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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 Osteogenic Differentiation of Human Mesenchymal Stem Cells | Luo et al

B
one tissue engineering involves the Materials and Methods the field’s homogeneity and stabilization
use of tissue engineering methods The following solutions, kits, and were acceptable. The coil was placed into
to promote the generation and dif- equipment were used in the experiments the cell incubator, and the field was set
ferentiation of bony cells. Using this ap- described herein: Percoll solution (Sigma- to different pulsed electromagnetic field
proach, new approaches can be explored Aldrich, St Louis, Missouri), HyClone frequencies, including 5, 25, 50, 75, 100,
to repair long segmental bone defects. Dulbecco’s Modified Eagle Medium: and 150 Hz, each with a field intensity
Mesenchymal stem cells are one of the Nutrient Mixture F-12 (DMEM/F12) (1:1) of 1.1 mT, for 30 minutes per day for 21
most widely used types of stem cells for (Thermo Fisher Scientific Inc, Waltham, days. The control groups of cells were
bone tissue engineering. Following in vitro Massachusetts), HyClone fetal calf serum incubated under the same experimental
amplification using chemical induction to (Thermo Fisher Scientific Inc), alkaline conditions with no exposure to the pulsed
induce mesenchymal stem cell osteoblast phosphatase test kit (Nanjing Jiancheng electromagnetic fields.
differentiation, the resulting osteoblasts Bioengineering Institute, Nanjing,
and support material can be combined to China), osteocalcin radioimmunity kit Morphological Observations
generate engineered bone tissue. (Beijing China Atomic Research Institute, Changes in cell morphology under the
In 1977, Bassett et al1 developed Beijing, China), mouse-anti-human os- different growth conditions were observed
pulsed electromagnetic field therapy and teocalcin antibody (Boshide, Wuhan, using an inverted microscope. Photos of
successfully treated a group of patients China), ultraviolet/visible spectrophotom- cell morphology were taken on days 1, 3,
with bone nonunion. Since then, several eter (UV751GD; the Shanghai Equipment 5, and 7 after serial subcultivation.
studies have reported that pulsed electro- Con-factory, Shanghai, China), and con-
magnetic fields accelerate the speed of trollable pulsed electromagnetic field ac- Ultramicrostructural Observations
mesenchymal stem cell amplification and tivator (Logistical Engineering University Following exposure to pulsed electro-
osteoblast induction, growth factor secre- of PLA, Chongqing, China). magnetic fields, when the cells covered
tion, and extracellular matrix synthesis; 80% of the area of the culture bottle, they
pulsed electromagnetic fields may also Separation and Cultivation of Human were digested, centrifuged, and washed
promote bone reconstruction and acceler- Mesenchymal Stem Cells twice with phosphate buffered saline. The
ate bone growth.2-7 These findings suggest Bony marrow (5-10 mL) from both cells were immobilized with 2.5% glutar-
that further studies of the effects of pulsed sides of the iliac crest was collected from aldehyde and subjected to gradient ace-
electromagnetic fields on mesenchymal healthy volunteers in a sterilized environ- tone anhydration, followed by embedding
stem cell osteoblast differentiation and ment using heparin as the anticoagulant. in epoxy resin. Extra thin sections were
bone tissue engineering are needed.8-13 Percoll solution (1.073 g/mL) was used generated and stained with uranyl acetate
These previous studies were performed to isolate the bony marrow. A total of and lead citrate. Transmission electron
using a specific pulsed electromagnetic 53105 cells/cm2 were inoculated into microscopy was used to observe the ul-
field frequency during the course of the a culture flask containing DMEM/F12 tramicrostructure of the experimental and
experiments; notably, the frequency is supplemented with 15% fetal calf se- control groups.
an important indicator for determining rum, and the cells were incubated under
the biological effects of pulsed electro- saturated humidity. After 3 generations, Alkaline Phosphoric Acid Enzyme Staining
magnetic fields. In the current study, we the cells were digested and centrifuged When using Gomori stain, cells posi-
investigated the effects of different pulsed to generate a cell suspension, which was tive for alkaline phosphatase expression
electromagnetic field frequencies on mes- divided into 6 experimental groups and 1 are brownish-black. Following 6 days of
enchymal stem cell induction in vitro control group. pulsed electromagnetic field stimulation,
with the overall goal of providing a new the human mesenchymal stem cells were
approach for mesenchymal stem cell in- Treatment of Human Mesenchymal removed from the incubator, rinsed with
duction in vitro and experimental support Stem Cells With Different Pulsed phosphate buffered saline, fixed in ice-
for a new type of bioreactor and the clini- Electromagnetic Field Frequencies cold acetone (220°C), incubated for 3
cal application of pulsed electromagnetic The 6 experimental cell groups were hours at 37°C in phosphoric acid enzyme
fields to promote bone fracture healing. treated with a Helmholtz coil with pulsed liquids, washed with distilled water, and
The results of the current study should electromagnetic fields, which was a dual subjected to ammonium sulfide process-
provide a strong theoretical foundation coil with a 10-cm space between the 2 ing using 2% nitric acid and 1% cobalt,
for future pulsed electromagnetic field- coils. The Hall effect was used to mea- 1 by 1 in order, followed by staining with
related research. sure the magnetic reactor to confirm that neutral and red contrast dye.

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n Feature Article

Alkaline Phosphatase Activity antibody, and a Y counter is used to mea- Results

Measurements sure the sediment counts per minute, which Inverted Phase Contrast Microscope
After 3, 6, 9, 12, and 15 days of pulsed is used to determine the osteocalcin content Observations
electromagnetic field exposure, cells of the sample according to a standard curve. At 4 hours post-inoculation, the third-
grown in 12-well flat-bottomed plates generation human mesenchymal stem cells
from each group were digested, centri- Staining With Alizarin Monosulfonate were adhered to the plates, and they began
fuged, collected, and washed twice with Calcium Dye to divide and grow 24 hours after they ad-
phosphate buffered saline. The cell den- After the appearance of oval-shaped hered. After 3 days, pulsed electromagnetic
sity was adjusted to 13105 cells/mL in nodules in the 6-well culture plate, the field group cells were larger than control
0.5% Triton-X100 (Sigma-Aldrich, St coverslips were removed, rinsed with group cells, and their morphology contin-
Louis, Missouri), and the cells were in- phosphate buffered saline, fixed with 75% ued to change; the cells eventually became
cubated at 4°C for 12 hours. Intermittent alcohol for 30 minutes, stained with 2% triangular and polygonal in shape, scales
ultrasound exposure on ice was used to alizarin monosulfonate, dehydrated us- formed, and the cytoplasm contained abun-
break up the cells and ensure complete ing an alcohol gradient, made transparent dant matrix and granular material. No ob-
cell lysis (150 W, 5 s). The absorbance of with xylene, and mounted using neutral vious differences were observed in the ap-
50 µL of the cell lysate was examined at resin. pearances of pulsed electromagnetic field
520 nm to determine the A value, and a group cells compared with control group
standard formula was used to calculate al- Staining With Von Kossa Calcium Dye cells. Over time, the cells became confluent
kaline phosphatase activity. After oval-shaped nodules appeared and began to exhibit overlapping growth.
in the 6-well culture plate, the coverslips After 3 weeks, mineralization of the matrix
Collagen Type I and Osteocalcin were removed, rinsed with phosphate led to the fusion of the oval-shaped calci-
Immunostaining buffered saline, fixed with 75% alcohol, fied nodules. Around the nodules, the cells
Coverslips were removed after 12 immersed in 2% silver nitrate aqua, ex- were distributed in an array-like pattern.
days, followed by phosphate buffered sa- posed to ultraviolet light for 30 minutes,
line rinses, fixation with 95% ethanol, and washed with distilled water, stained with Ultramicrostructural Observations
treatment with 3% hydrogen dioxide, fol- neutral red dye, treated with an alcohol Transmission electron microscopy
lowed by rinses with distilled water and gradient, made transparent with dimethyl analysis of human mesenchymal stem cells
incubation with 5% normal goat serum. benzene, and sealed using a neutral resin. in the pulsed electromagnetic field group
Cells were subjected to immunohisto- showed that they were more differentiated
chemical staining using the streptavidin- Staining With Achromycin Dye than the control group cells. The nuclear
biotin peroxidase complex method with a After oval-shaped nodules appeared matrix ratio of pulsed electromagnetic field
rat anti-collagen type I and anti-osteocalcin in the 6-well culture plate, the coverslips group cells was lower than that of control
monoclonal antibody. were removed, treated with 0.1 mg/mL group cells. The cytoplasm of pulsed elec-
achromycin culture solution for 30 min- tromagnetic field group cells contained
Quantification of Osteocalcin Levels utes, incubated with a common culture abundant organelles, including mitochon-
At 7, 14, and 21 days, 50 mL were re- solution for 30 minutes, rinsed with phos- dria, rough endoplasmic reticulum, and
moved from each culture, followed by phate buffered saline, fixed with 75% al- Golgi bodies. Control group human mes-
digestion with diastase vera. Following cohol, and observed using a fluorescence enchymal stem cells were more immature
centrifugation, a total of 13106 cells were microscope. with larger nuclei, a similar nuclear-matrix
resuspended in 1 mL of a 1:1 admixture of ratio, and fewer organelles.
phosphate buffered saline and Triton-X100, Statistical Analyses
followed by incubation at 4°C overnight. SPSS version 11.0 software (IBM, Alkaline Phosphatase Staining
The resulting samples were sent to the Armonk, New York) was used for statis- Cells that were not stimulated with
Atomic Medical Department of Southwest tical analyses. Self-paired t analysis was pulsed electromagnetic fields were nega-
Hospital to measure the osteocalcin content used to analyze each set of experimental tive for alkaline phosphatase expression,
in the cells using a radiographic-immunity data. One-factor analysis of variance was whereas cells subjected to pulsed elec-
method based on the radiographic-immunity used to analyze different groups stimulat- tromagnetic field stimulation were highly
competitive binding principle. In this assay, ed with different pulsed electromagnetic positive for alkaline phosphatase expres-
125
I-marked osteocalcin competes with free fields, and F analysis was used to com- sion, with brownish-black cytoplasm and
osteocalcin for binding to the osteocalcin plete the statistical analysis. black granulated precipitates.

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 Osteogenic Differentiation of Human Mesenchymal Stem Cells | Luo et al

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
1 groups expressed significantly higher lev-
Figure 1: Alkaline phosphatase (ALP) activity in human mesenchymal stem cells treated with different els of osteocalcin; although cells in the
frequencies of pulsed electromagnetic fields (PEMF). 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
2 stimulation for 21 days, matrix mineral-
Figure 2: Osteocalcin content of mesenchymal stem cells treated with different frequencies of pulsed
ization led to the formation of oval-shaped
electromagnetic fields (PEMF). calcium nodules, which stained orange
around the nodule with alizarin monosul-
fonate calcium, and the cells around the
Alkaline Phosphatase Activity phatase activity levels. Alkaline phospha- nodule were distributed in an array-like
Measurements tase activity in the 50-Hz pulsed electro- pattern. In contrast, no calcium nodules
Pulsed electromagnetic field group magnetic field group was higher than that were present in the control group cells.
cells exhibited stronger alkaline phospha- of the other pulsed electromagnetic field
tase activity than control group cells start- groups on days 9, 12, and 15. Von Kossa Calcium Dye Staining
ing on the third day. As time progressed, On day 21, cells stimulated with
alkaline phosphatase activity was higher in Collagen Type I and Osteocalcin pulsed electromagnetic fields began to
pulsed electromagnetic field group cells, Immunohistochemical Staining exhibit oval-shaped calcium nodules.
reaching a peak at 12 days. By day 15, After 12 days of pulsed electromagnetic The nodules changed from transparent to
no great change had occurred, and alka- field stimulation, human mesenchymal opaque, and then turned into a black mass.
line phosphatase activity remained stable, stem cells subjected to collagen type I im- After staining with a calcium dye, mas-
although it remained significantly higher munohistochemical staining exhibited high sive black crystal deposits were identi-
than that of the control groups (Figure 1). levels of yellow granulation and were high- fied, which were not present in the control
Furthermore, different frequencies were ly positive. In contrast, no yellow granula- group cells. The results of this analysis
associated with different alkaline phos- tion was detected in the control group cells. showed that pulsed electromagnetic field

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n Feature Article

treatment of human mesenchymal stem tromagnetic field frequencies exhibit a dif- Transmission electron microscopy analy-
cells can stimulate bone induction. ferential effect on cell differentiation was sis showed that human mesenchymal
not known. In the current study, we sought stem cells stimulated by pulsed electro-
Achromycin Fluorescent Labeling to determine whether a specific pulsed magnetic field were more mature than
Cells stimulated by pulsed electromag- electromagnetic field frequency is optimal control group cells. After 3 weeks in cul-
netic fields began to exhibit oval-shaped for the purpose of tissue-engineered bone ture, pulsed electromagnetic field group
calcium nodules after 3 weeks in culture, construction. The results of this study have cells exhibited oval and round nodules
and the nodules increased in size and important implications with regard to the and were positive for alizarin staining.
changed from transparent to an opaque- development of a new type of bioreactor Alkaline phosphatase is an enzyme that
black mass. After achromycin fluorescent and the clinical application of pulsed elec- hydrolyzes organic phosphate during the
labeling, the nodules appeared golden, tromagnetic fields to promote bone fracture process of bone construction; this process
and the light was equal to the size and healing. provides the phosphonic acid required for
shade under the inverted microscope. In Among the factors that affect electro- hydroxyapatite ceramic deposition and
contrast, no calcium nodules were present magnetic fields, frequency plays a major promotes bone formation. Alkaline phos-
in the control group. role. Bassett et al1 reported that different phatase expression is a marker for the
frequencies of pulsed electromagnetic initiation of bone formation and differen-
Discussion field influence bone fracture healing. tiation. Furthermore, osteocalcin is 1 type
The construction of tissue-engineered Research has shown that pulsed electro- of noncollagen protein that is secreted by
bone requires the establishment of seed cells magnetic fields in the frequency between osteoblasts. When calcium is present, os-
with strong characteristics. Mesenchymal 1 and 100 Hz allow the electromagnetic teocalcin combines with hydroxyapatite
stem cells, which are derived from meso- field to exert a biological effect.16 In the and stabilize the conformation, which is
derm, are adult stem cells that have strong skeletal system, the endogenous frequen- regarded as the most distinctive mark of
reproductive activity and are multipotent. cy ranges from 1 to 5 Hz (walk frequency) osteoblast differentiation. Thus alkaline
The differentiation of mesenchymal or from 10 to 100 Hz (muscle contraction phosphatase and osteocalcin are 2 impor-
stem cells into osteoblasts can be induced power frequency); Lee and McLeod17 tant indices reflecting the osteoblast dif-
by cytokines, hormones, physical meth- proposed that the most effective pulsed ferentiating into osteocyte and the matrix
ods, and by many other factors. Pulsed electromagnetic field frequency should becoming calcified.18
electromagnetic field is a method used to be similar to that associated with normal When we stimulated human mesen-
treat bony delayed union and bony non- body action frequency. In the current chymal stem cells with different pulsed
union. Pulsed electromagnetic field treat- study, we referred to a report focused on electromagnetic field frequencies in vitro,
ment is associated with satisfactory effects, the different frequency of pulsed electro- all of the cells in each frequency group
and has recently been used as a therapy to magnetic fields that affect the union of a exhibited detectable alkaline phosphatase
treat inborn bone defects, bone necrosis, bone fracture. In the frequency range of expression after 3 days, and with increas-
bone transplantation, and spinal fusion.1-6 1 to 150 Hz, we chose 5, 25, 50, 75, 100, ing time, alkaline phosphatase expression
Previous studies have shown that pulsed and 150 Hz to determine whether differ- continued to increase. Taken together, this
electromagnetic fields can mediate extra- ent effects occurred on the promotion of finding provided evidence that bone dif-
cellular matrix synthesis, increase alkaline human mesenchymal stem cell osteoblast ferentiation was initiated. After 1 week
phosphatase expression by osteoblasts, and differentiation and to identify the optimal in culture, cells stimulated with pulsed
promote the secretion of osteocalcin and pulsed electromagnetic field frequency. electromagnetic field expressed osteocal-
collagen.6,14,15 Mesenchymal stem cells Using an inverted phase contrast mi- cin, which reached peak levels in the third
are an important ancestor cell in the pro- croscope to analyze cell morphology, we week at the same time that mineralized
cess of bone growth. Based on these find- found that pulsed electromagnetic field nodules appeared. In control group cells,
ings, some researchers have proposed that treatment resulted in larger cells relative alkaline phosphatase activity was always
pulsed electromagnetic fields could induce to the control groups. Furthermore, the low, and we detected no osteocalcin ex-
a similar biological effect on human mes- shape of the cells in the pulsed electromag- pression. These findings provide evidence
enchymal stem cells and promote bone for- netic field group included triangular and that pulsed electromagnetic field stimula-
mation.8-13 This proposal has gained sup- polygonal cells with scales. The amount tion of human mesenchymal stem cells
port based on earlier studies performed in of cytosolic granulomaterial and secreted leads to bone differentiation. In the current
our laboratory. However, prior to the cur- matrix also increased significantly in the study, different pulsed electromagnetic
rent study, whether different pulsed elec- pulsed electromagnetic field group cells. field frequencies were associated with dif-

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 Osteogenic Differentiation of Human Mesenchymal Stem Cells | Luo et al

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