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The European Journal of Orthodontics Advance Access published online on September 18, 2007
The European Journal of Orthodontics, doi:10.1093/ejo/cjm064
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© The Author 2007. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org.

The effects of TBC3214, a selective endothelin ETA receptor antagonist, on orthodontic tooth movement in rats

S. Sprogar*, J. Volk**, M. Drevensek** and G. Drevensek*

* Institute of Pharmacology and Experimental Toxicology
** Department of Orthodontics, University of Ljubljana, Slovenia

Address for correspondence Spela Sprogar, Faculty of Medicine, Institute of Pharmacology and Experimental Toxicology, University of Ljubljana, Korytkova 2, 1000 Ljubljana, Slovenia, E-mail: spela.sprogar{at}mf.uni-lj.si


   Summary
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 Summary
Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 Funding
 References
 
Many chemical messengers are involved in the process of alveolar bone and periodontal ligament remodelling during orthodontic tooth movement. Among them is probably endothelin-1 (ET-1). Its role in this process has been partly explained using tezosentan, which affects endothelin A (ETA) and endothelin B (ETB) receptors. Tezosentan enhances orthodontic tooth movement. The aim of this study was to determine the possible effects of a highly selective ETA antagonist on orthodontic tooth movement in rats.

Thirty male Wistar rats, 11–12 weeks of age, divided into three equal groups. In group I, a closed-coil spring was used and they were treated daily with 15 mg/kg body weight of TBC3214, a highly selective ETA antagonist. A closed-coil spring was also used in group II and the animals were treated daily with a placebo. Group III were treated daily with a placebo. The coil spring delivered a force of 25 cN and was attached between the upper left first molar and upper left incisor. The distance between the teeth was measured with a digital calliper (accuracy ± 0.01 mm) on days 0, 7, 14, 21, 24, 32, 37, and 40. The differences in the distance between the teeth were calculated to determine the amount of tooth movement. Statistical analysis was performed using two-way analysis of variance, Bonferroni's correction, and paired t-tests.

The distance between the upper left first molar and the upper left incisor decreased in groups I and II. In group I, tooth movement was significantly less on days 32 and 37 (P < 0.01) and on day 40 (P < 0.001) compared with group II. In group III, the distance between the teeth increased during the study (P < 0.001). In animals treated daily with TBC3214, tooth movement was significantly less compared with the animals treated with a placebo. It is concluded that ET-1, which is the predominant form of endothelin isopeptides, is involved in orthodontic tooth movement in rats, probably by enhancing bone resorption via ETA receptors.


   Introduction
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 Summary
 Introduction
Materials and methods
 Results
 Discussion
 Conclusions
 Funding
 References
 
Remodelling of the periodontal ligament (PDL) and alveolar bone is one of the key processes of orthodontic tooth movement (Meikle, 2006Go). Many chemical messengers are involved in this process. Among them could be endothelin-1 (ET-1), which is released during intravascular shear stress, hypoxia, and ischaemia (Kourembanas et al., 1991Go; Rubanyi and Polokoff, 1994Go; Schmitz-Spanke and Schipke, 2000Go). All these phenomena are also present during force application, so ET-1 could be released in the PDL and alveolar bone during orthodontic tooth movement. An upregulation of ET-1 expression in the PDL microvasculature following acute tooth loading in marmoset monkeys has already been established (Sims et al., 2003Go).

The role of ET-1 in the process of orthodontic tooth movement has been partially explained using tezosentan, an endothelin A (ETA) and endothelin B (ETB) receptor antagonist. Tezosentan enhanced orthodontic tooth movement in rats after 25 days of treatment (Drevensek et al., 2006Go).

The endothelin system consists of four active endothelin isopeptides (ET-1–ET-4), and three specific endothelin receptors (ETA, ETB, and ETC). ET-1 is the predominant and most important isoform of endothelin in humans (Miyauchi and Masaki, 1999Go; Rich and McLaughlin, 2003Go). It has been established that it is produced by endothelial cells, fibroblasts, smooth muscle cells, leukocytes, macrophages, epithelial cells, and osteoblasts (Matsumura et al., 1989Go; Ehrenreich et al., 1990Go; Ohta et al., 1990Go; Scott-Burden et al., 1991Go; Sessa et al., 1991Go; Zeballos et al., 1991Go; Cybulsky et al., 1993Go; Levin, 1995Go).

Both ETA and ETB receptors have been found on osteoblasts (Stern et al., 1995Go; Kasperk et al., 1997Go). ET-1 stimulates their proliferation (Kasperk et al., 1997Go; von Schroeder et al., 2003Go) and differentiation and activity (Nelson et al., 1999Go; von Schroeder et al., 2003Go; Guise and Mohammad, 2004Go). ET-1 also stimulates the formation of extracellular bone matrix proteins (Stern et al., 1995Go) and bone (Tatrai et al., 1992Go), but inhibits osteoblast mineralization via ETA receptors (Hiruma et al., 1998Go). Long-term inhibition of bone ETA receptors causes less bone formation and osteopenia in growing rats (Tsukahara et al., 1998Go). While it appears that ET-1 stimulates bone formation predominantly via ETA receptors on osteoblasts, the mechanism of ET-1 acting on endothelin receptors on osteoclasts is unknown. The influence of ET-1 on bone resorption is controversial. ET-1 decreases bone resorption by inhibition of osteoclast motility (Alam et al., 1992Go; Nelson et al., 1999Go). A study performed on prostate cancer cells showed that osteoclastic bone resorption was blocked by the presence of ET-1 in a dose-dependent manner (Chiao et al., 2000Go). On the other hand, it was established that ET-1 stimulates prostaglandin-dependent bone resorption (Tatrai et al., 1992Go; Stern et al., 1995Go). The endothelin antagonist, tezosentan, which acts on ETA and on ETB receptors, has been shown to increase tooth movement (Drevensek et al., 2006Go).

The aim of the present study was to determine the involvement of ETA receptors in orthodontic tooth movement. Tooth movement modulated by applying an endothelin antagonist, acting only on ETA receptors, could help in understanding the role of ET-1 in the process of bone remodelling. A highly selective ETA receptor antagonist, TBC3214, was used in this study.


   Materials and methods
Top
 Summary
 Introduction
 Materials and methods
Results
 Discussion
 Conclusions
 Funding
 References
 

Animals

The investigation was approved by the Veterinary Administration of the Republic of Slovenia (No. 323-02-234/2005/2).

The study was carried out on 30 male Wistar rats (300–340 g, 11–12 weeks old). The animals were housed in groups of five in polycarbonate cages (Ehret, Emmendingen, Germany) under normal laboratory conditions [constant temperature (24–25°C) and humidity] with a 12-hour circadian cycle and fed with a diet of soaked standard laboratory rat chow (KZ Krka d.o.o., PE Krmila, Novo mesto, Slovenia) and water ad libitum. The rat chow was soaked in water to facilitate food intake.


Chemicals

A mixture of three anaesthetics was used to ensure general anaesthesia under which the placement of the closed-coil spring was performed. The anaesthetics were injected intraperitoneally: ketamine (Bioketan, Vetoquinol Zaklady Farmaceuticzne Biowet Gorozow, Poland, 50 mg/kg body weight), medetomidin hydrochloride (Domitor, Pfizer Animal Health, Louvain-la-Neuve, Belgium, 67 µg/kg body weight), and thiopental (Tiopental, Pliva, Zagreb, Croatia, 3 mg/kg body weight).


Orthodontic appliance

The orthodontic appliance consisted of a superelastic closed-coil spring (25 cN, wire diameter 0.15 mm, GAC International, Bohemia, New York, USA) placed between the first maxillary molar and the incisors. The closed-coil spring was attached to the upper left first molar with a stainless steel ligature wire (diameter 0.25 mm, Dentaurum, Ispringen, Germany) and to the incisors by surgical steel wire (4-0, multifilament, W310, Ethicon, Johnson & Johnson, New Jersey, USA). To improve fixation of the appliance, a 0.5-mm hole was made using a hard metal burr (HM 1, 204, 005, Meisinger, Neuss, Germany). The hole was drilled through the aproximal tooth surfaces, perpendicular to the longitudinal axis of the incisors at the gingival level. The steel wire was inserted through the hole and bent on the aproximal surface of the right incisor (Figure 1). Light curing bonding material (Tetric flow, Ivoclar Vivadent, Schaan, Lichtenstein) was used to protect the soft tissues from the sharp wire endings.


Figure 1
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  		Figure 1 Photograph showing the position of the drilled hole and of the orthodontic appliance.

 

Study protocol

The study protocol was set to 6 weeks since it takes a few weeks before tooth movement reaches the linear phase and ‘real’ tooth movement through bone occurs.

The animals were divided into three groups (n = 10):

Group I: a closed-coil spring was used and the animals were treated daily, at approximately the same time of day for 40 days, with 15 mg/kg body weight of TBC3214 subcutaneously. TBC3214 is an orally available, highly selective, ETA receptor antagonist (more than 100 000-fold selective, ETA versus ETB receptor) (Wu et al., 2001Go).
Group II: a closed-coil spring was used and the animals were injected daily with 0.1 ml of placebo (saline) subcutaneously, at approximately at the same time of day for 40 days.
Group III: the animals were injected daily with 0.1 ml of placebo (saline) subcutaneously, at approximately at the same time of day for 40 days.


Measurements and statistics

The distance between the most mesial point of the maxillary first molar and the most distal point of the ipsilateral incisor at the gingival level was measured on the experimental side (Figure 2). The measurements were undertaken using a digitronic calliper with an accuracy ±0.01 mm (144-15D, Wilson & Wolpert, Utrecht, The Netherlands) on days 0, 7, 14, 21, 24, 32, 37, and 40, while the animals were anaesthetized. All measurements were carried out twice by two investigators (JV and SS) independently within a few minutes. Inter-examiner reliability was tested with the intraclass correlation coefficient (ICC) and a paired t-test was used to assess systematic bias.


Figure 2
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  		Figure 2 Tooth movement on the experimental side. Tooth movement was significantly less in group I compared with group II.

 
In order to test the superelastic properties and delivered force of the closed-coil springs, the following method was used. Ten out of 20 closed-coil springs were randomly selected from eight boxes. The springs were activated from 1 to 20 mm and the reproducible force of 25 cN was determined over a range of 3–8 mm activation (Drevensek et al., 2006Go).

Statistical analysis was performed using two-way analysis of variance and Bonferroni's correction in GraphPad Prism 4.00 (GraphPad Software, San Diego, California, USA).


   Results
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 Summary
 Introduction
 Materials and methods
 Results
Discussion
 Conclusions
 Funding
 References
 
Systematic bias, which showed a value of P > 0.87, was tested using a paired t-test. The ICC was found to be 0.94 ± 0.02. Since reliability was within the standards, the mean value of the four measurements was used for further statistical analysis.

At the experimental side, a significant decrease was found in the measured distances between the incisors and molars between group I and group II on days 32 and 37 (P < 0.01) and on day 40 (P < 0.001). Changes in the distances significantly differed between groups I and II compared with group III on days 7, 14, 21, 24, 32, 37 and 40 (P < 0.001; Figure 2). In the control group, the distances on the experimental side significantly increased (P < 0.001) from day 0 to day 40.


   Discussion
Top
 Summary
 Introduction
 Materials and methods
 Results
 Discussion
Conclusions
 Funding
 References
 
The results of the present study demonstrate that TBC3214, a selective ETA antagonist, significantly reduces the rate of tooth movement in rats. A previous investigation has shown the possibility of ET-1 involvement in bone remodelling, using tezosentan, an ETA/ETB endothelin antagonist, which enhanced tooth movement in rats (Drevensek et al., 2006Go). Since tezosentan is an ETA/ETB endothelin antagonist, it could not be determined whether it enhanced tooth movement by acting on ETA and/or on ETB receptors.

Many studies have shown that ET-1 stimulates bone formation, acting predominantly on ETA receptors (Tatrai et al., 1992Go; Stern et al., 1995Go; Kasperk et al., 1997Go; Tsukahara et al., 1998Go; Nelson et al., 1999Go; von Schroeder et al., 2003Go; Guise and Mohammad, 2004Go). However, studies on the influence of ET-1 on bone resorption are contradictory. Some have shown that ET-1 inhibits (Alam et al., 1992Go; Nelson et al., 1999Go; Chiao et al., 2000Go), while others (Tatrai et al., 1992Go; Stern et al., 1995Go) have shown that ET-1 stimulates bone resorption. It is also not clear which endothelin receptor, if any, could be involved in bone resorption via ET-1. Tezosentan, a ETA/ETB receptor antagonist, was considered to increase bone resorption during the late phase of tooth movement (Drevensek et al., 2006Go).

Considering the existing data of the influence of ET-1 on bone resorption, two different explanations for the results of the present study can be postulated. On the one hand, it is possible that ET-1 enhances bone resorption via ETA receptors. Consequently, an ETA antagonist, such as TBC3214, would inhibit bone resorption and therefore decrease tooth movement in animals treated with ETA antagonist, as shown in the present study. On the other hand, it is possible that ET-1 inhibits bone resorption indirectly. This hypothesis can be explained by the fact that during ischaemic and hypoxic conditions, which can be expected during orthodontic tooth movement, the number of ETB receptors, which are responsible for endothelin clearance, increases (Kourembanas et al., 1991Go). Consequently, a greater activity of ETB versus ETA receptors would result in less tooth movement, because of the increased endothelin clearance, which is mediated via the ETB receptors.

There are several phases of orthodontic tooth movement. It takes from a few days to a few weeks before tooth movement reaches the linear phase and real tooth movement through bone occurs (Ren et al., 2004Go). As shown in Figure 2, there was a difference in tooth movement between groups I and II throughout the study period, but significant differences were only noted from day 32. This was also shown in a previous investigation (Drevensek et al., 2006Go), where a significant difference in tooth movement between the experimental and the control group was found on day 25. According to these results, it appears that ET-1 influences the late phase of orthodontic tooth movement.


   Conclusions
Top
 Summary
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
Funding
 References
 
Tooth movement was significantly less in animals treated with TBC3214, a highly selective ETA antagonist, compared with those treated with a placebo. Therefore, it is concluded that ET-1, which is the predominant isoform of endothelin in humans (Miyauchi and Masaki, 1999Go; Rich and McLaughlin, 2003Go), is involved in the mechanism of orthodontic tooth movement in rats. Further studies, including histological, immunohistochemical, and biomolecular techniques, will establish a more exact role for endothelin receptors in orthodontic tooth movement.


   Funding
Top
 Summary
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 Funding
References
 
Slovenian Ministry of Higher Education, Science, and Technology (P3-0067).


   Acknowledgement
 
We wish to thank Tommy A. Brock of Biological Sciences Encysive Pharmaceuticals for generously providing the ETA antagonist TBC3214 for this study.


   References
Top
 Summary
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 Funding
 References
 

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