- Email: [email protected]
Low dose propranolol decreases orthodontic movement
archives of oral biology 59 (2014) 1094–1100
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.elsevier.com/locate/aob
Low dose propranolol decreases orthodontic movement 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, Sa˜o Leopoldo Mandic Dental School and Research Center, Campinas/SP, Brazil b Laboratory of Orofacial Pain, Department of Physiology, Piracicaba Dental School, State University of Campinas, Piracicaba/SP, Brazil c Laboratory of Oral Radiology, Sa˜o Leopoldo Mandic Dental School and Research Center, Campinas/SP, Brazil
article info
abstract
Article history:
Objective: Low dose propranolol has previously been demonstrated to suppress bone remo-
Accepted 18 June 2014
delling. Therefore, its effect on orthodontic movement was tested. Design: Rats were assigned as follows (n = 5): animals with no orthodontic appliance (G1);
Keywords:
the remaining groups were fitted with a Ni-Ti closed-coil spring ligated to the upper left first
Propranolol
molar and connected to the incisors using metal and resin and received vehicle only (G2),
Tooth movement
0.1 mg/kg (G3) or 20 mg/kg (G4) of propranolol orally. Cone Beam Computed Tomography
Orthodontic
was performed using high resolution for image capture. The distance between the first and
Bone
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, Sa˜o Leopoldo Mandic Dental School and Research Center R. Jose´ Rocha Junqueira 13, Campinas, Sa˜o Paulo 13045-755, Brazil. Tel.: +55 19 3211 3627; fax: +55 19 3211 3636. E-mail addresses: [email protected], [email protected] (M.H. Napimoga). http://dx.doi.org/10.1016/j.archoralbio.2014.06.006 0003–9969/# 2014 Elsevier Ltd. All rights reserved.
archives of oral biology 59 (2014) 1094–1100
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 connects 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 microenvironment 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 b2adrenoceptors.4,5 It has been previously demonstrated that low doses of the b-blocker propranolol suppress alveolar bone resorption by inhibiting RANKL-mediated osteoclastogenesis 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 cardiovascular disease. Globally, the prevalence of obesity has been steadily increasing over recent decades. Hypertension is commonly present in the overweight and obese populations. 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 temperaturecontrolled 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,
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approved by the Council of the American Physiologic Society. The Animal Ethics Committee of Sa˜o Leopoldo Mandic Dental School approved this study (# 2012/0287).
2.2. Appliance placement and measurement of tooth movement 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 administration 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.
Fig. 1 – Photo of the orthodontic appliance placement used in the current study.
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2.3.
archives of oral biology 59 (2014) 1094–1100
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 humidity 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 homogenized in 300 ml of the appropriate protease inhibitor-containing buffer (RIPA Lysis and Extraction Buffer, Thermo Scientific,
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 temperature. 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 biotinconjugated 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 IL1b 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
Fig. 2 – CBCT image used for measuring orthodontic movement. (A) Cross-section image; (B) linear distance between the first and the second molars.
archives of oral biology 59 (2014) 1094–1100
nitrocellulose membrane (Bio-Rad Laboratories). A molecular weight standard (Bio-Rad Laboratories) was run in parallel to estimate molecular weight. Membranes were blocked overnight 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
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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).
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 propranolol (Fig. 3; 20 mg/kg) did not significantly reduce orthodontic movement (11%).
3.1. Low dose propranolol decreases orthodontic movement
3.2.
All animals gained weight during the study, however, the mean body weight was not significantly different between the
IL-1b and IL-6 are critical cytokines in bone biology, therefore, the levels of both cytokines present in the gingival tissue
Cytokine measurements from gingival tissue
Fig. 4 – Effects of propranolol in different doses on cytokines expression. Cytokine quantification was performed using the gingival tissue around the teeth orthodontically moved. Results are expressed as means (pg/mg of tissue) WSD (n = 5 per group). Different letters indicate statistical significance (One Way ANOVA followed by Bonferroni test).
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archives of oral biology 59 (2014) 1094–1100
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 expression of both cytokines ( p < 0.05) (Fig. 4A and B, respectively).
3.3.
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
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-associated 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).
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 blockade of sympathetic signalling with low dose propranolol inhibits orthodontic movement by reducing important molecules 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).
archives of oral biology 59 (2014) 1094–1100
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 periodontitis-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 movement. 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 somewhat 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. Interleukin-1b is known to be responsible for neutrophil recruitment, a complex process involving a sequence of molecular-mechanical 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 formation 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
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movement.24 Here, we have demonstrated that low dose of the betablocker propranolol decreases the amount of both molecules, IL-6 and RANKL, which further contributes towards the understanding of how this drug inhibits orthodontic movement. Likewise, previous data have shown that fenoterol (b2agonist) 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 antagonist, 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 Sa˜o Leopoldo Mandic (# 2012/0287).
references
1. Alves JB, Ferreira CL, Martins AF, Silva GA, Alves GD, Paulino TP, et al. Local delivery of EGF-liposome mediated bone modeling in orthodontic tooth movement by increasing RANKL expression. Life Sci 2009;85(19-20):693–9. 2. Krishnan V, Davidovitch Z. Cellular, molecular, and tissuelevel reactions to orthodontic force. Am J Orthod Dentofacial Orthop 2006;129(4):469.e1–e. 3. Diravidamani K, Sivalingam SK, Agarwal V. Drugs influencing orthodontic tooth movement: an overall review. J Pharm Bioallied Sci 2012;4(Suppl. 2):S299–303. 4. Takeda S, Elefteriou F, Levasseur R, Liu X, Zhao L, Parker KL, et al. Leptin regulates bone formation via the sympathetic nervous system. Cell 2002;111(3):305–17. 5. Bonnet N, Benhamou CL, Malaval L, Goncalves C, Vico L, Eder V, et al. Low dose beta-blocker prevents ovariectomyinduced bone loss in rats without affecting heart functions. J Cell Physiol 2008;217(3):819–27. 6. Rodrigues WF, Madeira MF, da Silva TA, ClementeNapimoga JT, Miguel CB, Dias-da-Silva VJ, et al. Low dose of propranolol down-modulates bone resorption by inhibiting inflammation and osteoclast differentiation. Br J Pharmacol 2012;165(7):2140–51. 7. Flegal KM, Carroll MD, Ogden CL, Curtin LR. Prevalence and trends in obesity among US adults, 1999–2008. JAMA 2010;303(3):235–41. 8. Wang YC, McPherson K, Marsh T, Gortmaker SL, Brown M. Health and economic burden of the projected obesity trends in the USA and the UK. Lancet 2011;378(9793):815–25. 9. Gameiro GH, Nouer DF, Pereira-Neto JS, Urtado MB, Novaes PD, de Castro M, et al. The effects of systemic stress on orthodontic tooth movement. Aust Orthod J 2008;24(2):121–8.
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10. Takeuchi T, Tsuboi T, Arai M, Togari A. Adrenergic stimulation of osteoclastogenesis mediated by expression of osteoclast differentiation factor in MC3T3-E1 osteoblast-like cells. Biochem Pharmacol 2001;61(5):579–86. 11. Elefteriou F, Ahn JD, Takeda S, Starbuck M, Yang X, Liu X, et al. Leptin regulation of bone resorption by the sympathetic nervous system and CART. Nature 2005;434(7032):514–20. 12. Kondo H, Nifuji A, Takeda S, Ezura Y, Rittling SR, Denhardt DT, et al. Unloading induces osteoblastic cell suppression and osteoclastic cell activation to lead to bone loss via sympathetic nervous system. J Biol Chem 2005;280(34):30192–200. 13. Kondo M, Kondo H, Miyazawa K, Goto S, Togari A. Experimental tooth movement-induced osteoclast activation is regulated by sympathetic signaling. Bone 2013;52(1):39–47. 14. Togari A. Adrenergic regulation of bone metabolism: possible involvement of sympathetic innervation of osteoblastic and osteoclastic cells. Microsc Res Tech 2002;58(2):77–84. 15. Sandhu HS, Kwong-Hing A, Herskovits MS, Singh IJ. The early effects of surgical sympathectomy on bone resorption in the rat incisor socket. Arch Oral Biol 1990;35(12):1003–7. 16. Hill EL, Turner R, Elde R. Effects of neonatal sympathectomy and capsaicin treatment on bone remodeling in rats. Neuroscience 1991;44(3):747–55. 17. Sherman BE, Chole RA. In vivo effects of surgical sympathectomy on intramembranous bone resorption. Am J Otol 1996;17(2):343–6.
18. Sherman BE, Chole RA. Effect of pharmacological sympathectomy on osteoclastic activity in the gerbilline auditory bulla in vivo. Ann Otol Rhinol Laryngol 1999;108(11 Pt 1):1078–87. 19. Pierroz DD, Bonnet N, Bianchi EN, Bouxsein ML, Baldock PA, Rizzoli R, Ferrari SL. Deletion of b-adrenergic receptor 1, 2, or both leads to different bone phenotypes and response to mechanical stimulation. J Bone Miner Res 2012;27(June (6)):1252–62. 20. Hogg JC, Walker BA. Polymorphonuclear leucocyte traffic in lung inflammation. Thorax 1995;50(8):819–20. 21. Nakamura K, Sahara N, Deguchi T. Temporal changes in the distribution and number of macrophage-lineage cells in the periodontal membrane of the rat molar in response to experimental tooth movement. Arch Oral Biol 2001;46(7):593– 607. 22. Uematsu S, Mogi M, Deguchi T. Interleukin (IL)-1 beta, IL-6, tumor necrosis factor-alpha, epidermal growth factor, and beta 2-microglobulin levels are elevated in gingival crevicular fluid during human orthodontic tooth movement. J Dent Res 1996;75(1):562–7. 23. Madureira DF, Taddei Sde A, Abreu MH, Pretti H, Lages EM, da Silva TA. Kinetics of interleukin-6 and chemokine ligands 2 and 3 expression of periodontal tissues during orthodontic tooth movement. Am J Orthod Dentofacial Orthop 2012;142(4):494–500. 24. Yamaguchi M. RANK/RANKL/OPG during orthodontic tooth movement. Orthod Craniofac Res 2009;12(2):113–9. 25. Huang HH, Brennan TC, Muir MM, Mason RS. Functional alpha1- and beta2-adrenergic receptors in human osteoblasts. J Cell Physiol 2009;220(1):267–75.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.elsevier.com/locate/aob
Low dose propranolol decreases orthodontic movement 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, Sa˜o Leopoldo Mandic Dental School and Research Center, Campinas/SP, Brazil b Laboratory of Orofacial Pain, Department of Physiology, Piracicaba Dental School, State University of Campinas, Piracicaba/SP, Brazil c Laboratory of Oral Radiology, Sa˜o Leopoldo Mandic Dental School and Research Center, Campinas/SP, Brazil
article info
abstract
Article history:
Objective: Low dose propranolol has previously been demonstrated to suppress bone remo-
Accepted 18 June 2014
delling. Therefore, its effect on orthodontic movement was tested. Design: Rats were assigned as follows (n = 5): animals with no orthodontic appliance (G1);
Keywords:
the remaining groups were fitted with a Ni-Ti closed-coil spring ligated to the upper left first
Propranolol
molar and connected to the incisors using metal and resin and received vehicle only (G2),
Tooth movement
0.1 mg/kg (G3) or 20 mg/kg (G4) of propranolol orally. Cone Beam Computed Tomography
Orthodontic
was performed using high resolution for image capture. The distance between the first and
Bone
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, Sa˜o Leopoldo Mandic Dental School and Research Center R. Jose´ Rocha Junqueira 13, Campinas, Sa˜o Paulo 13045-755, Brazil. Tel.: +55 19 3211 3627; fax: +55 19 3211 3636. E-mail addresses: [email protected], [email protected] (M.H. Napimoga). http://dx.doi.org/10.1016/j.archoralbio.2014.06.006 0003–9969/# 2014 Elsevier Ltd. All rights reserved.
archives of oral biology 59 (2014) 1094–1100
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 connects 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 microenvironment 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 b2adrenoceptors.4,5 It has been previously demonstrated that low doses of the b-blocker propranolol suppress alveolar bone resorption by inhibiting RANKL-mediated osteoclastogenesis 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 cardiovascular disease. Globally, the prevalence of obesity has been steadily increasing over recent decades. Hypertension is commonly present in the overweight and obese populations. 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 temperaturecontrolled 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,
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approved by the Council of the American Physiologic Society. The Animal Ethics Committee of Sa˜o Leopoldo Mandic Dental School approved this study (# 2012/0287).
2.2. Appliance placement and measurement of tooth movement 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 administration 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.
Fig. 1 – Photo of the orthodontic appliance placement used in the current study.
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2.3.
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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 humidity 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 homogenized in 300 ml of the appropriate protease inhibitor-containing buffer (RIPA Lysis and Extraction Buffer, Thermo Scientific,
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 temperature. 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 biotinconjugated 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 IL1b 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
Fig. 2 – CBCT image used for measuring orthodontic movement. (A) Cross-section image; (B) linear distance between the first and the second molars.
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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 overnight 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
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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).
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 propranolol (Fig. 3; 20 mg/kg) did not significantly reduce orthodontic movement (11%).
3.1. Low dose propranolol decreases orthodontic movement
3.2.
All animals gained weight during the study, however, the mean body weight was not significantly different between the
IL-1b and IL-6 are critical cytokines in bone biology, therefore, the levels of both cytokines present in the gingival tissue
Cytokine measurements from gingival tissue
Fig. 4 – Effects of propranolol in different doses on cytokines expression. Cytokine quantification was performed using the gingival tissue around the teeth orthodontically moved. Results are expressed as means (pg/mg of tissue) WSD (n = 5 per group). Different letters indicate statistical significance (One Way 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 expression of both cytokines ( p < 0.05) (Fig. 4A and B, respectively).
3.3.
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
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-associated 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).
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 blockade of sympathetic signalling with low dose propranolol inhibits orthodontic movement by reducing important molecules 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).
archives of oral biology 59 (2014) 1094–1100
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 periodontitis-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 movement. 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 somewhat 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. Interleukin-1b is known to be responsible for neutrophil recruitment, a complex process involving a sequence of molecular-mechanical 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 formation 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
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movement.24 Here, we have demonstrated that low dose of the betablocker propranolol decreases the amount of both molecules, IL-6 and RANKL, which further contributes towards the understanding of how this drug inhibits orthodontic movement. Likewise, previous data have shown that fenoterol (b2agonist) 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 antagonist, 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 Sa˜o Leopoldo Mandic (# 2012/0287).
references
1. Alves JB, Ferreira CL, Martins AF, Silva GA, Alves GD, Paulino TP, et al. Local delivery of EGF-liposome mediated bone modeling in orthodontic tooth movement by increasing RANKL expression. Life Sci 2009;85(19-20):693–9. 2. Krishnan V, Davidovitch Z. Cellular, molecular, and tissuelevel reactions to orthodontic force. Am J Orthod Dentofacial Orthop 2006;129(4):469.e1–e. 3. Diravidamani K, Sivalingam SK, Agarwal V. Drugs influencing orthodontic tooth movement: an overall review. J Pharm Bioallied Sci 2012;4(Suppl. 2):S299–303. 4. Takeda S, Elefteriou F, Levasseur R, Liu X, Zhao L, Parker KL, et al. Leptin regulates bone formation via the sympathetic nervous system. Cell 2002;111(3):305–17. 5. Bonnet N, Benhamou CL, Malaval L, Goncalves C, Vico L, Eder V, et al. Low dose beta-blocker prevents ovariectomyinduced bone loss in rats without affecting heart functions. J Cell Physiol 2008;217(3):819–27. 6. Rodrigues WF, Madeira MF, da Silva TA, ClementeNapimoga JT, Miguel CB, Dias-da-Silva VJ, et al. Low dose of propranolol down-modulates bone resorption by inhibiting inflammation and osteoclast differentiation. Br J Pharmacol 2012;165(7):2140–51. 7. Flegal KM, Carroll MD, Ogden CL, Curtin LR. Prevalence and trends in obesity among US adults, 1999–2008. JAMA 2010;303(3):235–41. 8. Wang YC, McPherson K, Marsh T, Gortmaker SL, Brown M. Health and economic burden of the projected obesity trends in the USA and the UK. Lancet 2011;378(9793):815–25. 9. Gameiro GH, Nouer DF, Pereira-Neto JS, Urtado MB, Novaes PD, de Castro M, et al. The effects of systemic stress on orthodontic tooth movement. Aust Orthod J 2008;24(2):121–8.
1100
archives of oral biology 59 (2014) 1094–1100
10. Takeuchi T, Tsuboi T, Arai M, Togari A. Adrenergic stimulation of osteoclastogenesis mediated by expression of osteoclast differentiation factor in MC3T3-E1 osteoblast-like cells. Biochem Pharmacol 2001;61(5):579–86. 11. Elefteriou F, Ahn JD, Takeda S, Starbuck M, Yang X, Liu X, et al. Leptin regulation of bone resorption by the sympathetic nervous system and CART. Nature 2005;434(7032):514–20. 12. Kondo H, Nifuji A, Takeda S, Ezura Y, Rittling SR, Denhardt DT, et al. Unloading induces osteoblastic cell suppression and osteoclastic cell activation to lead to bone loss via sympathetic nervous system. J Biol Chem 2005;280(34):30192–200. 13. Kondo M, Kondo H, Miyazawa K, Goto S, Togari A. Experimental tooth movement-induced osteoclast activation is regulated by sympathetic signaling. Bone 2013;52(1):39–47. 14. Togari A. Adrenergic regulation of bone metabolism: possible involvement of sympathetic innervation of osteoblastic and osteoclastic cells. Microsc Res Tech 2002;58(2):77–84. 15. Sandhu HS, Kwong-Hing A, Herskovits MS, Singh IJ. The early effects of surgical sympathectomy on bone resorption in the rat incisor socket. Arch Oral Biol 1990;35(12):1003–7. 16. Hill EL, Turner R, Elde R. Effects of neonatal sympathectomy and capsaicin treatment on bone remodeling in rats. Neuroscience 1991;44(3):747–55. 17. Sherman BE, Chole RA. In vivo effects of surgical sympathectomy on intramembranous bone resorption. Am J Otol 1996;17(2):343–6.
18. Sherman BE, Chole RA. Effect of pharmacological sympathectomy on osteoclastic activity in the gerbilline auditory bulla in vivo. Ann Otol Rhinol Laryngol 1999;108(11 Pt 1):1078–87. 19. Pierroz DD, Bonnet N, Bianchi EN, Bouxsein ML, Baldock PA, Rizzoli R, Ferrari SL. Deletion of b-adrenergic receptor 1, 2, or both leads to different bone phenotypes and response to mechanical stimulation. J Bone Miner Res 2012;27(June (6)):1252–62. 20. Hogg JC, Walker BA. Polymorphonuclear leucocyte traffic in lung inflammation. Thorax 1995;50(8):819–20. 21. Nakamura K, Sahara N, Deguchi T. Temporal changes in the distribution and number of macrophage-lineage cells in the periodontal membrane of the rat molar in response to experimental tooth movement. Arch Oral Biol 2001;46(7):593– 607. 22. Uematsu S, Mogi M, Deguchi T. Interleukin (IL)-1 beta, IL-6, tumor necrosis factor-alpha, epidermal growth factor, and beta 2-microglobulin levels are elevated in gingival crevicular fluid during human orthodontic tooth movement. J Dent Res 1996;75(1):562–7. 23. Madureira DF, Taddei Sde A, Abreu MH, Pretti H, Lages EM, da Silva TA. Kinetics of interleukin-6 and chemokine ligands 2 and 3 expression of periodontal tissues during orthodontic tooth movement. Am J Orthod Dentofacial Orthop 2012;142(4):494–500. 24. Yamaguchi M. RANK/RANKL/OPG during orthodontic tooth movement. Orthod Craniofac Res 2009;12(2):113–9. 25. Huang HH, Brennan TC, Muir MM, Mason RS. Functional alpha1- and beta2-adrenergic receptors in human osteoblasts. J Cell Physiol 2009;220(1):267–75.