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Effects of red and near-infrared LED light therapy on full-thickness skin graft
in rats

Article in Lasers in Medical Science · February 2020

DOI: 10.1007/s10103-019-02812-6

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Lasers in Medical Science
https://doi.org/10.1007/s10103-019-02812-6

ORIGINAL ARTICLE

Effects of red and near-infrared LED light therapy on full-thickness

skin graft in rats
Cintia Cristina Santi Martignago 1 & Carla Roberta Tim 2 & Lívia Assis 2,3 & Viviane Ribeiro Da Silva 1 &
Estefany Camila Bonfim Dos Santos 1 & Fabiana Nascimento Vieira 1 & Nivaldo Antonio Parizotto 1,2,4 &
Richard Eloin Liebano 1

Received: 15 January 2019 / Accepted: 21 May 2019

# Springer-Verlag London Ltd., part of Springer Nature 2019

Abstract
The aim of the present study was to evaluate the in vivo response of different wavelengths (red and near-infrared) of light-
emitting diode (LED) on full-thickness skin grafts (FTSG) in rats. Thirty rats were randomly allocated into three experimental
groups: control group (C); red LED treated group (R); and near-infrared LED group (NIR). Skin grafts were irradiated daily for
ten consecutive days, starting immediately after the surgery using a red (630 nm) or near-infrared (850 nm) LED. The results
showed that the red wavelength LED significantly enhanced the skin graft score in relation to the NIR group and increased
transforming growth factor beta (TGF-β) protein expression and density of collagen fibers compared with the other experimental
groups. These results suggest that the red wavelength LED was efficient to improve the dermo-epidermal junction and modulate
the expression proteins related to tissue repair.

Keywords Light-emitting diode (LED) . Light therapy . Photobiomodulation therapy . Full-thickness skin graft (FTSG) .
Rehabilitation . Plastic surgery

Introduction The major complication in tissue transplants, be it a flap or

A full-thickness skin graft (FTSG) is commonly used to cover
open wounds in orthopedic and plastic surgery. It includes the
epidermis, entire dermis, and subcutaneous tissue, and thus
provides robust wound coverage after healing and improved
texture and coloration of the skin [1, 2].
Cintia Cristina Santi Martignago and Carla Roberta Tim contributed
equally to this work.
* Cintia Cristina Santi Martignago
csantimartignago@yahoo.com.br
1
Department of Physiotherapy, Federal University of São Carlos, São
Carlos, SP, Brazil
2
Scientific Institute and Technological Department-University Brazil,
São Paulo-Itaquera, SP, Brazil
3
Federal University of São Paulo, UNIFESP, Santos, SP, Brazil
4
Biotechnology in Regenerative Medicine and Medical Chemistry,
University of Araraquara, Araraquara, SP, Brazil
graft, is necrosis [3]. To secure the survival of the graft, it is
important that the plasmatic imbibition and revascularization
with the recipient bed occurs after the graft transfer [4], and
vascular ingrowth is expected with the progression of the in-
tegration process and establishment of venous and lymphatic
drainage [5, 6]. When these processes do not occur, beyond
tissue necrosis, the contracture of the skin may also occur,
resulting in undesirable (aesthetically unpleasant) clinical con-
ditions that may lead to loss of function.
Treatments for reducing these complications or even pro-
moting full-thickness skin graft survival are based mainly with
the use of vasodilators, anticoagulants, and prostaglandin in-
hibitors [7, 8]. Recently, research involving more innovative
therapeutic approaches have been developed aiming to avoid
and reduce necrosis and enhance the safety and viability of
skin grafts. Among the innumerable interventions, highlighted
is photobiomodulation therapy (PBMT) by laser which has
positive effects on skin metabolism [9, 10].
PBMT consists of the use of non-ionizing light
sources such as lasers and light-emitting diodes (LEDs)
for therapeutic purposes [11]. In this therapeutic modal-
ity, the emitted photons are absorbed by specific

photoreceptors stimulating mitochondrial electron trans-
portation chain initiating a series of cytoplasmic or cell
membrane reactions [12] which lead to changes in mem-
brane potential, O2 consumption, and DNA and RNA
synthesis rates [13]. These molecular alterations acceler-
ate the process of cell differentiation and proliferation
[14], and increase protein synthesis [15], leading to the
main therapeutic effects of PBMT: analgesic, circulatory,
inflammatory processes modulator, and tissue repair [16,
17].
A recently published literature review [18] concluded that
LED light therapy and low-level laser therapy (LLLT) pro-
mote similar biological effects. Advantages of LEDs include
no laser safety considerations, ease of home use, ability to
irradiate a large area of tissue at once, possibility of wearable
devices, and lower cost [19].
In this context, it was hypothesized that the LED light
therapy may optimize the healing process and stimulate the
skin graft survival in rats, constituting a more suitable and
effective treatment to be used. Thus, the aim of the present
study was to evaluate the effectiveness of different LED light
therapy wavelengths using an experimental model of FTSG in
rats. For this purpose, morphological aspects of the grafts and
the expression of wound healing markers were evaluated.
Materials and methods
Experimental groups
Thirty adult male Wistar rats (Rattus norvegicus), weighing ±
245 g, 6 weeks old were used in the present study. Animals
were placed in plastic cages with sawdust bedding, with one
animal per cage, and were allowed to move freely in the cages
with free access to commercial food and water. The room had
a 12-h dark/light cycle and a controlled temperature (24 ±
2 °C). The present study was approved by the Animal Care
Committee guidelines at the Federal University of São Carlos
(protocol 8577280716) and it was conducted according to the
Guiding Principles for the Use of Laboratory Animals.
The experimental animals were randomly distributed into
three groups (n = 10 per group):
& Control group (C): the rats of this group were submit-
ted to skin grafting and to the simulation of the LED
application.
& Red treatment group (R): the rats of this group were sub-
mitted to skin grafting and treated with red LEDs with a
wavelength of 630 nm.
& Near-infrared LED treatment group (NIR): the rats of this
group were submitted to skin grafting and treated with
near-infrared LEDs at 850 nm wavelength.
Lasers Med Sci
Full-thickness skin graft experimental model
For the surgical procedure, the rats received intra-peritoneal
anesthesia with 40 mg/kg ketamine (Dopalen; Vetbrands; São
Paulo; Brazil) and 20 mg/kg xylazine (Anasedan; Vetbrands;
São Paulo; Brazil) and were then placed on a flat surface in a
ventral decubitus position and had their backs tricotomized,
and then the surgical procedure was performed to remove a
skin fragment from the dorsal region, measuring 3 cm wide
and 5 cm [20], with a reference point 0.5 cm below the lower
angle of the scapula, placing the midpoint of the mold on the
dorsal column of the animal. The skin fragment was drawn
from the deep fascia of the muscles using a no.11 scalpel
blade, and including the superficial fascia and skin being
outlined by means of a template trimmed in a standard mea-
surement (3 × 5 cm). The non-cutaneous tissues (fat and mus-
cle) adhered to the dermis were removed by means of a no. 11
scalpel until the fragment acquired a whitish and homoge-
neous appearance. After this procedure, the cutaneous tissue
was replaced in its own bed with 180° rotation. Sutures were
made with separate stitches of 4–0 monofilament nylon,
starting at the vertices and 1 cm apart. After suturing, the graft
was adequately manually compressed to avoid possible air
bubbles under it with gauze and physiological solution. In
order to minimize post-operative discomfort, the animals re-
ceived analgesia (i.m. 0.02 mg/kg buprenorfiine—Temgesic;
Reckitt Benckiser Health Care Ltd. Schering-Plow,
Hoddesdon, UK) directly after the operation and subcutane-
ously for 5 days after surgery.
LED protocol
LED light therapy was performed using a gallium–aluminum
arsenide (GaAlAs) 850-nm diode LED and arsenide gallium–
indium–aluminum–phosphorus (AsGaInAlP) 630-nm diode
LED (Ibramed, Brazil) with the following parameters: contin-
uous emission, 200 mW optical power output, 15 s irradiation
time, 0.5 cm2 spot area, energy density of 6 J/cm2, irradiance
of 0.4 W/cm2, and 3 J total energy per point. Both LEDs
irradiation were applied daily, at 15 points (3 points in the
FTSG surface and 12 surrounding it—Fig. 1), with the punc-
tual contact technique in all experimental groups. In this tech-
nique, the LED probe remains in contact with the animal
throughout the application period, must be maintained at a
90° perpendicular to the flap in the cast spaces of the mold
for 10 sessions with the first application immediately after the
FTSG.
Regardless of the group in which the animal belonged, the
plastic mold was placed on the back with the demarcation
point where the irradiation was carried with the purpose of
standardizing the application site with the probe remaining
in contact with the animal. For the animals of the C group,
the simulation was performed with the equipment device on
Lasers Med Sci
Figure 1 Schematic representation of LED irradiation point
off. The equipment was calibrated by the manufacturer both at
the beginning and at the end of the experiment in order to
obtain high reliability for the effective optical power emission
of the device. Ten days after surgery, all animals were eutha-
nized individually by anesthetic overdose (twofold anesthetic
dose) and the skin of each animal was removed for analysis.
Sample collection and preparation
After the euthanasia of the animals, a skin fragment of the
FTSG (1 cm2) was collected with the assistance of a plastic
mold to standardize the collected site, the site being the upper
border of the FTSG at the right extremity. After the material
was collected, it was fixed in formalin, embedded in paraffin,
and cut with a spinner microtome, Spencer 820 with 0.5-μm
thickness for histological analysis and immunohistochemistry.
Density of collagen fibers
The slides stained with Picrosirus Red (Sigma-Aldrich,
Darmstadt, Germany) were analyzed for the purpose of quan-
tifying collagen types I and III without differentiating them.
The images were captured in a polarized Optical Microscope
(OLYMPUS BX53) with × 400 magnification. These images
were analyzed using the ImageJ software, as proposed by
[21], where the images were subjected to a threshold such that
each nonwhite pixel was turned black and each white pixel
remained white. Next, the number of black pixels in each
image was used to calculate the percentage of the image area.
For each slide, the reading was performed in seven distinct
fields and the quantification mean was used for statistical
analysis.
Qualitative and semiquantitative histological
evaluation
The qualitative and semiquantitative histological analysis of
the FTSG were performed in histological slides stained with
hematoxylin and eosin (HE), (Merck, Darmstadt, Germany),
in an optical microscope (Carl Zeiss, Oberkochen, Germany)
at a magnification of × 400 by two evaluators blinded to the
group. These analyses were performed as proposed by
Basaran et al. [22] with modifications. This analysis consisted
of evaluating 5 items: epidermal integrity, dermo-epidermal
junction, collagen organization, graft adhesion, and inflamma-
tory infiltrate. Each of these items was scored from 0 to 2 (0
when the item had no characteristics, 1 had a significant pres-
ence of the characteristic, and 2 had similar characteristics to
normal); the results presented in each item were added up and
the higher the score, the better the prognosis of the FTSG.
Immunohistochemistry analysis
For the TGF-β (transformer growth factor beta) and bFGF
(basic fibroblastic growth factor) expression analysis, the
paraffin was removed with xylene from serial sections of
5 μm. After this procedure, the sections were rehydrated in
graded ethanol and pretreated in a microwave with 0.01 M
citric acid buffer (pH 6) for 3 cycles of 5 min each at
850 W for antigen retrieval. Subsequently, the material
was pre-incubated with 0.3% hydrogen peroxide in
phosphate-buffered saline (PBS) solution for 5 min to in-
activate the endogenous peroxidase and then blocked with
5% normal goat serum in PBS solution for 10 min. The
specimens were incubated with an anti-TGF-β polyclonal
primary antibody (Santa Cruz Biotechnology, USA) at a
concentration of 1:1000 and anti-bFGF polyclonal primary
antibody at a concentration of 1:500. Incubation was per-
formed overnight at 4 °C in the refrigerator and followed
by two washes in PBS for 10 min. Afterwards, the sections
were incubated with biotin-conjugated secondary antibody
anti-rabbit IgG (Vector Laboratories, Burlingame, CA,
USA) at a concentration of 1:200 in PBS for 1 h. The
sections were washed twice with PBS followed by the ap-
plication of avidin–biotin complex conjugated to peroxi-
dase for 45 min. The visualization of the bound complexes
was performed with the application of a 0.05% solution of
3–3′-diaminobenzidine and counterstained with Harris
Hematoxylin. Finally, for control analyses of the antibod-
ies, the serial sections were treated with rabbit IgG at a
concentration of 1:200 instead of the primary antibody.
Furthermore, internal positive controls were performed
with each staining bath. Digital images at × 200 magnifi-
cation were captured by an optical microscope. The results
were evaluated both qualitatively (presence of the
immunomarkers) and semiquantitatively according to a
previously described scoring scale from 1 to 4 (1 = absent,
2 = weak, 3 = moderate, and 4 = intense) for immunohisto-
chemical analysis. Two experienced observers performed
the scoring in a blinded manner.

Statistical analysis
Data are expressed as the mean ± standard error of the mean
(SEM). Shapiro-Wilk’s and Levene’s tests were applied to
evaluate the normality and homogeneity of the results, respec-
tively. For the variables that exhibited normal distribution,
comparisons between experimental groups were performed
by analysis of variance (one-way ANOVA), and the Tukey’s
post-test used to compare individual groups. For the variables
that exhibited non-normal distribution, the Kruskal-Wallis test
was used. A p value < 0.05 was considered significant. All
analyses were performed using the GraphPad Prism 6.0 pro-
gram (GraphPad Software, San Diego CA, USA).
Results
Histological semiquantitative analysis
The score of histological semiquantitative analysis was signif-
icantly higher in the R LED group (p = 0.0417) compared with
Fig. 2 a Morphometric analysis of graft performance. Values shown are
mean and standard deviation. Control group (C); red LED treated group
(R); near-infrared LED group (NIR). (indicated as *p = 0.0417versus
NIR). b Representative photomicrographs of morphological analysis of
FTSG cross-sections. Epidermal-dermal junction (asterisks); collagen
Lasers Med Sci
the NIR LED group. No other difference was observed
(Fig. 2a).
Histological descriptive analysis
Representative images of the skin graft sections are shown in
Fig. 2b. Histopathological analysis revealed that 10 days post-
surgery, the C and NIR groups demonstrated severe morpho-
logical modifications, characterized by the destruction of epi-
dermis integrity, discontinued epidermal-dermal junction, and
disturbed collagen organization loss. For the R group, mor-
phological modifications were also observed but less intense
compared with the C and NIR groups. Red-treated animals
showed a limited area of destroyed epidermis and epidermal-
dermal junction.
Density of collagen fibers
Figure 3 shows the morphometric evaluation of the density of
collagen fibers. The percentage of collagen fiber was signifi-
cantly higher in the R group compared with the NIR group
organization (arrow); presence of inflammatory process (arrowhead).
Control (C); red LED treated group (R); near-infrared LED group
(NIR). C and NIR groups demonstrated severe morphological modifica-
tions and R group showed less intense morphological modification
(Stain: HE; scale bar 20 μm)
Lasers Med Sci
Fig. 3 Graphic representation of
the morphometric analysis of
density of collagen fibers. Values
shown are mean and standard
deviation. Control group (C); red
LED treated group (R); near-
infrared LED group (NIR). (indi-
cated as *p = 0.0013 versus NIR;
#p = 0.0015 versus C), the per-
centage of collagen fiber was sig-
nificantly higher in the R group
compared with the NIR group
(p = 0.0015) and C group (p = 0.0013). No other difference
was observed.
Immunohistochemistry analysis
bFGF expression
The immunohistochemistry results revealed positive bFGF
staining in the fibroblasts and endothelial cells in all groups
(Fig. 4a). However, the semiquantitative analysis demonstrat-
ed no difference in bFGF immunoexpression among groups
(Fig. 4b).
TGF-β expression
Immunohistochemistry evaluation demonstrated that the
TGF-β expression observed mainly fibroblasts and endothe-
lial cells for all groups (Fig. 5a). Furthermore, the semiquan-
titative analysis exhibited a higher TGF-β expression in the R
group compared with the C group (p = 0.0121) and NIR (p =
0.0017; Fig. 5b).
Discussion
The present study aimed to evaluate the in vivo response of
two different wavelengths (red and near-infrared) of a LED
light therapy experimental model of FTSG in morphological
aspects and expression of wound healing markers. The histo-
logical findings demonstrated that red LED light therapy pro-
duced a significant increase of the semiquantitative histologi-
cal evaluation, higher TGF-β expression, and density of col-
lagen fibers compared with the other experimental groups.
It is of common knowledge that a FTSG leads to alterations
in the integumentary system, such as reduced blood flow,
ischemia conditions and impaired skin growth, skin contrac-
tures, and formation of partial or complete necrosis after an
injury [23].
In this context, the LED has been considered a promising
alternative to treat many skin diseases due to its repair process
positive effects [24]. The morphometric evaluation demon-
strated that the red LED irradiated animals presented a higher
semiquantitative histological evaluation when compared with
the near-infrared wavelength. Nishioka et al. [24], in a skin
flap model, observed that the animal irradiated with red LED
with the same wavelength utilized in our study obtained better
histological results than the control group, suggesting that the
energy offered to the cutaneous tissue was capable of improv-
ing the tissues’ quality in both researches.
Additionally, studies have shown that the impaired wound
healing in ischemic tissues such as a skin graft is associated
with reduced levels of fibroblast factors, including bFGF ex-
pression [25, 26]. It is known that bFGF induces fibroblast
proliferation and differentiation as well as improving survival
of the skin graft/flap via blood vessels formation [27].
Previously, Yu et al. [28] demonstrated that 660-nm wave-
length stimulated the production of bFGF from fibrolast cells
in cell culture. Interestingly, in the present study, no effect on
the expression of this immunomarker was found with the LED
light therapy. Possibly, the LED parameters used in the present
study were not able to offer sufficient energy to modulate
bFGF expression in the experimental period studied.
Furthermore, in the present study, the red LED presented an
increased TGF-β expression. TGF has been widely reported
as an important regulatory cytokine that is involved in large
number of cellular actives in wound healing [29]. In particular,

Fig. 4 Results of the qualitative and semiquantitative analysis of skin
tissue immunohistochemistry for bFGF. a Representative
photomicrograph of the qualitative results in the different groups
evaluated, Scale bar: 20 μm Immunolabeled fibroblastic cells (arrow). b
there is evidence indicating that TGF-β mediates fibroblasts
migration and proliferation, collagen production, and extracel-
lular matrix (ECM) deposition in the wound healing process
[30]. However, previous findings showed that several oxida-
tive exposures, as well as skin grafts, are responsible for a
downregulation of this growth factor, resulting in a reduced
regenerative capacity [31]. In the current study, the red LED
was able to increase the expression of these cytokines com-
pared with the other experimental groups. Recently, LED light
therapy has shown promise in modulating the growth factors
and cytokines expression related to skin repair in several treat-
ment parameters of LED [32]. Safavi et al. [33] demonstrated
that the red LLLT was able to accelerate the wound healing
process, with the up-immunoexpression of TGF-β and other
gene expressions in injured gingiva tissue of rats.
Using a skin excisional wound model, de Souza et al. [34],
found a downregulation of TGF-β immunoexpression after
LED irradiation (700 nm) at an early stage of the healing
process (2 days after injury); however, no differences were
observed at later times (4 and 6 days after injury). The increase
of the TGF-β expression 10 days after the FTSG evidenced in
the present study may suggest a tissue wavelength response
Lasers Med Sci
Results of semiquantitative analysis of the bFGF expression. Values
shown are mean and standard deviation. Control Group (C); red LED
treated group (R); near-infrared LED group (NIR). No difference in
bFGF immunoexpression among groups
dependency, with the best result being observed after red LED
irradiation. Thus, the modulated expression of TFG-β after
red LED irradiation may stimulate the formation of new der-
mal fibroblast and production of ECM proteins contributing to
the acceleration of skin graft healing.
Also, the increased collagen production in red LED
showed the healing effect of the electromagnetic treatment.
Collagen (mainly type I collagen) is the most abundant struc-
tural protein in connective skin tissue [30]. Collagen fibrils
depositions are responsible for the mechanical strength and
resiliency of skin graft. Possibly, the modulation of growth
factors produced by the red LED determined a stimulatory
effect on the dermal fibroblast cell metabolism, culminating
in a higher deposition of collagen, increasing the skin graft
integration. Therefore, the upregulation of TGF-β signaling
evidenced in the present study may increase collagen produc-
tion and, consequently, stimulate earlier wound healing in the
formation of the new epidermis after a FTSG.
One crucial point that needs to be determined in the field of
LED photobiomodulation is the importance of studies explor-
ing the effects of different wavelengths, in order to try to
establish the effective and safe treatment parameters of
Lasers Med Sci
Fig. 5 Results of the qualitative and semiquantitative analysis of skin
tissue immunohistochemistry for TGF-β. a Representative
photomicrograph of the qualitative results in the different groups
evaluated. Scale bar 20 μm. Immunolabeled fibroblastic cells (arrow). b
Results of semiquantitative analysis of the TGF-β expression. Values
LLLT for an optimal stimulation in tissue repair strategies
within the clinical setting. Since the present study was limited
to a relatively short-term evaluation of the effects of only one
set of LED doses, information on the long-term performance
of this therapy and higher amounts of LED energy remains to
be provided.
It is believed that a limitation in our research was the lack of
macroscopic evaluations that evaluated the FTSG survival, so
that it was not possible to correlate the microscopic findings
with the macroscopic findings.
Conclusion
In conclusion, the findings of the present study suggest that
the red LED was efficient in the deposition of collagen in the
cutaneous dermis for increasing expression of TGF-β growth
factors.
Funding information This study was financially supported by the
Brazilian funding agency FAPESP project # 2015/13501-3.
shown are mean and standard deviation. Control Group (C); red LED
treated group (R); near-infrared LED group (NIR). (indicated as *p =
0.0017 versus NIR; #p = 0.0121 versus C). Scale bar 20 μm. TGF-β
was higher in R group compared with the C group
Compliance with ethical standards The present study was
approved by the Animal Care Committee guidelines at the Federal
University of São Carlos (protocol 8577280716) and it was conducted
according to the Guiding Principles for the Use of Laboratory Animals.
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