ORIGINAL ARTICLE

S Fujita Low-energy laser stimulates tooth

M Yamaguchi
movement velocity via expression of
T Utsunomiya
H Yamamoto RANK and RANKL
K Kasai

Authors' affiliations: Structured Abstract

Shoji Fujita, Masaru Yamaguchi, Kazutaka
Kasai, Department of Orthodontics, Nihon
University School of Dentistry at Matsudo,
Chiba, Japan
Tadahiko Utsunomiya, Hirotsugu
Yamamoto, Department of Oral Pathology,
Nihon University School of Dentistry at
Matsudo, Chiba, Japan
Correspondence to:
Shoji Fujita
Department of Orthodontics
Nihon University School of Dentistry at
Matsudo
2-870-1 Sakaecho-Nishi, Matsudo City
Chiba 271-8587
Japan
E-mail: fujita.shouji@nihon-u.ac.jp
Authors – Fujita S, Yamaguchi M, Utsunomiya T, Yamamoto H, Kasai K
Objective – Recent studies have demonstrated that low-energy laser irradiation
stimulates bone formation in vitro and in vivo. However, very little is known about the
effects of laser irradiation on osteoclastogenesis. The receptor activator of the
nuclear factor-kB (RANK) ⁄ RANK ligand (RANKL) ⁄ osteoprotegerin (OPG) system is
essential and sufficient for osteoclastogenesis. The present study was designed to
examine the effects of low-energy laser irradiation on expressions of RANK, RANKL,
and OPG during experimental tooth movement.
Design – To induce experimental tooth movement in rats, 10 g of orthodontic
force was applied to the molars. Next, a Ga–Al–As diode laser was used to irradiate
the area around the moved tooth and the amount of tooth movement was measured
for 7 days. Immunohistochemical staining with RANK, RANKL, and OPG was
performed. Real time PCR was also performed to elucidate the expression of
RANK in irradiated rat osteoclast precursor cells in vitro.
Results – In the irradiation group, the amount of tooth movement was significantly
greater than in the non-irradiation group by the end of the experimental period. Cells
that showed positive immunoreactions to the primary antibodies of RANKL and
RANK were significantly increased in the irradiation group on day 2 and 3, compared
with the non-irradiation group. In contrast, the expression of OPG was not
changed. Further, RANK expression in osteoclast precursor cells was detected at an
early stage (day 2 and 3) in the irradiation group.
Conclusion – These findings suggest that low-energy laser irradiation stimulates
the velocity of tooth movement via induction of RANK and RANKL.

Key words: low-energy laser irradiation; orthodontics; osteoclast-like cells; RANK;

RANKL; tooth movement

Dates:
Accepted 16 January 2008
To cite this article:
Fujita S, Yamaguchi M, Utsunomiya T,
Yamamoto H, Kasai K:
Low-energy laser stimulates tooth movement
velocity via expression of RANK and RANKL
Orthod Craniofac Res 2008;11:143–155
Copyright  2008 The Authors.
Journal compilation  2008 Blackwell Munksgaard
Introduction
Accelerated orthodontic tooth movement is desirable from the patientÕs
point of view because normal treatment duration is long. Previous
methods to stimulate bone remodeling, such as drug injections (1),
electric stimulation (2), and ultrasound application (3) have been
reported. Recently, various bio-stimulatory effects of low-energy laser
irradiation have been shown, including effects on wound healing
(4), fibroblastic (5) and chondral (6) proliferation, collagen synthesis (7),
and nerve regeneration (8). In particular, the acceleration of bone
Fujita et al. Laser stimulates tooth movement velocity
regeneration by laser treatment has been the focus of
recent studies (9). In the field of orthodontics, low-
energy laser irradiation is utilized for several types of
orthodontic interventions, such as reduction of post-
adjustment pain (10) or treatment of traumatic ulcers
in the oral mucosa promoted by an appliance (11).
However, scant information is available concerning the
effects of low-energy laser irradiation on bone remod-
eling during orthodontic tooth movement.
Tooth movement is related to the response of applied
orthodontic forces that cause remodeling of peri-
odontal tissues, especially alveolar bone. We previously
reported the stimulatory effects of low-energy laser
irradiation on bone regeneration in the median pala-
tine suture area during rapid maxillary expansion in
rats (12). Further, Ozawa et al. (13) demonstrated that
laser irradiation stimulates cellular proliferation and
differentiation of osteoblast lineage nodule-forming
cells, especially in committed precursors, resulting in
an increase in the number of differentiated osteoblastic
cells as well as in bone formation. In addition,
Kawasaki and Shimizu (14) found that low-energy laser
irradiation stimulated the amount of tooth movement
and formation of osteoclasts on the side with pressure
during experimental tooth movement in vivo. As bone
remodeling is a physiological process that involves the
resorption of bone by osteoclasts and synthesis of bone
matrix by osteoblasts (15), those findings are not sur-
prising. Also, a recent study showed that low-energy
laser irradiation accelerated the velocity of orthodontic
movement by human teeth (16).
Osteoclasts, specific members of monocyte ⁄ macro-
phage lineage are derived from hematopoietic precur-
sors (17). The expression of tartrate-resistant acid
phosphatase (TRAP) is a characteristic feature of mac-
rophage ⁄ osteoclast lineage and often used as a lineage
marker (18). The activity of osteoclasts in vitro is
measured by counting excavation pits in bone or
dentin slices, which is the primary feature that
identifies mature, active osteoclasts (19).
According to Soedarsono et al. (20), recent advances in
bone cell biology have allowed elucidation of the crucial
roles of the receptor activator of nuclear factor-kB
(RANK) ⁄ RANK ligand (RANKL) ⁄ osteoprotegerin (OPG)
system in osteoclast differentiation and function.
RANKL is a cytokine that belongs to the Tumor necrosis
factor (TNF) family and is essential for the induction of
osteoclastogenesis. Osteoblasts and bone marrow
144 Orthod Craniofac Res 2008;11:143–155
stromal cells produce this cytokine, and its signals are
transduced by the specific receptor RANK, which is
localized on the cell surface of osteoclast progenitors.
OPG is also a cytokine that belongs to the TNF family
and is produced by osteoblasts and bone marrow
stromal cells. OPG inhibits osteoclastogenesis by
involvement in the competitive binding of RANK with
RANKL. Thus, RANKL and OPG regulate bone resorp-
tion by exerting a positive or negative control on the
activation of RANK on osteoclasts (21).
Very little is known about the effects of laser irradi-
ation on osteoclastogenesis via the RANK ⁄ RANKL ⁄ OPG
system. The present study was designed to examine the
effects of low-energy laser irradiation on expressions of
RANK, RANKL, and OPG during experimental tooth
movement, and on RANK expression in rat osteoclast
precursor cells in vitro.
Material and methods
Animals
Seventy-five male Wistar strain rats of 6-week-old were
obtained (Sankyo Labo Service Co, Tokyo, Japan) and
used for the experiments (mean weight, 180 ± 10 g).
They were kept at the Animal Center of Nihon
University School of Dentistry at Matsudo, in separate
cages in a 12-h light ⁄ dark environment at a constant
temperature of 23C and provided with food and water
ad libitum. The health status of each rat was evaluated
by daily body weight monitoring starting 1 week before
the beginning of the experiments.
Experimental tooth movement
The rats were divided into three groups of 25 each, the
laser irradiation, light emitting diode (LED) irradiation,
and non-irradiation groups. All operations were carried
out under general anesthesia using an intraperitoneal
injection of mixed ketamine hydrochloride and xyla-
zine hydrochloride. Experimental tooth movement was
performed using the method of Kawasaki and Shimizu
(14), in which a closed-coil spring (wire size: 0.005¢¢;
diameter: 1 ⁄ 12¢¢; Accurate Sales Co., Chiba, Japan) was
ligated to the maxillary first molar cleat by a stainless
steel ligature wire (wire size: 0.008¢¢, Tomy Interna-
tional Inc., Tokyo, Japan). The other side of the coil
spring was also ligated with the holes in the maxillary

Fig. 1. Diagram of method used for experimental tooth movement.
The upper first molar was moved mesially by a closed coil spring at
10 g of orthodontic force.
incisors drilled laterally just above the gingival papilla
with a round bur (#1 ⁄ 4) using the same ligature wire.
The upper first molar was moved mesially by the closed
coil spring at force of 10 g (Figs 1, 2). The force was
determined based on a preliminary study that showed
that the upper first molar could be moved by ortho-
dontic force without a decrease in body weight or
hyalinized degenerative tissues (22). The experiments
were performed for a period of 8 days (days 0–7; Fig. 3).
Fig. 2. Laser irradiation points. The laser beam was delivered by a
0.6-mm diameter optical fiber and irradiation was administered by
placing the end of the optical fiber tip in contact with the mesial,
buccal, palatal sides of the gingiva, located in the area of the moved
upper right first molar. Arrow, laser irradiation point.
Fujita et al. Laser stimulates tooth movement velocity
Fig. 3. Experimental time-schedule for each group.
The animal experimental protocol was approved by the
Ethics Committee for Animal Experiments of our
university (approval No. ECA-05-0025).
Laser irradiation
For the in vivo study, we used a gallium–aluminum–
arsenide (Ga–Al–As) diode laser (Osada Inc., Tokyo,
Japan) with a wavelength of 810 nm and continuous
waves at 100 mW of output power. These irradiation
conditions were determined by the results from previ-
ous experiments (14). The laser beam was delivered by
a 0.6-mm diameter optical fiber and irradiation was
applied under anesthesia by placing the end of the
optical fiber tip in contact with the mesial, buccal, and
palatal sides of the gingival, located in the area of the
upper right first molar (Fig. 2). Irradiation was per-
formed for 3 min at each point (total, 9 min) once a day
from day 0–7 (total eight times) (Fig. 3). Total energy
corresponding to a 9-min exposure was 54.0 J ⁄ cm2,
which was similar to the dose used by Kawasaki and
Shimizu (14). LED irradiation and non-irradiation
groups were used as controls.
For the in vitro study, we used a Ga–Al–As diode laser
(Ora-laser 2100, ORALIA Dentalproukte GmbH,
Konstanz, Germany) with a continuous wavelength of
810 nm and a maximum power output of 50 mW. The
laser beam was delivered by an optical fiber 5 mm in
diameter that was de-focused at the tip using concave
lenses and utilized to irradiate a uniform circular area
7 mm in diameter at the cell layer level. The power
density of the laser beam was measured using a laser
power meter and irradiation was performed from
14 mm above the cell layer. In this manner, 16-well
Orthod Craniofac Res 2008;11:143–155 145
Fujita et al. Laser stimulates tooth movement velocity
Lab-Tek chamber slides (Nunc, Naperville, IL, USA)
were simultaneously and uniformly irradiated for a
period of 8 days. The period of exposure was 3 min
each day, which corresponded to 27.99 J ⁄ cm2 per
exposure. When not being laser-irradiated, the cells
were kept in a 5% CO2 incubator at 37C. As controls,
chamber slides were placed on a clean bench for the
corresponding time points without irradiation.
LED irradiation
Ga–Al–As diode laser produces coherent light, while
that produced by the LED is incoherent. We investi-
gated the stimulatory effects of irradiation on osteo-
clast differentiation between those two types of light.
For the in vivo study, we used an infrared LED (Rohm
Co. Ltd., Kyoto, Japan) with a wavelength of 850 nm
and 75 mW of output power, which were the same as
the laser irradiation conditions (Fig. 2). LED was irra-
diated similar to laser irradiation group. Irradiation was
performed for 4 min at each point (total, 12 min) once
a day from day 0 to 7 (total eight times) (Fig. 3). Total
energy corresponding to a 12-min exposure was
54.0 J ⁄ cm2, which was similar to the dose used in the
laser irradiation experiment.
Measurement of tooth movement
To determine the amount of tooth movement, plaster
models of the maxillae were made using silicone
impression material (Dent Silicone-V, Shofu Inc., Kyoto,
Japan) before (day 0) and after initiating tooth movement
(day 1, 2, 3, 4, 7). The plaster models were scanned using a
contact-type three-dimensional measurement appara-
tus (3D-picza, Roland Co., Hamamatsu, Japan) by setting
the plane to pass through three points, which were the
bilateral interpapillary crests between the first and sec-
ond molars and the interpapillary crest between the
second and third molars. Using three-dimensional
morphological analysis software (3D-RUGLE, Medic Engi-
neering Inc., Kyoto, Japan), we measured the distance
between the first molar central fossa and second molar
mesial surface to determine tooth movement (Fig. 4).
Tissue preparation
The experimental periods were set at day 1, 2, 3, 4,
and 7 (total 8 days) after start of tooth movement
146 Orthod Craniofac Res 2008;11:143–155
Fig. 4. Measurement of tooth movement. Plaster models were scan-
ned using a contact-type three-dimensional measurement apparatus
(3D-picza, Roland Co.), setting the plane to pass through three points,
the bilateral interpapillary crests between the first molar and second
molar, and interpapillary crest between the second molar and third
molar. Using three-dimensional morphological analysis software
(3D-RUGLE, Medic Engineering Inc.), we measured the distance
between the first molar central fossa and second molar mesial surface
to determine tooth movement.
(Fig. 3). Each group of rats was further divided into
day 0–1, day 0–2, day 0–3, day 0–4, and day 0–7 sub-
groups, with five rats in each. Each rat was deeply
anesthetized using mixed ketamine hydrochloride and
xylazine hydrochloride and perfused with 10% for-
malin in 0.1 M phosphate buffer in a transcardial
manner, after which the maxilla was immediately
dissected and immersed in the same fixative overnight
at 4C. The specimens were decalcified in a 10% di-
sodium ethylenediamine tetraacetic acid (EDTA, pH
7.4) solution for 4 weeks, and the decalcified speci-
mens were dehydrated through an ethanol series and
embedded in paraffin.
Each sample was sliced into 4-lm continuous
sections in the horizontal direction and prepared
for hematoxylin and eosin (HE) immunohistochemical
staining for TRAP, RANK, RANKL, and OPG. The
periodontal tissues in the pressure area were quarter
of the mesial area facing the mesial root (MR), as
determined when linked with the center of the MR
and the distal buccal root (DBR) of the first molar
(Fig. 5). To evaluate the effect of laser irradiation on
the formation of multinucleate osteoclasts, we coun-
ted the number of multinucleate osteoclasts in the
laser irradiation, LED irradiation, and non-irradiation
groups.

Fig. 5. Photograph of labeled alveolar bone taken under a fluorescent
microscope (bar: 500 lm). The numbers of multinucleate osteoclasts,
TRAP positive cells, RANK positive cells, RANKL positive cells, and
OPG positive cells were measured in the pressure side (PS) quarter of
the mesial area (dots) and opposite of the measuring point on the
tension side (TS). Arrow, tooth movement direction; MR, mesial root;
DBR, distal buccal root; PDL, peridontal ligament.
Immunohistochemistry for TRAP, RANK, RANKL, and OPG
Immunohistochemical staining was performed as
follows. First, the sections were deparaffinized and
endogenous peroxidase activity was quenched by
incubation in 3% H2O2 in methanol for 15 min at room
temperature. After washing in Tris-buffered-saline
(TBS), the sections were incubated with polyclonal
anti-rabbit TRAP (SANTA CRUZ; working dilution,
1:400, SANTA CRUZ, CA, USA), polyclonal anti-rabbit
RANK (SANTA CRUZ; working dilution, 1:100), poly-
clonal anti-goat RANKL (SANTA CRUZ; working dilu-
tion, 1:100), and polyclonal anti-rabbit OPG (SANTA
CRUZ; working dilution, 1:100) overnight at 4C. TRAP,
RANK, and OPG were stained using a Histofine simple
stain rat MAX-Po(R) kit (Nichirei, Tokyo, Japan),
according to the manufacturerÕs protocol. RANKL was
Fujita et al. Laser stimulates tooth movement velocity
stained using a Histofine SAB-PO (G) kit (Nichirei, Ja-
pan) according to the manufacturerÕs protocol. The
sections were rinsed with TBS and final color reactions
were performed using the substrate reagent 3, 3¢-di-
aminobenzidine tetra-hydrochloride and aminoethyl
carbazole, then they were counter-stained with Mayer-
hematoxylin. As immunohistochemical negative con-
trols, some sections were incubated in the same way,
then incubated with either non-immune rabbit IgG or
0.01 M phosphate-buffered saline alone, instead of the
primary antibody. To evaluate the effects of laser and
LED irradiation on the expressions of TRAP, RANK,
RANKL, and OPG, we counted the numbers of TRAP-,
RANK-, and RANKL-positive cells in the laser irradia-
tion, LED irradiation, and non-irradiation groups.
Cell cultures
Rat osteoclast precursor cells that had been purified from
rat bone marrow cells (23) were purchased from Hokudo
Co. (Hokkaido, Japan). The cells were seeded into 16-well
Lab-Tek chamber slides (Nunc) at a density of 2 · 106
cells ⁄ ml and maintained in a commercial a-minimal
essential medium supplemented with 10 unit ⁄ ml of
penicillin, 10 lg ⁄ ml of gentamicin, and 10% fetal calf
serum, with both macrophage colony-stimulating factor
(M-CSF) and RANKL (both at 10 ng ⁄ ml) for 8 days. The
medium was changed every 3 days.
Analysis of real-time RT-PCR
We extracted RNA from rat osteoclast precursor cells
treated with RANK using an RNeasy mini kit (Qiagen
Co., Tokyo, Japan) following the manufacturerÕs pro-
tocol. RNA was amplified with an RT-PCR kit and 40 ll
of purified total RNA was obtained. Total RNA was
converted to cDNA using a PrimeScript RT reagent Kit
(Takara Co., Shiga, Japan). Real time PCR amplification
was performed using SYBR Premix Ex Taq (Takara Co.)
in a thermal cycler (TP-800 Thermal Cycler Dice,
Takara). The samples were denatured at 95C for 5 s,
then the primer was annealed at 60C for 40 cycles at
30 s each. PCR primers were purchased from Takara
Co. (Hokkaido, Japan) and the sequences were de-
signed with reference to the cDNA sequences reported
for RANK as follows: 5¢-GGTTATGTAATGAGCGGCAG-
CA-3¢ and 5¢-TTCTCATCGGCACTGTAGATCTGG-3¢.
The levels of the real-time PCR products corresponding
Orthod Craniofac Res 2008;11:143–155 147
Fujita et al. Laser stimulates tooth movement velocity
to b-actin were the same in the four experimental
groups, thus it was considered that the amount of real-
time PCR products reflected the level of each mRNA.
Statistical analysis
Values presented represent the mean ± SD for each
group. Intergroup comparisons of average values for
tooth movement as well as numbers of osteoclasts,
TRAP-, RANK-, and RANKL-positive cells were carried
out by Mann–Whitney U-test. A value of p < 0.05 was
considered to indicate a significant difference. One-
way ANOVA was also performed and the results indicated
in the results at p < 0.01.
Results
Tooth movement during experimental period
The body weights of the rats in the laser irradiation, LED
irradiation, and non-irradiation groups decreased
transiently on day 1 and then recovered. No significant
differences were found among the three groups (data
not shown). The amount of tooth movement was
significantly greater in the irradiation group on day 3
(2.2-fold), 4 (2.0-fold) and 7 (1.5-fold), compared with
the non-irradiation, and LED irradiation groups (Fig. 6).
Histopathological findings (HE staining)
At 1 day after the start of tooth movement (the day 2),
the arrangement of fibers and fibroblasts became coarse
and irregular, and blood capillaries were pressured.
Resorption lacunae with a few multinucleate osteo-
clasts were observed on the surface of the alveolar bone
Fig. 6. Effect of laser irradiation on tooth movement. *Significantly
different from corresponding non-irradiation group and LED irra-
diation group (p < 0.05). Values are shown as the mean ± SD of 5 rats.
148 Orthod Craniofac Res 2008;11:143–155
surface of the side with pressure [Fig. 7A (a–c)]. At day 2,
3, and 4 after initiation of tooth movement, the peri-
odontal ligament (PDL) was composed of a coarse
arrangement of fibers and expanded blood capillaries.
Many resorption lacunae with multinucleate osteo-
clasts also appeared on the alveolar bone surface of
pressure side [Fig. 7A (d–l)]. The number of multinu-
cleate osteoclasts in the laser irradiation group
was greater than that in the non-irradiation and LED
irradiation group. At day 7 after initiation of tooth
movement, fibroblasts in the PDL were increased. Fur-
ther, on the surface of the alveolar bone, bone resorp-
tion lacunae with multinucleate osteoclasts were rec-
ognized [Fig. 7A (m–o)]. In our quantitative evaluation,
the number of multinucleate osteoclasts was found to
be significantly increased in the irradiation group on
days 2 (2.1-fold) and 3 (1.6-fold) compared with the
non-irradiation, and LED irradiation groups (Fig. 7B).
Immunohistochemical findings of TRAP
At 1 day after the start of tooth movement, resorption
lacunae with a few TRAP-positive multinucleate oste-
oclasts were observed on the surfaces of the alveolar
bone and root [Fig. 8A (a–c)]. At day 2, 3, and 4, after
the start of tooth movement, many resorption lacunae
with TRAP-positive multinucleate osteoclasts
appeared on the alveolar bone surface of the pressure
side [Fig. 8A (d–l)]. The number of TRAP-positive cell
in the laser irradiation group was greater than that in
the non-irradiation and LED irradiation group. Seven
days after the start of tooth movement, bone resorp-
tion lacunae with multinucleate TRAP-positive osteo-
clasts were recognized on the surface of the alveolar
bone of pressure side [Fig. 8A (m–o)]. In our quanti-
tative evaluation, the number of TRAP-positive cell
was found to be significantly increased in the laser
irradiation group on day 2 (2.0-fold) and 3 (2.0-fold)
compared with the non-irradiation, and LED irradia-
tion groups (Fig. 8B).
Protein expressions of RANK, RANKL, and OPG
At 1 day after start of tooth movement, RANK-positive
multinucleate osteoclasts appeared on the alveolar
bone surface, while mononuclear RANK-positive cells
were present in the fibers of the PDL [Fig. 9A (a–c)].
Further, RANKL-positive fibroblasts in the PDL and
 Fujita et al. Laser stimulates tooth movement velocity

(A) HE
1 day 2 days 3 days 4 days 7 days
a d g j m

Non-
irradiation

b e h k n

LED
irradiation

c f i l o

Laser
irradiation

(B)

Fig. 7. (A) Effects of laser irradiation on multinucleate osteoclasts shown in light microscope images (HE, original magnification 400·) (bar:
50 lm). Basophilic multinucleate osteoclasts were increased on the alveolar bone surface of pressure side in the laser irradiation group (f, i, l
and o) on days 2, 3, 4 and 7. Further, basophilic multinucleate osteoclasts were detected on days 3, 4 and 7 (g–h, j–k and m–n) in the non-
irradiation and LED irradiation groups. (B) The number of multinucleate osteoclasts in laser irradiation group was greater than that in the non-
irradiation and and LED irradiation groups on days 2, 3, 4 and 7. *indicates p < 0.05. Values are shown as the mean ± SD of 5 rats.

osteoblasts on the bone surface were observed. The
number of RANKL-positive cell in the laser irradiation
group was greater than that in the non-irradiation and
LED irradiation group [Fig. 10A (a–c)].
At day 2, 3, and 4 after tooth movement, many RANK-
positive multinucleate osteoclasts and mononuclear
RANK-positive cells were observed on the mesial side
of the alveolar bone of pressure side [Fig. 9A (d–l)]. In
the PDL fibroblasts, the immunoreactivity of RANKL
was increased compared with the control. Further, a
number of RANKL-positive fibroblasts was observed
and many RANKL-positive osteoclasts were located on
the distal side of the alveolar bone surface facing the
mesial side of each tooth root of the lower first molar
[Fig. 10A (d–l)]. The number of RANK-positive cells and
RANKL-positive cells in the laser irradiation group
was greater than that in the non-irradiation and LED
irradiation group.
Seven days after movement, RANK immunoreactivity
was observed in multinucleate osteoclasts and
mononuclear osteoclasts [Fig. 9A (m–o)]. RANKL
immunoreactivity was also observed in fibroblasts and
osteoclasts on the alveolar bone surface of pressure
side [Fig. 10A (m–o)]. In our quantitative evaluation,
the number of RANK-positive cells was found to be
significantly increased in the laser irradiation group on
day 2 (1.42-fold) and 3 (1.41-fold) compared with the
non-irradiation, and LED irradiation groups (Fig. 9B).
In addition, RANKL-positive cells were found to be
significantly increased in the laser irradiation group on
day 2 (1.42-fold) and 3 (1.38-fold) compared with the
non-irradiation, and LED irradiation groups (Fig. 10B).

Orthod Craniofac Res 2008;11:143–155 149

Fujita et al. Laser stimulates tooth movement velocity

(A) TRAP
1 day 2 days 3 days 4 days 7 days
a d g j m

Non-
irradiation

b e h k n

LED
irradiation

c f i l o

Laser
irradiation

(B)

Fig. 8. (A) Effects of laser irradiation on TRAP positive osteoclasts as shown by immunohistochemistry (original magnification 400·) (bar:
50 lm). TRAP immunoreactivity was observed in osteoclasts on the alveolar bone surface of pressure side in the laser irradiation group (f, i, l
and o) on days 2, 3, 4 and 7, as well as on days 3, 4 and 7 (g–h, j–k and m–n) in the non-irradiation and LED irradiation groups. (B) The number
of TRAP positive osteoclasts in the laser irradiation group was greater than that in the non-irradiation and LED irradiation groups on days 2, 3,
4 and 7. *indicates p < 0.05. Values are shown as the mean ± SD of 5 rats.

Also RANKL-positive multinucleated osteoclasts in the
serial sections concomitantly expressed RANK.
As OPG is one of the crucial factors for regulation of
bone remodeling together with RANKL and RANK, we
examined the expression pattern of OPG in periodontal
tissues. OPG-positive signals were detected in nearly all
of the osteoblasts in the non-irradiation, LED irradia-
tion, and laser irradiation groups. In contrast,
OPG-positive osteoclasts were not observed in any of
the three groups (Fig. 11).
by laser irradiation, RANK mRNA expression in both
irradiated and non-irradiated cells was investigated
using real-time PCR. As the immunohistochemical
staining for RANK was increased on day 2 and 3 of
irradiation for 3 min ⁄ day, gene expression was inves-
tigated under the same conditions. When rat osteoclast
precursor cells were subjected to laser irradiation,
the expression of RANK was increased on day 2 and 3
(one-way ANOVA, p < 0.01; Fig. 12).

Effects of laser irradiation on RANK mRNA as shown Discussion

by real-time PCR
Luger et al. (24) reported that the scattering through
To elucidate the molecular mechanisms associated the skin reduces the energy level of laser beams
with alteration of RANK immunohistochemical staining between 3% and 6% of its original intensity. Infrared

150 Orthod Craniofac Res 2008;11:143–155

 Fujita et al. Laser stimulates tooth movement velocity

(A) RANK
1 day 2 days 3 days 4 days 7 days
a d g j m

Non-
irradiation

b e h k n

LED
irradiation

c f i l o

Laser
irradiation

(B)

Fig. 9. (A) Effects of laser irradiation on RANK positive cells as shown by immunohistochemistry (original magnification 400·) (bar: 50 lm).
RANK immunoreactivity was observed in osteoclasts on the alveolar bone surface of pressure side in the laser irradiation group (f, i, l and o) on
days 2, 3, 4 and 7, as well as on days 3, 4 and 7 (g–h, j–k and m–n) in the non-irradiation and LED irradiation groups. (B) The number of RANK
positive cells in the laser irradiation group was greater than that in the non-irradiation and LED irradiation groups on days 2, 3, 4 and 7.
*indicates p < 0.05. Values are shown as the mean ± SD of 5 rats.

radiation has a low absorption coefficient in hemo-
globin and water, and consequently, a high penetra-
tion depth in the irradiated tissue. It is well known
that infrared radiation 750 nm can penetrate more
than visible radiation at 650 nm into soft tissues (25).
As the objective of our study was to stimulate bone
cells which are placed deeply under the soft tissue
(e.g., gingival) in the PDL space, the infrared laser
(810 nm) was selected for our study. Further, previous
studies reported that laser irradiation stimulated bone
remodeling during orthodontic tooth movement
(830 nm; 14) and in mid-palatal suture exposed to
rapid palatal expansion (830 nm; 12). Therefore, we
assume that the energy of laser irradiation was deliv-
ered to the PDL through the mucosa ⁄ gingival and
alveolar bone in this study.
In the present study, the amount of tooth movement
was significantly greater in the laser irradiation group
on day 3 (2.2-fold), 4 (2.0-fold), and 7 (1.5-fold) com-
pared with the non-irradiation, and LED irradiation
groups (Fig. 6), while the number of multinucleate
osteoclasts in the laser irradiation group was also
greater (Figs 9 and 10). Recent studies have demon-
strated that low-energy irradiation accelerated ortho-
dontic tooth movement in rats (14) and humans (16).
Kawasaki and Shimizu (14) also showed that ortho-
dontic movement of laser irradiated rat teeth was 30%
faster than that of those in non-irradiated rats because
of increases in bone formation on the side where there
is pressure and number of osteoclasts on the com-
pression side as a result of cellular stimulation
promoted by low-energy laser irradiation. In our labo-

Orthod Craniofac Res 2008;11:143–155 151

Fujita et al. Laser stimulates tooth movement velocity

(A) RANKL
1 day 2 days 3 days 4 days 7 days
a d g j m

Non-
irradiation

b e h k n

LED
irradiation

c f i l o

Laser
irradiation

(B)

Fig. 10. (A) Effects of laser irradiation on RANKL positive cells as shown by immunohistochemistry (original magnification 400·) (bar: 50 lm).
RANKL immunoreactivity was observed in osteoclasts on the alveolar bone surface of pressure side in the laser irradiation group (f, i, l and o)
on days 2, 3, 4 and 7, while both RANKL and RANK were detected on days 3, 4 and 7 (g–h, j–k and m–n) in the non-irradiation and LED
irradiation groups. (B) The number of RANKL and RANK positive cells in the laser irradiation group was greater than that in the non-irradiation
and LED irradiation groups on days 2, 3, 4 and 7. *indicates p < 0.05. Values are shown as the mean ± SD of 5 rats.

ratory, Aihara et al. (26) reported that low-energy laser
irradiation facilitated differentiation and activation of
osteoclasts in vitro.
Limpanichkul et al. (27) reported that Ga–Al–As
low-level laser therapy (LLLT) was too low to express
either stimulatory effect or inhibitory effect on the
rate of orthodontic tooth movement. On the contrary,
Youssef et al. (25) and Cruz et al. (16) demonstrated
that LLLT stimulated the velocity of tooth movement.
With regard to the energy and wavelength of Ga–Al–As
low-level laser apparatus, the total energy (75 J ⁄ cm2)
of laser used by Limpanichkul et al. (27) was higher
than that of Youssef et al. (8 J ⁄ cm2) (25), Cruz et al.
(50 J ⁄ cm2) (16) and this study (54 J ⁄ cm2). Furthermore,
the wavelength of Limpanichkul et al. (860 nm) (27)
was also longer than that of Youssef et al. (809 nm) (25),
Cruz et al. (780 nm) (16) and this study (810 nm).
Therefore, it may be the optimal energy and wave-
length of laser for stimulating the rate of orthodontic
tooth movement.
As shown in Figs 9 and 10, RANK- and RANKL-
positive cells were significantly increased in the laser
irradiation group on day 2 (RANK: 1.42-fold, RANKL:
1.38-fold) and 3 (RANK: 1.42-fold, RANKL: 1.41-fold)
compared with the non-irradiation, and LED irradiation
groups (Figs 9 and 10). However, OPG-positive cells
were not different among the three groups (Fig. 11).
Further, the real-time PCR results showed that RANK
expression in osteoclast precursor cells was detected at
an early stage (day 2 and 3) in the laser-irradiated group
(Fig. 12). Ogasawara et al. (28) reported that no OPG-
positive osteoclasts were observed in cases of experi-

152 Orthod Craniofac Res 2008;11:143–155


OPG
1 day 2 days
a d
Non-
irradiation
b e
LED
irradiation
c f
Laser
irradiation
Fig. 11. Effects of laser irradiation on OPG positive osteoclasts as shown by immunohistochemistry (original magnification 400·) (bar: 50 lm).
OPG immunoreactivity was observed in osteoclasts on the alveolar bone surface of pressure side in the three groups. No significant differences
were observed among the three groups.
Fig. 12. RANK expression on rat osteoclast precursor cells. When rat
osteoclast precursor cells were subjected to laser irradiation, RANK
was increased in a dose-dependent manner (p < 0.01, by one-way
ANOVA).
mental tooth movement. Further, Kanzaki et al. (29)
found that OPG mRNA expression in human PDL
(hPDL) cells did not change, regardless of the amount of
compressive force or duration of compression in vitro.
On the contrary, Nishijima et al. (30) reported that
RANKL levels in gingival crevicular fluid during canine
retraction were significantly higher and the levels of
OPG significantly lower in the experimental canines
compared with control teeth at 24 h. Further, an
in vitro study indicated that compression force signif-
icantly increased the secretion of RANKL and
decreased that of OPG in hPDL cells in a time- and
force magnitude-dependent manner. Additional stud-
Fujita et al. Laser stimulates tooth movement velocity
3 days 4 days 7 days
g j m
h k n
i l o
ies are necessary to clarify these contrasting results. It is
considered that the acceleration of orthodontic tooth
movement by low-energy laser irradiation may be
affected by RANKL ⁄ RANK expression, though OPG was
also shown to have an effect in the present study.
Infrared LED irradiation did not stimulate the
velocity of tooth movement (Fig. 6) or osteoclastogen-
esis (Figs 7–11). Elke et al. (31) also reported that
infrared LED irradiation did not stimulate the prolif-
eration of fibroblasts obtained from chicken embryos.
Therefore, coherent light (Ga–Al–As diode laser), but
not incoherent light (infrared LED), may have an effect
on the proliferation and differentiation of fibroblast
and osteoclasts.
Macrophage colony-stimulating factor stimulates not
only the proliferation of osteoclast progenitors, but also
their differentiation into mature osteoclasts in vivo (32)
and in vitro (33). Yang et al. (34) reported that the
presence of the M-CSF receptor on pre-formed osteo-
clasts was carried over from developing cells and
played a functional role in the modulation of bone
resorption by mature cells. c-fms, the cellular homo-
logue of the feline transforming virus formerly McDo-
nough feline sarcoma viral (v-FMS) is the sole receptor
for M-CSF (35). The functional linkage between M-CSF
and its receptor has been established by the finding
Orthod Craniofac Res 2008;11:143–155 153
Fujita et al. Laser stimulates tooth movement velocity
that mice lacking csf1r, the gene coding for c-fms, ex-
hibit the same major phenotype as op ⁄ op mice,
namely, a marked decrease in tissue macrophages and
severe osteopetrosis because of a lack of osteoclasts
(36). Therefore, M-CSF and c-fms may also stimulate
the differentiation of osteoclasts induced by low-energy
laser irradiation.
Shimizu et al. (37) reported that low-power laser
irradiation significantly inhibited the production of
prostaglandin E (PGE2) and that interleukin (IL-1b) was
increased by mechanical stress in vitro. If low-energy
laser irradiation functions to inhibit these cytokines,
tooth movement may be slow. In regard to these con-
tradictory results, Katagiri and Takahashi (38) reported
that IL-1, RANKL, and M-CSF were stimulated via their
respective pathways during the differentiation of
dentary bone cells. Further, following the experiments
of tooth movement in rats, Kawasaki and Shimizu (14)
reported that the amount of tooth movement was
increased by low-energy laser irradiation. Moreover,
Aihara et al. (26) reported their in vitro results showing
that the gene expression of RANK in osteoclast pre-
cursor cells increased when the cells were irradiated
with a low-energy laser. In the present experiments, the
gene expression of RANK was increased in vitro, and
the expressions of RANK and RANKL proteins were
increased in vivo. Accordingly, we considered that the
activity of the RANK ⁄ RANKL system increased to
facilitate the differentiation of osteoclasts and acceler-
ate bone resorption, as the light stimulus from the low-
energy laser acted directly on osteoclasts, despite the
finding that PGE2 and IL-1b were decreased. Further
studies are necessary to confirm this apparent
phenomenon.
In examinations of the side with pressure during
orthodontic tooth movement, as well as the effects of
laser irradiation on proliferation and differentiation in
osteoblasts (39), Ozawa et al. (13) reported that laser
irradiation given during the early stages of formation of
osteoblast-like cells isolated from fetal rat calvariae
significantly stimulated cellular proliferation, alkaline
phosphatase (ALP) activity, and osteocalcin gene
expression. Further, laser irradiation in the earlier
stages of cell cultures significantly stimulated the pro-
liferation of osteoblasts, resulting in a greater number
(1.7-fold) and larger area (3.4-fold) of bone nodules that
developed in the culture dishes by day 21. These results
suggest that laser irradiation stimulates proliferation
154 Orthod Craniofac Res 2008;11:143–155
and differentiation, resulting in an increase in the
number of differentiated osteoblastic cells as well as in
bone formation. Also, Barushka et al. (40) reported that
low-energy laser (He–Ne) irradiation after injury af-
fected the populations of osteoblasts and osteoclasts in
the injured site. On the basis of the findings of the
present study, it is possible that low-energy laser irra-
diation accelerates the process of bone remodeling by
stimulating osteoblast and osteoclast differentiation
during orthodontic tooth movement.
In conclusion, low-energy laser irradiation stimu-
lated the velocity of tooth movement via RANK ⁄ RANKL
expression. Further, RANK expression in osteoclast
precursor cells was detected at an early stage (day 2
and 3) in the laser-irradiated group. Together, these
findings suggest that low-energy laser irradiation
accelerates bone remodeling, thus shortening the
orthodontic treatment period.
Acknowledgments: This research was supported in part by
a Grant-in-Aid for Scientific Research from the Japan Society
for the Promotion of Science (C: 18592252, C: 19592367),
and a Nihon University Individual Research Grant for 2005
(No. 05-119) to M. Yamaguchi and 2006 to K. Kasai
(No. 06-090).
References
1. Kobayashi Y, Takagi H, Sakai H, Hashimoto F, Mataki S,
Kobayashi K, et al. Effects of local administration of osteocal-
cin on experimental tooth movement. Angle Orthod
1998;68:259–66.
2. Spadaro JA. Mechanical and electrical interactions in bone
remodeling. Bioelectromagnetics 1997;18:193–202.
3. Hadjiargyrou M, McLeod K, Ryaby JP, Rubin C. Enhancement
of fracture healing by low intensity ultrasound. Clin Orthop
1998;355:216–29.
4. Mester E, Mester AF, Mester A. The biomedical effects of laser
application. Lasers Surg Med 1985;5:31–9.
5. Oudry M, Franquin JC, Pourreau-Schreider N, Martin PM.
Effect of a helium-neon laser on cellular growth: an in vitro
study of human gingival fibroblasts. J Biol Buccale 1988;16:129–
35.
6. Schultz RJ, Krishnamurthy S, Thelmo W, Rodriguez JE, Harvey G.
Effects of varying intensities of laser energy on articular cartilage:
a preliminary study. Lasers Surg Med 1985;5:577–88.
7. Balboni GC, Brandi ML, Zonefrati R, Repice F. Effects of
He–Ne ⁄ IR laser irradiation on two lines of normal human fibro-
blasts in vitro. Arch Ital Anat Embriol 1986;91:179–88.
8. Anders JJ, Borke RC, Woolery SK, Van de Merwe WP. Low power
laser irradiation alters the rate of regeneration of the rat facial
nerve. Lasers Surg Med 1993;13:72–82.
9. Trelles MA, Mayayo E. Bone fracture consolidates faster with
low-power laser. Lasers Surg Med 1987;7:36–45.

10. Lim HM, Lew KK, Tay DK. A clinical investigation of the efficacy
of low level laser therapy in reducing orthodontic postadjustment
pain. Am J Orthod Dentofacial Orthop 1995;108:614–22.
11. Rodrigues MTJ, Ribeiro MS, Groth EB, Zezell DM Evaluation of
effects of laser therapy (k = 830 nm) on oral ulceration induced
by fixed orthodontic appliances. Lasers Surg Med 2002;30 (Suppl.
14):15.
12. Saito S, Shimizu N. Stimulatory effects of low-power laser
irradiation on bone regeneration in midpalatal suture during
expansion in the rat. Am J Orthod Dentofacial Orthop
1997;111:525–32.
13. Ozawa Y, Shimizu N, Kariya G, Abiko Y. Low-energy laser
irradiation stimulates bone nodule formation at early stages of
cell culture in rat calvarial cells. Bone 1998;22:347–54.
14. Kawasaki K, Shimizu N. Effects of low-energy laser irradiation on
bone remodeling during experimental tooth movement in rats.
Lasers Surg Med 2000;26:282–91.
15. Lemaire V, Tobin FL, Greller LD, Cho CR, Suva LJ. Modeling the
interactions between osteoblast and osteoclast activities in bone
remodeling. J Theor Biol 2004;229:293–309.
16. Cruz DR, Kohara EK, Ribeiro MS, Wetter NU. Effects of low-
intensity laser therapy on the orthodontic movement velocity
of human teeth: a preliminary study. Lasers Surg Med
2004;35:117–20.
17. Roodman GD. Advances in bone biology: the osteoclast. Endocr
Rev 1996;17:308–32.
18. Roodman GD, Ibbotson KJ, MacDonald BR, Kuehl TJ, Mundy GR.
1,25-Dihydroxyvitamin D3 causes formation of multinucleated
cells with several osteoclast characteristics in cultures of primate
marrow. Proc Natl Acad Sci USA 1985;82:8213–7.
19. Chambers TJ, Revell PA, Fuller K, Athanasou NA. Resorption
of bone by isolated rabbit osteoclasts. J Cell Sci 1984;66:383–99.
20. Soedarsono N, Rabello D, Kamei H, Fuma D, Ishihara Y,
Suzuki M, et al. Evaluation of RANK ⁄ RANKL ⁄ OPG gene
polymorphisms in aggressive periodontitis. J Periodontal Res
2006;41:397–404.
21. Blair JM, Zheng Y, Dunstan CR. RANK ligand. Int J Biochem Cell
Biol 2007;39:1077–81.
22. Kirino Y, Tsuchiya T, Kurihara S, Chiba M, Miura F. A study of
tooth movement with super-elastic force. J Jpn Orthod Soc
1991;50:315–24.
23. Sccheven BA, Visser JW, Nijweide PJ. In vitro osteoclast genera-
tion from different bone marrow fractions, including a highly
enriched haematopoietic stem cell population. Nature
1986;321:79–81.
24. Luger JE, Rochkind S, Wollman Y, Kogan G, Dekel S. Effect of low-
power laser irradiation on the mechanical properties of bone
fracture healing in rats. Lasers Surg Med 1998;22:97–102.
25. Youssef M, Ashkar S, Hamade E, Gutknecht N, Lampert F,
Mir M. The effect of low-level laser therapy during orthodon-
tic movement: a preliminary study. Lasers Med Sci 2008;23:27–
33.
Fujita et al. Laser stimulates tooth movement velocity
26. Aihara N, Yamaguchi M, Kasai K. Low-energy irradiation stimu-
lates formation of osteoclast-like cells via RANK expression in
vitro. Lasers Med Sci 2006;21:24–33.
27. Limpanichkul W, Godfrey K, Srisuk N, Rattanayatikul C. Effects of
low-level laser therapy on the rate of orthodontic tooth move-
ment. Orthod Craniofacial Res 2006;9:38–43.
28. Ogasawara T, Yoshimine Y, Kiyoshima T, Kobayashi I, Matsuo K,
Akamine A, Sakai H. In situ expression of RANKL, RANK, osteo-
protegerin and cytokines in osteoclasts of rat periodontal tissue.
J Periodontal Res 2004;39:42–9.
29. Kanzaki H, Chiba M, Shimizu Y, Mitani H. Periodontal ligament
cells under mechanical stress induce osteoclastogenesis by
receptor activator of nuclear factor jB ligand up-regulation via
prostaglandin E2 synthesis. J Bone Miner Res 2002;17:210–20.
30. Nishijima Y, Yamaguchi M, Kojima T, Aihara N, Nakajima R, Kasai
K. Levels of RANKL and crevicular fluid during orthodontic tooth
movement and effect of compression force on releases from
periodontal ligament cells in vitro. Orthod Craniofacial Res
2006;9:63–70.
31. Elke MV, Barbara JC, Maria JC, Heidi AD, Dirk CC. Increased
fibroblast proliferation induced by light emitting diode and low
power laser irradiation. Lasers Med Sci 2003;18:95–9.
32. Felix R, Hofstetter W, Wetterwald A, Cecchini MG, Fleisch H.
Role of colony-stimulating factor-1 in bone metabolism. J Cell
Biochem 1994;55:340–9.
33. Tanaka S, Takahashi N, Udagawa N, Tamura T, Akatsu T, Stanley
ER, et al. Macrophage colony-stimulating factor is indispensable
for both proliferation and differentiation of osteoclast progeni-
tors. J Clin Invest 1993;91:257–63.
34. Yang S, Zhang Y, Rodriguiz RM, Ries WL, Key LL Jr. Functions of
the M-CSF receptor on osteoclasts. Bone 1996;18:355–60.
35. Stanley ER, Berg KL, Einstein DB, Lee PS, Pixley FJ, Wang Y, et al.
Biology and action of colony – stimulating factor-1. Mol Reprod
Dev 1997;46:4–10.
36. Dai XM, Ryan GR, Hapel AJ, Dominguez MG, Russell RG, Kapp S,
et al. Targeted disruption of the mouse colony-stimulating factor
1 receptor gene results in osteopetrosis, mononuclear phagocyte
deficiency, increased primitive progenitor cell frequencies, and
reproductive defects. Blood 2002;99:111–20.
37. Shimizu N, Yamaguchi M, Goseki T, Shibata Y, Takiguchi H,
Iwasawa T, et al. Inhibition of prostaglandin E2 and inter-
leukin 1-beta production by low-power laser irradiation in
stretched human periodontal ligament cells. J Dent Res
1995;74:1382–8.
38. Katagiri T, Takahashi N. Regulatory mechanisms of osteoblast
and osteoclast differentiation. Oral Dis 2002;8:147–59.
39. Martinasso G, Mozzati M, Pol R, Canuto RA, Muzio G. Effect of
superpulsed laser irradiation on bone formation in a human
osteoblast-like cell line. Minerva Stomatol 2007;56:27–30.
40. Barushka O, Yaakobi T, Oron U. Effect of low-energy laser
(He–Ne) irradiation on the process of bone repair in the rat tibia.
Bone 1995;16:47–55.
Orthod Craniofac Res 2008;11:143–155 155