Osteoprotegerin in Gingival Crevicular Fluid under Long-term Continuous Orthodontic Force Application

Bone remodeling is a dynamic interaction between bone-forming osteoblasts and bone-resorbing osteoclasts. The rate of remodeling is defined primarily by cells of the osteoblast lineage, which, in addition to bone formation, are also responsible for the activation and recruitment of osteoclast precursors.1 Recently, an intermediary factor, receptor activator of nuclear factor κB ligand (RANKL), presenting on the surface of osteoblasts was found to be responsible for the induction of osteoclastogenesis.2 Binding of RANKL to its cognate receptor, receptor activator of nuclear factor κB (RANK), expressed on the surface of osteoclast progenitor cells, induces osteoclastogenesis and activates osteoclasts, resulting in increased bone resorption.3 

On the other hand, RANKL has a capability to bind to osteoprotegerin (OPG), which is also a member of the tumor necrosis factor (TNF) receptor family. OPG is a soluble decoy receptor protein that competitively binds to cell surface membrane-bound RANKL proteins and inhibits RANKL activation of osteoclastogenesis.4 OPG is produced by human periodontal ligament cells,5 gingival fibroblasts, human pulp cells, and epithelial cells6 and has been found to be a key factor in the inhibition of osteoclast differentiation and activation.7 

Recent clinical studies have confirmed that both RANKL and OPG can be detected in human gingival crevicular fluid (GCF) and indicate that RANKL is elevated whereas OPG is decreased in periodontitis8,9 or during orthodontic tooth movement.10,11 Biochemical analysis of the GCF has provided a noninvasive model for investigating the cellular response of the underlying periodontal ligament during orthodontic tooth movement in vivo.12 In the literature, there are few studies in which GCF was used to determine the OPG expression in the periodontal tissue during orthodontic tooth movement.10,11 Furthermore, there is no information about the level of OPG during a continuous distalization of a canine toward an extracted premolar site that includes all phases of orthodontic tooth movement. In this context, this study was designed to investigate the level of OPG in GCF at baseline, 1 hour, 24 hours, 168 hours, 1 month, and 3 months after the application of mechanical stress in order to have orthodontic tooth movement.

Twelve orthodontic patients (7 girls with a mean age of 14.4 ± 1.5 years and 5 boys with a mean age of 15.0 ± 1.5 years) were selected to participate in this study according to the following criteria: (1) good general health, (2) no history of antimicrobial therapy within the past 6 months, (3) no use of anti-inflammatory drugs in the month preceding the study, (4) healthy periodontal tissues with generalized probing depths of ≤3 mm and no radiographic evidence of periodontal bone loss, and (5) first-premolar extraction and canine distal tooth movement as part of orthodontic treatment. All patients participating in the study were treated between January 2004 and July 2006 in the dental clinic of Baskent University, Adana Research and Medical Teaching Center, Adana, Turkey. The study protocol was reviewed and approved by the ethical committee of Baskent University School of Medicine. Informed consent was obtained from each participant and the guardian at the first visit.

For each subject, a canine undergoing distal movement was chosen as the test tooth, and the contralateral canine served as the control tooth. Tests and control sites were selected from the same subjects for eliminating individual differences. Orthodontic brackets including a vertical slot (GAC International Inc, Bohemia, NY) were placed on both the experimental and control teeth. Experimental canines were distalized on a segmental 0.016- × 0.022-inch stainless-steel arch wire. Force was approximated to the center of resistance of the canine by an auxiliary vertical wire (Figure 1A). Orthodontic force was applied by using a closed superelastic nitinol coil spring (GAC International Inc) exerting a continuous force of 150 g. In addition, a single brace, free from any orthodontic force, was bonded on the contralateral canine (control site; Figure 1B).

Tooth movement was measured on the lateral cephalometric radiographs taken at the beginning and the end of the experimental period (3 months). To differentiate between the test (distalized) and the control teeth, a guiding wire was inserted in the vertical slot of the test canine bracket before the cephalometric exposures.

All subjects underwent a session of supragingival scaling and received oral hygiene instructions to reach a level of meticulous plaque control. Remotivation was performed during the whole study, when necessary. The experiment was started a minimum of 2 months after the extraction of premolars. All patients were warned not to take any of the anti-inflammatory drugs during the study.

GCF was collected immediately before activation and at the following time periods after the activation: 1 hour, 24 hours, 168 hours, 1 month, and 3 months at the distal aspect of both test and control teeth. GCF was sampled by the method of Mogi et al.8 All clinically detectable supragingival plaque was removed without touching the gingiva to minimize contamination of the paper strips by the plaque. The teeth under study were gently washed with water, isolated with cotton rolls (to eliminate contamination from saliva), and gently dried with an air syringe. One paper strip was used for each site undergoing examination. Paper strips (Periopaper, Harco, Tustin, Calif) were carefully inserted 1 mm into the gingival crevice and allowed to remain for 30 seconds. Care was taken to avoid mechanical injury. Any strips visibly contaminated with blood were discarded.

The volume of GCF in the periopaper was measured with a calibrated Periotron 8000 (Periotron 8000, Oraflow Inc, New York, USA), and then the readings were converted to an actual volume (μL) by reference to the standard curve. All samples were lyophilized and stored at −80°C before laboratory analysis.

For analysis, 50 μL of phosphate-buffered saline (pH 7.2) containing protease inhibitors (Complete Protease Inhibitor Cocktail, Roche Diagnostics GmbH, Roche Applied Sciences, Penzberg, Germany) was used to re-elute the samples. The tubes were shaken gently for 1 minute and then centrifuged at 2000 × g for 15 minutes at 4°C before being processed on the enzyme-linked immunosorbent assay (ELISA) plate.

OPG was measured by a two-site sandwich ELISA kit (R&D Systems, Quantikine, Mouse OPG/ TNFRSF11B, Minneapolis, Minn). The assays were carried out in accordance with the manufacturer's instructions, and the levels of the biochemical compounds were reported as the total amount (pg). All samples and standards were assayed twice. Intra-assay and interassay coefficients of variation were <8%. These assays measure the total level of OPG present in the GCF, including both their unbound free forms. Calculation of the OPG concentration in each GCF sample was performed by dividing the total amount of OPG by the volume of the sample (OPG concentration [pg/μL] = total OPG [pg]/volume [μL]).

The data were processed and analyzed using the statistical package SPSS version 11.5 for Windows (release 11.50, standard version, SPSS, Chicago, Ill). Descriptive data are presented as counts, proportions, medians, and interquartile ranges. The distributions of OPG concentration (pg/μL) were investigated by using Shapiro Wilk's normality test, and the comparisons were done using nonparametric tests. The differences between the experiment and control group for each time point were tested using the Wilcoxon test; repeated measurements were evaluated using the Friedman test and post hoc tests. The proportional differences between the experiment and control groups were tested using a Z test for two proportions. Differences (two-tailed P) less than .05 were regarded as significant.

The test teeth underwent a mean distal movement of 4.5 ± 1.0 mm. No significant displacement was detected in the control teeth. There were no statistically significant differences in the OPG concentration (pg/ μL) between the test and control teeth (P > .05) at any time. The median OPG level changes were assessed based on the respective baseline levels. The longitudinal changes in the OPG levels in the compressed (distal) sites of the test teeth and control teeth, compared with baseline values, are shown in Table 1. Levels of OPG concentrations in distal sites of the test teeth were decreased in a time-dependent manner during the experimental period. Decreasing was statistically significantly at 1 hour, 24 hours, 168 hours, 1 month, and 3 months when compared with the baseline measurements (P = .038; Figure 2). Decreased levels of OPG were maintained throughout the 3 months of tooth movement in the test teeth. However, variability was detected in the levels of OPG concentration in the distal sites of the control teeth. OPG concentrations were observed to fall and rise throughout the experimental period when compared with baseline levels.

It is now well understood that osteoclast differentiation (bone resorption) is regulated by osteoblasts. RANK, RANKL, and the decoy receptor OPG are key molecules for osteoclast differentiation supported by osteoblasts.13 They coordinate in regulating bone density and structure by the well-balanced modulation of osteoclast differentiation.

There are only two studies in the literature determining the level of OPG in the GCF during orthodontic tooth movement. Nishijima et al10 have investigated OPG expression during a 1-week period: at baseline, 1 hour, 24 hours, and 168 hours. They found a decreased level of OPG in GCF samples collected from areas adjacent to teeth undergoing orthodontic tooth movement at 24 hours after applying a mechanical compression to the periodontal ligament (PDL). With a similar experimental design, Kawasaki et al11 compared the levels of OPG in GCF samples taken from juvenile and adult patients during orthodontic tooth movement. They also found significantly lower OPG values for the experimental teeth after 24 hours in both groups, but a greater decrease was reported in the juvenile patients.

On the other hand, in both aforementioned studies, the investigators indicated that the level of OPG had increased to approximately baseline levels by 168 hours. The experimental design used in those studies (force application with elastic chain) did not provide a continuous and consistent force over the entire experimental period; the authors also attributed the return in OPG to this factor. It is known that elastomeric chains generally lose 50% to 70% of their initial force during the first day of load application.14 

Continuous force application was attributed to present a constant responsive state in the cell biology system in opposition to intermittent force application, which probably creates a fluctuating environment of cellular activity/quiescence.15,16 Therefore, the experimental design in the present study comprised continuous force application by using superelastic nitinol coil springs that can exert a constant force over a wide range of activation.17 Conversely to the study of Nishijima et al10 and Kawasaki et al,11 the OPG level in the compressed sites of the test teeth decreased in a time-dependent manner. This might be related to the continuous force application throughout the entire experimental period.

Complete orthodontic tooth movement because of the alveolar bone–remodeling process involves several phases18 over a certain period of time. In most of the in vivo studies, different mediators produced from the PDL cells that are responsible for the bone-remodeling mechanism during orthodontic tooth movement were evaluated over a relatively short period of time.10,11,19,20 A 168-hour investigation might present data belonging to only the first two phases of the process (displacement and delay phases, respectively) in most circumstances. However, data belonging to the acceleration or linear phase, in which true orthodontic tooth movement is considered to take place, require an observation period of at least 1 month. Therefore, we investigated the levels of OPG in GCF over a 3-month period.

Very recently, Ren et al21 determined the cytokine profiles in GCF during orthodontic tooth movement over a 4-month period. They demonstrated that proinflammatory cytokines (interleukin-1β, -6, and -8 and TNF-α) were upregulated during the early stage of tooth movement, but in the linear phase, all of these cytokines were diminished to their baseline levels. The authors also noted that the number of osteoclasts increased during only the first couple of weeks; subsequently, the number decreased and remained at low levels with continuous orthodontic force and steady increments of tooth movement. In our study, the OPG level was significantly decreased at the first hour and remained at low levels during the 3-month experimental period.

There are conflicting results regarding the regulation of OPG expression in PDL cells stimulated by compressive forces in vitro. In their study, Kanzaki et al22 applied static compressive force to the precultured PDL cells to mimic the in vivo system applied in orthodontic treatment. The authors found that compressed PDL fibroblasts increased osteoclastogenesis in peripheral blood mononuclear cell cultures by upregulating RANKL but not by downregulating OPG expression. OPG expression did not change regardless of the amount of compression force or the duration of compression. Therefore, they assumed that the osteoclastogenic activity of PDL cells is lower than that of other stromal cell lines.

On the other hand, by using a similar in vivo method, Nishijima et al10 indicated that compression forces significantly decreased the secretion of OPG in a time- and magnitude-dependent manner in precultured human PDL cells. Moreover, Yamaguchi et al23 searched for the RANKL and OPG productions from the precultured PDL cells derived from the patients who presented with a severe external root resorption during their orthodontic therapy. The OPG level was found to decrease in PDL cells when stimulated by compressive force. Also, researchers found that a decrease in OPG level was greater in the severe root resorption group than in the nonresorption group. In our in vivo study, we found that continuous compressive force application to PDL resulted in a decrease of GCF OPG levels in the compressed sites of the distalizing teeth. Significant decreasing was detected at the first hour and was maintained at 24 hours, 168 hours, 1 month, and 3 months when compared with the baseline measurements. When the findings of the aforementioned in vivo studies and our present results are interpreted together, they suggest that compression of the periodontal tissues by an orthodontic force causes a downregulation of OPG release from the PDL cells.

Understanding the bone-remodeling metabolism more intensely might provide a therapeutic control during the orthodontic treatment approaches. Keles et al24 indicated that OPG injection, as an inhibitor of osteoclast recruitment, could have clinical utility in preventing undesired tooth movement. Moreover, Kanzaki et al25 discovered that OPG gene transfer to the periodontal tissue induced prolonged OPG expression and inhibited RANKL-mediated osteoclastogenesis and experimental tooth movement. Local OPG gene transfer25 significantly inhibited the osteoclastogenesis in the periodontium that was caused by experimental tooth movement.

The detection of local concentration of OPG and RANKL in GCF may give an opinion to the orthodontist about the convenience of the applied orthodontic force to achieve optimum tooth movement. The OPG/ RANKL ratio may change with the intensity of orthodontic forces. The orthodontist may use this information as a guide for detecting the optimum orthodontic treatment duration to avoid pathologic alveolar bone and root resorption and hyalinization in patients with bone metabolism pathologies such as hypoparathyrodism or hyperparathyrodism. At present, there are many questions about the local response of the bone to the orthodontic forces.

This is the first in vivo study to demonstrate the level of OPG concentration during a complete orthodontic tooth movement that comprises all long-term phases. The levels of OPG were significantly downregulated beginning from the first hour, and the levels remained low throughout the 3-month experimental period at the compressed site of distalizing tooth when compared with baseline levels.

• These results support the previous data demonstrating that OPG is one of the key mediators responsible for osteoclastogenesis (bone resorption) in alveolar bone remodeling during orthodontic tooth movement.

This study was supported by a Baskent University Research Foundation grant (D-KA04/02).