Effects of near-infrared laser exposure in a cellular model of wound healing

Abstract

Clinical studies have demonstrated beneficial outcomes for low-level laser therapy (LLLT) using near-infrared (NIR) wavelengths. It has been hypothesized that the benefits of NIR LLLT are due in part to the thermal effects of NIR exposure. However, it is not clear whether photochemical interactions between NIR light and superficial tissues contribute to beneficial outcomes. To investigate the photochemical effects of NIR exposure, the efficacy of 980 nm NIR LLLT on human fibroblast growth rates is investigated using an in vitro model of wound healing.
A small pipette is used to induce a wound in fibroblast cell cultures, which are imaged at specific time intervals over 48 h and exposed to a range of laser doses (1.5-66 J/cm(2)) selected to encompass the range of doses used during other in vivo and in vitro studies. For each image acquired, wound sizes were quantified using a novel application of existing image processing algorithms.
Cell growth rates were compared across different laser exposure intensities with the same exposure duration, and across different laser exposure durations with the same exposure intensity. Exposure to low- and medium-intensity laser light accelerates cell growth, whereas high-intensity light negated the beneficial effects of laser exposure. Cell growth was accelerated over a wide range of exposure durations using medium-intensity laser light, with no significant inhibition of cell growth at the longest exposure durations used in this study.
Low-level exposure to 980 nm laser light can accelerate wound healing in vitro without measurable temperature increases. However, these results also demonstrate the need for appropriate supervision of laser therapy sessions to prevent overexposure to NIR laser light that may inhibit cell growth rates observed in response to lower intensity laser exposure.

Full text

ORIGINAL ARTICLE
Effects of near-infrared laser exposure in a cellular model of wound
healing
Mark D. Skopin & Scott C. Molitor
Department of Bioengineering, University of Toledo, Toledo, OH, USA
Key words:
cellular model; image analysis; near-
infrared laser; photobiotherapy; wound
healing
Correspondence:
Scott C. Molitor, Ph.D., Department of
Bioengineering, University of Toledo, 5051 Nitschke
Hall MS 303, 2801 W. Bancroft St., Toledo, OH
43606-3390, USA.
Tel: 11 419 530 8168
Fax: 11 419 530 8076
e-mail: scott.molitor@utoledo.edu
Accepted for publication:
26 September 2008
Conflicts of interest:
None declared.
Summary
Background: Clinical studies have demonstrated beneficial outcomes for low-level laser
therapy (LLLT) using near-infrared (NIR) wavelengths. It has been hypothesized that the
benefits of NIR LLLTare due in part to the thermal effects of NIR exposure. However, it is not
clear whether photochemical interactions between NIR light and superficial tissues
contribute to beneficial outcomes. To investigate the photochemical effects of NIR
exposure, the efficacy of 980 nm NIR LLLT on human fibroblast growth rates is investigated
using an in vitro model of wound healing.
Methods: A small pipette is used to induce a wound in fibroblast cell cultures, which are
imaged at specific time intervals over 48 h and exposed to a range of laser doses (1.5–66 J/
cm
2
) selected to encompass the range of doses used during other in vivo and in vitro studies. For
each image acquired, wound sizes were quantified using a novel application of existing
image processing algorithms.
Results: Cell growth rates were compared across different laser exposure intensities with the
same exposure duration, and across different laser exposure durations with the same
exposure intensity. Exposure to low- and medium-intensity laser light accelerates cell
growth, whereas high-intensity light negated the beneficial effects of laser exposure. Cell
growth was accelerated over a wide range of exposure durations using medium-intensity
laser light, with no significant inhibition of cell growth at the longest exposure durations
used in this study.
Conclusion: Low-level exposure to 980 nm laser light can accelerate wound healing in vitro
without measurable temperature increases. However, these results also demonstrate the need
for appropriate supervision of laser therapy sessions to prevent overexposure to NIR laser
light that may inhibit cell growth rates observed in response to lower intensity laser
exposure.
Photobiotherapy is the clinical application of light for healing
decubitus ulcers and other superficial wounds. Previous
studies have demonstrated a significant clinical value for the use
of low-level laser therapy (LLLT) to accelerate the healing of
superficial wounds (1, 2). Although the cellular mechanisms of
this accelerated wound healing are not known, recent studies
have demonstrated that LLLT from the visible red spectrum
accelerates cell growth in a cellular model of wound healing and
improves cellular metabolism in a dose- (3, 4) and time-
dependent manner (5).
The use of near-infrared (NIR) light may have significant
advantages compared with visible red light for clinical
applications. Until recently, most studies have used mono-
chromatic visible red light to investigate the wound-healing
benefits of LLLT (3, 6, 7). In particular, the longer wavelength
NIR light minimizes scatter produced by superficial layers of the
skin, and allows for a penetration of the light into deeper layers of
skin that are most active during wound-healing processes (8, 9).
In addition, NIR light produces heating of deeper skin layers,
promoting increased blood flow, and to further accelerate
healing processes (10, 11).
Although NIR light has demonstrated a clinical value (12, 13),
the effects of NIR light at the cellular level have not been
characterized. In particular, it is not known whether photochemical
responses produced by NIR exposure contribute to improved
clinical outcomes. To determine whether NIR can improve cell
growth and recovery in the absence of thermal effects, we have
utilized a 980 nm clinical diode laser in a cellular model of wound
healing. Our results show that limited doses of NIR light can
increasetherateofcellgrowthin vitro within hours of light exposure.
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Journal compilation r2009 Blackwell Munksgaard Photodermatology, Photoimmunology & Photomedicine 25, 75–80

Materials and methods
Cell culture
Fetal human skin fibroblast cells (ATCC CCD1070SK) were
grown in 75 cm
2
flasks containing Dulbecco’s modification of
Eagle’s medium (DMEM) that was modified to contain 1 mM
L-glutamine, 1% penicillin–streptomycin and 3% fetal bovine
serum. The cultures were incubated at 37 1C with 5% CO
2
at
85% humidity. Cells were trypsinized using a 0.25% (w/v)
trypsin and 0.03% EDTA solution in DMEM and seeded into
sterile 35 mm polystyrene culture dishes at a density of 7.0 10
4
cells/cm
2
. Cells were incubated overnight to allow the cells to
recover from trypsinization and to adhere to the bottom of the
culture dishes.
Wound-healing model
To simulate a wound, the central scratch model was used, which
consists of confluent monolayers of fibroblasts scratched with a
sterile pipette approximately 1 mm in diameter (Fig. 1) (14–16).
After the induced scratch, all media were removed from the
culture dish to eliminate unattached fibroblasts and any light
reflection associated with phenol-containing media. The media
were immediately replaced with 3 ml of phenol-free DMEM
that was modified to contain 1 mM L-glutamine, 1%
penicillin–streptomycin and 3% fetal bovine serum.
Laser irradiation
Following wound induction, the output of a 7.5W, 980nm laser
used for clinical applications (VTR 75; Avicenna Laser
Technologies, West Palm Beach, FL, USA) was focused on a spot
12.5 mm in diameter centered on the wound with the visible red
aiming beam disabled. To attenuate the laser output and focus the
light on the 12.5 mm diameter spot, a 3 mm fiber approximately
1.5 m in length was coupled to the laser output and directed
toward the center of the 35 mm culture dish approximately
10 mm above the dish surface. The spot size was chosen to
provide NIR exposure to cells around the wound margin while
minimizing any temperature increase in the culture media
during NIR exposure. Initial measurements using a thermistor
probe showed a minimal temperature increase (o21C) in the
culture media at the maximal exposure intensity.
Cell growth into the wound region at time intervals up to 48 h
postexposure was compared with control dishes in which no
laser light was used. Two different methods were used to vary the
dose of the laser exposure: the first compared different exposure
intensities over the same exposure duration, and the second
compared different exposure durations at the same exposure
intensity. For the first set of experiments, the laser output was
varied from 1.5 to 7.5 W to produce measured exposure
densities of 26–120 mW/cm
2
for 2 min, resulting in exposure
doses from 3.1 to 14.4 J/cm
2
. For the second set of experiments,
the laser output was fixed at 4.5 W or 73 mW/cm
2
and the
exposure durations were varied from 20 s to 15 min, resulting in
exposure doses from 1.5 to 66 J/cm
2
.
Image acquisition and analysis
Wound closure was measured by manual image sampling (17).
To assess the growth of cells back into the wound region, images
were acquired at hourly intervals up to 8 h after wound induction
and laser exposure, and then at 24- and 48-h postexposure with
an inverted microscope equipped with relief contrast optics
(IX-71; Olympus America Inc., Center Valley, PA, USA), and
visualized using a 4, 0.13 numerical aperture objective. To
maintain a controlled environment, culture dishes were returned
to the incubator between image acquisition sessions. Images
were acquired using a Quantix 57 scientific-grade digital CCD
camera (Roper Scientific, Tucson, AZ, USA) using custom-made
software designed to run under MATLAB (Mathworks Inc.,
Natick, MA, USA). For each laser exposure dose and time
interval following wound induction, images were acquired from
seven different culture dishes to provide repeated data samples
for statistical analysis.
To assess cell growth, an automated analysis routine was
developed using the MATLAB Image Processing Toolbox to
measure the area of the imaged field that was covered by cells
(Fig. 2). Outlines of individual cells or groups of cells were
obtained using the edge detection function edge( ) with the
Canny algorithm option to detect lines of pixels along cell
edges (18). Once edges were detected, the MATLAB function
Fig. 1. Cell culture model of wound healing. Cells were exposed to 97mW/cm
2
of laser light for 2 min following wound induction for an exposure dose
of 11.7 J/cm
2
. A sterile pipette approximately 1 mm in diameter is used to induce a wound in a monolayer of fibroblast cells plated onto 35 mm culture
dishes (left image). Fibroblast cells exposed to 97 mW/cm
2
of laser light for 2 min (11.7 J/cm
2
) following wound induction grow back into the wound
region within 8 h (middle image) and the wound is completely overgrown within 24 h (right image) in this example. In many dishes, the wounds were
still visible after 24 h but were completely overgrown within 48 h. Scale bar, 250 mm.
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Skopin & Molitor

imclose( ) was utilized to fill in the gaps between edge lines using
circular structuring elements approximately one cell width across
(12–15 pixels). Images were manually inspected to verify the
accuracy of this method; the most common error was inclusion
of gaps between cells not in the wound region, which was
manually corrected during this post-processing inspection. The
result of this analysis was the detection of pixels in regions
covered by cells where edges were in proximity, whereas regions
such as the wound that had little or no cell coverage were left
undetected. Cell coverage area was then quantified at each time
interval following wound induction and laser exposure by
adding the number of detected pixels and comparing this with
the coverage area immediately following wound induction.
Statistical analysis
To separate the effects of elapsed time and laser exposure dose, a
two-factor analysis of variance (ANOVA) was performed on the
cell coverage area data obtained from images acquired at different
post-exposure elapsed times and from different laser exposure
doses. Without any laser exposure, a complete re-growth of
fibroblast cells into the wound region will occur over the 48-h
period, during which cells were imaged. Therefore, a statistically
significant increase in cell coverage area will occur over the
course of the experiment. However, the two-factor ANOVA
procedure allows for the separation of two experiment factors,
in this case laser exposure dose and elapsed time following
wound induction. Therefore, statistically significant effects
of laser exposure dose can be compared with the effects of
elapsed time, and can provide an estimate of the acceleration
of cell growth by the various laser exposures. The statistical
software package Minitab 14 (Minitab Inc., State College, PA,
USA) was utilized to perform the two-factor ANOVA; data were
entered as three columns with elapsed time, exposure dose and
percent increase in cell coverage area from images immediately
taken after wound induction.
Results
Our results demonstrate that exposure to light from a 980 nm
laser can enhance cell growth rates in an in vitro wound model. A
range of exposure doses was investigated by varying the laser
output power over a fixed exposure duration, or by varying
exposure duration at a fixed laser output power. Figure 3 shows
the results of the first experiment in which the laser output power
was varied from 1.5 to 7.5 W to produce an exposure intensity of
26–120 mW/cm
2
over a 2-min exposure, resulting in exposure
doses from 3.1 to 14.4 J/cm
2
. Regardless of the exposure
intensity, significant cell recovery was observed within 3 h of
wound induction; however, exposure to moderate intensities of
laser light (26–97 mW/cm
2
) appeared to enhance cell growth at
all time intervals relative to control experiments in which no laser
exposure was applied (Fig. 3, top panel). These results were
confirmed by the results of a two-factor ANOVA (Fig. 3, lower
right), which shows that significant increases in cell growth were
observed with 2-min exposures to 26–73 mW/cm
2
(Po0.01)
and 97 mW/cm
2
(Po0.05). These results also show that the
beneficial effects of laser exposure are negated by overexposure:
fibroblasts exposed to 120 mW/cm
2
of laser light for 2 min did
not show any significant increase in growth rates relative to
control experiments.
The two-factor ANOVA analysis also provided a measure of
how much cell growth was accelerated by laser exposure. Over
the first 8 h following wound induction, the average cell coverage
increased linearly by approximately 3.3%/h (Fig. 3, lower left);
the growth rate begins to slow before 24 h, when cells across the
wound margin begin to contact each other and completely fill the
area previously devoid of cells. When compared with the mean
cell growth measured at various time intervals following wound
induction, the 4–5% increase in cell growth produced by
49–73 mW/cm
2
of laser exposure over a 2-min duration
represented an acceleration of wound healing by approximately
1.5 h within the first 8 h of healing. This represents a sizeable
acceleration in cell growth, considering that the wounds from
our in vitro model were nearly completely healed within 24 h
following wound induction. Despite the significant increases in
cell growth across various time intervals and exposure intensities,
the two-factor ANOVA analysis did not find any significant
interaction between elapsed time and exposure intensity. In
other words, the various exposure intensities showed consistent
effects across all time intervals following wound induction, and
there were no exposure intensities whose effects were only
Fig. 2. Image analysis procedure. The image acquired with a CCD camera from cells exposed to 72 mW/cm
2
of laser light for 20 s (1.5J/cm
2
) 4 h after
wound induction (left image). The MATLAB edge detection function edge( ) with the Canny algorithm option is used to detect lines of pixels along cell
edges (middle image). Once edges were detected, the MATLAB function imclose( ) was utilized to fill in the gaps between edge lines using circular
structuring elements approximately one cell width across (12–15 pixels) (right image). Detected pixels are then used to calculate cell coverage area,
which is expressed as 0% at the initial wound size to 100% to indicate that the wound has healed completely.
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Cellular effects of near-infrared laser exposure

observed at a particular time interval or a subset of time intervals
following wound induction.
Figure 4 shows the results of the second experiment in which
exposure durations were varied from 20 s to 15 min, resulting in
exposure doses of 1.5–66 J/cm
2
at a constant laser output power
of 4.5 W, which provides an exposure intensity of 73 mW/cm
2
.
As with changes in exposure intensity, significant cell recovery
was observed within 3 h of wound induction regardless of the
exposure duration, and a wide range of exposure durations
appeared to enhance cell growth at all time intervals relative to
control experiments in which no laser exposure was applied
(Fig. 4, top panel). These results were confirmed by the results
of a two-factor ANOVA (Fig. 4, lower right), which shows
that significant increases in cell growth were observed with
73 mW/cm
2
exposures having durations of 50 s and 2 min to
produce exposure doses of 8.8–21.9 J/cm
2
(Po0.01). Note that
a long exposure of 15 min (65.7 J/cm
2
) did not demonstrate a
significant increase in cell growth, again suggesting that
excessive exposure to laser light can reverse the benefits of lower
exposure does. A comparison of these results with the mean cell
growth measured at various time intervals following wound
induction (Fig. 4, lower left) showed that the 4% increase in cell
growth produced by 73 mW/cm
2
of laser exposure over a 50-s
to 2-min period represented an acceleration of wound healing by
approximately 1.5 h within the first 8 h of healing. Furthermore,
the two-factor ANOVA analysis did not find any significant
interaction between elapsed time and exposure duration despite
the significant increases in cell growth across various time
intervals and various exposure durations.
Discussion
This study confirms the clinical observation that low-level
exposure to 980 nm of diode laser light can accelerate cell
growth in a wound-healing model. Many studies have
investigated the effects of visible red light on in vitro cell growth
rates (2–4, 6, 19–23). Beyond the clinical setting, few
experiments have studied the cellular progression of wound
healing after exposure from light in the NIR spectrum. Because
the measurements were obtained from an in vitro cell culture
model, these results also suggest that the mechanisms involved in
the acceleration of cell growth following laser exposure are
cellular or molecular in nature. The hypothesis that IR light
0
20
40
60
80
100
123456782448
elapsed time (hours)
% recovery
control
26 mW / cm
49 mW / cm
73 mW / cm
97 mW / cm
120 mW / cm
0 20 40 60 80 100
1
2
3
4
5
6
7
8
24
48
% recovery
elapsed time (hours)
**
**
**
**
**
**
**
**
24 26 28 30 32 34 36
0
26
49
73
97
120
% recovery
exposure (mW / cm2)
**
**
**
Fig. 3. Top panel: cell growth in the wound model as a function of time elapsed from wound induction and laser exposure intensity. Vertical bars show
percentage change in cell coverage area averaged across seven experiments in which cells were not exposed to laser light, or in which cells were exposed
to 26–120 mW/cm
2
of light during a 2-min exposure by varying the laser power from 1.5 to 7.5 W to giveexposure doses from 3.1 to 14.4J/cm
2
. Error
bars show SEM across seven experiments; error bars for images acquired 48h after wound induction are small and do not exceed the horizontal dashed
line at 100% recovery. Bottom left: confidence intervals from the two-factor analysis of variance (ANOVA) analysis demonstrate a significant increase in cell
coverage area observed as early as 3h after wound induction regardless of laser exposure (

Po0.01 for 3 h and beyond). Horizontal bars show 95%
confidence intervals with midline at the mean value; error bars show 99% confidence intervals. Bottom right: confidence intervals from the two-factor
ANOVA analysis demonstrate a significant increase in cell coverage area for low and moderate intensities of laser exposure when compared with no laser
exposure (

Po0.01 for 49–97 mW/cm
2
). No significant increase in cell coverage was observed at the lowest (26 mW/cm
2
) or the highest (120 mW/
cm
2
) exposure intensities.
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Skopin & Molitor

accelerates healing processes by heating skin and promoting
increased blood flow (10, 11) could not explain the
increased cell growth rates in an in vitro cell culture model. The
measurements suggested that IR exposure produced temperature
increases o21C, and the use of a controlled incubation
environment between image acquisition intervals further
minimizes the temperature variability in our experiments.
Previous researchers have suggested that light exposure increases
ATP levels by altering the energetic state of light-sensitive
cytochromes within the inner mitochondrial membrane that
participate in oxidative phosphorylation (19, 22, 23). Other
research has demonstrated increased levels of cytokines (24) or
growth factors (25) immediately following LLLT in similar in vitro
models. However, it is not clear whether increased cellular
signaling is due to a direct interaction of laser light and
enzymatic activity associated with signaling molecule synthesis
and release, or whether these effects are observed in response to
increases in cellular metabolism that may occur following light
exposure. Further experiments are needed to examine changes in
ATP, cytokines and other molecular processes following low-level
exposure to 980 nm laser light.
The results also demonstrate the importance of appropriate
supervision of laser light exposure in a clinical setting. In
particular, the average cell growth rates formed a non-
monotonic function of laser exposure intensities (Fig. 3, lower
right) and exposure doses (Fig. 4, lower right), with peak growth
rates at moderate exposures, and reduced benefit at higher
exposure intensities and doses. This result confirms the clinical
observation that excessive exposure to NIR light could have
potentially damaging effects that may negate any initial benefit
of NIR exposure. Although the harmful effects of NIR
overexposure are generally attributed to tissue heating, we did
not observe significant heating of cells and media within our
in vitro model following NIR exposures at the highest intensities
and durations used in this study. The reversal in the increased cell
growth observed with excessive light exposure could result from
the excess of reactive oxygen species observed following LLLT
in vitro at slightly lower wavelengths (26). Although the
mechanisms of NIR overexposure in vitro were not resolved in
the present study, the appropriate NIR exposure intensity and
duration must be selected in order to maximize cell growth rates
in vitro.
In addition to investigating the potential benefits of LLLT using
NIR laser light, a goal of this study was to develop a reproducible
and automated process for quantifying cell growth within in vitro
models of wound healing. These results demonstrate the
0
20
40
60
80
100
123456782448
elapsed time (hours)
% recovery
control
1.5 J / cm
3.7 J / cm
8.8 J / cm
21.9 J / cm
65.7 J / cm
0 20 40 60 80 100
1
2
3
4
5
6
7
8
24
48
% recovery
elapsed time (hours)
**
**
**
**
**
**
**
**
24 26 28 30 32 34 36
% recovery
0.0
1.5
3.7
8.8
21.9
65.7
dose (J / cm
2
)
*
*
Fig. 4. Top panel: cell growth in the wound model as a function of time elapsed from wound induction and laser exposure dose. Vertical bars show
percentage change in cell coverage area averaged across seven experiments in which cells were not exposed to laser light, or in which cells were exposed
to 73 mW/cm
2
of light during exposure durations that varied from 20 s to 15 min to give exposure doses from 1.5 to 65.7J/cm
2
. Error bars show SEM
across seven experiments; error bars for images acquired 48h after wound induction are small and do not exceed the horizontal dashed line at 100%
recovery. Bottom left: confidence intervals from the two-factor analysis of variance (ANOVA) analysis demonstrate a significant increase in cell coverage area
observed as early as 3 h after wound induction regardless of laser exposure (

Po0.01 for 3 h and beyond). Bottom right: confidence intervals from the
two-factor ANOVA analysis demonstrate a significant increase in cell coverage area for moderate laser exposure doses when compared with low-level laser
exposure (

Po0.05 for 8.8–21.9 J/cm
2
when compared with 1.5 J/cm
2
). No significant increase in cell coverage was observed at the highest dose
(65.7 J/cm
2
).
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Cellular effects of near-infrared laser exposure

feasibility of applying standard image processing methods in a
widely used software package to the analysis of in vitro wound
model data. This process allows investigators to quantify cell
growth following wound induction, and provides numerical data
for the statistical analysis of the effects of various wound-healing
therapies. In addition, this procedure provides a consistent and
reproducible basis for measuring wound size that facilitates the
analysis of multiple images that are obtained during the
progression of wound healing in this cellular model. A manual
inspection of processed images showed that detection errors
were relatively uncommon (o15% of processed images). The
most common detection error was the inclusion of gaps between
cells outside the wound region as part of the wound region being
measured. These errors could be minimized by only including
pixels that were detected in the wound region from the previous
image in the series, or by use of the MATLAB bwselect command
to invert all undetected pixels in a connected region selected by a
mouse click during a manual review of processed images. These
techniques allow researchers to obtain quantifiable data of cell
growth in vitro in an accurate and efficient manner, with little or
no manual processing required.
Acknowledgements
We would like to thank James Ohneck of Laser Therapy Services
in Cleveland, OH, for providing the laser for these studies and for
comments on this manuscript. We would also like to thank Dr
Brent Cameron for providing the optics for laser exposures and
for assistance in laser intensity measurements. We also wish to
acknowledge the comments and suggestions of an anonymous
reviewer during the revision of this manuscript. This research
was funded by a grant from the State of Ohio Third Frontier
Product Development Program.
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r2009 The Authors
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Skopin & Molitor