PAPER
www.rsc.org/loc | Lab on a Chip
A pneumatic micro cell chip for the differentiation of human mesenchymal
stem cells under mechanical stimulation{
Woo Young Sim,a Sin Wook Park,a Sang Hyug Park,b Byoung Hyun Min,bc So Ra Parkd and
Sang Sik Yang*a
Received 13th April 2007, Accepted 7th September 2007
First published as an Advance Article on the web 28th September 2007
DOI: 10.1039/b712361m
A new micro cell chip which can induce stem cells to differentiate into specific body cell types has
been designed and fabricated for tissue engineering. This paper presents the test results of a micro
cell stimulator which can provide a new miniaturized tool in cell stimulation, culture and analysis
for stem cell research. The micro cell stimulator is designed to apply compressive pressure to
the hMSCs (human mesenchymal stem cells) for inducing osteogenesis. The micro cell stimulator
is based on the pneumatic actuator with a flexible diaphragm which consists of an air chamber
and cell chambers. The hMSCs under cyclic compressive stimulation for one week were observed
and assessed by monitoring CD90 (Thy-1), actin, alkaline phosphatase (ALP) and alizarin red
expression. The results suggest that cyclic mechanical stimulation is attributed to the different
phenomenon of cultured hMSCs in cell proliferation and differentiation. These results are
important for the feasibility of the micro cell stimulator to provide the reduction of the necessary
quantity of cells, process cost and the increase of the throughput.
Introduction
Today, stem cell research is one of the most prominent areas in
both medicine and biology. Stem cell therapy has emerged as a
new way to treat degenerative disease and injury since two
independent research groups (Thomson and Gearhart, 1998)
reported success in growing human stem cells in culture.1–3
Stem cells have two unique characteristics that distinguish
them from other types of cells. First, they can theoretically
divide without limit to replenish other cells as long as the
person or animal is still alive. Secondly, under certain
physiological or experimental conditions, stem cells can be
induced to become specialized cells. When stem cells divide,
each new cell has the potential to either remain a stem cell or
become another type of cell with a specialized function.3–8
Among the types of adult stem cells which have no immune
rejection and ethical objections in clinical uses, bone marrowderived mesenchymal stem cells (MSCs) attract worldwide
attention as a source of easy to isolate, potentially regenerative
and genetically plastic stem cells.6,20,21 MSCs can be obtained
in quantities appropriate for clinical applications, making
them good candidates for use in tissue repair and disease
a
Department of Electrical and Computer Engineering, College of
Information Technology, Ajou University, Suwon, 443-749, Korea.
E-mail: [email protected]; Fax: +82 31 212 9531; Tel: +82 31 219 2481
b
Department of Molecular Science and Technology, Ajou University,
Suwon, Korea 443-749. E-mail: [email protected];
Fax: +82 31 215 0463; Tel: +82 31 215 0494
c
Cell Therapy Center/Department of Orthopaedic Surgery, School of
Medicine, Ajou University, Suwon, Korea 443-749.
E-mail: [email protected]; Fax: +82 31 215 0463; Tel: +82 31 215 0494
d
Department of Physiology, College of Medicine, Inha University,
Incheon, Korea 402-751. E-mail: [email protected];
Fax: +82 32 873 5992; Tel: +82 32 890 0922
{ Electronic supplementary information (ESI) available: Three figures
to explain Fig. 7 in more detail. See DOI: 10.1039/b712361m
This journal is ß The Royal Society of Chemistry 2007
management. Previous studies have shown that MSCs
reproducibly and predictably differentiate into bone, cartilage,
adipose, and even nerve in vitro and in vivo.5–10
In particular, the field of bone tissue engineering has been
at the forefront of research and product development in the
fields of stem cell biology, tissue engineering, and regenerative
medicine. The powerful combination of stem cell biology with
multiscale engineering tools has advanced the field of bone
tissue engineering to an unprecedented level with widespread
applications for the healing of damaged/diseased tissue (e.g.,
bone fracture, non-union, craniofacial/segmental bony defects,
arthritis, injuries, and spinal defects).32–34
Above all, biochemical and biophysical regulation in the
differentiation of stem cells are of great important, both for
biological research and for the screening and characterization
of cells for medical applications. For a long time, biochemical
factors (i.e., growth factors, cytokines, and other regulatory
molecules) have been widely used in regulation.10–12,29 Another
method is to use 3D scaffolds which provides an interim space
and mechanical stability for cell growth and integration.22,24
Several groups reported that physical and mechanical
stimuli (compression,13–17,23,24,35–38 shear stress,18,39,40 strain,19
stretch, hydraulic force,41 etc.) also play important roles in the
differentiation (chondrogenesis and osteogenesis) of MSCs.
Cells are very sensitive to mechanical (stress–strain) state, and
react directly to mechanical stimuli. Cyclic compression significantly enhanced a proteoglycan synthesis in cultured bovine
articular cartilage explants.36 Cross et al. demonstrated that
uniaxial–cyclic compression (1 kPa, 1 Hz, 30 min) increased
matrix metalloproteinase (MMP)-3 and MMP-13 gene expression at 2 h compared to unstimulated cells.37 These previous
studies suggested that the mechanical stimuli have been
described as enhancing mineralization and inducing chondrogenic/osteogenic differentiation both in vitro and in vivo.
Lab Chip, 2007, 7, 1775–1782 | 1775
In our previous studies, the micro cell stimulator actuated by
electromagnetic actuators has been developed and demonstrated.15,16 The cyclic loading (5–10 kPa) was given at 1 Hz
frequency for 10 min twice a day. The cyclic compressive
loading enhanced the synthesis of cartilage-specific matrix
proteins and the chondrogenic markers, such as aggrecan, type
II collagen, and Sox9. In general, intermittent stimulation
is more effective than continuous stimulation in the chondrogenesis/osteogenesis of MSCs because the resting time between
the mechanical stimulation is necessary for the stimulated cells
to adapt to the environment. Nerucci supported that the cyclic
loading was found to trigger a partial recovery in morphological and ultrastructural aspects of cells.42 The magnitude of
the applied stress in our previous works was significantly lower
than conventional mechanical loading protocols.
However, these experiments using conventional methods
require a large number of cell culture surfaces, samples,
bulky incubators, large fluid volumes, as well as costly labor
and equipment.18,25 Although the results of our previous
research using the electromagnetic cell stimulator were very
encouraging, there are still several problems with applying
stimulus, such as heating, handling, bubble trap, and electromagnetic disturbance.
Our current work addresses the development of a new micro
cell stimulator which overcomes the limitations mentioned
above. The new micro cell chip is designed and fabricated to
apply various amplitudes of pressures simultaneously and
remove trapped bubbles in the chamber. We investigate the
effects of mechanical stimulation as a specific physiological
stress on the differentiation of human mesenchymal stem cells
(hMSCs) using the new micro cell chip. Bone marrow-derived
hMSCs might be induced into osteogenesis by the mechanical
stimulation. To examine the influence of mechanical stimulation on the differentiation of hMSCs into osteoblast-like cells,
the expression of marker proteins and calcium concentration
were determined in mechanically stimulated and unstimulated
cultures. The changes of the hMSCs under the mechanical
stimulation are assessed by monitoring CD90 (Thy-1), actin,
alkaline phosphatase (ALP) and alizarin red expression.
This study using the micro cell stimulator could be useful to
investigate the role of mechanical signals on the osteogenic
turnover of bone marrow-derived MSCs and provide new
tools to design novel therapeutic approaches.
Experimental
Micro cell chip
A micro cell chip is designed to culture and apply the
mechanical stimulation to stem cells using a pneumatic force
without heating, handling and electromagnetic disturbing. We
focus on the development of a new platform to promote
the osteogenesis of hMSCs. Compared with conventional
methods, the micro cell chip in this paper has great potential
in the reduction of the necessary quantity of stem cells but
increases the throughput with various amplitudes of pressures.
The schematic diagram of a micro cell chip is shown
in Fig. 1. The micro cell stimulator is based on the
pneumatic actuator with a flexible diaphragm. It consists of
five layers of polydimethylsiloxane (PDMS) and borosilicate
1776 | Lab Chip, 2007, 7, 1775–1782
Fig. 1 Schematic diagram of a micro cell chip. A new micro cell
stimulator is designed to culture and apply the mechanical stimulation
to human mesenchymal stem cells using a pneumatic force. (A)
Exploded view: It consists of five layers of PDMS and glass substrates.
(B) Cross-sectional view (A-A9). The device is largely comprised of an
air chamber and five columns with three cell chambers (diameter 3 mm,
height 180 mm) connected by microchannels (y0.2 mm wide). To
apply the various pressures simultaneously, the hole diameters are
designed differently.
glass (Borofloat-33) substrates. To minimize the deformation
of PDMS structure at a moderate air-pressure (,10 kPa)
during the test, each PDMS substrate is placed among the
glass substrates. All materials are highly biocompatible and
transparent to facilitate cell culture and optical observation.
The dimensions of the device are 30 mm 6 20 mm 6 4.5 mm.
The device is largely comprised of an air chamber (19.4 6 16 6
2 mm3) and 5 6 3 array of equal-sized cell chambers (diameter
3 mm, height 180 mm). The cell chamber layer has five columns
with three chambers connected by microchannels (width
200 mm, height 180 mm) (Fig. 2). The compartmental volumes
of chamber and column are 1.26 mL and 4.95 mL, respectively.
The cell chamber is designed to have rounded corners for
minimizing the bubble trap (Fig. 2). The gasket and cell
chamber with a membrane are formed in PDMS by the
standard molding-process. An air chamber part, including
hole-plate and cover, is fabricated by the powder blasting
process. The 5 6 3 array of holes with different diameters
in the hole-plate are designed to apply the various pressures
This journal is ß The Royal Society of Chemistry 2007
Fig. 2 Pneumatic micro cell chip for the differentiation of human
mesenchymal stem cells under mechanical stimulation. The inserted
SEM image (bottom view) shows that the cell chamber is designed to
have rounded corners for minimizing the bubble trap.
simultaneously. The maximum hole (row 5 in Fig. 2) diameter
is the same as that of the cell chamber. As hole diameters are
decreased in five stages (from 3 mm to 0.3 mm), opened areas
of membrane also decrease logarithmically (from 7.07 mm2 to
0.14 mm2). The micro cell chip is able to stimulate stem cells
with various amplitudes of pressures simultaneously with a
single pressure source.
Fabrication
The fabrication process of the micro cell stimulator is shown in
Fig. 3 and starts with fabricating the top glass substrate with
an air inlet and media inlet–outlet holes by powder blasting.
A 500 mm thick glass wafer is coated with a 100 mm thick dry
film photoresist (BF410, Tokyo Ohka Kogyo, Japan), as an
erosion-resistant polymer mask, exposed to a UV light source
and developed in a sodium carbonate solution. The wafer is
then blasted using alumina particles (Al2O3) of 30 mm grain
size. Following the powder-blasting process, the dry film
photoresist was removed and cleaned in an acetone-containing
ultrasonic bath. The gasket and cell chamber with a membrane
are fabricated with a standard molding-process. The master
for each structure is created on a thermally-oxidized silicon
wafer via a lithography process with SU-8 (SU-8 2100,
MicroChem, MA, USA) negative thick photo-resist. A curing
agent and PDMS prepolymer (Sylgard 184, Dow Corning,
USA) are mixed in a 1 : 10 weight ratio and poured onto the
SU-8 masters. After curing at 60 uC for 3 h in a vacuum oven,
the PDMS prepolymer is stored overnight at room temperature. The PDMS replicas are peeled off from the masters and
cut out. Through-holes for fluidic connection are cored with a
puncher. Finally, each molded PDMS replica is bonded to the
glass substrates by oxidizing both surfaces in oxygen plasma
(10 s at 100 W and 100 mTorr). After bonding, the devices are
kept in an oven (over 6 h at 60 uC). The fabricated prototype
device is shown in Fig. 2. For the fluidic connections to the cell
chip, stainless steel tubing (Sunil metal, Seoul, Korea) fitted
into silicone tubing (Sewoon medical, Seoul, Korea) are
connected to the cell-media inlets and outlets. Finally, a glass
capillary tube for supplying the pneumatic pressure is glued to
This journal is ß The Royal Society of Chemistry 2007
Fig. 3 Fabrication of the device. An air chamber part, including holeplate and cover, is fabricated by the powder blasting process. The
gasket and cell chamber are formed in PDMS by the standard
molding-process. Each PDMS layer is bonded to the glass substrates
after oxygen plasma treatment.
the air inlet with sealant (Torr seal, Varian, CA, USA) at 60 uC
for 2 h.
Cell isolation and culture
Human mesenchymal stem cells (hMSCs) are used for this
experiment and bone marrow samples were obtained from five
hematologically normal patients undergoing routine total hip
replacement surgery with informed consent (approved by Ajou
University Hospital). The patients were aged 64 on average
ranging from 55 to 73. A primary culture of bone marrow
MSCs was established as previously described.26 In brief,
marrow cells were harvested in Dulbecco’s phosphate-buffered
saline (DPBS, Gibco, NY, USA) from trabecular bone
marrow samples and pelleted by centrifugation at 500 g for
5 min at room temperature. The cell pellet was resuspended
in 10 mL alpha-minimum essential medium (a-MEM, Sigma,
MO, USA) supplemented with 10% fetal bovine serum (FBS,
Gibco) and passed through nylon mesh (90 mm pore size,
Lockertex). Cell suspensions were stained with 0.4% (wt/vol)
Lab Chip, 2007, 7, 1775–1782 | 1777
trypan blue to determine the number and viability of cells.
Cells were then plated (1.5 6 107 cells) on a 150 mm culture
plate and incubated at 37 uC in a humidified atmosphere of
5% CO2. After 6 days, non-adherent hematopoietic cells were
removed and MSCs on the surface of culture plates were
replenished with fresh medium supplemented with 10% FBS.
MSCs were expanded in a monolayer culture with the culture
medium changed twice per week and passaged every 1–2 weeks.
tubings were clamped to prevent the flow of medium during
stimulation. Monolayer cultured hMSCs were exposed to
intermittent cyclic pressure with an amplitude of 5 kPa at a
frequency of 1 Hz for the duration of 10 min twice a day for
7 days. After stimulation, cell chips were kept in a humidified
incubator maintained at 37 uC in 5% CO2. A control group
was cultured under the same conditions except for the
application of pressure. The osteogenic medium without
growth factor was changed daily.
Experimental setup and operation
The experimental setup is shown in Fig. 4. The frequency and
duty ratio of pneumatic pressure are controlled with a fast
switching solenoid valve (MHE2 series, Festo) actuated by an
FET driving circuit. The square-wave (frequency: 1 Hz, duty
ratio: 50%) is applied from a function generator as a control
signal. The pressure of nitrogen is regulated with a low
pressure regulator and monitored with two pressure sensors
(digital pressure sensor: PG-208, Copal Electronics, ELP
sensor: GA100-001PD, ICS sensors, USA) in real-time.
Before the experiment began, devices and tubings were
sterilized with ethylene oxide (EO) gas at 65–70 uC. We placed
the devices and tubings in a vacuum desiccator over 48 hours
to remove the residual gas remaining after sterilization. The
device was initially filled with mixed solution, hMSCs and
osteogenic defined medium, by the syringe pump. Bubble
trapping hardly occurred during the filling process, because the
cell chamber has rounded corners and the culture medium
contains various electrolytes. The osteogenic medium contains
high glucose Dulbecco’s Modified Eagle’s Medium (DMEM,
Sigma), 1% penicillin streptomycin, 50 mg mL21 L-ascorbic
acid 2-phosphate, 100 nM dexamethasone and 10% FBS. The
cells (1 6 104 cells per chamber) were allowed to settle to the
bottom of the chamber for 12 h at 37 uC. After confirming that
cells were attached to the bottom of the chamber, all silicone
Cell proliferation and viability
Cell counts were determined at day 3 and 7. Cells were
stained with DAPI (49,6-diamidino-2-phenylindole, Santa
Cruz Biotechnology, CA, USA), fluorescent dye specific for
DNA. The sample-embedded slide was observed using a
fluorescent microscope (Nikon E600, Japan). The images from
the control and loaded samples were analyzed using the ImageProPlus (Ver. 5.0, Media Cybernetics, MD, USA) to count the
number of cells in the chamber. Data are presented as mean
and standard deviations for each time period and the
difference was analyzed statistically using a one-way analysis
of variance (ANOVA). The statistical significance between the
two groups is given as p , 0.05. The cell viability counted
with a trypan blue exclusion method was more than 80% on
average in both groups at day 7. There is no statistical
difference between the two groups. This indicates that the
present pneumatic mechanical stimulation had no influence on
the viability of hMSCs.
Immunofluorescence staining
Identification of cells typically relies on the use of cell surface
markers, cellular differentiation (CD) antigens, that denote the
expression of particular proteins associated with genomic
Fig. 4 Experimental setup. A nitrogen gas source connected to a low pressure regulator was used to pressurize an air chamber of a micro cell chip.
The frequency of the pneumatic pressure is controlled with a fast switching solenoid valve actuated by a driving circuit. The applied pressure of
nitrogen is monitored with two pressure sensors (digital pressure sensor, ELP sensor: extremely low pressure sensor) in real-time.
1778 | Lab Chip, 2007, 7, 1775–1782
This journal is ß The Royal Society of Chemistry 2007
activity related to a particular differentiation state of the
cell.17,18,27 The changes of the stimulated hMSCs were
examined by means of immunofluorescence staining on cells
in a monolayer using fluorescein isothiocyanate (FITC)-conjugated anti-human monoclonal antibodies, CD90 (Thy-1, BD
Biosciences) and actin–phalloidin (Invitrogen) staining. CD90
is useful as a differentiation marker following the development
of osteoblasts.19 Fluorescein-labeled phalloidin provides a
convenient method for staining cellular cytoskeletal structures
as an actin-specific fluorescent label. The actin cytoskeleton
has been reported to play an important role in the transmission
of mechanical stimuli into intracellular signals that upregulate
gene expression and stimulate new bone formation.40 Cells
were fixed for 5 min in 4% paraformaldehyde and washed
twice with phosphate buffered saline (PBS). For assessment of
the expression of proteins, monoclonal anti-CD90 and actin–
phalloidin were applied. Fluorescence images were captured
using a Carl Zeiss confocal microscope.
Staining for osteogenesis of MSCs
To analyze the osteogenic differentiation of MSCs, ALP
activity was determined by the p-nitrophenyl phosphate
(pNPP) hydrolysis method. The cells were collected by trypsin
treatment and were suspended in lysis buffer (0.2% IGEPAL
CA-630, 10 mM Tris-HCl, 1 mM MgCl2, pH 7.5). The
supernatant was assayed for ALP activity using pNPP as
substrate. To each well of 96-well multi-well culture plates, an
aliquot (3 mL) of supernatant was added to 25 mL of 56 mM
2-amino 2-methyl-1,3-propanediol (pH 9.8) containing 10 ml
pNPP with 1 mM MgCl2, and the mixture was incubated at
37 uC for 30 min. Then, 150 ml of 0.04 N NaOH was added to
the wells to stop the reaction before absorption was measured
with a spectrophotometer at 405 nm using an ELISA reader
(BIO-TEK Instruments Inc., Winooski, Vermont, USA). ALP
activity was extrapolated using a p-nitrophenol standard
solution in the range 0–10 mMol mL21. All products were
purchased from Sigma Co. (St Louis, MO, USA).
Alkaline phosphatase (ALP) staining for hMSCs in the
micro cell chip was assessed by histochemical analysis
using the staining kit (Sigma). Cells were incubated with
alkaline-mixture (2.4 mg fast violet B salt (Sigma) and 0.4 mL
naphthol AS-MX phosphate alkaline solution (Sigma) in
9.6 mL of distilled water) for 60 min at room temperature in
a dark room.
For alizarin red staining, cells were fixed in 4% formaldehyde after washing twice with PBS. After washing with
distilled water, cells were stained with 40 mM alizarin red S
(pH 4.2, Sigma) for 10 min.
Preliminary test: mechanical stimulation and ALP activity
In this study, we hypothesized that the stimulation of cells by
cyclic pressure will enhance their osteogenesis. We made a
preliminary experiment to verify the correlation between cyclic
compressive pressure and osteogenesis. We used the smallest
cell culture flask, 12.5 cm2 canted neck, plug seal cap, instead
of a micro cell chip. The stimulation experiment with cyclic
pressure was performed in the same setup (Fig. 4). ALP
activity is the method commonly used for analysis of
osteogenic differentiation. The stimulated groups with 5 kPa
and 20 kPa stimulus at day 3 presented the statistically significant difference compared with the control group. Especially,
the average ALP activity shows the highest level (0.211 ¡
0.023 mMol/5 6 104 cells) in the 5 kPa stimulation group at
day 5 (Fig. 5). The result represents that the cyclic pressure
enhances the osteogenesis of hMSCs. We chose 5 kPa as the
most appropriate pressure for the stimulation experiment.
Cell proliferation
hMSCs showed proper cell attachment and stretching on a
glass surface after cell seeding. To determine whether the
mechanical stimulus had an effect on the proliferation of
hMSCs, we counted the number of stained cells in each
chamber. Total cell counts in both experimental and control
groups generally increased with time. In particular, the cell
proliferation rate of the stimulation group was about 1.56
higher than the control group at day 7 (Fig. 6). There are two
possible reasons for the low proliferation in the whole cell
chambers. Firstly, gas exchange and mass transport are
degraded in the micro culture environment as compared to
in the macro-scale culture environment. Secondly, proliferation mostly tends to decrease when differentiation progresses.
However, the cell counts in the strongest stimulation
chamber (Ch 5) showed no proliferation even though the cell
morphology was not different to the others. The cessation of
self-renewal often marks the onset of lineage commitment in
stem cells indicating that these bio-physical conditions may
trigger mechanisms involved in stem cell differentiation to
another cell type.31 However, it would not be a case from this
Results and discussion
Human mesenchymal stem cells were cultivated with the
intermittent mechanical stimulus for 7 days. An experimental
group was mechanically stimulated twice a day for 10 min with
the cyclic compressive stimulus of 5 kPa. An unstimulated
group was cultured in the same conditions. At selected times
(1, 3 and 7 days), cells were assessed by monitoring CD90,
actin, ALP and alizarin expression.
This journal is ß The Royal Society of Chemistry 2007
Fig. 5 Alkaline phosphatase (ALP) activity vs. various pressures
(1, 5, 10, 20 kPa). The amount of calcium deposition was determined
on days 3 and 5. The result indicates that the cyclic pressure enhances
the osteogenesis of hMSCs.
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Fig. 6 Effect of mechanical stimulation on hMSCs proliferation.
Control and stimulation groups were stained with DAPI (nuclear
staining: blue). The cell proliferation rate of the stimulation group was
about 1.56 higher than the control group.
viewpoint, because there was no additional evidence (e.g. ALP
staining) to support the assumption at day 3.
The results revealed that cyclic compressive stimulus at
moderate levels can enhance the proliferation of hMSCs. This
phenomenon could be explained by the suggestion that the
mechanical stimulation might stimulate and modulate biosynthesis of extracellular matrix, and gene expression.22
Expression of CD90
Application of mechanical stimulation is known to change the
CD markers of cells.11 Therefore, CD 90 tested in this study
as a MSC marker. The expression of this 25–30 kDa
GPI-linked membrane protein, whose precise biological
function is not clear yet, was described to decline as the
osteoblast matures. Wiesmann reported that CD90 is
expressed during proliferation but the expression level declines
as the cells mature towards osteoblast-like cells. CD90 could
1780 | Lab Chip, 2007, 7, 1775–1782
Fig. 7 Micrographs of cultured human mesenchymal stem cells after
7 days. Control group (A–D): hMSCs were cultured in osteogenic
medium. Stimulated group (E–H): Cultures stimulated with cyclic
compressive stimuli (pressure: 5 kPa, frequency: 1 Hz, period: 10 min,
repetition: 26 day). (A and E: Immunofluorescence images of antiCD90; B and F: Immunofluorescence images of actin–phalloidin; C
and G: Alkaline phosphatase staining; D and H: Alizarin red staining).
The stimulated group resulted in enhanced expressions.{
then be considered as a transient marker for early MSC
differentiation towards osteogenic cells.19 However, the
expression of CD 90 in our experiment was increased in the
stimulated group (Fig. 7A and E). We speculated that
the early stage of osteogenesis in the stimulation system is
being induced. It is the basis on which osteoblasts express
Thy-1 antigen and that its expression is maximal at the earliest
stage of maturation, during the proliferative phase, and then
declines as the cells mature.28 Besides CD90, we also tried to
analyze CD105 (endoglin) and CD29 (Integrin-b1) expressions, but there is no distinct difference between the control
group and the stimulation group.
Actin cytoskeleton
It has been shown that the actin cytoskeleton and the integrin
family of cell adhesion molecules play important roles in
This journal is ß The Royal Society of Chemistry 2007
mechanotransduction.40 A change of cell cytoskeletal shapes
were observed by labeling the actin cytoskeleton. Actin is
recruited to adhesion sites to strengthen or stabilize integrin–
cytoskeletal linkages. The changes in the cells responding to
various stimulations are indistinguishable. The distribution of
actin cytoskeleton in the stimulation group is more uniform
and stable than that in the control group (Fig. 6B and 6F).
Normally, fluid shear induces development of prominent
F-actin stress fibers that are oriented roughly parallel to the
long axis of the cell, whereas stress fibers in cells not subjected
to flow are small and randomly oriented.40 In our experiment,
the observed stress fibers in cells are small and randomly
oriented. This evidence illustrates that there is no fluid shear in
the cell chamber of the micro cell chip. For further investigation, micro particle image velocimetry (Micro PIV) can be used
to verify whether there is fluid shear in the cell chamber.44
Osteogenic differentiation
Regarding osteogenic differentiation, alkaline phosphatase
activity (ALP) was assessed by histochemical analysis.
Differentiated stem cells are positive for ALP staining. There
was a significant rise in the expression of ALP staining from
day 1 to day 7. ALP staining was much higher in pneumatic
stimulation groups compared to the no stimulation groups
(Fig. 7C and G). The addition of mechanical stimulation
caused a significant increase in the expression of ALP staining.
The increased ALP is a marker of the commitment towards
an osteoblastic lineage and correlated with advanced matrix
mineralization and mature phenotype.29,43
In addition, alizarin red staining showed enhanced calcium
deposition in the stimulated groups. This staining is based on
the capacity of alizarin red to specifically stain matrix
containing calcium and its positive appearance is considered
an expression of bone matrix deposition.30 Alizarin red
positive nodular aggregate in stimulation groups was present
at day 7, indicating that a more extensive calcium deposition
had occurred (Fig. 7H). This would prove that the micro cell
stimulation system induced the early stage of osteogenesis
rather than the mature stage.
Conclusions
Demonstrations of hMSCs culture and stimulation in the
micro cell chip actuated by pneumatic force was performed.
The results demonstrate the feasibility of the micro cell
stimulator as a convenient and effective tool for stem cell
studies on the mechanical stimulus. We have previously
demonstrated the feasibility of the micro cell stimulator
actuated by electromagnetic actuators for chondrogenic
differentiation.15,16 In our current study, a new micro cell chip
which can apply various amplitudes of pressures simultaneously is developed and tested. Osteogenic differentiation
also was accelerated by the mechanical stimulation using the
new micro cell chip. The results of the study revealed that
dynamic compression force from the micro cell chip could
enhance the proliferation and osteogenic differentiation of
human MSCs in the absence of growth factors. This micro
cell stimulating system is promising for the many potential
applications for osteogenesis of MSCs.
This journal is ß The Royal Society of Chemistry 2007
In addition, the micro cell chip reduces the quantity of stem
cells, process cost and time, and increases the throughput on
various stimulation conditions. This system can be adopted
and applied to stimulate and analyze stem cells in tissue
engineering and regenerative medicine. The results presented in
this study suggest that cyclic compressive mechanical stimulation may play an important role in the differentiation of
MSCs. Mechanical stimulus affects many different physical
and biochemical phenomena at the cellular level, including
proliferation and biosynthetic activity.
As a next step, we plan to study the optimal condition for
osteogenic differentiation using an advanced micro cell chip
with embedded pressure sensors. Further studies are needed to
identify the primary osteogenic signal associated with cyclic
compressive mechanical stimulation and to determine the
mechanism by which it influences commitment to and
progression through the osteogenic lineage.
In order to develop stem cell therapy, scientists have been
intensively studying the fundamental properties of stem
cells. As scientists learn more, the potential of stem cell
treatment goes beyond cell-based therapies to screening
new drugs and understanding birth defects. This would
permit new understanding of cellular abnormalities, including
cancer, and new ways of steering cell differentiation in
desired paths.1
Acknowledgements
This work was supported by the Korea Science and
Engineering Foundation (KOSEF) grant funded by the
Korea government (MOST) (No. R01-2003-000-11614-0).
The authors thank Prof. Sang-Hoon Lee (Korea University,
Korea), Prof. Ok-Chan Jeong (Inje University, Korea), and
Dr Sang-Wook Lee (Tokyo University, Japan) for their
invaluable discussion and advice.
References
1 J. A. Thomson, J. I. Eldor, S. S. Shapiro, M. A. Waknitz,
J. J. Swiergiel, V. S. Marshall and J. M. Jones, Science, 1998, 282,
1145–1147.
2 J. Gearhart, Science, 1998, 282, 1061–1062.
3 Stem Cell Information: Stem Cell Basics, NIH (gov); http://
stemcells.nih.gov/info/basics.
4 R. M. Green, The Human Embryo Research Debates, Oxford
University Press, 2001.
5 P. A. Zuk, M. Zhu, P. Ashjian, D. A. D. Ugarte, J. I. Huang,
H. Mizuno, Z. C. Alfonso, J. K. Fraser, P. Benhaim and
M. H. Hedrick, Mol. Biol. Cell, 2002, 13, 4279–4295.
6 S. Kadereit, Adult Stem Cells, ISSCR (http://www.isscr.org).
7 N. Dewitt, R. L. Badge, P. J. Donovan, J. Gearhart, A. Spradling,
D. D. Barbosa, T. Kai, T. Reya, S. J. Morrison, M. F. Clarke,
I. L. Weissman, S. Temple, P. Bianco, P. G. Robey, M. A. Surani
and A. McLaren, Nature, 2001, 414, 87–132.
8 D. R. Marshak, R. L. Gardner and D. Gottlieb, Stem Cell Biology,
Cold Spring Harbor Laboratory Press, 2001.
9 M. F. Pittenger, A. M. Mackay, S. C. Beck, R. K. Jaiswal,
R. Douglas, J. D. Mosca, M. A. Moorman, D. W. Simonetti,
S. Craig and D. R. Marshak, Science, 1999, 284, 143–146.
10 F. P. Barry and J. M. Murphy, Int. J. Biochem. Cell Biol., 2004, 36,
568–584.
11 C. Perka, O. Schultz, R. S. Spitzer and K. Lindenhayn, J. Biomed.
Mater. Res. A, 2000, 52, 543–552.
12 E. K. F. Yim, A. C. A. Wan, C. L. Visage, I. C. Liao and
K. W. Leong, Biomaterials, 2006, 27, 6111–6122.
Lab Chip, 2007, 7, 1775–1782 | 1781
13 I. Takahashi, G. H. Nuckolls, K. Takahashi, O. Tanaka, I. Semba,
R. Dashner, L. Shum and H. C. Slavkin, J. Cell. Sci., 1998, 111,
2067–2076.
14 J. D. Kisiday, M. Jin, M. A. DiMicco, B. Kurz and
A. J. Grodzinsky, J. Biomech., 2004, 37, 595–604.
15 S. W. Park, W. Y. Sim, S. H. Park, B. H. Min, S. R. Park and S. S.
Yang, KIEE Int. Trans. Electrophys. Appl., 2004, 4(4), 176–180.
16 S. H. Park, W. Y. Sim, S. W. Park, S. S. Yang, B. H. Choi,
S. R. Park, K. D. Park and B. H. Min, Tissue Eng., 2006, 12(11),
3107–3117.
17 W. Y. Sim, S. W. Park, S. S. Yang, S. H. Park and B. H. Min,
IEEE Rev. Adv. Micro, Nano Mol. Syst., 2006, 1, 565–566.
18 D. Beebe and A. Folch, Lab Chip, 2005, 5, 10–11.
19 A. Wiesmann, H. J. Buhring, C. Mentrup and H. P. Wiesmann,
Head Face Med., 2006, 2, 8.
20 D. A. Prentice, L. R. Kass (chairman), et al., Monitoring Stem Cell
Research, The President’s Council on Bioethics, 2004, 309–346.
21 A. I. Caplan and S. P. Bruder, Trends Mol. Med., 2001, 7(6),
259–264.
22 C. Lee, S. Grad, M. Wimmer and M. Alini, Topics in Tissue
Engineering, ed. N. Ashammakhi and R. L. Reis, BTE: Expert
issues e-book, 2006, vol. 2.
23 S. H. Elder, J. H. Kimura, L. J. Soslowsky, M. Lavagnino and
S. A. Goldstein, J. Orthop. Res., 2000, 18(1), 78–86.
24 C. C. Huang, K. L. Hagar, L. E. Frost, Y. Sun and H. S. Cheung,
Stem Cells, 2004, 22, 313–323.
25 A. Tourovskaia, X. F. Masot and A. Folch, Lab Chip, 2005, 5,
14–19.
26 A. Alhadlaq and J. J. Mao, Stem Cells Dev., 2004, 13, 436–448.
27 J. P. Rodriguez, M. Gonzalez, S. Rios and V. Cambiazo, J. Cell.
Biochem., 2004, 93, 721–731.
28 X. D. Chen, H. Y. Qian, L. Neff, K. Satomura and M. C. Horowitz,
J. Bone Miner. Res., 1999, 14, 362–275.
29 N. Jaiswal, S. E. Haynesworth, A.I. Caplan and S. P. Bruder,
J. Cell Biochem., 1997, 64, 295–312.
1782 | Lab Chip, 2007, 7, 1775–1782
30 A. Gregory, W. G. Gunn, A. Peister and D. J. Prockop, Anal.
Biochem., 2004, 329, 77–84.
31 E. M. Kearney, P. J. Prendergast and V. A. Campbell, Proceedings
of the Bioengineering in Ireland Conference, 2006, p. 90.
32 J. J. Mao, Proceedings of the Workshop on Stem Cell Research for
Regenerative Medicine and Tissue Engineering, NSF, 2007.
33 L. Griffith and G. Naughton, Science, 2002, 295, 1009–1016.
34 G. Robling, A. B. Castillo and C. H. Turner, Annu. Rev. Biomed.
Eng., 2006, 8, 455–98.
35 M. Wong, M. Siegrist and X. Cao, Matrix Biology, 1999, 18,
391–399.
36 J. J. Parkkinen, M. Lammi, S. Karjalainen, J. Laakkonen,
E. Hyvarinen, A. Tiihonen, H. J. Helminen and M. Tammi,
J. Biomech., 1989, 22, 1285–1291.
37 J. N. De Croos, S. S. Dhaliwal, M. D. Grynpas, R. M. Pilliar and
R. A. Kandel, Matrix Biology, 2006, 25(6), 323–331.
38 B. Kurz, M. Jin, P. Patwari, D. M. Cheng, M. W. Lark and
A. J. Grodzinsky, J. Orthop. Res., 2001, 19(6), 1140–1146.
39 N. E. Ajubi, J. K. Nulend, P. J. Nijweide, T. V. Lammers,
M. J. Alblas and E. H. Burger, Biochem. Biophys. Res. Commun.,
1996, 225, 62–68.
40 M. P. Fredrick, X. C. Neal, H. T. Charles, B. B. David, A. Simon,
H. Y. Fang, Q. Jinya and L. D. Randall, Am. J. Physiol. Cell
Physiol., 1998, 275, C1591–C1601.
41 V. C. Mow, C. C. Wang and C. T. Hung, Osteoarthritis Cartilage,
1999, 7, 41–58.
42 F. Nerucci, A. Fioravanti, G. Collodel, D. Gambera, S. Carta,
E. Paccagnini, L. Bocchi and R. Marcolongo, Boll. Soc. Ital. Biol.
Sper., 1999, 75(9–10), 55–62.
43 R. K. Jaiswal, N. Jaiswal, S. P. Bruder, G. Mbalaviele,
D. R. Marshak and M. F. Pittenger, J. Biol. Chem., 2000,
275(13), 9645–9652.
44 H. S. Ko, S. S. Yang, J. S. Yoo and H. J. Kim, J. Electr. Eng.
Technol., 2006, 1(3), 387–395.
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