Hindawi Publishing Corporation

International Journal of Dentistry

Volume 2012, Article ID 719452, 6 pages
doi:10.1155/2012/719452

Research Article
In Vitro Wound Healing Improvement by Low-Level Laser
Therapy Application in Cultured Gingival Fibroblasts
Fernanda G. Basso,1 Taisa N. Pansani,2 Ana Paula S. Turrioni,2 Vanderlei S. Bagnato,3
Josimeri Hebling,2 and Carlos A. de Souza Costa2, 4
1 Faculdade

de Odontologia de Piracicaba, Universidade Estadual de Campinas (UNICAMP), 13414-903 Piracicaba, SP, Brazil
de Odontologia de Araraquara, Universidade de Estadual Paulista (UNESP), 14801-903 Araraquara, SP, Brazil
3 Instituto de F
sica de Sao Carlos, Universidade de Sao Paulo (USP), 13560-970 Sao Carlos, SP, Brazil
4 Departamento de Fisiologia e Patologia, Faculdade de Odontologia de Araraquara, Universidade Estadual Paulista,
Rua Humaita, 1680, Centro, Caixa Postal: 331, 14801903 Araraquara, SP, Brazil
2 Faculdade

Correspondence should be addressed to Carlos A. de Souza Costa, [email protected]

Received 4 April 2012; Revised 25 May 2012; Accepted 25 May 2012
Academic Editor: S. Nammour
Copyright 2012 Fernanda G. Basso et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
The aim of this study was to determine adequate energy doses using specific parameters of LLLT to produce biostimulatory
eects on human gingival fibroblast culture. Cells (3 104 cells/cm2 ) were seeded on 24-well acrylic plates using plain DMEM
supplemented with 10% fetal bovine serum. After 48-hour incubation with 5% CO2 at 37 C, cells were irradiated with a InGaAsP
diode laser prototype (LASERTable; 780 3 nm; 40 mW) with energy doses of 0.5, 1.5, 3, 5, and 7 J/cm2 . Cells were irradiated
every 24 h totalizing 3 applications. Twenty-four hours after the last irradiation, cell metabolism was evaluated by the MTT
assay and the two most eective doses (0.5 and 3 J/cm2 ) were selected to evaluate the cell number (trypan blue assay) and the
cell migration capacity (wound healing assay; transwell migration assay). Data were analyzed by the Kruskal-Wallis and MannWhitney nonparametric tests with statistical significance of 5%. Irradiation of the fibroblasts with 0.5 and 3 J/cm2 resulted in
significant increase in cell metabolism compared with the nonrradiated group (P < 0.05). Both energy doses promoted significant
increase in the cell number as well as in cell migration (P < 0.05). These results demonstrate that, under the tested conditions,
LLLT promoted biostimulation of fibroblasts in vitro.

1. Introduction
Tissue healing involves an intense activity of diverse cell
types, such as epithelial and endothelial cells, as well
as fibroblasts which play a key role in this process [1].
Fibroblasts secrete multiple growth factors during wound
reepitelialization and participate actively in the formation of
granulation tissue and the synthesis of a complex extracellular matrix after reepitelialization [1]. All these processes
directly involve the proliferation and migration capacity to
these cells [1]. The use of low-level laser therapy (LLLT) has
been proposed to promote biostimulation of fibroblasts and
accelerate the healing process [2].
Previous studies have evaluated the eect of LLLT on the
proliferation and migration of human gingival fibroblasts
as well as other cellular eects and responses, such as

protein production and growth factor expression [26].

Nevertheless, there is a shortage of studies investigating
irradiation parameters capable of promoting biostimulatory
eects on fibroblasts in order to establish an ideal irradiation
protocol for these cells [7]. Therefore, the aim of this study
was to determine the most adequate energy doses using
specific parameters of LLLT to produce biostimulatory eects
on human gingival fibroblast cultures in an in vitro wound
healing model.

2. Material and Methods

2.1. Gingival Fibroblast Cell Culture. All experiments were
performed using human gingival fibroblast cell culture
(continuous cell line; Ethics Committee 64/99-Piracicaba

2
Dental School, UNICAMP, Brazil). The fibroblast cells were
cultured in Dulbeccos Modified Eagles Medium (DMEM;
Sigma-Aldrich, St. Louis, MO, USA) supplemented with
10% fetal bovine serum (FBS; Gibco, Grand Island, NY,
USA), with 100 IU/mL penicillin, 100 g/mL streptomycin,
and 2 mmol/L glutamine (Gibco, Grand Island, NY, USA) in
an humidified incubator with 5% CO2 and 95% air at 37 C
(Isotemp; Fisher Scientific, Pittsburgh, PA, USA) [8]. The
cells were subcultured every 2 days in the incubator under
the conditions described above until an adequate number of
cells were obtained for the study. The cells (3104 cells/cm2 )
were then seeded on sterile 24-well acrylic plates using plain
DMEM supplemented with 10% FBS for 48 h.
2.2. LLLT on Fibroblast Culture. The LLLT device used in this
study was a near infrared indium gallium arsenide phosphide
(InGaAsP) diode laser prototype (LASERTable; 780 3 nm
wavelength, 0.04 W maximum power output), which was
specifically designed to provide a uniform irradiation of
each well (2 cm2 ) in which cultured cells are seeded [8, 9].
The power loss through the acrylic plate was calculated
using a potentiometer (Coherent LM-2 VIS High-Sensitivity
Optical Sensor, USA), which was placed inside the culture
plate. After this measure, the power loss of the plate was
determined as 5%. After that, the power of all diodes
was checked and standardized. Therefore, a final power of
0.025 W reached the cultured cells. This standardization was
performed as previously described in the literature [8, 9]. For
the evaluation of cell metabolism, the radiation originated
from the LASERTable was delivered on the base of each 24well plate with energy doses of 0.5, 1.5, 3, 5, and 7 J/cm2 , and
irradiation times of 40, 120, 240, 400, and 560 s, respectively.
The laser light reached the cells on the bottom of each
well with a final power of 0.025 W because of the loss of
optical power in each well due to the interposition of the
acrylic plate. The cells were irradiated every 24 h totalizing
3 applications during 3 consecutive days. The cells assigned
to control groups received the same treatment as that of
the experimental groups. The 24-well plates containing the
control cells were maintained at the LASERTable for the same
irradiation times used in the respective irradiated groups,
though without activating the laser source (sham irradiation)
[8, 9]. Twenty-four hours after the last irradiation (active
or sham), the metabolic activity of the cells was evaluated
using the MTT assay (described below). Based on cell
metabolism results, the two most eective irradiation doses
were selected to evaluate the cell number (trypan blue
assay), cell migration capacity by using the wound healing
assay (qualitative analysis) and the transwell migration assay
(quantitative analysis), as described below.

International Journal of Dentistry

Each well with the fibroblasts received 900 L of DMEM
plus 100 L of MTT solution (5 mg/mL sterile PBS). The
cells were incubated at 37 C for 4 h. Thereafter, the culture
medium (DMEM; Sigma Chemical Co., St. Louis, MO,
USA) with the MTT solution were aspirated and replaced
by 700 L of acidified isopropanol solution (0.04 N HCl) in
each well to dissolve the violet formazan crystals resulting
from the cleavage of the MTT salt ring by the SDH enzyme
present in the mitochondria of viable cells, producing a
homogenous bluish solution. Three 100 L aliquots of each
well were transferred to a 96-well plate (Costar Corp.,
Cambridge, MA, USA). Cell metabolism was evaluated by
spectrophotometry as being proportional to the absorbance
measured at 570 nm wavelength with an ELISA plate reader
(Thermo Plate, Nanshan District, Shenzhen, China) [8, 9].
The values obtained from the three aliquots were averaged
to provide a single value. The absorbance was expressed in
numerical values, which were subjected to statistical analysis
to determine the eect of LLLT on the mitochondrial activity
of the cells.
2.4. Viable Cell Counting (Trypan Blue Assay). Trypan blue
assay was used to evaluate the number of cells in the culture
after LLLT application. This test provides a direct assessment
of the total number of viable cells in the samples as the trypan
blue dye can penetrate only porous, permeable membranes
of lethally damaged (dead) cells, which is clearly detectable
under optical microscopy [11]. The LLLT protocol was
undertaken as previously described using energy doses of 0.5
and 3 J/cm2 . Cell counting was performed in the experimental and control groups 24 h after the last irradiation (active
or sham). The DMEM in contact with the cells was aspirated
and replaced by 0.12% trypsin (Invitrogen, Carlsbad, CA,
USA), which remained in contact with the cells for 10 min to
promote their detachment from the acrylic substrate. Then,
50 L aliquots of this cell suspension were added to 50 L
of 0.04% trypan blue dye (Sigma Aldrich Corp., St. Louis,
MO, USA), and the resulting solution was maintained at
room temperature for 2 min so that the trypan blue dye could
pass through the cytoplasmic membrane of the nonviable
cells, changing their color into blue. Ten microliters of the
solution were taken to a hemocytometer and examined with
an inverted light microscope (Nikon Eclipse TS 100, Nikon
Corporation, Tokyo, Japan) to determine the number of total
cells and nonviable cells. The number of viable cells was
calculated by deducting the number of nonviable cells from
the number of total cells [8]. The number of cells obtained
in the counting corresponded to n 104 cells per milliliter of
suspension.
2.5. Cell Migration

2.3. Analysis of Cell Metabolism (MTT Assay). Cell metabolism was evaluated using the methyltetrazolium (MTT)
assay [810]. This method determines the activity of succinic
dehydrogenase (SDH) enzyme, which is a measure of cellular
(mitochondrial) respiration and can be considered as the
metabolic rate of cells.

2.5.1. Wound Healing Assay. The wound healing assay was

used because it is a classic method of evaluation in vitro
tissue healing assays [12, 13]. After 48 h of cell culture, a
sterile 5 mL pipette tip was used to make a straight scratch
on the monolayer of cells attached to the acrylic substrate,
simulating a wound. Formation of the in vitro wound

2
Dental School, UNICAMP, Brazil). The fibroblast cells were
cultured in Dulbeccos Modified Eagles Medium (DMEM;
Sigma-Aldrich, St. Louis, MO, USA) supplemented with
10% fetal bovine serum (FBS; Gibco, Grand Island, NY,
USA), with 100 IU/mL penicillin, 100 g/mL streptomycin,
and 2 mmol/L glutamine (Gibco, Grand Island, NY, USA) in
an humidified incubator with 5% CO2 and 95% air at 37 C
(Isotemp; Fisher Scientific, Pittsburgh, PA, USA) [8]. The
cells were subcultured every 2 days in the incubator under
the conditions described above until an adequate number of
cells were obtained for the study. The cells (3104 cells/cm2 )
were then seeded on sterile 24-well acrylic plates using plain
DMEM supplemented with 10% FBS for 48 h.
2.2. LLLT on Fibroblast Culture. The LLLT device used in this
study was a near infrared indium gallium arsenide phosphide
(InGaAsP) diode laser prototype (LASERTable; 780 3 nm
wavelength, 0.04 W maximum power output), which was
specifically designed to provide a uniform irradiation of
each well (2 cm2 ) in which cultured cells are seeded [8, 9].
The power loss through the acrylic plate was calculated
using a potentiometer (Coherent LM-2 VIS High-Sensitivity
Optical Sensor, USA), which was placed inside the culture
plate. After this measure, the power loss of the plate was
determined as 5%. After that, the power of all diodes
was checked and standardized. Therefore, a final power of
0.025 W reached the cultured cells. This standardization was
performed as previously described in the literature [8, 9]. For
the evaluation of cell metabolism, the radiation originated
from the LASERTable was delivered on the base of each 24well plate with energy doses of 0.5, 1.5, 3, 5, and 7 J/cm2 , and
irradiation times of 40, 120, 240, 400, and 560 s, respectively.
The laser light reached the cells on the bottom of each
well with a final power of 0.025 W because of the loss of
optical power in each well due to the interposition of the
acrylic plate. The cells were irradiated every 24 h totalizing
3 applications during 3 consecutive days. The cells assigned
to control groups received the same treatment as that of
the experimental groups. The 24-well plates containing the
control cells were maintained at the LASERTable for the same
irradiation times used in the respective irradiated groups,
though without activating the laser source (sham irradiation)
[8, 9]. Twenty-four hours after the last irradiation (active
or sham), the metabolic activity of the cells was evaluated
using the MTT assay (described below). Based on cell
metabolism results, the two most eective irradiation doses
were selected to evaluate the cell number (trypan blue
assay), cell migration capacity by using the wound healing
assay (qualitative analysis) and the transwell migration assay
(quantitative analysis), as described below.

International Journal of Dentistry

Each well with the fibroblasts received 900 L of DMEM
plus 100 L of MTT solution (5 mg/mL sterile PBS). The
cells were incubated at 37 C for 4 h. Thereafter, the culture
medium (DMEM; Sigma Chemical Co., St. Louis, MO,
USA) with the MTT solution were aspirated and replaced
by 700 L of acidified isopropanol solution (0.04 N HCl) in
each well to dissolve the violet formazan crystals resulting
from the cleavage of the MTT salt ring by the SDH enzyme
present in the mitochondria of viable cells, producing a
homogenous bluish solution. Three 100 L aliquots of each
well were transferred to a 96-well plate (Costar Corp.,
Cambridge, MA, USA). Cell metabolism was evaluated by
spectrophotometry as being proportional to the absorbance
measured at 570 nm wavelength with an ELISA plate reader
(Thermo Plate, Nanshan District, Shenzhen, China) [8, 9].
The values obtained from the three aliquots were averaged
to provide a single value. The absorbance was expressed in
numerical values, which were subjected to statistical analysis
to determine the eect of LLLT on the mitochondrial activity
of the cells.
2.4. Viable Cell Counting (Trypan Blue Assay). Trypan blue
assay was used to evaluate the number of cells in the culture
after LLLT application. This test provides a direct assessment
of the total number of viable cells in the samples as the trypan
blue dye can penetrate only porous, permeable membranes
of lethally damaged (dead) cells, which is clearly detectable
under optical microscopy [11]. The LLLT protocol was
undertaken as previously described using energy doses of 0.5
and 3 J/cm2 . Cell counting was performed in the experimental and control groups 24 h after the last irradiation (active
or sham). The DMEM in contact with the cells was aspirated
and replaced by 0.12% trypsin (Invitrogen, Carlsbad, CA,
USA), which remained in contact with the cells for 10 min to
promote their detachment from the acrylic substrate. Then,
50 L aliquots of this cell suspension were added to 50 L
of 0.04% trypan blue dye (Sigma Aldrich Corp., St. Louis,
MO, USA), and the resulting solution was maintained at
room temperature for 2 min so that the trypan blue dye could
pass through the cytoplasmic membrane of the nonviable
cells, changing their color into blue. Ten microliters of the
solution were taken to a hemocytometer and examined with
an inverted light microscope (Nikon Eclipse TS 100, Nikon
Corporation, Tokyo, Japan) to determine the number of total
cells and nonviable cells. The number of viable cells was
calculated by deducting the number of nonviable cells from
the number of total cells [8]. The number of cells obtained
in the counting corresponded to n 104 cells per milliliter of
suspension.
2.5. Cell Migration

2.3. Analysis of Cell Metabolism (MTT Assay). Cell metabolism was evaluated using the methyltetrazolium (MTT)
assay [810]. This method determines the activity of succinic
dehydrogenase (SDH) enzyme, which is a measure of cellular
(mitochondrial) respiration and can be considered as the
metabolic rate of cells.

2.5.1. Wound Healing Assay. The wound healing assay was

used because it is a classic method of evaluation in vitro
tissue healing assays [12, 13]. After 48 h of cell culture, a
sterile 5 mL pipette tip was used to make a straight scratch
on the monolayer of cells attached to the acrylic substrate,
simulating a wound. Formation of the in vitro wound

International Journal of Dentistry

was confirmed under an inverted microscope (TS 100,
Nikon, Tokyo, Japan). The LLLT protocol was undertaken as
previously described using energy doses of 0.5 and 3 J/cm2 .
Twenty-four hours after the last irradiation, the cells were
fixed in 1.5% glutaraldehyde for 1 h, stained with 0.1% violet
crystal for 15 min, and washed twice with distilled water.
Wound repopulation was assessed with a light microscope
(Olympus BX51, Miami, FL, USA) equipped with a digital
camera (Olympus C5060, Miami, FL, USA).
2.5.2. Transwell Migration Assay. The capacity of human
gingival fibroblasts to migrate through a cell permeable
membrane was assessed using 6.5 mm-diameter transwell
chambers (Corning Costar, Cambridge, MA, USA) with
polycarbonate membrane inserts (8 m pore size) [14]. The
chambers were placed in 24-well plates containing 1 mL
of plain DMEM per well. The cells were seeded onto the
upper compartment of the chamber (1.5 104 cells/cm2 )
and incubated at 37 C for 48 h. After this period, the
LLLT protocol was undertaken as previously described using
energy doses of 0.5 and 3 J/cm2 . Twenty-four hours after the
last irradiation (active or sham), the cells that had migrated
through the membrane to the lower compartment of the
chamber were fixed in 1.5% glutaraldehyde for 1 h, incubated
with 0.1% violet crystal dye for 15 min, and washed twice
with distilled water. After the last wash, the stained cells
were viewed under a light microscope (Olympus BX51,
Miami, FL, USA) equipped with a digital camera (Olympus
C5060, Miami, FL, USA) and photomicrographs from three
randomly chosen fields were taken at 10 magnification
for counting the number of migrated cells using the imageanalysis J 1.45S software (Wayne Rasband, National Institutes
of Health, Bethesda, MD, USA). Two samples of each
group were evaluated and the experiment was performed in
triplicate.
2.6. Analysis of Migrated Cells by Scanning Electron Microscopy
(SEM). Part of the specimens used in the transwell migration assay was also used for the analysis of the cells by SEM.
Twenty-four hours after the last irradiation (active or sham),
the culture medium was aspirated and the transwell inserts
were fixed in 1 mL of 2.5% glutaraldehyde in PBS for 2 h.
Then, the glutaraldehyde solution was aspirated and the cells
adhered to the transwell inserts were washed with PBS and
distilled water two consecutive times (5 min each) and then
dehydrated in a series of increasing ethanol concentrations
(30, 50 and 70%, one time for 30 min each; 95 and 100%,
two times for 60 min each) and covered 3 times with 200 L
of 1,1,1,3,3,3-hexamethyldisilazane (HMDS; Sigma Aldrich
Corp., St. Louis, USA) [8]. The transwell inserts were stored
in a desiccator for 24 h, sputter-coated with gold, and
the morphology of the surface-adhered cells was examined
with a scanning electron microscope (JMS-T33A scanning
microscope, JEOL, Tokyo, Japan).
2.7. Statistical Analysis. Data from MTT, Trypan blue and
Transwell assay had a nonnormal distribution (KolmogorovSmirnov, P < 0.05) and were analyzed by the Kruskal-Wallis

3
Table 1: Succinate dehydrogenase enzyme (SDH) production by
human gingival fibroblasts detected by the MTT assay according to
the energy dose used in the low-level laser therapy.
Energy dose (J/cm2 )
0 (control)

MTT (%)
100 (96104) C

0.5

111 (110113) B

1.5

94 (9297) D

117 (113119) A

95 (81108) CD

92 (9196) D

Values expressed as medians of SDH production (P25P75) (n = 12).

Same letters indicate no statistically significant dierence (Mann-Whitney,
P > 0.05).

Table 2: Number of viable cells (%) detected by the trypan blue

assay, according to the energy doses used in the low-level laser
therapy.
Energy dose (J/cm2 )
0 (control)

Number of viable cells (%)

100 (95104) B

0.5

133 (112175) A

168 (149181) A

Values expressed as medians of SDH production (P25P75) (n = 8).

Same letters indicate no statistically significant dierence (Mann-Whitney,
P > 0.05).

and Mann-Whitney nonparametric tests. A significance level

of 5% was set for all analyses.

3. Results
3.1. Analysis of Cell Metabolism (MTT Assay). Data from
SDH production by human gingival fibroblast cultures
(MTT assay) after LLLT, according to the energy dose are
presented in Table 1.
Regarding the energy dose of 5 J/cm2 no statistically
significant dierence between the irradiated group and
the nonirradiated control group was observed (P > 0.05).
Conversely, irradiation of the fibroblast cultures with doses
of 0.5 J/cm2 and 3 J/cm2 resulted in 11% and 17% increases
in cell metabolism, respectively, diering significantly from
the control group (P < 0.05). The cells irradiated with
1.5 J/cm2 and 7 J/cm2 presented the lowest metabolic rate
compared with the nonirradiated control group (6% and 8%
decrease, resp., P < 0.05).
3.2. Viable Cell Counting (Trypan Blue Assay). The number
of viable cells (%) after LLLT application, according to the
energy dose, is presented in Table 2.
Comparison among the energy doses revealed that
irradiation of the human gingival fibroblast cultures with
0.5 J/cm2 and 3 J/cm2 increased the number of viable cells
by 31% and 66%, respectively, diering significantly from
the control (P < 0.05), but without statistically significant
dierence between each other (P > 0.05).

International Journal of Dentistry

0 J/cm 2

0.5 J/cm 2

3 J/cm 2

Figure 1: Photomicrographs showing human gingival fibroblast cultures seeded in 24-well plates after LLLT. The control group exhibits
a large cell-free area on acrylic surface. The group irradiated with 0.5 J/cm2 exhibits cell proliferation and migration, with consequent
reduction of the in vitro wound size. The group irradiated with 3.0 J/cm2 presented more intense cell proliferation and migration, resulting
in almost complete closure of the in vitro wound.

Table 3: Cell migration (%) by the transwell assay, according to the

energy dose used in the low-level laser therapy.
Energy dose (J/cm2 )
0 (control)
0.5
3

Cell migration (%)

100 (91107) B
118 (109123) A
120 (116122) A

Values expressed as medians of SDH production (P25P75) (n = 6).

Same letters indicate no statistically significant dierence (Mann-Whitney,
P > 0.05).

3.3. Fibroblast Migration

3.3.1. Wound Healing Assay. The analysis of the monolayer of
human gingival fibroblasts after irradiation of the in vitro
wound showed more intense cell migration, with consequent better coverage of the substrate (wound repopulation)
(Figure 1).
3.3.2. Transwell Assay. Data from the transwell assay after
LLLT, according to the energy dose are, presented in
Table 3.
Comparison among the energy doses revealed that
irradiation of the human gingival fibroblast cultures with
0.5 J/cm2 and 3 J/cm2 increased cell migration by 16% and
18%, respectively, diering significantly from the control
(P < 0.05), but without statistically significant dierence
between each other (P > 0.05).
3.4. Analysis of Migrated Cells by Scanning Electron Microscopy
(SEM). The SEM analysis of the transwell inserts, which
complemented the viable cell counting by the trypan blue
assay, revealed that the fibroblasts were capable of migrating
through the transwell membrane. The cells obtained from
human gengiva did not change their morphology after been
submitted to LLLT (Figure 2).

4. Discussion
Dierent LLLT modalities have been used for diverse treatments in the health fields. In Dentistry, LLLT has been

widely investigated and indicated for accelerating the healing

process, especially in the treatment of ulcerative oral mucosa
lesions [15, 16].
Several in vitro studies have evaluated the eect of
LLLT on healing [7, 17]. Nevertheless, current research
involving irradiation of cell cultures has not yet established
the irradiation patterns specific for the dierent cell lines.
Establishing the ideal irradiation parameters and techniques
is mandatory for the development of sequential studies that
can determine the potential biostimulatory eect of LLLT
on oral mucosa cells, such as keratinocytes and fibroblasts,
which are directly involved in the local healing process.
In the present study, the metabolic activity of human
gingival fibroblast cultures after LLLT with dierent energy
doses was evaluated to determine the adequate doses to
produce biostimulatory eects on these cells in vitro. The
results for SDH production showed that the 0.5 and 3 J/cm2
doses increased cell metabolism. Therefore, these two most
eective irradiation doses were selected to evaluate the
number of viable cells as well as the cell migration capacity.
The increase of SDH production after irradiation of gingival
fibroblasts has also been observed by Damante et al. [18],
using a similar laser prototype to the one used in the
present study. In the same way as in the present study, the
SDH production results also served as guide for subsequent
experiments that evaluated the expression of growth factors
by cultured fibroblasts.
In the present study, a significant increase in the number
of viable cells that presented normal morphological characteristics (SEM analysis) was observed after LLLT using
doses of 0.5 and 3 J/cm2 . These results confirm those of
previous laboratory investigations in which LLLT with the
same wavelength as that of the present study (780 nm)
increased the proliferation of gingival fibroblasts [19, 20].
Kreisler et al. [2] also reported increase of fibroblast cell
culture in vitro after direct and consecutive low level laser
irradiations. The mechanism by which LLLT can promote
biostimulation and induce proliferation of dierent cell
types remains a controversial subject [20, 21]. Some authors
[21, 22] claim that this mechanism is derived from light
absorption by the enzyme cytochrome c oxidase in the cells,
which participates in the cascade of oxidative respiration.

International Journal of Dentistry

0 J/cm 2

0.5 J/cm 2

3 J/cm 2

Figure 2: SEM micrograph showing cells with normal morphology that migrated through the transwell membrane. SEM 500.

Eells et al. [23] demonstrated the increase in the production

of this enzyme after dierent LLLT application of cell
cultures. It has also been suggested that the mechanism of
cell proliferation induced by LLLT might be derived from
the activation of singling pathways, such as the MAPK and
PI3K/Akt pathways, which control both cell proliferation and
regulation of gene expression [21, 24].
Fibroblast cell migration and proliferation are essential
events for tissue healing and are directly related with its
success [1, 3]. In the present study, the eect of LLLT
on the capacity of gingival fibroblast migration, using two
energy doses capable of increasing cell metabolism (0.5
and 3 J/cm2 ), was evaluated qualitatively, by the wound
healing assay, and quantitatively, by the transwell migration
assay. Both methodologies demonstrated that LLLT was able
to increase the migration capacity of fibroblasts and the
quantitative analysis of the results revealed no significant
dierence between the energy doses. These results are in
accordance with those of previous investigations [7, 17], but
studies using the transwell migration method to evaluate the
LLLT on cell cultures are still scarce. This methodology is
relevant because it measures the number of cells that can
pass through the transwell membrane inserts, demonstrating
their migration capacity after stimulation by LLLT.
Diverse mechanisms are involved in cell migration
during tissue healing, including expression and secretion of
growth factors [1]. Previous studies demonstrated that LLLT
may cause positive eects on cells by increasing growth factor
expression, which could be a form of action of specific laser
parameters on cell migration [2, 25]. A recent study of our
research group demonstrated that LLLT had a biostimulatory
eect on epithelial cells in vitro by increasing their metabolic
activity, number of viable cells and expression of growth
factors [8]. In the present paper, the biostimulation of human
gingival fibroblast cultures by LLLT with consequent increase
in the number of viable cells and cell migration capacity
demonstrates the ecacy of specific laser parameters and
irradiation technique on the healing process. In addition,
the obtained results are supportive to those of previous in
vivo studies in which acceleration of the healing process was
observed after LLLT [15, 16, 26], but the limitations of an in
vitro experiment should be considered.
In conclusion, the findings of the present study demonstrated that the preset laser parameters in combination with

the sequential irradiation technique caused biostimulation,

proliferation, and migration of human gingival fibroblast
cultures. These encouraging laboratory outcomes should
guide forthcoming studies involving tissue irradiation with
laser and its eects on in vivo tissue healing.

Acknowledgments
The authors acknowledge the Fundacao de Amparo a`
Pesquisa do Estado de Sao Paulo-FAPESP (Grants: 2009/
54722-1 and BP.DR: 2009/52326-1) and the Conselho

Nacional de Desenvolvimento Cientfico e Tecnologico

CNPq (Grant: 301029/2010-1) for the financial support.

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[3] W. Posten, D. A. Wrone, J. S. Dover, K. A. Arndt, S. Silapunt,
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