Universidade de São Paulo

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Departamento de Física e Ciências Materiais - IFSC/FCM Artigos e Materiais de Revistas Científicas - IFSC/FCM

2014-09

Effect of low-level laser therapy on

odontoblast-like cells exposed to bleaching
agent

Lasers in Medical Science, London : Springer, v. 29, n. 5, p. 1533-1538, Sept. 2014

http://www.producao.usp.br/handle/BDPI/50899

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Lasers Med Sci (2014) 29:1533–1538
DOI 10.1007/s10103-013-1309-2

ORIGINAL ARTICLE

Effect of low-level laser therapy on odontoblast-like cells

exposed to bleaching agent
Adriano Fonseca Lima & Ana Paula Dias Ribeiro &
Fernanda Gonçalves Basso &
Vanderlei Salvador Bagnato & Josimeri Hebling &
Giselle Maria Marchi & Carlos Alberto de Souza Costa

Received: 29 August 2012 / Accepted: 12 March 2013 / Published online: 23 March 2013
# Springer-Verlag London 2013

Abstract The aim of the present study was to evaluate the
effect of low-level laser therapy (LLLT) on odontoblast-like
MDPC-23 cells exposed to carbamide peroxide (CP 0.
01 %–2.21 μg/mL of H2O2). The cells were seeded in sterile
24-well plates for 72 h. Eight groups were established
according to the exposure or not to the bleaching agents
and the laser energy doses tested (0, 4, 10, and 15 J/cm2).
After exposing the cells to 0.01 % CP for 1 h, this bleaching
solution was replaced by fresh culture medium. The cells
were then irradiated (three sections) with a near-infrared
diode laser (InGaAsP—780±3 nm, 40 mW), with intervals
of 24 h. The 0.01 % CP solution caused statistically signif-
icant reductions in cell metabolism and alkaline phosphate
(ALP) activity when compared with those of the groups not
exposed to the bleaching agent. The LLLT did not modulate
cell metabolism; however, the dose of 4 J/cm2 increased the
ALP activity. It was concluded that 0.01 % CP reduces the
MDPC-23 cell metabolism and ALP activity. The LLLT in
the parameters tested did not influence the cell metabolism
of the cultured cells; nevertheless, the laser dose of 4 J/cm2
increases the ALP activity in groups both with and without
exposure to the bleaching agent.
Keywords Odontoblasts . Laser . Low-level laser therapy .
Bleaching treatment . Alkaline phosphatase
Introduction
Currently, at-home tooth bleaching may be performed with
gels having low concentrations of carbamide peroxide (CP,
10–22 %) or hydrogen peroxide (HP, 6–16 %). On the other

A. F. Lima : G. M. Marchi V. S. Bagnato

Department of Restorative Dentistry, Piracicaba Dental School, Physics Institute of São Carlos, USP—University of Sao Paulo,
University of Campinas—UNICAMP, Avenida Limeira, 901, C.P. 369( 13560-970 São Carlos, Sao Paulo, Brazil
13404-903 Piracicaba, Sao Paulo, Brazil

A. F. Lima
Department of Restorative Dentistry, Nove de Julho University,
Rua Vergueiro, 235,
01504-000 Sao Paulo, Sao Paulo, Brazil
A. P. D. Ribeiro
Department of Operative Dentistry, University of Brasília-UnB.
Campus Universitário Darcy Ribeiro, 70910-900,
Brasília, Federal District, Brazil
F. G. Basso
Department of Pathology, Piracicaba Dental School, University
of Campinas—UNICAMP, Avenida Limeira, 901,
13404-903 Piracicaba, Sao Paulo, Brazil
J. Hebling
Department of Orthodontics and Pediatric Dentistry, Araraquara
School of Dentistry, Univ. Estadual Paulista (UNESP),
Rua Humaitá, 1680,
14801-903 Araraquara, Sao Paulo, Brazil
C. A. de Souza Costa (*)
Department of Physiology and Pathology, Araraquara School
of Dentistry, Univ. Estadual Paulista (UNESP), Rua Humaitá, 1680,
14801-903 Araraquara, Sao Paulo, Brazil
e-mail: casouzac@foar.unesp.br
1534
hand, high-concentration gels of HP (30–35 %) or CP (35–
37 %) have been recommended for in-office bleaching ther-
apy. Studies have shown that tooth bleaching occurs due to
the breakdown of pigments located in enamel and/or dentin,
by the reactive oxygen species (ROS) released from the
bleaching agents, such as hydroxyl radicals (OH−) and sin-
glet oxygen (1O2) [1]. These pigments are broken into
smaller molecules, which absorb lower light or are diffused
from the dentin structure, promoting a clearer aspect of the
tooth [1].
Since the tooth-bleaching process is mediated by ROS,
which can diffuse through the enamel and dentin, reaching
the pulp space [2], these aesthetic procedures can promote
pulp alterations, such as mild inflammation or even partial
necrosis [3–5]. Due to the deleterious effects caused by
components of bleaching agents, which have been demon-
strated in both in vitro [6–10] and in vivo studies [3, 5, 11],
the evaluation of other bleaching techniques that can pre-
vent or reduce pulp damage is needed. Thus, laser is prom-
ising coadjutant treatment that can be used in the
photodynamic therapy [12, 13], stimulate cell differentia-
tion, reduction of inflammation, and tissue repair [14–17].
Previous studies demonstrated that the specific parameters
of low-level laser therapy LLLT can stimulate the deposition
of collagen by fibroblasts [18] and proliferation of osteo-
blasts [19], as well as increase the mitochondrial activity and
synthesis of ATP [20]. Once applied to cultured
odontoblast-like cells, the LLLT increased cell metabolism
as well as the synthesis of proteins and alkaline phosphate
activity [14–16]. The bio-stimulatory effects of LLLT on
pulp cells such as odontoblasts are important, since these
cells are responsible for dentin matrix deposition and its
mineralization. In addition, odontoblasts are the first pulp
cells to be reached by products released from dental mate-
rials capable of diffusing through enamel/dentin [6, 7]. In
this way, it may be suggested that the low-level laser irradi-
ation of odontoblasts, prior to or immediately after tooth-
bleaching therapies, may prevent pulp damage or improve
the pulpal healing caused by ROS or other sub-products
released from bleaching agents. Therefore, the aim of this
study was to evaluate the effects of LLLT on odontoblast-
like MDPC-23 cells exposed or not to bleaching agents.
Materials and methods
Cell culture, bleaching exposure, and LLL irradiation
Immortalized cells of the MDPC-23 cell line [21, 22] in
complete Dulbecco’s Modified Eagle’s Medium (DMEM;
Sigma Chemical Co., St. Louis, MO, USA) were seeded
(12,500 cells/cm2) in wells of sterile 24-well dishes (Costar
Corp., Cambridge, MA, USA) and maintained for 72 h in a
Lasers Med Sci (2014) 29:1533–1538
humidified incubator (Isotemp, Fisher Scientific, Pittsburgh,
PA, USA) with 5 % CO2 and 95 % air at 37 °C.
A 10 % carbamide peroxide (CP) bleaching gel (White-
ness, FGM, Joinville, Santa Catarina, Brazil) was diluted in
DMEM without fetal calf serum (FCS) to obtain an extract
with 0.01 % of CP (approximately 2.21 μg of hydrogen
peroxide) to be applied to the cultured cells [6, 8]. This
concentration of bleaching agent was chosen based on pre-
vious studies, and represents the amount of hydrogen per-
oxide that diffuses through enamel and dentin and reaches
the pulp chamber after the bleaching procedures [23]. The
experimental and control groups are presented in Table 1.
After exposing the cells to the extract for 1 h, the DMEM
with CP was replaced by fresh culture medium
supplemented with 1 % FCS. Thereafter, the cultured cells
were irradiated three times with different low-level param-
eters. The irradiations were performed every 24 h, according
to the specific groups shown in Table 1. The low-level laser
device (LASERTable) [14–17, 24] used in this study was
based on near-infrared indium gallium arsenide phosphide
(InGaAsP) diode lasers (LASERTable; 780±3 nm, 0.04 W).
Twenty-four hours after the last irradiation, the cell metab-
olism as well as the alkaline phosphatase activity was eval-
uated, as described below.
Analysis of cell metabolism (MTT assay)
In each group, ten wells were used to analyze cell metabo-
lism by the cytochemical demonstration of succinic dehy-
drogenase production, measuring the mitochondrial
respiration of the cells by the methyl tetrazolium (MTT)
assay, as described in several studies [6, 7, 14–16, 25].
Alkaline phosphatase (ALP) activity
Ten wells per group were also used to evaluate ALP activity.
A colorimetric endpoint assay (ALP Kit; Labtest
Diagnóstico S.A., Lagoa Santa, Minas Gerais, Brazil) with
a thymolphthalein monophosphate substrate was employed
for this purpose. This is a phosphoric acid ester substrate
that is hydrolyzed by ALP and releases thymolphthalein,
which gives a bluish color to the solution. The intensity of
the resulting color is directly proportional to the enzymatic
activity and is analyzed by spectrophotometry.
The culture medium was aspirated, and the cells were
washed with sterile PBS at 37 °C. A 1-mL quantity of 0.1 %
sodium lauryl sulfate (Sigma Chemical Co., St. Louis, MO,
USA) was added to each well and maintained for 30 min at
room temperature to produce cell lysis. Next, Falcon tubes
(test, standard, and blank) were properly labeled, and 50 μL of
substrate (thymolphthalein monophosphate, 22 mmol/L—
Kit’s reagent #1) and 500 μL of buffer (300 mmol/L, pH 10.
1—Kit’s reagent #2) were added to each tube. A 50-μL
Lasers Med Sci (2014) 29:1533–1538
Table 1 Experimental and control
groups according to the exposure to Groups CP 0.01 %
the bleaching agent and the differ-
ent energy doses tested G1 (negative control) Absent
G2 (positive control) Present
G3 Absent
G4 Absent
G5 Absent
G6 Present
G7 Present
G8 Present
quantity of the standard solution 45 U/L (Kit’s reagent #4) was
added only to the standard tube. Thirty minutes after cell lysis,
the tubes were placed in a double boiler (Fanem, Guarulhos,
Sao Paulo, Brazil) at 37 °C for 2 min. The samples were
homogenized, and a 50-μL quantity from each plate was
transferred to the test tubes and maintained in the double
boiler under gentle agitation. After 10-min incubation, a 2-
mL quantity of color reagent (sodium carbonate, 94 mmol/L,
and sodium hydroxide, 250 mmol/L—Kit’s reagent #3) was
added.
Absorbance was measured by spectrophotometry at a
590-nm wavelength (Thermo Plate, Nanshan District,
Shenzhen, China). ALP activity was calculated according
to a standard curve, with pre-determined values of the
enzyme.
Statistical analysis
After exploratory data analysis to evaluate the homogeneity
of variances and normality of errors, the data obtained from
MTT assay and ALP activity were subjected to two-way
analysis of variance (ANOVA). Multiple comparisons were
performed by Tukey’s test, at a significance level of 5 %.
Results
The cell metabolism data obtained from the MTT assay are
shown in Fig. 1. The exposure to 0.01 % CP solution
decreased the cell metabolism, which was statistically dif-
ferent from that observed in the groups not exposed to the
bleaching agent (two-way ANOVA and Tukey’s test, p<0.
05). The metabolism of cultured cells was not modulated by
the laser irradiation, whether or not they were exposed to
carbamide peroxide.
The ALP activity of cells exposed to the bleaching agent
was reduced compared with that in the groups without
exposure of cultured cells to 0.01 % CP solution. The laser
dose of 4 J/cm2 increased the ALP activity on the cells
exposed or not to the carbamide peroxide, which was
1535
Energy dose, J/cm2 Laser Number of applications
0 Absent 0
0 Absent 0
4 Present 3
10 Present 3
15 Present 3
4 Present 3
10 Present 3
15 Present 3
statistically different from that in the other groups (p<0.
05). The results of ALP activity are shown in Table 2.
Discussion
Researchers have worked hard to obtain dental bleaching
protocols that provide an effective aesthetic clinical out-
come without causing collateral deleterious effects to pulp
cells [8, 9, 26]. In a current study, Lima et al. [8, 9] demon-
strated that the administration of sodium ascorbate before
the dental bleaching treatment may reduce the cell damage
caused by toxic sub-products released from bleaching
agents. In the present study, low-level laser irradiation of
0.01 % CP-treated odontoblast-like cells was assessed to
determine if specific laser parameters positively modulated
the metabolism of cells damaged by a bleaching agent
widely used in dentistry for tooth whitening.
In the present investigation, the MDPC-23 cells exposed
to the CP bleaching agent had their metabolism reduced in
about 35 %. Previous studies demonstrated that components
released from bleaching agents, such as HP and other ROS
molecules, are capable of breaking down the double bonds
in the fatty acids present in the plasma membrane of cells,
which activate endonucleases and proteases as well as in-
crease cell permeability [27, 28]. In this study, a solution
(extract) with a very low concentration of bleaching agent
was obtained after diluting the 10 % CP gel directly in
culture medium without FCS. In this way, different factors
beyond the HP by itself, such as the pH of the final solution
as well as the presence of other CP bleaching agent compo-
nents may be responsible, at least in part, for the cytotoxicity
observed in the cultured MDPC-23 cells. Nevertheless,
since previous studies using similar methodology showed
that the toxic effects were reversed after concomitant use of
an antioxidizing agent [8, 9], the authors may suggest that
the HP released from the 10 % CP bleaching agent in the
culture medium played the main role in the cytotoxicity
observed in the present study. However, further investiga-
tions are needed to clarify this matter.
1536 Lasers Med Sci (2014) 29:1533–1538

Fig. 1 Graphic representation of

the reduction in cell metabolism
(absorbance) as a function of the
different treatments. Different
letters indicate statistical
differences (Tukey; p<0.05)

In the current study, different parameters of low-level laser
irradiation (4, 10, and 15 J/cm2) were applied to bio-stimulate
ROS-damaged pulp cells. However, the laser protocols tested
were not capable of influencing the metabolism of previously
damaged odontoblast-like cells. These data were not corroborat-
ed by results from in vitro studies in which osteoblasts, odonto-
blasts, or even fibroblasts exposed to bleaching agents were
positively modulated by low-level laser irradiation [26, 29, 30].
The differences observed when results of these investigations
were compared with those from the present study may be at least
partly due to the variable parameters of the laser protocols tested
(wavelength, potency, energy density). In addition, different
cultured cell types subjected to specific therapies may provide
distinct responses [31].
The bleaching agent evaluated in this study decreased
MDPC-23 cell metabolism as well as ALP activity. This last
negative collateral effect may have been caused by the interac-
tion among the ROS released from the bleaching agents and the
cell protein chains, breaking down the sequence of amino acids
near the active center, inactivating this molecule by protein
Table 2 Results of the alkaline phosphatase activity according to the
different experimental and control groups
Bleaching agent
Energy dose 0 0.01 % CP
0 J/cm2 1.97 (0.37) Ab 0.78 (0.31) Bb
4 J/cm2 2.26 (0.48) Aa 1.38 (0.3) Ba
10 J/cm2 1.74 (0.5) Ab 0.81 (0.14) Bb
15 J/cm2 1.46 (0.3) Ab 0.79 (0.22) Bb
Capital letters compare the bleaching treatment and lower case letters com-
pare the LLLT parameters (ANOVA two-way and Tukey’s test; α=0.05)
fragmentation [32]. However, in spite of the lack of LLLT
influence on cell metabolism, the dose of 4 J/cm2 was capable
of increasing the activity of ALP on the cells, whether or not
they were exposed to the CP bleaching agent. It may be
explained due to the fact that LLLT may affect cell activity in
various ways and different factors can induce specific cell
responses after laser irradiation [33]. It has been widely accept-
ed that the action of the LLLT starts in the mitochondria [33].
These organelles play a critical role in calcium homeostasis and
storage, sequestering calcium ions from the endoplasmic retic-
ulum or across the plasma membrane, to activate several pro-
cesses through releasing Ca2+ in subcellular regions [34]. After
the irradiation and light absorption by a photo acceptor, a
cascade of cellular signaling—such as dissociation of the nitric
oxide of the catalytic center of the cytochrome C oxidase, and
the increase of ATP synthesis—occurs, allied to an increase in
intracellular Ca2+ [35, 36]. Particularly in osteoblast-like cell
line (MC3T3-E1), high concentration of free ionized calcium
(Ca2+) may stimulate chemotaxis, proliferation, and matrix
maturation [37]. Other studies have shown that extracellular
Ca2+ increased the mineralization of osteoblasts and mesenchy-
mal stem cells [38, 39]. In addition, the extracellular free
ionized Ca2+ may promote an increase in intracellular Ca2+
through the calcium channels, resulting in the activation of
numerous targets [40]. In a current study, Gabusi et al. [41]
reported that a high concentration of Ca2+ intracellular in-
creased ALP activity as well as collagen-I, osteocalcin, and
sialoprotein expression in human osteoblasts. Therefore, based
on all these data, it may be speculated that, in the present study,
a laser dose of 4 J/cm2 was able to sensitize the photo acceptor
of the MDPC-23 cells, increase ATP synthesis, promoting
higher levels of intracellular Ca2+, and, consequently, increase
ALP activity in those groups, whether or not they were exposed
Lasers Med Sci (2014) 29:1533–1538
to bleaching agents. However, further studies are needed to
determine the mechanisms involved in the increase of ALP
activity caused by the specific parameter of cell laser
irradiation.
In the present study, the positive result regarding ALP cell
activity was obtained only with the laser irradiation of cells with
4 J/cm2. Nevertheless, doses of 10 and 15 J/cm2 were not capable
of producing any effect on the activity of the protein evaluated.
These results may be explained, at least in part, by a specific
phenomenon known as “biphasic dose response” or “hormesis”
(the Arndt–Schulz law) [42]. In this law, it is proposed that weak
stimuli can promote positive cell effects. Thus, relatively low
doses of laser irradiation, such as 4 J/cm2, can positively
biomodulate MDPC-23 cell behavior; however, when the peak
is reached, stronger stimuli can inhibit the positive effects or
even, in some situations, promote a negative response.
Analysis of the data obtained in the present study, and
other previous investigation [26] , indicated that low-level
laser irradiation may represent an alternative therapy for the
modulation of pulp cell responses against the toxic effects
caused by bleaching agent components capable of diffusing
across dental hard tissues, i.e., enamel and dentin. Never-
theless, other factors, such as the number of laser irradia-
tions and the energy doses used, need to be evaluated in
further studies.
Conclusions
Based on the methodology used and the results obtained in
the present study, it can be concluded that the concentration
of 0.01 % carbamide peroxide causes toxic effects in
odontoblast-like MDPC-23 cells, characterized by reduc-
tions in cell metabolism and ALP activity. Specific doses
of low-level laser therapy (4, 10, and 15 J/cm2) are incapa-
ble of modulating cell metabolism positively; however, the
laser dose of 4 J/cm 2 increased the ALP activity of
bleaching-agent-treated cultured MDPC-23 cells.
Acknowledgments This work is part of a thesis submitted to the
Piracicaba Dental School in partial fulfillment of the requirements for
the PhD degree. This study was supported by the Fundação de Amparo
à Pesquisa do Estado de São Paulo—FAPESP (grants: 2009/08992-7 -
8 and 2010/ 00645–3) and Conselho Nacional de Desenvolvimento
Científico e Tecnológico—CNPq (grant: 301291/2010-1).
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