Archives of Oral Biology 118 (2020) 104860

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Archives of Oral Biology

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Low magnitude high frequency vibration promotes chondrogenic T

differentiation of bone marrow stem cells with involvement of β-catenin
signaling pathway
Weiwei Houa,b, Denghui Zhanga,b, Xiaoxia Fenga,b, Yi Zhoua,b,*
a
The Affiliated Stomatology Hospital, Zhejiang University School of Medicine, China
b
Key Laboratory of Oral Biomedical Research of Zhejiang Province, China

A R T I C LE I N FO A B S T R A C T

Keywords: Objective: Mesenchymal stem cells (MSCs) are well known to have the capability to form bone and cartilage, and
Bone marrow stem cells chondrogenesis derived from MSCs is reported to be affected by mechanical stimuli. This research aimed to study
Low magnitude high frequency vibration the effects of low magnitude high frequency (LMHF) vibration on the chondrogenic differentiation of bone
Chondrogenesis marrow-derived MSCs (BMSCs) which were cultured with chondrogenic medium, and to investigate the role of
β-catenin
β-catenin cascade in this process.
Cytoskeleton
Methods: Rat bone marrow-derived MSCs (BMSCs) were isolated and randomized into vibration and static
cultures. The effect of vibration on BMSCs proliferation, differentiation and chondrogenic potential was assessed
at the protein level.
Results: LMHFV did not affect the proliferation of BMSCs. However, this was accompanied by increased markers
of chondrogenesis. The protein expression of chondrocyte-specific markers of Aggrecan, Sox9, and BMP7 were
upregulated and Collagen X was decreased by LMHF vibration introduced at the chondrogenic differentiation in
vitro. Specifically, thicker blue-stained particles were observed in Alcian Blue staining and the level of glyco-
saminoglycan were significantly increased respectively in the vibration culture group by 56.5 % and 93.6 % on
the 7d and 14d. The expression and nuclear translocation of β-catenin were activated in a significant manner.
And inhibition of GSK-3β activity with Licl rearranged and intensified the cytoskeleton affected by vibration
stimulation.
Conclusions: Our data demonstrated that LMHF mechanical vibration promotes BMSCs chondrogenic differ-
entiation and implies β-catenin signal acts as an essential mediator in the mechano-biochemical transduction
and subsequent transcriptional regulation in the process of chondrogenesis.

1. Introduction better therapeutic outcome, many biochemical and biophysical means

Osteoarthritis (OA) is the most common form of arthritis, a disease
that can affect all the structures of the joints. The extensive pathologic
changes in OA include degradation of the articular cartilage and the
subchondral bone (Venetis et al., 2011). Cartilage tissue has a poor
ability to self-repair. Although the incidence of osteoarthropathy is very
high, to date, various conventional therapies have had limited effects
on the clinical repair and regeneration of the osteochondral defect
(Nöth, Steinert, & Tuan, 2008). Cell-based tissue engineering is believed
as a promising treatment for damaged or diseased cartilages, and bone
marrow derived mesenchymal stromal cells (BMSCs) are widely con-
sidered as a prospective cell source due to their rapid self-renewing
capacity and chondrogenic differential potential. In order to attain a
have been applied to enhance chondrogenic potential of BMSCs cul-
tured in two or three-dimensional environment (Hosseini, Tafazzoli-
Shadpour, Haghighipour, Aghdami, & Goodarzi, 2015; Li, Ng et al.,
2012; Li, Wang et al., 2012; Yang & Men, 2018).
As an important biophysical signal in bone mechanics conduction,
mechanical vibration has been revealed to be able to regulate the
synthesis of DNA and protein oligosaccharide in particular chon-
drocytes, and promote cartilage formation (2001b, Liu et al., 2001a),
which is an effective method to promote the proliferation of chon-
drocytes while maintaining the phenotype of chondrocytes (Waldman
et al., 2003). Low amplitude, high frequency (LMHF) vibration
(LM: < 1xg, g =9.81 m/s2; HF:20−90 Hz) loading via whole body
vibration, as a special model of mechanical stimuli, has been

⁎
Corresponding author at: The Affiliated Stomatology Hospital, Zhejiang University School of Medicine, China.
E-mail addresses: 7311009@zju.edu.cn (W. Hou), 21818696@zju.edu.cn (D. Zhang), fengxiaoxia@zju.edu.cn (X. Feng), zyuthscsa@zju.edu.cn (Y. Zhou).

https://doi.org/10.1016/j.archoralbio.2020.104860
Received 25 July 2019; Received in revised form 22 July 2020; Accepted 24 July 2020
0003-9969/ © 2020 Elsevier Ltd. All rights reserved.
W. Hou, et al.
demonstrated to enhance bone formation and bone healing (Rubin,
Turner, Bain, Mallinckrodt, & McLeod, 2001; Sehmisch et al., 2009; Shi,
Cheung, Qin, Leung, & Leung, 2010). It provides non-invasive and
cyclic mechanical stimulation, so it has been introduced as a non-
pharmacological intervention for bone regeneration all over the world.
This alternating motion also promotes cartilage formation between
bone fragments (Liu et al., 2001b). Articular cartilage is a specialized
load-bearing connective tissue which absorbs mechanical stress and
distributes loads evenly across the underlying bone surface. However,
the role of LMHF vibration in controlling MSC differentiation of chon-
drogenesis and osteogenesis during skeletogenesis and the intermediary
mechanism of LMHF vibration through which mechanotransduction
occurs in chondrogenic differentiation has not been intensively studied.
The Wnt family of signaling molecules is involved in a multitude of
developmental processes, including chondrogenesis. Enomoto et al.
reported that Wnt was involved in the formation and metabolism of
cartilage and bone, and that β-catenin dependent signals, which were
the main transduction pathway for Wnt, played a role in the differ-
entiation of chondrocytes (Enomoto-Iwamoto et al., 2002). In classic
signaling pathways, Wnt combined with cell surface receptor Frizzled
family, through the Dishevelled proteins to form antagonist β-catenin
degradation compounds (APC-Axin) which allowed Gsk-3β-inactivated
and then blocked β-catenin phosphorylation and ubiquitin. After
reaching a certain concentration, β-catenin molecules accumulated in
cell cytoplasm and transcription factor Tcf-4/Lef form into the nuclear
transcription complex, which started the transcription and expression
of downstream target genes (Li, Ng et al., 2012; Li, Wang et al., 2012).
Our previous study have reported that Wnt signaling was involved in
mechanotransduction at low-magnitude vibration to mediate sub-
sequent transcriptional regulation in osteogenic responses in osteo-
blasts (Hou, Zhu, Zhou, Zhang, & Yu, 2011; Zhang et al., 2016).
In this study, we hypothesized that LMHF vibration signals would
influence BMSCs toward chondrogenic differentiation through Wnt/β-
catenin pathway. We applied vibration to primary Rat MSCs derived
from bone marrow daily and investigated its effect on BMSCs’ pro-
liferation and differentiation. As LMHFV (0.49 g, 40 Hz) has been
shown to have an impact on osteogenic differentiating (Hou et al.,
2011; Zhang et al., 2016), we examined whether it might be useful to
affect chondrogenic differentiation. Therefore, if LMHFV could also bias
the fate of MSCs to chondrogenesis lineage, it would be beneficial for
the repair and regeneration of articular cartilage and subchondral bone,
and would also support the application of LMHF vibration in clinical
therapies more reasonably and safely.
2. Material and methods
2.1. Primary culture of rat BMSCs
The Institutional Animal Care and Use Committee of Zhejiang
University, Hangzhou, China, approved the animal experiments in this
study. Three-week-old male Sprague-Dawley rats were used. Isolation
and culture of rBMSCs were performed as described elsewhere (Hou
et al., 2011). Briefly, rBMSCs were aspirated from the bone marrow,
and the obtained cells were cultured in Dulbecco's modified Eagle's
medium (DMEM; Gibco, Beijing, China) containing 10 % fetal bovine
serum (FBS; Gibco, USA), 0.272 g/L L-glutamine (Sigma, USA), and 1%
antibiotic solution (penicillin and streptomycin (Gibco, USA). The
rBMSCs were cultured for three passages and then used in follow-up
experiments.
2.2. LMHFV application
The BMSCs were seeded in 6-well plates (5 × 103 cells/cm2) and
randomly divided into two groups: i) chondrogenic medium (control
group) and ii) chondrogenic medium and LMHF vibration (Vibration
group). The culture medium contained 0.1μL/mL−1 dexamethasone,
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Archives of Oral Biology 118 (2020) 104860
3μL/mL−1 ascorbate, 10μL/mL−1 ITS + Supplement, 1μL/mL−1 so-
diumpyruvate, 1μL/mL−1 proline, and 10 μL/mL−1 TGF-μ3 (Cyagen
Biosciences Inc., Guangzhou, China), which was replaced every 2 days
for 21 days. For the Vibration group, the plates cultured with BMSCs
were mounted on the platform of a GJX-5 vibration sensor (Beijing
Sendig Technology, Beijing, China), as previously described (Hou et al.,
2011; Zhang et al., 2016; Zhou et al., 2011), and subjected to me-
chanical stimuli (magnitude, 0.49 g; frequency, 40 Hz) for 30 min every
24 h for a period of 1,3,5,7,14 and 21days. After 30 min of vibration,
the cells in the LMHF group received fresh chondrogenic medium. Vi-
bration and induction were carried out in the same period. And the
mechanical vibration device was in the incubator during the test.
2.3. Alcian blue staining
After 7 days vibration, according to the manufacturer’s instructions
(Cyagen Biosciences Inc., Guangzhou, China), a standard pellet culture
was performed. The culture medium was changed every three days. In
this 3D culture environment, chondrogenic induction for 28 days, then
the pellet was fixed in 4% paraformaldehyde, embedded in optimum
cutting temperature compound (OCT), and cut into slices. Then the
slices were stained with Alcian Blue for 30 min, washed three times
with 1x PBS, and observed under the phase-contrast microscope. The
static cultured-pellet as control.
2.4. Cell proliferation assay
Alamar blue assay is a colorimetric assay for the determination of
viable cell numbers. BMSCs with a density of 1500 cells/well were
seeded in 96 well plates, cultured in the chondrogenic medium. The
proliferation of BMSCs was assessed with the alamar blue assay (Bestbio
Laboratories, Shanghai, China), following manufacturer’s protocol. The
cell proliferation was assessed by 570 nm OD values with the micro-
plate reader (Varioskan Flash; Thermo Fisher Scientific, MA, USA).
2.5. Cell cycle assay
After1, 3, 5, 7 days of LMHF mechanical vibration stimulation, the
cells were detached from the culture plate 24 h after the last stimulation
cycle with trypsin and EDTA. The collected cells were then centrifuged
at 2000 g for 5 min with PBS; this step was repeated twice to wash the
cells. Then the cells were fixed with 70 % (w/v) ice- cold ethanol at 4–8
℃ overnight. The cells were washed with PBS and centrifuged at 1000 g
for 8 min prior to staining. Next, 100 mL of RNAase A was added for 30
min at 37.8℃, following which 300 mL of propidium iodide (PI)
staining solution (0.1 % Triton X-100, 10 mg/mL PI in PBS) was added
for 30 min at 4–8℃. The cell cycle in each sample was analysed using
flow cytometry (Beckman Coulter, Indianapolis, IN, USA).
2.6. Enzyme-linked immunosorbent assay for glycosaminoglycan
To quantify and compare the concentration of glycosaminoglycan
(GAG) in cell cultured supernatants, commercially available enzyme-
linked immunosorbent assay (ELISA) kits were used according to the
manufacturer’s recommendation. Constitutive GAG secretion was ana-
lyzed for 24 h after the last stimulation cycle of the 7th and 14th day in
culture, and expressed as ng/mL protein. A Rat glycosaminoglycan/
mucopolysaccharide ELISA kit (Cusabio, China) was used to evaluate.
2.7. Western blot analysis
Equal amounts of total cellular protein were resolved using 10 %
SDS-PAGE and transferred onto polyvinylidene difluoride membranes
(Millipore, Bedford, MA, U.S.A). The blots were incubated with 1:1000
dilutions of primary antibodies for 1 h. The murine monoclonal anti-
bodies (anti-Sox-9, anti-Collagen X, anti-β-catenin and anti-BMP7) were
W. Hou, et al.
acquired commercially (Abcam, UK). Visualization of the im-
munoreactive bands was achieved by incubation of the membranes
with horseradish peroxidase-conjugated secondary antibodies (Santa
Cruz Biotechnology, Inc) for 1 h. Immunoreactive protein bands were
visualized using a chemiluminescence detection kit (Millipore, Bedford,
MA, USA). Signal intensities were quantified by densitometry (Quantity
One, Bio-Rad, USA).
2.8. Immunofluorescence (IF) staining
Cells with or without LMHFV application, were seeded on 6-well
plate. After adherence, they were washed in PBS, fixed with 4% buf-
fered (0.01 mol/L PBS, pH 7.2, 0.1 % DEPC) paraformaldehyde for 30
min at room temperature, and blocked by 0.5 % bovine serum albumin
(BSA) for 15 min. After overnight incubation with rabbit anti-β-catenin
polyclonal antibody (1:60) (Abcam, Cambridge, UK) at 4℃, cells were
subsequently incubated with secondary antibody conjugated to
Rhodamine (Hebei Bio-high Technology Deve co., Hebei, China) for 20
min at 37℃. After being rinsed in PBS, cells were observed under the
Olympus IX 71 inversed fluorescent microscope (Olympus, Tokyo,
Japan). Images were captured by Image-Pro Plus version 6.0 (Media
Cybernetics) and analyzed by Image J software (National Institute of
Health, Maryland, USA).
2.9. FITC-phalloidin staining for detecting cytoskeletal organization
To confirm the involvement of β-catenin in the cytoskeletal orga-
nization of BMSCs, Licl (Byotime, Shanghai, China), a highly selective
inhibitor of GSK-3β (Clément-Lacroix et al., 2005) was used. BMSCs
were placed in chondrogenic medium with 10 u M Licl for 2 h before
exposure to LMHF vibration. The inhibitor was replenished together
with the medium in the following days.
BMSCs were washed twice with PBS, fixed with 4% paraformalde-
hyde for 15 min, permeabilized with PBS containing 0.2 % Triton X-100
for 15 min and blocked with 5% BSA. Then, after FITC-phalloidin
(Sigma) was incubated for 30 min, DAPI was used as nuclear staining.
All images were visualized and photographed with a fluorescence mi-
croscope under the same conditions of excitation and registration.
Multiple cells were categorized in each experimental point.
2.10. Statistical Analysis
All assays were repeated in three independent experiments with a
minimum of n = 3 per group. The results were expressed as
mean ± SD. Statistical analysis to compare the results between groups
was carried out by one-way ANOVA and the Independent-samples T test
with SPSS software, version 17.0 (SPSS Inc., Chicago, IL, USA).
Statistically significant values were defined as p < 0.05.
3. Results
3.1. Effects of LMHF vibration on morphogenesis of BMSCs
In order to elucidate the influence of vibratory load on the mor-
phological change of BMSCs during chondrogenic induction process,
these cells were subjected to periodic vibratory loading at 40 Hz, 0.49 g
for 30 min per day. After chondrogenic induction for 7 days, the mor-
phology of BMSCs showed asteroid or spindle shape with slim bodies
similar to fibroblasts in all experimental processes, and the vibrated
BMSCs exhibited more denser clusters than controls (Fig. 1).
3.2. Effect of LMHF mechanical vibration on BMSCs proliferation
To assess LMHFV’s effects on cell proliferation, A alamar blue-kit
was applied on 1, 3, 5, 7 and 14 days. The results showed that in both
group, cell proliferation was increased gradually as time increased until
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Archives of Oral Biology 118 (2020) 104860
the day 14 (Fig. 2). The proliferation of vibrate group was low at initial
time, quickly increased on day 3 (P < 0.05), while the proliferation of
control group increased significantly until the day 7 (P < 0.05). Both
groups attained the peak on day 7 (Control: P < 0.05; Vibration:
P < 0.01), and then decreased on the day 14. However, there was no
statistical difference between the two groups at every time points.
3.3. Effect of LMHF mechanical vibration on cell cycle progression
To further confirm the effects of LMHF mechanical vibration on
BMSCs proliferation, its impact on BMSCs’ cell-cycle was assessed by
flow cytometry. As shown in Fig. 3, compared to the 1th day, the
proliferative index significantly increased with time. These results
paralleled the results of alamar blue assay, which confirmed the same
trend in BMSCs under between vibration and control of the cells. The
increasement of PI (Fig. 3A) of the cells in the experimental group were
higher 25 % (P < 0.01) and 20 % (P < 0.05) than that of the static
control group on the 5th and 7th day, while the significant increase-
ment of SPF were on the 3th and 5th day (P < 0.0005; Fig. 3A). For
reference purposes, the DNA content histograms of different time points
were shown in Fig. 3C.
3.4. Effect of LMHF mechanical vibration on glycosaminoglycan during the
chondrogenic process of BMSCs
Increased glycosaminoglycan expression were markers of chondro-
genic differentiation. To investigate whether LMHF mechanical vibra-
tion affected BMSCs commitment to chondrogenic differentiation, gly-
cosaminoglycan levels was measured. As shown in Fig. 4A, total
amount of glycosaminoglycan was significantly increased respectively
in the vibration culture group by 56.5 % and 93.6 % on the day 7 and
day 14 (P < 0.0005).
In Alcian Blue staining, a visible cartilage sphere formed after
chondrogenic induction for 28 days. The pellet was cut into slices and
stained with Alcian Blue. This dye could stain glycosaminoglycans in
cartilages. Compared to the static culture group, thicker blue-stained
particles were observed in vibration culture group, which incubation
after 7 days vibration. Total areas of blue-stained were significantly
increased in the vibration culture group (Fig. 4B).
3.5. Western blot analysis expression of chondrogenesis related protein was
promoted by LMHFV during chondrogenic induction
We used Aggrecan and Sox9 to represent chondrogenesis related
protein. Western blot on day 1 revealed that Aggrecan and Sox9 re-
spectively increased 5.37 and 4.32 fold under LMHF vibration
(P < 0.0005; Fig. 5C and E). The expression of Sox9 was significantly
higher than the control at each point in time, and reached its peak on
the 7th day, while the peak of Aggrecan on the 3th day and began to
decline on the 21th days (P < 0.0005; Fig. 5E). Moreover, Collagen X
levels showed the opposite trend to Aggrecan levels, as expected.
Compared to the control group, vibration significantly reduced the le-
vels of the Collagen X protein on days 1, 3 and 5 (P < 0.0005; Fig. 5C).
Similarly, Vibration increased β-catenin levels during days 1–14 com-
pared to the control group (P < 0.0005; Fig. 5), while on the 21th day,
the protein expression was no significant difference. From the above
results, vibration could improve the early process of cartilage differ-
entiation in BMSCs.
To further confirm the effect of LMHF mechanical vibration on
BMSCs in the early stage of differentiation, we examined the levels of
the β-catenin and p-β-catenin protein (Fig. 6A). Compared to the con-
trol group, vibration also significantly decreased the levels of the
phosphorylated protein on days 1 (P < 0.0005), 3 (P < 0.05), 5
(P < 0.01) and 7 (P < 0.0005; Fig. 6B). The ration of p-β-catenin/ β-
catenin gradually decreased over time, which meant the activity of β-
catenin increased, while the expression of BMP7 significantly increased
W. Hou, et al. Archives of Oral Biology 118 (2020) 104860

Fig. 1. Effect of LMHF mechanical vibration on morphogenesis of BMSCs. (x100).

Fig. 2. Effect of LMHF mechanical vibration on BMSCs proliferation. A trend towards increased cell proliferation was gradually with increased time until on the 14th
day in both groups, though there is no significant difference compared with the control group at every time point. Bars represent the mean+ standard deviation (n =
3). * p < 0.05; **p < 0.01; *** p < 0.0005.

4
W. Hou, et al.
Fig. 3. Cell cycle analysis of BMSCs at different time points of vibration. Analysis by flow cytometry assay indicated that (A) proliferation index (PI) and (B) S-phase
fraction cell ratio (SPF) of experimental cells were lower than that of control cells (Co) on the 1th and 3th days, while higher on the 5th and 7th days. The DNA
content histograms for each group at different time points were shown. (C). Bars represent the mean + standard deviation (n = 3); * p < 0.05; **p < 0.01; ***
p < 0.0005.
Fig. 4. Glycosaminoglycan level in BMSCs cell lysates after different time points
of LMHF mechanical vibration. (A) Glycosaminoglycan activity (ng/mL) of vi-
brated rat BMSCs significant increased on the 7th and 14th day, as compared to
control cells (Co). (B) Alcian Blue staining of differentiated rat BMSCs on the
28th day. Each bar represents the mean + standard deviation (n = 5); *
p < 0.05; **p < 0.01; *** p < 0.0005. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this
article).
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Archives of Oral Biology 118 (2020) 104860
from the first day (P < 0.01; Fig. 6C), same as the result of other
chondrogenic protein.
3.6. LMHF vibration increased the nuclear translocation of β-catenin in
BMSCs
Next, we conducted immunofluorescence staining to explore whe-
ther the stimulatory effect of vibration on the chondrogenic of BMSCs
was related to the induction of the nuclear translocation of β-catenin
(Fig. 6D). The fluorescence microscopy images of immunolabeled cells
showed lower levels of β-catenin in the nucleus of the control group
than in vibration group (white arrow). The LMHF vibration obviously
induced the translocation of β-catenin (green) to the nucleus (blue).
3.7. LMHF vibration increased the rearrangement of the distribution of the
actin cytoskeleton
The effect of LMHF vibration on cytoskeletal organization was in-
vestigated on the day 3. As shown in Fig. 7, actin filaments were clearly
visible after stained with the F-actin antibody in vibration group, which
showed more prominent than that in the control group. After adding
Licl, the fluorescence intensity of control group was as strong as the
vibration group. However, the vibration with Licl group was the most
significant (P < 0.05).
4. Discussion
In our experiment using a sinusoidal acceleratory movement in the
horizontal planes, a two-dimensional, predominantly vibratory force
was conducted to cultures of BMSCs. Compared with the control group,
the vibration group showed a significant higher chondrogenic effect,
whether inducted in the three-dimensional pellet-forming culture or
continuous induction in the two-dimensional environment. The ob-
served effects were a direct result of BMSC cells being subjected to vi-
bration stress, as our experimental set-up eliminated fluid flow-induced
W. Hou, et al.
Fig. 5. The effect of LMHF mechanical vibration on chondrogenic protein expression in BMSCs. Sox9 and Aggrecan protein expression level increased while Collagen
X decreased compared to control (Co). β-catenin protein expression levels also increased. Each bar represents the mean + standard deviation (n = 3). * p < 0.05;
**p < 0.01; *** p < 0.0005.
effects from vibration effects by minimizing the movement of fluid in
the system. Additionally, fluid shear stress (i.e., contact stress primarily
acting on the cell membrane) might only be linearly rate-dependent at
low frequencies (< 10 Hz), because fluid flow might attenuate at high
frequencies (> 10 Hz) (Bacabac et al., 2006). However, the vitro model
might not replicate the in vivo environment as it lacks other cell types
and the fluid entirely filled with the bone marrow cavity is 40–400
times more viscous than water (Gurkan & Akkus, 2008).
The chondrogenesis process is characterized with expression of a
cascade of stage-specific markers in each stage. Aggrecan, a large ag-
gregating proteoglycan (PG) that binds to hyaluronan leading to a super
molecular complex, is one of the major structural components of car-
tilage (Watanabe, Yamada, & Kimata, 1998). BMP7 plays a key role in
the transformation of mesenchymal cells into bone and cartilage, pro-
moting the synthesis of Coll 2 and AGG in cartilage cells (Papanna et al.,
2012). Aggrecan and BMP7 are accepted as markers for phenotypic
expression of chondrocyte. Both of two genes are regulated by SRY-
related high mobility group-box gene 9 (Sox9), a master transcription
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Archives of Oral Biology 118 (2020) 104860
factor which is characterized by being expressed in mesenchymal
condensations both before and during the deposition of cartilage matrix
(Kawakami, Rodriguez-León, & Izpisúa Belmonte, 2006; Zhang et al.,
2015). Sox9 activates extracellular matrix genes such as Col2A1 and
regulates chondrocyte differentiation (Bell et al., 1997; Lefebvre,
Huang, Harley, Goodfellow, & de Crombrugghe, 1997). The application
of cyclic tensile strain or fluid flow was also reported to significantly
stimulate the expression levels of Sox9 in undifferentiated hMSCs
(Friedl et al., 2007) and C3H10T1/2 murine MSCs (Arnsdorf, Tummala,
Kwon, & Jacobs, 2009). Our studies found that LMHF vibration resulted
in an increase in chondrogenic markers Aggrecan (Acan), BMP7, SOX9
and a decrease in the expression of hypertrophic marker X-type Colla
(COL10A1), which is accepted as an important regulator for chon-
drocyte differentiation and maturation (Garvican, Vaughan-Thomas,
Innes, & Clegg, 2010; Mueller & Tuan, 2008).
During the development of chondrocytes, Sox9 promotes the dif-
ferentiation of mesenchymal cells into chondrocytes and inhibits the
differentiation of chondrocytes into hypertrophic chondrocytes
W. Hou, et al. Archives of Oral Biology 118 (2020) 104860

Fig. 6. The effect of LMHF mechanical vibration on the protein expression levels of β-catenin and BMP7 in BMSCs(A-C). p-β-catenin/β-catenin proteins ration
decreased and BMP7 protein levels increased. Representative IF staining showing that the translocation of β-catenin (green) to the nucleus (blue) was induced on the
3th day by vibration in BMSCs(D). Each bar represents the mean + standard deviation (n = 3). * p < 0.05; **p < 0.01; *** p < 0.0005. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article).

(Nalesso et al., 2011). This may explain the reason that the expression
of Sox9 decreased on 14th day in chondrogenic induction of BMSCs in
LMHF vibration. On the other hand, Aggrecan and BMP7 levels showed
the similar trend to Sox9 levels, but in contrast to Collagen X, as ex-
pected. From the above results, LMHFV promoted chondrogenic dif-
ferentiation of BMSCs and an inhibition effect of LMHFV on hyper-
trophy during chondrogenic differentiation. The protein result was
further confirmed by glycosaminoglycan activity assay and analysis of
the blue stained particles.
As an important regulatory factor for Wnt signal transduction, β-
catenin might act through specific mechanisms that allow for sequential
modification during the chondrogenic differentiation process. In our
study, the expression and the nuclear translocation of β-catenin in
BMSCs induced by LMHF vibration were promoted while the phos-
phorylation level of β-catenin was decreased and so promoted the total
β-catenin activity. Previously published studies also showed that
vibration activates the production of proteoglycan in 3-dimensional
cultured chondrocytes and stimulates β-catenin (Takeuchi et al., 2006).
The activation of β-catenin-mediated wnt signals causes increased
cartilage formation and a lack of β-catenin-mediated signals could in-
hibit chondrocytes differentiation (Chen et al., 2007). Our study further
suggested that chondrogenic markers expression was increased with the
β-catenin activity by LMHF vibration. But there were opposite studies
that showed that the activation of the WNT pathway resulted in a de-
crease in type II collagen (COL2A1), Aggrecan (Acan), SOX9, an in-
crease in the expression of X-type Colla (COL10A1), and had the effect
of promoting cell dedifferentiation (Nalesso et al., 2011). Therapeutic
inhibition wnt/β-catenin signal maintains phenotypic stability and
cartilage formation in chondrocytes (Niu et al., 2016). This discrepancy
is likely due to different microenvironments, in particular the inherent
cell-type and mechanical parameters.
The role of β-catenin signaling in mechanotransduction seems to be

Fig. 7. Cytoskeletal in BMSCs cell on the 3th day of LMHF mechanical vibration with or without Licl.(x100, x400).

7
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quite complicated and could be due to a wide range of potential me-
chanisms. As a component of cell-cell adhere junctions, β-catenin plays
an important role in linking E-cadherin to the actin cytoskeleton
(Kobielak & Fuchs, 2004). The cytoskeleton is suggested to be involved
in transducing mechanical signals applied to the cell surface into in-
tracellular signals necessary for increased gene expression and new
bone formation (Pavalko et al., 1998). Phalloidin is a specific marker
for actin fibers. In the present study, phalloidin staining revealed the
stimulation of BMSCs by LMHF vibration resulted in rearrangement of
the actin cytoskeleton with more prominent F-actin, and the fluores-
cence intensity was significantly stronger. A similar actin fiber pattern
has been observed in myoblasts following exposure to cyclic strain
(Zheng et al., 2008). This may be because β-catenin could strengthen
the attachment of E-cadherin to the cytoskeleton (Nagafuchi, 2001).
The development of actin fibers was the most notable in the group
treated with both Licl and vibration, of which morphologically ex-
hibited stretched and many thicker prominences. Licl evidently in-
tensified the stimulatory effect of vibration, as Licl almost completely
blocked the phosphorylation and degradation of β-catenin.
A change in the proliferation index could reflect an increase in the
rate of the cell cycle, the number of cells entering S phase during the
labeling period, or both (Stanford, Morcuende, & Brand, 1995). In our
LMHF vibration studies, the proliferation index was increased with
time, though with a maximum increase of only 25 % compared to
control (non-vibrated) cultures on the 5th day. This would explain the
result of colorimetric assay, in which there was no significant difference
between vibration and control groups. Lau et al. also reported that
LMHFV did not affect the proliferation of BMSCs (Lau et al., 2011).
Some studies found LMHFV increased BMSCs proliferation in the
growth medium (Kim, Song, Lee, & Hwang, 2012; Lu et al., 2018) while
inhibited proliferation in the osteogenic medium, in which most studies
have confirmed LMHF vibration promotes osteogenic differentiation of
MSCs (Chen et al., 2016; Lu et al., 2018; Prè et al., 2013; Zhou et al.,
2011). Given the culture medium has an important role for the cell
differentiation, LMHFV may not be the key factor to decide which
lineage BMSCs differentiate to, but it may act as a promotion factor of
BMSCs differentiation.
Taken together, this culture system seems to be suitable for a po-
tential application in chondrogenic differentiation of BMSCs. Whether
LMHF vibration can become a beneficial alternative strategy for the use
of chondrogenic medium, and the combination of mechanical stimu-
lation with growth factor treatment for different MSC sources, the op-
timal mechanical parameters and induction progress, further researches
should be needed for establishing more efficient, simpler, and safer
inducing methods to promote its utilization in MSC-based cartilage
tissue engineering and promote cartilage repair in an osteochondral
defect model.
Author contributions
Weiwei Hou put forward the conception and design of the study,
contributed to the acquisition of data, analysis and interpretation of
data and drafted the article. Denghui Zhang, Xiaoxia Feng contributed
to the acquisition of data and revised it critically for important in-
tellectual content. Yi Zhou directed and determined the topic, designed
the study, revised and finally approved the manuscript. All authors
have read and approved the final article.
Ethical approval
Research was approved by the Institutional Animal Care and Use
Committee of Zhejiang University, Hangzhou, China.
8
Archives of Oral Biology 118 (2020) 104860
Declaration of Competing Interest
All authors report no potential conflict of interest.
Acknowledgments
This work was supported by the Zhejiang Provincial Natural Science
Foundation of China (grant nos. LQ18H140002, LQ15H140003 and
LY19H140003) and the Medical Health Science and Technology Project
of Zhejiang Provincial Health Commission (Grant nos. 2018264007).
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