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Am J Stem Cell 2013;2(1):62-73
www.AJSC.us /ISSN:2160-4150/AJSC1301001
Original Article
Effect of extracorporeal shock wave on proliferation and
differentiation of equine adipose tissue-derived
mesenchymal stem cells in vitro
O Raabe1*, K Shell1*, A Goessl1, C Crispens1, Y Delhasse2, A Eva3, G Scheiner-Bobis3, S Wenisch4, S Arnhold1
1Institute of Veterinary -Anatomy, -Histology, and -Embryology, Justus-Liebig University of Giessen, Germany;
2Institute of Cardiovascular Research and Sport Medicine, Department of Molecular and Cellular Sport Medicine,
German Sport University Cologne, Germany; 3Institute for Veterinary Physiology and Biochemistry, Justus-Liebig
University of Giessen, Germany; 4Department of Veterinary Surgery, Justus-Liebig University of Giessen, Germany.
*Raabe O. and Shell K. contributed equally to this work.
Received January 7, 2013; Accepted February 22, 2013; Epub March 8, 2013; Published March 18, 2013
Abstract: Mesenchymal stem cells are regarded as common cellular precursors of the musculoskeletal tissue and
are responsible for tissue regeneration in the course of musculoskeletal disorders. In equine veterinary medicine
extracorporeal shock wave therapy (ESWT) is used to optimize healing processes of bone, tendon and cartilage. Nev-
ertheless, little is known about the effects of the shock waves on cells and tissues. Thus, the aim of this study was
to investigate the inuence of focused ESWT on the viability, proliferation, and differentiation capacity of adipose
tissue-derived mesenchymal stem cells (ASCs) and to explore its effects on gap junctional communication and the
activation of signalling cascades associated with cell proliferation and differentiation. ASCs were treated with differ-
ent pulses of focused ESWT. Treated cells showed increased proliferation and expression of Cx43, as detected by
means of qRT-PCR, histological staining, immunocytochemistry and western blot. At the same time, cells responded
to ESWT by signicant activation (phosphorylation) of Erk1/2, detected in western blots. No signicant effects on
the differentiation potential of the ASCs were evident. Taken together, the present results show signicant effects
of shock waves on stem cells in vitro.
Keywords: Horse, shock wave, stem cell, proliferation, differentiation
Introduction
Disorders of the musculoskeletal system, such
as osteoarthritis, tendinopathies, and frac-
tures, are the main reasons for the early retire-
ment and euthanasia of horses [1, 2]. A con-
ventional treatment option for these disorders
consists of a combination of stall rest, physio-
therapy, hoof care, and long-term pain manage-
ment using nonsteroidal anti-inammatory
drugs. Other, more recent treatment options
include implantation of cells and tissue-engi-
neered constructs in bone, cartilage and ten-
don [3, 4]. Mesenchymal stem cells are ideal
cells for implantation. Equine ASCs possess
excellent therapeutic potential for tissue regen-
eration, have self-regenerative abilities [5], and
have the potential to differentiate into several
musculoskeletal-related cell lineages [6-8]. In
recent years, several clinical investigations
have revealed promising results in the stem cell
treatment of a variety of orthopedic disorders in
horses [9-13].
An alternative treatment for musculoskeletal
disorders is the application of ESWT. The shock
wave is an acoustic wave that is characterized
by an extremely high amplitude and short rise
time and is followed by a longer, low-magnitude
negative wave [14]. Originally, ESWT was used
for lithotripsy of uroliths and gall stones in
humans and animals [15-18]. Since the early
1990s, ESWT has been commonly applied in
the treatment of musculoskeletal disorders,
such as tendinopathies and osteoarthritis, in
equine medicine [19-26].
The precise therapeutic and biological effects
of ESWT on tissues or cells, however, are not
yet completely understood. It has been sug-
Equine adipose tissue-derived mesenchymal stem cells
63 Am J Stem Cell 2013;2(1):62-73
gested that ESWT induces cell growth and
enzymatic activities [19], leads to an induction
of neovascularisation [27] has an osteostimu-
latory effect [28], and has a direct effect on
membrane permeability and viability of chon-
drocytes and on the structure of cartilage [29].
It is assumed that ESWT could possibly lead to
an activation of endogenous stem cells.
Implantation of stem cells in combination with
shock wave therapy could be considered as a
new, alternative therapy for orthopaedic dis-
eases. In order to evaluate this possibility, we
investigate in the work presented here whether
various doses of ESWT might affect the mor-
phological and biochemical properties of mes-
enchymal stem cells by analysing its effects on
proliferation, apoptosis, and differentiation of
equine ASCs.
Material and methods
The chemical reagents were obtained from PAA
(PAA, Germany) or Sigma (Sigma, Germany)
unless otherwise noted.
Tissue harvest and isolation of ASCs
Subcutaneous adipose tissue was collected
from the region localized above the dorsal glu-
teal muscles of three mixed-breed horses
(aged, mean ± SD, 7.8 ± 2.3 years) as previ-
ously described [7]. The samples were obtained
from horses being slaughtered at the local
abattoir. All samples were collected within 20
min post mortem. ASCs were isolated by colla-
genase type I (Biochrom AG, Germany) diges-
tion as previously described [7].
ESWT
ASCs from passages 1 to 4 were used for ESWT.
Since shock wave conditions have a strong
effect on the behaviour and the intercellular
contacts of the cells [30], adherent cells were
treated after reaching 80% conuence. Cells
with an optimal morphology (spindle-shaped)
and vitality were used for the experiments.
Hypertrophic cells were excluded from the
experiment, because an increased cell size can
be taken as evidence of senescence. The ASCs
with visible intracellular granules were exclud-
ed as well, because this can be a sign for dedif-
ferentiation. The proliferation capacities and
the differentiation potential were randomly
tested.
The control group did not receive any extracor-
poreal shock wave treatment, while
Experimental Group 1 (EG 1000/9) received 9
pulses of 1000 shock waves and Experimental
Group 2 (EG 2000/3) received 3 pulses of
2000 shock waves, according to standard
means of delivery in horse patients [31, 32].
Cell morphology
ASCs were examined using a phase contrast
microscope and the Axiovision image analysis
system (Carl Zeiss, Germany).
MTT assay
Cell viability was evaluated using a colorimetric
MTT assay measuring reduction power
(AppliChem, Germany). The cells (15 x 103 cell/
well) were cultured for 24 h and 48 h at 37°C
with 5% CO2. Thereafter, 0.5 mg/ml of MTT
reagent was added to the Dulbecco’s Modied
Eagle’s Medium (DMEM with 1000 mg/L
Glukose) and the cells were incubated for 4 h at
37°C with 5% CO2. DMSO (200 µl) was then
added to dissolve the water-insoluble formazan
salt. Quantication was performed with a spec-
trophotometer at 570 nm (ELISA reader; Tecan,
Germany).
Senescence assessment (β-galactosidase
assay)
Processes of cell aging can be indicated by
assessing β-galactosidase (β-Gal) activity. For
the determination of changes caused by senes-
cence, cells (15 x 103 cell/well) were harvested
immediately after shock wave application and
incubated for 24 h at 37°C with 5% CO2. Cells
were then washed in 0.1 M phosphate-buffered
saline (PBS) and subsequently lysed in 0.1%
Triton in PBS. After centrifugation at 4°C and
13.000 g for 15 min, samples were incubated
with β-Gal solution for 16 h at 37°C. The absor-
bance of the dye at 570 nm was measured with
a spectrophotometer.
Immunouorescence staining
The immunouorescence staining was per-
formed as previously described [8]. To visualize
the actin cytoskeleton, cells were incubated
with Alexa Fluor 594 phalloidin (6.6 µM nal
concentration). Cell nuclei were counterstained
using the Hoechst nuclear stain H3334. Cell
proliferation was assessed by the detection of
Equine adipose tissue-derived mesenchymal stem cells
64 Am J Stem Cell 2013;2(1):62-73
Ki67 antigen. For the detection of apoptotic
cells, cells were stained with propidium iodide
(1.5 mM nal concentration).
The method of detection of connexin 43 (Cx43)
was similar to that for the detection of Ki67
antigen [8]. The cells were incubated with the
primary antibody (mouse anti-connexin43,
diluted 1:100 by vol.; DAKO, Germany) over-
night at 4°C and were then exposed to the sec-
ondary antibody (goat anti-mouse, diluted
1:200 by vol.; DAKO, Germany) for 30 min.
Fluorescence images of ASCs were obtained
using the Axiovision image analysis system
(Carl Zeiss, Germany).
Preparation of cell lysates
Cells were lysed using a commercially available
cell lysis buffer according to the manufacturer’s
protocol (Cell Signaling Technology, Germany).
All lysis steps were carried out on ice. After cen-
trifugation at 4°C and 13.000 for 15 min, pro-
tein content in the supernatant was determined
using the bicinchoninic acid (BCA) protein assay
reagent kit (Pierce, IL, USA).
SDS-PAGE and immunodetection of Cx43 and
total and activated Erk1/2
A total of 10 µg of proteins from cell lysates
were separated by SDS-PAGE using slab gels
containing 10% acrylamide and 0.3% N,
N′-methylene-bis-acrylamide. Biotinylated
molecular weight markers (Cell Signaling
Technology, Germany) were run parallel. After
electrophoresis, the proteins were electro-blot-
ted at 500 mA for 30 min onto nitrocellulose
membranes (Schleicher & Schuell, Germany).
After blotting, the membranes were incubated
with the appropriate primary antibody in a dilu-
tion of 1:500 (phosphorylated (p-Erk1/2) or
total Erk1/2 (t-Erk1/2) or anti-Cx43 (mouse
monoclonal anti-Cx43, Invitrogen) overnight at
4°C. The visualization of the ERK1/2 proteins
was performed using the appropriate horserad-
ish peroxidase-conjugated IgG (1:2000 in
TBS-T containing 5% nonfat milk) provided by
the enhanced chemiluminescence (ECL) kit
(Amersham-Pharmacia, Germany). The mem-
branes incubated with the C43 antibody were
further incubated with a biotinylated secondary
antibody (goat-anti-mouse IgG, in a dilution of
1:1000, DAKO Hamburg, Germany). Then the
peroxidase conjugated avidine (ABC)-complex
(Vector, Burlinghame, CA, USA) was applied. A
horseradish peroxidase-conjugated anti-biotin
IgG (Cell Signaling Technology, Germany) to
detect the biotinylated molecular weight mark-
ers was included in the incubation medium at a
dilution of 1:2000. After lm exposure, the den-
sity of the resultant bands was analyzed using
a digital documentation system (Adobe
Photoshop CS5, Ireland). As a negative control,
the primary antibody was replaced by non-
immune serum.
In vitro differentiation
Adipogenic and osteogenic differentiation was
performed in a monolayer as described previ-
ously [8]. Cells were plated at a density of 3 x
103 cells/cm2. All cells were incubated in a
humidied atmosphere at 37°C with 5% CO2 for
various periods. The medium was changed
three times a week.
Adipogenic differentiation
ASCs were cultured in DMEM supplemented
with 10% fetal calf serum (FCS), 1% antibiotics
(penicillin 100 U/ml, streptomycin 0.1 mg/ml;
P/S), 1 μM dexamethasone, 10 μM insulin, 0.5
mM 3-isobutyl-1-methylxanthine, and 100 μM
indomethacin. Adipogenic differentiation was
conrmed on day 14 using an Oil Red O stain as
an indicator of intracellular lipid accumulation
and adipocyte-specic gene expression.
Osteogenic differentiation
The cells used for osteogenic differentiation
were cultured in osteogenic medium. DMEM
was supplemented with 10% FCS, 1% P/S, 0.05
mM ascorbic acid-2-phosphate, 10 mM
β-glycerophosphate and 0.1 μM dexamethasone
for 3 weeks. Osteogenesis was demonstrated
by accumulation of mineralized calcium phos-
phate (calcied extracellular matrix, ECM)
assessed by von Kossa stain.
Chondrogenic differentiation
Chondrogenic differentiation was performed in
a 3D culture with a cell density of 3 x 105 cells/
pellet. For determination of chondrogenesis,
the cells were centrifuged in 15 ml Falcon tubes
at 200 g for 5 min. Cells were incubated over-
night at 37°C, 5% CO2 in a humidied atmo-
sphere. Formed cell pellets were cultivated in
chondrogenic differentiation medium supple-
Equine adipose tissue-derived mesenchymal stem cells
65 Am J Stem Cell 2013;2(1):62-73
mented with DMEM, 1% FCS, 1% P/S, 0.05%
ITSx100, 50 µM ascorbic acid, 100 nM dexa-
methasone and 10 ng/ml TGF β1 for 3 weeks.
After culture, pellets were xed in 4% parafor-
maldehyde, embedded in parafn, and cut into
5 µm thin sections. The sections were deparaf-
nised with xylene and ethanol. To detect pro-
teoglycan synthesis as an indicator of cartilage
production, sections were stained with Alcian
blue. Counterstaining was performed with
nuclear fast red.
Quantitative realtime-RT-PCR (qRT-PCR)
Total RNA was extracted using TRI® Reagent
according to the manufacturer’s protocol
(Sigma, Germany). The RNA concentration was
adjusted to 200 ng/µl, treated with a recombi-
nant DNAse I (Roche, Germany), and subse-
quently reverse-transcribed using GeneAmp®
Gold RNA PCR Core Kit according to the
manufacturer’s protocol (Applied Biosystems,
Germany). The PCR primers and annealing con-
ditions are listed in Table 1. Real-time RT-PCR
was carried out on a CFX96 Realtime Cycler
(Bio-Rad, Germany) using IQ SybrGreen
Supermix (Bio-Rad) according to the following
protocol: after an initial 3 min at 90°C ampli-
cation was accomplished by 40 cycles of heat-
ing for 15 sec at 90°C followed by 1 min 60°C,
with a subsequent melting curve. Expression of
gene of interest was mormalized to GAPDH
expression. Data were analyzed using the CFX
Manager software 1.6 (Bio-Rad) applying the
ΔΔCT-method for relative gene expression.
Statistical analysis of data
One-way ANOVA was performed for comparison
of groups. Subsequently, the groups were com-
pared pairwise with the control by the Mann-
Whitney U test followed by Dunnett’s test. For
all tests the statistical software program,
SPSS19.0, was used (IBM, Germany).
Results
Cell morphology
The ASCs showed elongated, spindle-shaped
cell bodies with 2-3 long, slim processes (Figure
1). Only a minor percentage of the shock wave-
treated cells changed their morphology (approx-
imately 10% within EG 1000/9 and approxi-
mately 20% within EG 2000/9). The cell
processes shortened and the cell bodies
gained volume, which may have been caused
by a swelling process (Figure 1).
The visualization of the f-actin cytoskeleton
was accomplished by phalloidin staining, where
the alignment of directed and undirected bers
was assessed. A slightly increased alignment of
Figure 1. Cell morphology of ASCs. Phase-contrast microscopy of untreated ASCs revealed highly spindle-shaped,
broblastic cell morphology. The cells of EG 1000/9 and EG 2000/3 were less spindle-shaped and the cell exten-
sions were shortened. The cytoskeleton of the ASCs was stained with phalloidin.
Equine adipose tissue-derived mesenchymal stem cells
66 Am J Stem Cell 2013;2(1):62-73
the f-actin bers in the shock wave-treated
cells was observed. However, no signicant
alterations could be detected (Figure 1).
Cell proliferation and viability
There was a signicantly increased prolifera-
tion of the stem cells, which was determined
utilizing the proliferation marker Ki67. Thus, the
proliferation in cells of the control group it was
about 25.01 ± 2.5%, in the cells of EG 1000/9
at 33.08 ± 2.4% and in EG 2000/3 at 40.95 ±
4.3% (Figures 2, 3A). The results of the prolif-
eration were conrmed by the MTT assay, so
that a signicant increase of the number of
shock wave-treated cells compared with the
untreated cells was clearly recognizable. Thus,
in the cells of the control group, the absorbance
after 24 h was about 0.45 ± 0.06 and after 48
h 0.98 ± 0.14; in the cells of EG 1000/9 after
24 h 0.73 ± 0.16 and after 48 h 1.28 ± 0.24; in
EG 2000/3 after 24 h 0.84 ± 0.25 and after 48
h 1.92 ± 0.61 (Figure 3B).
Cell senescence and apoptosis
The effect of mechanotransduction of the
shock wave on cell apoptosis was also exam-
ined. The cells of EG 2000/3 showed a signi-
cantly higher amount of cell apoptosis (12 ±
4.7%) than the cells of EG 1000/9 (7.72 ± 2.2%)
or the untreated cells (3.34 ± 0.28%) (Figures
2, 3C).
Cell senescence can be assessed by measur-
ing β-Gal activity. Following, the density of the
dye was measured. Relating to the untreated
cells, the results of the β-Gal assay revealed no
changes for the shock wave application at any
time. Thus, the senescence in cells of the con-
trol group was 0.19 ± 0.01, in the cells of EG
1000/9 at 0.19 ± 0.03 and in EG 2000/3 at
0.2 ± 0.04 (Figure 3D).
In vitro differentiation
The analysis of the differentiation potential
indicates to what extent stem cells can be
directed towards the adipogenic, osteogenic, or
chondrogenic lineage; this can be determined
by applying specic media and factors. Shock
wave-treated cells showed a slightly higher
potential for differentiation than untreated
cells. The adipogenic differentiation in the
treated groups was more pronounced in terms
of both numbers of differentiated cells and size
of fat vacuoles. Fat vacuoles were noted within
3-4 days of shock wave treatment in treated
cells and within 5 days in untreated cells (Figure
4). Following induction of osteogenic differenti-
ation, we observed a clear change according to
the morphology. The shock wave-treated cells
Figure 2. Cell proliferation and apoptosis. Cell proliferation was assessed by immunouorescence with the Ki67
antibody. Upper panels: Turquoise-stained nuclei indicate proliferation. Lower panels: apoptosis was stained with
propidium iodide; pink stained nuclei indicate apoptosis.
Equine adipose tissue-derived mesenchymal stem cells
67 Am J Stem Cell 2013;2(1):62-73
of both treated groups acquired a polygonal
shape within three days and cell aggregates
and nodule formation were observed after 3-5
days of culture; in contrast, this was observed
in the untreated cells after 5-7 days. Matrix
mineralization and calcium accumulation were
also more prominent in the treated groups
(Figure 4). Within a day after seeding the ASCs
in culture for chondrogenesis, three-dimension-
al aggregates were observed in all groups. Both
treated and untreated cell pellets stained posi-
tive for alcian blue, indicating the presence of
sulfated proteoglycans (Figure 4). No positive
staining (for all three differentiation lineages)
was observed in the cells cultured in growth
medium (data not shown). However, the analy-
sis of the adipo-, osteo- and chondrogenesis-
relevant mRNA expression did not show any
signicant differences between the samples
(Figure 5).
Effect of ESWT on Erk1/2
ESW treatment did not affect the expression of
total Erk1/2 (Figure 6; t-Erk1/2). The expres-
sion of p-Erk1/2 (activated Erk1/2), however,
was signicantly different between EG 2000/3
and EG 1000/9 and between treated cells of
the both groups and the untreated cells (Figure
6). In EG 1000/9 cells, p-Erk1/2 was signi-
cantly higher than p-Erk1/2 in untreated or in
EG 2000/3 cells.
Cx43 expression
Cx43 was detected by immunouorescence
staining in the untreated and both treated
groups (Figure 7A). This can be conrmed by
Western blot experiments (Figure 7C) showing
a marginally higher expression of Cx43 in both
treated groups (48.76% and 49.29%) com-
pared to the control group (46.01%). However,
looking at Cx43 mRNA expression a signicant
increase in Cx43 expression was observed in
EG 1000/9 in comparison with the untreated
cells and cells treated by EG 2000/3 (Figure
7B).
Discussion
ASCs have a high therapeutic potential and are
therefore used in various areas of regenerative
Figure 3. Quantitative analysis of cell proliferation, viability, apoptosis, and senescence. The treated cells showed
a better proliferative capability as well as a higher apoptotic rate than the untreated cells. The data were compiled
from triplicate determinations. The MTT assay showed a signicantly higher cell proliferation of the treated cells
than the untreated cells. The β-Gal assay showed that the cells of the three groups had nearly the same level of
senescence. All values reect the arithmetic mean ± standard deviation; a: signicantly higher than control, b: sig-
nicantly higher than EG 1000/9, a, b, c: all samples are signicant with each other.
Equine adipose tissue-derived mesenchymal stem cells
68 Am J Stem Cell 2013;2(1):62-73
medicine. However, the success of this therapy
is limited by the small number of stem cells that
reach the target area. Therefore, the cells have
to be conditioned for their in vivo application or
to be modied for the transplantation niche.
Recently, various forms of shock wave therapy
have been applied in addition to a stem cell
therapy. The mechanisms involved in shock
wave therapy, however, are not yet completely
understood and are often the subject of contro-
versy. Thus, the aim of the present study was to
examine whether behaviour and characteris-
tics of equine ASCs can be modied under the
inuence of shock wave therapy. For that pur-
pose the effects of ESWT on the cytoskeleton
of ASC and on their proliferation and differenti-
ation potential were analyzed. The results
obtained show a distinct mechano-sensitive
reaction of the equine ASCs to the application
of shock wave treatment. Signicantly different
effects were observed between the treated
groups and the untreated group, which were
consistent with results reported by other
researchers [33-35]. Additionally, there are
numerous reports about the effect of ESWT on
different cells (chondrocytes, marrow stromal
cells, tumor cells etc) [36-40]. Similar to our
ndings the authors of these papers report
about an increase in the proliferation rate as
well as about a dose-and impulse dependent
cytotoxic effect. However, the increased prolif-
eration rate especially of stem cells observed
after shock wave application, as investigated in
our study, suggests that the transplantation
results might be inuenced in a positive way if
division- and reproduction speed of stem cells
(proliferation rate) can be stimulated by ESWT
ex vivo prior to transplantation.
It is well known that the cytoskeleton plays an
active role in the process of apoptosis [41].
Thus, loosening the cells from their focal adhe-
Figure 4. Cell differentiation. The adipogenic differentiation was detected by the formation of lipid droplets in the cy-
toplasm of cells stained with Oil Red O. Osteogenic differentiation was demonstrated by calcium deposition stained
with von Kossa stain. Chondrogenic differentiation of ASCs was determined by a transformation from the broblastic
to a chondrocyte-like appearance; formation of GAG-rich matrix and numerous chondrone-like areas were observed.
The center of the pellet from EG 2000/3 became increasingly subjected to necrosis.
Equine adipose tissue-derived mesenchymal stem cells
69 Am J Stem Cell 2013;2(1):62-73
sion complexes as well as reorganization of the
f-actin bers and inhibition of actin dynamics
following cell contraction are some of the rst
signs of apoptosis [18]. Our investigation
showed signicant effects of the application of
shock wave treatment on cell apoptosis with
increasing severity of the shock wave condi-
tions. Furthermore, an alignment of the f-actin
bers was demonstrated by phalloidin staining
after shock wave application. Therefore, a cor-
relation between the dynamics of the f-actin
bre formation and apoptosis can be assumed
that is in accordance with the observations of
other research groups [18]. Furthermore, differ-
ent structures of the ASCs are altered during
shock wave application, and these ndings con-
rm that the use of the wrong shock wave con-
ditions can result in cell death.
Figure 5. qRT-PCR after differentiation. mRNA expression of adipogenic (Pparγ2), osteogenic (AP, OC, SSP1, Runx2),
and chondrogenic markers (Col I, Col III, and Col X). All values reect the arithmetic mean ± standard deviation. RGE:
relative gene expression.
Equine adipose tissue-derived mesenchymal stem cells
70 Am J Stem Cell 2013;2(1):62-73
Aging processes are reected in the morpholo-
gy of cells as well as in the activity of
β-galactosidase [42]; therefore, we measured
β-Gal activity to assess effects of the shock
wave treatment on cellular aging. There were
no differences in β-Gal activity between control
and treated cells, thus allowing to assume that
the treatment did not cause any senescence on
ASCs.
A better differentiation potential of the cells
after shock wave treatment was demonstrated
for the adipogenic, osteogenic, and chondro-
genic lineages (Figure 4) without any detect-
able changes in the mRNA expression of corre-
sponding markers (Figure 5). The shock waves
possibly stimulate the translation of the marker
mRNAs into protein without any direct effect on
mRNA synthesis. These results are strength-
ened by the fact that shock wave treatment
caused an almost 2-fold stimulation of Erk1/2
in the EG 1000/9 group when compared with
the untreated group. This effect was not
observed in the EG 2000/3 group, however,
demonstrating once again the importance of
the correct choice of treatment. Since p-Erk1/2
is involved in the regulation of cell growth and
differentiation, it is justied to assume that the
observed activation of the kinase might be of
signicance for the mitogenic and differentia-
tion responses of the ASCs. Activation of
Erk1/2 in connection with differentiation and
proliferation of mesenchymal stem cells has
been described previously [43, 44].
The analysis of the cell-cell contacts was per-
formed by examining the presence of Cx43, the
most common Cx in mammals [45]. Cx43 has a
Figure 6. Erk1/2 expression. A: Western blot analysis of total- and phosphorylated-Erk1/2 expression in untreated
(1), EG 1000/9 (2), and EG 2000/3 (3) groups. The p-Erk1/2 expression is signicantly higher in the EG 1000/9
than in the control or EG 2000/3 group (n=6; mean ± SEM). The value for the EG 2000/3 group is signicantly
smaller than that of the control group (n=6; mean ± SEM); a: signicantly higher than control, b: signicantly higher
than EG 2000/3.
Equine adipose tissue-derived mesenchymal stem cells
71 Am J Stem Cell 2013;2(1):62-73
role in the regulation of cell differentiation and
cell proliferation [46, 47]. On the protein level,
Cx43 was detected in the cells of untreated as
well as slightly increased in both shock wave-
treated groups, with a slightly higher expres-
sion in the EG 2000/3 group. Cx43-specic
mRNA expression, however, was higher in the
EG 1000/9 group than in EG 2000/3 or in
untreated cells. These ndings actually indicate
that shock wave treatment has the potency to
alter the expression of cell-cell contact proteins
as shown here for the gap junction proteins.
Furthermore, these ndings also conrm the
results for proliferation, and differentiation,
and reveal new aspects for fundamental
research, which might be translated to clinical
applications. Since the multiplication and culti-
vation of ASCs ex vivo allow for the production
of a high number of stem cells that can be sub-
jected to shock waves under ideal conditions
without affecting other cells in the surrounding
tissue, ex vivo pre-conditioning of equine stem
cells by shock wave application followed by the
re-implantation of these cells into tissue lesions
might help to improve the treatment of ortho-
paedic disorders.
Acknowledgments
This work was supported by Richard and
Annemarie Wolf-Foundation.
Address correspondence to: Dr. Stefan Arnhold,
Institute of Veterinary -Anatomy, -Histolog y and –
Embryology, Justus-Liebig University of Giessen,
Frankfurterstrasse 98, 35392 Giessen, Germany.
E-mail: stefan.arnhold@vetmed.uni-giessen.de
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