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Effect of cetirizine, a histamine (H1) receptor antagonist, on bone modeling during orthodontic tooth movement in rats
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Effect of cetirizine, a histamine (H1) receptor antagonist, on bone modeling during orthodontic tooth movement in rats € r,d Gorazd Drevens ek,e Janja Marc,f Sprogar,b Tomaz Vaupotic,c Andrej Co Alja Meh,a Spela g and Martina Drevensek Ljubljana, Slovenia Introduction: Histamine (H1) receptor antagonists are widely used drugs for treatment of allergic conditions. Although histamine was shown to be involved in bone remodeling, the aim of this study was to determine the effects of cetirizine, an H1 receptor antagonist, on bone modeling processes during orthodontic tooth movement. Methods: We used 3 groups of Wistar rats: control group (n 5 16), appliance-only group (n 5 16) and cetirizine group (n 5 16). Each animal of the last 2 groups was fitted with a superelastic closed-coil spring appliance and treated daily with saline solution or cetirizine. Tooth movement was measured weekly from day 0 to day 42. Gene expression levels for bone turnover markers cathepsin K and osteocalcin were determined by means of real-time polymerase chain reaction. Histologic samples were analyzed by using histomorphometry. Results: Cetirizine decreased the amount of tooth movement from day 28 onward (P \0.01), and it also decreased osteoclast volume density (P \0.001). An increase in alveolar bone volume density was observed in the cetirizine group (P \0.01) compared with the appliance-only group. No statistically significant differences were observed in osteoclast activity, osteoblast volume density, and osteoblast activity between the cetirizine and the appliance-only groups. Conclusions: Cetirizine influences bone modeling, mainly by inhibiting bone resorption. Therefore, H1 receptor antagonists could interfere with orthodontic treatment. (Am J Orthod Dentofacial Orthop 2011;139:e323-e329)
A
ntihistamines are widely prescribed drugs. Histamine (H1) receptor antagonists are mostly used in the treatment of allergic rhinitis1 and
a Postgraduate student, Department of Orthodontics, University Medical Centre, Ljubljana, Slovenia. b Postgraduate student, Institute of Pharmacology and Experimental Toxicology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia. c Research scientist, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia. d Professor, Institute of Histology and Embryology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia. e Research professor, Institute of Pharmacology and Experimental Toxicology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia. f Professor, Department of Clinical Biochemistry, Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia. g Assistant professor, Department of Orthodontics, University Medical Centre Ljubljana, Ljubljana, Slovenia. The authors report no commercial, proprietary, or financial interest in the products or companies described in this article. Supported by grants P3-0067 and P3-0293 from the Slovenian Research Agency and the Ministry of Higher Education, Science and Technology, Slovenia. Reprint requests to: Martina Drevensek, Department of Orthodontics, University Medical Centre Ljubljana, Zaloska 2, 1000 Ljubljana, Slovenia; e-mail, martina. [email protected]. Submitted, July 2009; revised and accepted, November 2009. 0889-5406/$36.00 Copyright Ó 2011 by the American Association of Orthodontists. doi:10.1016/j.ajodo.2009.11.013
chronic idiopathic urticaria.2 These chronic diseases are common in the general population; allergic rhinitis affects up to 60 million people in the United States,3 whereas chronic idiopathic urticaria affects up to 3% of the population at some time in their lives.2 Therefore, it is highly possible that some orthodontic patients are prescribed H1 receptor antagonists during their orthodontic therapy. Histamine is involved in bone remodeling—osteoclasts first resorb bone, and then osteoblasts deposit bone at the same location—and in bone modeling, the process in which the shape of the bone is changed.4 Both osteoclasts and osteoblasts were found to express H1 receptors.5 The role of histamine in bone remodeling is more pronounced in situations of high bone turnover but has little or no impact on basal bone remodeling.6 Histamine promotes the formation of osteoclasts in vitro indirectly via H1 receptors on osteoblasts.7 H1 receptors are also expressed on mature osteoclasts.5 In-vitro studies agree with in-vivo studies. In a study of postmenopausal women with pollen allergy, it was observed that H1 receptor antagonists have beneficial effects on the risk of bone fractures.8 In a synchronized resorption rat model, it was shown that mepyramine, an H1 receptor antagonist, transiently inhibits bone e323
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resorption.9 Although the effect of histamine on bone resorption has been well studied, less is known about histamine involvement in bone formation. Mice lacking the capacity to synthesize histamine exhibited increased cortical bone thickness and mineral content, because of increased bone formation and reduced bone resorption. Histamine deficiency also increases bone formation, partly by increasing serum calcitriol levels, which consequently stimulate osteoblast activity.10 These findings contrast with those from an in-vitro study, in which it was found that histamine stimulates the maturation and the differentiation of osteoblasts.11 Bone modeling is the key process in orthodontic tooth movement. It starts 20 to 30 days after force application to the tooth when sterile necrosis is removed. At the pressure side, osteoclasts degrade the alveolar bone to create space for the moving tooth. At the tension side, new bone is formed by activated osteoblasts that produce new extracellular matrix, which subsequently mineralizes. During orthodontic tooth movement, different mediators, which induce the bone-modeling processes, are produced by the periodontal ligament (PDL) and bone cells12; histamine is one of them. It has already been shown that cetirizine, an H1 receptor antagonist, affects orthodontic tooth movement in rats. H1 receptor gene expression level increased on days 14 and 42 after force application to the teeth.13 The purpose of this study was to further evaluate the effects of cetirizine, an H1 receptor antagonist, on bone resorption and bone formation during orthodontic tooth movement in rats. MATERIAL AND METHODS
All animal procedures and the study protocol were approved by the Veterinary Administration of the Republic of Slovenia (number 323-02-234/2005/2) and are in compliance with the guiding principles in the “Care and Use of Animals.” The study was performed with 48 male Wistar rats (320-340 g, 13-14 weeks old). The animals were housed in groups of 4 in polycarbonate cages under normal laboratory conditions at constant temperature (24 C-25 C) and humidity, with a 12-hour circadian cycle. They were fed with soaked standard laboratory rat chow diet (Krka, Novo mesto, Slovenia) and water ad libitum. The rat chow was soaked in water to facilitate food intake that was impaired by the orthodontic appliance. General anesthesia was used for placing the orthodontic appliance. To ensure general anesthesia, a mixture of 3 anesthetics was used. The anesthetics were injected intraperitoneally: ketamine at 50 mg per kilogram 1 of body weight (Bioketan, Vetoquinol Biowet, Gorz ow Wielkopolski, Poland), medetomidin hydrochloride at 67 mg per kilogram 1 of body weight (Domitor, Pfizer, Brooklyn,
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NY), and thiopental at 3 mg per kilogram 1 of body weight (Tiopental, Pliva, Zagreb, Croatia).14 The orthodontic appliance consisted of a superelastic closed-coil spring (25 cN; wire diameter, 0.15 mm; GAC International, Bohemia, NY), placed between the maxillary left first molar and the incisors. The superelasticity of the coil spring ensured a constant force (25 cN) during the whole time of coil-spring activation.14 The closedcoil spring was attached to the maxillary left first molar with a stainless steel ligature wire (diameter, 0.25 mm; Dentaurum, Ispringen, Germany) and to the incisors by a surgical steel wire (4-0, multifilament, W310, Ethicon; Johnson & Johnson, New Brunswick, NJ). To improve the fixation of the appliance, a 0.5-mm hole was made by using a hard metal burr (HM 1, 204, 005, Meisinger, Neuss, Germany). The hole was drilled from the distal surfaces of the incisors, through the teeth, and perpendicular to the longitudinal axis of the incisor at the gingival level. The steel wire was inserted through the hole and bonded to the proximal surface of the right incisor.15 The animals were divided into 3 groups, 1 control and 2 appliance groups (appliance-only and cetirizine). In the control group (n 5 16), no orthodontic appliance was placed, and each animal received 0.1 mL of saline solution daily. In the appliance-only group (n 5 16), each animal was fitted with an orthodontic appliance and received 0.1 mL of saline solution daily. In the cetirizine group (n 5 16), each animal was fitted with an orthodontic appliance and received 3 mg of kilogram 1 of cetirizine (Krka) daily. All animals received saline solution or cetirizine at approximately the same time for 42 days. The orthodontic appliance was placed under general anesthesia at the beginning of the study and replaced to the correct position every 7 days to ensure its proper activation, and consequently a constant force was exerted on the teeth.16 On day 42, all animals were killed. The distance between the most mesial point of the maxillary left first molar and the most palatal point of the ipsilateral incisor at the gingival level was measured in all animals. Measurements were made weekly with a digitronic caliper with an accuracy of 6 0.01 mm (Wilson & Wolpert, Utrecht, The Netherlands) while the animals were anesthetized. All measurements were made twice by 2 investigators (A.M., S.S.) independently within a few minutes. Tooth movement was calculated by subtracting the distance between the teeth on each day of measurement (days 7, 14, 21, 28, 35, and 42) from the distance between the teeth measured the previous week. Cathepsin K gene expression level was used to determine osteoclast activity,17 and osteocalcin gene expression level was used to determine osteoblast activity.18 On
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day 42, 8 animals from each of the 3 groups were killed with an intraperitoneal injection of anesthetics and carbon dioxide. Tissue samples of alveolar bone, with all 3 molars and their PDLs, were excised and frozen in liquid nitrogen and mechanically powdered. The total ribonucleic acid (RNA) content from 120 6 30 mg of each powdered sample was isolated by using TRIzol reagent (Invitrogen, Carlsbad, Calif), according to the manufacturer’s instructions. RNA concentrations and quality were determined by the Agilent 2100 Bioanalyzer with an RNA microfluidics chip (Agilent, Santa Clara, Calif). To prepare primary cDNA, 10 ng of total RNA was used along with a high-capacity cDNA archive kit, with the addition of RNase inhibitor (Applied Biosystems, Foster City, Calif). The reaction was conducted according to the manufacturer’s recommendations at a final volume of 100 mL. It proceeded at 25 C for 10 minutes and 37 C for 2 hours, and was stopped by inactivation at 99 C for 5 minutes. Oligonucleotides for cathepsin K and osteocalcin were chosen from predesigned assays (TaqMan Gene Expression Assays, Applied Biosystems, Rn00580723_m1, Rn01455285_g1, respectively). Thermal cycling comprised initial steps at 50 C for 2 minutes and at 95 C for 10 minutes, followed by 40 cycles at 95 C for 15 seconds and at 60 C for 1 minute. A standard curve was constructed with serial dilutions of a mix of a few samples. The cDNA was amplified and quantified by using a sequence detection system (SDS 7000, Applied Biosystems, Foster City, Calif). To exclude variations from different inputs of total mRNA to the reaction, data on cathepsin K and osteocalcin were normalized to an internal housekeeping gene, GAPDH, for which data was obtained by using TaqMan GAPDH predesigned assays (TaqMan Gene Expression Assays, Applied Biosystems, Rn01462662_g1). All reactions for standard samples and for samples from all 3 groups (control, appliance-only, and cetirizine) were performed in duplicate. The data were averaged from the values obtained in each reaction. Bone histomorphometry was used to determine alveolar bone volume density, osteoclast volume density, and osteoblast volume density in all 3 groups. On day 42, the remaining 8 animals from each group were killed by intraperitoneal injection of anesthetics and carbon dioxide. Tissue samples of the maxilla containing all 3 molars were taken. Tissue specimens were prepared as described.15 Histomorphometry was performed by using a point-counting method. For this purpose, the stereologic cycloid grid system incorporated into the ocular of a light microscope (BX-60, Olympus, Tokyo, Japan) was used. All histomorphometric measurements and calculations were made according to the standard nomenclature.19 The alveolar bone area density, expressed
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Fig 1. A horizontal section through the mid third of the roots of all 3 maxillary left molars at 10-times magnification. The alveolar bone volume density (BV/TV) around all 5 roots of the maxillary left first molar (A 1 B) and the osteoclast and osteoblast volume densities (Oc.V/BV and Ob.V/BV) around the mesial root of the maxillary left first molar (B) were determined histomorphometrically.
as the percentage of the tissue area consisting of tooth, PDL, connective tissue, and bone marrow spaces (B.Ar/ T.Ar), was determined at 10-times magnification. Osteoclast and osteoblast area densities, defined as the alveolar bone area covered with either osteoclasts (multi-nucleated cells at the alveolar bone surface) or osteoblasts (“round” cells at the alveolar bone surface) as a fraction of the total alveolar bone area (Oc.Ar/B.Ar or Ob.Ar/B.Ar), were determined at 40-times magnification. The borders of the tissue area examined were determined as described by Sprogar et al.15 Figure 1 shows the examined area. Since 20 sections were examined from each part (apical, middle, and cervical) of the root of the maxillary left first molar, the 2-dimensional term “area density” was extrapolated to the 3-dimensional term “volume density.” Alveolar bone area density, osteoclast area density, and osteoblast area density were extrapolated to alveolar bone volume density (BV/TV), osteoclast volume density (Oc.V/BV), and osteoblast volume density (Ob.V/BV). Statistical analysis
The data were expressed as means 6 standard errors and calculated for each parameter for all animals of all groups (tooth movement, gene expression levels of cathepsin K and osteocalcin, alveolar bone volume density, and osteoclast and osteoblast volume densities). Tooth movement was calculated from measurements of the distance between the teeth. Interexaminer reliability was tested by using the intraclass correlation coefficient (ICC), and a paired t test was used to assess the systemic bias. Analysis of systemic bias showed a P value of .0.88, and the ICC was 0.91 6 0.02. The mean value of 4 measurements was used for statistical analysis. Within-group and between-group comparisons were made for gene expression levels of cathepsin K and osteocalcin, and for the alveolar bone volume density
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Fig 2. Tooth movement in all groups during the experiment. Tooth movement was significantly less in the cetirizine group on days 28, 35, and 42 (**P \0.01 and ***P \0.001) compared with the applianceonly group.
(BV/TV), osteoclast volume density (Oc.V/BV), and osteoblast volume density (Ob.V/BV) with 1-way analysis of variance (ANOVA). A P value less than 0.05 was considered statistically significant. RESULTS
Tooth movement decreased in the cetirizine group, relative to the appliance-only group, from day 28 onward. In the control group, the distance between the teeth increased relative to the other 3 groups because of the distal drift of the molars (Fig 2). The constitutive gene expression levels for cathepsin K and osteocalcin and the response of the cells to the orthodontic force in both experimental groups were assessed by determination of gene expression levels for both markers. The gene expression level for cathepsin K on day 42 was up-regulated in the appliance-only and the cetirizine groups compared with the control group (Fig 3). In the appliance-only and cetirizine groups, the gene expression levels were up-regulated equally relative to the control group.
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The gene expression level for osteocalcin on day 42 was up-regulated only in the appliance-only group relative to the control group. No significant difference was observed in gene expression level of osteocalcin in the cetirizine group compared with the control group. Alveolar bone volume density was decreased in both experimental groups relative to the control group (Fig 4). It was increased in the cetirizine group relative to the appliance-only group. Osteoclast volume density in the cetirizine group was significantly decreased relative to the appliance-only group. No differences in the osteoblast volume density were observed between the groups (Fig 5). DISCUSSION
Many studies have shown that histamine is involved in bone turnover processes. Regarding bone modeling in orthodontic tooth movement, it has only been shown that histamine receptors are transcriptionally induced during the bone modeling stage of orthodontic tooth movement.13,20 In our study, cetirizine, an H1 receptor antagonist, decreased orthodontic tooth movement in
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Fig 3. Gene expression levels of cathepsin K and osteocalcin in alveolar bone and PDL after orthodontic tooth movement. The gene expression level of cathepsin K was up-regulated in the appliance-only and cetirizine groups, whereas the gene expression level of osteocalcin was up-regulated only in the appliance-only group. Levels of the indicated genes during orthodontic tooth movement are expressed relative to that of GAPDH. The results are given as means and standard errors from 3 independent experiments (*P \0.05 and **P \0.01).
its late phase, when alveolar bone modeling takes place at a constant rate.21 These data suggest that cetirizine reduces the rate of bone turnover during orthodontic tooth movement. Cetirizine significantly decreased osteoclast volume density but did not affect osteoclast activity during orthodontic tooth movement. H1 receptors are expressed on osteoclast precursors and are also involved in osteoclast maturation via RANKL, which is secreted by osteoblasts.5 It is possible to assume that the decrease in osteoclast volume density observed in this study might be the consequence of the decrease in differentiation of osteoclasts. H1 receptors are expressed on mature osteoclasts as well.5 Therefore, decreased osteoclast activity would be expected.9 In contrast, in this study, osteoclast activity was the same as in the applianceonly group. This indicates the possible involvement of a compensatory mechanism. Several other studies have also reported decreased bone resorption after administration of antihistamines. Reduced bone resorption
was observed in ovariectomized rats treated with H1 receptor antagonist.22 In a synchronized resorption rat model, it was shown that H1 receptor antagonists reduced the numbers of osteoclasts, but the effect was only transient.9 In the latter study, the authors observed the bone resorption process after tooth extraction, which is a 1-time event, and bone modeling lasted for 16 days. In contrast, constant force application to the tooth during orthodontic tooth movement causes continuous bone modeling; this might be the reason for decreased osteoclast volume density in the cetirizine group observed in our study. The studies on the effects of antihistamines on bone turnover mostly concentrated on osteoclasts, but little is known about their effect on osteoblasts. Histamine was reported to stimulate maturation and differentiation of osteoblasts in vitro.11 On the other hand, an in-vivo study showed increased bone formation without histamine.10 In our study, there was no change in osteoblast volume density after cetirizine administration, whereas
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Fig 4. Horizontal sections through the midpart of the roots of the maxillary left first molar in all groups. In B, the appliance-only group, and C, the cetirizine group, the percentage of cancellous bone of alveolar bone (CnB) vs all other tissues: roots of teeth, PDL, and bone marrow spaces (BMaS) were decreased when compared with A, the control group (M, mesial root; MP and MB, mesiopalatal and mesiobuccal roots; DP and DB, distopalatal and distobuccal roots).
Fig 5. Histomorphometric analysis of alveolar bone volume density, expressed as alveolar bone volume vs tissue volume; tooth, PDL, connective tissue, and bone marrow spaces (BV/TV) were significantly higher in the cetirizine group than in the appliance-only group (**P \0.01). BV/TV was significantly lower in the appliance-only and cetirizine groups than in the control group (**P \0.01 and ***P \0.001). Osteoclast volume density, expressed as alveolar bone volume covered with osteoclasts vs alveolar bone volume (Oc.V/BV), was significantly lower in the cetirizine group than in the appliance-only group (***P \0.001) and significantly higher in the appliance-only group than in the control group (***P \0.001). Osteoblast volume density, expressed as alveolar bone volume covered with osteoblasts vs alveolar bone volume (Ob.V/BV), showed no significant differences between the control group and both appliance groups.
a nonsignificant decrease in osteoblast activity was observed, as determined by the gene expression level for osteocalcin, a marker for osteoblast activity.18 Cetirizine increased alveolar bone volume density during orthodontic tooth movement. Alveolar bone volume density shows the balance between bone resorption
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and bone formation. During orthodontic tooth movement, when force is applied to the tooth, the alveolar bone volume density is reduced presumably because of the increase in bone resorption. In our study, alveolar bone volume density increased after the application of H1 antagonist. Since H1 antagonist did not increase
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osteoblast volume density, the increase in alveolar bone volume density might be attributed to inhibited bone resorption. Immature osteoblasts expressing H1 receptors support proliferation and differentiation of osteoclasts.5 Therefore, by blocking H1 receptors on osteoblasts, H1 antagonists block recruitment and differentiation of osteoclasts. It seems that H1 receptor antagonists mainly affect bone resorption during orthodontic tooth movement and therefore reduce the amount of tooth movement. CONCLUSIONS
We showed the effects of H1 receptor antagonists on bone modeling during orthodontic tooth movement. Cetirizine reduced the amount of tooth movement during force application. The observed effect was probably due to decreased alveolar bone resorption. Accordingly, it is reasonable to assume that chronic use of antihistamines in orthodontic patients might suppress tooth movement and therefore prolong treatment times. This should be considered by orthodontists during treatment planning. The cetirizine was kindly provided by Krka, Novo mesto, Slovenia. REFERENCES 1. Badorrek P, Dick M, Schauerte A, Hecker H, Murdoch R, Luettig B, et al. A combination of cetirizine and pseudoephedrine has therapeutic benefits when compared to single drug treatment in allergic rhinitis. Int J Clin Pharmacol Ther 2009;47:71-7. 2. Najib U, Sheikh J. The spectrum of chronic urticaria. Allergy Asthma Proc 2009;30:1-10. 3. Meltzer EO, Blaiss MS, Derebery MJ, Mahr TA, Gordon BR, Sheth KK, et al. Burden of allergic rhinitis: results from the Pediatric Allergies in America survey. J Allergy Clin Immunol 2009; 124(3 Suppl):S43-70. 4. Seeman E. Structural basis of growth-related gain and age-related loss of bone strength. Rheumatology (Oxford) 2008;47(Suppl 4): iv2-8. 5. Biosse-Duplan M, Baroukh B, Dy M, de Vernejoul MC, Saffar JL. Histamine promotes osteoclastogenesis through the differential expression of histamine receptors on osteoclasts and osteoblasts. Am J Pathol 2009;17:1426-34. 6. Lesclous P, Schramm F, Gallina S, Baroukh B, Guez D, Saffar JL. Histamine mediates osteoclastic resorption only during the acute phase of bone loss in ovariectomized rats. Exp Physiol 2006;9: 561-70. 7. Deyama Y, Kikuiri T, Ohnishi G, Feng YG, Takeyama S, Hatta M, et al. Histamine stimulates production of osteoclast differentiation factor/receptor activator of nuclear factor-kappaB ligand by osteoblasts. Biochem Biophys Res Commun 2002;298:240-6.
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8. Ferencz V, Meszaros S, Csupor E, Toth E, Bors K, Falus A, et al. Increased bone fracture prevalence in postmenopausal women suffering from pollen-allergy. Osteoporos Int 2006;17:484-91. 9. Dobigny C, Saffar JL. H1 and H2 histamine receptors modulate osteoclastic resorption by different pathways: evidence obtained by using receptor antagonists in a rat synchronized resorption model. J Cell Physiol 1997;173:10-8. 10. Fitzpatrick LA, Buzas E, Gagne TJ, Nagy A, Horvath C, Ferencz V, et al. Targeted deletion of histidine decarboxylase gene in mice increases bone formation and protects against ovariectomy-induced bone loss. Proc Natl Acad Sci U S A 2003;100:6027-32. 11. Ikawa Y, Yonekawa T, Ohkuni Y, Kuribayashi M, Fukino K, Ueno K. A comparative study of histamine activities on differentiation of osteoblasts and osteoclasts. J Toxicol Sci 2007;32: 555-64. 12. Krishnan V, Davidovitch Z. Cellular, molecular, and tissue-level reactions to orthodontic force. Am J Orthod Dentofacial Orthop 2006;129:469.e1-32. 13. Kriznar I, Sprogar S, Drevensek M, Vaupotic T, Drevensek G. Cetirizine, a histamine H1 receptor antagonist, decreases the first stage of orthodontic tooth movement in rats. Inflamm Res 2008;57 (Suppl):S29-30. 14. Drevensek M, Sprogar S, Boras I, Drevensek G. Effects of endothelin antagonist tezosentan on orthodontic tooth movement in rats. Am J Orthod Dentofacial Orthop 2006;129:555-8. 15. Sprogar S, Vaupotic T, C€ or A, Drevensek M, Drevensek G. The endothelin system mediates bone modeling in the late stage of orthodontic tooth movement in rats. Bone 2008;43:740-7. 16. Drevensek M, Volk J, Sprogar S, Drevensek G. Orthodontic force decreases the eruption rate of rat incisors. Eur J Orthod 2009; 31:46-50. 17. Zhang Y, Dong XL, Leung PC, Wong MS. Differential mRNA expression profiles in proximal tibia of aged rats in response to ovariectomy and low-Ca diet. Bone 2009;44:46-52. 18. Owen TA, Aronow M, Shalhoub V, Barone LM, Wilming L, Tassinari MS, et al. Progressive development of the rat osteoblast phenotype in vitro: reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix. J Cell Physiol 1990; 143:420-30. 19. Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, et al. Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res 1987; 2:595-610. 20. Sprogar S, Kriznar I, Drevensek M, Vaupotic T, Drevensek G. Famotidine, a H2 receptor antagonist, decreases the late phase of orthodontic tooth movement in rats. Inflamm Res 2008;57 (Suppl):S31-2. 21. Von B€ ohl M, Maltha J, Von den Hoff H, Kuijpers-Jagtman AM. Changes in the periodontal ligament after experimental tooth movement using high and low continuous forces in beagle dogs. Angle Orthod 2004;74:16-25. 22. Rico H, Gomez M, Revilla M, Gonzalez-Riola J, Seco C, Hernandez ER, et al. Effects of promethazine on bone mass and on bone remodeling in ovariectomized rats: a morphometric, densitometric, and histomorphometric experimental study. Calcif Tissue Int 1999;65:272-5.
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Effect of cetirizine, a histamine (H1) receptor antagonist, on bone modeling during orthodontic tooth movement in rats € r,d Gorazd Drevens ek,e Janja Marc,f Sprogar,b Tomaz Vaupotic,c Andrej Co Alja Meh,a Spela g and Martina Drevensek Ljubljana, Slovenia Introduction: Histamine (H1) receptor antagonists are widely used drugs for treatment of allergic conditions. Although histamine was shown to be involved in bone remodeling, the aim of this study was to determine the effects of cetirizine, an H1 receptor antagonist, on bone modeling processes during orthodontic tooth movement. Methods: We used 3 groups of Wistar rats: control group (n 5 16), appliance-only group (n 5 16) and cetirizine group (n 5 16). Each animal of the last 2 groups was fitted with a superelastic closed-coil spring appliance and treated daily with saline solution or cetirizine. Tooth movement was measured weekly from day 0 to day 42. Gene expression levels for bone turnover markers cathepsin K and osteocalcin were determined by means of real-time polymerase chain reaction. Histologic samples were analyzed by using histomorphometry. Results: Cetirizine decreased the amount of tooth movement from day 28 onward (P \0.01), and it also decreased osteoclast volume density (P \0.001). An increase in alveolar bone volume density was observed in the cetirizine group (P \0.01) compared with the appliance-only group. No statistically significant differences were observed in osteoclast activity, osteoblast volume density, and osteoblast activity between the cetirizine and the appliance-only groups. Conclusions: Cetirizine influences bone modeling, mainly by inhibiting bone resorption. Therefore, H1 receptor antagonists could interfere with orthodontic treatment. (Am J Orthod Dentofacial Orthop 2011;139:e323-e329)
A
ntihistamines are widely prescribed drugs. Histamine (H1) receptor antagonists are mostly used in the treatment of allergic rhinitis1 and
a Postgraduate student, Department of Orthodontics, University Medical Centre, Ljubljana, Slovenia. b Postgraduate student, Institute of Pharmacology and Experimental Toxicology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia. c Research scientist, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia. d Professor, Institute of Histology and Embryology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia. e Research professor, Institute of Pharmacology and Experimental Toxicology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia. f Professor, Department of Clinical Biochemistry, Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia. g Assistant professor, Department of Orthodontics, University Medical Centre Ljubljana, Ljubljana, Slovenia. The authors report no commercial, proprietary, or financial interest in the products or companies described in this article. Supported by grants P3-0067 and P3-0293 from the Slovenian Research Agency and the Ministry of Higher Education, Science and Technology, Slovenia. Reprint requests to: Martina Drevensek, Department of Orthodontics, University Medical Centre Ljubljana, Zaloska 2, 1000 Ljubljana, Slovenia; e-mail, martina. [email protected]. Submitted, July 2009; revised and accepted, November 2009. 0889-5406/$36.00 Copyright Ó 2011 by the American Association of Orthodontists. doi:10.1016/j.ajodo.2009.11.013
chronic idiopathic urticaria.2 These chronic diseases are common in the general population; allergic rhinitis affects up to 60 million people in the United States,3 whereas chronic idiopathic urticaria affects up to 3% of the population at some time in their lives.2 Therefore, it is highly possible that some orthodontic patients are prescribed H1 receptor antagonists during their orthodontic therapy. Histamine is involved in bone remodeling—osteoclasts first resorb bone, and then osteoblasts deposit bone at the same location—and in bone modeling, the process in which the shape of the bone is changed.4 Both osteoclasts and osteoblasts were found to express H1 receptors.5 The role of histamine in bone remodeling is more pronounced in situations of high bone turnover but has little or no impact on basal bone remodeling.6 Histamine promotes the formation of osteoclasts in vitro indirectly via H1 receptors on osteoblasts.7 H1 receptors are also expressed on mature osteoclasts.5 In-vitro studies agree with in-vivo studies. In a study of postmenopausal women with pollen allergy, it was observed that H1 receptor antagonists have beneficial effects on the risk of bone fractures.8 In a synchronized resorption rat model, it was shown that mepyramine, an H1 receptor antagonist, transiently inhibits bone e323
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resorption.9 Although the effect of histamine on bone resorption has been well studied, less is known about histamine involvement in bone formation. Mice lacking the capacity to synthesize histamine exhibited increased cortical bone thickness and mineral content, because of increased bone formation and reduced bone resorption. Histamine deficiency also increases bone formation, partly by increasing serum calcitriol levels, which consequently stimulate osteoblast activity.10 These findings contrast with those from an in-vitro study, in which it was found that histamine stimulates the maturation and the differentiation of osteoblasts.11 Bone modeling is the key process in orthodontic tooth movement. It starts 20 to 30 days after force application to the tooth when sterile necrosis is removed. At the pressure side, osteoclasts degrade the alveolar bone to create space for the moving tooth. At the tension side, new bone is formed by activated osteoblasts that produce new extracellular matrix, which subsequently mineralizes. During orthodontic tooth movement, different mediators, which induce the bone-modeling processes, are produced by the periodontal ligament (PDL) and bone cells12; histamine is one of them. It has already been shown that cetirizine, an H1 receptor antagonist, affects orthodontic tooth movement in rats. H1 receptor gene expression level increased on days 14 and 42 after force application to the teeth.13 The purpose of this study was to further evaluate the effects of cetirizine, an H1 receptor antagonist, on bone resorption and bone formation during orthodontic tooth movement in rats. MATERIAL AND METHODS
All animal procedures and the study protocol were approved by the Veterinary Administration of the Republic of Slovenia (number 323-02-234/2005/2) and are in compliance with the guiding principles in the “Care and Use of Animals.” The study was performed with 48 male Wistar rats (320-340 g, 13-14 weeks old). The animals were housed in groups of 4 in polycarbonate cages under normal laboratory conditions at constant temperature (24 C-25 C) and humidity, with a 12-hour circadian cycle. They were fed with soaked standard laboratory rat chow diet (Krka, Novo mesto, Slovenia) and water ad libitum. The rat chow was soaked in water to facilitate food intake that was impaired by the orthodontic appliance. General anesthesia was used for placing the orthodontic appliance. To ensure general anesthesia, a mixture of 3 anesthetics was used. The anesthetics were injected intraperitoneally: ketamine at 50 mg per kilogram 1 of body weight (Bioketan, Vetoquinol Biowet, Gorz ow Wielkopolski, Poland), medetomidin hydrochloride at 67 mg per kilogram 1 of body weight (Domitor, Pfizer, Brooklyn,
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NY), and thiopental at 3 mg per kilogram 1 of body weight (Tiopental, Pliva, Zagreb, Croatia).14 The orthodontic appliance consisted of a superelastic closed-coil spring (25 cN; wire diameter, 0.15 mm; GAC International, Bohemia, NY), placed between the maxillary left first molar and the incisors. The superelasticity of the coil spring ensured a constant force (25 cN) during the whole time of coil-spring activation.14 The closedcoil spring was attached to the maxillary left first molar with a stainless steel ligature wire (diameter, 0.25 mm; Dentaurum, Ispringen, Germany) and to the incisors by a surgical steel wire (4-0, multifilament, W310, Ethicon; Johnson & Johnson, New Brunswick, NJ). To improve the fixation of the appliance, a 0.5-mm hole was made by using a hard metal burr (HM 1, 204, 005, Meisinger, Neuss, Germany). The hole was drilled from the distal surfaces of the incisors, through the teeth, and perpendicular to the longitudinal axis of the incisor at the gingival level. The steel wire was inserted through the hole and bonded to the proximal surface of the right incisor.15 The animals were divided into 3 groups, 1 control and 2 appliance groups (appliance-only and cetirizine). In the control group (n 5 16), no orthodontic appliance was placed, and each animal received 0.1 mL of saline solution daily. In the appliance-only group (n 5 16), each animal was fitted with an orthodontic appliance and received 0.1 mL of saline solution daily. In the cetirizine group (n 5 16), each animal was fitted with an orthodontic appliance and received 3 mg of kilogram 1 of cetirizine (Krka) daily. All animals received saline solution or cetirizine at approximately the same time for 42 days. The orthodontic appliance was placed under general anesthesia at the beginning of the study and replaced to the correct position every 7 days to ensure its proper activation, and consequently a constant force was exerted on the teeth.16 On day 42, all animals were killed. The distance between the most mesial point of the maxillary left first molar and the most palatal point of the ipsilateral incisor at the gingival level was measured in all animals. Measurements were made weekly with a digitronic caliper with an accuracy of 6 0.01 mm (Wilson & Wolpert, Utrecht, The Netherlands) while the animals were anesthetized. All measurements were made twice by 2 investigators (A.M., S.S.) independently within a few minutes. Tooth movement was calculated by subtracting the distance between the teeth on each day of measurement (days 7, 14, 21, 28, 35, and 42) from the distance between the teeth measured the previous week. Cathepsin K gene expression level was used to determine osteoclast activity,17 and osteocalcin gene expression level was used to determine osteoblast activity.18 On
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day 42, 8 animals from each of the 3 groups were killed with an intraperitoneal injection of anesthetics and carbon dioxide. Tissue samples of alveolar bone, with all 3 molars and their PDLs, were excised and frozen in liquid nitrogen and mechanically powdered. The total ribonucleic acid (RNA) content from 120 6 30 mg of each powdered sample was isolated by using TRIzol reagent (Invitrogen, Carlsbad, Calif), according to the manufacturer’s instructions. RNA concentrations and quality were determined by the Agilent 2100 Bioanalyzer with an RNA microfluidics chip (Agilent, Santa Clara, Calif). To prepare primary cDNA, 10 ng of total RNA was used along with a high-capacity cDNA archive kit, with the addition of RNase inhibitor (Applied Biosystems, Foster City, Calif). The reaction was conducted according to the manufacturer’s recommendations at a final volume of 100 mL. It proceeded at 25 C for 10 minutes and 37 C for 2 hours, and was stopped by inactivation at 99 C for 5 minutes. Oligonucleotides for cathepsin K and osteocalcin were chosen from predesigned assays (TaqMan Gene Expression Assays, Applied Biosystems, Rn00580723_m1, Rn01455285_g1, respectively). Thermal cycling comprised initial steps at 50 C for 2 minutes and at 95 C for 10 minutes, followed by 40 cycles at 95 C for 15 seconds and at 60 C for 1 minute. A standard curve was constructed with serial dilutions of a mix of a few samples. The cDNA was amplified and quantified by using a sequence detection system (SDS 7000, Applied Biosystems, Foster City, Calif). To exclude variations from different inputs of total mRNA to the reaction, data on cathepsin K and osteocalcin were normalized to an internal housekeeping gene, GAPDH, for which data was obtained by using TaqMan GAPDH predesigned assays (TaqMan Gene Expression Assays, Applied Biosystems, Rn01462662_g1). All reactions for standard samples and for samples from all 3 groups (control, appliance-only, and cetirizine) were performed in duplicate. The data were averaged from the values obtained in each reaction. Bone histomorphometry was used to determine alveolar bone volume density, osteoclast volume density, and osteoblast volume density in all 3 groups. On day 42, the remaining 8 animals from each group were killed by intraperitoneal injection of anesthetics and carbon dioxide. Tissue samples of the maxilla containing all 3 molars were taken. Tissue specimens were prepared as described.15 Histomorphometry was performed by using a point-counting method. For this purpose, the stereologic cycloid grid system incorporated into the ocular of a light microscope (BX-60, Olympus, Tokyo, Japan) was used. All histomorphometric measurements and calculations were made according to the standard nomenclature.19 The alveolar bone area density, expressed
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Fig 1. A horizontal section through the mid third of the roots of all 3 maxillary left molars at 10-times magnification. The alveolar bone volume density (BV/TV) around all 5 roots of the maxillary left first molar (A 1 B) and the osteoclast and osteoblast volume densities (Oc.V/BV and Ob.V/BV) around the mesial root of the maxillary left first molar (B) were determined histomorphometrically.
as the percentage of the tissue area consisting of tooth, PDL, connective tissue, and bone marrow spaces (B.Ar/ T.Ar), was determined at 10-times magnification. Osteoclast and osteoblast area densities, defined as the alveolar bone area covered with either osteoclasts (multi-nucleated cells at the alveolar bone surface) or osteoblasts (“round” cells at the alveolar bone surface) as a fraction of the total alveolar bone area (Oc.Ar/B.Ar or Ob.Ar/B.Ar), were determined at 40-times magnification. The borders of the tissue area examined were determined as described by Sprogar et al.15 Figure 1 shows the examined area. Since 20 sections were examined from each part (apical, middle, and cervical) of the root of the maxillary left first molar, the 2-dimensional term “area density” was extrapolated to the 3-dimensional term “volume density.” Alveolar bone area density, osteoclast area density, and osteoblast area density were extrapolated to alveolar bone volume density (BV/TV), osteoclast volume density (Oc.V/BV), and osteoblast volume density (Ob.V/BV). Statistical analysis
The data were expressed as means 6 standard errors and calculated for each parameter for all animals of all groups (tooth movement, gene expression levels of cathepsin K and osteocalcin, alveolar bone volume density, and osteoclast and osteoblast volume densities). Tooth movement was calculated from measurements of the distance between the teeth. Interexaminer reliability was tested by using the intraclass correlation coefficient (ICC), and a paired t test was used to assess the systemic bias. Analysis of systemic bias showed a P value of .0.88, and the ICC was 0.91 6 0.02. The mean value of 4 measurements was used for statistical analysis. Within-group and between-group comparisons were made for gene expression levels of cathepsin K and osteocalcin, and for the alveolar bone volume density
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Fig 2. Tooth movement in all groups during the experiment. Tooth movement was significantly less in the cetirizine group on days 28, 35, and 42 (**P \0.01 and ***P \0.001) compared with the applianceonly group.
(BV/TV), osteoclast volume density (Oc.V/BV), and osteoblast volume density (Ob.V/BV) with 1-way analysis of variance (ANOVA). A P value less than 0.05 was considered statistically significant. RESULTS
Tooth movement decreased in the cetirizine group, relative to the appliance-only group, from day 28 onward. In the control group, the distance between the teeth increased relative to the other 3 groups because of the distal drift of the molars (Fig 2). The constitutive gene expression levels for cathepsin K and osteocalcin and the response of the cells to the orthodontic force in both experimental groups were assessed by determination of gene expression levels for both markers. The gene expression level for cathepsin K on day 42 was up-regulated in the appliance-only and the cetirizine groups compared with the control group (Fig 3). In the appliance-only and cetirizine groups, the gene expression levels were up-regulated equally relative to the control group.
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The gene expression level for osteocalcin on day 42 was up-regulated only in the appliance-only group relative to the control group. No significant difference was observed in gene expression level of osteocalcin in the cetirizine group compared with the control group. Alveolar bone volume density was decreased in both experimental groups relative to the control group (Fig 4). It was increased in the cetirizine group relative to the appliance-only group. Osteoclast volume density in the cetirizine group was significantly decreased relative to the appliance-only group. No differences in the osteoblast volume density were observed between the groups (Fig 5). DISCUSSION
Many studies have shown that histamine is involved in bone turnover processes. Regarding bone modeling in orthodontic tooth movement, it has only been shown that histamine receptors are transcriptionally induced during the bone modeling stage of orthodontic tooth movement.13,20 In our study, cetirizine, an H1 receptor antagonist, decreased orthodontic tooth movement in
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Fig 3. Gene expression levels of cathepsin K and osteocalcin in alveolar bone and PDL after orthodontic tooth movement. The gene expression level of cathepsin K was up-regulated in the appliance-only and cetirizine groups, whereas the gene expression level of osteocalcin was up-regulated only in the appliance-only group. Levels of the indicated genes during orthodontic tooth movement are expressed relative to that of GAPDH. The results are given as means and standard errors from 3 independent experiments (*P \0.05 and **P \0.01).
its late phase, when alveolar bone modeling takes place at a constant rate.21 These data suggest that cetirizine reduces the rate of bone turnover during orthodontic tooth movement. Cetirizine significantly decreased osteoclast volume density but did not affect osteoclast activity during orthodontic tooth movement. H1 receptors are expressed on osteoclast precursors and are also involved in osteoclast maturation via RANKL, which is secreted by osteoblasts.5 It is possible to assume that the decrease in osteoclast volume density observed in this study might be the consequence of the decrease in differentiation of osteoclasts. H1 receptors are expressed on mature osteoclasts as well.5 Therefore, decreased osteoclast activity would be expected.9 In contrast, in this study, osteoclast activity was the same as in the applianceonly group. This indicates the possible involvement of a compensatory mechanism. Several other studies have also reported decreased bone resorption after administration of antihistamines. Reduced bone resorption
was observed in ovariectomized rats treated with H1 receptor antagonist.22 In a synchronized resorption rat model, it was shown that H1 receptor antagonists reduced the numbers of osteoclasts, but the effect was only transient.9 In the latter study, the authors observed the bone resorption process after tooth extraction, which is a 1-time event, and bone modeling lasted for 16 days. In contrast, constant force application to the tooth during orthodontic tooth movement causes continuous bone modeling; this might be the reason for decreased osteoclast volume density in the cetirizine group observed in our study. The studies on the effects of antihistamines on bone turnover mostly concentrated on osteoclasts, but little is known about their effect on osteoblasts. Histamine was reported to stimulate maturation and differentiation of osteoblasts in vitro.11 On the other hand, an in-vivo study showed increased bone formation without histamine.10 In our study, there was no change in osteoblast volume density after cetirizine administration, whereas
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Fig 4. Horizontal sections through the midpart of the roots of the maxillary left first molar in all groups. In B, the appliance-only group, and C, the cetirizine group, the percentage of cancellous bone of alveolar bone (CnB) vs all other tissues: roots of teeth, PDL, and bone marrow spaces (BMaS) were decreased when compared with A, the control group (M, mesial root; MP and MB, mesiopalatal and mesiobuccal roots; DP and DB, distopalatal and distobuccal roots).
Fig 5. Histomorphometric analysis of alveolar bone volume density, expressed as alveolar bone volume vs tissue volume; tooth, PDL, connective tissue, and bone marrow spaces (BV/TV) were significantly higher in the cetirizine group than in the appliance-only group (**P \0.01). BV/TV was significantly lower in the appliance-only and cetirizine groups than in the control group (**P \0.01 and ***P \0.001). Osteoclast volume density, expressed as alveolar bone volume covered with osteoclasts vs alveolar bone volume (Oc.V/BV), was significantly lower in the cetirizine group than in the appliance-only group (***P \0.001) and significantly higher in the appliance-only group than in the control group (***P \0.001). Osteoblast volume density, expressed as alveolar bone volume covered with osteoblasts vs alveolar bone volume (Ob.V/BV), showed no significant differences between the control group and both appliance groups.
a nonsignificant decrease in osteoblast activity was observed, as determined by the gene expression level for osteocalcin, a marker for osteoblast activity.18 Cetirizine increased alveolar bone volume density during orthodontic tooth movement. Alveolar bone volume density shows the balance between bone resorption
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and bone formation. During orthodontic tooth movement, when force is applied to the tooth, the alveolar bone volume density is reduced presumably because of the increase in bone resorption. In our study, alveolar bone volume density increased after the application of H1 antagonist. Since H1 antagonist did not increase
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osteoblast volume density, the increase in alveolar bone volume density might be attributed to inhibited bone resorption. Immature osteoblasts expressing H1 receptors support proliferation and differentiation of osteoclasts.5 Therefore, by blocking H1 receptors on osteoblasts, H1 antagonists block recruitment and differentiation of osteoclasts. It seems that H1 receptor antagonists mainly affect bone resorption during orthodontic tooth movement and therefore reduce the amount of tooth movement. CONCLUSIONS
We showed the effects of H1 receptor antagonists on bone modeling during orthodontic tooth movement. Cetirizine reduced the amount of tooth movement during force application. The observed effect was probably due to decreased alveolar bone resorption. Accordingly, it is reasonable to assume that chronic use of antihistamines in orthodontic patients might suppress tooth movement and therefore prolong treatment times. This should be considered by orthodontists during treatment planning. The cetirizine was kindly provided by Krka, Novo mesto, Slovenia. REFERENCES 1. Badorrek P, Dick M, Schauerte A, Hecker H, Murdoch R, Luettig B, et al. A combination of cetirizine and pseudoephedrine has therapeutic benefits when compared to single drug treatment in allergic rhinitis. Int J Clin Pharmacol Ther 2009;47:71-7. 2. Najib U, Sheikh J. The spectrum of chronic urticaria. Allergy Asthma Proc 2009;30:1-10. 3. Meltzer EO, Blaiss MS, Derebery MJ, Mahr TA, Gordon BR, Sheth KK, et al. Burden of allergic rhinitis: results from the Pediatric Allergies in America survey. J Allergy Clin Immunol 2009; 124(3 Suppl):S43-70. 4. Seeman E. Structural basis of growth-related gain and age-related loss of bone strength. Rheumatology (Oxford) 2008;47(Suppl 4): iv2-8. 5. Biosse-Duplan M, Baroukh B, Dy M, de Vernejoul MC, Saffar JL. Histamine promotes osteoclastogenesis through the differential expression of histamine receptors on osteoclasts and osteoblasts. Am J Pathol 2009;17:1426-34. 6. Lesclous P, Schramm F, Gallina S, Baroukh B, Guez D, Saffar JL. Histamine mediates osteoclastic resorption only during the acute phase of bone loss in ovariectomized rats. Exp Physiol 2006;9: 561-70. 7. Deyama Y, Kikuiri T, Ohnishi G, Feng YG, Takeyama S, Hatta M, et al. Histamine stimulates production of osteoclast differentiation factor/receptor activator of nuclear factor-kappaB ligand by osteoblasts. Biochem Biophys Res Commun 2002;298:240-6.
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8. Ferencz V, Meszaros S, Csupor E, Toth E, Bors K, Falus A, et al. Increased bone fracture prevalence in postmenopausal women suffering from pollen-allergy. Osteoporos Int 2006;17:484-91. 9. Dobigny C, Saffar JL. H1 and H2 histamine receptors modulate osteoclastic resorption by different pathways: evidence obtained by using receptor antagonists in a rat synchronized resorption model. J Cell Physiol 1997;173:10-8. 10. Fitzpatrick LA, Buzas E, Gagne TJ, Nagy A, Horvath C, Ferencz V, et al. Targeted deletion of histidine decarboxylase gene in mice increases bone formation and protects against ovariectomy-induced bone loss. Proc Natl Acad Sci U S A 2003;100:6027-32. 11. Ikawa Y, Yonekawa T, Ohkuni Y, Kuribayashi M, Fukino K, Ueno K. A comparative study of histamine activities on differentiation of osteoblasts and osteoclasts. J Toxicol Sci 2007;32: 555-64. 12. Krishnan V, Davidovitch Z. Cellular, molecular, and tissue-level reactions to orthodontic force. Am J Orthod Dentofacial Orthop 2006;129:469.e1-32. 13. Kriznar I, Sprogar S, Drevensek M, Vaupotic T, Drevensek G. Cetirizine, a histamine H1 receptor antagonist, decreases the first stage of orthodontic tooth movement in rats. Inflamm Res 2008;57 (Suppl):S29-30. 14. Drevensek M, Sprogar S, Boras I, Drevensek G. Effects of endothelin antagonist tezosentan on orthodontic tooth movement in rats. Am J Orthod Dentofacial Orthop 2006;129:555-8. 15. Sprogar S, Vaupotic T, C€ or A, Drevensek M, Drevensek G. The endothelin system mediates bone modeling in the late stage of orthodontic tooth movement in rats. Bone 2008;43:740-7. 16. Drevensek M, Volk J, Sprogar S, Drevensek G. Orthodontic force decreases the eruption rate of rat incisors. Eur J Orthod 2009; 31:46-50. 17. Zhang Y, Dong XL, Leung PC, Wong MS. Differential mRNA expression profiles in proximal tibia of aged rats in response to ovariectomy and low-Ca diet. Bone 2009;44:46-52. 18. Owen TA, Aronow M, Shalhoub V, Barone LM, Wilming L, Tassinari MS, et al. Progressive development of the rat osteoblast phenotype in vitro: reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix. J Cell Physiol 1990; 143:420-30. 19. Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, et al. Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res 1987; 2:595-610. 20. Sprogar S, Kriznar I, Drevensek M, Vaupotic T, Drevensek G. Famotidine, a H2 receptor antagonist, decreases the late phase of orthodontic tooth movement in rats. Inflamm Res 2008;57 (Suppl):S31-2. 21. Von B€ ohl M, Maltha J, Von den Hoff H, Kuijpers-Jagtman AM. Changes in the periodontal ligament after experimental tooth movement using high and low continuous forces in beagle dogs. Angle Orthod 2004;74:16-25. 22. Rico H, Gomez M, Revilla M, Gonzalez-Riola J, Seco C, Hernandez ER, et al. Effects of promethazine on bone mass and on bone remodeling in ovariectomized rats: a morphometric, densitometric, and histomorphometric experimental study. Calcif Tissue Int 1999;65:272-5.
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