Photobiomodulation, Photomedicine, and Laser Surgery

Volume XX, Number XX, 2020 Original Research

ª Mary Ann Liebert, Inc.
Pp. 1–6
DOI: 10.1089/photob.2019.4754

The Effect of Photobiomodulation Therapy (660 nm)

on Wound Healing of Rat Skin Infected by Staphylococcus

Zi-Xuan Wang, PhD, MD,1,2 and Seong-Hwan Kim, PhD, MD2

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Abstract

Objective: To assess the impact of photobiomodulation (PBM) therapy on healing of infected wounds and
document the microscopic findings throughout the recovery process.
Background: Previous studies have suggested that PBM accelerates wound healing and reduces inflammation
and pain. However, the ideal protocol and ultimate value of PBM treatment for infected wounds are contro-
versial.
Materials and methods: Eight-month-old male rats were randomly divided into the control group, the non-
irradiation group, or the irradiation group. A 1 cm diameter skin excision was made. The wounds of the nonir-
radiated and irradiated rats were inoculated with a suspension of Staphylococcus aureus. We then performed 7
days of PBM therapy at a wavelength of 660 nm for 35 min/day. On day 8, the rats were sacrificed for histological
assessment. Sections were stained with hematoxylin and eosin, Masson trichrome, and a proliferating cell nuclear
antigen (PCNA) kit. Defect diameter was calculated using the Visus Image Analysis System.
Results: The irradiated group had more epithelial cells and richer granulation tissue compared to those in the
other groups. The irradiation group had a significantly smaller defect size than the nonirradiation group
( p < 0.01) and the control group ( p < 0.05). The amount of collagen was highest in the irradiation group and was
graded as 3, 2, and 3+ in the control, nonirradiation, and irradiation groups, respectively. The percentage of
PCNA in the control group was significantly lower than that in the other two groups ( p < 0.05).
Conclusions: PBM therapy (660 nm) promoted cell proliferation and collagen synthesis, thereby improving the
wound healing response to an S. aureus infection.

Keywords: 660 nm, photobiomodulation therapy, wound healing, infection, Staphylococcus

Introduction surgery use.5 A number of studies have suggested that spe-

W ound healing is a complex cellular and molecular
process involving blood clotting, inflammation, cel-
lular proliferation, and extracellular matrix remodeling.1–3
Infections may prolong the wound healing phase or impede
normal wound healing.4 Administration of antibiotics is im-
portant for managing wound infections, but there are several
limitations to their full efficacious use, such as difficulty
achieving adequate wound penetration and the development
of antibiotic resistance.4 Therefore, promoting wound repair
and the regeneration process using another method could
potentially benefit burn patients and other cases of tissue loss
and scarring.
Lasers have been used widely in the medical field, with
applications ranging from dermatologic, aesthetic, and plastic
cific wavelengths of light accelerate the healing of wounds
and reduce inflammation and pain.6–15 Gonçalves et al.8
compared the effects of a gallium-aluminum-arsenide diode
laser and healing oil, and found that photobiomodulation
(PBM) was more effective for stimulating angiogenesis and
maturing scar tissue. Silva et al.11 performed an experi-
mental study using male Wistar rats and demonstrated that
a red diode laser (wavelength 658 nm) reduces bacterial
proliferation.
However, Nussbaum et al.12 inoculated rats with Pseu-
domonas aeruginosa and found that, while a single treatment
with 808 nm light improved wound healing, repeated
treatments with the 808 nm light might actually increase
growth of Staphylococcus aureus. Therefore, the value of
PBM on infected wound healing remains controversial. In this

1
Department of Interventional Radiology, Qingdao Municipal Hospital, Qingdao, Shandong, P.R. China.
2
Department of Surgery, School of Medicine, Chosun University, Gwangju, Korea.

1
 2 WANG AND KIM
study, we assessed the role of PBM on infected wound healing
and evaluated the microscopic findings during the healing
process.
Materials and Methods
Protocol
This protocol was approved by the Clinical Trial Ethics
Committee of Qingdao Municipal Hospital, and experiments
were performed in accordance with all applicable interna-
tional, national, and institutional guidelines for the care and
use of laboratory animals.
Subjects and randomization
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Eighteen 8-month-old male rats (NTacSam: SD; Samtako
Bio, Seoul, Korea) weighing 250–300 g were randomly di-
vided into three groups of six rats each using a random
numbers table. The three experimental groups were desig-
nated as the control, nonirradiation, and irradiation groups.
All rats were caged separately in a room held on a day/night
light cycle and a temperature of 21–23C. The animals were
fed a standard laboratory diet and water was provided ad
libitum. Blood samples were taken from the tail vein and a
complete blood count was performed just before injury and
after injury on days 1, 4, and 7. Rats with an abnormal white
blood cell count at baseline were excluded.
Wound inducement and inoculation
All rats were anesthetized with sevoflurane (2.2%), and
the dorsal region was shaved. A 1 cm diameter skin excision
was created using a round blade to expose the muscle fascia
just above the thoracic spine. A sterile swab was used to
inoculate the wounds of the nonirradiation and irradiation
groups with a suspension of S. aureus at a concentration of
2 · 106 colony-forming U/mL.
Photobiomodulation
Light intensity was calibrated and matched using an op-
tical power meter (TQ8210; Advantest Co., Tokyo, Japan)
before the experiment. The PBM therapy (Bioarteco;
Chosun University, Gwangju, Korea) irradiation was initi-
ated 24 h after injury and was performed 35 min/day for 7
days a week using a laser diode matrix and a 660 nm wave-
length (Table 1). The irradiation was applied in a continuous
wave at a radiant exposure of 15 J/cm2.
Table 1. Laser Parameters for the Treatments
Characteristics Parameters
Wavelength 660 nm
Wave type Continuous wave
Irradiance density 0.001 – 10% W/cm2
Irradiation time 2100 sec/day for 7 days
Energy density 2.1 – 10% J/cm2
Cumulative dose density 15 J/cm2
Gross wound observations
The wound was observed by the naked eye and docu-
mented with a digital camera every 24 h for 8 consecutive
days, beginning immediately on the day of wounding. The
digital camera was fixed on a special support above the an-
imal experiment table to ensure a consistent shooting angle
and distance.
Histological assessment
On day 8, that is, 24 h after the last irradiation, the ani-
mals were sacrificed and wound tissue was collected for a
histological assessment. Specimens, including the original
wound plus the surrounding normal tissue, were excised,
fixed in 10% neutral buffered formalin solution, and em-
bedded in paraffin blocks. The embedded tissue sample was
sliced at a thickness of 4 lm and mounted on X-tra slides
(Surgipath, Richmond, CA). The sections were stained and
examined using optical microscopy (Olympus Optical Co.,
Tokyo, Japan). The tissue slides were stained with hema-
toxylin and eosin (Merck, Darmstadt, Germany) to measure
defect size, Masson’s trichrome for the collagen analysis,
and immunohistochemistry was used for the proliferating
cell nuclear antigen (PCNA) analysis.
Defect size measurements
The tissue slides were stained and photographed using a
Magna Fire digital camera system (Optronics, Goleta, CA).
The diameter of the wound was calculated using the Visus
Image Analysis System (Image & Microscope Technology,
Daejon, Korea).
Collagen analysis by Masson’s trichrome staining
The paraffin-embedded sections were stained and exam-
ined microscopically to evaluate the regenerative tissue in
the wound, with special attention to the morphological
features of collagen. The amount of collagen was evaluated
and rated on a scale from 0 to 4 as follows6: ‘‘0’’ no col-
lagen observed; ‘‘1’’ several dim collagen fibers; ‘‘2’’ small
collagen fiber-forming bundles; ‘‘3’’ large collagen fiber-
forming bundles; and ‘‘4’’ collagen fibers forming a dense
matrix.
PCNA analysis by immunohistochemical staining6
PCNA expression (antibody diluted 1:400) was evaluated
using immunohistochemical staining of paraffin-embedded
tissue sections. We also evaluated pan-cytokeratin and actin
by immunohistochemical staining. A stained nucleus was
defined as positive, and a brown appearance of pan-
cytokeratin and actin was defined as positive. The percent-
age of PCNA and the percentage of positive cells among all
cells were calculated in four to five of the strongest stained
areas per slide.
Statistical analysis
Data are expressed as mean – standard deviation and the
enumerated data are presented as proportions. Differences
between means of continuous variables were tested using
one-way analysis of variance with the Bonferroni post-hoc
test. A p value £0.05 was considered significant for all
 PBM THERAPY ON INFECTED WOUND 3

FIG. 1. Typical gross

changes in wound repair ac-
cording to the time variation.
(A) Induced wound; (B) first
post-operative day; (C) sec-
ond post-operative day;
(D) third post-operative day;
(E) fourth post-operative
day; (F) fifth post-operative
day; (G) sixth post-operative
day; and (H) seventh post-
operative day.

analyses. The Statistical Package for the Social Sciences process, such as pavementing by epithelial cells, newly
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(SPSS version 15.0 for Windows; SPSS, Inc., Chicago, IL)
was used to perform all statistical analyses.
Results
Gross observations and evaluation
No rat was excluded from the study, and all rats survived
until they were killed on day 8. There were obvious indi-
vidual features in the formation, development, and elimi-
nation of scabs and edema during wound healing. Therefore,
no specific trend was apparent from the gross observations,
except gradual formation of new tissue in the vicinity of the
wound and contraction of the wound area. In addition, no
significant difference was observed in the gross observations
of the wounds across each group. The typical gross changes
in wound repair according to time are shown in Fig. 1.
Histological findings and defect size
on microphotographs
Histological specimens from post-operative day 8
showed the crust and cell components of the wound repair
formed granulation tissue, and blood vessels. The repair
of epithelial tissue in the irradiation group was better than
that in the other two groups. Granulation tissue was richer
in the irradiation group than in the nonirradiation group
(Fig. 2). A difference in average wound size was observed
in each group (F = 8.461, p = 0.004 < 0.01). The irradiation
group had significantly smaller defects (2439 – 813 lm)
relative to the nonirradiation group (4360 – 713 lm,
p = 0.003 < 0.01) and the control group (3700 – 926 lm,
p = 0.018 < 0.05). Although defect size in the control
group was smaller than that in the nonirradiation group,
this difference was not significant ( p = 0.185 > 0.05)
(Fig. 3).
Collagen formation evaluated by Masson’s
trichrome staining
Masson’s trichrome staining revealed that the formation
of collagen was significantly different in the nonirradiation
(grade 2), control (grade 3), and irradiation groups (grade
3+) (Fig. 4).

FIG. 2. Histological findings under op-

tical microscopy (hematoxylin and eosin
stain, 12.5 · ). Photomicrograph showed
crust (black triangle), granulation tissue
(white triangle), and wound defect margin
(black arrow). (A) Case of control group
(defect size 3782 lm); (B) case of non-
irradiation group (defect size 4095 lm);
and (C) case of irradiation group (defect
size 2697 lm).
 4 WANG AND KIM
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PCNA expression
The quantitative analysis of PCNA expression showed
that the percentage of PCNA in the control group was the
lowest, with an average of 88%. PCNA expression was
significantly lower than that in the other two groups
( p < 0.05). The percentage of PCNA in the nonirradiation
group was the same as that in the irradiation group, with an
average of 91% (Fig. 5, Table 2).
Discussion
Since its original development by Ohshiro and Calder-
head16 in 1988, PBM has seen significant clinical applica-
tions among many medical specialties, particularly in
dermatology. Skin wounds are a common type of injury.
Several therapeutic approaches are efficacious for improv-
ing skin wound healing, including PBM. During the irradi-
FIG. 3. Diameter of wound defect.
ation procedure, energy is transferred from the absorbed
photons directly to cells, causing photoactivation and
changes in cellular activity,5 but not causing a significant
increase in tissue temperature.17 By this mechanism, PBM
therapy induces analgesia, and improves cell metabolism,
the inflammatory response, and wound healing.18,19
Infection is one of the most significant complications in
unhealed wounds, often causing delayed wound healing.20
Impaired wound repair is usually reflected at the cellular
level, including vascular and cellular responses in the
wound microenvironment. S. aureus is the most common
etiology for wound infection.21,22 Therefore, we used a
wound model of rats locally inoculated with S. aureus to
evaluate the efficacy of PBM at the tissue and cytological
levels.
Previous studies have indicated that different laser
wavelengths are suitable for specific tissues or diseases.
FIG. 4. Collagen formation in each
group (Masson’s trichrome stain, 12.5 · ).
(A) Control group was graded as 3; (B)
nonirradiation group was graded as 2; and
(C) irradiation group was graded as 3+.
 PBM THERAPY ON INFECTED WOUND 5

lengths of 630–680 nm should be used to heal wounds.24

Therefore, we investigated the effect of a 660 nm wave-
length in our study.
Previous studies have proposed that PBM positively en-
hances wound healing by increasing proliferation and me-
tabolism of cells.19 However, the presence of infection
prolongs the healing process due to impaired wound repair.
The infectious process and removal of necrotic tissue are
typical precursors to tissue repair. Therefore, what is the
efficacy of PBM on healing during the complex infectious
wound process? We found no difference among the control,
nonirradiation, and irradiation groups according to the gross
observations. However, the microscopic findings indicated
significant effects of PBM at the histological and cellular
levels, specifically with findings of more epithelial cells,
richer granulation tissue, and smaller defect diameters in the
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irradiation group. These differences indicated more ad-

vanced wound repair in the irradiation group and the posi-
tive effect of PBM on wound healing, which was in good
agreement with previous reports.
Since the initial research by Saperia et al., many studies
have demonstrated that collagen synthesis and fibroblast
proliferation are promoted by PBM.6,20 However, fibroblasts
are inhibited in infected wounds due to competition with
other cells and microorganisms for nutrients and oxygen in
the tissue microenvironment. In addition, infection increases
FIG. 5. Expression of PCNA in each group (immunohis- collagen lysis due to increased release of lysosomal en-
tochemical stain, 12.5 · and 100 · ). (A, B) Case of control zymes from leukocytes. Therefore, wound infection reduces
group; (C, D) case of nonirradiation group; and (E, F) case synthesis and metabolism, but increases lysis of collagen in
of irradiation group. infected wounded tissue.20 In this study, collagen formation
in the wounds of the nonirradiated rats was less compared
with the control rats. This finding indicates that infection
negatively affected collagen synthesis. However, the amount
Yousef et al.23 evaluated the efficacy of a 660 nm PBM for of collagen in the PBM-treated group was the largest, sug-
healing pemphigus lesions and revealed that PBM resulted gesting that both the amount and quality of collagen might be
in impressive healing of ulcers. Mun et al.6 performed ani- increased by PBM therapy.
mal experiments to assess the effect of 655, 785, and 850 nm PCNA, as a protein influencing DNA polymerase delta,
wavelengths of PBM, and found that PBM has beneficial plays an important role in the wound healing process. DNA
effects on collagen that resulted in better wound healing. polymerase delta regulates the synthesis, repair, and re-
Nussbaum et al.12 and Silva et al.11 conducted further generation of DNA, and PCNA is considered to be an in-
studies in animals to explore the effect of PBM on bacterial dicator of dividing nuclei. In this study, the amount of
infection and growth. They found that 808 nm PBM may PCNA expressed was quantitatively analyzed and used to
have stimulatory effects on S. aureus growth,12 and that the evaluate the rate of tissue regeneration. The tissues of an
658 nm wavelength PBM reduces bacterial proliferation.11 infected wound exhibited more active regeneration than
Most other studies have suggested that therapeutic wave- the control group according to the percentage of PCNA.
However, there was no difference in the percentage of
PCNA between the two infected wound groups (i.e., the
nonirradiation group and the irradiation group). This un-
Table 2. The Percentage of PCNA in Each Group expected result might be due to well-developed cell pro-
(Mean – Standard Deviation) liferation and collagen in the healing area. Therefore, it
might be more suitable to measure PCNA expression be-
p fore collagen synthesis.
vs. control vs. non-
Groups n % group irradiation group
Conclusions
Control group 6 88 – 2
Nonirradiation 6 91 – 2 0.020 PBM therapy (660 nm) promoted cell proliferation and
group collagen synthesis, thereby improving the healing response
Irradiation 6 91 – 2 0.020 1.000 in wounds infected by S. aureus. We anticipate that PBM
group could have similar positive effects in wounds infected by
Analysis of variance (ANOVA) of three groups: F = 4.500, other pathogens. We expect that future studies to refine the
p = 0.030 < 0.05. specific PBM regimen (dose, frequency, and wavelength)
 6 WANG AND KIM

could lead to additional improvements in the beneficial ef-
fects of PBM therapy.
Author Disclosure Statement
No competing financial interests exist.
Funding Information
This study was supported, in part, by research funds from
Chosun University (Kwangju, Korea).
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