Photomedicine and Laser Surgery

Volume 36, Number 12, 2018

ª Mary Ann Liebert, Inc.
Pp. 647–652
DOI: 10.1089/pho.2018.4532

Laser Photobiomodulation Over Teeth Subjected

to Orthodontic Movement

Joseli M. Cordeiro, PhD, DDS,1 Marcelo G. Sahad, PhD, DDS,1 Marcos F.X.B. Cavalcanti, DDS,2,3
Rodrigo L. Marcos, PhD,4 Francesca Diomede, PhD, DDS,5 Oriana Trubiani, PhD, DDS,5
Durvanei A. Maria, PhD,6 Ernesto C.P. Leal-Junior, PhD,7 and Lucio Frigo, PhD, DDS8,9
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Abstract

Background: Orthodontics of the 21st century requires aesthetic, painless, predictable, and quick treatments.
This demand for faster results generated orthodontic movement acceleration protocols (OMAPs); among other
OMAPs we present low-level laser (LLL) as a candidate.
Objective: To evaluate levels of interleukin (IL)-1, IL-10, and type 1 collagen in the periodontal ligament of
first molars of rats subjected to orthodontic traction with and without LLL irradiation, compared with untreated
controls (CO), and to evaluate whether the dose of LLL used in this work is eligible as an OMAP.
Materials and methods: A total of 35 male Wistar rats were distributed into three groups: group 1 NI
(nonirradiated) n = 15, group 2 IR (laser irradiated using 5 J, 177 J/cm2, and 100 mW applied in contact to the
vestibular mesial, vestibular distal, and palatal faces of gum tissue around molar region for 50 sec each point,
for 3 consecutive days, immediately 24 and 48 h after orthodontic device placement.) n = 15, and group 3 CO
n = 5; groups 1 and 2 were subjected to orthodontic force and each group was divided into three subgroups that
were sacrificed after 3, 5, and 7 days, IL-1/10 and COL-1 levels were analyzed.
Results: In the IR group, levels of IL-1/10 and COL-1 showed peak anticipation after LLL irradiation compared
with those in the NI and CO groups.
Conclusions: These results can also infer that this dose of LLL can be used as an OMAP.

Keywords: tooth movement, photobiomodulation, low-level laser therapy, IL-1, IL-10, collagen type 1

Introduction The primary goal to accelerate tooth movement must be

to understand, control, and accelerate microenvironment

O rthodontics of the 21st century requires aesthetic,

painless, predictable, and quick treatments. These
requirements were at first satisfied by the introduction of
aligners (Invisalign) in the orthodontic practice and its
virtual planning (Clincheck).1
Despite the predictability of the results and the reduction
of chair time and total treatment time using aligners, patients
demand even faster results, which generated orthodontic
movement acceleration protocols (OMAPs).2
inflammation reactions inside periodontal tissues.
Biochemical reactions in the periodontal tissues around
the moving tooth confirm that teeth move, when a force
system is applied to it, due to a stretch–compression
system in the periodontal ligament. Bone marrow stem
cells increase their differentiation rate into osteoblasts
around stretched periodontal fibers and macrophages
start bone resorption around compressed periodontal
fibers.3

1
Post Graduation Program in Orthodontics and Dentofacial Orthopedics, Cruzeiro do Sul University, São Paulo, Brazil.
2
Biophotonics Laboratory and 3Dental Clinic, Nove de Julho University, São Paulo, Brazil.
4
Post Graduation Program in Biophotonics at Nove de Julho University, São Paulo, Brazil.
5
Researcher at Laboratory of Stem Cells and Regenerative Medicine, Department of Medical, Oral and Biotechnological Sciences,
‘‘G. d’Annunzio’’ University of Chieti–Pescara, Chieti, Italy.
6
Laboratory of Biochemistry and Biophysics, Institute Butantan, Butantan, São Paulo, Brazil.
7
Post Graduation Program in Biophotonics at Nove de Julho University, Center for Research and Innovation in Laser, Nove de Julho
University (UNINOVE), São Paulo, Brazil.
8
Movement Laboratory of Nove de Julho University, Santana, São Paulo, Brazil.
9
Graduation Program in Dentistry at Faculdade de Odontologia da Associação Paulista de Cirurgiôes Dentistas (FAOA), Santana, São Paulo, Brazil.

647
 648 CORDEIRO ET AL.

Interleukin (IL)-1 is a cytokine present at the first steps of

inflammation; it controls macrophage recruitment inside
affected tissues, such as periodontal tissues subjected to
orthodontic forces, preparing the organism response.4 At the
later phase of inflammation, IL-10 is upregulated controlling
(reducing) macrophage activity,5,6 pointing where and how
much bone resorption will take place in the affected tissue.
COL-1 is the most frequent protein found in the extracel-
lular matrix of living tissues; at periodontal stressed fibers it
indicates increase of extracellular bone matrix deposition
indicating repair is in course.7
Among other movement acceleration protocols, we
present low-level laser (LLL) as a candidate; it can change
cell cycle and increase cell proliferation,8 it acts in the re-
spiratory chain of mitochondria and in the cytoplasm Ca++
influencing the redox ambience modulating inflammation
and reducing pain.9
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FIG. 1. Orthodontic device placed on upper teeth, first

Objective
To evaluate levels of IL-1, IL-10, and COL-1 in the
periodontal ligament of first molars of rats subjected to or-
thodontic traction with and without LLL irradiation, com-
pared with untreated controls (CO), and to evaluate whether
the dose of LLL used in this work is eligible as an OMAP.
Materials and Methods
This study was approved by the ethics committee of
Cruzeiro do Sul University (protocol number 011/07), which
respects laws of using animals for scientific purposes.
Sample
A total of 35 male Wistar rats (Rattus norvegicus)
*200 g each, from Butantan Institute, were kept at the vi-
varium of Cruzeiro do Sul University, at room temperature
between 21C and 23C, in specific cages, with free access
to water and food (Labina Purina) (Table 1).
molar and central incisors, covered by orthodontic com-
posite.
sphoric acid at 37% (ref. 282085), water washed, air dried,
covered by Fill Magic orthodontic composite (ref. 141; São
Paulo, Brazil), and light activated for 30 sec using Gnatus
Opty 600 (cod. 30050918; Gnatus, São Paulo, Brazil) to
increase retention and prevent device displacement; borders
of the lower incisors were reduced by diamond bur and hand
piece to avoid occlusal interferences. The spring was acti-
vated to 250g by a calibrator (ref. 7502006; Morelli). No
other activations were made until sacrifice.
LLL protocol
LLL device. Infrared wavelength 808 nm, GaAlAs,
Photon lase III–DMC–São Carlos–Brazil, operation voltage:
127–220 V, irradiance: 1.2 W/cm2, and stick area 0.2 mm2.

Orthodontic treatment
Application protocol. Laser characteristics 5 J, 177 J/cm2,
and 100 mW were applied in contact to the vestibular me-
Anesthesia. Animals were anesthetized before all pro- sial, vestibular distal, and palatal faces of gum tissue around
cedures to avoid anxiety and/or pain using ketamine hy- molar region for 50 sec each point, for 3 consecutive days,
drochloride and xylazine (2:1), 1.5 cc/100 g per animal,
intramuscular injection.
Table 1. Distribution of Animals Used
Orthodontic device. Seven millimeter-long nickel– in the Experiment
titanium traction spring (ref. 35.20.064; Morelli, São Paulo,
Groups Sacrifice Number of animals
Brazil) was distended between the upper incisors and the
first upper right molar to move it to mesial, (Fig. 1) creating Group 1 (NI) A: 72 h (3 days) 5
pressure and traction areas in the periodontal ligament. Group 1 (NI) B: 120 h (5 days) 5
Group 1 (NI) C: 168 h (7 days) 5
Orthodontic device placement. To a perforation, made Group 2 (IR) A: 72 h (3 days) 5
with a diamond bur (KG1090; KG. Sorensen, São Paulo, Group 2 (IR) B: 120 h (5 days) 5
Brazil) and a hand piece (605C; Kavo, Joinvile, Brazil) in Group 2 (IR) C: 168 h (7 days) 5
the mesial of the upper incisors, was attached the front side Group 3 (CO) 0h 5
of the spring, using a 0.25 mm ligature wire (ref. 5501210; Group 1 (NI): 15 animals with orthodontic treatment, without
Morelli). The rear part of the spring was attached to the first LLL application in the periodontal tissues around the tooth.
molar using the same ligature wire passed around the first Group 2 (IR): 15 animals with orthodontic treatment with LLL
molar crown under the contact point between first and sec- application in the periodontal tissues around the tooth.
Group 3 (CO): 5 animals without orthodontic treatment and
ond molars. without LLL application in the periodontal tissues around the tooth.
Dental surfaces around ligature wires were cleaned with CO, controls; IR, irradiated; LLL, low-level laser; NI, nonirradiated.
cotton hubs, washed, air dried, conditioned with orthopho- A–C indicates sacrifice date after treatment.
 LASER PHOTOBIOMODULATION AND ORTHODONTIC MOVEMENT
Table 2. Low-Level Laser Treatment Parameters
Each Each session Total treatment
point (three points) (three sessions)
Dose, ( J/cm2) 177 531 1593
Time exposure, (sec) 50 150 450
Energy, ( J) 5 15 45
LLL treatment parameters: dose energy density or fluence ( J/
cm2), time exposure (sec), energy ( J), gallium aluminum arsenide
(GaAlAs), infrared wavelength 808 nm, irradiance or power density
1.2 W/cm2. LLL, low-level laser.
immediately, 24, and 48 h after orthodontic device place-
ment8 (Table 2).
Histology
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Animals were sacrificed with analgesic overdose. Upper
jaws were removed and fixed in 10% buffered formalin
(Sigma Chemical Co., St. Louis, MO) for 48 h, dehydrated
in increasing concentrations of ethanol (Sigma Chemical
Co.), and embedded in paraffin (Sigma Chemical Co.). Para-
sagittal sections (4–5 lm) of the upper jaw, including first
molars, were made (microtome Leica-RM2145) and col-
lected in silanized glass slides (3-aminopropil-trietoxi-silano;
Sigma Chemical Co.).
IL-1/IL-10 immunohistochemistry
Semiserial (1/3) histological slides were rinsed twice in
xylene (Sigma Chemical Co.): first at 60C–55C for 5 min
and second at room temperature for 20 min. Slides were
rehydrated with decreasing concentrations of ethanol (100%
to 70%), water washed, PBS pH 7.4, citric acid 10 mM pH 6,
H2O2 3%, casein PBS pH 7.4 (Synth, São Paulo, Brazil) for
5 min at room temperature.
Slide sections were incubated with IL-1/IL-10 antibody
for 24 h, (1:100; Santa Cruz Biotechnology, Inc., TX) in
albumin solution (1%), then incubated with ABC Kit Vec-
FIG. 2. IL-1 behavior during treatment. Y-
axis: percentage of IL-1 per analyzed area,
X-axis: time (days). IL, interleukin.
649
tastain (Vector LabCA), using diaminobenzidine (Sigma-
Aldrich Chemie, Steinheim, Germany).
Photographs were taken by Sony Cybershot DSC-W17,
7.2, with Nikon Eclipse-E800 microscope at 200 · magni-
fication, and analyzed at Image J software Media Cyber-
netics, Inc.6
COL-1 quantification protocol (picrosirius red staining)
Histological sections received two baths of xylene, first at
60C–55C for 5 min and the second at room temperature
for 20 min. Sections were rehydrated in decreasing con-
centrations of ethanol (100% to 70%), washed in tap water,
bathed for 30 min in a picrosirius red solution (1%; Sigma
Chemical Co.), and washed again in tap water. Sections
were dried and counter-stained with methylene blue solution
(3%; Sigma Chemical Co.).10
Photographs of marked areas were taken by Sony Cy-
bershot DSC-W17, 7.2, with Nikon Eclipse-E800 micro-
scope with 200 · image amplification, and analyzed at
Image J software Media Cybernetics, Inc.
Statistical analysis
Results were analyzed using GraphPad Prism 5.0 soft-
ware, IL-1 and IL-10 values were subjected to analysis of
variance (ANOVA) and Mann–Whitney test ( p < 0.05),
COL-1 values were subjected to ANOVA, Tukey test, and t-
test ( p < 0.05).
Results
Interleukin-1
Levels of IL-1 in the traction and in the compression sides
showed peak anticipation after LLL irradiation (IR, day 3)
compared with nonirradiated (NI) groups (day 5); moreover,
a reduction of *50% of the IL-1 level was also observed,
inferring a reduction of the inflammation response at the
peak. After peaks both groups (IR and NI) presented lower
levels of IL-1 compared with CO (Fig. 2).
 650
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Interleukin-10
Levels of IL-10 peaked at days 3 (LLL IR) and 5 (NI)
suggesting that the macrophage activity has started first but
with less intensity in the IR group compared with the NI
group. After peaks, both groups (IR and NI) also presented
lower levels of IL-10 compared with the CO group (Fig. 3).
COL-1
Levels of COL-1 in the compression side peaked at day 7
(NI), and at day 3 (LLL IR), returning to the untreated level
after day 5. In the traction side, levels of COL-1 peaked at
FIG. 4. COL-1 behavior during treatment. Y-axis: percentage of COL-1 per analyzed area, X-axis: time (days).
CORDEIRO ET AL.
FIG. 3. IL-10 behavior during treatment. Y-
axis: percentage of IL-10 per analyzed area, X-
axis: time (days).
day 7 (LLL IR) but did not peak in the NI group, suggesting
that the repair process has started earlier in the LLL IR
group (Fig. 4).
Discussion
It is possible to optimize microenvironment reactions
inside periodontal tissues subjected to orthodontic forces
using LLL.
LLL can increase orthodontic treatment speed.11 Inside
irradiated cells LLL fastens oxygen binding into the respi-
ratory chain of mitochondria and also affects intracellular
 LASER PHOTOBIOMODULATION AND ORTHODONTIC MOVEMENT
Ca++, activating mRNAs.9 This reaction can modulate in-
flammation and change cell cycle, inducing cell proliferation.8
Looking at this scenario, our work focused on investigating
microenvironment indicators of inflammation process4,5 inside
tissues subjected to orthodontic forces,1,3,7 IR and NI with LLL
compared with a CO.2
After receiving orthodontic forces, fibers of the peri-
odontal ligament of the dental root stretch at the side where
force is applied and compress at the opposite side.3
At the compressed fibers side, there is inflammation with
affluence of osteoclasts and macrophages that reabsorb bone
matrix and dead cells, cleaning the affected region and
preparing bone and periodontal ligament repair.3,12
At the stretched fibers side, the affluence of osteopro-
genitor cells that differentiate into osteoblasts increases
levels of Col I, a protein necessary to fix bone extracellular
matrix that indicates repair has started.12
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Interleukin-1
IL-1 is an interleukin produced by osteoclasts, macro-
phages, B lymphocytes, and NK cells.13 Its chemotactic
properties cause macrophage affluence to the affected tis-
sue,4 LLL can influence levels of IL-1 at damaged tissues,14
modulating inflammation.14–16 Our work found that levels
of IL-1 were lower at IR tissues. Surprisingly both com-
pressed and stretched fibers sides reacted similarly; more-
over, the peak of IL-1 was at day 5 at NI and at day 3 at IR
tissues, inferring an anticipation of inflammation peak.
Interleukin-10
IL-10 is an interleukin produced by B lymphocytes,
monocytes, and macrophages.5,17 Its chemotactic properties
cause macrophage inactivation reducing resorption of the
affected tissues. This is an important tool to control bone
remodeling.5,6 LLL can affect levels of IL-10 at damaged
tissues,15 controlling bone resorption.15,16 Our work found
that levels of IL-10 followed IL-1 results, they were lower at
IR tissues, and also here both compressed and stretched
fibers sides reacted similarly; moreover, the peak of IL-10
was at day 5 at NI and at day 3 at IR tissues, also inferring a
peak anticipation of the acute phase of inflammation.
COL-I
COL-I is the most abundant protein of the extracellular
matrix produced by cells of the human body. Inside bone
tissues it is produced by osteoblasts to form bone extracel-
lular matrix. Its presence in the analyzed tissue infers bone
repair is in course.12
LLL can also affect levels of COL-I at damaged tis-
sues.18,19 Our results showed that in the traction side, there
was a small peak at day 3, followed by a short reduction at
day 5 and a significant increase in the COL-I content at day
7. These findings infer that tissue repair is in course and
can be explained by collagen recycling dynamics. Initially
COL-1 is degraded through metalloproteinases, aiming a
new collagen protein synthesis. LLL therapy can modulate
metalloproteinases and the balance COL-I/COL-III, as
demonstrated in different models of tissue injury.20,21
651
Conclusions
These data can infer that this dose of LLL affected living
tissues subjected to orthodontic forces, modulating inflam-
mation and anticipating its peak that anticipates also the
beginning of the repair process.
These results can also infer that this dose of LLL can be
used as a movement acceleration protocol.
Acknowledgments
J.M.C. received funding as a doctorate scholarship
from the Brazilian Government ‘‘CAPES-coordenadoria de
aperfeiçoamento de pessoal de nı́vel superior’’ to conduct
this work.
Author Disclosure Statement
Professor E.C.P.L.J. receives research support from Multi
Radiance Medical (Solon, OH), a laser device manufacturer.
Multi Radiance Medical had no role in the planning of this
study, and the laser device used was not from Multi Ra-
diance Medical. Multi Radiance Medical had no influence
on study design, data collection and analysis, decision to
publish, or preparation of the article. The remaining authors
declare that they have no conflict of interests.
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