Am J Physiol Cell Physiol 299: C528–C534, 2010.
First published May 5, 2010; doi:10.1152/ajpcell.00504.2009.
Effects of negative pressures on epithelial tight junctions and migration in
wound healing
Chih-Chin Hsu,1,2,3 Wen-Chung Tsai,2,3,4 Carl Pai-Chu Chen,4 Yun-Mei Lu,3 and Jong-Shyan Wang3
1
Department of Physical Medicine and Rehabilitation, Chang Gung Memorial Hospital at Keelung, Keelung; 2School of
Traditional Chinese Medicine, Chang Gung University, Taoyuan; 3Graduate Institute of Rehabilitation Science, College of
Medicine, Chang Gung University, Taoyuan, and 4Department of Physical Medicine and Rehabilitation, Chang Gung
Memorial Hospital at Linkou, Taoyuan, Taiwan
Submitted 12 November 2009; accepted in final form 30 April 2010
negative-pressure wound therapy; electric impedance; cell junctions;
cell movement
individuals are believed to have
chronic wounds and billions of dollars are spent on treatment
each year in the United States (12). The lifetime risk of
developing foot ulcers in the diabetic patient may be as high as
25% (26). Venous ulcers or wounds resulting from arterial
disease may also lead to chronic wound problems (14). Different types of dressings are developed but few of them have
convincingly been shown to provide higher wound closure
rates than traditional wet gauze dressings (31). Despite the
advance of current treatments for chronic wounds, many of
them fail to heal and persist for months to years (25). To
accelerate wound healing, the negative-pressure wound ther-
AN ESTIMATED THREE MILLION
Address for reprint requests and other correspondence: J.-S. Wang, Graduate
Institute of Rehabilitation Science, Chang Gung Univ., 259, Wen-Hwa 1st
Road, Kwei-Shan, Taoyuan 333, Taiwan (e-mail: s5492@mail.cgu.edu.tw).
C528
apy (NPWT) has been developed (20) and has become widely
used in chronic wound cares (30).
A significant reduction of the partial foot amputation wound
size in patients treated with NPWT has been reported in a
randomized control trial (1). Another trial has demonstrated
that this kind of therapy could increase granulation tissue
formation in chronic nonhealing wounds (12). The negative
pressure of 125 mmHg has been used as a therapeutic dosage
in clinical practice based on the subcutaneous flow measurements in a swine study (20). However, investigators have
concluded that negative pressure of 125 mmHg might not be
the optimal pressure for wound healing based on the cutaneous
blood flow measurement in 10 healthy subjects (29). A negative pressure of 75 mmHg has also been found to work on
holding a skin graft in place (11). Thus exploring mechanisms
for effects of the negative pressure on wound healing is
required to clarify the controversial findings.
With the aid of a commercialized NPWT device, different
negative pressures have been applied on human endothelial cells
(2) and human dermal fibroblasts (19). Conflicted results regarding cell viability and migration response to negative pressure have
been observed in the previous reports. These laboratory findings
did not provide strong support for the encouraging clinical observations of NPWT for chronic wounds (1, 12).
Wound-healing assays have been carried out in cell cultures for
many years to monitor the healing process. A new method of
obtaining cell migration information using an electrical means to
assess the wound-healing process is referred as electric cellsubstrate impedance sensing (ECIS). As cells grow and move on
the ECIS measure electrode, the impedance fluctuates until the
cell layer becomes confluent. The value is directly proportional to
the cell coverage (9) and intercellular resistance (34). The ECIS
technique has been used to continuously monitor Madin-Darby
canine kidney (MDCK) epithelial cell attachment and spreading
(34), the relationship between calcium signaling and coherent
changes in adhesion properties of hormone-stimulated MDCK
cells (7), and effects of exercise on natural killer cell activities
(33). Previous studies have also used the technique to evaluate the
impedance change along time of human keratinocytes to different
chemical toxicants (6), the adhesion capacity of normal human
keratinocytes to different adhesive peptides (21), and the keratinocyte migration response to the epidermal growth factor (37).
Therefore, the ECIS can be an ideal choice for in-depth analysis
of cell behaviors under various conditions. The purpose of the
present work is to integrate a negative pressure incubator (NPI)
and the ECIS technique to continuously observe the healing
process of wounded monolayer cells at different negative pressures.
0363-6143/10 Copyright © 2010 the American Physiological Society
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Hsu CC, Tsai WC, Chen CP, Lu YM, Wang JS. Effects of
negative pressures on epithelial tight junctions and migration in
wound healing. Am J Physiol Cell Physiol 299: C528 –C534, 2010.
First published May 5, 2010; doi:10.1152/ajpcell.00504.2009.—Negative-pressure wound therapy has recently gained popularity in
chronic wound care. This study attempted to explore effects of
different negative pressures on epithelial migration in the woundhealing process. The electric cell-substrate impedance sensing (ECIS)
technique was used to create a 5 ⫻ 10⫺4 cm2 wound in the MadinDarby canine kidney (MDCK) and human keratinocyte (HaCaT) cells.
The wounded cells were cultured in a negative pressure incubator at
ambient pressure (AP) and negative pressures of 75 mmHg (NP75),
125 mmHg (NP125), and 175 mmHg (NP175). The effective time (ET),
complete wound healing time (Tmax), healing rate (Rheal), cell diameter, and wound area over time at different pressures were evaluated.
Traditional wound-healing assays were prepared for fluorescent staining of cells viability, cell junction proteins, including ZO-1 and
E-cadherin, and actins. Amount of cell junction proteins at AP and
NP125 was also quantified. In MDCK cells, the ET (1.25 ⫾ 0.27 h),
Tmax (1.76 ⫾ 0.32 h), and Rheal (2.94 ⫾ 0.62 ⫻ 10⫺4 cm2/h) at NP125
were significantly (P ⬍ 0.01) different from those at three other
pressure conditions. In HaCaT cells, the Tmax (7.34 ⫾ 0.29 h) and
Rheal (6.82 ⫾ 0.26 ⫻ 10⫺5 cm2/h) at NP125 were significantly (P ⬍
0.01) different from those at NP75. Prominent cell migration features
were identified in cells at the specific negative pressure. Cell migration activities at different pressures can be documented with the
real-time wound-healing measurement system. Negative pressure of
125 mmHg can help disassemble the cell junction to enhance epithelial migration and subsequently result in quick wound closure.
CELLULAR RESPONSE TO NEGATIVE PRESSURE
MATERIALS AND METHODS
NPI. To create a negative pressure circumstance for cell culture, air
had to be removed from the incubator. The pressure and water content
would decrease persistently in building up the negative pressure
environment. Thus an NPI should have a capability to keep a dynamic
balance between the removal and the supply of the air and water at
different air pressures. An NPI (NPI1000, Linston Advanced Technology, Longtan, Taoyuan, Taiwan) was constructed based on the
above principles. An airtight chamber was used as the incubator to
harvest cells. The air in the incubator was continuously removed by a
vacuum pump with a speed of 46 l/min and the pressure drop rate of
12.5 mmHg/s. A manual knob was used to adjust the pressure level in
the airtight incubator. Adequate amount of room air was introduced
into the chamber to maintain the incubator O2 tension constantly at
20% of incubator air pressure. Mist, produced by an ultrasound mist
generator in a water container, was brought into the incubator to
maintain the relative humidity ⬎60%. The CO2 was constantly
supplied to maintain the incubator CO2 tension at 5%. A gas analyzer
was used to detect the O2 and CO2 tensions in the incubator. The
temperature inside the chamber was maintained at 37°C by a heating
system surrounding the chamber wall. The ECIS (1600R, Applied
Biophysics, Troy, NY) was integrated into the incubator to continuously monitor cell behaviors at different pressures. The equipment
was presented in the Fig. 1.
Cell culture procedures. MDCK cells (Bioresource Collection and
Research Center, Hsinchu, Taiwan) and human skin keratinocyte
HaCaT cells (kindly provided by the Institute of Pharmacology,
National Yang-Ming University, Taiwan) were cultured in DMEM
(Sigma, St. Louis, MO) solution containing 10% FBS and 10 mg/ml
streptomycin-penicillin.
Inoculation of cells on electrode arrays. In assessment of cell
migration, a confluent monolayer is first formed on the glass in the
AJP-Cell Physiol • VOL
traditional wound-healing assay. Scratching the layer with a needle or
micropipette tip then disrupts the layer. Once the wound is achieved,
the open area is then intermittently observed over time with a
microscope or after special staining to assess the healing process (22).
In comparison with the traditional wound-healing assay, the ECIS has
been developed to record cell migration activities during wound
healing without interruption. With this technique, the wound size can
be induced with high reproducibility and the order of the resolution
for cells motion is nanometers (17).
Each ECIS electrode array (8W1E) consisted of eight individually addressable wells. The insulating film covering the gold film
electrode with an exposed measure area of 5 ⫻ 10⫺4 cm2 with the
approximate diameter of 250 ␮m is deposited on the bottom of
each well. We followed the previously developed procedure to
inoculate cells on the electrode array (13). Each well contained a
final concentration of 1.25 ⫻ 105 cells/cm2 and a medium volume
of 400 ␮l. Thereafter, eight electrodes were prepared and were
incubated at ambient pressure for 24 h before experiments in different
pressures. The impedance fluctuations of cell attachment and spread
were continuously monitored. In ECIS normal mode, an alternative
current (AC) of 1 ␮A at 4 kHz on the measure electrode was applied.
The impedance in each well was measured. The impedance at different wells ranged from 5,000 to 1,200 Ohms. Although we had loaded
constant amount of cells into each well, the cell spread and attachment
in each well might be different. The variation could lead to the
different degree of cell-cell contact and finally result in different
initial impedance (34). Diameters of 20 randomized selected cells in
the electrode area were measured with a phase-contrast and fluorescent microscope (Leica DMIRB, Leica Microsystems, Wetzlar, Germany), and the average value of the selected cells was treated as the
original cell diameter (Do) in each well.
Cellular response at different pressures. An AC of 1 mA at 40 kHz
with duration of 200 ms was applied to electroporate the confluent
monolayer cells. A wound area equal to the measured electrode size
was produced. Cell movement started immediately after the injury.
The impedance in each well increased gradually during the healing
process. When the wounded monolayer cells became confluent, the
impedance arrived at the maximum plateau level, which was similar
as the prewounded impedance value (Fig. 2). The impedance at this
stage of both cell lines in different wells at different pressures varied
Fig. 2. Impedance time-course graph at ambient pressure. The X-axis represented the time in hours and the Y-axis represented the normalized impedance,
i.e., impedance of different time divided by the maximum impedance. As cells
migrated from the periphery of the wound, the impedance (solid line) increased
gradually during the wound-healing process. The effective time (ET) was
determined when the impedance was about half of the maximum impedance
(dotted line). The complete wound healing time (Tmax) was determined when
the measured impedance arrived at the maximum level (dashed line).
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Fig. 1. The integrating system of a negative pressure incubator and electric cellsubstrate impedance sensing (ECIS) equipment. A: airtight chamber, B: pressure meter,
C: air flow meter, D: LED chamber temperature display, E: relative
humidity meter, F: ECIS, G: gas analyzer, H: water container and ultrasound mist generator, I: vacuum pump, J: CO2 tank.
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Fig. 4. The mean ET, Tmax, and healing rate (Rheal) of wounded monolayer
madin-Darby canine kidney (MDCK) (open bars) and HaCaT (solid bars) cells
at different pressures. Each error bar represented 1 SD. Significant difference
of wound healing response could be observed between the AP and NP125 (*),
AP and NP75 (†), and the NP75 and NP125 (‡).
Fig. 3. Healing process in both cell lines at different pressures. Each well of the
electrode array was fully covered by the cells at ambient pressure (AP). The
circle area was the exposed measure electrode with the diameter of 250 ␮m.
An elevated alternative current (AC) current was applied to electroporate the
confluent monolayer cell. Then the electrode array was harvested at AP,
negative pressure of 75 mmHg (NP75), 125 mmHg (NP125), and 175 mmHg
(NP175). The wound healing condition was checked at ET and Tmax.
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each well. Six prepared coverglasses were then harvested at AP and
NP125, respectively, for 3 h. Two of them were prepared for cells vital
stain. The remaining four coverglasses were fixed with 4% paraformaldehyde for 5 min and prepared for tight junction and actin
filaments stains at room temperatures. There were eight wells in each
stain, respectively, at AP and NP125.
Vital staining. The Live/Deads Viability/Cytotoxicity kit (Invitrogen, Carlsbad, CA) was used for vital staining of cells in two
coverglasses at the two different pressures. The kit differentially stains
live and dead cells. Green fluorescence is indicative of live cells, and
red fluorescence indicates dead cells (23). Random selections of four
fields of view at ⫻200, about 8,000 cells in each view, were counted
per well with MetaMorph 5.0 software (Molecular Devices, Sunnyvale, CA). The dead cell percentage was determined as numbers of
dead cells divided by total cells in each view.
Cell junction and nucleus staining. To observe the tight junction,
we stained the MDCK and HaCaT cells with antibodies to ZO-1
conjugated by Alexa Fluor 594 (Invitrogen). Primary rat monoclonal
anti-(E-cadherin) antibodies (Abcam, Cambridge, UK) were used to
observe the cell adherent junction. FITC-conjugated AffiniPure Goat
anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove,
PA) was used as the secondary antibodies for fluorescent microscopic
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from 5,500 to 15,000 Ohms. To compare resistance between different
wells at different pressures, the relative degree of impedance, i.e.,
impedance at certain point during the wound-healing process was
divided by the maximum impedance at complete wound healing stage,
was used in the study. The effective time (ET), representing the
sensitivity of wounded cells to the applied pressure, was defined as the
time between the immediately postwounded point and the time at 50%
of the maximum impedance. The time from the beginning of wound
healing to the time of the maximum impedance (Tmax) was defined as
complete wound healing time. The shorter the two parameters were
the faster the wound healed. The wounded cells in 16 wells in two
control electrode arrays were cultured in our self-constructed incubator at ambient pressure (AP). Every two of the other six electrode
arrays were harvested under the negative pressure of 75 (NP75), 125
(NP125), and 175 (NP175) mmHg, respectively. Therefore, the tested
sample number was 16 in each group. Healing processes at different
pressures were shown in the Fig. 3. The monolayer cell wound healing
rate (Rheal) was estimated as 5 ⫻ 10⫺4/Tmax cm2/h. Each well at ET
and Tmax was examined with the microscope. The cell diameter in
each well at the complete wound-healing stage (Dend) at different
pressures was determined as the measurement for Do. The strain (ε) of
cells at different pressures could be derived as (Dend ⫺ D0)/D0. The
wound area (A) in each well at ET in the four groups was measured
with Photoshop CS4 extended (Adobe System, San Jose, CA). The A
was further normalized with the measure electrode area (AN) and was
calculated as A/5 ⫻ 10⫺4. Therefore, the AN at initial and end
wound-healing process would always be 1 and 0, respectively. The
ET, Tmax, Rheal, Do, Dend, ε, and AN in the four groups were recorded.
Traditional wound healing assay. A 400-␮l DMEM medium containing 1.25 ⫻ 105 cells/cm2 was inoculated on each well in the
chambered coverglass system (Lab-Tek, Nalge Nunc International,
Naperville, IL), and then the system was incubated overnight. Every
coverglass has eight wells. A pipette tip (0.1–10 ␮l, Labcon North
America, Petaluma, CA) was later used to create a scratch wound in
CELLULAR RESPONSE TO NEGATIVE PRESSURE
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examination. Counterstaining of the nucleus was performed with
4=,6-diamidino-2-phenylindole solution (Invitrogen).
Actin staining. Alexa 488-conjugated phalloidin (Sigma) was used
to stain actin filaments in the MDCK and HaCaT cells permeabilized
by 0.5% (wt/vol) Triton X-100 solution.
Preparation of cell lysates and protein analysis. The MDCK cells
of 2 ⫻ 106 were seeded on a 60-mm culture dish (Corning, Corning,
NY). A pipette tip (0.1–10 ␮l, Labcon North America, Petaluma, CA)
was used to create a scratch wound in four culture dishes. The two
dishes were incubated at AP and NP125, respectively. The cells at
different pressures were washed with ice-cold lysate buffer (pH 7.4)
Fig. 7. MDCK cell vitality in the traditional wound-healing assay at AP and
NP125. The numbers of live cells (green) and dead cells (red) were similar
between the two pressure circumstances (Magnification ⫻200).
Fig. 6. The average cell diameter before wound creation (open bars) and after
wound healing (solid bars) in both cell lines at different pressures. Each error
bar represented 1 SD. The cell diameter was similar before wound creation in
all tested samples. Although the cell diameter was also similar at complete
wound-healing stage at four different pressures, increased cell diameter was
observed in cells at NP125.
before incubation and after incubation of 3 h. Each lysis solution was
centrifuged at 12,000 g for 1 min at room temperature for three times
and the supernatant was used for protein quantification. Protein
samples of 20 ␮g in each lane were separated on a 7% SDSpolyacrylamide gel for ZO-1 and were transferred to polyvinylidene
difluoride membranes [Immobilon(TM)-P, Millipore, Bedford, MA].
The membranes were incubated with primary mouse monoclonal
anti-(ZO-1) antibodies at 4°C overnight and then incubated with the
anti-mouse IgG antibodies for more than 1 h. The immunoreactive
protein bands were developed by enhanced chemiluminescence
method (ECL, Amersham Pharmacia Biotech, Freiburg, Germany).
The other one dish with confluent and undamaged monolayer cells
was cultured at AP and treated as a control for ZO-1 protein analysis.
The analysis of E-cadherin in cells at different pressures and the
control group followed the above procedure. SDS-polyacrylamide gel
(10%), primary mouse monoclonal anti-(E-cadherin) antibodies, and
secondary anti-mouse IgG antibodies were used for quantifying Ecadherin. Three groups of cells were used for cell junction proteins
analysis. Protein amounts in cells at the control, AP, and NP125
status were measured by Gelpro32 Analyzer (Media Cybemetics,
Bethesda, MD).
Kruskall-Wallis test was used to compare the differences of ET,
Tmax, Rheal, AN, Do, Dend, and ε among the four groups of cells. The
analysis was also conducted to estimate the differences of ZO-1 and
E-cadherin expression between cells at the control, AP, and NP125.
The differences between any two of the above four groups of cells was
estimated by multiple comparisons Dunn’s test. The difference of the
dead cell percentage between cells at the AP and NP125 were compared with Student t-test. A P value ⬍0.05 was regarded as statistical
significance.
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Fig. 5. Wound healing curves at different pressures. The normalized wound
area (AN) changed over time. Each error bar represented 1 SD. The AN
measured at ET was similar at the four pressures. The nonlinear wound-healing
characteristics were observed in the wound-healing process.
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CELLULAR RESPONSE TO NEGATIVE PRESSURE
RESULTS
Fig. 9. MDCK cell junction protein amounts analysis in injured cells harvested
at negative pressure of 125 mmHg (NP) for 3 h and at AP for 3 h, respectively.
The uninjured confluent monolayer cells were treated as the control (C). Reduced
expression of ZO-1 and E-cadherin in injured cells at NP was observed when
compared with those incubated at AP and the control. The cell junction protein
amounts at AP and NP were divided by those in the control. Thus the protein
amounts in the control were 1 and the relative protein amounts at the AP and
NP were shown in the bar graph.
to the control were measured. Although ZO-1 (P ⫽ 0.148) and
E-cadherin (P ⫽ 0.095) expression in cells at NP125 was not
significantly different from cells at AP, a trend of decreased cell
junction proteins at NP125 was observed (Fig. 9). Prominent
Fig. 8. Immunocytochemical staining of ZO-1 (red)
and E-cadherin (green) in MDCK cell junctions and
4=,6-diamidino-2-phenylindole nucleic staining (blue).
A: prominent fluorescence of ZO-1 in tight junction
and nucleus was observed in cells (white arrow) at AP.
B: almost no ZO-1 fluorescence was observed in cells
(white arrowhead) at NP125. B: similar findings were
observed in E-cadherin activities in cells (arrow) at AP
and cells (arrowhead) at NP125. C and D: prominent
E-cadherin fluorescence (open arrow) in cells at AP
and decreased E-cadherin fluorescence (open arrowhead) in cells at NP125. (Magnification ⫻200).
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The ET (1.25 ⫾ 0.27 h), Tmax (1.76 ⫾ 0.32 h), and Rheal
(2.94 ⫾ 0.62 ⫻ 10⫺4 cm2/h) in MDCK cells cultured at NP125
were significantly (P ⬍ 0.01) different from those harvested at
AP and NP75. The ET (7.85 ⫾ 1.9 h) in HaCaT cells cultured
at NP75 was significantly (P ⬍ 0.01) different from those
harvested at AP. The Tmax (7.34 ⫾ 0.29 h) and Rheal (6.82 ⫾
0.26 ⫻ 10⫺5 cm2/h) in HaCaT cells at NP125 were significantly
(P ⬍ 0.01) different from those at NP75. The detail information
was presented in the Fig. 4. Measured AN in both MDCK and
HaCaT cells at ET ranged from 0.11 ⫾ 0.02 to 0.19 ⫾ 0.05 h
under different pressures during the wound-healing process.
The wound-healing process was similar in both cell lines at
four different pressure conditions (Fig. 5). The cell diameter
was similar during the wound-healing process in both cell lines
(Fig. 6). An increase of cell diameter after complete wound
healing was observed in MDCK cells at NP125 with the
positive strain of 8.04 ⫾ 6.0%. Similar findings could be
identified in HaCaT cells and the strain was 10.8 ⫾ 5.9%.
Wounded MDCK and HaCaT cells at NP125 had the significantly (P ⬍ 0.01) greatest wound-healing rate when compared
with cells at the other three pressure conditions. Therefore, viability, tight junction, and actin fluorescent staining were done in
cells treated with AP and NP125. The percentage of dead cells in
each well ranged from 0.05% to 1.8% at AP and ranged from
0.1% to 1.6% at NP125 in both cell lines. Since the cell viability
was not significantly different (P ⬎ 0.05) between AP and NP125
in both cell lines, only the MDCK cells vital stain results were
presented in the Fig. 7. Decreased ZO-1 and E-cadherin fluorescent intensities in MDCK cells at NP125 were observed (Fig. 8).
Similar findings could also be observed in HaCaT cells and thus
the stained cells pictures were not shown in Fig. 8. Relative
amounts of ZO-1 and E-cadherin in MDCK cells at AP and NP125
CELLULAR RESPONSE TO NEGATIVE PRESSURE
lamellipodium and filopodia were identified in MDCK cells harvested at NP125 (Fig. 10). Similar changes could be observed in
HaCaT cells and were not additionally presented in the figure.
DISCUSSION
Fig. 10. Immunicytochemical staining of actin in MDCK cells at AP and
NP125. The wound was at the lower portion of each panel. A: cells at AP did
not demonstrate prominent lamellipodia. B: cells at NP125 enlarged and
deformed prominently. The dense green portion (arrow) was the lamellum, the
more distant thin plasma sheet (open arrow) was the lamellipodium and many
digit protrusions (arrowhead) were filopodia (Magnification ⫻200).
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recommended dosage of negative pressure of 125 mmHg (20).
Suction force induced by the negative pressure can exert a centripetal effect at wound edges and may change cell migratory
behaviors (35). Greater cell coverage did not result in the increase
of measured impedance in cells at negative pressure ⬎125
mmHg. Alteration of intercellular resistance, reflecting degree of
contact in cell-cell junction, is assumed at the specific pressure
environment. Fluorescent intensities of tight junctions and adherent junctions decreased in cells harvested at NP125 have been
documented in the study. Cells have spent 60 –70% of complete
healing time to cover 80 –90% of the wound. The healing process
seems to slow down at late wound-healing stage, because it takes
30 – 40% of complete healing time to cover the remaining 10 –
20% of wound size. The healing pattern is commensurate with the
exponential wound-size decay model based on clinical observations of the healing of venous leg ulcers (3). It is speculated that
the negative pressure greater than 125 mmHg may help loosen
connections between cells during wound healing. The alteration
occurs mainly in the early wound-healing stage and may help the
wound to close quickly. Increased cell volume at late woundhealing process in a limited space may slow down cell migration
because of cell-cell contact inhibition (27).
Disassembly of cell-cell junctions to facilitate cell migration
has a central role in wound closure (5). Matrilysin (matrix metalloproteinase-7) has been reported to facilitate migration by
shedding E-cadherin ectodomain (18). Stimulation of the proximal tubular cell line by transforming growth factor-␤ (TGF-␤)
can induce cell junction disassembly thus allowing cell migration.
Reduction in the expression of both ZO-1 and E-cadherin has
been identified after the TGF-␤ stimulation (28). It is considered
that the negative pressure may activate cell migration through
cytokine effects on cell junctions. The first step in crawling cell
motility is the forward protrusion of the cell front, generally as a
thin sheet of membrane-enclosed cytoplasm, lamellipodia, and
finger-like protrusions, filopodia. Actin filaments assemble to
form these structures from actin monomers in response to different stimuli (4). Prominent lamellipodia and filopodia have also
been identified in cells at NP125. Cells enlarged with the strain of
about 8% in MDCK cells and about 10% in keratinocytes at
NP125, which may be associated with cytoskeletons induced cell
migration activities. The value ranges in the 5% to 20% of cell
strain reported by the finite element model study for wound
histology examination after NPWT treatment (24). Negative pressure greater than 125 mmHg may dissociate the cell adhesion to
the extracellular matrix. The effect may slow down the cell
movement.
Limitation of the study is that cells are cultured at various
subnormal O2 tensions in the three different negative pressure
conditions. Hypoxia is a microenvironmental stress in wounded
skin. This disturbance can facilitate wound healing by promoting
cell motility. Human dermal fibroblast migration can be enhanced
at 1% O2 with the aid of hypoxia-inducible factor-1␣ (HIF-1␣)
(16). On the contrary, the clinical observation has suggested that
poorly healed chronic wounds are frequently associated with low
tissue oxygen (8). Thus effects of O2 tension on wound healing
are still controversial. Although acute exposure to 12% O2 may
promote leukocytes transmigration activity, this phenomenon
does not occur at 12–21% O2 tension (32). A previous report has
found that the microvascular inflammatory response would not
occur in rats when inspired O2 tension is ⬎70 Torr (36). The O2
tension in the study ranged from 15% to 18%. It is considered that
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Basic science for the effects of NPWT on wound healing,
including creating a moist wound-healing environment (10), removing fluid containing an imbalance of matrix metalloproteinase
and their inhibitors (15), enhancing subcutaneous blood flow at
the wound bed (20), reducing bacterial loads (20), and translating
physical stress to cell proliferation (24) has been proposed. However, benefits of negative pressure on cell migration are still
debated. Fibroblasts in a three-dimensional fibrin matrix were
treated with either negative pressure plus dressings or without
negative pressure. Less migration and higher apoptosis have been
detected in cells at negative pressure (19). Effects of the negative
pressure on endothelial cells migration into the dermal substitute
has been studied in a polycarbonate negative pressure chamber
(2). From the study, both negative pressure of 40 and 125 mmHg
could enhance endothelial cell migration, and the effects were
similar between the two different negative pressures. On the
contrary of the above fibroblast three-dimensional culture study,
there was no significant loss of cell viability during the negative
pressure treatment period. Therefore, further investigation is still
indicated to define the debated issue.
A significant increase of wound-healing rate occurred only at
NP125 in our measurements. The findings support the clinically
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ACKNOWLEDGMENTS
We thank the Department of Industrial Technology, Ministry of Economic
Affairs in our country for building the negative pressure incubator, the Chang
Gung Memorial Hospital at Keelung for granting (CMRPG280181) the academic research, and Dr. Shu-Er Chow in Center of General Education, Chang
Gung University for quantifying protein amount.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
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neither hypoxia nor HIF-1␣ play a major role in enhancing the
epithelial migration in the experiment. The changes of cell behaviors are largely owing to the applied negative pressure. Our model
can mimic the regional milieu of the superficial wound during
NPWT. Local environment in the NPI may be different from
tissues deep in the real wound because there is no blood flow in
the study design.
The present work has used the ECIS technique to continuously
document cell behaviors during wound healing at different pressures in the NPI. The measured impedance is an integrative
performance of cell junctions and migration activities in the
wound-healing process. Decreased cell junction activities at negative pressure in wound healing are observed and cells migrate to
heal the wound thereafter. Negative pressure of 125 mmHg for
wound treatment can activate such healing cascade to accelerate
the wound closure without interfering cells viability. Findings
concerning cell migration obtained with this model at different
interventions may help refine the contemporary modality and
encourage the development of new facility in the future.