Photomedicine and Laser Surgery
Volume 27, Number 2, 2009
© Mary Ann Liebert, Inc.
Pp. 261–267
DOI: 10.1089/pho.2008.2237
Effect of Visible and Infrared Polarized Light
on the Healing Process of Full-Thickness Skin Wounds:
An Experimental Study
Panagiota Iordanou, Ph.D.,1 Efstathios G. Lykoudis, Ph.D., M.D.,2 Athanasios Athanasiou, Ph.D., M.D.,3
Efthymios Koniaris, M.D.,4 Maria Papaevangelou, Ph.D., M.D.,4 Theodora Fatsea, M.D.,3
and Panagiota Bellou, Ph.D.1
Abstract
Objective and Background Data: Polarized light has already been experimentally and clinically used in an effort to promote wound healing, but the findings have been equivocal. The aim of this study was to evaluate
the effect of visible and infrared polarized light of a specific range of wavelength (580–3400 nm) on the secondary healing of full-thickness skin wounds in rats. Materials and Methods: Forty male Wistar rats were used,
divided in two groups of 20 animals each. A standardized open full-thickness skin wound was created on the
back of each animal. In the first group the rats were exposed to polarized light (40 mW/cm2 and 2.4 J/cm2) for
7 min on a daily basis (total daily dose 16.8 J/cm2), while the second group acted as controls. Clinical and
histological evaluation of wound healing were performed on days 5, 10, 15, and 20 post-wound. The size of
the wounds was measured with the use of planimetry, whereas epithelialization, inflammatory response, neovascularization, and collagen formation were histologically assessed. Results: According to our findings, the
group exposed to light therapy showed statistically significantly faster epithelialization seen on days 10 and 15
post-wound compared to controls, as well as better quality of the healing process (although not statistically significantly) at all time points. Conclusion: In conclusion, this specific fraction of polarized light seems to have
beneficial effects on wound healing, leading to faster epithelialization and qualitatively better wound healing.
Introduction
N
ONHEALING ACUTE OR CHRONIC WOUNDS represent a
source of pain and disability for thousands of patients,
and have a great impact on their quality of life. In addition,
they represent a significant economic burden to society due
to loss of income and increased health care costs. The development of an effective, fast-acting, and low-cost treatment
for these kinds of wounds would have enormous benefits
for both patient quality of life and the economic burden.1,2
To date, a wide range of local and systemic treatment modalities have been used, both experimentally and clinically,
in an effort to improve and accelerate wound healing.
Among these, certain physical applications have been tried,
such as pulsed electromagnetic fields, low-power laser therapy, and ultrasound, which have in common the delivery of
energy to the target tissues.3–5
It is generally accepted that low-power laser light promotes wound healing, and this is attributed to its biomodulating effect, which depends on several parameters, such as
wavelength, dose, coherence, and power density. There are
also some studies that support the beneficial effects of noncoherent light on wound healing.6,7–13 The data indicating
the potential beneficial effects of polychromatic polarized
light on wound healing are limited and controversial, and
few experimental and clinical studies have been conducted
using this spectrum of polarized light.7–9,14–17
The aim of this study was to investigate the effect of polarized polychromatic light of a specific range of wavelength
(580–3400 nm), which represents the yellow, orange, red, and
near-middle infrared portions of the spectrum, on secondary
skin wound healing in a rat model. This modality uses the
fraction of visible light that is capable of deeper tissue penetration, and the fraction of infrared that has only minimal
1Technological Educational Institute of Athens, 2Department of Plastic Surgery and Burns, Ioannina University School of Medicine, Ioannina, 3Department of Plastic Surgery, and 4Department of Pathology, Hellenic Anticancer Institute “St. Savvas,” Athens, Greece.
261
262
IORDANOU ET AL.
TABLE 1.
Score
0
1
2
3
4
5
SCORING SYSTEM USED
Epithelialization
None
Minimal
Focal
Wide
Nearly complete
Complete
TO
ASSESS
THE
HISTOPATHOLOGICAL PARAMETERS
Inflammatory
response
Neoangiogenesis
Collagen formation
None
Minimal
Mild
Moderate
Severe
None
Minimal
Mild
Moderate
Considerable
None
Minimal
Mild
Moderate
Considerable
harmful effects. To the best of our knowledge, this specific
fraction of polarized light has not yet been investigated for
this purpose.
Materials and Methods
Forty male Wistar rats, 17 weeks old and weighing 200 30 g, were used and cared for according to Greek and European guidelines regulating animal research. The animals
were acclimated for a period of 3 d prior to the experiments,
during which time they were examined for any signs of disease. Throughout the study period the animals were kept
under stable conditions (temperature 22° C, humidity
30%–70%, and light cycles on a 12/12-h light/dark schedule), and nourished with dried pellets and tap water.
On day 0, a standard full-thickness skin defect was created on the back of each rat. Under intraperitoneal anesthesia (ketamine 3.5 mg/kg body weight and midazolam 7
mg/kg body weight) and aseptic conditions, all animals underwent en block excision of the skin and underlying panniculus carnosus of a square-shaped area measuring 2 2
cm from their back.
The rats were randomly divided in two groups of 20 rats
each. The animals in the first group (experimental group:
EG) were exposed to polarized light, and those of the second group (control group: CG) acted as controls. A polarized light source (Bioptron III, Bioptron-AG, Wellezau, Germany) with the following characteristics was used:
wavelength 480–3400 nm, degree of polarization 95%,
power density 40 mW/cm2, and light energy 2.4 J/cm2.
However, the wavelength of the polarized light delivered to
the animals was modified to 580–3400 nm via the use of a
Wratten color gelatin filter (number 25, Eastman Kodak
Company, Rochester, NY, USA) placed over the light source.
Beginning on day 0 and then on a daily basis, each animal in the EG was placed in restraints for 7 min in a specially-constructed wooden cage (16 8 cm). No metallic
parts were used to build the cage to avoid any interference
with the polarized light. The light source was vertically centered over the box at a distance of 10 cm from the wound
surface. In the EG the animals were exposed to polarized
light (total daily dose: 2.4 J/cm2 7 min 16.8 J/cm2). In
the control group, although the animals were caged for the
same time period, the light source was not activated. From
the beginning until the end of the experiment the rats were
housed individually to avoid cannibalistic behavior. Dressings were not used and antibiotics were not administered.
On days 5, 10, 15, and 20 after wound creation, five rats
from each group were sacrificed to evaluate progression of
the healing process. Clinical evaluation comprised measure-
ment of the size of each wound via the use of a high-precision (1 mm2) polar planimeter (model no. 317 E; HAFF
Planimeters, Pfrenten, Germany) by tracing its borders onto
a plastic film. Also, the presence of purulent discharge or
any other signs of infection of the wound were recorded. As
for the histological evaluation, each wound with some surrounding healthy tissue was harvested, fixed in 10% formalin, paraffin-embedded, and cut into 5-mm-thick sections
perpendicularly to the skin surface. Hematoxylin and eosin
and van Gieson stains were used. An immunohistochemical
study was also performed via the use of vascular endothelial growth factor Ab-1 (1:100; Lab Vision, Fremont, CA,
USA), smooth muscle actin Ab-1 (1:200; Lab Vision), and
Vimentin Ab-2 (1:400; Lab Vision). The tissues were evaluated for epithelialization, inflammatory response, neoangiogenesis, and collagen formation. The scoring system we used
to assess the histopathological parameters are shown in
Table 1.
Statistical analysis
Comparison of wound closure between the groups was
performed with the use of the Student’s t-test, and the nonparametric Kruskall-Wallis test was used to compare the
data from the semi-quantitative analysis. For both p 0.05
was set as the level of statistical significance.
Results
Clinical findings
Throughout the entire experiment, all rats in both groups
remained healthy and no complications were noted. Also, all
the wounds healed without signs of infection or purulent
discharge.
The findings of the planimetric evaluation of the total
wound area showed a statistically significant acceleration of
TABLE 2. WOUND AREA (IN CM2)
AS ASSESSED BY PLANIMETRY
Experimental
group
Control
group
Day
Mean
SD
Mean
SD
p Value
5
10
15
20
2.53
0.95
0.15
0.00
0.3211
0.4268
0.1151
0.0000
3.03
185
0.48
0.05
0.2860
0.2008
0.1984
0.0150
0.059
0.017
0.036
0.194
VISIBLE AND INFRARED POLARIZED LIGHT AND WOUND HEALING
263
4.5
3.5
3
Wound area (cm2)
2.5
2
*
1.5
1
0.5
*
0
0
DAY 5
DAY 10
DAY 15
DAY 20
CONTROL
EXPERIMENT
FIG. 1. Wound surface area in the experimental and control groups (* statistically significant difference between the two
groups at days 10 and 15).
wound epithelialization in the experimental group compared to the controls, on days 10 and 15 (p 0.05). For the
rest of the assessment period, although epithelialization was
faster in the experimental group, there were no statistically
significant differences between the two groups. The findings
are listed in detail in Table 2 and the difference between the
two groups is shown in Fig. 1.
Histopathological findings
The findings of the histological evaluation, which included
assessment of the degree of epithelialization, inflammatory
TABLE 3.
Day
5
10
15
20
aStatistically
Epithelialization. The first complete wound epithelialization was noted on day 15 in the experimental group, and all
the wounds were healed by day 20 in this group. In the control group on day 20 the wounds were still not completely
healed. Statistical analysis showed significantly enhanced
epithelialization in the experimental group at 10 and 15 days
post-wound (p 0.05). On days 5 and 20, although the epithelialization scores were higher in the experimental group,
HISTOLOGICAL FINDINGS EXPRESSED
Epithelialization
Experimental group
Control group
Experimental group
Control group
Experimental group
Control group
Experimental group
Control group
response, neoangiogenesis, and collagen formation, are
listed in detail in Table 3 and illustrated in Figs. 2, 3, 4,
and 5.
1.6
1.0
2.8
1.4
4.6
3.4
5.0
4.6
(1–2)
(0–1)
(2–3)a
(1–2)
(4–5)a
(3–4)
(5)
(4–5)
AS
MEAN SCORE (RANGE)
Inflammatory
response
2.2
2.3
1.6
1.8
0.4
0.6
0
0.4
(2–3)
(2–3)
(1–2)
(1–2)
(0–1)
(0–1)
(0)
(0–1)
significant difference between the two groups by Kruskall-Wallis testing.
Neoangiogenesis
4.6
4.2
3.8
3.2
2.4
2.2
1.4
1.2
(4–5)
(3–5)
(2–4)
(2–4)
(2–3)
(2–3)
(1–2)
(1–2)
Collagen formation
1.8
1.4
2.8
2.4
3.6
3.2
3.8
3.6
(1–3)
(1–2)
(2–4)
(2–3)
(3–4)
(3–4)
(3–4)
(3–4)
264
IORDANOU ET AL.
lagen around the margins of the wounds in the controls. On
day 10, the amount of collagen formation by fibroblasts was
increased in both groups. On day 15, a moderate number of
fibroblasts producing abundant bundles of collagen was
found in both groups. Finally, on day 20, more mature fibroblasts creating a denser collagen layer beneath the epithelial layer were seen in the experimental group compared
to the controls. However, no statistically significant differences were found between the two groups at any time point.
Discussion
Wound healing is a complex process, which involves several local and systemic tissue responses regulated by many
different cellular and humoral factors.19–21 It normally proceeds in four overlapping phases: inflammation, granulation
tissue formation, matrix formation, and remodeling.22
It is well known that low-power lasers, but also other light
sources, can promote wound healing, and this is mainly attributable to the biomodulating effects of light energy. With
regard to the mechanisms involved in biophotomodulation,
several models have been suggested by the existing literature, such as stimulation of mitochondria, changes in the cell
FIG. 2. Photomicrographs of skin wounds at day 5 (vascular endothelial growth factor, 40). (A) Control group. Immature endothelial cells can be seen in the newly formed
capillaries (arrow). (B) Experimental group. A denser capillary network with more mature endothelial cells can be seen
(arrows).
the difference was not statistically significant compared to
controls. The histopathologic findings were in accordance
with those of planimetry.
Inflammatory response. Inflammation scores were constantly lower in the experimental group; however, no statistically significantly differences were found at any time point,
compared to those of the control group.
Neoangiogenesis. In the experimental group, a denser,
newly-formed capillary network was noted on days 5 and
10, which became more mature by days 15 and 20, and was
confirmed by the use of vascular endothelial growth factor
stain and smooth muscle actin/Vimentin stain for endothelial cells and pre- and post-capillary vascular smooth muscle cells, respectively. Vascularization scores, although constantly higher in the experimental group compared to the
controls, did not reach a statistically significant level at any
time point.
Collagen formation. On day 5, the presence of several fibroblasts producing collagen bundles was evident in experimental group, but there were few fibroblasts producing col-
FIG. 3. Photomicrographs of skin wounds at day 10 (hematoxylin and eosin, 10). (A) Control group. Slight epithelialization (gray arrow) and dense connective tissue (black
arrow) can be seen. (B) Experimental group. Better epithelialization (gray arrow), and fibroblasts organized parallel to
the surface of the wound (black arrow) can be seen.
VISIBLE AND INFRARED POLARIZED LIGHT AND WOUND HEALING
265
lease of growth factors and cytokines, and enhanced neovascularization and blood flow to the wounded area and the
surrounding skin. Nevertheless, the possible role of polarized light in phototherapy is not fully understood and further experiments are needed.7,8,14,18,24,34,39,40
With regard to the protocol of this study, the effect of visible and infrared light on secondary healing of full-thickness
skin wounds was investigated in a rodent model. The light
used was polarized and of low power, similar to that of the
low-powered laser, in order to prevent thermal damage to
regenerating tissues. But it was polychromatic and incoherent, since it combined visible light at 580–760 nm and infrared light at 760–3400 nm. This range of wavelength
(580–3400 nm) was used in order to: (1) avoid the use of ultraviolet irradiation, which has some dangerous effects; (2)
to benefit from the deeper tissue penetration of the yelloworange-red fraction of visible light (580–760 nm); and (3) to
use only near- to middle-infrared irradiation (760–1500 nm
and 1500–3400 nm, respectively), thus avoiding the harmful
effects of far-infrared irradiation (5600–10,000 nm).39–41
The use of polychromatic light also has certain benefits
over the low-power laser, since it is relatively inexpensive,
easier to use, safer, and able to irradiate a larger treatment
area. In addition, the range of wavelengths used is wider,
and can be easily changed at low cost through the use of filters placed over the light source. In addition to the light
FIG. 4. Photomicrographs of skin wounds at day 15
(smooth muscle actin, 2). (A) Control group. Slight epithelialization (gray arrow) and immature fibroblasts within
abundant collagen bundles (black arrow) can be seen. (B)
Experimental group. A moderate amount of surface epithelialization (gray arrow) and mature fibroblasts (black arrow)
can be seen.
membrane due to light absorption, and the photophysical
action of light on hydrogen bonds. All of these lead to a similar photoresponse, characterized by increased cell energy
and activation of nucleic acid synthesis that are essential for
wound healing, but each of these factors acts on the metabolic cascade at a different cellular level.6,23–28
In vitro and in vivo data demonstrate that phototherapy
acts during all four healing phases, and has positive effects
on each of them. During the inflammatory phase, cellular
modulation regulates the local inflammatory response (proliferation and activation of lymphocytes and monocytes, and
an increase in phagocytotic capacity of neutrophils), and increases the release of growth factors, initiating the proliferative phase. In the second phase, neovascularization is enhanced by photostimulation of endothelial cells. Also,
increased proliferation of fibroblasts and keratinocytes, as
well as collagen synthesis and deposition contribute towards
granulation tissue formation and epithelialization. During
the phase of matrix formation and remodeling, phototherapy induces denser collagen deposits with better bundle orientation and maturation.14,29–38
As for the effects polarized light, the results of several
studies support the fact that it has a beneficial effect on
wound healing. This is primarily attributable to increased re-
FIG. 5. Photomicrographs of skin wounds at day 20 (Vimentin, 20). (A) Control group. A completely epithelialized wound (arrow) is seen here. (B) Experimental group. A
completely epithelialized wound with a layer of mature keratinocytes (arrow) is seen here.
266
source we used in our study, light-emitting diodes represent
another source of polychromatic light, and their use in phototherapy is still in its infancy.44
Our results demonstrate that the visible and infrared light
used in this study enhance wound healing as evidenced by
statistically significantly accelerated epithelialization at days
10 and 15 in the light-treated animals as compared to the
control group. Also, the quality and organization of the epithelium was significantly better for at the same time points,
as evidenced by the histologic findings. One possible explanation for the faster and better epithelialization could be increased keratinocyte proliferation and migration due to a local or systemic effect of photomodulation. As for the other
histological findings, although no statistically significant difference was found between the two groups, the results in the
light-treated group were consistently better than those of the
control animals, and a possible explanation could be limited
light penetration into the tissues.34,45,46
IORDANOU ET AL.
9.
10.
11.
12.
13.
Conclusion
To summarize, the results of the present study support a
positive biomodulatory effect of the visible and infrared light
used (580–3400 nm) on the healing process of full-thickness
skin wounds through faster re-epithelialization. Nevertheless, further studies are warranted to define the optimal
wavelength, dose, and direction of polarized polychromatic
light to optimally improve wound healing, and to elucidate
the mechanisms behind photomodulation, since the improvements seen in some of the histologic parameters we
evaluated were not statistically significant.
14.
15.
16.
17.
Acknowledgment
This study was supported by a grant from the Technological Educational Institute (T.E.I.) of Athens, Greece.
18.
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Address reprint requests to:
Dr. Efstathios G. Lykoudis, M.D.
Department of Plastic Surgery and Burns
Ioannina University School of Medicine
University Campus
45110 Ioannina, Greece
E-mail: [email protected]
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