Effects of Mechanical Stretch on Cell Proliferation and Matrix Formation of Mesenchymal Stem Cell and Anterior Cruciate Ligament Fibroblast

Abstract

Mesenchymal stem cells (MSCs) and fibroblasts are two major seed cells for ligament tissue engineering. To understand the effects of mechanical stimulation on these cells and to develop effective approaches for cell therapy, it is necessary to investigate the biological effects of various mechanical loading conditions on cells. In this study, fibroblasts and MSCs were tested and compared under a novel Uniflex/Bioflex culture system that might mimic mechanical strain in ligament tissue. The cells were uniaxially or radially stretched with different strains (5%, 10%, and 15%) at 0.1, 0.5, and 1.0 Hz. The cell proliferation and collagen production were compared to find the optimal parameters. The results indicated that uniaxial stretch (15% at 0.5 Hz; 10% at 1.0 Hz) showed positive effects on fibroblast. The uniaxial strains (5%, 10%, and 15%) at 0.5 Hz and 10% strain at 1.0 Hz were favorable for MSCs. Radial strain did not have significant effect on fibroblast. On the contrary, the radial strains (5%, 10%, and 15%) at 0.1 Hz had positive effects on MSCs. This study suggested that fibroblasts and MSCs had their own appropriate mechanical stimulatory parameters. These specific parameters potentially provide fundamental knowledge for future cell-based ligament regeneration.

Full text

Research Article
Effects of Mechanical Stretch on Cell Proliferation and Matrix
Formation of Mesenchymal Stem Cell and Anterior Cruciate
Ligament Fibroblast
Liguo Sun,1,2 Ling Qu,3Rui Zhu,4Hongguo Li,1Yingsen Xue,1Xincheng Liu,1
Jiabing Fan,5and Hongbin Fan1
1DepartmentofOrthopedicSurgery,XijingHospital,eFourthMilitaryMedicalUniversity,Xi’an710032,China
2Tianjin Sanatorium, Beijing Military Region, Tianjin 300381, China
3Department of Clinical Laboratory, Xijing Hospital, e Fourth Military Medical University, Xi’an 710032, China
4CollegeofScience,AirForceEngineeringUniversity,Xi’an710051,China
5Division of Advanced Prosthodontics, School of Dentistry, University of California, Los Angeles, CA 90095, USA
Correspondence should be addressed to Hongbin Fan; fanhb@fmmu.edu.cn
Received  December ; Accepted  June 
Academic Editor: Renke Li
Copyright ©  Liguo Sun et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Mesenchymal stem cells (MSCs) and broblasts are two major seed cells for ligament tissue engineering. To understand the eects of
mechanical stimulation on these cells and to develop eective approaches for cell therapy, it is necessary to investigate the biological
eects of various mechanical loading conditions on cells. In this study, broblasts and MSCs were tested and compared under a novel
Uniex/Bioex culture system that might mimic mechanical strain in ligament tissue. e cells were uniaxially or radially stretched
with dierent strains (%, %, and %) at ., ., and . Hz. e cell proliferation and collagen production were compared to
nd the optimal parameters. e results indicated that uniaxial stretch (% at . Hz; % at . Hz) showed positive eects on
broblast. e uniaxial strains (%, %, and %) at .Hz and % strain at .Hz were favorable for MSCs. Radial strain did not
have signicant eect on broblast. On the contrary, the radial strains (%, %, and %) at . Hz had positiveeec ts onMSCs. is
study suggested that broblasts and MSCs had their own appropriate mechanical stimulatory parameters. ese specic parameters
potentially provide fundamental knowledge for future cell-based ligament regeneration.
1. Introduction
Anterior cruciate ligament (ACL) is an important intra-
articular structure to maintain the stability of knee joint.
However, it cannot heal spontaneously aer severe injury
due to poor vascularization [–]. Allogras or autogras
(hamstring or patella tendon) are now frequently used to
reconstruct ACL because of the poor results of synthetic
gras. Although the promising results such as subjective
satisfaction and partial stability restoration are acquired by
allo/auto gra transplantation, no reliable and functional
tissue repair is achieved in long-term follow-up studies.
e increased concerns including ligament laxity, donor
site morbidity, and pathogen transfer are observed in clin-
ical treatments [–]. Recently tissue-engineered ligament
provides a new approach to the solution of aforementioned
problems.
Tissue-engineered ligament has the potential to provide
an alternative gra that could be readily available. However,
construction of a viable and biomechanically equivalent
ligament requires a fundamental understanding of ACL
biology including broblast matrix synthesis and remodel-
inginresponsetothelocalmechanicalenvironment[].
e properties of ligament including structure, function,
heal capability, and development are signicantly aected
by mechanical stimulus. With daily activities, the ACL is
subjected to varying amounts of tensile strain, which is
crucial for ligament homeostasis. Mechanical loads induce
changes in the structure, composition, and function of living
tissues. It is now well recognized that mechanical forces play
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Stem Cells International
Volume 2016, Article ID 9842075, 10 pages
https://doi.org/10.1155/2016/9842075

Stem Cells International
a fundamental role in the regulation of cell functions, includ-
ing gene induction, protein synthesis, cell growth, death,
and dierentiation, which are essential to maintain tissue
homeostasis []. Another study also showed that mechanical
loads aect cellular functions such as cell proliferation and
collagen synthesis [].
To reconstruct a functional tissue-engineered ligament,
selection of cell source is of great importance. Due to
dierences in phenotype and function, dierent seed cell
will greatly inuence the properties of tissue-engineered
ligament. ACL broblasts are load-sensitive cells and their
complexstructurechangesinresponsetomechanicalforces.
Furthermore,thecollagenproducedbybroblastsisthemain
component of ligament and has great tensile strength [].
eoretically, ACL broblast should be the primary choice
for potential ligament tissue engineering, because especially
theycouldbeeasilyharvestedindiagnosticarthroscopy
procedure. In addition to ACL broblasts, mesenchymal stem
cell (MSC) isolated from bone marrow is another potential
cell source for ligament repair due to their multipotent and
proliferate capabilities. e scaold fabricated from woven
silk bers has mechanical properties similar to the native
ACL, showing the abilities to enhance MSCs attachment,
proliferation, and dierentiation []. To potentially improve
the functionality and structure of tissue-engineered ligament,
broblasts forming ACL and medial collateral ligament
(MCL) tissues were compared with MSCs in previous studies.
e proliferation rate and collagen excretion of MSCs were
further shown to be higher than ACL and MCL broblasts
[]. Although many studies investigated the inuence of
cyclic mechanical stimulation on gra incorporation, cell
morphology, collagen production, and cellular dierentia-
tion, few literatures have characterized the optimal parameter
of mechanical stimulation [–].
In an eort to better understand the eects of mechan-
ical stimulation on dierent cells and to develop eective
approaches for cell therapy, it is necessary to study the
biological eects of various mechanical loading conditions
on cells. In this study, broblasts and MSCs were tested and
compared under a novel Uniex/Bioex culture system that
may mimic mechanical strain in ligament tissue. e objec-
tive is to nd the optimal parameters (magnitude, frequency,
and duration of strain) required for cell proliferation and
collagen production, which potentially provides fundamental
knowledge for future cell-based ligament regeneration.
2. Materials and Methods
2.1. Isolation and Expansion of MSC and Fibroblast. MSCs
andbroblastswere,respectively,isolatedfrombonemarrow
aspirates and ligament tissues of New Zealand White rabbits
( weeks old, .–. kg) following the methods previously
reported []. In general, mononuclear cells from bone
marrow were separated by centrifugation in a Ficoll-Hypaque
gradient (Sigma Co., St. Louis) and suspended in  mL
of Dulbecco’s Modied Eagle Medium (DMEM) supple-
mented with % fetal bovine serum (FBS) (HyClone Logan,
Utah), l-glutamine ( mg/L), and penicillin-streptomycin
( U/mL). Cultures were incubated at ∘Cand%CO
2.
Aer  h, nonadherent cells were removed by changing
medium. When reaching –% conuence, adherent cells
were freed from the ask with .% trypsin and subcultured.
A homogenous MSCs population was obtained aer  weeks
of culture and MSCs (passage ) were harvested for further
use.
For broblasts isolation, the collected rabbit ACL was
excised under sterile condition. e ligament tissue was
minced and washed twice in % antibiotic medium for  min.
e minced ligament tissue was then placed in a solution
of .% collagenase at ∘Candagitatedovernightfor–
 h. Fibroblasts were isolated by straining the digest through
a𝜇m lter. e cell-containing solution was centrifuged
at  g for  min, the supernatant removed, and the pellet
resuspended in % antibiotic medium and recentrifuged. e
supernatant was removed and the cells suspended in culture
medium with % antibiotic, % glutamine, and % fetal
bovine serum (FBS) and cultured in T- asks at ∘C, %
humidity, and % CO2. Conuence was achieved in  weeks
and subculture was performed. e broblasts (passage )
were collected for further evaluation.
2.2. Cell Culture in Uniex/Bioex Plate. Cells were
trypsinized by adding  mL of .% trypsin solution to a
Taskwithconuentcellsfollowedbyminincubation
at ∘C with regular gentle shaking. e trypsin reaction
was stopped by adding  mL of culture medium containing
% FBS. e cell suspension was then centrifuged at  g
for  min at ∘C. e cell pellet was resuspended in  mL
of medium (% antibiotic, % glutamine, and % FBS) and
thoroughlymixedbyrepeatedpipetting.×6cells were
seeded in each well of the Uniex/Bioex culture plates and
incubated at ∘C, % humidity, and % CO2.
2.3. Mechanical Loading
2.3.1. Uniaxial Strain. e broblasts and MSCs were, respec-
tively,loadedineachwellofUniexcultureplatesat
∘C,
% humidity, and % CO2until it reached conuence. A
. cm gap was made on each side of the cell seeding area
for allowing cell migration and proliferation (Figure (a)).
e cells were uniaxially loaded by placing loading rectangle
posts (Flexcell International) beneath each well of the Uniex
culture plates in a gasketed baseplate and applying vacuum
to deform the exible membranes downward. e exible
membrane deformed downward along the long sides of the
loading posts thus applying uniaxial strain to loaded cells
(Figure (b)). e loading regimen was for  days,  h/day
(with  min rest every  h) at , , and % strain and ., .,
and  Hz, using a Flexcell Strain Unit (Flexcell International).
2.3.2. Radial Strain. e broblasts and MSCs from T ask
were trypsinized and cultured in medium (% antibiotic,
% glutamine, and % FBS) in each well of Bioex culture
plates at ∘C, % humidity, and % CO2until it reached
conuence. A . cm gap was made around the cell seeding
area allowing space for cell migration and proliferation (Fig-
ure (a)). e cells were radially loaded by placing cylindrical
loading posts (Flexcell International) beneath each well of the

Stem Cells International 
Cells
0.5 cm gap 0.5 cm gap
(a)
Medium
Cells
Loading post
Rubber
membrane
Anchor
Uniaxial elongation
Loading post
Vacu u m
(b)
F : (a) Formation of cell sheet construct on Uniex culture plate; (b) diagram of side view of uniaxial stretch system.
Bioex culture plates in a gasketed baseplate and applying
vacuum to deform the exible membranes downward. e
exible membrane deformed downward along the circum-
ference of the cylindrical loading posts thus applying radial
strain to ACL broblast (Figure (b)). e loading regimen
was for  days,  h/day (with  min rest every  h) at , ,
or % strain and ., ., or  Hz, using a Flexcell Strain Unit
(Flexcell International).
2.4. Cell Viability/Proliferation. Alamar Blue (AB, Sacra-
mento. CA) was added into the culture media in the -well
plate at a nal concentration of % and was incubated for  h
at ∘C (AB mixture should turn to a purplish/reddish shade).
Aer incubation for  h, triplicates of 𝜇LABmixture
from each well were transferred and placed in a -well plate.
Optical density of the AB mixture was measured at  and
 nm with a standard spectrophotometer.
e oxidized form of AB is nonuorescent and blue
(𝜆max =  nm), whereas the reduced form is uorescent
and red (𝜆max =  nm). e proposed mechanism by
which the dye detects living cells involves metabolic-based
reduction via reactions of the respirator chain. e number
of viable cells correlates with the magnitude of dye reduction
and is expressed as percentage of AB reduction []. e
percentage of AB reduction (% AB reduction) was calculated
according to the manufacturer’s protocol. It was corrected for
background values of negative controls containing medium
without cells.
2.5. Collagen Production Assay. e culture medium was
completely removed from the -well plates. e seeded cells
were washed twice with PBS solution. e pepsin (.%)
was then added to the wells and incubated with cells for  h
to digest all synthesized collagen. e solubilized collagen was
neutralized with  M NaOH and aliquot to microcentrifuge
tubes.  𝜇L of Sircol Dye reagent was added to  𝜇Lof
solubilized collagen and was shaken for  min. During this
period the Sircol Dye will bind to soluble collagen. e dye
reagent is designed so that the collagen-dye complex will
precipitate out of solution. e microcentrifuge tubes were
spun at , ×g for a  min. It is important to rmly pack
the insoluble pellet of the collagen-dye complex at the bottom
of the tubes, so as to avoid any loss during draining. e
unbound dye solution is removed by carefully inverting and
draining the tubes. Alkali Reagent ( 𝜇L) was added to each
tubeandvortexedtoreleasethebounddyeintosolution.
 𝜇L aliquots of the released bound dye were transferred
into a microtitter plate. e absorbance was read at  nm
and reference wavelength at  nm.
2.6. Statistical Analysis. Unpaired t-test was used for sta-
tistical data analysis of the stretching eects on cells at a
signicancelevelof.andsamplesizeof.
3. Results
3.1. Uniaxial Stretch for Fibroblasts. Aer %, %, and %
stretching at . Hz,  hrs/day for  days, the ACL broblast

Stem Cells International
Cells
0.5 cm 0.5 cm
(a)
Medium
Loading post
Gasket
Bioblex
well Radial enlongation
Loading post
Vacu u m
Rubber
membrane
(b)
F : (a) Formation of cell sheet construct on Bioex culture plate; (b) diagram of side view of radial stretch system.
proliferation decreased signicantly by .%, .%, and .%,
respectively (𝑝 < 0.05). e collagen production was
decreased signicantly by %, %, and .%, respectively.
(𝑝 < 0.05).
% and % stretching of the ACL broblast at . Hz
signicantly increased cell proliferation by % and %,
respectively (𝑝 < 0.05). % stretch at . Hz signicantly
decreased cell proliferation by % (𝑝 < 0.05). Collagen
production was signicantly decreased by .% when the
cells are stretched at % and . Hz (𝑝 < 0.05). However,
whenthecellsarestretchedat%and%withthesame
frequency, collagen production was increased by .% (𝑝<
0.05) and .% (𝑝 < 0.05), respectively.
Cyclic stretching of ACL broblast at  Hz with magnitude
of either % or % showed a decrease in cell proliferation
by .% and .%, respectively (𝑝 < 0.05). Similarly,
the collagen production was decreased by .% and .%,
respectively (𝑝 < 0.05). On the other hand, % stretch at  Hz
increased cell proliferation by .% (𝑝 < 0.05)andcollagen
production by % (𝑝 < 0.05) (Figures  and ).
3.2. Uniaxial Stretch for MSCs. e proliferation of MSCs
showed similar trend with broblasts. Aer %, %, and
% stretching at . Hz,  hrs/day for  days, the MSCs
proliferation all decreased signicantly (𝑝 < 0.05). However,
when stretching at . Hz with %, %, and % strain, the
proliferation all increased by %, %, and % (𝑝 < 0.05).
When the frequency increased to  Hz, only % strain could
enhance proliferation (Figure ).
0
20
40
60
80
(%)
100
120
140
0.1 0.5 1
5% uniaxial strain
10% uniaxial strain
15% uniaxial strain
ACL broblasts
(Hz)
F : e proliferation of broblasts aer uniaxial stretch
stimulation.

Stem Cells International 
(%)
0
20
40
60
80
100
120
140
160
ACL broblats
0.1 0.5 1
5% uniaxial strain
10% uniaxial strain
15% uniaxial strain
(Hz)
F : e collagen production of broblasts aer uniaxial
stretch stimulation.
MSCs showed the decreased collagen production at
. Hz with magnitude of either %, %, or % (𝑝 < 0.05).
On the contrary, the collagen production increased by %,
%, and %, respectively, at . Hz with %, %, and %
strain (𝑝 < 0.05). At  Hz, only % stretch increased collagen
production by % (𝑝 < 0.05) (Figure ).
3.3. Radial Stretch for Fibroblasts. Aer % stretching at
. Hz, hrs/day for  days, the ACL broblast proliferation
increased by % (𝑝 < 0.05). No signicant dierence was
detected in cells with % and % stretching as compared
to unstretched cells (Figure ). However, there was a signif-
icantly increased collagen production by .%, .%, and
.% in %, %, and % radial strain groups, respectively
(Figure ).
At . Hz, % stretch group showed a decrease in
proliferation by .% (𝑝 < 0.05)andcollagenproductionby
.% (𝑝 < 0.05). In % stretch group, an increase in collagen
production by .% (𝑝 < 0.05) was observed although the
cell proliferation showed no signicant dierence compared
with nonstretch group. No signicant change was observed
in cell proliferation and collagen production in % stretch
group (Figures  and ).
Cyclic stretching of ACL broblast at  Hz with magnitude
of % and % showed an increase cell proliferation by .%
and .% (𝑝 < 0.05), respectively. However, at % stretch
cell proliferation decreased by .% (𝑝 < 0.05). ere was no
signicant change in collagen production at %, %, and %
stretch group (Figures  and ).
(%)
0
20
40
60
80
100
120
140 MSCs
0.1 0.5 1
5% uniaxial strain
10% uniaxial strain
15% uniaxial strain
(Hz)
F : e proliferation of MSCs aer uniaxial stretch stimula-
tion.
(%)
0
20
40
60
80
100
120
140
MSCs
0.1 0.5 1
5% uniaxial strain
10% uniaxial strain
15% uniaxial strain
(Hz)
F : e collagen production of MSCs aer uniaxial stretch
stimulation.

Stem Cells International
(%)
0
20
40
60
80
100
120
5% radial strain
10% radial strain
15% radial strain
ACL broblasts
0.1 0.5 1
(Hz)
F : e proliferation of broblasts aer radial stretch stimu-
lation.
3.4. Radial Stretch for MSCs. In comparison with non-
stretched group, the MSCs proliferation increased signi-
cantlyby%,%,and%in%,%,and%radialstrain
groups, respectively, at . Hz, hrs/day for  days (𝑝 < 0.05).
e amounts of collagen production in all stretching groups
were signicantly higher than those of control group (Figures
and).
At . Hz, the proliferation decreased signicantly by
.%, %, and % in %, %, and % strain groups,
respectively (𝑝 < 0.05). Correspondingly, the collagen
production also decreased by .%, .%, and .% (𝑝<
0.05) (Figures  and ).
At . Hz, the cell proliferation and collagen production
showednosignicantdierencein%stretchand%
stretch groups. However, at % stretch the cell proliferation
decreased by .% and collagen production decreased by
.% (𝑝 < 0.05) (Figures  and ).
4. Discussion
Ligament is a strong, dense structure made of connective
tissue. It connects bone to bone across the joint to keep
the dynamic and stable movement. e ACL is one of the
most important four strong ligaments connecting the bones
of knee joint. e function of ACL is to provide stability to
knee and minimize stress across the knee joint. However,
it has a poor self-regenerative capacity due to ligament’s
low cellularity and vascularity. erefore, it is important
to determine the eects of mechanical loading on ACL
broblast in order to better understand ACL mechanobiology
(%)
0
20
40
60
80
100
120
140
160
ACL broblast
5% radial strain
10% radial strain
15% radial strain
0.1 0.5 1
(Hz)
F : e collagen production of broblasts aer radial stretch
stimulation.
as well as pathophysiology. In addition, the tissue-engineered
ligament has been extensively studied in recent years as an
alternative gra in preclinical study. Mesenchymal stem cells
(MSCs)areamongthemostpromisingandsuitablestemcell
types for ligament tissue engineering. e microenvironment
of ACL not only contains biochemical factors but also
exerts hemodynamic forces, such as shear stress and cyclic
strain, which may inuence the dierentiation of MSCs
[].Althoughmanystudiesinvestigatedtheinuenceof
cyclic mechanical stimulation on gra incorporation and
cellular dierentiation, few literatures have characterized the
optimal parameter. In current study, using an in vitro system
(Flexcell) that can control the magnitude and frequency of
thestretching,theproliferationandcollagenproductionof
broblast and MSCs were compared to explore the optimal
strain condition.
Appropriate mechanical loads at physiological levels
wouldpositivelyinuencetheexpressionofECMandthere-
fore the mechanisms of tendon regeneration. However, while
excessive mechanical loading caused anabolic changes in
tendons, it also induced dierentiation of tendon stem cells
into nontenocytes, which may lead to the development of
degenerative tendinopathy frequently seen in clinical settings
[].emechanicalstrainusedincurrentstudyranged
from % to % elongation, which was within the physi-
ological range experienced by human tendons, given that
tendons can elongate by –% []. When broblasts were
uniaxially stretched, the optimal frequency for proliferation
and collagen production was .Hz (Figures  and ). ACL
broblasts showed an increase in either proliferation or

Stem Cells International 
(%)
0
20
40
60
80
100
120
MSCs
5% radial strain
10% radial strain
15% radial strain
0.1 0.5 1
(Hz)
F : e proliferation of MSCs aer radial stretch stimulation.
collagen production when they are stretched at dierent
strains (%, %, and %).
% uniaxial strain at . Hz and % uniaxial strain at
 Hz both stimulated broblast proliferation and collagen
production. e results indicated that as the frequency
increased, lower magnitude of stretch is more favorable for
cell proliferation and collagen production. Collagen type I,
collagen type III, decorin, and tenascin-C are fundamental
proteins in the ECM of tendons []. Lohberger et al.
[] stimulated human rotator cu broblast using Flexcell
tension system with % elongation and a frequency of
. Hz. e total soluble collagen was measured in cell
culture supernatants. Cyclic strain signicantly increased
thecollagenproductionondaysand.eexpression
of tenascin-C and scleraxis increased signicantly in the
mechanically stimulated groups at both time points. ere
results were correlated with our ndings in current study.
Uniaxial strain at .Hz is the least favorable for broblast
proliferation and collagen production. e cells showed a
decrease proliferation and collagen production when they are
stretched at . Hz at dierent strains (%, %, and %)
(Table ).
In contrast to uniaxial strain, . Hz was least favorable
for cell proliferation. Radial strains (% and %) at . Hz did
not have signicant eect on cell proliferation. e % radial
strain showed negative eect and decreased cell proliferation.
e strains (% and %) at Hz and % strain at . Hz
all stimulated cell proliferation. Interestingly, the collagen
production under these conditions showed no signicant
dierence compared to that of nonstretched group. Although
the strains (% and %) at . Hz and % strain at . Hz had
(%)
0
20
40
60
80
100
120
140
MSCs
5% radial strain
10% radial strain
15% radial strain
0.1 0.5 1
(Hz)
F : e collagen production of MSCs aer radial stretch
stimulation.
no eect on cell proliferation, the cells under these conditions
showed signicantly increased collagen production (Table ).
For MSCs under uniaxial stretch condition, . Hz is
favorable for cell proliferation and collagen production.
Dierent strains (%, %, and %) all showed positive
eects. In addition, % strain at .Hz also upregulated cell
proliferation and collagen synthesis. Interestingly, for radial
stretch groups, MSCs showed an increase in both prolifer-
ation and collagen production when they are stretched at
. Hz at dierent strains (%, %, and %) (Table ).
In summary, uniaxial stretch (% at . Hz; % at
. Hz) showed positive eects on broblast. e uniaxial
strains (%, %, and %) at . Hz and % strain at . Hz
showed positive eects on MSCs. Radial strain did not have
signicant eect on broblast. On the contrary, all radial
strains (%, %, and %) at .Hz had positive eects on
MSCs.
5. Conclusion
is study suggested that exposing broblasts and MSCs
to uniaxial or radial strains promoted cell proliferation and
collagen production. e broblasts and MSCs had their
own appropriate mechanical stimulatory parameters. ese
specic parameters had great parental application in cell
expansion to fabricate tissue engineering products.
Competing Interests
e authors declare that there is no conict of interests
regarding the publication of this paper.

Stem Cells International
T : Eects of strains at various frequencies on broblasts.
Function
Uniaxial stretch Radial stretch
. Hz .Hz . Hz . Hz . Hz . Hz
% % % % % % % % % % % % % % % % % %
(strain) (strain) (strain) (strain) (strain) (strain)
Proliferation ↓↓↓↑↓↑↓↑ ↓—— ↑—↓—↑↑ ↓
Collagen ↓↓ ↓↓↑↑↓↑↓↑↑—↑↓——— ↓
“↑”: increase; “↓”: decrease; “—”: no dierence (𝑝 < 0.05).

Stem Cells International 
T : Eects of strains at various frequencies on MSCs.
Function
Uniaxial stretch Radial stretch
. Hz .Hz . Hz . Hz . Hz . Hz
% % % % % % % % % % % % % % % % % %
(strain) (strain) (strain) (strain) (strain) (strain)
Proliferation ↓↓↓↑↑ ↑↓↑ ↓↑↑↑↓↓ ↓—— ↓
Collagen ↓↓ ↓↑↑↑↓↑↓↑↑ ↑↓↓↓—— ↓
“↑”: increase; “↓”: decrease; “—”: no dierence (𝑝 < 0.05).

 Stem Cells International
Authors’ Contributions
LiguoSun,LingQu,andRuiZhucontributedequallytothis
work and were regarded as co-rst authors.
Funding
is work was supported by grants from National Science
Foundation of China (nos.  and ).
Acknowledgments
e authors gratefully acknowledge the funding support from
the National Natural Science Foundation of China (nos.
 and ).
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