ORIGINAL RESEARCH ARTICLE

Journal of
1935
Cellular
Physiology
Cell Proliferation is Promoted by
Compressive Stress During
Early Stage of Chondrogenic
Differentiation of Rat BMSCs
YATING WANG,1 JUN WANG,1 DING BAI,1 JINLIN SONG,2 RUI YE,1 ZHIHE ZHAO,1 LEI LEI,1
JIN HAO,1 CHUNMIAO JIANG,1 SHANBAO FANG,1 SHU AN,1 QIAN CHENG,1 AND JUAN LI1*
1
Department of Orthodontics, State Key Laboratory of Oral Diseases, West China Hospital of Stomatology,
West China School of Stomatology, Sichuan University, Chengdu, 610041, China
2
Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences, The Affiliated Hospital of Stomatology,
Chongqing Medical University, Chongqing, 401147, China

The presence of an appropriate number of viable cells is prerequisite for successive differentiation during chondrogenesis. Chondrogenic
differentiation has been reported to be influenced by mechanical stimuli. This research aimed to study the effects of cyclic compressive
stress on cell viability of rat bone marrow-derived MSCs (BMSCs) during chondrogenesis as well as its underlying mechanisms. The results
showed that dynamic compression increased cell quantity and viability remarkably in the early stage of chondrogenesis, during which the
expression of Ihh, Cyclin D1, CDK4, and Col2a1 were enhanced significantly. Possible signal pathways implicated in the process were
explored in our study. MEK/ERK and p38 MAPK were not found to function in this process while BMP signaling seemed to play an
important role in the mechanotransduction during chondrogenic proliferation. In conclusion, dynamic compressive stress could enhance
cell viability during chondrogenesis, which might be achieved by activating BMP signaling.
J. Cell. Physiol. 228: 1935–1942, 2013. ß 2013 Wiley Periodicals, Inc.

Cartilage is recalcitrant to endogenous repair and regeneration
(Ryan et al., 2009). Cell-based tissue engineering is believed as a
promising treatment for damaged cartilage. It’s a phased
process, which begins with aggregation and condensation of
mesenchymal chondroprogenitor cells, following a progression
toward proliferation, prehypertrophy, and hypertrophy
(Goldring et al., 2006; Zuscik et al., 2008). The presence of
an appropriate number of viable cells is prerequisite of
successive chondrogenesis. Richter’s study (Dexheimer
et al., 2012) suggests that high cell metabolism could guarantee
more cells survive the shift from 2D-proliferation to
condensation and determination to the chondrogenic
lineage. However, due to high cell density, low nutrient,
and oxygen conditions in culture, unwanted cell loss restricts
tissue engineering of cartilage in vitro. Thus prevention of
early apoptosis and stimulation of cell proliferation, especially
cell viability, are attractive goals to achieve better
chondrogenesis.
Efforts have been made for the improvement of the
chondrocyte differentiation, such as modification of culture
system (Park et al., 2010; Wei et al., 2012), supplement with
chondrogenic cytokines (Opolka et al., 2012) as well as
extrinsic physical stimuli (Ebisawa et al., 2004; Li et al., 2012b).
Among them, mechanic loading has been widely accepted as an
effective enhancement for chondrogenesis. Studies (Wu
et al., 2001; Fanning et al., 2003; Haudenschild et al., 2009; Kelly
and Jacobs, 2010) show that Ihh, MAPKs, and JNKs act as
essential mediators in the mechano-biochemical transduction
and subsequent transcriptional regulation in the process of
chondrogenesis. Our previous research (Li et al., 2009) also
indicates p38MAPK signaling is important in transduction of
mechanical signals during chondrogenesis. Besides, by inter-
acting with the TGF-b1/Smads signaling pathway, MAPK
subtypes seems to regulate chondrogenesis with a delicate
balance (Li et al., 2010b).
Meanwhile, it’s reported (Ryan et al., 2009) that prolifera-
tion of chondrocyte is regulated by ERK1/2 and p38MAPK,
which play opposing roles. The ERK pathway may be a negative
regulator of Sox9 mRNA expression in hydrostatic pressure-
induced mechanotransduction (Mio et al., 2007). Indian
hedgehog (Ihh) in prehypertophic chondrocytes has also
been shown to be indispensible in regulation of chondrocyte
proliferation (Long et al., 2001; Minina et al., 2002). In parallel
to Ihh, BMP signaling induces chondrocyte proliferation, which
is mediated by heteromeric complexes of type I (BM PR1a and
Abbreviations: BMP, bone morphogenetic protein; CDK4, cyclin-
dependent kinase 4; Col2a1, collage type II alpha 1; Cyclin D1, D-
type cyclins D1; ERKs, extracellular-signal-regulated kinases; Ihh,
Indian hedgehog; JNKs, c-Jun N-terminal kinases; MEK, mitogen-
activated protein kinase kinase; p38MAPK, p38 Mitogen-activated
protein kinase; rBMSCs, rat bone marrow-derived mesenchymal
stem cells
Contract grant sponsor: National Natural Science Foundation of
China.
Contract grant sponsor: Science and Technology Fund of Sichuan
Province;
Contract grant numbers: 30900286, 81030034, 2011SZ0096.
* Correspondence to: Juan Li, Department of Orthodontics, State
key laboratory of oral disease, West China School of Stomatology,
Sichuan University, 14#, 3rd Section, Renmin South Road, Chengdu
610041, PR China. E-mail: lijuan@scu.edu.cn
Manuscript Received: 10 November 2012
Manuscript Accepted: 11 March 2013
Accepted manuscript online in Wiley Online Library
(wileyonlinelibrary.com): 29 April 2013.
DOI: 10.1002/jcp.24359

2 0 1 3 W I L E Y P E R I O D I C A L S , I N C .
1936 WANG ET AL.
BMPR1b) and type II (BMPR2) receptors. Loss of both BMPR1a
and 1b leads to hypoplastic cartilage anlagen, indicating BMPs as
positive regulators of cell cycle progression (Wuelling and
Vortkamp, 2011).
Relevance of mechanic loading to chondrocyte proliferation
might exist as the same cellular pathways and regulators are
shared by cell proliferation and mechano-biochemical
transduction. Indeed, a number of previous studies have
demonstrated the sensitivity of chondrocyte proliferation to
mechanical perturbation with divergences among them. The
application of static compression inhibits the proliferation of
chondrocytes cultured in vivo (Takahashi et al., 1998) and in
vitro (Lee and Bader, 1997; Elder et al., 2000) whereas dynamic
compression (De Witt et al., 1984; Lee and Bader, 1997)
increases DNA synthesis. However, proliferation and differ-
entiation are phase-specific and linked closely during chon-
drogenesis, in which proliferation would be restrained once
the differentiation initiated. It’s reported by Saitoh et al. (2000)
that compressive force could promote chondrogenic differ-
entiation and hypertrophy. If the mechanic stress brought
forward the differentiation, it would be still unable to achieve
cartilage engineering in vitro due to unbalanced proliferation
and differentiation.
Therefore, the purpose of this study is to find out that
whether dynamic compression would have any effect on the
cell viability in the stage of proliferation during chondrogenesis
of BMSCs, as well as the underlying mechanisms.
Materials and Methods
Primary culture of rat BMSCs
Cell collection and culture were conducted according to previous
study (Li et al., 2010a). Briefly, rat BMSCs were harvested from
bone marrow of posterior tibias and femurs of 2-week-old SD rats.
After centrifugation at 200g for 10 min, cells were resuspended in
a-MEM (Gibco, Grand Island, NY) supplemented with 10% FBS
(fetal bovine serum, Gibco). Non-adherent cells were removed
from plates 48 h later by changing the medium. At confluence,
primary cultures were detached using trypsin treatment and
subcultured at 8
103 cells/cm2. All experiments were performed
according to institutionally approved guidelines for animal welfare.
BMSCs were identified by detecting expression of CD34, CD44,
CD54, and CD105 and multipotent differentiation (data not
shown).
In vitro chondrogenic induction
Chondrogenic differentiation of the BMSCs was initiated in
micromass culture system (Li et al., 2010a). Briefly, passage two
cells were harvested by a treatment of 0.25% trypsin and 0.02%
EDTA. Suspension at a density of 2
106 cells/ml was seeded in
96-well plate with 2 l in each well to form cell aggregates at 37˚C for
2 h; then they were supplemented with a-MEM containing 10%
FBS. 24 h later, culture medium was replaced with serum-free
high-glucose DMEM (Gibco) supplemented with 10 ng/ml
recombinant human TGF-b1 (Peprotech, Rocky Hill, NJ), 107 M
dexamethasone (Sigma, St.Louis, MO), 50 mg/ml ascorbic acid
phosphate (Sigma), and 1% ITS-A (Gibco). Control cells were
cultured in high-glucose DMEM supplemented with 1% FBS.
Micromass cultures were performed for 14 days with culture
media replacement of every 3 days.
Chondrocyte proliferation assays
Cell number was measured directly by cell counting. Briefly, cells
were trypsinized with 0.05% trypsin and collagenase, and cell
numbers were counted directly by a hemocytometer (American
Optical Corp., Buffalo, NY) in triplicate. The viable cells were
confirmed by trypan blue exclusion assay. Cell numbers at 1st, 3rd,
JOURNAL OF CELLULAR PHYSIOLOGY
5th, 7th, 10th, 12th, and 14th day were counted and compare to
those at the first day of chondrogenesis. The cell number at the
first day was ascribed 100%.
Cell viability was measured in 3-[4,5-dimethylthiazol-2-yl]-2, 5-
diphenyl tetrazolium bromide (MTT) assay. At the same time on
the 1st, 3rd, 5th, 7th, 10th, 12th, and 14th day, a 20 l volume of
MTT (Sigma) solution was added in each well at the concentration
of 5 mg/ml (pH 7.4). After 4 h of incubation for MTT formazane
formation, supernatant was removed and 150 l dimethyl
sulphoxide (DMSO) was added with fully vibration to dissolve the
formazane. Absorbance was measured at a major wavelength of
570 nm and OD value was obtained for each well. Each group
included five plates and the data were repeated three times.
[3H]-thymidine incorporation was also used to evaluate cell
proliferation. Briefly, at the same time on the 1st, 3rd, 5th, 7th,
10th, 12th, and 14th day, 20 l Ci (1Ci ¼ 37 GBq) [3H]-Tdr at the
concentration of 50 Ci/ml (1 Ci) was added in each well in the
micromass culture system. After incubation for 12 h, cells were
gathered and transferred to filters, following stabilizing with 5%
trichloroacetic acid, decolorizing with absolute ethanol and
drying for 30 min at 80˚C. Then the cells were transferred into
scintillation fluid, and counted by liquid scintillation counter
(counts/min, cpm). Each group included ten wells and the data
were repeated three times. Cpm at the first day was ascribed
100%.
Dynamic compressive loading
Dynamic compressive stimulation was delivered to cells via a
custom-made, computer-operated pressure system, in which
compressive pressure was implemented by increasing gaseous
tension above the supernatant media in a sealed chamber. The
details of the device were described in our previous reports
(Li et al., 2009, 2012a). Cells under chondrogenic induction
were exposed to sinusoidal dynamic force at 10–40 kPa. A daily
regimen was delivered at 1 h per day for the loading group, and the
loading experiments were conducted for 1, 3, 5, 7, 10, 12, and 14
consecutive days. Cultures without mechanic loading were served
as controls.
Before the mechanical loading experiment, a preliminary test of
frequency gradient was performed to choose optimal frequency
for compressive loading: 0.125, 0.25, 0.5, and 1 Hz were
implemented, respectively. All experiments were repeated three
times for reliable data.
Pharmacological inhibition test
The participations of MEK/ERK, p38MAPK, and BMP in cell
proliferation were explored in this study. The pharmacological
inhibitors of MEK/ERK (PD98059, Sigma), p38 MAPK (SB203580,
Sigma) and BMP (recombinant human noggin, Prospec) were
applied. Cells after chondrogenic induction were divided into four
groups: (a) no-load without inhibitor; (b) no-load with inhibitor;
(c) loaded without inhibitor; and (d) loaded with inhibitor. The
inhibitors were added 2 h before mechanic loading. The final
concentration of PD98059 was 10 mM; SB203580, 2 mM; and
noggin, 1 g/ml. Each groups contained five samples. Tests were
conducted at 1st, 3rd, 5th, and 7th day respectively and repeated
for three times.
Real-time quantitative PCR (RT-PCR) for
Col2a1 and Ihh
Expression of Col2a1 and Ihh were determined by RT-PCR as
described previously (Li et al., 2009). Briefly, cells were harvested
at indicated time points by trypsin and dissolved into Trizol
Reagent (Invitrogen, Carlsbad, CA). Total mRNA was extracted
and reverse transcribed into cDNA by Mmlv reverse transcriptase
(Invitrogen) according to the manufacturer’s protocol. Each real-
 CELL PROLIFERATION IN CHONDROGENESIS 1937

time PCR was conducted using the SYBR Prime ScriptTM RT-PCR
kit (Takara, Dalian, China) in an ABI PRISM 7300 Real-Time PCR
System. The primers and reaction system were the same with our
previous study (Li et al., 2010a). The sterilized ddH2O was used as
negative control. The housekeeping gene GAPDH was amplified in
each sample and used as a control for RNA loading. The cDNA of
control cells at the first detected time point was ascribed a fold
induction of one.

Western blot assay

Expression of cyclin D1 and CDK4 were determined by western
blotting. Briefly, cells were harvested at indicated time points, and
then dissolved into a lysis buffer supplemented with inhibitors of
proteases and phosphatases (CST, Boston). Protein concentration
was determined by a BCA Protein Assay kit (Pierce, Rockford, IL).
Equal amounts of proteins were transferred onto a PVDF
membrane (Millipore, Billerica, MA). After transfer, membranes
were blocked and then incubated overnight at 4˚C with
appropriate antibodies Endogenous levels of cyclin D1 and CDK4
were determined by anti-cyclin D1 (ab1224, Abcam, HK),
Anti-CDK4 antibody (ab7955, Abcam). All primary antibodies
were applied at a 1:1,000 dilution. Blots were further incubated
with appropriate horseradish peroxidase (HRP)-conjugated
secondary antibodies (Cell signaling) at 37˚C for 1 h. The immune
complexes were detected using a chemiluminescence kit
(Millipore) and visualized via the ChemiDoc XRS Gel
documentation system (Bio-rad, CA). The gray intensity was
measured by the software of Quantity One (Bio-Rad).

Statistical analysis
Data of evaluation parameters were expressed as mean  SE, and
analyzed by factorial analysis of variance (ANOVA) followed by
Newman–Keuls test. P < 0.05 was considered statistically
significant.

Results
Cell proliferation during chondrogenesis
To define the proliferative stage of chondrocytes in vitro, cell
number was counted from 1st to 14th day after seeding. In
control group, the cell number increased immediately after
seeding. After a sharp increase in the first 5 days, the growth
slowed down and remained in a high level, about two times of
the initial level. Cells under chondrogenic induction remained
in a remarkably lower level than the control group throughout
the 14 days of culture. After a mild increase during the first
3 days, a sharp increase followed until reaching the peak on the
7th day, increasing by 70%. Then a gradual decrease followed.
The result revealed that the quick increasing period of cell
number distributed from the 1st to 7th day regardless of
chondrogenic induction (Fig. 1A).
The result of [3H]-thymidine incorporation indicated that
there was a gradual decrease of DNA replication activity since
chondrogenic induction initiated, compared to cells in control
group. However, it remained constant after the 7th day, at the
level of 70% of the first day (Fig. 1B).
We also tested the cell viability by MTT assay. For cells in
Fig. 1. The temporal profiles for cell proliferation in the process
control group, cell viability fluctuated to reach to the peak on of chondrogenic induction in vitro. When chondrogenic induction
the 7th day, after that, it decreased gradually until the 14th day, initiates, the number and viability of cells as well as proliferation are
at almost the same level of 1st day. In the meantime, viability of lower than those in control group. Subpart (A) shows the changes of
chondrogenic cells remained in a low level throughout the cell number and (B) shows that the activity of DNA replication was
lower than those in control group; and (C) shows the changes of cell
period. After reaching a peak on the 3rd day, cell viability fell viability in chondrogenic induction.
back to the 1st day’s level on the 7th day. Slight increase was
observed at the 9th day, though, the cell number decreased
gradually after that (Fig. 1C).

JOURNAL OF CELLULAR PHYSIOLOGY

1938 WANG ET AL.

Cell proliferation upon mechanical regulation

A preliminary frequency-gradient test was conducted.
Different frequencies of dynamic compression as well as static
compression were applied to the chondrocytes in medium
respectively to choose the optimal frequency of dynamic
loading. Culture without loading was served as control group.
MTT assay demonstrated that under static compression (0 Hz)
cell viability decreased slightly compared to the control group,
whereas dynamic compression enhanced cell viability to
various degrees relative to different frequencies. Among them,
cells viability was the highest under dynamic compression of
0.5 Hz. Thus 0.5 Hz was chosen as the optimal frequency of
stress in the following experiment (Fig. 2A).
After application of cyclic stress of 0.5 Hz, cells under
chondrogenic induction showed increased cell viability than
those in control group (Fig. 2B). Both groups showed similar
tendency with peak on the 3rd day, then a decline followed
throughout the period, except a sudden rise on the 10th day in
control group. [3H]-thymidine incorporation showed DNA
replication was more active in loaded group than those without
stress (Fig. 2C). These results indicated that dynamic
compression could enhance the proliferation of cells under
chondrogenic induction significantly.

Col2a1expression increased under dynamic stress

The immunohistochemical staining in chondrocyte showed the
expression of Col2a1 increased significantly under dynamic
compression. As the culture time prolonged, cell morphology
turned round, and increased cells overlapped in micromass
with deepening staining color (Fig. 3A).
Gene expression of Col2a1 in both groups increased
gradually from the 1st day to the 14th day (Fig. 3B). Compared
to control samples, dynamic compression enhanced Col2a1
expression significantly (P < 0.05), especially on the 10th and
14th day (P < 0.01).

Cell cycle regulators increased under stress

Western blotting showed that in control group without stress,
protein expression of Cyclin D1 as well as Cyclin-dependent
kinase 4 (CDK4) increased in early time, after achieving to a
peak in the 5th day, it declined gradually. Dynamic compression
increased their expression significantly with similar trends
(Fig. 4A).
In control group without stress, expression of Ihh increased
quickly when chondrogenic induction initiated. After reaching
the peak on the 5th day, it falls gradually to the 14th day.
Dynamic stress increased Ihh expression significantly with
similar tendency (P < 0.05), especially on the 1st and 5th day
(P < 0.01). After the 7th day, Ihh expression under dynamic
compression fell to the same level as the control group
(Fig. 4B).

MEK/ERK and p38 MAPK were not involved in cell

proliferation
MEK/ERK and p38MAPK were accepted as important
regulators for chondrogenic differentiation, so we tested
whether cell proliferation was regulated by the two key MAPK
pathways in stress-induced chondrogenic process. Dynamic
compression increased cell viability significantly compared to
the non-load groups (P < 0.01), and this increase was not
influenced by the supplement of PD98059 (P > 0.05). Besides,
this inhibitor did not bring any significant difference between
groups under the same loading conditions. The results indicate
MEK/ERK may not participate in the chondrocyte proliferation
(Fig. 5A).
Fig. 2. Cell viability under dynamic compression of different
frequencies during chondrogenic induction. Subpart (A) shows cell
viability is the highest under dynamic compression of 0.5 Hz and (B)
demonstrates that dynamic compression could significantly enhance
the proliferation of cells under chondrogenic induction. (C) shows
that dynamic compression could increase the activity of DNA
synthesis.  Indicates significant difference from corresponding
control group, P < 0.05;   Indicates P < 0.01.

JOURNAL OF CELLULAR PHYSIOLOGY

 CELL PROLIFERATION IN CHONDROGENESIS
Fig. 3. Morphology changes of cells in micromass (200
) and
Col2a1 expression in chondrogenic induction at 3rd, 7th, and 14th
day under dynamic compression. The expression of Col2a1 is up-
regulated by dynamic compression. Subpart (A) shows
immunohistochemical staining of Col2a1 and (B) demonstrates
gene expression of Col2a1 in the process of chondrogenic induction.

Indicates significant difference from corresponding control group,
P < 0.05;  Indicates P < 0.01.
Similar situation was also found in groups adding SB203580,
the inhibitor of p38 MAPK. Mechanic loading seemed to be the
factor making the cell viability different regardless of the
presence of SB203580 (Fig. 5B). This inhibitor could decrease
cell viability in groups under the same loading condition;
however, these differences were insignificant (P > 0.05).
BMP inhibitor partially inhibited proliferation of cells
Noggin decreased cell viability significantly in all groups
(Fig. 6A). The inhibitory effect of Noggin was observed since
the first day and it maintained throughout the period. Dynamic
stress boosted cell viability remarkably, but this increase was
offset by the supplement of Noggin, which decreased the raised
cell viability to almost the same level as control group.
However, cell viability in group-(d) was still higher than that in
group-(b).
Gene expression of Ihh increased steadily to the peak on the
5th day and then declined on the 7th day, and it was not
influenced by Noggin in non-load groups (Fig. 6B). Dynamic
compression raised Ihh expression level significantly (P < 0.01),
JOURNAL OF CELLULAR PHYSIOLOGY
1939
though; this increase was offset by Noggin. Noggin significantly
reduced Ihh expression in loading group to almost at the same
level as that in control group (P < 0.01). However, in presence
of Noggin, Ihh expression stimulated by stress was still higher
than that in no-load groups.
Discussion
Our study shows that appropriately applied dynamic
compressive stress could increase cell viability by increasing
the expression of Col2a1, Ihh, Cyclin D1, and CDK4 in early
stage of chondrogenic induction, in which BMP signaling might
play a role.
The chondrogenic differentiation is a phased process in
which proliferation is in early stage. We found that when
chondrogenic induction initiated, cell proliferation was slightly
increased, however, it is lower than cells without chondrogenic
induction. The presence of appropriate number of viable cells is
prerequisite of successful tissue engineering, thus promotion of
cell viability is important while maintaining its capacity of
chondrogenesis. Previous studies have demonstrated the
sensitivity of chondrocyte to mechanical perturbation. Wang
and Mao (2002) study shows that application of cyclic
mechanical stimuli is sufficient to up-regulate chondrocyte
proliferation in vivo. Dynamic compression and cyclic stretch
at physiological frequencies induces DNA synthesis of articular
chondrocytes (Lee and Bader, 1997). The results of our study
demonstrate that dynamic compression increases cell number
and viability, as well as the expression of Ihh, Cyclin D1 and
CDK4. As one of the key regulators of endochondral bone
formation, Ihh is expressed in prehypertophic chondrocytes,
which directly activates proliferation. Increased Ihh expression
resulted in an upregulation of Cyclin D1, a positive regulator of
cell cycle (Minina et al., 2002). Cyclin D1 belongs to the D-type
cyclins (D1, D2, and D3), which controls progression through
the G1 phase of the cell cycle in complexes with CDKs 4 and 6
(Beier et al., 2001). The increased expression of Ihh, Cyclin D1,
and CDK4 indicates that cell proliferation is active during the
early stage of chondrogenesis, and it is promoted by dynamic
compression. The gene of collagen type II (Col2a1) is
expressed primarily in chondrocytes, which is accepted as
important markers for phenotypic chondrocyte in numerous
previous studies (Krebsbach et al., 1996; Lu et al., 2008; Seki
et al., 2003; Szabova et al., 2009). It is suggested that
chondrogenesis involves a transient proliferation phase
appearing simultaneously with start of collagen type II
deposition (Dexheimer et al., 2012). Previous studies suggest
that the cyclic compression (Park et al., 2006) as well as cyclic
tension (Wong et al., 2003) could up-regulate the expression of
type II collagen. In our study, the expression of Col2a1 has
increased gradually since the chondrogenic induction initiated,
which implies these cells are in determination to chondrogenic
lineage, and dynamic stress could promote its expression in the
early stage. Furthermore, it is found in our study that dynamic
stress did not shift the peaks of cell number and viability
forward or backward, as well as gene expression between the
groups, implying that the dynamic stress does not break the
balance between proliferation and differentiation during
chondrogenesis. This is coincident with Wu and Chen (2000)
study that mechanical signals stimulate the differentiation
extent by increasing the peak of marker synthesis instead of
altering the speed of differentiation.
It is observed in our study that inhibition of both MEK/ERK
and p38 MAPK do not bring significant differences to cell
viability in non-loaded condition, as well as to the boosted cell
viability brought by dynamic stress. It is suggested by
Lemonnier et al. (2000) that ERK and p38 MAPK mediates
proliferation and differentiation of chondrocytes respectively,
indicating this two pathways function in a time-specific way. It is
1940 WANG ET AL.

Fig. 4. Effects of compressive stress on the expression of Cyclin D1 and CDK4 as well as Ihh. When the cells are exposed to compressive
stress during the first 7 days of chondrogenic induction, the expression of cell cycle regulators Cyclin D1 and CDK4 increase (A), and
the expression of Ihh is also raised by compressive stress (B).  Indicates significant difference from corresponding control group, P < 0.05;

Indicates P < 0.01.

also argued by Ryan et al. (2009) that ERK1/2 inhibitor could
block the increased chondrocyte proliferation induced by
acute static mechanical compression, However, our result
indicates that cellular pathways of MEK/ERK and p38 MAPK
may not function during early proliferation of chondrogenesis,
and the enhanced cell proliferation induced by dynamic
compression may not take effect via the two pathways.
Different frequencies and magnitudes of stress as well as cell
sources may be responsible for the divergences between our
results and previous studies.
Our study found that inhibitor of BMP signaling could
remarkably decrease cell viability, and dynamic stress could
make up for this reduction to a certain extent. It’s suggested by
Wuelling and Vortkamp (2011) that maintaining a normal
proliferation rate requires BMP and Ihh acting in parallel, and
the role of BMP signaling is independent of the Ihh/PTHrP
pathway (Minina et al., 2002). This is evidenced by our study
that Ihh expression is not influenced by noggin under
conditions without stress; however, the increased Ihh
expression induced by stress is partially offset by BMP
antagonist. It is speculated that BMP signaling not only
participate in chondrogenic proliferation, but also may
intervene in the expression of Ihh signaling under compression.
It is suggested by Wu et al. (2001) that Ihh mediates the

JOURNAL OF CELLULAR PHYSIOLOGY

 CELL PROLIFERATION IN CHONDROGENESIS
Fig. 5. Influence of inhibitors of MEK/ERK and p38MAPK on cell
viability under compressive conditions. The medium is added with
10 mM PD98059 and 2 mM SB203580, respectively before
compressive loading. The MTT assay shows viability of cells under
chondrogenic induction is not influenced by PD98059 (A) nor
SB20358 (B).  Indicates significant difference from corresponding
control group, P < 0.05;  Indicates P < 0.01.
mechanotransduction process in BMP-dependent and para-
thyroid hormone-related peptide-independent manner. BMP
antagonist might inhibit mechanical stimulation of chondrocyte
proliferation through the induction of Ihh. Furthermore, the
inhibition of Noggin could not completely neutralize the
increased Ihh expression induced by stress, which implies that
other cellular pathway might implicate in transduction of
mechanic signaling to Ihh expression. The mechanical stimulus
transmitted from extracellular to intracellular depends on
various pathways (Wu and Chen, 2000). Studies show that
mechanical signals are transduced from extracellular matrix to
inside of the cell via cation channels (Duncan and
Turner, 1995).
In summary, the present study is focused on rapid
proliferative stage in chondrogenic differentiation, which spans
from 1st to 7th day. During this period, dynamic stress of
0.5 Hz could remarkably promote cell proliferation, evidenced
by increased cell viability, expression of chondrogenic-specific
markers and cell cycle regulators. MEK/ERK and p38 MAPK
may not participated in early stage of chondrogenesis while
BMP signaling plays an important role in stress-induced cell
proliferation, which might be achieved by regulating the
expression of Ihh.
JOURNAL OF CELLULAR PHYSIOLOGY
1941
Fig. 6. Effects of BMP antagonist on cell viability and gene
expression of Ihh under compressive stimulation. Noggin at
concentration of 1 çg/ml is added into cell culture 2 h before
mechanic loading for 1 and 7 days. The MTT assay shows that cell
viability is affected by noggin in different degrees under compressive
conditions (A). RT-PCR analysis shows Ihh expression was not
influence by noggin in non-loaded condition whereas it is suppressed
under compression (B).  Indicates significant difference from
corresponding control group, P < 0.05.  Indicates P < 0.01.
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