low dose propranolol decreases orthodontic movement
TRANSCRIPT
Low dose propranolol decreases orthodonticmovement
Erika Lira de Oliveira a, Fabiana Furtado Freitas b, Cristina Gomes de Macedo b,Juliana Trindade Clemente-Napimoga b, Milena Bortolotto Felippe Silva c,Luiz Roberto Coutinho Manha es-Jr c, Jose Luiz Cintra Junqueira c,Marcelo Henrique Napimoga a,*a Laboratory of Immunology and Molecular Biology, Sao Leopoldo Mandic Dental School and Research Center,
Campinas/SP, Brazilb Laboratory of Orofacial Pain, Department of Physiology, Piracicaba Dental School, State University of Campinas,
Piracicaba/SP, Brazilc Laboratory of Oral Radiology, Sao Leopoldo Mandic Dental School and Research Center, Campinas/SP, Brazil
a r c h i v e s o f o r a l b i o l o g y 5 9 ( 2 0 1 4 ) 1 0 9 4 – 1 1 0 0
a r t i c l e i n f o
Article history:
Accepted 18 June 2014
Keywords:
Propranolol
Tooth movement
Orthodontic
Bone
a b s t r a c t
Objective: Low dose propranolol has previously been demonstrated to suppress bone remo-
delling. Therefore, its effect on orthodontic movement was tested.
Design: Rats were assigned as follows (n = 5): animals with no orthodontic appliance (G1);
the remaining groups were fitted with a Ni-Ti closed-coil spring ligated to the upper left first
molar and connected to the incisors using metal and resin and received vehicle only (G2),
0.1 mg/kg (G3) or 20 mg/kg (G4) of propranolol orally. Cone Beam Computed Tomography
was performed using high resolution for image capture. The distance between the first and
second upper molars, both with and without the orthodontic appliance, was measured in
millimetres. Gingival tissue was harvested and assessed for IL-1b and IL-6 using ELISA and
for ICAM-1 and RANKL by Western blotting.
Results: The orthodontic appliance induced a significant tooth movement in G2 when
compared to the animals without an orthodontic appliance (G1) ( p < 0.05). The animals
from G3 showed a significantly reduction in tooth movement ( p < 0.05) when compared
with rats from G2. Animals treated with 20 mg/kg of propranolol (G4) showed tooth
movement similar to that of G2. The reduced tooth movement observed in the animals
treated with 0.1 mg/kg of propranolol (G3) occurred due to decreased amounts of IL-1b and
IL-6, in addition to lower ICAM-1 and RANKL expression.
Conclusions: Low dose propranolol inhibits bone remodelling and orthodontic movement.
# 2014 Elsevier Ltd. All rights reserved.
* Corresponding author at: Laboratory of Immunology and Molecular Biology, Sao Leopoldo Mandic Dental School and Research Center R.Jose Rocha Junqueira 13, Campinas, Sao Paulo 13045-755, Brazil. Tel.: +55 19 3211 3627; fax: +55 19 3211 3636.
E-mail addresses: [email protected], [email protected] (M.H. Napimoga).
Available online at www.sciencedirect.com
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journal homepage: http://www.elsevier.com/locate/aob
http://dx.doi.org/10.1016/j.archoralbio.2014.06.0060003–9969/# 2014 Elsevier Ltd. All rights reserved.
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Fig. 1 – Photo of the orthodontic appliance placement used
in the current study.
1. Introduction
During orthodontic movement, force is applied to the teeth,
and areas of pressure and tension are formed in the
periodontal ligament (PDL), the connective tissue that con-
nects the tooth to its surrounding alveolar bone.1 Mechanical
loading also alters periodontal tissue vascularity and blood
flow, which results in the synthesis and release of various
molecules, locally, such as neurotransmitters, cytokines,
growth factors, and arachidonic acid metabolites. These
molecules evoke a cellular response in the different cell types
surrounding the teeth, providing a favourable microenviron-
ment for bone deposition and resorption.2
Molecules present in drugs that are regularly consumed
by patients reach the mechanically stressed paradental
tissue via the circulation and interact with local target cells.
The combined effect of the mechanical forces and one or
more of these agents can be inhibitory, additive, or
synergistic3. The regulation of bone metabolism by the
sympathetic nervous system has been demonstrated in
studies showing that osteoblasts and osteoclasts express b2-
adrenoceptors.4,5 It has been previously demonstrated that
low doses of the b-blocker propranolol suppress alveolar
bone resorption by inhibiting RANKL-mediated osteoclasto-
genesis and inflammatory markers, with no affect on
haemodynamic parameters.6
b-Blockers have been classified as a first-line drug in the
treatment of hypertension and are widely used in cardio-
vascular disease. Globally, the prevalence of obesity has
been steadily increasing over recent decades. Hypertension
is commonly present in the overweight and obese popula-
tions. The US leads the developed world in terms of obesity
rates, with a prevalence of 34% and 17% in adults and
children aged 2–19 years, respectively.7 Projections based on
the current obesity trends predict that by 2030 there will be
another 65 million obese adults in the US,8 which will
consequently increase hypertension rates. Propranolol is
used in hypertension, angina, and migraine, amongst other
conditions. Considering that the number of hypertensive
patients is increasing, including in young people who are
the main target of orthodontic treatment, it is hypothesized
that a common b-blocker (propranolol) may influence
bone remodelling during orthodontic movement. The
objective of the present study was to investigate the effects
of low and high doses of propranolol on tooth movement
in rats.
2. Materials and methods
2.1. Animals
Three-month old male Wistar rats (200–250 g) were used. The
animals were kept in appropriate cages in a temperature-
controlled room under a 12-h dark/light cycle. Free access to
water and food was provided and they were acclimatized over
a period of approximately 7 days in the laboratory prior to the
experiment. All animals were manipulated in accordance with
the Guiding Principles in The Care and Use of Animals,
approved by the Council of the American Physiologic Society.
The Animal Ethics Committee of Sao Leopoldo Mandic Dental
School approved this study (# 2012/0287).
2.2. Appliance placement and measurement of toothmovement
The appliance placement was adapted from Gameiro et al.9
The animals were anaesthetized using xylazine (10 mg/kg)
and maintained with ketamine (50 mg/kg). A closed coil
nickel-titanium (NiTi) spring (Morelli1, Campinas, Brazil),
calibrated to provide a force of 0.49 N, measured by a
tensiometer (Morelli1, Campinas, Brazil) was ligated to the
upper left first molar and connected to incisors by
orthodontic stainless steel wire (.00800) (Morelli1, Campinas,
Brazil) and light-cured resin (Transbond XT, 3M1, Campinas,
Brazil) as illustrated in Fig. 1. NiTi springs were used to
provide a relatively constant force over the course of the
experiment. To limit the influence of inter-animal variation
a split-mouth design was used and the contralateral
untreated side served as the intra-animal control. After 10
days of tooth movement the rats were decapitated, and the
maxillae excised.
After the orthodontic appliance was positioned, the
animals were randomly assigned to one of the following
groups: 1) sham-appliance animals receiving administration
of saline (vehicle control) (n = 5); 2) animals with orthodontic
appliance receiving administration of vehicle (n = 5); 3)
animals with orthodontic appliance receiving administra-
tion of oral propranolol (0.1 mg/kg/day) (n = 5); 4) animals
with orthodontic appliance receiving administration of
oral propranolol (20 mg/kg/day) (n = 5). Saline (vehicle)
or propranolol was orally administered daily to each
animal.
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2.3. Measurement of tooth movement
After scout acquisition and protocol selection, Cone Beam
Computed Tomography (CBCT) equipment was calibrated
according to the manufacturer’s instructions to avoid humid-
ity and temperature variations, therefore maintaining image
quality. Classic I-Cat (Imaging Science International, Hatfield,
USA), model 914040000-0000R, was used to acquire images of
the maxillae of the rats. Voxel with a 0.25 mm � 6.00 cm field
of view and an exposure time of 40 s was selected for all
images captured. The X-ray settings were established using
equipment at 120 kV and 5–7 mA in accordance with the
resolution. The maxillae of the rats were positioned with the
occlusal plane directed upwards and held in place by wax. All
images were processed using XoranCatTM software (Xoran
Technologies Inc, Ann Arbor, USA). All anatomical plane slice
corrections and measurements were performed. In addition,
the Angio-Sharpen-Medium 5 � 500 filter was applied to all
images, and the contrast and brightness were adjusted in
order to give a more detailed image.
In the Multiplanar Reconstruction (MPR) images, the plane
correction arrows were used to construct the occlusal plane of
the maxillae of the rats in tangent to the coronal, sagittal and
axial planes. After correction, MPR plane cross-sections were
performed using the oblique tool (Fig. 2A), whilst the ruler tool
was used for measurements. The linear distance was
measured in the antero-posterior direction on the treated
side only and performed between the distal surface of the first
molar to the mesial surface of the second molar (Fig. 2B).
2.4. Protein extraction from gingival tissue
The marginal gingival tissue around the maxillary first molars
was surgically harvested (approximately 100 mg), rinsed with
cold sterile saline solution (0.9%) and triturated and homoge-
nized in 300 ml of the appropriate protease inhibitor-contain-
ing buffer (RIPA Lysis and Extraction Buffer, Thermo Scientific,
Fig. 2 – CBCT image used for measuring orthodontic movement.
and the second molars.
Rockford, IL, USA) and then centrifuged for 10 min at
10,000 � g. The total extracted protein was colorimetrically
measured using the micro BCA protein assay kit (Thermo
Scientific). The supernatant was stored at �70 8C until further
analysis. In addition, the maxillae were removed and fixed in
4% neutral formalin for 48 h for Cone Beam Computed
Tomography image acquisition, in order to determine the
amount of tooth movement.
2.5. Enzyme-linked immunosorbent assay (ELISA)
The levels of IL-1b and IL-6 were determined via capture
enzyme-linked immunosorbent assays (ELISA) using protocols
supplied by the manufacturer (R&D Systems Minneapolis,
USA). Fifty ml of gingival homogenate samples were applied in
duplicate, and the plates incubated for 2 h at room tempera-
ture. All experiments included serial dilutions (800, 400, 200,
100, 50, 25, 12.5 and 6.25 pg/ml) of a standard sample of mouse
IL-1b or IL-6 protein. The secondary antibody was biotin-
conjugated at a dilution of 1:1000. After incubation with a
solution of avidin–peroxidase for 30 min at room temperature,
a further series of washes was performed and 100 ml of
3,30,5,50-Tetramethylbenzidine substrate (TMB) was added and
incubated for 15 min. Absorbance values (A450 nm) were
obtained using an ELISA plate reader (Microplate Reader/
Model 3550, Bio Rad). Negative controls did not include
gingival homogenate. Absorbance values were plotted against
the standard curve obtained for the serial dilutions of the
purified mouse standard within a linear range to determine IL-
1b and IL-6 concentrations. ELISA was carried out in a blind
fashion.
2.6. Western blotting
Equal amounts of protein (20 mg) from the gingival tissue of the
animals were separated using 10% sodium dodecyl sulfate–
polyacrylamide gel electrophoresis and transferred to a
(A) Cross-section image; (B) linear distance between the first
Fig. 3 – Orthodontic tooth movement. The tooth movement
following orthodontic pressure was measured using the
XoranCatTM software. The distance is presented in
millimetres. Data are representative of 3 independent
experiments in duplicate (n = 5 per group). Different letters
indicate statistical significance (One Way ANOVA followed
by Bonferroni test).
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nitrocellulose membrane (Bio-Rad Laboratories). A molecular
weight standard (Bio-Rad Laboratories) was run in parallel to
estimate molecular weight. Membranes were blocked over-
night at 4 8C in Tris-buffered saline-Tween (20 mM Tris–HCl,
pH 7.5, 500 mM NaCl, 0.1% Tween 20; TBST) containing 5%
dried milk. The membranes were then incubated at 4 8C
overnight with anti-RANKL (Receptor activator of nuclear
factor kappa-B ligand; 1:1000), anti-ICAM-1 (Intercellular
Adhesion Molecule-1; 1:2000) or a-tubulin (1:1000) (Santa Cruz
Biotechnology, Santa Cruz, CA, USA), and diluted in TBST
containing 5% dried milk. Membranes were then incubated at
room temperature for 60 min with a secondary antibody
conjugated with peroxidase (1:5000), also diluted in TBS-T
containing 5% dried milk. The bands recognized by the specific
antibody were visualized using a chemiluminescence-based
ECL system (Amersham Biosciences, Piscataway, NJ) and
exposed to an X-ray film for 30 min (Eastman Kodak,
Rochester, NY). A computer-based imaging system (Image J)
was used to measure the intensity of optical density of the
bands.
2.7. Statistical analyses
Statistical analysis was performed using the GraphPad Prism
4.0 software (La Jolla, CA, USA). Data were reported as
means � SD, with five animals per group. The means from
different treatments were compared using ANOVA. When a
significant difference was identified, individual comparisons
were subsequently performed using Bonferroni’s t-test for
unpaired values. Statistical significance was set at p < 0.05.
3. Results
3.1. Low dose propranolol decreases orthodonticmovement
All animals gained weight during the study, however, the
mean body weight was not significantly different between the
Fig. 4 – Effects of propranolol in different doses on cytokines ex
gingival tissue around the teeth orthodontically moved. Results
group). Different letters indicate statistical significance (One Wa
groups at the end of the experimental period (data not shown).
The orthodontic appliance significantly increased tooth
movement (4.1-fold) compared to the control group (Fig. 3;
p < 0.05). Oral administration of low dose propranolol (0.1 mg/
kg) markedly reduced orthodontic movement in 41% (Fig. 3;
p < 0.05) when compared to orthodontic movement in animals
receiving the vehicle. On the other hand, high dose proprano-
lol (Fig. 3; 20 mg/kg) did not significantly reduce orthodontic
movement (11%).
3.2. Cytokine measurements from gingival tissue
IL-1b and IL-6 are critical cytokines in bone biology, therefore,
the levels of both cytokines present in the gingival tissue
pression. Cytokine quantification was performed using the
are expressed as means (pg/mg of tissue) WSD (n = 5 per
y ANOVA followed by Bonferroni test).
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subjected to the orthodontic appliance following 10 days of
propranolol treatment was evaluated. Expression of both IL-1b
and IL-6 was significantly increased in the group using the
orthodontic appliance, when compared to the sham-appliance
group. Interestingly, only the animals treated with 0.1 mg/kg
of propranolol demonstrated a significant decrease in expres-
sion of both cytokines ( p < 0.05) (Fig. 4A and B, respectively).
3.3. RANKL and ICAM-1 expression
The expression of two important molecules was analyzed using
Western blotting: RANKL, a key molecule in the activation of
osteoclasts; and ICAM-1, an endothelial- and leucocyte-associ-
ated transmembrane protein. As shown in Fig. 5, RANKL was
upregulated ( p < 0.05) in the gingival tissue of the molar teeth
subjected to orthodontic forces, when compared to the control
group. In contrast, the animals that received 0.1 mg/kg showed
a significantly decreased expression ( p < 0.05), whilst 20 mg/kg
of propranolol showed no difference in expression of this
molecule. ICAM-1 expression was also raised in the gingival
tissue of the molars only subjected to orthodontic forces
( p < 0.05), whilst the animals treated with 0.1 mg/kg of
propranolol in addition to orthodontic forces had statistically
Fig. 5 – Effects of propranolol on RANKL expression during
orthodontic tooth movement. Protein expression was
analyzed via Western blotting. The intensity of the bands
in terms of optical density was measured and normalized
against a-tubulin expression. Protein band intensity is
represented as arbitrary units. The results are expressed
as mean W SD of five animals per group. Different letters
indicate statistical significance (One Way ANOVA followed
by Bonferroni test).
decreased levels of ICAM-1 expression ( p < 0.05). On the other
hand, animals subjected to orthodontic forces during treatment
with 20 mg/kg of propranolol showed the same ICAM-1
expression as demonstrated in the aforementioned group, for
which only an orthodontic appliance was used (Fig. 6).
4. Discussion
Studies have indicated that b2-adrenergic receptors mediate
signalling in osteoblasts, which inhibits bone formation and
increases osteoclastogenesis via receptor activation of nuclear
factor kappa-B ligand (RANKL) expression.10,11 Kondo et al.12
reported that bone loss induced by mechanical unloading is
regulated by the sympathetic nervous system. The present
study corroborate their results by demonstrating that block-
ade of sympathetic signalling with low dose propranolol
inhibits orthodontic movement by reducing important mole-
cules responsible for bone remodelling.
It has been demonstrated that sympathetic signalling via
osteoclast activation controls the mechano-adaptive response
induced by experimental tooth movement.13 In the present
study, the use of low dose propranolol, a b-adrenergic
antagonist, decreased orthodontic movement. This may be
explained by the fact that osteoblasts and osteoclasts are well
equipped with adrenergic and peptidergic receptors, indicating
Fig. 6 – Effects of propranolol on ICAM-1 expression during
orthodontic tooth movement. Protein expression was
analyzed via Western blotting. The intensity of the bands
in terms of optical density was measured and normalized
against a-tubulin expression. Protein band intensity is
represented as arbitrary units. The results are expressed
as mean W SD of five animals per group. Different letters
indicate statistical significance (One Way ANOVA followed
by Bonferroni test).
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that they are influenced by sympathetic neurotransmitters.14
Additionally, osteoclast number, surface, and activity are
increased after sympathectomy, whereas osteoblastic activity
is decreased.15–18 Rodrigues et al. demonstrated that at low
doses, propranolol can inhibit bone resorption in a periodonti-
tis-induced rat model via inhibition of osteoclast differentiation
and resorptive activity due to suppression of the nuclear factor
of activated T cells (NFATc)1 pathway and the expression of
tartrate-resistant acid phosphatase (TRAP), cathepsin K and
MMP-9.6 It is important to highlight that the effect observed
using low dose propranolol did not evoke a haemodynamic
effect in the animals.
The present study demonstrated that only low dose
propranolol was able to decrease orthodontic tooth move-
ment. This data is in accordance with the literature, which
demonstrated that b1- and b2-adrenergic signalling exerts
opposing effects on bone, with b1 being predominantly an
anabolic stimulus in response to mechanical stimulation and
during growth, and b2 mainly regulating bone resorption.
Interestingly, mice lacking the adrenoreceptor b-2 (Adrb2R)
present a high bone mass phenotype, whilst Adrb 1 and 2R
deficient mice have reduced trabecular and cortical bone
mass. This suggests that high dose propranolol may some-
what imitate the double deletion phenotype,19 whilst low dose
propranolol may act mainly via the Adrb 2R, therefore exerting
beneficial effects.6 Hence, in the present study, low dose
propanolol may have blocked signalling by Adrb 2R and
consequently inhibited orthodontic movement, whereas high
doses may have blocked both Adbr 1 and 2 and therefore
prevented this effect.
Mechanical forces during orthodontic treatment can cause
an increased production of different cytokines by the
periodontal ligament cells, including IL-1b and IL-6. Interleu-
kin-1b is known to be responsible for neutrophil recruitment, a
complex process involving a sequence of molecular-mechani-
cal events on leukocytes and endothelial cells that depend on
distinct cell-cell adhesion molecules, such as ICAM-1, and
increased cytokines/chemokines production.20 Additionally,
IL-1b attracts macrophages and supports their differentiation
into osteoclasts, which carry out bone resorption. They also
inhibit the activity of osteoblasts, thus preventing bone
formation.21 This guarantees that necrotic tissue is resorbed
due to the initial application of force, and that tooth
movement occurs with bone remodelling. Therefore, the
inhibitory effect of low dose propranolol on IL-1b production
and ICAM-1 expression, explained in part the diminished
orthodontic movement observed in the present study.
Interleukin-6 regulates immune responses at sites of
inflammation, as well as an autocrine/paracrine activity that
stimulates osteoclast formation and bone-resorbing activity.22
It plays an important role in local regulation of bone remodelling
and is produced at the beginning of orthodontic tooth
movement (until 12 days after orthodontic activation), and its
expression decreases over time. A physiological homeostasis is
probably reached through downregulation via a feedback
mechanism.23 Furthermore, it is currently known that IL-6
can upregulate RANKL, indirectly supporting osteoclast forma-
tion via the interaction with mesenchymal cells. The cytokine
RANKL is essential for osteoclast development in bone and is
abundantly found at the pressure site during orthodontic
movement.24 Here, we have demonstrated that low dose of the
betablocker propranolol decreases the amount of both mole-
cules, IL-6 and RANKL, which further contributes towards the
understanding of how this drug inhibits orthodontic move-
ment. Likewise, previous data have shown that fenoterol (b2-
agonist) stimulated RANKL mRNA expression by nearly twofold
and this was suppressed by propranolol (b-blocker), suggesting
that b-adrenoceptors may play a role in modulating bone
turnover via the sympathetic nervous system.25
Based on the aforementioned results, it is possible to
conclude that low dose propranolol, a b-adrenergic antago-
nist, decreases orthodontic movement.
Funding
There is no governmental or private funding for this research.
Competing interest
There is no conflict of interest to declare.
Ethical approval
This study was approved by the Animal Ethics Committee of
the Faculty Sao Leopoldo Mandic (# 2012/0287).
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