition on
chondrogenic differentiation and TRPC1 expression during repeated PEMF
exposure, whereas blocking TRPC1 channels was unable to revert the
inhibition on differentiation and expression of TRPV4 in response to
repeated PEMF exposure, suggesting that TRPV4-mediated calcium entry
antagonizes TRPC1 expression and is an essential step in initiating
differentiation (Figs 6B and ?and7).7).
TRPV4-mediated calcium entry may thus increase after the induction of
differentiation (>2 days) serving to curtail TRPC1 expression and
thereby promote differentiation by inhibiting TRPC1-medited
proliferation (Suppl. Fig. 2).
An involvement of voltage-gated calcium channels was more
difficult to establish. A predominant role for L-type VGCCs (CACNA1,
CACNA2D1) in transducing PEMF’s effects was not supported given that a
chondrogenically-effective dose of, Nifedipine, a L-type VGCC antagonist53–55, had no significant effect on the PEMF-induced upregulation of MSC chondrogenesis (Fig. 4B). Moreover, the expression level of the L-type channel was not correlated with changes in calcium (Suppl. Fig. 3). Ca2+ influx via the low-threshold T-type VGCC had been previously implicated in tracheal chondrogenesis56.
The expression of T-type VGCC (CACNA1H) was induced by lone early
exposure to PEMFs or transient calcium administration, and was
suppressed by repeated exposures to PEMF or extracellular calcium,
mirroring the expression pattern of chondrogenic markers under identical
conditions (Suppl. Fig. 3A).
The induction of the T-type calcium channel in response to PEMF/calcium
exposure more likely reflects chondrogenic differentiation, rather than
a fully determinant role in PEMF-induced chondrogenesis, as its
expression was in the majority of conditions unchanged (relative to
control) by removal of extracellular calcium during PEMF exposure
(Suppl. Fig. 3B).
Our strongest data support the interpretation that TRPC1 and TRPV4 play
a more predominant role, although not necessarily exclusive, in
transducing the chondrogenic effects of PEMFs. Further work will require
to fully disentangle the intricasy of calcium homeostasis during the
chondrogenic developmental process.
In summary, we have provided
comprehensive characterization of the effects of PEMFs over MSC
chondrogenic differentiation. MSCs undergoing chondrogenic induction are
preferentially responsive to a well-defined window of PEMF stimulation
of particular amplitude (2?mT), duration (10?min) and dosage (once on
day 1 induction). By contrast, treatment with higher amplitude PEMFs,
longer exposure durations or repeated expositions, as are more common in
the field, are generally counterproductive, helping explain the lack of
resolution in the field. Our results indicate that PEMFs mediate their
effect by activating calcium influx through mechanosensitive calcium TRP
channels. The unprecedented efficacy of our low amplitude,
exceptionally brief and non-invasive PEMF-exposure protocol over MSC
chondrogenesis has broad clinical and practical implications for the
ultimate translation of related PEMF-based therapeutic strategies for
stem cell-based cartilage regeneration.
Human bone marrow MSCs culture and chondrogenic differentiation
Primary human mesenchymal stem
cells (MSCs) were purchased from RoosterBio Inc. (Frederick, MD),
supplied at passage 3. The MSCs was further expanded in MSC High
Performance Media (RoosterBio Inc.) at 37?°C in 5% CO2 atmosphere.
The expanded MSCs were used at passage 5–6. Chondrogenic
differentiation of MSCs was induced through 3D pellet culture as
previously described16, 69. Briefly, 2.5?×?105 cells
were centrifuged to form pellets and cultured in a chondrogenic
differentiation medium containing high glucose DMEM supplemented with
4?mM proline, 50?µg/mL ascorbic acid, 1% ITS-Premix (Becton-Dickinson,
San Jose, CA), 1?mM sodium pyruvate, and 10?7?M dexamethasone
(Sigma-Aldrich, St Louis, MO), in the absence of antibiotics, for up to
7 or 21 days in the presence of 10 ng/mL of transforming growth
factor-?3 (TGF?3; R&D Systems, Minneapolis, MN). To investigate an
involvement of calcium influx or of calcium channels in transmitting the
effects of PEMFs, cells were pre-incubated in chondrogenic media
supplemented with elevated calcium, EGTA or particular calcium channel
antagonist for 10?minutes prior to pulsing. Ten minutes after exposure
to PEMFs the supplemented chondrogenic media was replaced with
age-matched chondrogenic media (0?mT) cultures. To attenuate
extracellular calcium influx, 2?mM ethylene-bis(oxyethylenenitrilo)
tetraacetic acid (EGTA; Sigma) was added to the bathing media as noted.
To promote extracellular Ca2+ influx the bathing media was supplemented with 5?mM CaCl2 (Sigma).
To block calcium permeation through dihydropyridine-sensitive, L-type
voltage-gated calcium channel (VGCC), Nifedipine (1?µM, Sigma) was added
to the bathing media. 2-aminoethoxydiphenyl borate (2-APB, 100?µM,
Sigma) and Ruthenium Red (10?µM, Merck Millipore) were administered as
indicated to block calcium entry via TRPC and TRPV channels,
respectively. Aminoglycoside antibiotics such as streptomycin were
excluded in all MSC expansion and chondrogenic differentiation media to
avoid interference with mechanosensitive ion channels70.
PEMF Exposure system
The ELF-PEMF (extremely low frequency – pulsed magnetic field) delivery system has been described previously43.
For the purposes of this study a barrage of magnetic pulses of 6 ms
duration was applied at a repetition rate of 15?Hz and at flux densities
between 1–4?mT. Each 6 ms burst consisted of a series of 20 consecutive
asymmetric pulses of 150?µs on and off duration with an approximate
rise time of 17?T/s. The background magnetic flux density measured in
the chamber was below 1?µT between 0?Hz to 5?kHz. The coil size,
position and individual number of windings were numerically optimized by
a CST low frequency solver for low field non-uniformity over a wide
frequency range taking into consideration the shielding capacity of the
µ-metallic chassis. The measured field non-uniformity did not exceed 4%
within the uniform exposure region of the coils.
To investigate the optimum dosage
of PEMF, MSCs in a 3D pellet culture were exposed to PEMFs of different
exposure durations, dosage and the magnetic flux amplitude. MSCs were
subjected to PEMFs of 1–4?mT amplitude with exposure times ranging
between 5 to 60?min on the day of chondrogenic induction, applied once
or multiple times as indicated in the respective figure legend. Cell
pellets to be treated once with PEMFs (1x) were exposed on first day of
chondrogenic induction. Two scenarios of multiple exposures were
administrated (Fig. 2).
Firstly, multiple exposures were administrated during the course of a
week; double exposures (2x) were applied on days 1 and 2; triple
exposures (3x) on days 1, 2 and 4. Alternatively, multiple exposures
were applied on a once a week basis, for up to three week. Non-exposed
(control) cells were placed within the PEMF device without current flux
to produce a magnetic field to ensure that all cells were subject to the
same climatic and mechanical conditions.
Real time PCR analysis
Chondrogenic cell pellets were
digested in 0.25% Type II collagenase (Gibco, Life Technologies)
followed by centrifugation. Total RNA was extracted using the RNeasy®
Mini Kit (Qiagen, Germany). Reverse transcription was performed with 100
ng total RNA using iScript™ cDNA synthesis kit (Bio-Rad, USA).
Real-time PCR was conducted using the SYBR®green assay on ABI
7500 Real-Time PCR System (Applied Biosystems, Life Technologies, USA).
Real-time PCR program was set at 95?°C for 10?min, followed by 40
cycles of amplifications, consisting of a 15?s denaturation at 95?°C and
a 1?min extension step at 60?°C. Primer sequences used in this study
were according to previous publication16 and presented as Supplementary Table 1. The level of expression of the target gene, normalized to GAPDH, was then calculated using the 2???Ct formula
with reference to the undifferentiated MSC. Results were averaged from
triplicate samples of two independent experiments.
ECM and DNA quantification
Samples harvested were digested
with 10?mg/mL of pepsin in 0.05?M acetic acid at 4?°C, followed by
digestion with elastase (1?mg/mL). A Blyscan sulfated glycosaminoglycan
(sGAG) assay kit (Biocolor Ltd., Newtownabbey, Ireland) was used to
quantify sGAG deposition according to manufacturer’s protocol.
Absorbance was measured at 656?nm and sGAG concentration was
extrapolated from a standard curve generated using a sGAG standard. Type
II Collagen (Col 2) content was measured using a captured enzyme-linked
immunosorbent assay (Chondrex, Redmond, WA). Absorbance at 490?nm was
measured and the concentration of Col 2 was extrapolated from a standard
curve generated using a Col 2 standard. Values for sGAG and Col 2
content obtained were normalized to the total DNA content of respective
samples, measured using Picogreen dsDNA assay (Molecular Probes, OR,
USA). Quadruplicates of each group were analyzed from two independent
Histological and immunohistochemical evaluation
Samples were fixed in formalin,
dehydrated, paraffin embedded, and cut into sections of 5?µm. For
Safranin-O staining, the sections were incubated in hematoxylin
(Sigma-Aldrich), washed and stained with fast green (Sigma-Aldrich),
before staining with Safranin-O solution (AcrosOrganics). For
immunohistochemistry, ultra-vision detection kit (Thermo scientific) was
used. Endogenous peroxidase in the sections was first blocked with
hydrogen peroxide before pepsin treatment for 20?min. Samples were
treated with monoclonal antibodies of collagen type II (Clone 6B3;
Chemicon Inc.) followed by incubation with biotinylated goat anti-mouse
(Lab Vision Corporation). A mouse IgG isotype (Zymed Laboratories Inc.)
was used as control for immunohistochemistry studies.
All experiments were performed in
biological replicates (n?=?3 or 4) and results reported as
mean?±?standard deviation (SD). Statistical analysis was carried out by
Students t-test for comparison between two groups using the Microsoft
Excel software. The level of significance was set at p?<?0.05. All
quantitative data reported here were averaged from at least two
The study was supported by University of Malaya HIR-MoE
Grant (Reference number – UM.C/625/1/HIR/MOHE/MED/32 account number –
H20001-E000071) and Singapore-MIT Alliance for Research and Technology
(SMART) Foundation (ING14085-BIO). Dinesh Parate was supported by NUS
D.P. performed experiments, analyzed data and drafted the
manuscript. A.F.O., J.F. and C.B. provided technological expertise and
contributed to the fabrication the PEMF facility. A.A.A., T.K., J.H.P.H.
and A.F.O. provided funding and critical reading of the manuscript.
A.F.O. and Z.Y. designed the study, analyzed data and provided critical
revision of the manuscript. All authors reviewed the manuscript.
The authors declare that they have no competing interests.
Electronic supplementary material
Supplementary information accompanies this paper at doi:10.1038/s41598-017-09892-w
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ASAIO J. 2018 Mar/Apr;64(2):253-260. doi: 10.1097/MAT.0000000000000631.
Synergism of Electrospun Nanofibers and Pulsed Electromagnetic Field on Osteogenic Differentiation of Induced Pluripotent Stem Cells.
Ardeshirylajimi A1, Khojasteh A.
From the Department of Tissue Engineering and Applied Cell Sciences,
School of Advanced Technologies in Medicine, Shahid Beheshti University
of Medical Sciences, Tehran, Iran.
According to the current therapies failure for bone fractures and
lesions, tissue engineering showed a great potential to help solve these
challenges. Because the use of growth factors is very limited in the
clinic, it could be very useful that could be introducing an alternative
to it. Extremely low frequency pulsed electromagnetic fields (PEMF, 1 mT, 50 Hz) were used for achieving this aim. The PEMF potential
in combination with electrospun polycaprolactone (PCL) nanofibers was
used to investigate the osteogenic potential of human induced
pluripotent stem cells (iPSCs). Several
relevant osteogenic markers, such as Alizarin red staining, alkaline
phosphatase activity, calcium content, gene expression, and
immunocytochemistry, were used to evaluate osteoinductivity of PEMF. Results were shown that PEMF alone
can induce osteogenic differentiation, but this capability increased
when used in combination with PCL nanofibers significantly. In addition,
simultaneous use of osteogenic medium, PEMF and PCL surprisingly increased osteogenic differentiation potential of iPSCs. According to the results, PEMF alone,
iPSCs-seeded PCL, and both of them could be considered as a promising
candidate for use in bone tissue engineering applications.
J Physiol Pharmacol. 2017 Apr;68(2):253-264.
Changes in viability of rat adipose-derived stem cells isolated from abdominal/perinuclear adipose tissue stimulated with pulsed electromagnetic field.
Department of Pathophysiology, Jagiellonian University Medical College, Cracow, Poland.
Department of Immunology, Jagiellonian University Medical College, Cracow, Poland.
Institute of Electron Technology, Cracow, Poland.
Department of Pathophysiology, Jagiellonian University Medical College, Cracow, Poland. email@example.com.
Previous experiments demonstrated that low-frequency electromagnetic
field (LF-EMF) may activate cellular death pathways in proliferating cells. Therefore, we hypothesized that LF-EMF may also influence viability of highly proliferating undifferentiated adipose-derived stem cells.
Obesity is classified as a civilization disease; its etiopathogenesis
is presumed to include both genetic predisposition and influence of
modified environmental factors, such as unbalanced diet with excess
calories and/or too low physical activity. Obesity may lead to a number
of metabolic disorders, including type 2 diabetes mellitus,
cardiovascular diseases (associated with atherosclerosis) related to
primary hypertension and ischemic heart disease, myocardial infarction
and other complications. The aim of this study was to verify if LF-EMF
alters viability parameters of adipose-derived stem cells (ADSCs) isolated from rats, cultured in vitro and exposed to pulsed electromagnetic field (PEMF;
7 Hz, 30 mT). ADSCs were obtained from healthy rats and animals with
experimentally-induced obesity, both males and females, pups and adults.
The animals were fed with chow with either low (LF diet) or high fat
content (HF diet) for 21 days. Then, ADSCs were isolated from extracted
adipose tissue and used to establish cell cultures. ADSCs from the first
passage were exposed to PEMF three times, 4 hours per exposure, at 24-h intervals (experimentally developed protocol of PEMF stimulation). 24 hours after the last exposure to PEMF,
viability parameters of ADSCs were analyzed by flow cytometry (FCM).
The study demonstrated that LF diet exerted a protective effect on PEMF-exposed ADSCs, especially in the case of male and female pups. In turn, the proportion of early apoptotic cells in PEMF-treated
ADSC cultures from adult female rats maintained on HF diet turned out
to be significantly higher than in other experimental groups.
Pulsed Electromagnetic Field Regulates MicroRNA 21 Expression to Activate TGF-? Signaling in Human Bone Marrow Stromal Cells to Enhance Osteoblast Differentiation
Nagarajan Selvamurugan, 1 Zhiming He, 2 Daniel Rifkin, 3 Branka Dabovic, 3 and Nicola C. Partridge 2 , *
Author information ? Article notes ? Copyright and License information ? Disclaimer
This article has been cited by other articles in PMC.
Pulsed electromagnetic fields
(PEMFs) have been documented to promote bone fracture healing in
nonunions and increase lumbar spinal fusion rates. However, the
molecular mechanisms by which PEMF stimulates differentiation of human
bone marrow stromal cells (hBMSCs) into osteoblasts are not well
understood. In this study the PEMF effects on hBMSCs were studied by
microarray analysis. PEMF stimulation of hBMSCs’ cell numbers mainly
affected genes of cell cycle regulation, cell structure, and growth
receptors or kinase pathways. In the differentiation and mineralization
stages, PEMF regulated preosteoblast gene expression and notably, the
transforming growth factor-beta (TGF-?) signaling pathway and
microRNA 21 (miR21) were most highly regulated. PEMF stimulated
activation of Smad2 and miR21-5p expression in differentiated
osteoblasts, and TGF-? signaling was essential for PEMF stimulation of alkaline phosphatase mRNA expression. Smad7, an antagonist of the TGF-? signaling
pathway, was found to be miR21-5p’s putative target gene and PEMF
caused a decrease in Smad7 expression. Expression of Runx2 was increased
by PEMF treatment and the miR21-5p inhibitor prevented the PEMF
stimulation of Runx2 expression in differentiating cells. Thus, PEMF
could mediate its effects on bone metabolism by activation of the TGF-? signaling pathway and stimulation of expression of miR21-5p in hBMSCs.
Abundant reports describe the effects of
electricity on bone growth and fracture repair, and a variety of pulsed
electromagnetic field (PEMF) devices have been developed to produce
electromagnetic fields at the fracture site. These widespread PEMF
devices utilize noninvasive inductive coupling and can be used along
with every method of fracture fixation [1, 2].
The stimulation of bone at the fracture site by the introduction of
electromagnetic fields may be similar to the resulting stimulation from
mechanical loading . The beneficial therapeutic effects of such selected low energy, time varying PEMF promote fracture healing in nonunions , increase lumbar spinal fusion rates [4, 5], and have been found to affect bone metabolism by decreasing bone resorption and increasing bone formation [6–8]. PEMFs have also been reported to stimulate the synthesis of extracellular matrix (ECM) proteins 
and may also affect several membrane receptors including those for
parathyroid hormone, low density lipoprotein, insulin-like growth
factor-2, insulin, and calcitonin . Several growth factors such as bone morphogenetic proteins 2 and 4 (BMP-2, BMP-4) and transforming growth factor-beta (TGF-?) have been reported to be secreted from osteoblasts upon PEMF treatment . It has been shown that electromagnetic stimulation could raise net Ca2+ flux in human osteoblast-like cells, and the increase in the cytosolic Ca2+ concentration could initiate activation of signaling pathways resulting in regulation of expression of bone matrix genes [12, 13]. Accelerated osteogenesis has been found in bone marrow-derived mesenchymal stem cells by PEMF treatment  and this promotion of ECM deposition was more efficient compared with adipose-tissue mesenchymal stem cells .
Previously we have reported that both
BMP-2 and PEMF (Spinal-Stim® by Orthofix, Inc., Lewisville, TX)
separately stimulated proliferation of rat primary calvarial
osteoblastic cells and stimulated expression of early osteoblast
differentiation genes in culture .
In this study, we investigated the effects of PEMF (Cervical-Stim® by
Orthofix, Inc., Lewisville, TX) on human bone marrow stromal cells
(hBMSCs) proliferating and differentiated to osteoblastic cells. In
addition, the underlying molecular mechanisms by which PEMF stimulates
differentiation of hBMSCs into osteoblasts are not well understood.
Thus, we also aimed to investigate the PEMF effects on proliferation,
differentiation, and mineralization of hBMSCs by Affymetrix microarray
analysis. The TGF-? signaling pathway and microRNA 21 (miR21)
were most highly regulated by PEMF. Thus, in this study we
systematically investigated the mechanism of action of PEMF effects on
osteogenesis via TGF-? and miR21 using hBMSCs.
2. Materials and Methods
2.1. Cell Culture
Fresh human bone marrows from
21–68-year-old women were used. These were either purchased from Lonza
(Walkersville, MD) or left over tissue from surgical procedures at New
York University Hospital for Joint Diseases. Since these were
deidentified, this is not considered Human Subjects Research by the New
York University School of Medicine Institutional Review Board. In both
cases, the bone marrows were freshly collected, never frozen, and
immediately diluted 1?:?1 in Hank’s Balanced Salt Solution (HBSS; GIBCO
Laboratories, Grand Island, NY) containing 20?IU/mL of sodium heparin
(Sigma Chemical Co., St. Louis, MO). The diluted bone marrow was layered
over an equal volume of Ficoll-Paque Plus (GE Healthcare, Piscataway,
NJ) and centrifuged at 400g for 40?min at 18°C. The mononuclear
cells at the interface layer were collected, washed three times with
HBSS, resuspended and seeded into a tissue culture flask, and incubated
at 37°C in the presence of 5% CO2 overnight. The next day,
nonadherent cells were removed from the culture flask. Adherent cells
(BMSCs) were grown to confluence then placed in 6-well plates at 6.4 ×
104?cells/well for exposure to PEMF or control. All cells were incubated at 37°C in the presence of 5% CO2. The medium used for culturing these cells was ?-MEM
(Corning, Tewksbury, MA) containing 15% fetal bovine serum (FBS; GIBCO,
Grand Island, NY) and Penicillin-Streptomycin (GIBCO, Grand Island,
2.2. PEMF Exposure
The PEMF was generated as previously described 
but was set to have similar waveform characteristics to a commercial,
clinically approved proprietary device (Cervical-Stim by Orthofix Inc.,
Lewisville, TX). Cervical-Stim is the only device approved by the FDA
for cervical fusion use and has been reported to be safe and effective . The specific differences from our previous publication 
were a burst frequency of 15?Hz and a burst period of 67?ms. The
induced magnetic field was vertical relative to the surface of the
plates. The PEMF waveform was routinely checked for its consistency
using a field probe and oscilloscope. The first PEMF exposure was
initiated 24?h after seeding cells in wells (day 1) and continued
through the entire experiment. Control plates were placed in an
identical incubator on Plexiglas shelves. The CO2 concentration,
humidity, and temperature of the control and treatment incubators
(upper and lower chambers of the same double incubator) were identical
and were not affected by the PEMF.
2.3. Cell Number
Cells were grown in normal growth
medium and were trypsinized, resuspended, and counted using a
hemocytometer when they reached 70–80% confluence on day 10 or 20 of
culture, respectively, for the BMSCs from the younger (21–30) women
versus those from the 31–65-year-old women.
2.4. Osteoblast Differentiation
Human BMSCs were seeded at 6.4 × 104 cells/well in 6-well cell culture plates and cultured for 10 days or 20 days in normal cell culture medium (?MEM
+ 15% FBS + 1% Penn/Strep) before they reached confluence. They were
then cultured for an additional 13 (differentiation) or 23
(mineralization) days in osteogenic medium [normal growth medium
supplemented with 10?4?M L-ascorbic acid, 10?8?M dexamethasone, and 1.80?mM potassium phosphate monobasic (Sigma, St. Louis, MO)]. The medium was changed three times/week.
2.5. Von Kossa Staining
For Von Kossa staining, 6 replicates
of BMSCs were treated with PEMF or control daily from day 1 of culture.
On day 23, 33, or 43, the cells were fixed with 95% ethanol for 15?min
at 37°C, then rinsed and rehydrated through 80%, 50%, and 20% ethanol
and then water, and incubated with 5% silver nitrate solution for 1?h at
37°C. The cells were rinsed with water, exposed to UV light for 10?min,
and photographed. Von Kossa staining was analyzed by computer based
morphometry (ImageJ: NIH, Bethesda, Maryland).
2.6. Extracellular Regulated Kinases Activation and Western Blot Analyses
Human BMSCs treated with control or
PEMF for 5 and 10 days in the proliferation phase were washed with cold
phosphate buffered saline (PBS) and lysed in Cell Lysis Buffer
(Invitrogen, Grand Island, NY) containing protease and phosphatase
inhibitor cocktails (Sigma). Cell lysates were centrifuged at 10,000?rpm
for 10 minutes at 4°C and supernatants were saved and used for Western
blot analysis. Twenty ?g of total cell protein was loaded per
well and separated on 4–15% Mini-Protean TGX precast gels (Bio-Rad,
Hercules, CA), followed by transferring to nitrocellulose membranes
(Bio-Rad, Hercules, CA). The membranes were blocked and incubated with
primary rabbit antibodies (Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204),
p44/42 MAPK (Erk1/2) (137F5), or Cdk2 (sc-163; Cyclin dependent kinase
2, loading control)) overnight at 4°C. The membranes were then probed
with secondary antibody conjugated with horseradish peroxidase. Finally,
the bands were visualized by adding Super Signal West Dura Extended
Duration Substrate (Thermo Scientific, Pittsburgh, PA) according to the
manufacturer’s instructions. The primary antibodies to total ERKs and
phosphorylated ERKs were obtained from Cell Signaling Technology
(Danvers, MA), while the antibody to Cdk2 was obtained from Santa Cruz
Biotechnology, Inc. (Dallas, TX). The secondary antibody
(goat-anti-rabbit) conjugated with horseradish peroxidase (HRP) was
obtained from Santa Cruz Biotechnology. Results were captured and
quantitated by ChemiDoc XRS+ software (Bio-Rad, Hercules, CA). Both the
Phospho-ERK1/2 and total ERK 1/2 were normalized to Cdk2 and then
expressed as a percent of the values obtained in untreated control
2.7. Microarray Assays
Human BMSCs of a 27-year-old
healthy female donor were used for microarray experiments. Only hBMSCs
expanded from the second to sixth passages were used for the
experiments. PEMF treatment (Cervical-Stim) was initiated 24?h after
hBMSCs were seeded, with 4?h daily exposure every day throughout the
experimental period. Quadruplicate cell samples from both PEMF-treated
and control groups were collected simultaneously at time points of hBMSC
proliferation, osteoblast differentiation, and mineralization phases.
Total RNA was isolated from cells by using TRIzol reagent (Thermo
Scientific, Pittsburgh, PA) and then purified with RNeasy mini kit from
Qiagen (Valencia, CA). Prior to microarray analysis, the RNA integrity
was assessed by Agilent 2100 Bioanalyzer (Santa Clara, CA) and the best
quality triplicate samples were chosen for the subsequent analyses.
Microarrays and data analyses with Affymetrix Human U133 plus 2.0 Gene
Chips (Santa Clara, CA) were performed at University of Medicine and
Dentistry of New Jersey Genome Center according to the manufacturer’s
instructions. In the case of gene expression where it was significantly
found to be above 1.5-fold after PEMF treatment, gene ontology analysis
was carried out by DAVID Bioinformatics Resources 6.7 software (NIAID,
2.8. Real-Time RT-PCR
Total RNA was isolated from cells using
the total RNA isolation kit from Qiagen (Valencia, CA). For
determination of expression of genes other than miR21-5p, 100?ng of
total RNA from each sample was used for cDNA synthesis using TaqMan
Reverse Transcription Reagents (Roche, Indianapolis, IN). Quantitative
(q)PCR reactions were performed according to the real-time thermocycler
machine (Realplex) manufacturer’s instructions (Eppendorf, Hauppauge,
NY), which allowed real-time quantitative detection of the PCR products
by measuring the increase in SYBR green fluorescence caused by binding
of SYBR green to double-stranded DNA. The Power SYBR green master mix
kit for PCR reactions was purchased from Invitrogen. The qPCR was
performed in triplicate with reaction conditions of 95°C, 10?min, 1
cycle; 95°C, 15?sec; and 58.5°C, 1?min, for 40 cycles. Gene expression
was analyzed with threshold cycle (CT) values averaged from triplicate
samples and normalized to their CT values of housekeeping gene RPL13A.
Primers were designed by NCBI primer Blast software. Table 1 lists the human-specific primers used for PCR amplification.
Primers used in this study.
Forward primer (5? > 3?)
Reverse primer (5? > 3?)
For miR21-5p and snoR10-1, the reagents
and primer sets for RT-qPCR were purchased from Qiagen. One ug of total
RNA was reverse-transcribed into cDNA using the miScript II kit with
miScript HiSpec Buffer according to the manufacturer’s instructions. The
cDNA was then diluted 10 times and utilized as a template to amplify
miR21-5p and snoR10-1 with the miScript SYBR Green PCR kit using the
appropriate primers. snoR10-1 was used as normalizing gene control. The
qPCR was performed in triplicate with reaction conditions of 95°C 15?min
for Taq DNA polymerase activation, 94°C 15?sec denaturation, 55°C
30?sec annealing, and 70°C 30?sec extension for 40 cycles. Gene
expression results of miR21-5p from either control or PEMF-treated
groups were normalized to their relative snoR10-1 results.
2.9. TGF-? Signaling
Human BMSCs were cultured and treated with control or PEMF as described above. For TGF-? and BMP signaling assays, osteoblasts were treated with PEMF at days 23 and 33 and were also treated with TGF-?2 (R&D System, Minneapolis, MN) as a positive control for the TGF-? pathway.
The day before assay, the cells were starved overnight (0.1% FBS
medium) to reduce endogenous signaling activity. At day 23 at the same
time as PEMF exposure started, 5?ng/mL TGF-?2 was added to the
medium of positive control wells. Cell lysates from different groups
were collected at 0, 2, and 4?h time points after treatment to examine
Smad2, Smad3, and Smad1/5/8 protein phosphorylation by Western blot
analysis as described above. Phospho-Smad2 (Ser465/467, 138D4)/Smad2
(D43B4), phospho-Smad3 (Ser423/425, C25A9)/Smad3 (C67H9), and
phospho-Smad1/5/8/Smad1/5 antibodies were obtained from Cell Signaling
Technology (Danvers, MA). In TGF-? neutralization experiments, 30?ug/mL normal rabbit IgG or TGF-? pan
antibody (R&D System, Minneapolis, MN) was added to osteogenic
medium during the entire differentiation period. At day 23, two
non-PEMF-treated cell groups were also included with 5?ng/mL of TGF-?2 as positive controls. After 2?h of PEMF exposure, all sample groups were collected for Western blots and RT-qPCR assays.
2.10. Transient Transfection
Cells were seeded in growth medium in 6-well plates at a density of 105?cells/well
on the day before the transfection. miR-21 is now referred as
miR-21-5p, based on the latest miRBase release (V.21). miR21-5p
inhibitor (Applied Biosystems: 4464084) designed to bind with endogenous
miR21, when introduced into cells, inhibits its activities. miR21-5p
mimic (Applied Biosystems: 4464066) was designed to be similar to that
of endogenous miR21. A negative control miRNA (Applied Biosystems:
4464076) was included in the study. The X-treme Gene transfection
reagent obtained from Roche, USA, was mixed with 50?nM of negative
control miRNA, miR21 mimic, or miR21 inhibitor, and transient
transfection was carried out  for 3 or 6 days along with PEMF treatment every day for 4?h.
2.11. Statistical Analysis
Statistical analysis was done by one-way ANOVA, Student’s t-test, or Wilcoxon Ranking. Significant difference is p < 0.05. All data are shown as mean ± standard deviation with n as indicated.
3.1. PEMF Effects on Proliferation and Differentiation of Human BMSCs from Subjects of Different Ages
We previously reported that PEMF
generated by Spinal-Stim stimulated cell proliferation and expression of
early differentiation marker genes in rat primary calvarial
osteoblastic cultures .
In the present study we used PEMF (Cervical-Stim) to determine its
effect on osteoblasts using human bone marrow cells. PEMF significantly
stimulated the cell number of preosteoblasts from BMSCs of young women
(21–30 years old) while not stimulating those of BMSCs from
31–65-year-old women (Figures 1(a) and 1(b)).
It should also be noted that the hBMSCs from aged individuals (58, 59,
and 65 years old) also required much longer time (20 days) to approach a
similar cell culture density to those from the younger women. Since
PEMF had an effect on preosteoblastic cell number from the younger women
and cell proliferation involves activation of intracellular signaling
pathways, especially extracellular regulated kinases (ERKs), we
determined activation of these enzymes by PEMF. As shown in Figure 2(a),
PEMF increased ERK activation (phosphorylation) after 15?min on day 5
in BMSCs from a 24-year-old woman. A similar effect was also found in
hBMSCs from other younger female subjects (24- and 27-year-old women’s
cells) and the quantitative analysis of ERK activation (phosphorylated
ERKs) from the three individuals after normalization to total ERKs
confirmed the above result (Figure 2(b)). There was no significant activation of ERKs from any of these cells on the 10th day of PEMF treatment (Figures 2(c) and 2(d)).
Effect of PEMF on hBMSC preosteoblastic cell number. (a)
Human preosteoblasts derived from bone marrow stromal cells of
21–36-year-old women were treated with PEMF for 10 days; cells from 58-,
59-, and 65-year-old women were treated with PEMF for 20 days. Cell
number/well was calculated using a hemocytometer (n = 3–6 wells). (b) Aggregation of the data into the two age groups, n = 5-6. The statistical p value for the younger versus older samples is shown using Student’s t-test analysis.
Effect of PEMF on ERK activation. Human BMSCs from two
different 24-year-old women and a 27-year-old woman were subjected to
4?h daily PEMF treatment for either 4 days or 9 days. On the 5th (a) or
10th day (c), their cells were treated with PEMF for different time
periods as indicated and whole cell lysates were obtained and subjected
to Western blot analyses; cells from a 24-year-old woman are shown as an
example. ((b) day 5, (d) day 10) The quantitation of activated or
phosphorylated ERKs for cells from 2 separate 24-year-old women and a
27-year-old woman was determined by normalization of phosphorylated ERKs
to total ERKs after normalization to Cdk2 as a loading control and
expressed as a percent of untreated control cells. The results are shown
for the cells of the 3 individuals. ? indicates significant
increase compared to control. # indicates significant increase compared
to 0 times of PEMF on the 5th day. The p value ? 0.05 is considered as significant using one-way ANOVA.
To determine the role played by PEMF in
osteoblast differentiation and mineralization of hBMSCs, experiments
were carried out at molecular and cellular levels. At the molecular
level, the mRNA expression of alkaline phosphatase (ALP), type I
collagen (COL1A1), and osteocalcin (OC), which are known osteoblast
differentiation and mineralization marker genes, was determined using
qRT-PCR analysis. PEMF significantly increased mRNA expression of ALP
and Col1 but not OC in BMSCs that had been allowed to proliferate,
differentiate, and mineralize (Figure 3(a)). We next determined the effect of PEMF on mineralization in BMSCs by Von Kossa staining (Figures 3(b) and 3(c)). PEMF significantly stimulated mineralization of BMSCs in the mineralization phase and did not in the differentiation phase.
Effect of PEMF on expression of osteoblastic marker genes
and mineralization in hBMSCs. Differentiating preosteoblasts from
24–68-year-old women were grown in the presence of osteoblast
differentiation medium after confluence was reached and were treated
with PEMF for 33 days or 43 days (59–68-year-old samples) of culture.
(a) Total RNA was isolated and subjected to qRT-PCR using specific
primers for human ALP, type I collagen, OC, and RPL13A. n = 9. (b) Cells were then subjected to Von Kossa staining and the mineralized calcium deposits were quantified. n = 9. Statistical analyses were conducted using Wilcoxon signed rank test. The p value
? 0.05 is considered as significant compared with the controls. (c) An
example of Von Kossa staining and mineralized calcium deposits for
hBMSCs of a 24-year-old female after 33 days of osteogenic culture in
the presence or absence of daily Cervical-Stim PEMF.
3.2. PEMF Regulation of Genes during hBMSC Proliferation by Microarray Analysis
A sample from a young individual
was used for microarray analyses because PEMF significantly enhanced
cell growth for young individuals compared to old individuals as shown
in Figure 1.
For assessment of the effect of PEMF on gene expression during hBMSC
proliferation, on the 5th day of PEMF treatment 2?h after initiating the
PEMF signal (pilot studies had shown significant PEMF stimulation of
Cyclin gene expression at this time and day, data not shown), total RNA
was isolated and used for the subsequent test with Affymetrix Human U133
plus 2.0 Gene Chips. After identifying significantly regulated genes,
gene ontology analyses were performed by DAVID Bioinformatics Resources
6.7 software. The results indicated that PEMF stimulation of
proliferating hBMSCs mainly affected genes of cell cycle regulation,
cell structure, extracellular matrix (ECM), and some growth receptors or
kinase pathways. There were a total of 114 known genes upregulated and
17 known genes downregulated at this time point (partially listed in Table 2).
We have also included the decrease in fibrillin 2, even though it was
not ?1.5-fold, since this sequesters members of the TGF-? family and is the subject of our later research in this report.
Genes regulated by PEMF during hBMSCs proliferation by
microarray analysis. Cells were from a normal 27-year-old female. Total
RNA was isolated at day 5 after 2?h of PEMF treatment and used for
microarray assays as described in Materials and Methods. Analysis by
(Avg PEMF versus avg controls)
Cell adhesion and binding and cytoskeletal and structural proteins
3.3. PEMF Regulation of Genes in Differentiated and Mineralized hBMSCs by Microarray Analysis
In the differentiation (day 23) and
mineralization stages (day 33) after daily 4?h PEMF treatment, a total
of 37 (partially listed in Table 3) and 173 (partially listed in Table 4)
known genes, respectively, were identified as significantly regulated.
In these two stages, PEMF regulated preosteoblast gene expression and
most genes were downregulated including transcriptional regulators,
metabolism, proteases, and regulators and also cell adhesion and binding
proteins and cytoskeletal and structural proteins. Changes in gene
transcription of candidate genes chosen from microarray analyses were
verified and confirmed by RT-qPCR on RNA from differentiated hBMSCs from
3 females, aged 24, 27, and 31 years (Table 5). Notably, the TGF-? signaling
pathway seems to be most highly regulated by PEMF. In particular,
RT-qPCR showed that fibrillin 2 (FBN2) was significantly decreased in
expression by 65 ± 14%, while TGF-?2 mRNA significantly increased to 155 ± 44% and TGF-? regulator 1 (TBRG1) mRNA significantly increased to 143 ± 23%, relative to controls. In contrast, in mineralizing cells (Table 6), there was no decrease in FBN2 expression and a lesser significant increase in TGF-?2. It appears that PEMF stimulated a number of components of the TGF-? pathway
in differentiating and mineralizing osteoblasts. It is notable that no
components of the BMP pathway were seen to be regulated.
Genes regulated by PEMF in differentiating hBMSCs. Cells
were from a normal 27-year-old female. Total RNA was isolated at day 23
of PEMF treatment. Analysis by Student’s t-test.
(Avg PEMF versus avg controls)
Transcriptional regulator, RNA metabolism, and RNA transport
Real-time RT-PCR analysis of three different female donor
samples’ hBMSCs, aged 24, 27, and 31 years in the mineralization stage.
Analysis by Student’s t-test.
Average PEMF/control %
Cadherin 11, type 2, OB-cadherin (osteoblast)
130 ± 36%
1.10E ? 01
Collagen, type I, alpha 1
144 ± 47%
9.20E ? 02
Chemokine (C-X-C motif) ligand 12
146 ± 20%
8.48E ? 03
149 ± 54%
9.82E ? 02
FBJ murine osteosarcoma viral oncogene homolog B
165 ± 27%
6.97E ? 03
128 ± 25%
6.23E ? 02
162 ± 68%
9.49E ? 02
130 ± 19%
2.53E ? 02
Matrix metallopeptidase 16 (membrane-inserted)
135 ± 68%
2.07E ? 01
SRY- (sex determining region Y-) box 11
137 ± 37%
7.60E ? 02
Secreted phosphoprotein 1
132 ± 52%
1.74E ? 01
Transforming growth factor, beta 2
128 ± 21%
3.99E ? 02
Transforming growth factor beta regulator 1
113 ± 14%
9.71E ? 02
142 ± 58%
1.37E ? 01
3.4. PEMF Activation of TGF-? Signaling via Smad2 in Differentiated and Mineralizing Osteoblasts
To validate the PEMF effect on activation of the TGF-? signaling
pathway, hBMSCs were subjected to differentiation (day 23) and
mineralization (day 33) as described. During differentiation and
mineralization, the cells were continuously treated with PEMF for 4?h
each day. At days 23 and 33, cells were subjected to control, TGF-?2, or PEMF treatments for 0, 2, and 4?h. TGF-?2 was used as a positive control for activation of TGF-? signaling.
Whole cell lysates were prepared and subjected to Western blot analyses
using the antibodies for phosphorylated and total Smad2. The results
show that PEMF stimulated activation of Smad2 by increased
phosphorylation at day 23 in differentiated osteoblasts (Figure 4(a)) and less at day 33 in mineralizing osteoblasts (Figure 4(b)). To determine the specificity of activation of the TGF-?signaling by PEMF, osteoblasts were pretreated with pan-TGF-? antibody
before PEMF treatment. The results show that the PEMF-stimulated Smad2
activation in differentiated osteoblasts (day 23) was blocked when cells
were pretreated with pan-TGF-? antibody (Figure 4(c)). Since a recent paper has described a different PEMF signal as acting through the BMP pathway on rat calvarial osteoblasts , we examined whether Smad1/5/8 was phosphorylated in response to the Cervical-Stim signal in hBMSCs (Figure 4(d)).
We were unable to observe any stimulation of this pathway, in contrast
to the activation of the Smad2 pathway, even though the strong positive
control, TGF-?2, slightly stimulated Smad1/5/8 phosphorylation, as has been observed by others [19, 20].
PEMF resulted in activation of the TGF-? signaling
pathway in human osteoblastic cells during differentiation and
mineralization. (a) Whole cell lysates after PEMF treatment of hBMSCs of
a 24-year-old female at day 23 (differentiation) and (b) at day 33
(mineralization) were subjected to Western blot analysis using the
antibodies as indicated for Smad2 and Cdk2. TGF-?2 (5?ng/mL) was added to control (non-PEMF-treated) cells on days 23 and 33 as positive controls. (c) The pan-TGF-? neutralizing
antibody (30?ug/mL) was added to the osteogenic medium of hBMSCs from a
24-year-old female during the entire differentiation period and lysates
were prepared on day 23 of PEMF treatment, 2?h after PEMF was started
or TGF-?2 was added and subjected to Western blot analysis. TGF-?2
(5?ng/mL) was added to control (non-PEMF-treated) cells on day 23 as a
positive control. Cdk2 was used as loading control. (d) The same lysates
were subjected to Western blot analysis for phosphorylation of
Smad1/5/8 as indicated.
3.5. PEMF Stimulates Osteoblast Marker Gene Expression by Activation of the TGF-? Signaling Pathway
To determine if TGF-? signaling
is responsible for the PEMF effect on expression of osteoblast
differentiation marker genes such as ALP and type I collagen, this
pathway was inhibited and RNA collected from differentiated osteoblasts
at day 23 and subjected to RT-qPCR analysis. We found that PEMF
significantly stimulated mRNA expression of ALP (Figure 5(a)) and type I collagen (Figure 5(b)) in differentiated osteoblasts. When cells were pretreated with pan-TGF-? antibody, PEMF stimulation of expression of these genes was significantly decreased (Figures 5(a) and 5(b)). Thus, this result indicates that the osteogenic effect of Cervical-Stim PEMF on hBMSCs is mediated via the TGF-? signaling pathway.
PEMF resulted in stimulation of expression of osteoblast differentiation marker genes via the TGF-? signaling
pathway. Differentiated human osteoblasts derived from hBMSCs from a
30-year-old female were used. Total RNA was isolated after incubation
with IgG or pan-TGF-? antibody (Pan-Anti) and treatment with
control (Ctr) or PEMF and subjected to RT-qPCR using the primers for (a)
ALP and (b) collagen 1A1 genes. RPL13 was used to normalize gene
expression. n = 3. ? indicates significant increase
compared with control IgG. # indicates significant decrease compared to
all groups with ALP mRNA expression; ## indicates significant decrease
compared to control or PEMF treatment with IgG incubation with collagen
1A1 mRNA expression; analysis by one-way ANOVA.
3.6. PEMF Stimulation of miR21-5p Expression in Differentiating Osteoblasts
MicroRNAs are considered to be
regulators of osteogenesis and bone formation. The microarray analysis
of hBMSCs subjected to differentiation at day 23 identified the
stimulation of expression of miR21 (Table 3).
To verify this, total RNA was obtained with differentiated hBMSCs from
females aged 24 × 2, 27, 29, and 30 (young individuals) and 31, 36, 58,
and 68 (older individuals) years and subjected to RT-qPCR. The result
shows that the expression of miR21-5p was 155% increased in cells from
the younger women but not significantly increased in cells from the
older individuals after PEMF treatment (Figure 6).
PEMF stimulated expression of miR21-5p in differentiated
human osteoblasts. Total RNAs from control or PEMF-treated hBMSCs of
females (24 × 2, 27, 29, and 30 years old, n = 5) at day 23 of
differentiation or (31, 36, 58, and 68 years old) at day 23 or 33 of
differentiation were isolated and subjected to RT-qPCR using the
miScript II kit with miScript HiSpec Buffer and miScript SYBR Green PCR
Kit. snoR10-1 was used to normalize miR21-5p expression and the
expression is shown as a percentage of the relevant control samples. ?indicates significant increase compared to control using one-way ANOVA.
3.7. PEMF and miR21-5p Stimulation of Osteoblast Differentiation Marker Gene Expression
It is evident that PEMF stimulated miR21-5p expression in differentiated osteoblasts from younger individuals (Figure 6)
which strongly suggested a role for miR21-5p in promotion of osteoblast
differentiation. To determine this role, hBMSCs were transiently
transfected with negative control miRNA or miR21-5p mimic for 3 days and
concurrently subjected to 4?h PEMF treatment every day for 6 days.
Total RNA was isolated and subjected to RT-qPCR analysis. When cells
were treated with PEMF, there was significantly increased ALP mRNA
expression. The miR21-5p mimic alone had no effect but together with
PEMF treatment caused a significant increase in ALP mRNA expression
compared with PEMF treatment alone (Figure 7(a)).
With type I collagen mRNA expression, no significant effect was seen
with respect to PEMF, miR21-5p mimic, or both treatments under these
conditions (Figure 7(b)).
PEMF resulted in stimulation of expression of ALP mRNA and
its effect was further enhanced by miR21-5p. Human BMSCs from a
31-year-old female were transiently transfected with 50?nM of negative
control miRNA or miR-21-5p mimic for 72?h in osteogenic medium and PEMF
treatment was carried out for 4?h each day for a total of 6 days. Total
RNA was isolated and RT-qPCR was carried out using the primers for ALP
(a) and collagen 1A1 (b) genes. Expression of the mRNAs is shown
relative to the RPL13 gene. n = 3. ?? indicates
significant increase compared to negative control miRNA transfection. #
indicates significant increase compared to all treatments. Analysis by
3.8. PEMF Regulation of Smad7 via miR21-5p in Differentiating Osteoblasts
In silico analysis (http://www.microrna.org/microrna/home.do)
was used to identify the putative target genes of miR21-5p for its
functional importance towards osteogenic commitment. Among them some
antagonistic effectors of osteogenesis such as Smad7, Smurf1, and Crim1
were found. The 3?UTR regions of Smad7, Smurf1, and Crim1 held at least
6-nt perfect complementarities to the miR21-5p seed region (Figure 8(a)).
To validate these putative target genes of miR21-5p, hBMSCs were
transiently transfected with either negative control miRNA or miR21-5p
inhibitor and concurrently treated with PEMF for 4?h each day for 3
days. To determine the expression level of these target genes, total RNA
was isolated, followed by RT-qPCR analysis. There was no significant
change in mRNA expression of Smurf2 (Figure 8(b)) and Crim1 (Figure 8(c))
in the cells in the presence of PEMF treatment, miR21-5p inhibitor, or
both. In the case of Smad7, there was a significant decrease in its mRNA
expression after PEMF treatment, and inclusion of miR21-5p inhibitor
reversed the PEMF effect resulting in increased Smad7 mRNA expression (Figure 8(d)). From these results we suggest that Smad7, an antagonist of TGF-?signaling,
is likely to be miR21-5p’s target gene and PEMF downregulates its mRNA
expression via miR21-5p in differentiating osteoblasts. In fact, at
least two groups have shown that the 3?-UTR of Smad7 is, indeed, a
target for miR21-5p, resulting in a decrease in Smad7 protein levels [21, 22].
Putative target genes of miR21-5p and PEMF decreases Smad7
mRNA through miR21-5p. (a) The putative target region analysis was
performed for Smurf2, Crim1, and Smad7 mRNAs 3? UTR by miR21-5p seed
sequence. ((b)–(d)) Human BMSCs from a 27-year-old female were
transiently transfected with 50?nM of negative control miRNA or miR21-5p
inhibitor for 72?h in osteogenic medium and PEMF treatment was carried
out concurrently for 4?h each day for 3 days. Total RNA was isolated and
RT-qPCR was carried out using the primers for (a) Smurf2, (b) Crim1,
and (c) Smad7 genes. Expression of mRNAs is shown relative to that of
the RPL13 gene. n = 3. ? indicates significant
increase compared to negative control miRNA transfection or PEMF
treatment with negative control miRNA transfection. # indicates
significant decrease compared to PEMF treatment with miR21-5p inhibitor
transfection. Analysis by one-way ANOVA.
3.9. PEMF Regulation of Runx2 Expression via miR21-5p and Smad7 in Differentiating Osteoblasts
Since Runx2 is required for
osteoblast differentiation and PEMF stimulated expression of osteoblast
differentiation marker genes (Figure 3),
we next examined the PEMF stimulation of expression of Runx2 in
differentiating hBMSCs and the role played by miR21-5p. Human BMSCs were
transiently transfected with either negative control miRNA or miR-21-5p
inhibitor, followed by PEMF treatment. Total RNA was isolated and
subjected to RT-qPCR analysis. The result showed that there was a
significant increase in expression of Runx2 mRNA in response to PEMF
treatment and this effect was blocked by miR21-5p inhibitor in
differentiating osteoblasts (Figure 9).
From these results, we suggest that PEMF promotes its osteogenic effect
via stimulation of miR21-5p expression and activation of TGF-? signaling
in hBMSCs. A figure summarizing that the mechanisms we conclude are
involved in PEMF stimulation of BMSCs and osteoblast differentiation is
shown in Figure 10.
PEMF stimulated Runx2 expression and its effect was
downregulated by miR21-5p inhibitor. Human BMSCs from a 27-year-old
female were transiently transfected with 50?nM of negative control miRNA
or miR21-5p inhibitor for 3 days in osteogenic medium and PEMF
treatment was carried out concurrently for 4?h each day for 3 days.
Total RNA was isolated and RT-qPCR was carried out using the primers for
Runx2. Expression of Runx2 mRNA is shown relative to that of the RPL13
gene. n = 3. ? indicates significant decrease compared
to negative control miRNA transfection or PEMF treatment with negative
control miRNA transfection. # indicates significant increase compared to
control treatment with negative control miRNA transfection. Analysis by
Schema of the mechanisms involved in PEMF stimulation of
BMSC proliferation and osteoblast differentiation. The magnetic field
(B) is thought to elicit Eddy Currents that act on BMSCs and cause
activation of ERKs that are then involved in increased BMSC
proliferation. After the BMSCs reach confluence and they are switched to
differentiation medium, the magnetic field (B) and the resultant Eddy
Currents cause a decrease in fibrillin 2 expression and an increase in
TGF-?2 and miR21-5p expression. The decrease in fibrillin 2 would lead to an increase in the amount of available TGF-?2. The increase in miR21-5p appears to cause a decrease in inhibitory Smad7 expression, thus, enhancing TGF-?2
activation of Smad2 with resulting increase in Runx2, collagen I, and
alkaline phosphatase expression in the cultures, that is, increased
Numerous studies have shown that mechanical stimulation of bone progenitors including ultrasound , mechanical strain [24, 25],
and compression as well as shear forces has a stimulatory effect on
bone progenitors involved in bone healing of critical size defects and
nonunions in vivo. A broad set of investigations has aimed to unravel
potential underlying molecular mechanisms and growth factor pathways
involved with sophisticated in vitro methods .
A number of mechanisms have been proposed by which mechanical cues on
different physical scales and identities can incorporate into growth
factor signaling . In particular, the major TGF-? growth factor superfamily of ligands (including TGF-? 1 and 2 as well as BMPs) and their downstream signaling via Smad2/3 and Smad1/5/8 transcription factors, respectively [28, 29],
appears to be affected by mechanical stimulation in a diverse set of
cells, with the majority of research focussing on bone progenitors, for
example, BMSCs, osteoblasts, osteocytes, and chondrocytes. This is a
large and ongoing field of study.
The molecular mechanisms responsible for the effect of PEMF on bone formation [14, 30]
have not been completely elucidated. We found that PEMF promoted
preosteoblast proliferation from hBMSCs from individuals up to age 30,
but not older individuals, and stimulated differentiation marker gene
expression of mineralizing hBMSCs of all ages. To dissect the
mechanisms, PEMF effects on proliferation, differentiation, and
mineralization of hBMSCs were examined by Affymetrix microarray
analyses. We found that PEMF stimulation of hBMSC proliferation mainly
affected genes of cell cycle regulation, cell structure, ECM, and some
growth receptors or kinase pathways (Table 2).
At the cellular and molecular levels, PEMF has been reported to promote
the synthesis of ECM proteins and exert a direct effect on the
production of proteins that regulate gene transcription. PEMF may affect
several membrane receptors and stimulate osteoblasts to secrete several
growth factors such as BMP-2 and BMP-4 and TGF-?. PEMF has
been reported to affect osteoblast cellular proliferation and
differentiation of bone cells in vitro by enhancing DNA synthesis [14, 31], increasing the expression of bone marker genes during differentiation and mineralization ,
and enhancing calcified matrix production. Several experimental studies
also demonstrated that PEMF stimulation could potently promote
osteogenesis and enhance bone mineralization both in vivo and in vitro [32–34].
The microarray data for PEMF regulation of differentiation
and mineralization of hBMSCs showed regulation of transcriptional
regulators, metabolism, proteases, cytokines and growth factors, and
also cell adhesion and binding proteins and cytoskeletal and structural
proteins (Tables ?(Tables33 and ?and4).4).
Identifying the signaling pathways and their associated regulatory
mechanisms of PEMF action on osteogenesis might further promote its use
in clinical applications. Thus, PEMF regulated preosteoblast gene
expression during the differentiation and mineralization stages, and
candidate genes chosen from microarray analyses were confirmed by
RT-qPCR (Tables ?(Tables55 and ?and6).6). Notably, the TGF-? signaling
pathway and miR21 seem to be most highly regulated by PEMF. Thus, in
the present study, we systematically investigated the mechanism of
action of PEMF effects on osteogenesis via activation of TGF-? signaling and miR21-5p expression using hBMSCs.
The TGF-?/BMP signaling pathway plays a
fundamental role in the regulation of bone organogenesis through the
activation of receptor serine/threonine kinases. Perturbations of TGF-?/BMP activity are almost invariably linked to a wide variety of clinical outcomes including skeletal anomalies . Phosphorylation of TGF-? (I/II) or BMP receptors activates intracellular downstream Smads, the transducer of TGF-?/BMP
signals. In our studies, PEMF (Cervical-Stim) treatment activated only
the Smad2 signaling component in differentiated hBMSCs (Figure 4)
and activation of this signaling pathway appeared to be essential for
PEMF stimulation of early osteoblast differentiation marker genes such
as ALP and type I collagen (Figure 5). It is notable that it did not appear to activate the BMP pathway through Smad1/5/8 phosphorylation. The TGF-?/BMP
signaling effect may be complex and highly time- and space-specific
during skeletal development and bone formation. Very recently, Xie et
have described a different PEMF signal as operating through the BMP
receptor on the primary cilium of rat calvarial osteoblasts in culture.
Our accumulated data do not indicate that the BMP pathway is involved in
the signaling mechanism of either Spinal-Stim or Cervical-Stim but we
cannot rule out that it may have a role if investigated further. This
signaling cascade can be modulated by various factors and other pathways
[35, 36]. Activation of Wnt/Lrp5/?-catenin or calcium-related mechanisms by PEMF treatment for osteogenic activity have also been reported [37, 38].
Osteoblast differentiation is tightly controlled by several regulators including miRNAs [17, 39, 40]
that can regulate expression of genes during differentiation of MSCs
towards osteoblasts, resulting in the osteogenic lineage. Differential
expression of miRNAs could be responsible for activation of several
signaling pathways such as TGF-?/BMP, Wnt/?-catenin, and transcription factors . PEMF stimulated miR21-5p expression in differentiated hBMSCs from younger females (Figure 6)
suggesting one of the ways PEMF mediates its osteogenic effect on these
cells is via miR21-5p. MicroRNA 21 was one of the first miRNAs detected
in the human genome and it was found to be overexpressed in several
types of cancer tissues . A role for miR21 in cell proliferation and apoptosis has been reported .
With regard to the regulation of bone formation, a number of miRNAs are
expressed in the developing skeletal system and miRNA-dependent
modulation of gene function can alter skeletal phenotypes across
individuals and also within the same individual over time . MicroRNAs might have direct or indirect effects for their regulatory functions in osteoblast differentiation.
To study the functional role of miR21-5p during osteoblast
differentiation by PEMF treatment, it was necessary to alter its
endogenous expression/activity. Overexpression of miR21-5p (mimic) in
differentiated hBMSCs had no effect on mRNA expression of ALP and type I
collagen (Figure 7)
but required PEMF to have an enhanced effect on ALP mRNA expression
which suggests that PEMF could also involve other pathways and molecules
in addition to miR21-5p for its osteogenic effects in these cells. The
putative targets of miR21-5p can be classified according to their
negative contribution in osteogenic differentiation or positive
contribution to other lineages using online software. Among them are
some key regulators or antagonistic effectors of osteogenesis such as
Smad7, Smurf2, and Crim1 and these genes are well documented in their
antagonistic roles in osteogenesis [29, 45].
Expression of the putative target genes in the presence of the miR21-5p
inhibitor showed a significant increase in Smad7 mRNA expression in
differentiated hBMSCs (Figure 8). The inhibitory Smads (Smad6, Smad7) potentially act as suppressors of bone formation. While Smad7 inhibits TGF-?/BMP signaling, Smad6 is less effective in inhibiting TGF-?signaling. It has been reported that Smad7 can inhibit ALP activity and suppress type I collagen mRNA and protein levels . MicroRNA 21 has been shown to be a key regulator of TGF-? signaling  and Smad7 was found to be one of its target genes [21, 40, 43]. Other target genes such as PTEN and STAT3 have also been reported for miR21 [48, 49]. Based on our results (Figures ?(Figures77 and ?and8),8), we suggest that Smad7 is a target gene for miR21-5p during PEMF regulation of osteoblast differentiation.
Since PEMF stimulates miR21-5p expression in differentiated hBMSCs (Figure 6) and miR21-5p targets Smad7 (Figure 8(d)),
the PEMF action on osteogenesis via miR21-5p and Smad7 was further
investigated. Runx2 is essential for the commitment of multipotent
mesenchymal cells to the osteoblastic lineage. In general, Runx2
activity can be altered by its interacting proteins and/or
posttranslational modifications [17, 50–54].
The steady-state protein level of Runx2 can be regulated by E3
ubiquitin ligases, Smurf1 and Smurf2, and it has been reported that the
degradation of endogenous Runx2 can be blocked by a proteasomal
inhibitor or by Smurf2 siRNA .
PEMF stimulated Runx2 mRNA in differentiated hBMSCs, and miR21-5p
inhibitor prevented the PEMF stimulation of Runx2 expression (Figure 9). It has already been reported that Smad7 interacts with Smurf2 but it does not interact with Runx2 .
Hence, targeting Smad7 through miR21-5p by PEMF could possibly decrease
the Smad7-dependent Smurf2 activity, resulting in stabilization of
Runx2 protein, and feedback to increased transcription of Runx2 in
differentiated hBMSCs. A figure summarizing that the mechanisms we
conclude are involved in PEMF stimulation of BMSCs and osteoblast
differentiation is shown in Figure 10.
We can only speculate as to how PEMF regulates miR21-5p, but others
have shown that this microRNA is regulated by transcriptional
mechanisms, such as by myocardin-related transcription factor-A (58) or
by STAT3 (59), and such mechanisms could possibly be implicated in
Our results show that PEMF
significantly stimulated the cell number of preosteoblasts from BMSCs of
young women while not stimulating those from women older than 30. We
also showed that PEMF regulates a range of genes in hBMSCs to stimulate
their proliferation, differentiation, and mineralization. Our further
investigation suggests a novel regulatory mechanism of PEMF action
during differentiation and mineralization of hBMSCs by activation of the
TGF-? signaling pathway. PEMF appears to activate this pathway
in hBMSCs of younger women by inhibiting Smad7 expression through
miR21-5p and in turn PEMF controls the function of Runx2 resulting in
promotion of its osteogenic effect.
This work was supported by a research contract to Dr.
Partridge from Orthofix, Inc., Lewisville, TX, USA. Dr. Partridge also
received honoraria and travel support from Orthofix, Inc. Dr.
Selvamurugan and Mr. He received travel support from Orthofix, Inc. Drs.
Rifkin and Dabovic received a subcontract from Dr. Partridge’s Orthofix
research contract. Drs. James T. Ryaby, Nianli Zhang, and Erik I.
Waldorff of Orthofix, Inc., critically reviewed the paper.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
Nagarajan Selvamurugan and Zhiming
He contributed equally to the work; Nagarajan Selvamurugan drafted the
paper; Nagarajan Selvamurugan, Zhiming He, Daniel Rifkin, and Branka
Dabovic contributed to the research design, acquisition, analysis, and
interpretation of data; Nicola C. Partridge designed the research,
contributed to the analysis and interpretation of the data, and
critically revised the paper. All authors have read and approved the
final submitted manuscript.
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Noninvasive magnetic stimulation has
been widely used in autonomic disorders in the past few decades, but
few studies has been done in cardiac diseases. Recently, studies showed
that low-frequency electromagnetic field (LF-EMF) might suppress atrial
fibrillation by mediating the cardiac autonomic nervous system. In the
present study, the effect of LF-EMF stimulation of left stellate
ganglion (LSG) on LSG neural activity and ventricular arrhythmia has
been studied in an acute myocardium infarction canine model. It is shown
that LF-EMF stimulation leads to a reduction both in the neural
activity of LSG and in the incidence of ventricular arrhythmia. The
obtained results suggested that inhibition of the LSG neural activity
might be the causal of the reduction of ventricular arrhythmia since
previous studies have shown that LSG hyperactivity may facilitate the
incidence of ventricular arrhythmia. LF-EMF stimulation might be a novel
noninvasive substitute for the existing implant device-based electrical
stimulation or sympathectomy in the treatment of cardiac disorders.
Previous studies have demonstrated that
the activation and remodeling of left stellate ganglion (LSG) induced by
myocardial infarction1,2 might be the immediate triggering mechanisms of ventricular arrhythmia (VA) and sudden cardiac death3,4, and suppressing LSG neural activity might be a feasible antiarrhythmic therapy5. In the past decades, LSG denervation and blocking have been shown to be benefit for reducing VA6.
However, undesirable side effects, such as cervical injury and Horner’s
syndrome, have limited the clinic use of LSG denervation or blocking.
Therefore, exploring a novel noninvasive approach is necessary.
Transcranial magnetic stimulation (TMS), a
neurostimulation and neuromodulation technique based on the principle of
electromagnetic induction of an electric field in the brain, has been
proposed for treatment of a variety of neurological disorders. Previous
studies has shown that TMS might mediate the cardiac rhythm by
modulating the autonomic nervous system7. Scherlag et al.8 showed
that exposure the vagal trunks or the chest to the low-frequency
magnetic field (LF-EMF) might suppress atrial fibrillation, whereas
exposure to the high-frequency field might induce atrial fibrillation by
autonomic modulating. Recently, Yu et al.9 further
demonstrated that LF-EMF stimulation of the vagal trunks or chest might
suppress atrial fibrillation by inhibiting the neural activity of
atrial ganglionated plexus. In this study, we hypothesized that exposure
LSG to the LF-EMF might inhibit the LSG neural activity, thereby
reducing VAs after acute myocardial infarction6.
LSG was exposed to intermittent LF-EMF stimulation before left anterior descending artery occlusion in LF-EMF group (Fig. 1A–C).
Both the blood pressure and heart rate were kept at a stable level
during the LF-EMF stimulation. No visible damage was shown in LSG or
cardiac tissue after 90?min LF-EMF treatment. All dogs developed ECG
ST-segment and/or T-wave changes acutely after ligating the left
anterior descending artery.
Schematic representation of the position of the LF-EMF (A), stimulus pattern (B) and the experimental design flow chart (C).
LSG, left stellate ganglion; LF-EMF, low-frequency electromagnetic
field; LAD, left anterior descending artery; MAP, monophasic action
potential; HRV, heart rate variability; VA, ventricular arrhythmia.
Effect of LF-EMF stimulation on myocardial infarction-induced VAs
Figure 2A shows
the representative examples of VAs in the Control group and LF-EMF
group. As compared to the Control group, both the number of ventricular
premature beat (VPB) and the number of non-sustained ventricular
tachycardia (VT) were significantly decreased (Fig. 2B,C). Furthermore, the incidence of sustained VT/VF was significantly suppressed (75.0% vs 12.5%, P?<?0.05, Fig. 2D) in the LF-EMF group.
Representative examples (A) and the incidence (B–E)
of AMI-induced VAs in the Control group (n?=?8) and EMF group (n?=?8).
*P?<?0.05 and **P?<?0.05 as compared to the Control group. AMI,
acute myocardial infarction; VPB, ventricular premature beats; VT,
ventricular tachycardia; VF, ventricular fibrillation; other
abbreviations as in Fig. 1.
Effect of LF-EMF stimulation on MAP
Figure 3A–F demonstrates the effect of LF-EMF on action potential duration at 90% repolarization (APD90, Fig. 3A–C), pacing cycle length of action potential duration alternans (PCL, Fig. 3D–F) and the maximal slope of the restitute curve (Smax, Fig. 3G–I). As compared to group baseline, no significant change was shown in APD90, PCL or Smax obtained from different sites of left ventricle in the Control group, whereas a significant change was shown in APD90, PCL and Smax of those sites both at 30?min and 90?min after LF-EMF stimulation in the LF-EMF group (Fig. 3A–F).
Effect of LF-EMF stimulation on APD90 (A,B), PCL (C,D) and Smax (E,F) in the Control group (n?=?8) and EMF group (n?=?8). *P?<?0.05 and **P?<?0.05 as compared to the group baseline; #P?<?0.05 and ##P?<?0.05
as compared to the Control group. LVA, left ventricular apex; LVM, the
median of left ventricle; LVB, left ventricular base; MAP, monophasic
action potential; APD, action potential duration; APD90,
monophasic action potential duration determined at 90% of
repolarization; PCL, pacing cycle length of APD alternans; BH, baseline;
Smax, the maximal slope of the restitution curve, other abbreviations
are identical to Fig. 1.
Effect of LF-EMF stimulation on heart rate variability
Figure 4 demonstrates
that both low frequency component (LF) and the ratio between LF the
high component (LF/HF) were significantly decreased by LF-EMF
stimulation both at 30?min and 90?min later but not by sham LF-EMF
stimulation as compared to group baseline. In comparison with group
baseline, acute myocardial infarction resulted in a significant change
in LF (2.54?±?0.23?ms2 vs 1.72?±?0.12?ms2, P?<?0.01, Fig. 4A), high frequency component (HF, 1.01?±?0.08?ms2 vs 1.43?±?0.18?ms2, P?<?0.01, Fig. 4B) and LF/HF (2.51?±?0.34 vs 1.20?±?0.20, P?<?0.01, Fig. 4C) in the Control group, whereas those were kept at a normal level in the LF-EMF group (LF, 1.52?±?0.1?1?ms2 vs 1.68?±?0.10?ms2; HF, 1.43?±?0.12?ms2 vs 1.48?±?0.13?ms2; LF/HF, 1.06?±?0.10 vs 1.14?±?0.19, all P?>?0.05, Fig. 4A–C).
Effect of LF-EMF stimulation on LF (A), HF (B) and LF/HF (C) in the Control group (n?=?8) and EMF group (n?=?8). *P?<?0.05 and **P?<?0.01 vs group baseline; #P?<?0.05 and ##P?<?0.05
as compared to the Control group. LF, low frequency; HF, high
frequency; LF/HF, the ratio between LF and HF; BH, baseline. Other
abbreviations are identical to those in Fig. 1.
Effect of LF-EMF stimulation on serum norepinephrine and LSG function
In comparison with group baseline,
serum norepinephrine was decreased from 180.3?±?6.8?pg/ml to
162.5?±?5.8?pg/ml at 30?min later and to 160.3?±?5.2?pg/ml at 90?min
later in the LF-EMF group, whereas kept a stable level in the Control
group (Fig. 5A).
Furthermore, the systolic blood pressure increase in response to LSG
stimulation was kept a baseline level in the Control group (Fig. 5B), whereas significantly attenuated by LF-EMF in the LF-EMF group at a voltage of 20–30?V as compared to group baseline (Fig. 5C).
Take 25?V for example, the maximal systolic blood pressure increase
induced by LSG stimulation was decreased from 88.3?±?15.4% to
43.1?±?6.2% (P?<?0.01) at 90?min later, whereas kept at about 90% in
the Control group (Fig. 5B,C).
Effect of LF-EMF stimulation on serum NE (A) and LSG function (B,C)
in the Control group (n?=?8) and EMF group (n?=?8). NS, P?>?0.05,
*P?<?0.05 and **P?<?0.01 as compared to the Control group at the
same time point. NE, norepinephrine. Other abbreviations are alike to
those in Fig. 1.
Effect of LF-EMF stimulation on the neural activity of LSG
Figure 6A shows
the representative examples of LSG neural activity at baseline, 30?min
after LF-EMF stimulation, 90?min after LF-EMF stimulation and 15?min
after acute myocardial infarction. Figure 6B,Cdemonstrates
that no significant difference was shown both in the frequency and the
amplitude of LSG neural activity between the Control group and the
LF-EMF group. As compared to group baseline, LF-EMF stimulation resulted
in a significant decrease in LSG neural activity at 30?min and 90?min
later, whereas no significant change was caused by sham LF-EMF
stimulation (Fig. 6B,C).
Furthermore, as compared to baseline, the neural activity was
significantly increased after acute myocardial infarction in the Control
group (Frequency: 62.5?±?5.2impulse/min vs 112.2?±?8.1impulse/min,
P?<?0.01; Amplitude: 0.18?±?0.03?mV vs 0.33?±?0.05?mV, P?<?0.01)
but kept at a comparable level in the LF-EMF group (Frequency:
60.8?±?4.8impulse/min vs 65.6?±?4.8impulse/min, P?>?0.05; Amplitude:
0.19?±?0.02?mV vs 0.18?±?0.02?mV, P?>?0.05).
Representative examples (A) and quantitative analysis (B,C) of LSG neural activity in the Control group (n?=?8) and EMF group (n?=?8). **P?<?0.01 as compared to group baseline; #P?<?0.05 and ##P?<?0.05 as compared to the Control group. All abbreviations are identical to Figs 1 and ?and22.
In the present study, we applied LF-EMF at the body surface of LSG. Both the ventricular electrophysiological parameters (APD90,
PCL, Smax) and autonomic neural activity (serum norepinephrine, LSG
function and LSG neural activity) were significantly affected by LF-EMF
stimulation. Furthermore, the acute myocardial infarction-induced
increased neural activity of LSG was significantly attenuated and the
VAs was significantly reduced by LF-EMF. These findings suggested that
exposure the LSG to LF-EMF might significantly reduce the neural
activity of LSG, therefore reducing the incidence of VAs.
Previous studies have shown that activation of LSG facilitates, whereas inhibition of LSG protects against VAs4,10. In the past two decades, TMS has been widely used in clinical neurology11,12.
Amounts of studies have shown that high-frequency stimulation increases
cortical excitability, whereas low-frequency stimulation decreases
neuronal excitability11,12. Recently, studies also demonstrated that TMS might affect the cardiac rhythm by modulating the autonomic nervous system7. Scherlag et al.8 showed
that high-frequency magnetic stimulation of the vagal nerves might
induce atrial tachycardia and atrial fibrillation, which was eliminated
after propranolol and atropine injection. Low-frequency stimulation of
the vagal nerves, however, reduced the heart rate and decreased the
voltage required to induce atrioventricular conduction block8.
Furthermore, recent study demonstrated that exposure the heart to the
LF-EMF might significantly suppress atrial fibrillation and the
mechanism might be by modulating the neural activity of atrial
In the present study, we found that exposure the LSG to the LF-EMF
significantly reduced the serum norepinephrine, neural activity of LSG
and VAs. All these indicate that noninvasive LF-EMF might reduce VAs by
facilitating the autonomic rebalance, but what underlie the beneficial
effects of LF-EMF on LSG was poorly defined.
In the present study, we suggested some possible
mechanisms underlying the suppressing of LSG neural activity. Firstly,
TMS, as an effective treatment for patients with neural disorders, has
been implicated long-lasting therapeutic effects after the cessation of
Most researchers have contributed these effects to be long-term
depression (LTD) and long-term potentiation (LTP) cause the duration of
the effects seemed to implicate changes in synaptic plasticity13.
LTD is caused by low-frequency stimulation or the stimulation of a
postsynaptic neuron, whereas LTP is caused by high-frequency stimulation
or the stimulation of a presynaptic neuron13.
Ca++ signal, which is known to regulate membrane excitability and
modulate second messengers related to multiple receptors and signal
transduction pathways, has been shown to be the major determinant
whether LTD or LTD arises14,15. Recently, Scherlag et al.8 also
suggested that LTP or LTD was existed cause exposure the chest to the
low-frequency electromagnetic field for 35?mins might result in the
suppression of atrial fibrillation for 3 to 4?hours after the
application of LF-EMF. In the present study, we also found that
pretreatment with LF-EMF might significantly attenuated the acute
myocardial infarction-induced activation of LSG neural activity and VAs,
suggesting that LTP or LTD might be a potential explain for the
salutary effects of LF-EMF stimulation. Secondly, previous studies have
shown that TMS might also affect the expression levels of various
receptors and other neuromediators, such as ?-adrenoreceptors, dopamine11,16,17.
In the present study, serum norepinephrine was significantly decreased
after exposure to the LF-EMF, indicating that modulating the
neurotransmitters might be one of the underlying mechanisms underlying
the salutary effects of LF-EMF stimulation. Thirdly, previous studies
also showed that TMS might also modulate dentritic sprouting (axon
growth) and the density of synaptic contacts, and the authors suggested
that these results are associated with the Brain-derived neurotrophic
factor (BDNF)-tyrosine kinase B (TrkB) signaling system18,19.
BDNF, as the most abundant neurotrophin in the brain, was reported to
be a major contributor to the N-methyl-D-aspartate receptor-dependent
LTP and LTD processes20. Wang et al.21 demonstrated
that low-frequency TMS might reduce BDNF levels. High-frequency
stimulation, however, might increase serum BDNF levels and the affinity
of BDNF for TrkB receptors. Furthermore, previous studies also showed
that trancranial stimulation might result in the changes in
neural-related proteins, such as c-fos and tyrosine hydroxylase, which
are closely related with the neural remodeling processes6,13,20. Autonomic neural remodeling, however, plays a key role in the initiation and maintenance of VAs4,10.
All these implicate that modulating autonomic neural remodeling might
be another mechanism of the antiarrhythmic effect of LF-EMF stimulation.
Fourthly, the above mainly shows the underlying mechanisms of LF-EMF
stimulation, but how can the LSG perceive the LF-EMF remains unknown.
During the past few decades, many mechanisms, which might provide the
basis for how the animals detect magnetic fields, have been proposed22.
However, the magnetoreceptors have not been identified with certainty
in any animal, and the mode of transduction for the magnetic sense
remains unknown23. Recently, Xie et al.
hypothesized that the putative magnetoreceptor, the iron-sulphur
cluster protein, might combine with the magnetoreception-related
photoreceptor cryptochromes to form the basis of magnetoreception in
animals and this was corroborated in pigeon retina24. Furthermore, Zhang et al.
further showed that the cells which had been transfected iron-sulphur
cluster protein might response to the remote magnetic stimulation25.
All these indicate that the iron-sulphur cluster protein might be the
potential magnetoreceptor for the animals to detect the magnetic fields.
Though the present study showed wonderful results, but
there are some limitations in this study. First, anesthesia with
pentobarbital might affect the autonomic nervous system. However, this
could be counteracted cause anesthesia was maintained continuously
during the whole surgery and conducted in a same fashion in both groups.
Second, the coil used in this study is too large to achieve
LSG-targeted stimulation without affecting the surrounding tissues. It
would be a great step forward if the coils could be technically
improved. Third, we only observed the effect of LF-EMF in acute canine
model. Fourth, we mainly focused on the autonomic nervous system
imbalance, one of the major contributors of post-infarction VAs, cause
we intervened the LSG with LF-EMF in this study. It’s a great limitation
that some other major factors, like area at risk, infarct size, degree
of collateral flow and the possibility of any preconditioning pathway
were not involved in this study. However, previous studies have shown
that LSG activation might facilitate the incidence of VAs, whereas
pre-emptive or post-ischemic/infarction LSG inhibition by blockage or
denervation might decrease the incidence of VAs and improve the infarct
size, collateral flow, contractile force both in animals26,27,28,29 and patients30,31.
Furthermore, studies have shown that LSG stimulation might increase the
likelihood of early or delayed afterdepolarization development and the
initiation of reentry, thereby resulting in the incidence of VAs32,33,34.
In this study, LF-EMF stimulation of the LSG might significantly
inhibit the neural activity of LSG, thereby reducing the incidence of
VAs. Therefore, it’s reasonable to refer that improving the above
factors might also be the potential mechanisms underlying the beneficial
effects of LF-EMF stimulation, but further studies with optimized
parameters and all-round considerations are required in the future.
In conclusion, the present study showed
that LF-EMF stimulation might significantly reduce the neural function
and neural activity of LSG. Exposure the LSG to the LF-EMF might be a
feasible method for preventing the acute myocardial infarction-induced
VAs. However, larger studies with optimized parameters should be done in
the chronic models to verify the beneficial effect of LF-EMF
Sixteen canines weighing between 20
and 25?kg were included in this study. The experiments were approved by
the Animal Ethics Committee of Wuhan University under approval number
2015–0445 and followed the guidelines outlined by the Care and Use of
Laboratory Animals of the National Institutes of Health. All surgeries
were performed under anesthesia with sodium pentobarbital at an initial
dose of 30?mg/kg and a maintenance dose of 60?mg/h. The depth of
anesthesia was evaluated by monitoring corneal reflexes, jaw tone, and
alterations in cardiovascular indices. The body surface
electrocardiogram was recorded throughout the experiment with a
computer-based Lab System (Lead 2000B, Jingjiang Inc., Wuhan, China).
The core body temperature of the dogs was kept at 36.5?±?1.5?°C. Left
thoracotomy was conducted at the fourth intercostal space. At the end of
the experiment, canines were a lethal dose of pentobarbital (100?mg/kg,
Repeated LF-EMF was supplied by the
magnetic stimulation machine (YRD CCY-I, YiRuiDe Inc., Wuhan, China)
with the curve 8 coil located at the body surface of the LSG (Fig. 1A).
The LSG was stimulated by intermittent (8?s ON, 10?s OFF) LF-EMF
stimulation with the frequency set at 1?HZ and intensity at
approximately 90% of motor threshold (Fig. 1B).
Motor threshold was defined as the lowest electromagnetic intensity
that induced muscle contractions in the proximal forepaw and shoulder.
Monophasic action potential recording
Monophasic action potentials from
the left ventricle were recorded with a custom-made Ag–AgCl catheter. A
dynamic steady state pacing protocol (S1S1) was performed to determine
action potential duration alternans35.
The pulse train was delivered at an initial cycle length slightly
shorter than the sinus cycle length and the drive train of stimuli was
maintained for 30?s to ensure a steady state, and then a 2-min
interruption was taken to minimize the pacing memory effects. After
that, another pulse train with the PCL decreased by 10?ms was delivered
until action potential duration alternans appeared. Action potential
duration alternans was defined as ?APD90?10?ms for ?5 consecutive beats36.
The monophasic action potential recordings were analyzed by the LEAD
2000B work station system (Lead 2000B, Jingjiang Inc. China). The APD90 was defined as the 90% repolarization duration and the diastolic interval was the time interval from the previous APD90 point
to the activation time of the following beat. As described in previous
studies, the dynamic action potential duration restitution curves were
constructed from (Diastolic interval, APD90) pairs using Origin 8.0 (OriginLab, Co., Northampton, MA, USA)35,37. Slope of the shortest diastolic interval was defined as Smax.
Measurements of heart rate variability
Spectral power for heart rate
variability was analyzed on 5-minute electrocardiogram recording
segments and an autoregressive algorithm was used to analyze digitized
signals from the electrocardiographic recordings. The following power
spectral variables were determined: HF, LF and LF/HF38.
Neural recording from the LSG
To record the neural activity of
the LSG, one tungsten-coated microelectrode was inserted into the fascia
of the LSG and one ground lead was connected to the chest wall. The
signal of the LSG was recorded with a PowerLab data acquisition system
(8/35, AD Instruments, Australia) and amplified by an amplifier (DP-304,
Warner Instruments, Hamden, CT, USA). The band-pass filters were set at
300?Hz to 1?kHz and the amplification ranges from 30 to 50 times39.
The neural activity, deflections with a signal-to-noise ratio greater
than 3:1, was manually determined as described in our previous studies39,40,41.
LSG function was measured as the
LSG stimulation-induced maximal change in systolic blood pressure as
described in our previous study38.
High frequency stimulation (20?Hz, 0.1?ms pulse duration) was applied
to the LSG using a stimulator (Grass-S88; Astro-Med, West Warwick, RI,
USA). The voltage ranged from 20?V to 30?V and increased by 5?V. To
eliminate the residual effect of the LSG stimulation, each stimulation
should be less than 30?s and the next stimulation should be not be taken
until the blood pressure returned to a normal level.
Venous blood samples were
collected. Serum was separated by centrifuging at 3000?rpm for 15?min at
4?°C, and stored at ?80?°C until assayed. The serum norepinephrine
level was measured with a canine-specific high sensitivity ELISA kit
(Nanjing Jiancheng Bioengineering Institute, Nanjing City, China)38.
Measurement of the acute myocardial infarction-induced VAs
The left anterior descending
coronary artery was ligated at approximately 2.5?centimeters away from
its origin to induce acute myocardial infarction. The incidence and
duration of the VAs induced by acute myocardial infarction during the
first hour was analyzed. The VAs recorded on the ECG were defined as
VPBs, identifiable premature QRS complexes; VT, three or more
consecutive VPBs; non-sustained VT, VT terminating spontaneously within
30?s; sustained VT, VT sustained for more than 30?s; and VF, a
tachycardia with random ECG morphology and an associated loss of
arterial blood pressure that degenerates into ventricular asystole.
Sixteen dogs were randomly divided
into LF-EMF group (n?=?8, with LF-EMF) and Control group (n?=?8, with
sham LF-EMF). LF-EMF (1?HZ; stimulation time 8?s; interstimulus
interval, 5?s) was delivered to the surface area of LSG for 90?minutes.
As shown in Fig. 1C,
monophasic action potential, heart rate variability, serum
norepinephrine, LSG function and LSG neural activity were measured at
baseline, 30?min and 90?min after LF-EMF treatment. Measurements of
heart rate variability and LSG neural activity were repeated at 15?min
after acute myocardial infarction. Furthermore, the incidence of VAs was
recorded during the first hour after acute myocardial infarction.
Continuous variables are presented
as the mean?±?SEM and were analyzed by t test, one-way ANOVA, or two-way
repeated-measures ANOVA with a Bonferroni posthoc test. To compare the
incidence of VF between groups, Fisher’s exact test was used. All data
was analyzed by GraphPad Prism version 5.0 software (GraphPad Software,
Inc., San Diego, CA), and two-tailed P???0.05 was considered
How to cite this article: Wang, S. et al.
Noninvasive low-frequency electromagnetic stimulation of the left
stellate ganglion reduces myocardial infarction-induced ventricular
arrhythmia. Sci. Rep.6, 30783; doi: 10.1038/srep30783 (2016).
This work was supported by the grants from National
Natural Science Foundation of China No. 81270339, No. 81300182, No.
81530011, No. 81570463, grant from the Natural Science Foundation of
Hubei Province No. 2013CFB302, and grants from the Fundamental Research
Funds for the Central Universities No. 2042014kf0110 and No.
Author Contributions S.W.
and X.Z. wrote the main manuscript text and prepared figures; B.H.,
Z.W., L.Z. and M.W. performed experiments and anlalyzed data; L.Y. and
H.J. designed the project and revised the paper. All authors reviewed
and approved the final version.
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Articles from Scientific Reports are provided here courtesy of Nature Publishing Group
Spine J. 2008 May-Jun;8(3):436-42. Epub 2007 Jul 17.
Randomized, prospective, and controlled clinical trial of pulsed electromagnetic field stimulation for cervical fusion.
Foley KT, Mroz TE, Arnold PM, Chandler HC Jr, Dixon RA, Girasole GJ,
Renkens KL Jr, Riew KD, Sasso RC, Smith RC, Tung H, Wecht DA, Whiting
Department of Neurosurgery, University of Tennessee Health Science
Center and Semmes-Murphey Neurologic and Spine Institute, Memphis,
Tennessee 38104, USA. firstname.lastname@example.org
BACKGROUND CONTEXT: Multilevel fusions, the use of allograft bone,
and smoking have been associated with an increased risk of nonunion
after anterior cervical discectomy and fusion (ACDF) procedures. Pulsed
electromagnetic field (PEMF) stimulation has been shown to increase
arthrodesis rates after lumbar spine fusion surgery, but there are
minimal data concerning the effect of PEMF stimulation on cervical spine
PURPOSE: To determine the efficacy and safety of PEMF stimulation as
an adjunct to arthrodesis after ACDF in patients with potential risk
factors for nonunion.
STUDY DESIGN: A randomized, controlled, prospective multicenter clinical trial.
PATIENT SAMPLE: Three hundred and twenty-three patients with
radiographic evidence (computed tomography-myelogram [CT-myelo] or
magnetic resonance imaging [MRI]) of a compressed cervical nerve root
and symptomatic radiculopathy appropriate to the compressed root that
had failed to respond to nonoperative management were enrolled in the
study. The patients were either smokers (more than one pack per day)
and/or were undergoing multilevel fusions. All patients underwent ACDF
using the Smith-Robinson technique. Allograft bone and an anterior
cervical plate were used in all cases.
OUTCOME MEASURES: Measurements were obtained preoperatively and at
each postoperative interval and included neurologic assessment, visual
analog scale (VAS) scores for shoulder/arm pain at rest and with
activity, SF-12 scores, the neck disability index (NDI), and radiographs
(anteroposterior, lateral, and flexion-extension views). Two orthopedic
surgeons not otherwise affiliated with the study and blinded to
treatment group evaluated the radiographs, as did a blinded radiologist.
Adverse events were reported by all patients throughout the study to
determine device safety.
METHODS: Patients were randomly assigned to one of two groups: those
receiving PEMF stimulation after surgery (PEMF group, 163 patients) and
those not receiving PEMF stimulation (control group, 160 patients).
Postoperative care was otherwise identical. Follow-up was carried out at
1, 2, 3, 6, and 12 months postoperatively.
RESULTS: The PEMF and control groups were comparable with regard to
age, gender, race, past medical history, smoking status, and litigation
status. Both groups were also comparable in terms of baseline diagnosis
(herniated disc, spondylosis, or both) and number of levels operated
(one, two, three, or four). At 6 months postoperatively, the PEMF group
had a significantly higher fusion rate than the control group (83.6% vs.
68.6%, p=.0065). At 12 months after surgery, the stimulated group had a
fusion rate of 92.8% compared with 86.7% for the control group
(p=.1129). There were no significant differences between the PEMF and
control groups with regard to VAS pain scores, NDI, or SF-12 scores at 6
or 12 months. No significant differences were found in the incidence of
adverse events in the groups.
CONCLUSIONS: This is the first randomized, controlled trial that
analyzes the effects of PEMF stimulation on cervical spine fusion. PEMF
stimulation significantly improved the fusion rate at 6 months
postoperatively in patients undergoing ACDF with an allograft and an
anterior cervical plate, the eligibility criteria being patients who
were smokers or had undergone multilevel cervical fusion. At 12 months
postoperatively, however, the fusion rate for PEMF patients was not
significantly different from that of the control group. There were no
differences in the incidence of adverse events in the two groups,
indicating that the use of PEMF stimulation is safe in this clinical
Ann Biomed Eng. 2008 Feb;36(2):195-203. Epub 2007 Nov 27.
Why do electromagnetic pulses enhance bone growth?
Bowen SP, Mancini JD, Fessatidis V, Grabiner M.
Department of Chemistry and Physics, Chicago State University, Chicago, IL 60628, USA. email@example.com
The excitation probability of substrate molecules involved in the
production of growth factors influencing the division of chondrocytes in
the growth layer of bone under the influence of pulsed electromagnetic
fields is studied theoretically in a quantum mechanical model
calculation. In this model matrix elements and anti-bonding energy
levels are assumed known and the dynamics of the interaction with pulsed
electromagnetic fields is derived. The derivation makes it clear that
continuous pulsing or large driving currents can overwhelm local
diffusive transport to the growth plane resulting in a loss of its
enhancement properties. Optimal locations within a pair of Helmholtz
coils for enhancement of bone growth are also investigated and found to
be close to the coils. The work presented here is believed to be the
first derivation in a model calculation of a physical basis for the
effects of pulsed electromagnetic fields on bone growth and fusion.
outh Med J. 2004 May;97(5):519-24.
Reversal of delayed union of anterior cervical fusion treated with pulsed electromagnetic field stimulation: case report.
Mackenzie D, Veninga FD.
Department of Surgery, Medical Center of Plano, Plano, TX, USA.
We present a case report of anterior cervical fusion non-union that
was successfully treated with pulsed electromagnetic field (PEMF)
stimulation. In this case, a C6-C7 nonunion was identified clinically
and radiographically 1 year after surgery. Imaging revealed nonunion
with partial resorption of the bone graft compared with imaging studies
performed 8 months earlier. The patient wore a PEMF stimulation device
for 3 h/d for 10 months. After 3 months of treatment, the patient’s
symptoms were resolved. X-rays obtained after 15 weeks of stimulation
showed improvement in bone fusion, and x-rays obtained at 31 weeks after
stimulation showed even bone density around the C7 screws. The patient
remained symptom-free 13 months after the termination of PEMF
stimulation at last assessment. PEMF stimulation demonstrated its
clinical potential in healing established nonunion of anterior cervical
spine fusion. Its use is noninvasive and can be considered an
alternative to surgical intervention in selected patients.
Am J Orthop (Belle Mead NJ). 2004 Jan;33(1):27-30.
Pseudarthrosis after lumbar spine fusion: nonoperative salvage with pulsed electromagnetic fields.
Simmons JW Jr, Mooney V, Thacker I.
UTMB, Galveston, Texas, USA.
We studied 100 patients in whom symptomatic pseudarthrosis had been
established at more than 9 months after lumbar spine fusion. All
patients were treated with a pulsed electromagnetic field device worn
consistently 2 hours a day for at least 90 days. Solid fusion was
achieved in 67% of patients. Effectiveness was not statistically
significantly different for patients with risk factors such as smoking,
use of allograft, absence of fixation, or multilevel fusions. Treatment
was equally effective for posterolateral fusions (66%) as with interbody
fusions (69%). For patients with symptomatic pseudarthrosis after
lumbar spine fusion, pulsed electromagnetic field stimulation is an
effective nonoperative salvage approach to achieving fusion.
J Am Acad Orthop Surg. 2003 Sep-Oct;11(5):344-54.
Use of physical forces in bone healing.
Nelson FR, Brighton CT, Ryaby J, Simon BJ, Nielson JH, Lorich DG, Bolander M, Seelig J.
Henry Ford Hospital, Detroit, MI, USA.
During the past two decades, a number of physical modalities have
been approved for the management of nonunions and delayed unions.
Implantable direct current stimulation is effective in managing
established nonunions of the extremities and as an adjuvant in achieving
spinal fusion. Pulsed electromagnetic fields and capacitive coupling
induce fields through the soft tissue, resulting in low-magnitude
voltage and currents at the fracture site. Pulsed electromagnetic fields
may be as effective as surgery in managing extremity nonunions.
Capacitive coupling appears to be effective both in extremity nonunions
and lumbar fusions. Low-intensity ultrasound has been used to speed
normal fracture healing and manage delayed unions. It has recently been
approved for the management of nonunions. Despite the different
mechanisms for stimulating bone healing, all signals result in increased
intracellular calcium, thereby leading to bone formation.
Adv Ther. 2001 Jan-Feb;18(1):12-20.
Outcomes after posterolateral lumbar fusion with instrumentation in
patients treated with adjunctive pulsed electromagnetic field
Medical Center of Delaware, Newark, USA.
Fusion success and clinical outcome were determined in 48 high-risk
patients who underwent posterolateral lumbar fusions with internal
fixation and were treated with adjunctive pulsed electromagnetic field
(PEMF) stimulation postoperatively. An independent radiographic
assessment demonstrated a success rate of 97.9%. Following treatment,
59% of the working patients returned to their employment. Overall
clinical assessment was excellent in 4.2% of patients, good in 79.2%,
and fair in 16.7%; no patient had a poor clinical assessment.
J Spinal Disord. 2000 Aug;13(4):290-6.
Prospective comparison of the effect of direct current electrical
stimulation and pulsed electromagnetic fields on instrumented
posterolateral lumbar arthrodesis.
Jenis LG, An HS, Stein R, Young B.
New England Baptist Spine Center, Boston, Massachusetts 02120, USA.
The purpose of this prospective study was to compare the effect of
adjunctive direct current (DC) electrical stimulation and pulsed
electromagnetic field therapy (PEMF) on augmentation of instrumented
lumbar fusion. Sixty-one patients undergoing lumbar spine fusion were
enrolled in the study and randomized to one of three treatment
protocols: 1) adjunctive PEMF group (n = 22) fitted with Spinal-Stim
model 8212(AME) within 30 days of surgery; 2) DC group (n = 17) had a
SpF-2T stimulator(EBI) implanted at the time of surgery; or 3) control
group (n = 22). The fusion mass bone mineral density (BMD) assessment
was performed on 3-month and 1-year radiographs for each patient.
Lateral flexion-extension and anteroposterior radiographs were evaluated
at 1 year to determine the presence of fusion. Clinical outcome patient
analyses were performed at 1 year. At 1-year follow-up, radiographic
fusion and fusion mass bone density were not significantly different
among the groups. In the nonstimulated group, there were 43% excellent,
43% good, and 14% fair results. In the PEMF group, there were 35%
excellent, 50% good, 10% fair, and 5% poor results. In the DC group,
there were 32% excellent, 37% good, and 31% fair results. The results of
the current study suggest that electrical stimulation does not
significantly enhance fusion rate in instrumented lumbar arthrodesis,
although we observed a statistically insignificant trend toward
increased fusion mass BMD in the electrically stimulated groups. The
significance of increased BMD remains unknown.
Adv Ther. 2000 Mar-Apr;17(2):57-67.
Spine fusion for discogenic low back pain: outcomes in patients
treated with or without pulsed electromagnetic field stimulation.
Richardson Orthopaedic Surgery, Texas 75080, USA.
Sixty-one randomly selected patients who underwent lumbar fusion
surgeries for discogenic low back pain between 1987 and 1994 were
retrospectively studied. All patients had failed to respond to
preoperative conservative treatments. Forty-two patients received
adjunctive therapy with pulsed electromagnetic field (PEMF) stimulation,
and 19 patients received no electrical stimulation of any kind. Average
follow-up time was 15.6 months postoperatively. Fusion succeeded in
97.6% of the PEMF group and in 52.6% of the unstimulated group (P <
.001). The observed agreement between clinical and radiographic outcome
was 75%. The use of PEMF stimulation enhances bony bridging in lumbar
spinal fusions. Successful fusion underlies a good clinical outcome in
patients with discogenic low back pain.
Spine (Phila Pa 1976). 1997 Oct 15;22(20):2351-6.
Use of electromagnetic fields in a spinal fusion. A rabbit model.
Glazer PA, Heilmann MR, Lotz JC, Bradford DS.
Department of Orthopaedic Surgery, University of California, San Francisco, USA.
STUDY DESIGN: The biomechanical and histologic characteristics of
posterolateral spinal fusion in a rabbit model with and without the
application of a pulsed electromagnetic field were analyzed in a
prospective, randomized trial. In addition, fusion rate with and without
a pulsed electromagnetic field in this model was assessed by
biomechanical testing, radiographs, and manual palpation.
OBJECTIVES: To evaluate the influence of a pulsed electromagnetic
field on the spinal fusion rate and biomechanical characteristics in a
SUMMARY OF BACKGROUND DATA: Previous studies performed to assess the
benefits of a pulsed electromagnetic field in spinal fusion have been
complicated by the use of instrumentation, and the animal models used do
not have a pseudarthrosis rate comparable to that seen in humans. In
contrast, the posterolateral intertransverse process fusion in the
rabbit is uncomplicated by the use of instrumentation and has been shown
to have a pseudarthrosis rate similar to that found in humans (5-35%).
METHODS: Ten New Zealand white rabbits each were randomly assigned to
undergo spinal fusion using either 1) autologous bone with
electromagnetic fields, or 2) autologous bone without electromagnetic
fields. A specially designed plastic constraint was used to focus the
pulsed electromagnetic field over the rabbits’ lumbar spine 4 hours per
day. Animals were killed at 6 weeks for biomechanical and histologic
RESULTS: The rate of pseudarthrosis, as evaluated radiographically
and manually in a blinded fashion, decreased from 40% to 20% with the
pulsed electromagnetic field, but this decrease in the nonunion rate was
not statistically significant given the number of animals per group.
Biomechanical analysis of the fusion mass showed that a pulsed
electromagnetic field resulted in statistically significant increases in
stiffness (35%), area under the load-displacement curve (37%), and load
to failure of the fusion mass (42%). Qualitative histologic assessment
showed increased bone formation in those fusions exposed to a pulsed
CONCLUSIONS: This study demonstrates the reproducibility of a rabbit
fusion model, and the ability of a pulsed electromagnetic field to
induce a statistically significant increase in stiffness, area under the
load-displacement curve, and load to failure of the fusion mass. This
investigation provides a basis for continued evaluation of biologic
enhancement of spinal arthrodesis with the use of a pulsed
Spine (Phila Pa 1976). 1997 Feb 15;22(4):382-8.
The effect of pulsed electromagnetic fields on instrumented posterolateral spinal fusion and device-related stress shielding.
Ito M, Fay LA, Ito Y, Yuan MR, Edwards WT, Yuan HA.
Department of Orthopaedics, Hokkaido University School of Medicine, Sapporo, Japan.
STUDY DESIGN: This study was designed to examine stress-shielding
effects on the spine caused by rigid implants and to investigate the
effects of pulsed electromagnetic fields on the instrumented spine.
OBJECTIVES: To investigate the effects of pulsed electromagnetic
fields on posterolateral spinal fusion, and to determine if osteopenia
induced by rigid instrumentation can be diminished by pulsed
SUMMARY OF BACKGROUND DATA: Although device-related osteopenia on
vertebral bodies is of a great clinical importance, no method for
preventing bone mineral loss in vertebrae by stiff spinal implants has
METHODS: Twenty-eight adult beagles underwent L5-L6 destabilization
followed by posterolateral spinal fusion. The study was divided into
four groups: 1) Group CNTL: without instrumentation, without pulsed
electromagnetic fields, 2) Group PEMF: without Steffee, with pulsed
electromagnetic fields, 3) Group INST: with Steffee, without pulsed
electromagnetic fields, 4) Group PEMF + INST: with Steffee, with pulsed
electromagnetic fields. At the end of 24 weeks, the dogs were killed,
and L4-L7 segments were tested biomechanically without instrumentation.
Radiographs and quantitative computed tomography assessed the condition
of the fusion mass.
RESULTS: Stress shielding was induced in the anterior vertebral
bodies of L6 with the Steffee plates; bone mineral density was increased
with the addition of pulsed electromagnetic fields, regardless of the
presence or absence of fixation. A decrease in flexion and bending
stiffness was observed in the Group INST; pulsed electromagnetic fields
did increase the flexion stiffness regardless of the presence or absence
of fixation, although this was not statistically significant.
CONCLUSIONS: Use of pulsed electromagnetic fields has the potential to minimize device-related vertebral-bone mineral loss.
Spine (Phila Pa 1976). 1994 Mar 15;19(6):705-9.
The effect of electromagnetic pulsing on posterior lumbar spinal fusions in dogs.
Kahanovitz N, Arnoczky SP, Nemzek J, Shores A.
Anderson Clinic, Arlington, Virginia.
This study evaluated the effect of pulsed electromagnetic fields
(PEMF) on the healing of lumbar spinal fusions. Bilateral posterior
facet fusions were performed at L1-2 and L4-5 in 24 adult mongrel dogs.
After surgery, eight animals were stimulated with a pulse burst type
signal (PEMF) for 30 minutes a day, and eight animals were stimulated
with the same PEMF for 60 minutes a day. The remaining eight animals
received no active PEMF stimulation and served as controls. Four animals
from each group were euthanatized at 6 and 12 weeks, and the facet
fusions were evaluated using high resolution radiographs and routine
histology. No statistical difference in the radiographic or histologic
appearance of the fusion mass could be detected between the stimulated
and control groups at either 6 or 12 weeks. The results of this study
suggest that PEMF stimulation had no effect on the healing of the
primary posterior spinal fusions in this controlled experimental canine
Spine (Phila Pa 1976). 1990 Jul;15(7):708-12.
A randomized double-blind prospective study of the efficacy of pulsed electromagnetic fields for interbody lumbar fusions.
Division of Orthopaedic Surgery, University of California, Irvine.
A randomized double-blind prospective study of pulsed electromagnetic
fields for lumbar interbody fusions was performed on 195 subjects.
There were 98 subjects in the active group and 97 subjects in the
placebo group. A brace containing equipment to induce an electromagnetic
field was applied to patients undergoing interbody fusion in the active
group, and a sham brace was used in the control group. In the active
group there was a 92% success rate, while the control group had a 65%
success rate (P greater than 0.005). The effectiveness of bone graft
stimulation with the device is thus established.
J Bone Miner Res. 1989 Apr;4(2):227-33.
Stimulation of experimental endochondral ossification by low-energy pulsing electromagnetic fields.
Aaron RK, Ciombor DM, Jolly G.
Department of Biochemistry and Biophysics, University of Rhode Island, Providence.
Pulsed electromagnetic fields (PEMFs) of certain configuration have
been shown to be effective clinically in promoting the healing of
fracture nonunions and are believed to enhance calcification of
extracellular matrix. In vitro studies have suggested that PEMFs may
also have the effect of modifying the extracellular matrix by promoting
the synthesis of matrix molecules. This study examines the effect of one
PEMF upon the extracellular matrix and calcification of endochondral
ossification in vivo. The synthesis of cartilage molecules is enhanced
by PEMF, and subsequent endochondral calcification is stimulated.
Histomorphometric studies indicate that the maturation of bone
trabeculae is also promoted by PEMF stimulation. These results indicate
that a specific PEMF can change the composition of cartilage
extracellular matrix in vivo and raises the possibility that the effects
on other processes of endochondral ossification (e.g., fracture healing
and growth plates) may occur through a similar mechanism.
Orthop Clin North Am. 1984 Jan;15(1):61-87.
The development and application of pulsed electromagnetic fields (PEMFs) for ununited fractures and arthrodeses.
This article deals with the rational and practical use of surgically
noninvasive pulsed electromagnetic fields (PEMFs) in treating ununited
fractures, failed arthrodeses, and congenital pseudarthroses (infantile
nonunions). The method is highly effective (more than 90 per cent
success) in adult patients when used in conjunction with good management
techniques that are founded on biomechanical principles. When union
fails to occur with PEMFs alone after approximately four months, their
proper use in conjunction with fresh bone grafts insures a maximum
failure rate of 1 to 1.5 per cent. Union occurs because the weak
electric currents induced in tissues by the time-varying fields effect
calcification of the fibrocartilage in the fracture gap, thereby setting
the stage for the final phases of fracture healing by endochondral
ossification. The efficacy, safety, and simplicity of the method has
prompted its use by the majority of orthopedic surgeons in this country.
In patients with delayed union three to four months postfracture, PEMFs
appear to be more successful and healing, generally, is more rapid than
in patients managed by other conservative methods. For more challenging
problems such as actively infected nonunions, multiple surgical
failures, long-standing (for example, more than two years postfracture)
atrophic lesions, failed knee arthrodeses after removal of infected
prostheses, and congenital pseudarthroses, success can be expected in a
large majority of patients in whom PEMFs are used. Finally, as
laboratory studies have expanded knowledge of the mechanisms of PEMF
action, it is clear that different pulses affect different biologic
processes in different ways. Selection of the proper pulse for a given
pathologic entity has begun to be governed by rational processes
similar, in certain respects, to those applied to pharmacologic agents.
OBJECTIVE: Review the literature on non-pharmacological prevention and treatment of osteoporosis after spinal cord injury (SCI).
METHODS: PubMed, EMBASE and the Cochrane Controlled Trials Register
were searched. All identified papers were read by title, abstract and
full-length article when relevant. Hand search of the articles’ sources
identified additional papers. For included studies, the level of
evidence was determined.
RESULTS: No studies conclusively showed an effective intervention.
However, there are few randomized controlled trials (RCTs), and those
that exist assess interventions and outcome measures that could be
improved. Five studies on weight-bearing early post-injury are
conflicting, but standing or walking may help retain bone mineral. In
the chronic phase, there was no effect of weight bearing (12 studies).
One study found that an early commencement of sports after SCI improved
bone mineral, and the longer the period of athletic career, the higher
the (leg) bone mineral. Early after SCI, there may be some effects of
electrical stimulation (ES) (five studies). Chronic-phase ES studies
vary (14 studies, including mixed periods after injury), but improvement
is seen with longer period of training, or higher frequency or stimulus
intensity. Improvements correspond to trabecular bone in the distal
femur or proximal tibia. Impact vibration and pulsed electromagnetic
fields may have some positive effects, whereas pulsed ultrasound does
not. Six studies on the influence of spasticity show inconsistent
CONCLUSIONS: Bone mineral should be measured around the knee; the
length and intensity of the treatment should be sufficiently long and
high, respectively, and should commence early after SCI. If bone mineral
is to remain, the stimulation has to be possibly continued for long
term. In addition, RCTs are necessary.
Spine. 2003 Dec 15;28(24):2660-6.
Exposure to pulsed magnetic fields enhances motor recovery in cats after spinal cord injury.
Neuroscience Research Laboratories, The Clement J. Zablocki VA Medical Center, Milwaukee, WI 53295, USA. firstname.lastname@example.org
STUDY DESIGN: Animal model study of eight healthy commercial cats was conducted.
OBJECTIVE: To determine whether pulsed electromagnetic field (PMF)
stimulation results in improvement of function after contusive spinal
cord injury in cats. SUMMARY OF
BACKGROUND DATA: PMF stimulation has been shown to enhance nerve
growth, regeneration, and functional recovery of peripheral nerves.
Little research has been performed examining the effects of PMF
stimulation on the central nervous system and no studies of PMF effects
on in vivo spinal cord injury (SCI) models have been reported.
MATERIALS AND METHODS: PMF stimulation was noninvasively applied for
up to 12 weeks to the midthoracic spine of cats with acute contusive
spinal cord injury. The injury was produced using a weight-drop
apparatus. Motor functions were evaluated with the modified Tarlov
assessment scale. Morphologic analyses of the injury sites and
somatosensory-evoked potential measurements were conducted to compare
results between PMF-stimulated and control groups.
RESULTS: There was a significant difference in locomotor recovery
between the PMF-stimulated and control groups. Although not
statistically significant, PMF-stimulated spinal cords demonstrated
greater sparing of peripheral white matter and smaller lesion volumes
compared to controls. Somatosensory-evoked potential measurements
indicated that the PMF-stimulated group had better recovery of preinjury
waveforms than the control group; however, this observation also was
not statistically significant because of the small sample size.
CONCLUSIONS: This preliminary study indicates that pulsed magnetic
fields may have beneficial effects on motor function recovery and lesion
volume size after acute spinal cord injury.
J Spinal Cord Med. 1999 Winter;22(4):239-45.
The effect of pulsed electromagnetic fields on osteoporosis at the knee in individuals with spinal cord injury.
Garland DE, Adkins RH, Matsuno NN, Stewart CA.
Rancho Los Amigos Medical Center, Downey, California 90242, USA.
The purpose of this study was to determine the effects of pulsed
electromagnetic fields on osteoporotic bone at the knee in individuals
with chronic spinal injury. The study consisted of 6 males with complete
spinal cord injury at a minimum of 2 years duration. Bone mineral
density (BMD) was obtained at both knees at initiation, 3 months, 6
months, and 12 months using dual energy X-ray absorptiometry. In each
case, 1 knee was stimulated using The Bone Growth Stimulator Model 3005
from American Medical Electronics, Incorporated and the opposite knee
served as the control. Stimulation ceased at 6 months. At 3 months BMD
increased in the stimulated knees 5.1% and declined in the control knees
6.6% (p < .05 and p < .02, respectively). By 6 months the BMD
returned to near baseline values and at 12 months both knees had lost
bone at a similar rate to 2.4% below baseline for the stimulated knee
and 3.6% below baseline for the control. There were larger effects
closer to the site of stimulation. While the stimulation appeared useful
in retarding osteoporosis, the unexpected exaggerated decline in the
control knees and reversal at 6 months suggests underlying mechanisms
are more complex than originally anticipated. The authors believe a
local as well as a systemic response was created.
Zh Nevropatol Psikhiatr Im S S Korsakova. 1990;90(7):108-12.
Regional cerebral angiodystonia in the practice of a neurologist and therapist.
[Article in Russian]
Pokalev GM, Raspopina LA.
Altogether 108 patients with regional cerebral angiodystonia were
examined using rheoencephalography, measurements of temporal and venous
pressure and functional tests (nitroglycerin and bicycle ergometry).
Three variants of abnormalities connected with regional cerebral
angiodystonia were distinguished: dysfunction of the inflow, derangement
of the venous outflow, and initial functional venous hypertonia. The
patients were treated with nonmedicamentous therapy (electroanalgesia,
Pulsed subcutaneous electrical stimulation in spinal cord injury: preliminary results.
The treatment of long-term, stable para- and quadriplegics with
pulsed electrical stimulation for pain control resulted in, anecdotally,
a significant number of these individuals showing increased motor
function as well as sensory awareness. This small pilot study was
conducted in order to assess the hypothesis that pulsed electrical
fields can effect diseased neurological function. Thirteen para- and
quadriplegic subjects with 18 months of stable neurological signs and
symptoms were exposed daily to pulsed electrical stimulation for a
6-month period and assessed for any improvement in motor function or
sensory perception. The hypothesis is that pulsed electromagnetic fields
can normalize viable but dysfunctional neuronal structures. Results
Surg Neurol. 1987 Oct;28(4):269-76.
Effect of alternating current stimulation of the spinal cord on recovery from acute spinal cord injury in rats.
Wallace MC, Tator CH, Gentles WM.
The therapeutic value of electrical stimulation of the spinal cord
was studied in rats injured by acute compression of the spinal cord.
Twenty adult Wistar rats underwent cord compression at T6-7 by the
extradural clip compression technique at a force of 125 g for 1 minute.
After injury and group randomization, stimulating electrodes were placed
extradurally, proximal and distal to the injury site, and attached to a
small, implantable receiver-stimulator. The receiver was secured to the
paraspinal muscles and implanted subcutaneously, overlying the thoracic
spine. The animals were maintained in specially designed cages with
encircling antennae attached to radio frequency transmitters. The 10
treatment animals were subjected to a 460-kHz electromagnetic field,
pulsed at a frequency of 10 Hz. The receivers converted the pulsed radio
frequency into square-wave pulses at the cord electrodes (width 1 ms,
frequency 10 Hz). The 10 control animals were exposed to a similar field
but with a frequency below the range of the tuned receiver, and
therefore they did not receive the square-wave pulse. Clinical recovery
was assessed by the inclined plane technique which measures the maximum
angle of inclination attained without falling. After 15 weeks of
continuous spinal cord stimulation, the inclined plane performance was
not significantly different between the two groups (treatment group
mean, 44.4 +/- 5.4; control group mean, 41.7 +/- 7.9). This is the first
experimental study of the effect of long-term continuous electrical
stimulation on spinal cord recovery in mammals. The methods required and
the technical aspects involved in achieving continuous stimulation, and
the guidelines for future study of this modality are discussed.
Paraplegia. 1976 May;14(1):12-20.
Experimental regeneration in peripheral nerves and the spinal cord
in laboratory animals exposed to a pulsed electromagnetic field.
Wilson DH, Jagadeesh P.
Peripheral nerve section and suture was performed in 132 rats.
Postoperatively half the animals were exposed to a pulsed
electromagnetic field each day and half were kept as controls. Nerve
conduction studies, histology and nerve fibre counts all indicated an
increased rate of regeneration in the treated animals. A similar
controlled study of spinal cord regeneration following hemicordotomy in
cats has been started, and preliminary results indicate that when the
animals are sacrificed three months after the hemicordotomy, the pulsed
electromagnetic therapy has induced nerve fibre regeneration across the
region of the scar.
Neurosci Behav Physiol. 1998 Sep-Oct;28(5):594-7.
Magnetic and electrical stimulation in the rehabilitative treatment of patients with organic lesions of the nervous system.
Tyshkevich TG, Nikitina VV.
A. L. Polenov Russian Science Research Neurosurgical Institute, St. Petersburg.
Studies were performed on 89 patients with organic lesions of the
nervous system in which the leading clinical symptoms consisted of
paralysis and pareses. Patients received complex treatment, including
pulsed magnetic fields and an electrical stimulation regime producing
multilevel stimulation. A control group of 49 patients with similar
conditions was included, and these patients received only sinusoidal
currents. Combined treatment with magnetic and electrical stimulation
was more effective, as indicated by radiographic and electromyographic
Static magnetotherapy for the treatment of insomnia.
Shieh YY, Tsai FY.
Department of Radiological Sciences, School of Medicine, UCIrvine Medical Center, Orange CA 92868, USA. email@example.com
Magnets have been used for centuries to treat a number of physical
disorders. The vast majority of research, however, on static magnet
therapy for insomnia has been confined to the auricular type of therapy,
with publications limited to Chinese journals. Most of these studies
have depended on the subjective self-assessment of participants rather
than objective scientific measurements. In this study, the authors
report the positive preliminary results of insomnia treatment using
pillows with embedded magnets, magnetic insoles and TriPhase bracelets.
The analysis is based on objective actigraphic and polysomnographic
data. A theory of accelerated transition from wakefulness to sleep is
proposed to explain the process of insomnia relief through low-strength
static magnetic fields. Analysis by functional Magnetic Resonance
Imaging (fMRI) is used to further investigate the theory.
J Sleep Res 16(3) 253-8 (2007)
Pulsed radio-frequency electromagnetic fields: dose-dependent effects on sleep, the sleep EEG and cognitive performance
Regel SJ, Tinguely G, Schuderer J, Adam M, Kuster N, Landolt HP, Achermann P
To establish a dose-response relationship between the strength of
electromagnetic fields (EMF) and previously reported effects on the
brain, we investigated the influence of EMF exposure by varying the
signal intensity in three experimental sessions. The head of 15 healthy
male subjects was unilaterally exposed for 30 min prior to sleep to a
pulse-modulated EMF (GSM handset like signal) with a 10 g-averaged peak
spatial specific absorption rate of (1) 0.2 W kg(-1), (2) 5 W kg(-1), or
(3) sham exposed in a double-blind, crossover design. During exposure,
subjects performed two series of three computerized cognitive tasks,
each presented in a fixed order [simple reaction time task, two-choice
reaction time task (CRT), 1-, 2-, 3-back task]. Immediately after
exposure, night-time sleep was polysomnographically recorded for 8 h.
Sleep architecture was not affected by EMF exposure. Analysis of the
sleep electroencephalogram (EEG) revealed a dose-dependent increase of
power in the spindle frequency range in non-REM sleep. Reaction speed
decelerated with increasing field intensity in the 1-back task, while
accuracy in the CRT and N-back task were not affected in a
dose-dependent manner. In summary, this study reveals first indications
of a dose-response relationship between EMF field intensity and its
effects on brain physiology as demonstrated by changes in the sleep EEG
and in cognitive performance.
Conf Proc IEEE Eng Med Biol Soc. 2005;6:6214-6.
Influence of time-varying magnetic field on the release of neurotransmitters in raphe nuclei of rats.
Zhang J, Wang X, Wang M.
Mayo Clinic Rochester, MN 55905, USA.
A specially-designed time-varying magnetic field was developed to
treat insomnia. Clinical results showed that this method could shorten
the time to go to sleep and prolong the sleep duration. However, the
mechanism of this method is still not well understood. In this study,
the effect of magnetic stimulation on the release of serotonin (5-HT),
noradrenaline (NE), dopamine (DA) in raphe nuclei of rats, which are
known to play an important role in the sleep-wake regulation, was
investigated. It was shown that there was a significant difference in
the release of serotonin between control group and experimental group
(p<0.01). The release of serotonin of the experimental group
increased significantly. No obvious release changes of NE and DA are
found (p>0.05). The results indicates that one possible mechanism of
inducing sleep using specially designed magnetic field is to change the
release of sleep-related neurotransmitters.
Neurosci Behav Physiol. 2005 Feb;35(2):165-70.
Actions of pulsed ultra-broadband electromagnetic irradiation on the EEG and sleep in laboratory animals.
Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, 117865 Moscow, Russia.
Irradiation of animals with ultrashort impulses of ultra-broadband
magnetic irradiation with an impulse repetition frequency of 6 Hz for 1 h
induced changes in the spectral composition of cerebral cortex
electrical activity in rats, measured over the 5 min immediately after
irradiation, as compared with controls. In particular, there was
suppression of frequencies close to the impulse sequence frequency,
along with a decrease in interhemisphere coherence. Continuous recording
of polygrams for 22 h from rabbits after irradiation revealed a
“delayed” effect–a significant increase in paradoxical sleep, starting
16 h after the end of irradiation and persisting to the end of the
recording period. It is suggested that irradiation has a direct action
both on the mechanisms of generation of the theta rhythm
(septohippocampal) and on the system controlling circadian rhythms (the
suprachiasmatic nucleus-epiphysis system).
Eur J Neurosci. 2004 Jun;19(11):3099-104.
Intracortical inhibition and facilitation upon awakening from different sleep stages: a transcranial magnetic stimulation study.
De Gennaro L, Bertini M, Ferrara M, Curcio G, Cristiani R, Romei V, Fratello F, Pauri F, Rossini PM.
Intracortical facilitation and inhibition, as assessed by the
paired-pulse transcranial magnetic stimulation technique with a
subthreshold conditioning pulse followed by a suprathreshold test pulse,
was studied upon awakening from REM and slow-wave sleep (SWS). Ten
normal subjects were studied for four consecutive nights. Intracortical
facilitation and inhibition were assessed upon awakening from SWS and
REM sleep, and during a presleep baseline. Independently of sleep stage
at awakening, intracortical inhibition was found at 1-3-ms interstimulus
intervals and facilitation at 7-15-ms interstimulus intervals. Motor
thresholds were higher in SWS awakenings, with no differences between
REM awakenings and wakefulness, while motor evoked potential amplitude
to unconditioned stimuli decreased upon REM awakening as compared to the
other conditions. REM sleep awakenings showed a significant increase of
intracortical facilitation at 10 and 15 ms, while intracortical
inhibition was not affected by sleep stage at awakening. While the
dissociation between motor thresholds and motor evoked potential
amplitudes could be explained by the different excitability of the
corticospinal system during SWS and REM sleep, the heightened cortical
facilitation upon awakening from REM sleep points to a cortical motor
activation during this stage.
Neurosci Lett. 2004 May 13;362(1):31-4.
Changes of motor cortical excitability in human subjects from
wakefulness to early stages of sleep: a combined transcranial magnetic
stimulation and electroencephalographic study.
Manganotti P, Fuggetta G, Fiaschi A.
Section of Neurological Rehabilitation, Department of Neurological
and Visual Sciences, Giambattista Rossi Hospital, University of Verona,
Verona, Italy. firstname.lastname@example.org
The effect of sleep on human motor cortical excitability was
investigated by evaluating the latency and amplitude of motor evoked
potentials in ten subjects using transcranial magnetic stimulation.
Motor evoked potentials and electroencephalographic data were recorded
simultaneously and analyzed. Recordings were performed before, during
and after a sleep period. A significant decrease in motor evoked
potentials amplitude and a slight change in motor evoked potentials
latency were noted in the recordings during the different sleep stages
with a return to baseline values on awakening. A decrease in motor
cortical excitability is suggested as explaining the effect of sleep.
Ross Fiziol Zh Im I M Sechenova. 2003 Jul;89(7):786-94.
Effect of impulse extrabroad-band electromagnetic radiation on electroencephalogram and sleep in laboratory animals.
Institute of Higher Nervous Activity and Neurophysiology, Russian Acad. Sci., Russia, 117865, Moscow.
1-hour exposure to ultra-short impulse low-frequency (6 Hz)
superbroad band electromagnetic radiation altered cortical EEG in rats
just after the exposure and increased the paradoxical sleep in rabbits
within 16-22 hours following the radiation.
Advances in Therapy, Volume 18, Number 4 / July, 2001
Impulse magnetic-field therapy for insomnia: A double-blind, placebo-controlled study.
Rainer B. Pelka1 , Christof Jaenicke2 and Joerg Gruenwald2
(1) Universität der Bundeswehr München, Werner-Heisenberg-Weg 39,
85577 Neubiberg/München, Germany. (2) PhytoPharm Consulting Institute
for Phytopharmaceuticals, Berlin, Germany
This 4-week double-blind, placebo-controlled study assessed the
efficacy of impulse magnetic-field therapy for insomnia. One hundred one
patients were randomly assigned to either active treatment (n = 50) or
placebo (n = 51) and allocated to one of three diagnostic groups: (1)
sleep latency; (2) interrupted sleep; or (3) nightmares. Efficacy
endpoints were intensity of sleep latency, frequency of interruptions,
sleepiness after rising, daytime sleepiness, difficulty with
concentration, and daytime headaches. In the active-treatment group, the
values of all criteria were significantly lower at study end (P <
.00001). The placebo group also showed significant symptomatic
improvement (P < .05), but the differences between groups were highly
significant (P < .00001). Seventy percent (n = 34) of the patients
given active treatment experienced substantial or even complete relief
of their complaints; 24% (n = 12) reported clear improvement; 6% (n = 3)
noted a slight improvement. Only one placebo patient (2%) had very
clear relief; 49% (n = 23) reported slight or clear improvement; and 49%
(n = 23) saw no change in their symptoms. No adverse effects of
treatment were reported.
J Sleep Res. 2002 Dec;11(4):289-95.
Electromagnetic fields, such as those from mobile phones, alter regional cerebral blood flow and sleep and waking EEG.
Huber R, Treyer V, Borbély AA, Schuderer J, Gottselig JM, Landolt HP, Werth E, Berthold T, Kuster N, Buck A, Achermann P.
Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland.
Usage of mobile phones is rapidly increasing, but there is limited
data on the possible effects of electromagnetic field (EMF) exposure on
brain physiology. We investigated the effect of EMF vs. sham control
exposure on waking regional cerebral blood flow (rCBF) and on waking and
sleep electroencephalogram (EEG) in humans. In Experiment 1, positron
emission tomography (PET) scans were taken after unilateral head
exposure to 30-min pulse-modulated 900 MHz electromagnetic field
(pm-EMF). In Experiment 2, night-time sleep was polysomnographically
recorded after EMF exposure. Pulse-modulated EMF exposure increased
relative rCBF in the dorsolateral prefrontal cortex ipsilateral to
exposure. Also, pm-EMF exposure enhanced EEG power in the alpha
frequency range prior to sleep onset and in the spindle frequency range
during stage 2 sleep. Exposure to EMF without pulse modulation did not
enhance power in the waking or sleep EEG. We previously observed EMF
effects on the sleep EEG (A. A. Borbély, R. Huber, T. Graf, B. Fuchs, E.
Gallmann and P. Achermann. Neurosci. Lett., 1999, 275: 207-210; R.
Huber, T. Graf, K. A. Cote, L. Wittmann, E. Gallmann, D. Matter, J.
Schuderer, N. Kuster, A. A. Borbély, and P. Achermann. Neuroreport,
2000, 11: 3321-3325), but the basis for these effects was unknown. The
present results show for the first time that (1) pm-EMF alters waking
rCBF and (2) pulse modulation of EMF is necessary to induce waking and
sleep EEG changes. Pulse-modulated EMF exposure may provide a new,
non-invasive method for modifying brain function for experimental,
diagnostic and therapeutic purposes.
Neurosci Biobehav Rev. 2001 May;25(3):235-60.
A detailed ethological analysis of the mouse open field test:
effects of diazepam, chlordiazepoxide and an extremely low frequency
pulsed magnetic field.
Choleris E, Thomas AW, Kavaliers M, Prato FS.
Room 9222D, Department of Psychology, Social Science Center, University of Western Ontario, London, Ontario, Canada N6A 5C2. email@example.com
The open field test (OFT) is a widely used procedure for examining
the behavioral effects of drugs and anxiety. Detailed ethological
assessments of animal behavior are lacking. Here we present a detailed
ethological assessment of the effects of acute treatment with the
benzodiazepines, diazepam (DZ, 1.5mg/kg) and chlordiazepoxide (CDP, 5.0
and 10.0mg/kg), as well as exposure to a non-pharmacological agent, a
specific pulsed extremely low frequency magnetic field (MAG) on open
field behavior. We examined the duration, frequency and time course of
various behaviors (i.e. exploration, walk, rear, stretch attend, return,
groom, sit, spin turn, jump and sleep) exhibited by male mice in
different regions of a novel open field. Both DZ and CDP consistently
reduced the typical anxiety-like behaviors of stretch attend and
wall-following (thigmotaxis), along with that of an additional new
measure: ‘returns’, without producing any overall effects on total
locomotion. The drugs also differed in their effects. CDP elicited a
shift in the locomotor pattern from a ‘high explore’ to a ‘high walk’,
while DZ mainly elicited alterations in sit and groom. The MAG treatment
was repeated twice with both exposures reducing horizontal and vertical
(rearing) activity and increasing grooming and spin turns. However, the
anxiety-like behaviors of stretch attend and return were marginally
reduced by only the first exposure. We conclude that a detailed
ethological analysis of the OFT allows not only the detection of
specific effects of drugs and non-pharmacological agents (i.e. pulsed
magnetic field) on anxiety-like behaviors, but also permits the
examination of non-specific effects, in particular those on general
Clin Neurophysiol. 2000 Nov;111(11):1936-41.
Nocturnal magnetic field exposure: gender-specific effects on heart rate variability and sleep.
OBJECTIVE: To determine if controlled exposure to power-frequency
magnetic fields alters heart rate variability (HRV) and polysomnographic
endpoints in healthy men (n=22) and women (n=24), 40-60 years of age.
METHODS: A randomized, double-blind, crossover design was used. Study
endpoints collected during all-night exposure to 60 Hz magnetic fields
at an occupational intensity (resultant flux density=28.3 microTesla,
microT) were compared to similar endpoints obtained under equivalent,
counterbalanced, no-exposure (< or =0.2 microT) control conditions.
RESULTS: Older men, but not women, exposed to the magnetic fields
showed power reductions in the LF band of the HRV frequency spectrum,
which is associated with sympathetically-mediated blood pressure and
thermoregulatory control (P<0.04). Older women, but not men, exposed
to the fields showed a pattern of disrupted sleep, with reductions in
the duration of REM sleep (P=0.03), and strong trends for reductions in
sleep efficiency (P=0.06) and total sleep time (P=0.06).
CONCLUSIONS: The gender-specific effects seen here with older
volunteers replicate the results of previous exposure studies with
younger men and women.
Neuroreport. 2000 Oct 20;11(15):3321-5.
Exposure to pulsed high-frequency electromagneticf field during waking affects human sleep EEG.
Huber R, Graf T, Cote KA, Wittmann L, Gallmann E, Matter D, Schuderer J, Kuster N, Borbely AA, Achermann P.
Institute of Pharmacology and Toxicology, University of Zurich, Switzerland.
The aim of the study was to investigate whether the electromagnetic
field (EMF) emitted by digital radiotelephone handsets affects brain
physiology. Healthy, young male subjects were exposed for 30 min to EMF
(900 MHz; spatial peak specific absorption rate 1 W/kg) during the
waking period preceding sleep. Compared with the control condition with
sham exposure, spectral power of the EEG in non-rapid eye movement sleep
was increased. The maximum rise occurred in the 9.75-11.25 Hz and
12.5-13.25 Hz band during the initial part of sleep. These changes
correspond to those obtained in a previous study where EMF was
intermittently applied during sleep. Unilateral exposure induced no
hemispheric asymmetry of EEG power. The present results demonstrate that
exposure during waking modifies the EEG during subsequent sleep. Thus
the changes of brain function induced by pulsed high-frequency EMF
outlast the exposure period.
Neurosci Lett. 1999 Nov 19;275(3):207-10.
Pulsed high-frequency electromagnetic field affects human sleep and sleep electroencephalogram.
Borbely AA, Huber R, Graf T, Fuchs B, Gallmann E, Achermann P.
To investigate whether the electromagnetic field (EMF) emitted by
digital radiotelephone handsets affects the brain, healthy, young
subjects were exposed during an entire night-time sleep episode to an
intermittent radiation schedule (900 MHz; maximum specific absorption
rate 1 W/kg) consisting of alternating 15-min on-15-min off intervals.
Compared with a control night with sham exposure, the amount of waking
after sleep onset was reduced from 18 to 12 min. Spectral power of the
electroencephalogram in non-rapid eye movement sleep was increased. The
maximum rise occurred in the 10-11 Hz and 13.5-14 Hz bands during the
initial part of sleep and then subsided. The results demonstrate that
pulsed high-frequency EMF in the range of radiotelephones may promote
sleep and modify the sleep EEG.
Human sleep under the influence of pulsed radiofrequency
electromagnetic fields: a polysomnographic study using standardized
To investigate the influence of radiofrequency electromagnetic fields
(EMFs) of cellular phone GSM signals on human sleep
electroencephalographic (EEG) pattern, all-night polysomnographies of 24
healthy male subjects were recorded, both with and without exposure to a
circular polarized EMF (900 MHz, pulsed with a frequency of 217 Hz,
pulse width 577 micros, power flux density 0.2 W/m2. Suppression of
rapid eye movement (REM) sleep as well as a sleep-inducing effect under
field exposure did not reach statistical significance, so that previous
results indicating alterations of these sleep parameters could not be
replicated. Spectral power analysis also did not reveal any alterations
of the EEG rhythms during EMF exposure. The failure to confirm our
previous results might be due to dose-dependent effects of the EMF on
the human sleep profile.
Effects of pulsed high-frequency electromagnetic fields on human sleep.
Mann K, Roschke J.
Department of Psychiatry, University of Mainz, Germany.
In the present study we investigated the influence of pulsed
high-frequency electromagnetic fields of digital mobile radio telephones
on sleep in healthy humans. Besides a hypnotic effect with shortening
of sleep onset latency, a REM suppressive effect with reduction of
duration and percentage of REM sleep was found. Moreover, spectral
analysis revealed qualitative alterations of the EEG signal during REM
sleep with an increased spectral power density. Knowing the relevance of
REM sleep for adequate information processing in the brain, especially
concerning mnestic functions and learning processes, the results
emphasize the necessity to carry out further investigations on the
interaction of this type of electromagnetic fields and the human
Neuropsychobiology. 1998 Nov;38(4):251-6.
No effects of pulsed high-frequency electromagnetic fields on heart rate variability during human sleep.
Mann K, Roschke J, Connemann B, Beta H.
Department of Psychiatry, University of Mainz, Germany.
The influence of pulsed high-frequency electromagnetic fields emitted
by digital mobile radio telephones on heart rate during sleep in
healthy humans was investigated. Beside mean RR interval and total
variability of RR intervals based on calculation of the standard
deviation, heart rate variability was assessed in the frequency domain
by spectral power analysis providing information about the balance
between the two branches of the autonomic nervous system. For most
parameters, significant differences between different sleep stages were
found. In particular, slow-wave sleep was characterized by a low ratio
of low- and high-frequency components, indicating a predominance of the
parasympathetic over the sympathetic tone. In contrast, during REM sleep
the autonomic balance was shifted in favor of the sympathetic activity.
For all heart rate parameters, no significant effects were detected
under exposure to the field compared to placebo condition. Thus, under
the given experimental conditions, autonomic control of heart rate was
not affected by weak-pulsed high-frequency electromagnetic fields.
Nocturnal exposure to intermittent 60 Hz magnetic fields alters human cardiac rhythm.
Heart rate variability (HRV) results from the action of neuronal and
cardiovascular reflexes, including those involved in the control of
temperature, blood pressure and respiration. Quantitative spectral
analyses of alterations in HRV using the digital Fourier transform
technique provide useful in vivo indicators of beat-to-beat variations
in sympathetic and parasympathetic nerve activity. Recently, decreases
in HRV have been shown to have clinical value in the prediction of
cardiovascular morbidity and mortality. While previous studies have
shown that exposure to power-frequency electric and magnetic fields
alters mean heart rate, the studies reported here are the first to
examine effects of exposure on HRV. This report describes three
double-blind studies involving a total of 77 human volunteers. In the
first two studies, nocturnal exposure to an intermittent, circularly
polarized magnetic field at 200 mG significantly reduced HRV in the
spectral band associated with temperature and blood pressure control
mechanisms (P = 0.035 and P = 0.02), and increased variability in the
spectral band associated with respiration (P = 0.06 and P = 0.008). In
the third study the field was presented continuously rather than
intermittently, and no significant effects on HRV were found. The
changes seen as a function of intermittent magnetic field exposure are
similar, but not identical, to those reported as predictive of
cardiovascular morbidity and mortality. Furthermore, the changes
resemble those reported during stage II sleep. Further research will be
required to determine whether exposure to magnetic fields alters stage
II sleep and to define further the anatomical structures where
field-related interactions between magnetic fields and human physiology
should be sought.
Ann Biomed Eng. 1996 May-Jun;24(3):424-9.
Electroencephalographic changes following low energy emission therapy.
Lebet JP, Barbault A, Rossel C, Tomic Z, Reite M, Higgs L, Dafni U, Amato D, Pasche B.
Symtonic SA., Renens, Switzerland.
Low energy emission therapy (LEET) is a novel approach to delivering
low levels of amplitude-modulated electromagnetic fields to the human
brain. The sleep electroencephalogram (EEG) effects of a 15-min LEET
treatment were investigated in a double-find cross-over study to assess
sleep induction. Fifty-two healthy volunteers were exposed to both
active and inactive LEET treatment sessions, with a minimum interval of 1
week between the two sessions. Baseline EEGs were obtained, and 15-min
posttreatment EEGs were recorded and analyzed according to the Loomis
classification. A significant increase in the duration of stage B1 sleep
(0.58 +/- 2.42 min [mean +/- SD], p = 0.046), decreased latency to the
first 10 sec epoch of sleep (-1.23 +/- 5.32 min, p = 0.051) and
decreased latency to sleep stage B2 (-1.21 +/- 5.25 min, p = 0.052) were
observed after active treatment. Additionally, establishment of slow
waves with progression from stages B to C was significantly more
pronounced after active LEET treatment (p = 0.040). A combined analysis
of these results with those of an identical study performed in Denver
showed that LEET had a significant effect on afternoon sleep induction
and maintenance with shorter sleep latencies (decreased latency to the
first 10 sec epoch of sleep; -1.00 +/- 5.51 min, p = 0.033; decreased
latency to sleep stage B2; -1.49 +/- 5.40 min, p = 0.003), an increased
duration of stage B2 (0.67 +/- 2.50 min, p = 0.003), an increase in the
total duration of sleep (0.69 +/- 4.21 min, p = 0.049), and a more
prominent establishment of slow waves with progression to a deeper sleep
stage (p = 0.006). It is concluded that the intermittent 42.7 HZ
amplitude modulation of 27.12-MHz electromagnetic fields results in EEG
changes consistent with shorter sleep latencies, longer sleep duration,
and deeper sleep in healthy subjects.
Sleep. 1996 May;19(4):327-36.
Effects of low energy emission therapy in chronic psychophysiological insomnia.
Pasche B, Erman M, Hayduk R, Mitler MM, Reite M, Higgs L, Kuster N, Rossel C, Dafni U, Amato D, Barbault A, Lebet JP.
Symtonic USA, Inc., New York, New York 10162, USA.
The treatment of chronic psychophysiological insomnia presents a
challenge that has not been met using currently available
pharmacotherapy. Low energy emission therapy (LEET) has been developed
as a potential alternative therapy for this disorder. LEET consists of
amplitude-modulated electromagnetic fields delivered intrabuccally by
means of an electrically conducting mouthpiece in direct contact with
the oral mucosa. The effect of LEET on chronic psychophysiological
insomnia was assessed with polysomnography (PSG) and sleep rating forms
on a total of 106 patients at two different centers. Active or inactive
LEET was administered for 20 minutes in late afternoon three times a
week for a total of 12 treatments. Primary efficacy endpoints evaluating
the results were changes from baseline in PSG-assessed total sleep time
(TST) and sleep latency (SL). Secondary endpoints were changes in sleep
efficiency (SE), sleep stages, and reports by the subjects of SL and
TST. There was a significant increase in TST as assessed by PSG between
baseline and post-treatment values for the active treatment group (76.0
+/- 11.1 minutes, p = 0.0001). The increase for the inactive treatment
group was not statistically significant. The TST improvement was
significantly greater for the active group when compared to the inactive
group (adjusted for baseline TST; p = 0.020. R1 = 0.20). There was a
significant decrease in SL as assessed by PSG between baseline and
post-treatment values for the active treatment group (-21.6 +/- 5.9
minutes, p = 0.0006), whereas the decrease noted for the inactive
treatment group was not statistically significant. The difference in SL
decrease between the two treatment groups was marginally significant
(adjusted for baseline SL and center, p = 0.068, R2 = 0.60). The number
of sleep cycles per night increased by 30% after active treatment (p =
0.0001) but was unchanged following inactive treatment. Subjects did not
experience rebound insomnia, and there were no significant side
effects. The data presented in this report indicate that LEET
administered for 20 minutes three times a week increased TST and reduced
SL in chronic psychophysiological insomnia. LEET is safe and well
tolerated and it effectively improved the sleep of chronic insomniacs
given 12 treatments over a 4-week period by increasing the number of
sleep cycles without altering the percentage of the various sleep stages
during the night. The therapeutic action of LEET differs from that of
currently available drug therapies in that the sleep pattern noted in
insomniacs following LEET treatment more closely resembles nocturnal
physiological sleep. This novel treatment may offer an attractive
alternative therapy for chronic insomnia.
Sleep inducing effect of low energy emission therapy.
Reite M, Higgs L, Lebet JP, Barbault A, Rossel C, Kuster N, Dafni U, Amato D, Pasche B.
Department of Psychiatry, University of Colorado Health Sciences Center, Denver.
The sleep inducing effect of a 15 min treatment with either an active
or an inactive Low Energy Emission Therapy (LEET) device emitting
amplitude-modulated electromagnetic (EM) fields was investigated in a
double-blind cross-over study performed on 52 healthy subjects. All
subjects were exposed to both active and inactive LEET treatment
sessions, with an interval of at least 1 week between the two sessions.
LEET consists of 27.12 MHz amplitude-modulated (sine wave) EM fields
emitted intrabuccally by means of an electrically conducting mouthpiece
in direct contact with the oral mucosa. The estimated local peak SAR is
less than 10 W/kg in the oral mucosa and 0.1 to 100 mW/kg in brain
tissue. No appreciable sensation is experienced during treatment, and
subjects are therefore unable to tell whether they are receiving an
active or an inactive treatment. In this study the active treatment
consisted of EM fields intermittently amplitude-modulated (sine wave) at
42.7 Hz for 3 s followed by a pause of 1 s during which no EM fields
were emitted. During the inactive treatment no EM fields were emitted.
Baseline EEGs were obtained and 15 min post-treatment EEGs were recorded
and analyzed according to the Loomis classification. A significant
decrease (paired t test) in sleep latency to stage B2 (-1.78 +/- 5.57
min, P = 0.013), and an increase in the total duration of stage B2 (1.15
+/- 2.47 min, P = 0.0008) were observed on active treatment as compared
with inactive treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
Application of variable magnetic fields in medicine–15 years experience.
[Article in Polish]
Sieron A, Cieslar G.
Katedra i Klinika Chorob Wewnetrznych, Angiologii i Medycyny Fizykalnej SAM, ul. Batorego 15, 41-902 Bytom.firstname.lastname@example.org
The results of 15-year own experimental and clinical research on
application of variable magnetic fields in medicine were presented. In
experimental studies analgesic effect (related to endogenous opioid
system and nitrogen oxide activity) and regenerative effect of variable
magnetic fields with therapeutical parameters was observed. The
influence of this fields on enzymatic and hormonal activity, free oxygen
radicals, carbohydrates, protein and lipid metabolism, dielectric and
rheological properties of blood as well as behavioural reactions and
activity of central dopamine receptor in experimental animals was
proved. In clinical studies high therapeutic efficacy of magnetotherapy
and magnetostimulation in the treatment of osteoarthrosis, abnormal
ossification, osteoporosis, nasosinusitis, multiple sclerosis,
Parkinson’s disease, spastic paresis, diabetic polyneuropathy and
retinopathy, vegetative neurosis, peptic ulcers, colon irritable and
trophic ulcers was confirmed.
Otolaryngol Pol. 1997;51 Suppl 25:299-302.
Clinical application of magnetic pulsing fields in paranasal sinusitis treatment.
[Article in Polish]
Kantor I, Szamborski R, Misztela A.
Kliniki Otolaryngologicznej Centralnego Szpitala Klinicznego WAM w Warszawie.
In this work were described advantages of the pulsating magnetic
fields in sinusitis treatment as independent and helping method.
Vestn Otorinolaringol. 1994 Mar-Apr;(2):27-31.
The clinico-immunological assessment of the efficacy of magnetic-laser therapy in patients with chronic maxillary sinusitis.
Magneto-laser therapy (80 Hz, 0.01 mW/cm2, 12 min) consisting of 10
sessions was performed in patients with maxillary sinusitis (MS).
Therapeutic action on the sinus mucosa was conducted through the sinus
anterior wall. The magneto-optic attachment coupled with semiconductor
laser in the Uzor apparatus was tightly pressed to the facial skin in
the sinus projection area. Positive clinical responses associated with
stimulation of neutrophil phagocytic activity and with a rise in the
portion of rosette-forming neutrophils and Ig A were achieved in 90% of
the patients with catarrhal MS and in 83% of those with purulent MS. A
1.5-2-year follow-up registered the effect persistence in 80% and 69%
of them, respectively. Compared to standard methods (microwave
therapy), magneto-laser treatment was superior both in short-term and
long-term effects obtained in purulent MS. The method can be applied in
mono- and polytherapy.
Vestn Otorinolaringol. 1991 May-Jun;(3):60-3.
Clinical effectiveness of magnetolaser therapy of vasomotor rhinitis.
[Article in Russian]
Magnet-laser therapy was used to treat vasomotor rhinitis in 90
patients. A constant magnet of 50 mT was applied to the exterior of the
nose, and an infrared laser beam was applied via a light-guide to the
reflexogenic zones of concha inferior. The radiation power density was 5
mW/cm2. The magnetic exposure time was 6-10 min and the laser exposure
time was 3-5 min for each half of the nose. The therapeutic course was
8-12 sessions. The therapeutic results depended on the type of
vasomotor rhinitis, clinical disorders, and duration of the disease.
Good results were seen in 84 (93.3%) patients out of 90 cases. Stable
remission was recorded in 61.1% patients, significant improvement of
clinical manifestations of vasomotor rhinitis was observed in 32.2%
cases. Best results were reported in patients with autonomic forms of
vasomotor rhinitis who suffered from short-term disease and
The new magnetic therapy TAMMEF in the treatment of simple shoulder pain.
[Article in Italian]
Battisti E, Bianciardi L, Albanese A, Piazza E, Rigato M, Galassi G, Giordano N.
Centro TAMMEF, Sezione di Fisica Medica, Dipartimento di Fisica, Università degli Studi di Siena, Italia.
AIMS: Numerous studies have demonstrated the utility of extremely low
frequencies (ELF) electromagnetic fields in the treatment of pain.
Moreover, the effects of these fields seems to depend on their
respective codes (frequency, intensity, waveform). In our study we want
to assess the effects of the TAMMEF (Therapeutic Application of a
Musically Modulated Electromagnetic Field) system, whose field is
piloted by a musical signal and its parameters (frequency, intensity,
waveform) are modified in time, randomly varying within the respective
ranges, so that all possible codes can occur during a single
PATIENTS AND METHODS: Sixty subjects, affected by shoulder
periarthritis were enrolled in the study and randomly divided into three
groups of 20 patients each: A exposed to TAMMEF, B exposed to ELF, C
exposed to a simulated field. All subjects underwent a cycle of 15 daily
sessions of 30 minutes each and a clinical examination upon enrollment,
after 7 days of therapy, at the end of the cycle and at a follow-up 30
RESULTS: All the patients of groups A and B completed the therapy
without the appearance of side effects: they presented a significant
improvement of the subjective pain and the functional limitation, which
remained stable at the follow-up examination. In group C, there was no
improvement of the pain symptoms or articular functionality.
CONCLUSIONS: This study suggests that the TAMMEF system is
efficacious in the control of pain symptoms and in the reduction of
functional limitation in patients with shoulder periarthritis. Moreover,
the effects of the TAMMEF system cover those produced by the ELF field.
Radiologe. 2004 Jun;44(6):597-603.
Conservative treatment and rehabilitation of shoulder problems
The shoulder joint has an important influence on arm- and hand
function. Therefore, activities of daily living, working and leisure
time can be negatively influenced by diseases of the shoulder joint.
Problems of the shoulder joint can be induced by muscular dysbalance and
poor body posture. There is a strong relationship between shoulder
function and body posture. Conservative treatment and rehabilitation of
the shoulder joint aims at improving the local dysfunction of the
shoulder joint as well as at improving function and social
participation. Antiinflammatory and pain medication, exercise,
occupational, electro-, ultrasound and shock wave therapy, massage,
thermotherapy and pulsed electromagnetic fields are used as conservative
treatments. Exercise therapy aims at improving muscular performance,
joint mobility and body posture. Occupational therapy aims at improving
functional movements for daily living and work. Electrotherapy is
primarily used to relieve pain. Shock wave and ultrasound therapy proved
to be an effective treatment for patients with calcific tendinitis. The
subacromial impingement syndrome can be effectively treated by
J Spinal Cord Med. 2004;27(2):138-42.
Treatment of myofascial shoulder pain in the spinal cord injured population using static magnetic fields: a case series.
Panagos A, Jensen M, Cardenas DD.
Department of Rehabilitation Medicine, University of Washington, Seattle, Washington 98195-6490, USA. email@example.com
OBJECTIVE: Magnetic therapy has been used in the treatment of a wide
variety of chronic pain syndromes. It has not been studied in the
treatment of myofascial shoulder pain in persons with spinal cord injury
(SCI). Because this type of pain is commonly refractory to traditional
therapy, alternative treatments often are considered. The primary
objective is to determine whether myofascial shoulder pain in persons
with SCI can be temporarily ameliorated with static magnetic fields.
DESIGN: Case series.
SETTING: Clinic of a university hospital system.
\PARTICIPANTS: A volunteer sample of 8 participants with SCI; 3
women, 5 men; mean age = 45 years; mean duration of injury = 12.3 years.
INTERVENTIONS: Placement of a commercially available magnet with a
static magnetic field of 500 gauss on the affected shoulder for 1 hour.
MAIN OUTCOME MEASURES: Pretreatment and posttreatment scores on the
short-form McGill Pain Questionnaire and pressure algometry were
RESULTS: The short-form McGill Pain Questionnaire descriptors
demonstrated significant decreases: stabbing, 0.75 +/- 0.71 (P <
0.02); sharp, 0.50 +/- 0.53 (P < 0.033); and tender, 0.88 +/- 0.83 (P
< 0.021). They also demonstrated a significant decrease in the
present pain intensity of 0.63 +/- 0.52 (P < 0.011). Participants
demonstrated a nonsignificant decrease of 0.813 +/- 0.998 (P < 0.55)
on the visual analog scale. Pressure algometry was nonsignificant with a
difference of 0.062 +/- 1.17 (P < 0.885).
CONCLUSION: Static magnetic fields may decrease the sensory
dimensions and intensity of myofascial shoulder pain in persons with
Cochrane Database Syst Rev. 2003;(2):CD004258.
Physiotherapy interventions for shoulder pain
Green S, Buchbinder R, Hetrick S.
Australasian Cochrane Centre, Monash University, Australasian Cochrane Centre, Locked Bag 29, Clayton, Victoria, Australia. firstname.lastname@example.org
BACKGROUND: The prevalence of shoulder disorders has been reported to
range from seven to 36% of the population (Lundberg 1969) accounting
for 1.2% of all General Practitioner encounters in Australia (Bridges
Webb 1992). Substantial disability and significant morbidity can result
from shoulder disorders. While many treatments have been employed in the
treatment of shoulder disorders, few have been proven in randomised
controlled trials. Physiotherapy is often the first line of management
for shoulder pain and to date its efficacy has not been established.
This review is one in a series of reviews of varying interventions for
shoulder disorders, updated from an earlier Cochrane review of all
interventions for shoulder disorder.
OBJECTIVES: To determine the efficacy of physiotherapy interventions
for disorders resulting in pain, stiffness and/or disability of the
SEARCH STRATEGY: MEDLINE, EMBASE, the Cochrane Clinical Trials
Regiter and CINAHL were searched 1966 to June 2002. The Cochrane
Musculoskeletal Review Group’s search strategy was used and key words
gained from previous reviews and all relevant articles were used as text
terms in the search.
SELECTION CRITERIA: Each identified study was assessed for possible
inclusion by two independent reviewers. The determinants for inclusion
were that the trial be of an intervention generally delivered by a
physiotherapist, that treatment allocation was randomised; and that the
study population be suffering from a shoulder disorder, excluding trauma
and systemic inflammatory diseases such as rheumatoid arthritis.
DATA COLLECTION AND ANALYSIS: The methodological quality of the
included trials was assessed by two independent reviewers according to a
list of predetermined criteria, which were based on the PEDro scale
specifically designed for the assessment of validity of trials of
physiotherapy interventions. Outcome data was extracted and entered into
Revman 4.1. Means and standard deviations for continuous outcomes and
number of events for binary outcomes were extracted where available from
the published reports. All standard errors of the mean were converted
to standard deviation. For trials where the required data was not
reported or not able to be calculated, further details were requested
from first authors. If no further details were provided, the trial was
included in the review and fully described, but not included in the
meta-analysis. Results were presented for each diagnostic sub group
(rotator cuff disease, adhesive capsulitis, anterior instability etc)
and, where possible, combined in meta-analysis to give a treatment
effect across all trials.
MAIN RESULTS: Twenty six trials met inclusion criteria.
Methodological quality was variable and trial populations were generally
small (median sample size = 48, range 14 to 180). Exercise was
demonstrated to be effective in terms of short term recovery in rotator
cuff disease (RR 7.74 (1.97, 30.32), and longer term benefit with
respect to function (RR 2.45 (1.24, 4.86). Combining mobilisation with
exercise resulted in additional benefit when compared to exercise alone
for rotator cuff disease. Laser therapy was demonstrated to be more
effective than placebo (RR 3.71 (1.89, 7.28) for adhesive capsulitis but
not for rotator cuff tendinitis. Both ultrasound and pulsed
electromagnetic field therapy resulted in improvement compared to
placebo in pain in calcific tendinitis (RR 1.81 (1.26, 2.60) and RR 19
(1.16, 12.43) respectively). There is no evidence of the effect of
ultrasound in shoulder pain (mixed diagnosis), adhesive capsulitis or
rotator cuff tendinitis. When compared to exercises, ultrasound is of no
additional benefit over and above exercise alone. There is some
evidence that for rotator cuff disease, corticosteroid injections are
superior to physiotherapy and no evidence that physiotherapy alone is of
benefit for Adhesive Capsulitis
REVIEWER’S CONCLUSIONS: The small sample sizes, variable
methodological quality and heterogeneity in terms of population studied,
physiotherapy intervention employed and length of follow up of
randomised controlled trials of physiotherapy interventions results in
little overall evidence to guide treatment. There is evidence to support
the use of some interventions in specific and circumscribed cases.
There is a need for trials of physiotherapy interventions for specific
clinical conditions associated with shoulder pain, for shoulder pain
where combinations of physiotherapy interventions, as well as,
physiotherapy interventions as an adjunct to other, non physiotherapy
interventions are compared. This is more reflective of current clinical
practice. Trials should be adequately powered and address key
methodological criteria such as allocation concealment and blinding of
J Med Eng Technol. 2002 Nov-Dec;26(6):253-8.
Comparison between the analgesic and therapeutic effects of a
musically modulated electromagnetic field (TAMMEF) and those of a 100 Hz
electromagnetic field: blind experiment on patients suffering from
cervical spondylitis or shoulder periarthritis.
Rigato M, Battisti E, Fortunato M, Giordano N.
Department of Physics, Section of Medical Physics University of Sienna, Italy. email@example.com
The analgesic-therapeutic efficacy and tolerability of a
low-frequency electromagnetic field (ELF), modulated at a frequency of
100 Hz with a sinusoidal waveform and mean induction of a few gauss, has
been demonstrated by the authors in numerous previous studies of
various hyperalgic pathologies, particularly of the locomotor apparatus.
In the present study, the authors tested a new type of all-inclusive
field, denoted TAMMEF, whose parameters (frequency, intensity, waveform)
are modified in time, randomly varying within the respective ranges, so
that all the possible codes can occur during a single application. For
the comparison, 150 subjects (118 women and 32 men, between 37 and 66
years of age) were enrolled. They were affected by cervical spondylosis
(101 cases) or shoulder periarthritis (49 cases). Unbeknownst to them,
they were randomly divided into three groups of 50 subjects. One group
was exposed to the new TAMMEF, another group to the usual ELF, and the
third group to simulated treatment. The results show that the effects of
the new TAMMEF therapy are equivalent to those obtained with the ELF.
The combined use of electromagnetic decimeter waves and deresinated
naphthalan in patients with vertebrogenic humeroscapular periarthrosis
(its experimental and clinical validation).
[Article in Russian]
Musaev AV, Guse?nova SG, Mamedov AP.
Physicochemical and experimental studies on pond snail neuron were
made to validate combined or simultaneous usage of decimeter microwaves
and deresinified naphthalane. Clinical and neurophysiological trials in
133 patients with vertebrogenic scapulohumeral periarthritis revealed
that the above treatment is clinically beneficial and corrects
functional activity of segmental-peripheral neuromotor system.
The cerebral hemodynamics in patients with humeroscapular
periarthritis under the influence of decimeter waves and deresinated
[Article in Russian]
Musaev AV, Guse?nova SG.
110 patients with scapulohumeral periarthritis of vertebrogenic
origin were exposed to decimetric waves or received salt-free
naphthalan. There were also patients who got combined treatment with
decimetric waves and salt-free naphthalan. REG recorded positive shifts
in cerebral hemodynamics due to these factors utilization.
Arch Phys Med Rehabil. 1991 Apr;72(5):284-7.
Electromagnetic treatment of shoulder periarthritis: a randomized
controlled trial of the efficiency and tolerance of magnetotherapy.
Leclaire R, Bourgouin J.
Rehabilitation Medicine Service, Notre Dame Hospital, Montreal, Quebec, Canada.
The potential benefit of magnetotherapy was investigated in 47
consecutive outpatients with periarthritis of the shoulder. Using a
controlled triple-blind study design, one group of patients received hot
pack applications and passive manual stretching and pulley exercises;
the other group received the same therapy plus magnetotherapy. Treatment
was administered three times a week. For a maximum of three months, a
standardized treatment protocol was used. There was no significant
improvement in pain reduction or in range of motion with electromagnetic
field therapy. After 12 weeks of therapy, the patients who received
magnetotherapy showed mean pain scores of 1.5 (+/- .61 SD) at rest, 2.2
(+/- .76 SD) on movement, and 1.9 (+/- .94 SD), on lying, compared to
scores for the control group of 1.4 (+/- .65 SD), 2.2 (+/- .7 SD), and
1.9 (+/- .95 SD), respectively. Linear pain scale scores improved from
71 to 21 for both groups. At 12 weeks the gain in range of motion was
mean 109 degrees +/- 46.8 in patients receiving electromagnetic field
therapy, compared to 122 degrees +/- 33.4 for the controls (not
significant). At entry, the functional handicap score was 53.5 for both
groups. At 12 weeks, it was 24 for the magnetotherapy group and 17 for
the control group (difference not significant). In conclusion, this
study showed no benefit from magnetotherapy in the pain score, range of
motion, or improvement of functional status in patients with
periarthritis of the shoulder.
Lancet. 1984 Mar 31;1(8379):695-8.
Pulsed electromagnetic field therapy of persistent rotator cuff tendinitis. A double-blind controlled assessment.
Binder A, Parr G, Hazleman B, Fitton-Jackson S.
The value of pulsed electromagnetic fields (PEMF) for the treatment
of persistent rotator cuff tendinitis was tested in a double-blind
controlled study in 29 patients whose symptoms were refractory to
steroid injection and other conventional conservative measures. The
treated group (15 patients) had a significant benefit compared with the
control group (14 patients) during the first 4 weeks of the study, when
the control group received a placebo. In the second 4 weeks, when all
patients were on active coils, no significant differences were noted
between the groups. This lack of difference persisted over the third
phase, when neither group received any treatment for 8 weeks. At the end
of the study 19 (65%) of the 29 patients were symptomless and 5 others
much improved. PEMF therapy may thus be useful in the treatment of
severe and persistent rotator cuff and possibly other chronic tendon
Vopr Onkol. 1988;34(2):213-7.
Use of physical therapy measures in the rehabilitation of patients having undergone radical mastectomy.
[Article in Russian]
Gerasimenko VN, Voinarevich AO, Grushina TI.
Low-frequency electrotherapy, magnetotherapy, massage, exercise
therapy and drugs were used in 90 patients who after radical treatment
for breast cancer suffered pain and limited mobility in the shoulder
joint. These procedures were intended to treat said complications, to
normalize reflexes and to cut down the period of rehabilitative therapy.
The treatment proved effective. No untoward effects on the course of
the disease were observed within the first 3-5 years.
Pulsed magnetic fields. Observations in 353 patients suffering from chronic pain.
[Article in Italian]
Di Massa A, Misuriello I, Olivieri MC, Rigato M.
Three hundred-fifty-three patients with chronic pain have been
treated with pulsed electromagnetic fields. In this work the Authors
show the result obtained in the unsteady follow-up (2-60 months). The
eventual progressive reduction of benefits is valued by Spearman’s test.
We noted the better results in the group of patients with post-herpetic
pain (deafferentation) and in patients simultaneously suffering from
neck and low back pain.