n

Feature Article

Effects of Pulsed Electromagnetic Field

Frequencies on the Osteogenic Differentiation
of Human Mesenchymal Stem Cells
Fei Luo, PhD; Tianyong Hou, PhD; Zehua Zhang, PhD; Zhao Xie, PhD; Xuehui Wu, PhD;
Jianzhong Xu, PhD

abstract
Full article available online at Healio.com/Orthopedics. Search: 20120327-11

The purpose of this study was to evaluate the effect of different frequencies of pulsed
electromagnetic fields on the osteogenic differentiation of human mesenchymal stem
1
cells. Third-generation human mesenchymal stem cells were irradiated with different
Figure 1: Alkaline phosphatase (ALP) activity in
frequencies of pulsed electromagnetic fields, including 5, 25, 50, 75, 100, and 150 human mesenchymal stem cells treated with dif-
Hz, with a field intensity of 1.1 mT, for 30 minutes per day for 21 days. Changes in hu- ferent frequencies of pulsed electromagnetic fields
man mesenchymal stem cell morphology were observed using phase contrast micros- (PEMF).
copy. Alkaline phosphatase activity and osteocalcin expression were also determined
to evaluate human mesenchymal stem cell osteogenic differentiation.

Different effects were observed on human mesenchymal stem cell osteoblast induc-
tion following exposure to different pulsed electromagnetic field frequencies. Levels
of human mesenchymal stem cell differentiation increased when the pulsed electro-
magnetic field frequency was increased from 5 hz to 50 hz, but the effect was weaker
when the pulsed electromagnetic field frequency was increased from 50 Hz to 150 2
hz. The most significant effect on human mesenchymal stem cell differentiation was Figure 2: Osteocalcin content of mesenchymal
stem cells treated with different frequencies of
observed at of 50 hz.
pulsed electromagnetic fields (PEMF).

The results of the current study show that pulsed electromagnetic field frequency is an
important factor with regard to the induction of human mesenchymal stem cell differ-
entiation. Furthermore, a pulsed electromagnetic field frequency of 50 Hz was the most
effective at inducing human mesenchymal stem cell osteoblast differentiation in vitro.

Drs Luo, Hou, Zhang, Xie, Wu, and Xu are from the Department of Orthopaedics, South-West
Hospital, The Third Military University, Chongqing, China.
Drs Luo, Hou, Zhang, Xie, Wu, and Xu have no relevant financial relationships to disclose.
This study was supported by the National High Technology Research and Development Program
(863 Project, Grant No.: 2006AA02A122) and the Chongqing Natural Sciences Foundation (Grant No.:
2010JJ0379).
Correspondence should be addressed to: Jianzhong Xu, PhD, Department of Orthopaedics,
South-West Hospital, The Third Military University, No. 29 Gaotanyan Rd, Chongqing 400038, China
(xjzslw@hotmail.com).
doi: 10.3928/01477447-20120327-11

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B
one tissue engineering involves the
use of tissue engineering methods
to promote the generation and dif-
ferentiation of bony cells. Using this ap-
proach, new approaches can be explored
to repair long segmental bone defects.
Mesenchymal stem cells are one of the
most widely used types of stem cells for
bone tissue engineering. Following in vitro
amplification using chemical induction to
induce mesenchymal stem cell osteoblast
differentiation, the resulting osteoblasts
and support material can be combined to
generate engineered bone tissue.
In 1977, Bassett et al1 developed
pulsed electromagnetic field therapy and
successfully treated a group of patients
with bone nonunion. Since then, several
studies have reported that pulsed electro-
magnetic fields accelerate the speed of
mesenchymal stem cell amplification and
osteoblast induction, growth factor secre-
tion, and extracellular matrix synthesis;
pulsed electromagnetic fields may also
promote bone reconstruction and acceler-
ate bone growth.2-7 These findings suggest
that further studies of the effects of pulsed
electromagnetic fields on mesenchymal
stem cell osteoblast differentiation and
bone tissue engineering are needed.8-13
These previous studies were performed
using a specific pulsed electromagnetic
field frequency during the course of the
experiments; notably, the frequency is
an important indicator for determining
the biological effects of pulsed electro-
magnetic fields. In the current study, we
investigated the effects of different pulsed
electromagnetic field frequencies on mes-
enchymal stem cell induction in vitro
with the overall goal of providing a new
approach for mesenchymal stem cell in-
duction in vitro and experimental support
for a new type of bioreactor and the clini-
cal application of pulsed electromagnetic
fields to promote bone fracture healing.
The results of the current study should
provide a strong theoretical foundation
for future pulsed electromagnetic field-
related research.
APRIL 2012 | Volume 35 • Number 4
Osteogenic Differentiation of Human Mesenchymal Stem Cells | Luo et al
Materials and Methods
The following solutions, kits, and
equipment were used in the experiments
described herein: Percoll solution (Sigma-
Aldrich, St Louis, Missouri), HyClone
Dulbecco’s Modified Eagle Medium:
Nutrient Mixture F-12 (DMEM/F12) (1:1)
(Thermo Fisher Scientific Inc, Waltham,
Massachusetts), HyClone fetal calf serum
(Thermo Fisher Scientific Inc), alkaline
phosphatase test kit (Nanjing Jiancheng
Bioengineering Institute, Nanjing,
China), osteocalcin radioimmunity kit
(Beijing China Atomic Research Institute,
Beijing, China), mouse-anti-human os-
teocalcin antibody (Boshide, Wuhan,
China), ultraviolet/visible spectrophotom-
eter (UV751GD; the Shanghai Equipment
Con-factory, Shanghai, China), and con-
trollable pulsed electromagnetic field ac-
tivator (Logistical Engineering University
of PLA, Chongqing, China).
Separation and Cultivation of Human
Mesenchymal Stem Cells
Bony marrow (5-10 mL) from both
sides of the iliac crest was collected from
healthy volunteers in a sterilized environ-
ment using heparin as the anticoagulant.
Percoll solution (1.073 g/mL) was used
to isolate the bony marrow. A total of
53105 cells/cm2 were inoculated into
a culture flask containing DMEM/F12
supplemented with 15% fetal calf se-
rum, and the cells were incubated under
saturated humidity. After 3 generations,
the cells were digested and centrifuged
to generate a cell suspension, which was
divided into 6 experimental groups and 1
control group.
Treatment of Human Mesenchymal
Stem Cells With Different Pulsed
Electromagnetic Field Frequencies
The 6 experimental cell groups were
treated with a Helmholtz coil with pulsed
electromagnetic fields, which was a dual
coil with a 10-cm space between the 2
coils. The Hall effect was used to mea-
sure the magnetic reactor to confirm that
the field’s homogeneity and stabilization
were acceptable. The coil was placed into
the cell incubator, and the field was set
to different pulsed electromagnetic field
frequencies, including 5, 25, 50, 75, 100,
and 150 Hz, each with a field intensity
of 1.1 mT, for 30 minutes per day for 21
days. The control groups of cells were
incubated under the same experimental
conditions with no exposure to the pulsed
electromagnetic fields.
Morphological Observations
Changes in cell morphology under the
different growth conditions were observed
using an inverted microscope. Photos of
cell morphology were taken on days 1, 3,
5, and 7 after serial subcultivation.
Ultramicrostructural Observations
Following exposure to pulsed electro-
magnetic fields, when the cells covered
80% of the area of the culture bottle, they
were digested, centrifuged, and washed
twice with phosphate buffered saline. The
cells were immobilized with 2.5% glutar-
aldehyde and subjected to gradient ace-
tone anhydration, followed by embedding
in epoxy resin. Extra thin sections were
generated and stained with uranyl acetate
and lead citrate. Transmission electron
microscopy was used to observe the ul-
tramicrostructure of the experimental and
control groups.
Alkaline Phosphoric Acid Enzyme Staining
When using Gomori stain, cells posi-
tive for alkaline phosphatase expression
are brownish-black. Following 6 days of
pulsed electromagnetic field stimulation,
the human mesenchymal stem cells were
removed from the incubator, rinsed with
phosphate buffered saline, fixed in ice-
cold acetone (220°C), incubated for 3
hours at 37°C in phosphoric acid enzyme
liquids, washed with distilled water, and
subjected to ammonium sulfide process-
ing using 2% nitric acid and 1% cobalt,
1 by 1 in order, followed by staining with
neutral and red contrast dye.
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n Feature Article
Alkaline Phosphatase Activity
Measurements
After 3, 6, 9, 12, and 15 days of pulsed
electromagnetic field exposure, cells
grown in 12-well flat-bottomed plates
from each group were digested, centri-
fuged, collected, and washed twice with
phosphate buffered saline. The cell den-
sity was adjusted to 13105 cells/mL in
0.5% Triton-X100 (Sigma-Aldrich, St
Louis, Missouri), and the cells were in-
cubated at 4°C for 12 hours. Intermittent
ultrasound exposure on ice was used to
break up the cells and ensure complete
cell lysis (150 W, 5 s). The absorbance of
50 µL of the cell lysate was examined at
520 nm to determine the A value, and a
standard formula was used to calculate al-
kaline phosphatase activity.
Collagen Type I and Osteocalcin
Immunostaining
Coverslips were removed after 12
days, followed by phosphate buffered sa-
line rinses, fixation with 95% ethanol, and
treatment with 3% hydrogen dioxide, fol-
lowed by rinses with distilled water and
incubation with 5% normal goat serum.
Cells were subjected to immunohisto-
chemical staining using the streptavidin-
biotin peroxidase complex method with a
rat anti-collagen type I and anti-osteocalcin
monoclonal antibody.
Quantification of Osteocalcin Levels
At 7, 14, and 21 days, 50 mL were re-
moved from each culture, followed by
digestion with diastase vera. Following
centrifugation, a total of 13106 cells were
resuspended in 1 mL of a 1:1 admixture of
phosphate buffered saline and Triton-X100,
followed by incubation at 4°C overnight.
The resulting samples were sent to the
Atomic Medical Department of Southwest
Hospital to measure the osteocalcin content
in the cells using a radiographic-immunity
method based on the radiographic-immunity
competitive binding principle. In this assay,
125
I-marked osteocalcin competes with free
osteocalcin for binding to the osteocalcin
e528
antibody, and a Y counter is used to mea-
sure the sediment counts per minute, which
is used to determine the osteocalcin content
of the sample according to a standard curve.
Staining With Alizarin Monosulfonate
Calcium Dye
After the appearance of oval-shaped
nodules in the 6-well culture plate, the
coverslips were removed, rinsed with
phosphate buffered saline, fixed with 75%
alcohol for 30 minutes, stained with 2%
alizarin monosulfonate, dehydrated us-
ing an alcohol gradient, made transparent
with xylene, and mounted using neutral
resin.
Staining With Von Kossa Calcium Dye
After oval-shaped nodules appeared
in the 6-well culture plate, the coverslips
were removed, rinsed with phosphate
buffered saline, fixed with 75% alcohol,
immersed in 2% silver nitrate aqua, ex-
posed to ultraviolet light for 30 minutes,
washed with distilled water, stained with
neutral red dye, treated with an alcohol
gradient, made transparent with dimethyl
benzene, and sealed using a neutral resin.
Staining With Achromycin Dye
After oval-shaped nodules appeared
in the 6-well culture plate, the coverslips
were removed, treated with 0.1 mg/mL
achromycin culture solution for 30 min-
utes, incubated with a common culture
solution for 30 minutes, rinsed with phos-
phate buffered saline, fixed with 75% al-
cohol, and observed using a fluorescence
microscope.
Statistical Analyses
SPSS version 11.0 software (IBM,
Armonk, New York) was used for statis-
tical analyses. Self-paired t analysis was
used to analyze each set of experimental
data. One-factor analysis of variance was
used to analyze different groups stimulat-
ed with different pulsed electromagnetic
fields, and F analysis was used to com-
plete the statistical analysis.
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Results
Inverted Phase Contrast Microscope
Observations
At 4 hours post-inoculation, the third-
generation human mesenchymal stem cells
were adhered to the plates, and they began
to divide and grow 24 hours after they ad-
hered. After 3 days, pulsed electromagnetic
field group cells were larger than control
group cells, and their morphology contin-
ued to change; the cells eventually became
triangular and polygonal in shape, scales
formed, and the cytoplasm contained abun-
dant matrix and granular material. No ob-
vious differences were observed in the ap-
pearances of pulsed electromagnetic field
group cells compared with control group
cells. Over time, the cells became confluent
and began to exhibit overlapping growth.
After 3 weeks, mineralization of the matrix
led to the fusion of the oval-shaped calci-
fied nodules. Around the nodules, the cells
were distributed in an array-like pattern.
Ultramicrostructural Observations
Transmission electron microscopy
analysis of human mesenchymal stem cells
in the pulsed electromagnetic field group
showed that they were more differentiated
than the control group cells. The nuclear
matrix ratio of pulsed electromagnetic field
group cells was lower than that of control
group cells. The cytoplasm of pulsed elec-
tromagnetic field group cells contained
abundant organelles, including mitochon-
dria, rough endoplasmic reticulum, and
Golgi bodies. Control group human mes-
enchymal stem cells were more immature
with larger nuclei, a similar nuclear-matrix
ratio, and fewer organelles.
Alkaline Phosphatase Staining
Cells that were not stimulated with
pulsed electromagnetic fields were nega-
tive for alkaline phosphatase expression,
whereas cells subjected to pulsed elec-
tromagnetic field stimulation were highly
positive for alkaline phosphatase expres-
sion, with brownish-black cytoplasm and
black granulated precipitates.

Figure 1: Alkaline phosphatase (ALP) activity in human mesenchymal stem cells treated with different
frequencies of pulsed electromagnetic fields (PEMF).
Figure 2: Osteocalcin content of mesenchymal stem cells treated with different frequencies of pulsed
electromagnetic fields (PEMF).
Alkaline Phosphatase Activity
Measurements
Pulsed electromagnetic field group
cells exhibited stronger alkaline phospha-
tase activity than control group cells start-
ing on the third day. As time progressed,
alkaline phosphatase activity was higher in
pulsed electromagnetic field group cells,
reaching a peak at 12 days. By day 15,
no great change had occurred, and alka-
line phosphatase activity remained stable,
although it remained significantly higher
than that of the control groups (Figure 1).
Furthermore, different frequencies were
associated with different alkaline phos-
APRIL 2012 | Volume 35 • Number 4
Osteogenic Differentiation of Human Mesenchymal Stem Cells | Luo et al
1 groups expressed significantly higher lev-
2 stimulation for 21 days, matrix mineral-
phatase activity levels. Alkaline phospha-
tase activity in the 50-Hz pulsed electro-
magnetic field group was higher than that
of the other pulsed electromagnetic field
groups on days 9, 12, and 15.
Collagen Type I and Osteocalcin
Immunohistochemical Staining
After 12 days of pulsed electromagnetic
field stimulation, human mesenchymal
stem cells subjected to collagen type I im-
munohistochemical staining exhibited high
levels of yellow granulation and were high-
ly positive. In contrast, no yellow granula-
tion was detected in the control group cells.
Osteocalcin immunohistochemical
staining of human mesenchymal stem cells
showed that they exhibited high levels of
yellow granulation and were highly posi-
tive. In contrast, no yellow granulation was
detected in the control group cells.
Quantification of Osteocalcin Levels
On day 7, cells in all of the different
pulsed electromagnetic field groups ex-
pressed osteocalcin slightly, whereas the
control group cells did not. On day 14,
cells in the pulsed electromagnetic field
els of osteocalcin; although cells in the
control group also expressed osteocalcin
on day 14, the osteocalcin levels of con-
trol cells were significantly lower than
the pulsed electromagnetic field groups.
Osteocalcin levels varied among the
pulsed electromagnetic field groups treat-
ed with different frequencies, and osteo-
calcin levels in the pulsed electromagnetic
field group treated with a frequency of 50
Hz were higher than the others on day 21
(Figure 2).
Alizarin Monosulfonate Calcium Nodule
Staining
Following pulsed electromagnetic field
ization led to the formation of oval-shaped
calcium nodules, which stained orange
around the nodule with alizarin monosul-
fonate calcium, and the cells around the
nodule were distributed in an array-like
pattern. In contrast, no calcium nodules
were present in the control group cells.
Von Kossa Calcium Dye Staining
On day 21, cells stimulated with
pulsed electromagnetic fields began to
exhibit oval-shaped calcium nodules.
The nodules changed from transparent to
opaque, and then turned into a black mass.
After staining with a calcium dye, mas-
sive black crystal deposits were identi-
fied, which were not present in the control
group cells. The results of this analysis
showed that pulsed electromagnetic field
e529
n Feature Article
treatment of human mesenchymal stem
cells can stimulate bone induction.
Achromycin Fluorescent Labeling
Cells stimulated by pulsed electromag-
netic fields began to exhibit oval-shaped
calcium nodules after 3 weeks in culture,
and the nodules increased in size and
changed from transparent to an opaque-
black mass. After achromycin fluorescent
labeling, the nodules appeared golden,
and the light was equal to the size and
shade under the inverted microscope. In
contrast, no calcium nodules were present
in the control group.
Discussion
The construction of tissue-engineered
bone requires the establishment of seed cells
with strong characteristics. Mesenchymal
stem cells, which are derived from meso-
derm, are adult stem cells that have strong
reproductive activity and are multipotent.
The differentiation of mesenchymal
stem cells into osteoblasts can be induced
by cytokines, hormones, physical meth-
ods, and by many other factors. Pulsed
electromagnetic field is a method used to
treat bony delayed union and bony non-
union. Pulsed electromagnetic field treat-
ment is associated with satisfactory effects,
and has recently been used as a therapy to
treat inborn bone defects, bone necrosis,
bone transplantation, and spinal fusion.1-6
Previous studies have shown that pulsed
electromagnetic fields can mediate extra-
cellular matrix synthesis, increase alkaline
phosphatase expression by osteoblasts, and
promote the secretion of osteocalcin and
collagen.6,14,15 Mesenchymal stem cells
are an important ancestor cell in the pro-
cess of bone growth. Based on these find-
ings, some researchers have proposed that
pulsed electromagnetic fields could induce
a similar biological effect on human mes-
enchymal stem cells and promote bone for-
mation.8-13 This proposal has gained sup-
port based on earlier studies performed in
our laboratory. However, prior to the cur-
rent study, whether different pulsed elec-
e530
tromagnetic field frequencies exhibit a dif-
ferential effect on cell differentiation was
not known. In the current study, we sought
to determine whether a specific pulsed
electromagnetic field frequency is optimal
for the purpose of tissue-engineered bone
construction. The results of this study have
important implications with regard to the
development of a new type of bioreactor
and the clinical application of pulsed elec-
tromagnetic fields to promote bone fracture
healing.
Among the factors that affect electro-
magnetic fields, frequency plays a major
role. Bassett et al1 reported that different
frequencies of pulsed electromagnetic
field influence bone fracture healing.
Research has shown that pulsed electro-
magnetic fields in the frequency between
1 and 100 Hz allow the electromagnetic
field to exert a biological effect.16 In the
skeletal system, the endogenous frequen-
cy ranges from 1 to 5 Hz (walk frequency)
or from 10 to 100 Hz (muscle contraction
power frequency); Lee and McLeod17
proposed that the most effective pulsed
electromagnetic field frequency should
be similar to that associated with normal
body action frequency. In the current
study, we referred to a report focused on
the different frequency of pulsed electro-
magnetic fields that affect the union of a
bone fracture. In the frequency range of
1 to 150 Hz, we chose 5, 25, 50, 75, 100,
and 150 Hz to determine whether differ-
ent effects occurred on the promotion of
human mesenchymal stem cell osteoblast
differentiation and to identify the optimal
pulsed electromagnetic field frequency.
Using an inverted phase contrast mi-
croscope to analyze cell morphology, we
found that pulsed electromagnetic field
treatment resulted in larger cells relative
to the control groups. Furthermore, the
shape of the cells in the pulsed electromag-
netic field group included triangular and
polygonal cells with scales. The amount
of cytosolic granulomaterial and secreted
matrix also increased significantly in the
pulsed electromagnetic field group cells.
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Transmission electron microscopy analy-
sis showed that human mesenchymal
stem cells stimulated by pulsed electro-
magnetic field were more mature than
control group cells. After 3 weeks in cul-
ture, pulsed electromagnetic field group
cells exhibited oval and round nodules
and were positive for alizarin staining.
Alkaline phosphatase is an enzyme that
hydrolyzes organic phosphate during the
process of bone construction; this process
provides the phosphonic acid required for
hydroxyapatite ceramic deposition and
promotes bone formation. Alkaline phos-
phatase expression is a marker for the
initiation of bone formation and differen-
tiation. Furthermore, osteocalcin is 1 type
of noncollagen protein that is secreted by
osteoblasts. When calcium is present, os-
teocalcin combines with hydroxyapatite
and stabilize the conformation, which is
regarded as the most distinctive mark of
osteoblast differentiation. Thus alkaline
phosphatase and osteocalcin are 2 impor-
tant indices reflecting the osteoblast dif-
ferentiating into osteocyte and the matrix
becoming calcified.18
When we stimulated human mesen-
chymal stem cells with different pulsed
electromagnetic field frequencies in vitro,
all of the cells in each frequency group
exhibited detectable alkaline phosphatase
expression after 3 days, and with increas-
ing time, alkaline phosphatase expression
continued to increase. Taken together, this
finding provided evidence that bone dif-
ferentiation was initiated. After 1 week
in culture, cells stimulated with pulsed
electromagnetic field expressed osteocal-
cin, which reached peak levels in the third
week at the same time that mineralized
nodules appeared. In control group cells,
alkaline phosphatase activity was always
low, and we detected no osteocalcin ex-
pression. These findings provide evidence
that pulsed electromagnetic field stimula-
tion of human mesenchymal stem cells
leads to bone differentiation. In the current
study, different pulsed electromagnetic
field frequencies were associated with dif-

ferent levels of bone induction. From 5 to
50 Hz, as the frequency increased, the in-
ductive effect on bone differentiation also
increased. However, from 50 to 150 Hz, as
the frequency increased, the inductive ef-
fect on bone differentiation decreased.
Taken together, the results of the cur-
rent study show that pulsed electromag-
netic field frequency is an important factor
for the induction of human mesenchymal
stem cell bone differentiation. Different
frequencies of pulsed electromagnetic
field have different effects on the induc-
tion of bone differentiation, and 50 Hz
was an optimal frequency for the in vitro
induction of human mesenchymal stem
cell bone differentiation. The results of
this study may help promote the osteo-
genic differentiation of seed cells for tis-
sue engineering for bone production and
provide new insights and parameters for
clinical applications aimed at bone frac-
ture healing. The biological mechanisms
responsible for pulsed electromagnetic
field-mediated promotion of bone differ-
entiation by human mesenchymal stem
cells remain to be determined and will be
the focus of future studies. layer culture to ELF electromagnetic fields.
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