Radial shockwave treatment promotes human mesenchymal stem cell self-renewal and enhances cartilage healing
Zhang et al. Stem Cell Research & Therapy
Radial shockwave treatment promotes human mesenchymal stem cell self-renewal and enhances cartilage healing
Hao Zhang 1 3
Zhong-Li Li 1 3
Fei Yang 2
Qiang Zhang 1 3
Xiang-Zheng Su 1 3
Ji Li 1 3
Ning Zhang 1 3 4
Chun-Hui Liu 1 3
Ning Mao 0
Heng Zhu 0
0 Department of Cell Biology, Institute of Basic Medical Sciences , Tai Ping Road 27, Beijing , China
1 Department of Orthopedics, Sports Medicine Center, People's Liberation Army General Hospital , Beijing 100853 , China
2 BNLMS, State Key Laboratory of Polymer Physics & Chemistry, Institute of Chemistry , Beijing , China
3 Department of Orthopedics, Sports Medicine Center, People's Liberation Army General Hospital , Beijing 100853 , China
4 Department of Orthopedics, People's Liberation Army Rocket Force General Hospital , Beijing , China
Background: Shockwaves and mesenchymal stem cells (MSCs) have been widely accepted as useful tools for many orthopedic applications. However, the modulatory effects of shockwaves on MSCs remain controversial. In this study, we explored the influence of radial shockwaves on human bone marrow MSCs using a floating model in vitro and evaluated the healing effects of these cells on cartilage defects in vivo using a rabbit model. Methods: MSCs were cultured in vitro, harvested, resuspended, and treated with various doses of radial shockwaves in a floating system. Cell proliferation was evaluated by growth kinetics and Cell Counting Kit-8 (CCK-8) assay. In addition, the cell cycle and apoptotic activity were analyzed by fluorescence activated cell sorting. To explore the “stemness” of MSCs, cell colony-forming tests and multidifferentiation assays were performed. We also examined the MSC subcellular structure using transmission electron microscopy and examined the healing effects of these cells on cartilage defects by pathological analyses. Results: The results of growth kinetics and CCK-8 assays showed that radial shockwave treatment significantly promoted MSC proliferation. Enhanced cell growth was also reflected by an increase in the numbers of cells in the S phase and a decrease in the numbers of cells arrested in the G0/G1 phase in shockwave-treated MSCs. Unexpectedly, shockwaves caused a slight increase in MSC apoptosis rates. Furthermore, radial shockwaves promoted self-replicating activity of MSCs. Transmission electron microscopy revealed that MSCs were metabolically activated by shockwave treatment. In addition, radial shockwaves favored MSC osteogenic differentiation but inhibited adipogenic activity. Most importantly, MSCs pretreated by radial shockwaves exhibited an enhanced healing effect on cartilage defects in vivo. Compared with control groups, shockwave-treated MSCs combined with bio-scaffolds significantly improved histological scores of injured rabbit knees. Conclusions: In the present study, we found that radial shockwaves significantly promoted the proliferation and self-renewal of MSCs in vitro and safely accelerated the cartilage repair process in vivo, indicating favorable clinical outcomes.
Radial shockwave; Mesenchymal stem cell; Cartilage repair
Articular cartilage is a nonvascularized tissue that contains
only a sparse population of chondrocytes [
collagen–proteoglycan matrix surrounding the chondrocytes
gives the tissue its anticompressive features and enables
frictionless motion during habitual loading. However,
these features also make it difficult for cartilage to
regenerate after injury. Therefore, various interventions have
been developed to facilitate the regeneration of cartilage
]. Since chondrocytes have a limited ability to
renew and limited matrix production following cell
expansion, stem cells have been the optimal choice for
facilitating cartilage regeneration [
Mesenchymal stem cells (MSCs) were originally
identified in bone marrow and have since been found in
numerous tissues, including bone, fat, and tendon [
6, 8, 9
Under certain conditions, MSCs can differentiate into
numerous tissue cell types and promote tissue repair [
Moreover, MSCs are capable of repopulating themselves,
continuously supplying seed cells for tissue regeneration.
Given the innate ability of MSCs to promote tissue
repair, there is rising interest in utilizing MSCs solely or
MSCs combined with bio-scaffolds for the treatment of
osteochondral disorders, including osteoarthritis,
cartilage defects, and rheumatoid arthritis [
1, 5, 10
Extracorporeal shockwaves (ESWs), as a type of transient
pressure fluctuation, have been accepted as an effective
and safe method for the treatment of several diseases,
especially for a wide range of musculoskeletal disorders
]. In 2006, the US Food and Drug Administration
(FDA) approved ESW devices for the treatment of plantar
fasciitis. Based on the increasing accumulation of
shockwave applications and improved shockwave-based therapy,
researchers have begun to explore the regulatory effects
of shockwaves on seed cells for regenerative medicine
]. However, the biological changes of seed cells
following shockwave stimulation, especially the changes
of stem cell characteristics, remain controversial.
Therefore, in the present study, we explored the
biological characteristics of MSCs before and after direct
treatment with radial shockwaves. Based on the
shockwave literature and our previous findings of shockwaves
in cartilage repair, we hypothesized that radial
shockwaves may have a positive effect on MSC proliferation
and repopulation, and that the promoting effect would
accelerate the process of osteochondral healing in vivo.
Human bone marrow-derived MSCs were harvested from
the posterior iliac crest under local anesthetic with a core
biopsy needle aspiration system following our previous
]. Briefly, mononuclear cells were isolated
by gradient centrifugation at 900 × g for 30 min on Percoll
(Amersham Biosciences, Uppsala, Sweden) at a density of
1.073 g/ml and cultured at 2 × 105–5 × 105 cells/cm2 in
alpha-modified Eagle’s medium (α-MEM; Invitrogen,
Carlsbad, CA, USA) supplemented with 10% fetal bovine
serum (FBS; HyClone, Logan, UT, USA). Nonadherent
cells were removed by changing the culture medium after
the initial 72 h. The adherent cells were trypsinized (0.05%
trypsin at 37 °C for 5 min) when adherent cells were
approximately 80% confluent. MSCs at passages 3–6 were
used for experiments unless otherwise stated.
Experimental animals were provided by the Experimental
Animals Center of the Chinese People’s Liberation Army
(PLA) General Hospital. Rabbits were kept in a controlled
clean environment and received professional care. All of
the experimental protocols were in compliance with the
Animal Welfare Act and were approved by the Animal
Care and Use Committee of the Laboratory Animal
Research Center at the PLA General Hospital (Reference
Shockwave-MSC preparation in a floating model
Compared with traditional adherent stem cell culture
systems, recent studies reported that floating culture
systems are regarded as more physiologically relevant.
Thus, a floating shockwave treatment system was
constructed in the present study. In brief, a total of 2.5 ×
107 MSCs were harvested and resuspended in 25 ml of
culture medium in 100-mm cell culture dishes. The
radial shockwave applicator treated the floating MSCs
below the surface of the liquid level. Radial shockwaves
were generated by a Swiss DolorClast Master (Electro
Medical Systems SA, Switzerland). Radial shockwave
treatment was conducted at the following rates:
continuous pulse, 1000 impulses, and 5 Hz (total treatment
time, 200 s). Four groups were treated at different
pressures as follows: 0 bar served as the control, whereas 1
bar, 2 bars, and 3 bars served as experimental groups.
The radial-shockwave-treated MSCs were used for
further biological experiments in vitro and in vivo.
Growth kinetics and CCK-8 assays
The growth kinetics of radial-shockwave-treated MSCs
and MSCs in the control groups were determined using
trypan blue exclusion cell counting. In brief, MSCs were
cultured in 48-well plates at 2 × 104 cells/well and
harvested every 2 days for hemocytometer cell counting
during a period of 17 days.
The Cell Counting Kit-8 (CCK-8; Dojindo) was also
used to evaluate MSC proliferation. In accordance with
the manufacturer’s protocol, all MSCs were seeded in
96well plates at 2 × 103 cells/well (five wells in each group),
cultured in α-MEM supplemented with 10% FBS, and
added to the CCK-8 solution in a ratio of 100 ml/1 ml
before incubation at 37 °C for 1 h. Absorbance was then
measured at a wavelength of 450 nm using a microplate
reader. In the present study, the CCK-8 experiments were
performed over a time period of 17 days.
Cell cycle and apoptosis analysis
Radial-shockwave-treated MSCs were collected, and the
cell concentration was adjusted to 1 × 106 cells/ml.
MSCs in every group were then centrifuged at 300G for
15 min and fixed with 70% ethanol at 4 °C for 2 h. Fixed
cells were washed and incubated in PBS supplemented
with 100 mg/ml propidium iodide (PI) (Sigma-Aldrich),
100 mg/ml Annexin V (Sigma-Aldrich), and 20 ng/ml
RNase (Sigma-Aldrich) for 20 min. Flow cytometry (BD
FACSCanto) was used to measure stem cell fluorescence
at an excitation wavelength of 488 nm. FacsDiva and
MODFIT software were used to analyze the results.
Colony-forming unit fibroblast formation assay
Passage 4 MSCs were trypsinized, harvested, and
prepared before the shockwave treatment was applied. The
MSCs in each group were adjusted to different cell
numbers (1 × 103, 5 × 103, and 1 × 104 cells /well) following
the application of shockwave treatments. The method of
shockwave stimulation followed previous protocols [
Aliquots of cell suspensions were added to six-well culture
plates and were maintained in culture for 10 days. Crystal
violet was used to stain the colonies, and their vertical
gross appearances were imaged by digital photography.
MSC pluripotency differentiation assay
To investigate the effects of radial shockwaves on
multilineage differentiation capacity, MSCs in all groups were
induced to differentiate into osteoblasts, adipocytes, and
chondrocytes according to published protocols [
For osteogenic differentiation, MSCs were cultured in
24well plates at a density of 5 × 10 3cells/cm2 with
highglucose DMEM (HG-DMEM; Invitrogen) supplemented
with 10% FBS, 10 mM β-glycerol phosphate, 10−7 M
dexamethasone, and 50 μM ascorbate-2-phosphate. Alkaline
phosphatase (ALP) activity in MSCs was determined after
14 days of induction using an ALP assay kit
(SigmaAldrich) following the manufacturer’s instructions. For
adipogenic differentiation, MSCs were cultivated in
24-well plates at a density of 1 × 104/cm2 with
HGDMEM containing 10% FBS, 0.5 μM
3-isobutyl-1methylxanthine (IBMX), 10−7 M dexamethasone, and
10 ng/ml insulin for 14 days. Intracellular
accumulation of adipocyte lipids in MSCs was determined by
in-situ Oil-Red-O staining. For chondrogenic
differentiation, MSCs were seeded in 24-well plates at a
density of 1 × 104/cm2 and induced by HG-DMEM
containing 1% insulin, transferrin, and selenium (ITS),
10−7 M dexamethasone, 1 mM sodium pyruvate, 50 μ g/ml
proline, 50 μM ascorbate-2-phosphate, and 20 ng/ml
human transforming growth factor beta (TGF-β3) for 3 weeks.
The differentiated cells were identified by in-situ
Real-time quantitative PCR analysis
Total RNA was extracted from radial-shockwave-treated
and nontreated MSCs with TRIzol reagent (Fermentas)
and reverse transcribed using the mRNA Selective PCR
Kit (TaKaRa). In some experiments, the MSCs were
cultured in osteogenic, adipogenic, and chondrogenic
induction medium for 10 days before gene analysis.
Human Nanog, Oct-4, Sox-2, Runx-2, Osterix, CEBP/α,
PPARγ, Sox-9, and collagen type II (Col-II) cDNA were
amplified by real-time PCR using a SYBR PCR Master
Mix Kit (Sigma-Aldrich). The primer sequences are
presented in Additional file 1: Table S1.
Transmission electron microscopy analysis
To observe the ultrastructure changes of MSC
postshockwave treatment, transmission electron microscopy
analysis was performed. After the radial shockwave
treatment, MSCs were fixed with 2% glutaraldehyde in PBS
at 4 °C. Veronal acetate buffer (pH 7.4) was then applied,
and the samples were postfixed in 1% osmium tetroxide
for 1 h at 25 °C. Samples were stained with uranyl
acetate (5 mg/ml) and dehydrated in acetone, embedded in
Epon 812 (EMbed812; Electron Microscopy Science,
Hatfield, PA, USA). A Morgagni 268D transmission
electron microscope (FEI, Hillsboro, OR, USA) was
used to examine the ultrathin sections. The microscope
was equipped with an AMT TEM camera system
(AMT, Woolpit, UK), and the data were analyzed with
AnalySIS software (SIS, Soft Imaging System GmbH,
The healing effects of radial-shockwave-treated MSCs in vivo
To assess the healing effects of radial-shockwave-treated
MSCs in vivo, cells were combined with
polylacticcoglycolic acid (PLGA) scaffolds and implanted into the
cartilage defects of a rabbit model. To prepare
MSCPLGA constructs, shockwave-treated MSCs were first
seeded onto sterilized PLGA films to allow the MSCs to
adhere to the films. The MSC-PLGA constructs were
cultured in MSC medium for 8 h to improve cell
attachment before implantation.
The model of the rabbit knee cartilage defect was
designed following our previous protocol [
]. In brief,
rabbits were anesthetized by peritoneal injection of
ketamine/xylazine/buprenorphine. After shaving and
disinfecting, the knee joint was opened under sterile
conditions by medial parapatellar incision. The lateral
patella was then exposed followed by 120° joint flexion.
A round cartilage defect (4.5 mm in diameter) was
created in the weight-bearing area of the femoral trochlear
with a sterile trephine (external diameter 4.5 mm). To
make a full-thickness cartilage lesion, the subchondral
bone must be completely exposed.
Twenty skeletally mature and healthy New Zealand
White rabbits (male or female, 3–4 months old, 2–2.5
kg body weight) were used to prepare the model. The
animals were randomly divided into four different
groups as presented in Table 1. In brief, in group A,
cartilage defects were created on rabbit knees and received
no therapy. In group B, the same cartilage defects were
created and PLGA scaffolds were implanted into the
bone lesion. In group C, untreated MSCs were
combined with PLGA scaffolds and were implanted into the
cartilage lesion. In group D, scaffolds were combined
with radial-shockwave-treated MSCs and were
implanted into the cartilage lesion. In groups C and D, 1 ×
106 MSCs were implanted into each rabbit. In all groups,
the patella was repositioned after the operation, followed
by closure of the knee capsule with precise suturing.
Finally, the soft tissue flap and skin were closed in sutured
layers. After recovery from anesthesia, all animals were
allowed unrestrained daily activity in cages. In total,
80,000 U of penicillin were injected daily into all rabbits
for 3 days following surgery.
Pathological analysis of repaired tissues
Rabbits were sacrificed 8 weeks after surgery by
intravenous injection of a lethal dose of barbiturate. The
process was in compliance with the Animal Welfare Act.
The femoral condyles, including the trochlear defects,
were resected and harvested. After gross examination,
the samples were fixed in 10% neutral buffered formalin.
All of the samples were decalcified in
ethylenediaminetetraacetic acid (EDTA) for 30–45 days, and the defects
were dissected at an angle perpendicular to the surface
of the lesion. After decalcification, all of the samples
were dehydrated by successive concentrations of alcohol
(ranging from 70% to absolute). After washing in xylene,
the samples were embedded in paraffin and cut with a
microtome (RM2016; Leica Microsystems). Paraffin
blocks were cut into 2-μm sections and stained with
histochemical stain including hematoxylin and eosin
(H&E), Alcian blue, and Safranin O/Fast Green. To
observe the cell nucleus in the repair area,
4′,6-diamidino2-phenylindole (DAPI) staining was performed.
Type II collagen and proliferating cell nuclear antigen
(PCNA) immunohistochemical stains were used to further
identify the type and histologic origin of newly developed
tissues. The sample sections were deparaffinized,
rehydrated, and immersed in 5% H2O2 for 15 min at room
temperature. The sections were then put in a pressure
cooker with an EDTA retrieval solution at 140 °C for 3
min. After washing with phosphate-buffered saline (PBS)
for 5 min (repeated three times), the slides were incubated
at 37 °C with goat serum, followed by incubation with
anti-IgG (Abcam Biotechology, USA). Subsequently, the
slides were rinsed in PBS for 5 min (repeated three times)
and incubated with antibodies (Beijing, China) for 20 min
at 37 °C. Before incubation with horseradish peroxidase
(HRP), the slides were washed again in PBS three times.
Finally, the slides were incubated in
3,3′-diaminobenzidine tetrahydrochloride (DAB) solution and were
observed under the microscope.
The stained sections were scored using the Modified
International Cartilage Repair Society (ICRS) II histology
scoring system for assessment of cartilage repair (Table 2).
For assessment of vascularization in the layer of the
subchondral bone, the modified version was applied. All
histologic sections were observed using a dual-view
microscope. Two senior doctors examined the specimens
independently in a double-blind fashion, and a
doubleblind analyst calculated the final scores.
Data are presented as mean values with standard
deviations. Statistical significance was analyzed using Student’s
t test. P < 0.05 was considered significant.
Radial shockwave treatment promoted MSC proliferation but caused cell apoptosis
Research has increasingly demonstrated that multipotent
stem cells grown in floating culture systems exhibit
enhanced angiogenic, multipotent, and tissue regenerative
effects in vivo and in vitro. Therefore, in the current
study MSCs were stimulated by radial shockwaves in
suspension as shown in Fig. 1.
Cell proliferation assays and cell cycle analyses were
performed to explore the effects of radial shockwaves on
MSC growth. Trypan blue exclusion cell counting assays
(Fig. 2a) and CCK-8 cell growth assays (Fig. 2b)
demonstrated that radial-shockwave-treated MSCs exert
stronger proliferative effects than MSCs in control groups. In
addition, a dose–response relationship was observed
with shockwave doses ranging between 0 and 2 bars,
aDefect is defined as the area of the original cartilage defect adjacent to the
surface of the cartilage with 2 mm surrounding the defect and surrounding
the sides and bottom of the defect
bScores assigned on a 0–100 scale, 100 being normal
Fig. 1 Floating system of radial shockwave stimulation. MSCs harvested
and resuspended in 25 ml of culture medium in 100-mm cell culture
dishes received the energy of shockwaves directly. ESW extracorporeal
shockwave, MSC mesenchymal stem cell
with the 2 bars group displaying the highest vitality at 11
days. The data indicated that radial shockwaves enhance
MSC cell proliferation.
Enhanced cell growth is also reflected in the results of
the flow cytometry analyses, which showed an increased
number of cells in the S phase and a decreased number
of cells arrested in the G0/G1 phase (Fig. 3). Similar to
the results of the trypan blue exclusion cell counting
and CCK-8 assays, significant dose-dependent effects
were observed for shockwaves and the percentage of cells
in the S phase. There were significant differences between
specific treated groups and control groups (Table 3) (0 bar
vs 2 bars, P < 0.01; 0 bar vs 3 bars, P < 0.01). Thus, radial
shockwave treatment might promote MSC proliferation
by advancing the G1/S cell cycle transition. Moreover, the
rate of apoptosis increased with increased doses of
radial shockwaves. The MSCs in the 3 bars group showed
the highest percentage of the apoptosis phenomenon
Radial shockwave treatment increases MSC self-renewal in a dose-dependent fashion
MSCs can be distinguished from other seed cells that
are involved with skeletal regeneration primarily by
their multidifferentiation capacity and consistent
selfrenewal. These biological properties are also referred to
To explore the effect of radial shockwaves on MSC
self-renewal, the colony-forming unit fibroblast (CFU-F)
formation test was performed. The results showed
that radial-shockwave-treated MSCs generated more
abundant and larger cell colonies than untreated cells
(Fig. 4a, b). The promoting effects relied significantly on
the shockwave dose used in the treatment (Fig. 4a, b).
To investigate the underlying mechanisms of radial
shockwaves on the promotion of MSC self-renewal,
MSCs and radial-shockwave-treated MSCs were
analyzed with transmission electron microscopy. As shown
in Fig. 5, the MSCs possessed metabolically active
appearances after shockwave treatment at a dose of 2 bars.
In a gross view, the cell volume is seen to have become
larger, and the number of organelles is seen to have
increased (Fig. 5a). In addition, the Golgi apparatus and
endoplasmic reticulum were active in the treated group,
and the endoplasmic reticulum became increasingly well
ordered and extended (Fig. 5b). Associated with the
increased nuclear diameter was a significant increase of
nuclear content, and the kernel exhibited chromosome
replication (Fig. 5c). In addition to the modified Golgi
apparatus and endoplasmic reticulum, there was an
apparent increase in the number of mitochondria in the
treated group compared with the control (Fig. 5d).
In accordance with changes in CFU-F formation and
subcellular structures, the results of real-time quantitative
PCR showed that the transcript levels of the
selfreplication genes Nanog, Oct-4, and Sox-2 were markedly
higher in the radial-shockwave-treated MSCs than those
in the untreated MSCs (Additional file 2: Figure S1). This
suggests that radial shockwave treatment enhances the
self-renewing ability of MSCs.
Radial shockwave exhibited different effects on osteogenic, adipocytic, and chondrocytic differentiation of MSCs
To further explore the impact of radial shockwaves on
MSC stemness, multidifferentiation tests of MSCs were
performed. As shown in Fig. 6, strong ALP activity was
observed in radial-shockwave-treated MSCs following a
2-week period of osteogenic induction. In contrast,
weaker ALP expression was observed in untreated MSCs
(Fig. 6a). However, few Oil Red-O-positive lipid droplets
were observed in radial-shockwave-treated MSCs, while
many lipid droplets formed in MSCs of the control
group (Fig. 6a). In addition, no significant differences
were observed in the two cohorts of cells after
chondrogenic induction (Fig. 6a).
To further determine the effects of radial shockwaves
on MSC multipotency, the mRNA levels of Runx-2,
Osterix, CEBP/α, PPARγ, Sox-9, and Col-II were
examined by real-time PCR. Consistent with the results of the
histochemical staining, the radial-shockwave-treated
MSCs exhibited increased mRNA expression of Runx-2
and Osterix after osteogenic induction and decreased
mRNA expression of CEBP/α and PPARγ after
adipogenic induction. No significant change in mRNA
expression of Sox-9 and Col-II was observed after chondrogenic
induction (Fig. 6b). These data indicate that radial
shockwaves differentially regulate MSC differentiation into
osteocytes, adipocytes, and chondrocytes.
Radial-shockwave-treated MSCs enhanced the healing effect on rabbit cartilage defects
All animals were sacrificed at 8 weeks following surgery.
As shown in Fig. 7, the cartilage repair effect of each group
was clearly apparent as seen by the gross morphology.
The sham surgery group showed a completely unhealed
lesion and appeared to have full-thickness chondral
defects. In the scaffold-only group, the base of the lesions
was covered with fibrous tissues. When the scaffold
was seeded with untreated MSCs, there were marked
cartilage-repairing responses in the area of the lesions,
but the healing processes were relatively slow and the
lesions exhibited uneven articular surfaces. In the
scaffold seeded with radial-shockwave-treated MSCs, the
cartilage defects were completely healed. Regenerated
cartilage tissue had completely filled the defect, as seen
by the smooth glossy surface of the femoral trochlear
Further pathological analyses demonstrated the
different cartilage-repairing activities of each group in detail.
H&E and DAPI staining was employed to identify the
general characteristics of the repaired tissues (Fig. 8).
Alcian blue, Safranin O/Fast Green, and type II collagen
staining showed chondrocyte differentiation, and PCNA
staining revealed cell proliferation activity in vivo (Fig. 9).
The histological data showed that no cartilage appeared in
the defects of the sham surgery group and only a thin film
of chondrocyte-like connective tissue covered the defect
area implanted with the blank scaffold. In contrast,
chondrocyte-like tissue filled the defects engrafted by both
MSC-PLGA constructs. Most importantly, compared
with the other groups, the scaffold seeded with
radialshockwave-treated MSCs showed abundant mature
chondrocytes and a considerable amount of
proteoglycans in newly formed cartilage tissue. The regenerative
cartilage tissues in the defects of the
radial-shockwavetreated group were similar to normal cartilage tissues,
and the margins of the lesions were nearly absent.
Moreover, the results of the PCNA staining indicated
that radial shockwave treatment may result in more
proliferative tissues in vivo.
In addition, we evaluated the repair of cartilage defects
by the ICRS II histology scoring system for cartilage
repair. Table 4 shows that new cartilage formation in the
defects that were implanted with radial-shockwave-treated
MSC-PLGA constructs was significantly improved
compared to cartilage formation in the defects that were
implanted with the untreated MSC-PLGA constructs.
In the current study we demonstrated the promoting
effects of radial shockwaves on cell proliferation and
self-renewal. In addition, we found that radial shockwave
treatment enhanced the healing effects of MSCs on
cartilage defects in a rabbit model.
Although numerous reports have demonstrated that
shockwaves influence MSC biological properties, the
regulatory effects of shockwaves on MSCs are still
]. The controversial data in various
Fig. 4 Radial shockwaves promote colony-forming unit fibroblast formation of MSCs. a Comparisons of colony-formation efficiency. b Transformed
data prepared using ImageJ software. Cell number in each subgroup shown above. When cell number is 1 × 103 cells, there was no significant
difference among the groups. There was a slight difference when cell number is 5 × 103 cells. However, there is a significant difference when cell
number is 1 × 104 cells; the 2 bars group showed the highest colony-formation efficiency. CFU-F formation assay performed at least three times
independently; representative data from a single experiment shown. **Statistically significant difference compared with control groups, P < 0.001
studies may be due to highly heterogeneous cell origins
and variable shockwave application methods. To
exclude the potential impact of biological media such as
bio-gel and skin, a floating system was used in the
current study. Suspended MSCs were stimulated by
shockwaves to minimize energy loss and to reveal direct
effects on MSCs. In the present study, shockwave
treatment followed the previous protocol [
continuous pulse, 1000 impulses, and 5 Hz (total treatment
time 200 s).
Using this novel system, we found that radial
shockwave treatment significantly enhanced MSC
proliferation. Proliferation effects were confirmed by growth
kinetics assay, CCK-8 assay, and cell cycle analysis.
Notably, a significant dose-dependent shockwave effect
was observed for doses between 0 and 2 bars; MSCs in
the 2 bars group displayed the highest vitality after 11
days. In addition, the promoting effect lasted for nearly
12 h after the shockwave treatment according to previous
studies. These findings suggest that it is possible to
upregulate the growth efficiency of various seed cells that
are associated with regenerative medicine using
shockwave treatment in appropriate systems. However, our data
showed that direct application of shockwaves on MSCs
resulted in cell apoptosis. This result is consistent with
previous reports using equine adipose tissue-derived MSCs,
indicating that shockwave therapy may be a double-edged
sword and inappropriate applications of shockwaves may
lead to cell death.
Self-replication or self-renewal controls the stem cell
pool and determines the number of skeletal progenitors.
However, little information is available about the
effects of shockwaves on human bone marrow MSCs. In
the current study, MSC self-renewal was assessed by
the CFU-F formation assay, transcription expression
levels of key factors, and ultrastructural analyses of
MSCs. Compared with the expression of stem cell
surface markers, the CFU-F formation test assesses the
colony-forming function of the cells and provides a
more convincing evaluation of MSC self-renewal. Our
data demonstrated that radial shockwave treatment
augments colony formation of MSCs in a
dosedependent manner, strongly suggesting that these
mechanistic stimuli enhance MSC self-repopulation.
As expected, radial shockwave treatment resulted in
increased mRNA levels of the self-renewal genes
Nanog, Oct-4, and Sox-2, which supports the
hypothesis that radial shockwaves enhance the innate
selfrenewing activity of MSCs [
]. Although previous
studies were highly concerned about changes in the
biological behavior of stem cells after shockwave
stimulation, little is known about the changes in the
ultrastructure of these cells after treatment. In this
study, we found many changes of organelle structure
using transmission electron microscopy. The activation
of the Golgi apparatus and endoplasmic reticulum
suggests that protein synthesis increases after treatment,
and the enlargement of nucleoli and mitotic figures is
consistent with thriving proliferative phenomena. In
addition, there was a significant increase in the number of
Data presented as mean ± standard deviation. The International Cartilage Repair Society II scoring system was modified to assess cartilage repair and
vascularization in subchondral bone. A score of 100 indicates normal and 0 represents complete loss of normal architecture. Student’s t test applied to compare
the S + untMSC and S + tMSC group
Sham sham surgery group, S only scaffold only group, S + untMSC scaffold + untreated MSC group, S + tMSC scaffold + treated MSC group, MSC mesenchymal
*Statistically significant difference compared with control groups, P < 0.05
**Statistically significant difference compared with control groups, P < 0.01
mitochondria after shockwave stimulation, which also
indicates that stem cell metabolism had increased. Taken
together, the data indicate that radial shockwaves
modulate the stemness of MSCs by promoting self-replicating
Increasing evidence suggests that shockwaves direct
stem cell differentiation; however, this hypothesis has
not been fully investigated. Raabe et al. [
that in-vitro shockwave treatment promotes the
osteogenic, adipogenic, and chondrogenic differentiation of
equine adipose tissue-derived MSCs, but our analysis of
the adipogenesis-relevant, osteogenesis-relevant, and
chondrogenesis-relevant mRNA expression did not show
any significant differences between samples. In contrast,
Suhr et al. [
] found that while the adipogenic
differentiation was unaffected by shockwave treatment, the
osteogenic differentiation potential was slightly reduced
in vitro. In addition, they observed a more effective
chondrogenic differentiation of human bone marrow
MSCs after shockwave application. Moreover, Priglinger
et al. [
] demonstrated that shockwave therapy
significantly improved osteogenic and adipogenic
differentiation of human adipose tissue-derived cells, but no
difference in chondrogenic differentiation was visible
between control and shockwave-treated cells. In our
study, the MSC multilineage differentiation potential
was evaluated after direct treatment with radial
shockwaves. We found that radial shockwaves favored MSC
osteogenic differentiation but inhibited the adipogenic
activity of these cells. In addition, the chondrogenic
capacity of MSCs was not impaired by shockwave stimuli.
Our findings suggest that direct shockwave treatment is
helpful for maintaining MSC differentiation capacity for
bone and cartilage but might limit the regeneration of
Although in-vitro data demonstrated that radial
shockwave treatment significantly increased the number of
MSCs and did not impair their regenerative capacity for
skeletal tissues, it remains unknown whether treated
MSCs were sufficiently potent to improve osteochondral
formation in vivo. For improved healing in situ, porous
PLGA scaffolds were used in our study to provide a
surface and void volume upon which MSCs were able
to attach, proliferate, and differentiate as reported
previously. The general observations and histological
analyses of rabbit cartilage defects indicated that
radialshockwave-treated MSCs significantly enhanced and
promoted tissue repair. These results lead us to
reconsider the underlying mechanisms of shockwave therapy.
The data also suggest ideas for the development of novel
strategies in cartilage repair by virtue of boosting MSC
self-renewal. Although the mechanisms of
shockwavepromoting effects on MSC proliferation and
differentiation have been investigated in previous studies, the
underlying mechanisms of our findings need to be
revealed in further study.
There are still some limitations to the present study.
Only pressure and frequency can be regulated with the
radial shockwave generator that we used, thereby
limiting the different dose-dependent studies to these
parameters. In addition, we found that there is minor
cell loss during shockwave treatment. Moreover,
further investigations into the mechanisms of cell signal
transduction are needed to strengthen the findings.
Our studies demonstrate that radial shockwave
treatment is a potential tool for regulating MSC behavior and
show that these effects could be involved with MSC
cartilage regeneration. Therefore, this strategy appears to
have promising clinical relevance and may provide a
low-cost and effective treatment for cartilage repair.
Additional file 1: Table S1. Presenting primer sequences. (DOC 34 kb)
Additional file 2: Figure S1. Showing radial shockwaves increase
transcript levels of stemness markers. Transcript levels of
selfreplication genes Nanog, Oct-4, and Sox-2 were significantly higher in
the radial-shockwave-treated MSCs than in untreated MSCs (*P < 0.05).
Representative data from three separate experiments shown. (TIFF 1329 kb)
ALP: Alkaline phosphatase; CCK-8: Cell Counting Kit-8; CFU-F: Colony-forming
unit fibroblast; Col-II: Collagen type II; DAB: 3,3′-Diaminobenzidine
tetrahydrochloride; DAPI: 4′,6-Diamidino-2-phenylindole; DMEM: Dulbecco’s
modified Eagle medium; EDTA: Ethylenediaminetetraacetic acid;
ESW: Extracorporeal shockwave; FACS: Fluorescence activated cell sorting;
FDA: Food and Drug Administration; H&E: Hematoxylin and eosin;
HRP: Horseradish peroxidase; IBMX: 3-Isobutyl-1-methylxanthine;
ICRS: International Cartilage Repair Society; ITS: Insulin, transferrin, and
selenium; MEM: Minimum essential medium; MSC: Mesenchymal stem cell;
PBS: Phosphate-buffered saline; PCNA: Proliferating cell nuclear antigen;
PI: Propidium iodide; PLA: Chinese People’s Liberation Army; PLGA:
Polylacticcoglycolic acid; TGF-β3: Transforming growth factor beta
This work was supported by National Natural Sciences Grants (No. 81572159,
81371945) and the Beijing Natural Sciences Foundation (No. 7182123).
Availability of data and materials
The datasets used and/or analyzed during the current study are available
from the corresponding author on reasonable request.
HaZ performed the experiments and wrote the manuscript supervised by
Z-LL, HeZ, and QZ, who edited the manuscript. HaZ, X-ZS, JL, and NZ
performed the cell primary cell preparation. HaZ and FY performed scaffold
preparation and animal experiments. C-HL performed data interpretation
and manuscript editing supervised by NM. All authors read and approved
the final manuscript.
Informed consent was obtained from all patients for research purposes, and
experiments were approved by the Ethics Review Committee of the PLA
General Hospital. All animal experimental protocols were in compliance with
the Animal Welfare Act and were approved by the Animal Care and Use
Committee of the Laboratory Animal Research Center at the PLA General
Hospital (Reference number: 2015-X11-10).
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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