The regulatory role of the RANKL/RANK/OPG signaling pathway in the mechanisms of tooth eruption in patients with impacted teeth

The study was registered with the research center of Bogomolets National Medical University. Ethical approval for the study was obtained from the Ethics and Research Committee of Bogomolets National Medical University (Protocol No 95 from 05/05/2016). Written informed consent was obtained from each patient. Any uncooperative patients and patients who had not provided consent to participate in the study were excluded.

A total of 18 participants were analyzed. Calibrated dental hygienists performed whole-mouth oral examinations before orthodontic treatment. Table 1 shows patient’s characteristics including age, gender, type of orthodontic treatment, and the principle оf group formation. Among 18 participants, the gender distribution was as follows: 9 males (25.44 ± 3.43) years and 9 females (25.11 ± 3.06). The inclusion criteria of patients in the control group were the obligatoriness to remove аn erupted third molar and orthodontic prescriptions for the installation of the stainless steel brackets simultaneously. The inclusion criteria in the tooth impaction group were the presence of tooth impaction (maxillary third molars) and orthodontic prescriptions for installation of the stainless steel brackets. The same brand and model of stainless steel brackets (Dentaurum, Germany) were used for all patients. Bone samples from control patients and patients with tooth impaction were divided into three groups: 1 – bone tissue of healthy persons from the area of the normally eruthird molar that has been removed according to orthodontic prescriptions (control group); all teeth were normally erupted in patients of this group (based on the data of X-ray diagnostics); 2 – bone tissue of patients with tooth impaction, that was taken from the area of normally erupted third molar that has been removed according to orthodontic prescriptions; 3 – bone tissue of patients with tooth impaction, collected near the impacted third molar.

The surgical approach was used to collect bone samples near the maxillary third molars from the control group. Under the tuberal and greater palatine nerve block (sol. Ubistesin 4%, 2 ml) the third molar was removed by the traditional method. During the inspection, free bone tissue fragments of the alveolar socket were collected. To take the samples of bone tissue from patients with tooth impaction a surgical approach was used as well. After infraorbital and nasopalatine nerve block (sol. Ubistesin 4%, 2 ml) an incision of the mucous membrane was carried out followed by separation of the mucoperiosteal flap. The bone fragment that covered (or partially covered) the crown of the tooth was removed (osteoectomy) by bone nipper in the area of the impacted tooth. After collection bone samples were frozen in liquid nitrogen to prepare total protein lysates.

Levels of RANKL, RANK, OPG, osteocalcin (OC), NF-κB p65 subunit, NFATc1, and caspase-3 in bone tissue were determined by western blot analysis. Bone tissue samples were ground in a porcelain mortar with liquid nitrogen. After that samples were lysed by an incubation on ice for 20 min in the appropriate volume of RIPA buffer 1:6 weight-to-volume ratio (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% Triton X-100; 1 mM EGTA; 0.1% SDS; 1% sodium deoxycholate; 10 mM sodium pyrophosphate) containing protease inhibitor cocktail (PIC, Sigma, USA), sonicated and then centrifuged at 14000 g for 20 min at + 4 °C. Protein concentrations in the lysates were measured by Lowry’s method. 30 μg of protein were loaded onto 10% SDS polyacrylamide gels. After SDS-PAGE, the proteins were transferred onto a nitrocellulose membrane (350 mA, 1 h). Immunoblotting was conducted with primary antibodies against RANKL and OPG (1:250; Santa Cruz, USA), RANK, OC and caspase-3 (1:500; Santa Cruz, USA), NF-κB p65 subunit (1:500; Thermo Fisher Scientific, USA), NFATc1 (1:1000; Thermo Fisher Scientific, USA) and β-actin (1:10000; Sigma-Aldrich, USA). Primary-antibody-bound membranes were incubated with peroxidase-conjugated secondary antibodies: anti-mouse IgG (Fab Specific)-Peroxidase (1:2500; Sigma, USA), anti-rabbit IgG (H + L)-HRP conjugate (1:4000; Bio-Rad Laboratories, Inc., USA) or anti-goat IgG (H + L) (1:2500; Invitrogen, USA). Thereafter the membranes were developed with chemiluminescent agents: p-coumaric acid (Sigma-Aldrich, USA) and luminol (AppliChem GmbH). Tissue levels of all proteins were normalized to β-actin. The immunoreactive bands were quantified using Gel-Pro Analyzer32 software.

As the RANKL/RANK/OPG signaling axis regulates the interplay between osteoclasts and osteoblasts that can influence bone remodeling [6], we first determined the content of the RANKL/RANK/OPG system components in the bone tissue of patients with tooth impaction. The results of the study presented in Fig. 1 show that there is no difference in the relative protein content of RANK (Fig. 1 a, b, Tukey’s test, p-value > 0.05) and osteocalcin (Fig. 1 a, d, Tukey’s test, p-value > 0.05) between the samples of healthy persons (control group) and samples, that were taken near the normally erupted third molar in patients with tooth impaction. At the same time, it was shown a slight tendency to decreased content of osteoprotegerin (by 1.2 times) in the bone tissue near the healthy tooth in patients with tooth impaction compared with the control (Fig. 1c, Tukey’s test, p-value 0.048) and we observed a significant decrease in the RANKL content (membrane-bound form – by 1.6 folds, Tukey’s test, p-value 0.021, and soluble form – by 10 folds, Tukey’s test, p-value 0.003) (Fig. 2c, d).

Completely different results were obtained when studying the area of ​​the impacted third molars. Western blot analysis data revealed a 1.73-fold elevation of the RANK level in the IT area vs. control (Fig. 1b, Tukey’s test, p-value 0.0008), indicating an accumulation of osteoclast precursors. Moreover, a significant increase in the relative content of OPG (Fig. 1c, Tukey’s test, p-value 0.012), osteocalcin (Fig. 1d, Tukey’s test, p-value 0.021) and total RANKL (Fig. 2b, Tukey’s test, p-value 0.009) proteins (by 1.48, 1.42, and 1.46 folds, respectively) was demonstrated as well, that could correlate, at least partially, with the high activity of osteoblasts and/or its precursors in the area of IT.

Physiological effects of OPG and RANKL on bone tissue metabolism are known to be largely determined by the ratio of their synthesis [13]. Based on the measurement of protein levels of RANKL/RANK/OPG signaling pathway components by western blot analysis, we obtained the ratios applied for assessing the status of local bone remodeling in the areas near the healthy and impacted teeth in patients with tooth impaction. It was established a significant decrease in the RANK/RANKL (2.6-fold) ratio and an increase in RANKL/OPG (2.5-fold) ratio in the area near the impacted tooth compared with the healthy tooth (Table 2). Declined RANK/RANKL ratio in the IT area may reflect the disturbances in the ligand-to-receptor binding and indicate the reduced effectiveness of signal transduction in the RANKL/RANK/OPG axis in addition to elevated RANKL/OPG ratio. Based on these observations we can assume that the local interrelation between bone resorption and bone formation in the IT area may lead to delayed tooth eruption/tooth impaction.

Binding RANKL to RANK leads to the further activation of NF-κB and translocation of this transcription factor to the nucleus to stimulate osteoclastogenesis [14]. To address whether excessive RANKL and RANK levels in bone tissue near the area of tooth impaction are engaged in the downstream transcriptional activation, we explored the involvement of NF-κB in the mechanisms of tooth eruption.

We demonstrated a slight decrease in the NF-κB (Fig. 3a, b, Tukey’s test, p-value 0.043) and NFATc1 (Fig. 3a, c, Tukey’s test, p-value 0.038) levels by 17 and 20% respectively in the bone tissue selected near the healthy tooth compared vs. control patients. Interestingly, it was demonstrated for the first time that despite the activation of the RANKL/RANK/OPG system in the impaction area, the content of both NF-κB and NFATc1 did not significantly change compared with the control group (Tukey’s test, p-value > 0.05), indicating a blocked or delayed process of osteoclastogenesis near the IT although an accumulation of osteoclast precursors in this area was observed.

Since caspase cascades play a key role in the activation of cell apoptosis [15] and may be triggered by NF-κB, we determined the levels of procaspase-3 (32 kDa) and its active subunit p17 (17 kDa). It was shown a decrease in the content of procaspase-3 (32 kDa) in the area of ​​a healthy tooth of patients with tooth impaction by 28% (Tukey’s test, p-value 0.031), while in the area of impacted tooth its content was increased by 1.32 folds compared with control (Fig. 3e, Tukey’s test, p-value 0.028). Сonsidering the active caspase-3 enzyme is a heterodimer consisting of two p17 and two p11 caspase-3 subunits [16], it was also important to evaluate the content of the activated p17 form. We detected an increase of its protein level in the area of the healthy tooth by 2.26 folds compared vs. control (Tukey’s test, p-value 0.0007), and an elevation in the area of the impacted tooth by 1.78 folds (Fig. 3f, Tukey’s test, p-value 0.001) on the background of an accumulation of inactive procaspase-3 32 kDa. These observations indicate the cleavage of procaspase-3 and its activation in the area of the healthy tooth of patients with IT, that promote the tooth eruption process. We also suggest the impairment of caspase-3 activation and the accumulation of an inactive form in the area of tooth impaction may lead to tooth eruption failure.

Our data demonstrate that tooth impaction may be associated with blocked NF-κB signaling and NFATc1 expression and with the disturbances in the cascade activation of caspase-3.