Indian Journal of Experimental Biology

Vol. 49, May 2011, pp. 357-361

Effect of low-level laser therapy on experimental wounds of hard palate

mucosa in mice
Farahnaz Fahimipour
Shahid Beheshti University MC, Tehran, Iran

Mohsen Nouruzian
Department of Anatomy and Biology, Shahid Beheshti University MC, Tehran, Iran

Morteza Anvari.1, Mahmood Akhavan Tafti.2 & Mehdimalek Yazdi.3

Anatomy1, Pathology2 & Medical Engineering3 Department
Shahid Sadoghi University of Medical Science, Yazd, Iran

Mahsa Khosravi & Zahra Dehghannayeri

Medical Faculty, Shahid Sadoghi University of Medical Faculty, Yazd, Iran

Shabnam S Sabounchi
Dental Faculty, Shahid Beheshti University MC, Tehran, Iran

and
Mohammad Bayat *
Department of Anatomy and Biology, Medical Faculty, Shahid Beheshti University MC, Tehran 1985717443, Iran
Received 8 November 2010; revised 9 February 2011
Under general anesthesia and sterile conditions, incision wound was induced in the hard palate mucosa of adult male
mice. The wounds of groups 1 and 2 were irradiated daily with He-Ne laser at 3 and 7.5 J/cm2 for 120 and 300 s,
respectively, while the incision wound of group 3 not exposed served as controls. On day 3 of injury, the laser-treated
wounds contained significantly lower neutrophils than the wounds in the control group. By day 7 after injury, the lasertreated wounds contained significantly more fibroblasts and at the same time contained significantly fewer macrophages. In
conclusion, an acceleration of the wound healing process of experimental wounds in the hard palate mucosa of mice at lowlevel laser therapy with a He-Ne laser at energy densities of 3 and 7.5 J/cm2 was observed.
Keywords: Hard palate mucosa, Low-level laser therapy, Wound healing

Craniomaxillofacial injuries occur in a significant

proportion of trauma patients, either in isolation or in
combination with other serious injuries1. Transverse
maxillary deficiencies are relatively common
dentofacial deformity in combination with other
maxillary and/or mandibular deformities2. Orofacial
clefts are defined as congenital defects in which the
fusion between two or more of the following
structures have failed: the palatal shelves, the
maxillary prominences, and the medial nasal
prominences. The clefts are surgically closed to
restore the integrity of the oral and nasal cavity and to
allow for normal feeding and speech development3.

*Correspondent author
Telephone/Fax: 98 21 22439949.
E-mail: bayatenator@gmail.com

Healing of these wounds is associated with

the disadvantageous effects in maxillary growth
and dento-alveolar development often seen in cleft
palate patient3-5.
Low-level laser therapy (LLLT) has been used for
more than 25 years in clinical practice and is known
to modulate various biological processes6-9. LLLT is a
non-thermal modality; usually the temperature
changes associated with treatment are negligible10. A
number of different laser light sources, including
helium-neon, ruby, and gallium arsenide have been
used to deliver LLLT in different treatments and
schedules.
In patients, biostimulation with low-level laser
radiation has accelerated the healing of gingival
incisions and gingivectomy11-13, reduced the severity

358

INDIAN J EXP BIOL, MAY 2011

of conditioning-induced oral mucositis in bone

marrow transplantation patients14. Low-level laser
radiation induced formation of new periodontal
ligament, cementum, and bone in dogs15, and also
improved macromolecular clearance via the lymph
flow in the hamster gingiva16. At the cellular level,
studies on cultured human gingival fibroblasts and
human oral mucosal fibroblasts have suggested a
number of LLLT effects: increased DNA synthesis17,
induced rapid generation of myofibroblasts from
fibroblasts7, enhanced proliferation18,19 and attachment
of human gingival fibroblasts20.
The effects of LLLT on the gingiva have been
studied quite extensively in vivo and in vitro; however
there is a dearth of data on the effect of LLLT on the
mucosa of soft and hard palates14. Barasch et al.14
irradiated He-Ne laser to patients' oral mucositis,
including the mucosa of the soft palate. Oral
mucositis and pain scores were significantly lower for
the treated versus the untreated side. They concluded
that He-Ne laser treatment was well tolerated and
reduced the severity of oral mucositis in patients14. Be
that as it may, clinical trial studies have failed to show
beneficial effects from LLLT on wound healing after
gingivoplasty in humans21 and also have failed to
show beneficial effects from LLLT on the protein
synthesis of human gingival fibroblasts in vitro22.
The use of LLLT has still not been widely accepted
by physicians and a number of controlled studies13,
and the FDA withdrew its approval for its therapeutic
use in the USA in 198323.
The present study sought to address the problem by
evaluating whether He-Ne laser radiation at energy
densities of 3 and 7.5 J/cm2 accelerated the healing of
the experimental wounds of the hard palate mucosa in
mice.
Materials and Methods
Ninety adult male Albino N mari mice (3 months
old, weighing 6010g) were used. The study protocol
was approved by the Medical Ethics Committees of
Shahid Sadoghi Medical University,Yazd, Iran. The
mice were provided with a standard animal food and
water ad libitum. On day zero, all the mice were
anesthetized with 50 mg/kg ketamine hydrochloride
injected intramuscularly along with 5 mg/kg
diazepam. In the hard palate mucosa, 2 mm posterior
to the incisive tooth of the upper jaw a 4-mm long and
2-mm deep incision wound was made under sterile
conditions. The animals were randomly divided into

three groups, of 30 each. The incisions in groups 1

and 2 were treated daily with a He-Ne laser tube with
3 and 7.5J/cm2 energy densities, respectively. LLLT
was commenced 24 h after surgery under general
anesthesia. The laser used was a He-Ne laser
(Meredith Co, Turkey) rated at 25 mW of power.
Groups 1 and 2 were exposed to 120 and 300s of
laser light, respectively. LLLT was applied to one
point, covering the whole length of the incision in
each session (day). The laser was held 1 cm away
from the surface of the target tissue.
The incision wounds of group 3 were treated with
the LLLT probe without the laser being switched on,
and the group was thus considered the control group.
Thereafter, 10 mice of each group were sacrificed 3
days after surgery, 10 mice of each group were
sacrificed 7 days after surgery, and the remaining 10
mice were sacrificed on the 15 th postoperative day.
The mice were sacrificed via the inhalation of
chloroform in a closed space.
For histological examinations, a sample was
excised from the wound bed and the normal adjacent
mucosa of each mouse and was fixed in formaldehyde
saline before it was embedded in paraffin blocks.
Sagittal sections (5 m thickness) were cut and stained
with the hematoxylin and eosin method. Ten zones
from each sample were examined morphometrically
using a calibrated ocular on a Nikon light microscope
at a magnification of 400X for counting the fibroblasts,
macrophages, neutrophils, and endotheliums of the
blood vessels. The histological examinations were
performed in a blind fashion.
The data were subjected to the one way analysis of
variance (ANOVA) and were expressed as mean
SE. Multiple comparisons were performed by the
least significant difference (LSD) test. P values <0.05
were considered statistically significant.
Results
None of the mice showed any sign of swelling or
exudation at the surgical site.
Day 3 after surgeryThe mean number of
fibroblasts in group 1 was higher than those of the
other groups, the mean number of blood vessel
endotheliums in group 3 was higher than those of the
other groups, and the mean number of macrophages in
group 2 was higher than those of the other groups.
Nevertheless, there were no significant differences in
the above-mentioned histological parameters between
the studied groups. The ANOVA test showed that the

FAHIMIPOUR et al.: LASER THERAPY OF WOUNDS OF HARD PALATE MUCOSA

mean number of neutrophils in group 3 was

significantly higher than those of the other groups
(P<0.05). The LSD test revealed that there were
significant differences between group 3 and groups 1
and 2 (both P<0.05) (Table 1).
Day 7 after surgeryThe mean numbers of
neutrophils and blood vessel endotheliums were
higher in group 3 than those of the other groups.
However, there were no significant differences in the
above histological parameters between the studied
groups. The ANOVA test demonstrated that the mean
number of fibroblasts in group 3 was significantly
lower than those of the other groups (P<0.05). The
LSD test revealed significant differences between
group 3 and groups 1 and 2 (both P<0.05). The
ANOVA test also showed that the mean number of
macrophages in group 3 was significantly higher than
that of the other groups (P<0.05). The LSD test
revealed significant differences between group 3 and
groups 1 and 2 (both P<0.05) (Table 2 ).
Day 15 after surgeryThe mean numbers of
macrophages, neutrophils, and blood vessel
endotheliums of group 3 were higher than those of the
other groups. The mean number of fibroblasts in
group 1 was higher than those of the other groups.
Nonetheless, the ANOVA test showed no significant
differences in the aforementioned histological
parameters between the studied groups (Table 3).
Discussion
The mean number of the neutrophils of the wound
in the mice treated with a He-Ne laser was
significantly lower than that of the control group. A
significant increase in the fibroblasts of the lasertreated groups compared to the control group and also
a significant decrease in the number of the
macrophages of the laser-treated groups compared to
the control group on day 7 after surgery were noted.
Table 1Fibroblasts, macrophages, neutrophils and blood
vessel endotheliums in ten zones of wound of studied group
at three days after surgery
[Values are meanSE from 10 animals in each group]
Group

Fibroblasts Macrophages Neutrophils Blood vessel

endothelium

Gr.1(First
749.530.7
laser group)
Gr. 2(Second 72818.8
laser group)
Gr.3(Control 707.724.2
group)
*

P < 0.05

81.45.4

827.1

117.16.1

97.18.5

83.96.1

114.1 5.2

94.37

105.65.7* 123.7 5.3

359

Beukelman et al.24 suggested that reducing

components of inflammation may positively affect the
wound healing process. Young and Dyson25 studied
the effect of therapeutic ultrasound on the healing of
the dermis of full-thickness excised lesions made in
the skin of rats. They quantitatively assessed the
healing by means of differential cell counts made in
sections of the wound bed 5 and 7 days after injury.
The wounds were either control group or exposed to
pulsed ultrasound. By day 5 after injury, the
ultrasound treated wounds contained more extensive
granulation
tissue,
fewer
polymorphonuclear
leucocytes and macrophages, and more fibroblasts
than the control wounds. Young and Dyson25
suggested that ultrasound therapy could be useful in
accelerating the inflammatory and early proliferative
stages of repair.
The decrease in the number of inflammatory cells
and the increase in the number of fibroblasts in
the lasertreated wounds in the present study on
days 3 and 7 after injury chime in with the Beukelman
et al.24 and Young and Dyson25 studies. Bisht et al.6
studied the biostimulatory effect of He-Ne laser
irradiation on various parameters of the healing
process in open skin wounds of rats. They produced
two full thickness skin wounds on either side of the
Table 2Fibroblasts, macrophages, neutrophils, and blood
vessel endotheliums of the wounds of the studied groups on the
7th postoperative day.
[Values are meanSE from 10 animals in each group]
Group

Fibroblasts Macrophages Neutrophils Blood vessel

endothelium

Gr.1(First
834.830.1 31.53.9
laser group)
Gr. 2(Second 827.625.1 73.84.4
laser group)
Gr.3(Control 739.729.1* 88.15.9*
group)

77.46.2

102.45.6

79.06.5

101.15.5

96.46.6

112.25.3

*P < 0.05
Table 3Fibroblasts, macrophages, neutrophils, and blood
vessel endotheliums of the wounds of the studied groups on
the 15th postoperative day.
[Values are meanSE from 10 animals in each group]
Group

Fibroblasts Macrophages Neutrophils Blood vessel

endothelium

Gr.1(First
897.518.6
laser group)
Gr. 2(Second 852.343.1
laser group)
Gr.3(Control 822.127
group)

55.93.9

47.79.2

91.45.8

57.64.5

79.23.2

96.119.4

65.55.4

52.05.1

98.24.7

INDIAN J EXP BIOL, MAY 2011

360

midline. The wounds on the left side were irradiated

daily with He-Ne laser at 4J/cm2, whereas the wounds
on the right side were not exposed and thus served as
controls. Early epithelialisation with increased
fibroblastic reaction and neovascularization was seen
in the lasertreated wounds. Wound histology also
showed a significant increase in leukocyte infiltration
until day 9. Bisht et al. 6 reported that their results had
established the biostimulatory effects of low-intensity
laser radiation on the healing of the skin wound.
Increased leukocytic reaction, reported by Bisht et al.,
is in disagreement with decreased neutrophil and
macrophage counts, observed in the present study.
The
literature,
however,
shows
that
photobioactivation accelerates inflammation and
promotes fibroblast proliferation, and even accelerates
the healing process26.
Recent investigations using advanced experimental
techniques have tried to define the precise
biostimulatory effects of LLLT. Recently, an
upregulation of genes leading to an inverse in the
proliferation of laser-treated fibroblasts has been
demonstrated27.
Irradiation of human fibroblasts at 628 nm was
reported to have caused an increase in 7 categories of
genes already known to play roles in the enhancement
of cell proliferation and suppression of apoptosis27.
Another related study succeeded in demonstrating in
real time that LLLT could cause intracytosolic
increase in pH and calcium as well as changes in
reactive oxygen species28.
These finding have laid a new groundwork for
attempting to understand the comprehensive effects of
LLLT on the biochemical milieu of the injured tissue29.
To conclude, acceleration in the healing of
experimental wounds in the hard palate mucosa of
mice at LLLT with He-Ne laser at energy densities of
3 and 7.5 J/cm2 was observed.
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