Introduction

Endothelin 1 (ET-1) plays an important role in the regulation of bone metabolism in physiological as well as in pathophysiological processes1,2,3,4. It has a known role in the maintenance of bone homeostasis and the regulation of osteoblastic function. ET-1 stimulates the proliferation, differentiation and activity of osteoblasts5,6 and inhibits osteoblast apoptosis, promoting osteoblastic growth7. ET-1 acts through both endothelin A (ETA) and endothelin B (ETB) receptors. Specifically, it has been shown that the ET-1/ETA axis is an important regulator of osteoblast activity; targeted inactivation of ETA in mature osteoblasts induced lower tibial trabecular bone volume in vivo8. However, less is known about the role of ETB in osteogenesis. In one study, treatment with the dual ETA and ETB antagonist Macitentan showed decreased vertebral bone mass in mice, potentially from decreased osteoblast activity as well as from the increased osteoclast activity9.

Several studies also indicate the involvement of endothelins during orthodontic tooth movement (OTM)10,11,12. Orthodontic movement is a consequence of applying a force to the teeth. It is a mechanism that involves the biomechanical adaptation of the alveolar process and supporting periodontium. Alterations in the vascularity within the periodontal ligament (PDL), the connective tissue that connects a tooth to its surrounding alveolar bone, may trigger responses at the cellular level, such as alveolar bone modelling13,14. During OTM areas of pressure and tension are formed in the PDL. At pressure areas, which appear in the direction of the application of force, alveolar bone is resorbed, and at tension areas, which appear on the opposite side, new bone is formed (Fig. 1). Therefore the animal model of OTM can be used to study the accelerated bone modelling15,16. OTM consists of three different phases—the initial, lag and late phase. In the late phase linear teeth movement can be observed17.

Figure 1
figure 1

Model of the OTM. Applying of orthodontic force to the tooth compresses the PDL. At the compression side of the tooth, which appears in the direction of the orthodontic force, bone resorption takes place, carried out mainly by osteoclasts. At the tension side, osteoblasts are responsible for the bone formation process.

Full size image

ET-1 release in the PDL begins after 3 h of continuous loading of a rat molar18. However, in the alveolar bone ET-1 gene expression levels increased for the following 4 weeks after the start of the application of orthodontic force and predominated in the late phase of OTM, when the ETB expression rate was also upregulated11. A selective ETA antagonist significantly increased alveolar bone volume and decreased osteoclast volume and the amount of OTM, indicating decreased bone resorption in the late stage of OTM, and confirming the role of ETA in accelerated bone modelling11. On the other hand, a dual-selective endothelin antagonist (ETA/ETB) increased the amount of OTM in rats after 25 days of treatment12. Furthermore, the gene expression levels of both ETA and ETB were increased in the late phase of OTM11, when bone formation on the tension side and bone resorption on the pressure side of the loaded tooth were reported17, suggesting that both receptor subtypes could be involved in the process of the late stage of OTM.

The multipotent stem cells in the alveolar bone marrow, the PDL and the periosteum all participate in the regulation of bone remodelling and tooth movement. Mesenchymal stem cells (MSCs), when stimulated by a mechanical strain, differentiate into an osteo-chondrogenic lineage with increased expression of osteoblastic and chondrogenic markers19. Periodontal ligament stem cells (PDLSCs) are tissue-specific MSCs in PDL that play an important role during OTM20. It has been shown that both tension and compression can regulate the osteogenic differentiation of PDLSCs16,21. Interestingly, it has also been reported that ET-1 promotes the osteogenic differentiation of bone marrow-derived MSCs22 and PDLSCs23. Moreover, some studies mention an important role of both ETA and ETB receptors in the differentiation of different types of MSCs into different cellular phenotypes24,25.

ET-1 has been shown to have anti-apoptotic activity in numerous tissues. For example, it inhibits bone degradation by the induction of anti-apoptotic activity, and stimulates bone formation via endothelin ETA receptors in vitro and in vivo26. ET-1 is also known as an anti-apoptotic factor in osteoblasts7, and it promotes osteosarcoma cell invasion and survival against cisplatin-induced apoptosis through the ETA receptor4. The anti-apoptotic effects of ET-1 have been described in several other tissues: neurons27, cardiomyocytes28, and airway smooth muscle cells29.

The role of ETB has mostly been studied in cancer cell lines and in cancer tissue cultures30. The activation of ETB receptors by ET-1 has been shown to affect the processes involved in the inhibition of cancer, inducing cell death by apoptosis and promoting ET-1 clearance31,32. ETB expression has also been reported in many tumour types, including prostate cancer33,34 melanomas35, and oligodendrogliomas36. For example, in prostate cancer, the downregulation of ETB results in a higher local concentration of ET-1 which, through the stimulation of ETA receptors, facilitates cancer progression, including proliferation, escape from apoptosis and new bone formation34. ETB has been predominantly classified as a “clearance receptor”, and the role of circulatory ET-1 clearance by ETB has been confirmed in several studies32,37,38. In healthy men an injection of a selective ETB antagonist increased the plasma concentration of ET-1 and confirmed the crucial role of ETB in the clearance of endothelins in humans39.

Despite the fact that the role of ETA in bone modelling during OTM has been studied, the role of ETB in bone modelling in OTM is not well understood at present. The aim of this study was to determine the role of ETB in bone modelling in OTM using ETB–KO rats and to evaluate its effect on osteoclastogenesis and osteoblastogenesis in comparison to bone modelling in ETB–WT rats.

Material and methods

Laboratory animal model

The study was performed on ETB knockout (ETB-KO −/−) and wild type (ETB-WT +/+) rats. The ETB-KO rat line and its control line ETB-WT was established at the laboratory of Masashi Yanagisawa, PhD, at Howard Hughes Medical Institute (University of Texas Southwestern Medical Centre, Dallas, Texas, USA). The ETB-KO animals were incorporated with a dopamine beta-hydroxylase (DβH) transgene to enable the development of a normal enteric nervous system. The DβH transgene was also inserted into the control animals40. The rats were bred in a homozygous line at the Faculty of Medicine, University of Ljubljana (Slovenia). In this study we used 32 male ETB-KO rats (285 ± 27 g, 13–15 weeks old) and 32 male ETB-WT rats (286 ± 30 g, 13–15 weeks old). The animals were housed as well as procedures were identical to our previous studies11. The part of daily intake of rat chow (Teklad 2016 Global rodent diet, Harlan, The Netherlands) was soaked in water to facilitate food intake due to its mild impairment during orthodontic force application.

To ensure general anaesthesia, the anaesthetics were injected intraperitoneally: ketamine 50 mg/kg body weight (Bioketan, Vetoquinol Biowet, Gorzów Wielkopolski, Poland), medetomidine hydrochloride 67 mg/kg body weight (Domitor, Pfizer, Brooklyn, NY, USA), and thiopental 3 mg/kg body weight (Tiopental, Pliva, Zagreb, Croatia)12.

All the animal procedures and the study protocol were approved by the Ethics Committee for Animal Experiments of the Administration of the Republic of Slovenia for Food Safety, Veterinary Sector and Plant Protection (34401-62/2008/20), and complied with the guiding principles in ''The Care and Use of Animals''.

Study protocol

Thirty-two rats were divided into the following four groups: (1) ETB-KO appliance group (n = 8); (2) ETB-KO control group (n = 8); (3) ETB-WT appliance group (n = 8); (4) ETB-WT control group (n = 8).

The orthodontic appliance consisted of a superelastic closed coil spring (25 cN; wire diameter, 0.15 mm; GAC Dentsply International, York, PA, USA) which was placed between the first and second left maxillary molars and the incisors by a stainless-steel ligature in the ETB-KO and ETB-WT appliance groups11. The coil spring was attached to a steel thread placed around the first and second molar on one side and through a drilled hole into the upper incisors on the other side (Fig. 2). The hole was drilled laterally in the incisors through the area where the vivid tooth structures were unaffected12. The orthodontic appliance was placed in each animal under general anaesthesia at the beginning of the study, and replaced to the correct position every 7 days, ensuring its proper activation and the exertion of constant force on the teeth41.

Figure 2
figure 2

Schematic view of the orthodontic appliance.

Full size image

Tooth movement measurements

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 sides in all four groups. The measurements were obtained using a digital calliper (Digitronic Calliper, 144–15 D (Wilson & Wolpert, Utrecht, The Netherlands)) while the animals were under general anaesthesia at days 0 and 35. All the measurements were carried out twice by two investigators independently within a period of few minutes and the reliability of the measurements was assessed by using the intraclass correlation coefficient (ICC) as described in previous studies11,42. Tooth movement was calculated by subtracting the distance between the teeth on day 35 from the distance between the teeth on day 0.

Preparation of histology samples and bone histomorphometry

On day 35, all the animals were sacrificed by intraperitoneal injection of anaesthetics and carbon dioxide. Tissue samples of the maxilla containing all 3 molars were taken. Samples were prepared in vertical section perpendicular to the occlusal plane of the molars. Paraffin blocks were then prepared and stained with haematoxylin and eosin (Fig. 3) as previously published in Plut et al.42.

Figure 3
figure 3

(A) Schematic representation of the examined areas in part of the maxilla with all three molars (M1, M2, M3). Alveolar bone volume was determined around the first and second maxillary molars (area surrounded by dashed line). Osteoblast and osteoclast volumes were determined along the mesial and distal roots of the second maxillary molar (green areas surrounded by dashed line). The arrow shows direction of tooth movement. (B) Examples of osteoclast (left picture) and osteoblast (right picture) under 40-fold magnification. Al.b—alveolar bone; Ob—osteoblast; Oc—osteoclast; PDL—periodontal ligament; T—tooth.

Full size image

Bone histomorphometry was used to determine alveolar bone, osteoclast and osteoblast volume density in all four groups. 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. As described by Sprogar et al.11, the alveolar bone area was expressed as the percentage of alveolar bone area versus the tissue area consisting of tooth, PDL, connective tissue and bone marrow spaces. In addition, the osteoblast and osteoclast areas were defined as the alveolar bone area covered with osteoblasts or osteoclasts versus alveolar bone area. The cells were counted in the alveolar bone alongside the mesial and distal roots of the second molar42. However, because 20 sections from each specimen were examined, alveolar bone area, osteoblast area and osteoclast area were extrapolated to alveolar bone volume, osteoblast volume and osteoclast volume, as we already described in previous studies11,42.

Endothelin 1 (ET-1) determination

Endothelin 1 levels were measured in rat serum in all 4 groups using the commercially available kit Endothelin 1 ELISA Kit (ab133030, Abcam, Cambridge, MA, USA), according to the manufacturer’s instructions. Briefly, standards and samples were added to the designated wells of a microplate, precoated with an endothelin-1 specific antibody. The plate was incubated at room temperature for 1 h, washed and HRP-conjugated. The endothelin 1 detection antibody was added to each well. After incubation, the unbound detection antibodies were removed and TMB was added to visualise the HRP enzymatic reaction. After incubation, a stop solution was added and the absorbance was read at 450 nm, with correction at 570 nm, using Tecan Safire (Tecan Group Ltd., Switzerland).

RNA isolation and semiquantitative RT-PCR

Osteocalcin and DMP1 gene expression levels were used to determine osteoblast activity, and the cathepsin K gene expression level was used to determine osteoclast activity in all 4 groups43,44. The maxillary bones with all 3 molars and their PDLs were excised and immediately frozen in liquid nitrogen. RNA isolation and semiquantitative RT-PCR was performed as described by Plut et al.42. Oligonucleotides for cathepsin K, osteocalcin and DMP1 were chosen from predesigned assays (TaqMan Gene Expression Assays, Applied Biosystems). Thermal cycling, construction of standard curve and cDNA amplification and quantification were performed as we reported in a previous study43. In order to exclude variations from different inputs of total mRNA to the reaction, data on cathepsin K, osteocalcin and DMP1 were normalised to an internal housekeeping gene, GAPDH, for which data was obtained by using TaqMan GAPDH predesigned assays (TaqMan Gene Expression Assays, Applied Biosystems). All the reactions for standard samples and for samples from all 4 groups were performed in duplicate. The data were averaged from the values obtained in each reaction43. For all the transcripts tested, a time-course-dependent gene expression consensus profile was observed after normalisation to the expression of the housekeeping gene GAPDH.

Statistical analyses

The data were expressed as means ± standard error of the mean (SEM) and calculated for each parameter for all the animal groups. The evaluated parameters were tooth movement, alveolar bone volume density, osteoclast and osteoblast volume densities, serum ET-1 level, and gene expression levels of cathepsin K, DMP1 and osteocalcin. Comparisons within and between the groups were performed using analysis of variance (ANOVA), followed by the Tukey multiple comparison test. A P value less than 0.05 was considered statistically significant.

Interexaminer reliability for the measurements of the distance between the teeth was tested using the intraclass correlation coefficient (ICC).

In the results, not all the groups contained the initial number of rats (n = 8 per group). During the experiment some of the rats had to be excluded. The number of samples in each group is explicitly stated in the Figures.

Results

Orthodontic tooth movement and physiologic distal drift measurements after 35 days

The overall mean value of the ICC for all the measurements of the distances was 0.938. In the ETB-KO appliance group the amount of OTM (1.67 mm ± 0.10 mm) was significantly less pronounced than in the ETB-WT appliance group (2.28 mm ± 0.17 mm) on day 35 (P = 0.0255). The physiologic distal drift did not significantly differ between the ETB-KO and ETB-WT groups (Fig. 4).

Figure 4
figure 4

Amount of total tooth movement after 35 days. Significant differences were observed between the ETB-KO and ETB-WT appliance groups (P = 0.0255) and between appliance and control groups (P < 0.0001). The physiological distal drift was non-significantly decreased in the ETB-KO control group in comparison to the ETB-WT control group. The data are presented as mean ± SEM and analysed by the one-way ANOVA and Tukey post hoc tests.

Full size image

Histomorphometric analyses

The histomorphometric analysis showed that after 35 days of OTM the alveolar bone volume was significantly lower in the ETB-KO appliance group (35.26% ± 1.47%) in comparison to ETB-KO control group (44.45% ± 1.63%) (P = 0.0004). Furthermore, the alveolar bone volume was significantly less in the ETB-KO appliance group than in the ETB-WT appliance group (45.26% ± 1.74%) (P = 0.0001) (Fig. 5a). No significant differences in the osteoblast volume were observed between the groups. Osteoblast volume was non-significantly increased in the ETB-KO control group in comparison to the other three groups (Fig. 5b). The osteoclast volume was significantly increased in the ETB-KO (1.08% ± 0.13%) and ETB-WT appliance (1.30% ± 0.10%) groups compared to their controls (0.47% ± 0.06% and 0.69% ± 0.06%, respectively) (P < 0.0001). The osteoclast volumes in the ETB-KO appliance and the ETB-KO control groups were less than in the ETB-WT groups, but the differences were not significant (Fig. 5c).

Figure 5
figure 5

Histomorphometric analyses of the maxillary bone specimens after 35 days. The alveolar bone volume in the ETB-KO appliance group was significantly less than in the ETB-WT appliance group (P = 0.0001) (a); no significant (NS) differences in the osteoblast volume were observed between the groups (b); the osteoclast volume was significantly increased in the ETB-KO and ETB-WT appliance group compared to the control groups (P < 0.0001) (c). The data are presented as mean ± SEM and analysed by the one-way ANOVA and Tukey post hoc tests. (−)—control, ( +)—appliance.

Full size image

Serum endothelin 1 levels

The serum endothelin level (ET-1) on day 35 was significantly higher in the ETB-KO control rats (3.04 pg/ml ± 0.43 pg/ml) compared to the WT control rats (1.46 pg/ml ± 0.18 pg/ml) (P = 0.0002) and between the ETB-KO appliance rats (2.78 pg/ml ± 0.13 pg/ml) and the ETB-WT appliance rats (1.70 pg/ml ± 0.12 pg/ml) (P = 0.0023) (Fig. 6).

Figure 6
figure 6

Blood serum endothelin level (ET-1) during the experimental period (35 days). The serum endothelin level (ET-1) was significantly higher in the ETB-KO control rats than in the WT control rats (P = 0.0002), and in the ETB-KO appliance group compared to the WT appliance group (P = 0.0023). The data are presented as mean ± SEM and analysed by the one-way ANOVA and Tukey post hoc tests. (−)—control; ( +)—appliance.

Full size image

RT-PCR analysis

The gene expression level of osteocalcin on day 35 was significantly downregulated in the ETB-KO appliance group compared to the ETB-WT appliance group (P = 0.0157). A significant difference in the gene expression level of osteocalcin was also determined between the ETB-KO appliance group and the ETB-KO control group (P = 0.0288) (Fig. 7a). Similarly, the gene expression of DMP1 was significantly downregulated in the ETB-KO appliance group in comparison to the WT appliance group (P = 0.0040) (Fig. 7b). The gene expression level of cathepsin K was downregulated in both appliance groups compared to the control groups. Significant differences were determined between the ETB-KO control group compared to the ETB-KO appliance group (P = 0.0314) (Fig. 7c).

Figure 7
figure 7

Relative gene expression levels of osteocalcin, DMP1 and cathepsin K in the alveolar bone and periodontal ligament after 35 days of OTM. Osteocalcin (a) and DMP1 (b) gene expression levels were significantly downregulated in the ETB-KO appliance group compared to the ETB-WT appliance group (P = 0.0157 and P = 0.0040, respectively). A significant difference was observed in the gene expression level of cathepsin K between the ETB-KO groups (P = 0.0314), but not between the ETB-WT groups (c). All gene expressions were normalised to the reference gene GAPDH. The data are presented as mean ± SEM and analysed by the one-way ANOVA and Tukey post hoc tests. (−)—control, ( +)—appliance.

Full size image

Discussion

The results of this study showed that ETB is involved in bone modelling during the late stage of OTM in the rat animal model. The amount of OTM after 35 days of the experiment was significantly less in the ETB-KO appliance group than in the ETB-WT group (P = 0.0255). There was a significant difference in the alveolar bone volume in the ETB-KO appliance group compared to the ETB-WT appliance group (P = 0.0004), probably due to diminished osteoblast activity in the ETB-KO appliance group.

Some of the ETB antagonists showed significant inhibition of ET-1 effects in vitro45,46; however, none of the available antagonists have an established pharmacological and toxicological profile. Furthermore, they have to be administered intravenously and daily intravenous application over a longer period of time represents a great deal of stress for the animals. To study the effects of ET-1 in a reduced ETB response, the most suitable model is to use ETB knock-out animals. The ETB-KO strain of rats used in the present study is described as a natural mutation in the progeny of a Wistar rat. Mutations in the ETB gene have been linked to Hirschsprung’s disease in humans, a congenital disease characterised by aganglionic megacolon, an absence of enteric ganglia, and a lack of innervations to the lower gastrointestinal tract. The disease is associated with polymorphism and several missense mutations in the ETB gene which lead to decreased expression, changes in cell signalling, and loss of endothelin ET receptor function47,48. The animals used in this study were incorporated with a transgene (DβH) to enable the development of a normal enteric nervous system. The resulting transgenic rats are healthy but present with a total absence of ETB in all non-adrenergic tissues40. The animal model of OTM used in this study had already been confirmed as appropriate for studying the role of endothelin system in bone modelling10,11,12. The advantage of the model was the minimally invasive placement of the coil spring between the molars and incisors to maximally avoid injuries to the vital structures in the incisors and surrounding structures which may otherwise cause an inflammatory response and interfere with the results of the OTM. The force used in the experiment was constant and continuous41,49. The duration of the experiment is important in studying the processes in bone modelling during OTM. Bone formation on the tension side and bone resorption on the pressure side has been reported in the late stage of OTM; a time period usually around 2–4 weeks after the force has been applied17.

Both appliance groups showed lower alveolar bone volume compared to their control groups. Furthermore, the alveolar bone volume in the ETB-KO appliance group was significantly less than that of the ETB-WT appliance group. Differences in alveolar bone volume during physiological and pathological processes depend on the relationship between bone formation and bone resorption. Similarly to our study, histomorphometric analyses in previous studies have shown that alveolar bone volume was less in appliance groups than in their control groups11,42,43. We studied bone formation by determining osteoblast volume and osteoblast activity using osteocalcin and DMP1. There were no significant differences in osteoblast volumes between the four groups, but there was a significant decrease in osteoblast activity as determined by the gene expression levels for osteocalcin and DMP1 in the ETB-KO appliance group compared to the ETB-WT appliance group after 35 days of OTM. It has been shown that bone formation after the application of orthodontic force is increased predominantly by stimulating the differentiation of osteoblasts, and to a lesser extent by an increase in the number of these cells50. This is in concordance with our results, which show no considerable difference in osteoblast volume between the groups, but significant changes in osteoblast activity. Osteocalcin plays an important role in the maturation of mineral species51, and modulates osteogenic differentiation of MSCs52; DMP1 has a similar role in osteoblast differentiation and matrix mineralisation. Interestingly, it has also been reported that recombinant DMP1 induces the osteogenic differentiation of human periodontal ligament cells53, and appears to play an important role in the osteogenic differentiation of dental follicle stem cells54.

In a study on Bone Marrow-Derived Stem Cells (BMSCs) it was shown, using specific antagonists, that both receptors ETA and ETB are involved in the differentiation of BMSCs into active osteoblasts, and the osteogenesis of BMSCs was attenuated by blocking ETA and/or ETB receptors. The findings of this study reveal that both ETA and ETB receptors and downstream AKT and ERK signalling are involved in ET-1 primed lineage specification of MSCs24. Similarly, a study on ETB-KO mice showed less fibroblast activation and myofibroblast formation in response to bleomycin or ET-125. Therefore, in our study, the lower expression levels of osteoblast activity markers, DMP1 and osteocalcin could be a result of attenuated osteoblast maturation and/or osteogenic differentiation of MSCs, a process that is mediated through both receptors ETA and ETB. In the absence of ETB in the ETB-KO appliance group, osteogenesis is attenuated, resulting in a lower alveolar bone volume and a decreased amount of OTM (Fig. 8).

Figure 8
figure 8

Possible mechanisms in PDL space are represented in this Figure, ultimately leading to decreased alveolar bone volume and a lower amount of OTM in the ETB-KO rats. The crossed-out triangles indicate the absence of ETB receptors. ET-1—Endothelin-1; ETA—Endothelin receptor A; ETB—Endothelin receptor B; EC—Endothelial cell; PDLSC—Periodontal Ligament Stem Cell.

Full size image

The bone resorption process was studied by determining osteoclast volume and osteoclast activity using cathepsin K, a marker of bone resorption. A considerable increase in osteoclast volume was determined in both appliance groups compared to the control groups after 35 days of OTM. Similar results were obtained in a previous study, where osteoclast volume increased in all animal appliance groups11. In the late stage of OTM, we expected the upregulation of the cathepsin K gene expression level in the ETB-KO and ETB-WT appliance groups, whereas the osteoclast volume increased significantly in both appliance groups43. However, a significant downregulation of the gene expression level of cathepsin K appeared in the ETB-KO appliance group compared to ETB-KO control group. Several lines of evidence suggest that changes in osteoclast function and volume/number do not always happen simultaneously55. In a previous study it was shown that the absence of ETB in osteoclast specific ETB-KO mice impairs the formation of mature osteoclasts and impairs bone resorption activity with no influence on the expression of osteoclastogenic genes56. ET-1/ETB axis was shown to enhance osteoclast differentiation via co-stimulation of RANKL and M-CSF signalling and ETB deficiency impaired bone resorption activity and formation of mature osteoclasts56. However, in the present study, there were no significant differences between the ETB-KO and ETB-WT appliance groups in terms of osteoclast volume and osteoclast activity. The applied force due to the coil spring used in the OTM model induced osteoclast activity independently of the presence or absence of ETB or increased circulatory ET-1.

In the present study, the amount of OTM in the ETB-KO appliance group was significantly less than in the ETB-WT appliance group. Because of the diminished alveolar bone volume and lower osteoblast activity, a higher amount of OTM would initially be expected. However, we observed a lower amount of OTM, and similar results were also reported in Plut et al.42. During OTM, due to the lower osteoblast activity, bone formation at the tension site cannot keep up with bone resorption at the compression site, and the consequences are a widening of the PDL space and increased tooth mobility57. Tooth movement requires a coupling of bone resorption and bone formation. Due to the delayed bone formation in the absence of ETB there may be a further aggravation of the uncoupling of bone formation and bone resorption, expressing as suppressed bone turnover and resulting in lower amount of OTM. In rats, molars naturally drift distally. Therefore, there is predominately bone resorption on the distal side and bone formation on the mesial side of molar roots58. In the present study, the distal drift was smaller in the ETB-KO control group than in the ETB-WT control group, but there was no significant difference. These results suggest a different mechanism of alveolar bone turnover under physiological conditions in comparison to OTM, shown in the ETB-KO groups.

In the ETB-KO control group the serum ET-1 level was significantly higher than in the ETB-WT control group. It was also significantly elevated in the ETB-KO appliance group in comparison to ETB-WT appliance group. This is in concordance with many studies that confirmed the role of ETB as a ET-1 clearance receptor32,37,38,39,59. The lower amount of OTM in this study could be assigned to several processes, among them attenuated osteoblast maturation and increased ET-1 due to a lack of circulatory ET-1 clearance. High ET-1 levels are normally almost exclusively cleared by endothelial ETB, which was lacking in our animal model. It has been shown that chronic exposure of ET receptors to increased plasma ET-1 levels results in a significantly reduced density of ETA in mice aortas, correlating with a reduction in functional response to ET-160. Similarly, ETB-KO mice were found to have a 45% lower ETA density and significantly reduced expression (lower ETA mRNA) in peripheral tissues with no change in receptor affinity. A potential mechanism of reduced ETA density in ETB-KO models was proposed as the compensatory downregulation of the receptor in response to higher circulating ET-1 levels as a result of a lack of ETB clearing receptors or some other role of ETB in the development of cells expressing ETA receptors60,61. It is therefore possible that in our study high serum ET-1 in the ETB-KO groups resulted in downregulation of the ETA receptors, which already have an established role in bone modelling, resulting in diminished alveolar bone volume and lower amount of OTM (Fig. 8).

Many studies report interactions (cross talk) between ETA and ETB receptors, which means that the activation or inhibition of one receptor subtype can alter the function of another receptor subtype. For example, using ETB receptor-deficient rats it was reported that both ETA and ETB are involved in ET-1-induced DNA synthesis in astrocytes, accompanied by MAPK activation62. Similarly, it was shown that both ETA and ETB regulate lung myofibroblast proliferation, indicating possible interactions between receptor subtypes63. In addition, endothelin receptors (ETA and ETB) also exist in the form of homo- and heterodimers64, and for many receptor heterodimers, co-expression of both receptor subtypes is crucial for functional receptor activity, pharmacological proprieties, maturation, and proper cell-surface trafficking65,66. Another example of cooperation between receptor subtypes are HEK 293 cells which, transfected with both receptors, display a considerably prolonged increase in intracellular Ca2+ concentration in response to ET-1 or the selective ETB receptor agonist Sarafotoxin 6c, in comparison to more transitory responses of cells transfected with either receptor subtype alone67. It is therefore possible that similar interactions between the two receptor subtypes are also necessary for normal bone modelling during the late stage of OTM.

Another potential explanation for reduced OTM is anti-apoptotic activity of elevated ET-1 in the two ETB-KO groups. Specifically, apoptosis of osteocytes is critical for osteoclast activation and resorption at the compression site of the tooth during OTM68,69. Moreover, in a study of OTM with micro-osteoperforations it was shown that the rate of OTM is increased by increased apoptosis and cell proliferation of PDL cells70. Therefore, the lower amount of OTM in the absence of ETB could be a result of several different processes, including the anti-apoptotic activity of ET-1 on numerous cells in periodontal tissues. One limitation of the study, however, are only two time points in the experiment. Thus, no exact mechanism of the action can be elucidated. We rather propose possible mechanisms through which ETB-KO modulated bone modelling during OTM (Fig. 8).

Conclusion

Our results showed for the first time that ETB is involved in bone modelling in the late stage of OTM. ETB-KO resulted in lower osteoblast activity and therefore decreased alveolar bone volume and lower amount of OTM. This could be due to the role of ETB in osteoblast maturation and/or the differentiation of mesenchymal precursor cells, the adaptive downregulation of ETA as a response to high levels of circulating ET-1 or anti-apoptotic activity of ET-1. Further research is needed to explain the exact mechanism by which ETB modulates bone modelling in OTM, and which of the proposed mechanisms is predominant.