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Table of Contents
Year : 2019  |  Volume : 2  |  Issue : 1  |  Page : 3-7

Evaluation of the receptor activator of nuclear factor-κb ligand/osteoprotegerin ratio in the gingival crevicular fluid of patients under periodontal maintenance

1 Post-Graduation Program in Dentistry, Fluminense Federal University, Niteroi, Brazil
2 Department of Periodontology, School of Dentistry, Fluminense Federal University, Niteroi, Brazil
3 Post-Graduation Program in Science and Biotechnology, Fluminense Federal University, Niteroi, Brazil
4 Clinical Research Unit, Antonio Pedro Hospital, Fluminense Federal University, Niteroi, Brazil
5 Laboratory of Bioengineering, National Institute of Metrology, Quality and Technology, Duque de Caxias, Rio De Janeiro, Brazil

Date of Web Publication22-Apr-2019

Correspondence Address:
Dr. Gutemberg Gomes Alves
Av. Marquês Do Paraná, 303 - Centro, Niterói, Rio De Janeiro
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/GFSC.GFSC_4_19

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Context: Periodontitis is an infectious disease characterized by an inflammatory response and bone resorption, which can cause tooth loss. The bone destruction may be caused by deregulation of the receptor activator of nuclear factor κB/receptor activator of NF-κB ligand/osteoprotegerin (RANK/RANKL/OPG) system, stimulating osteoclastogenesis. After the treatment of the active disease, a formal program of periodontal maintenance is implemented according to each patient needs. Aims: This study aimed to evaluate the RANKL/OPG relation in patients under periodontal maintenance. Setting and Design: Thirty individuals were selected and divided into the test group, comprised of patients who received periodontal treatment and were under periodontal maintenance showing active sites (n = 15), and nonactive sites of the disease (n = 15), and the control group, comprised of healthy periodontium (n = 15). Materials and Methods: The GCF was collected by absorption into paper cones. The measurement of RANKL and OP was performed by xMAP technology and a Luminex 200 flow luminometer system. Statistical Analysis: Data were compared through Kruskal–Wallis test, at 5% significance. Results: There was no intrapatient difference on the RANKL/OPG ratio, but it was significantly lower in the active sites of patients treated of periodontitis, under a maintenance program, compared with the control (P < 0.05), even tough all ratios were under the range reported for healthy individuals. Conclusions: Patients in periodontal maintenance have a significantly lower RANKL/OPG ratio than healthy indivduals, demonstrating its importance for patients' periodontal health.

Keywords: Luminex, periodontal disease, periodontal maintenance, receptor activator of NF-κB ligand

How to cite this article:
Da Costa Reis MM, Casado PL, Batista Brochado AC, Alves GG, Granjeiro J, Porto Barboza ED. Evaluation of the receptor activator of nuclear factor-κb ligand/osteoprotegerin ratio in the gingival crevicular fluid of patients under periodontal maintenance. Int J Growth Factors Stem Cells Dent 2019;2:3-7

How to cite this URL:
Da Costa Reis MM, Casado PL, Batista Brochado AC, Alves GG, Granjeiro J, Porto Barboza ED. Evaluation of the receptor activator of nuclear factor-κb ligand/osteoprotegerin ratio in the gingival crevicular fluid of patients under periodontal maintenance. Int J Growth Factors Stem Cells Dent [serial online] 2019 [cited 2022 May 16];2:3-7. Available from: https://www.cellsindentistry.org/text.asp?2019/2/1/3/256788

  Introduction Top

Periodontitis is an inflammatory disease affecting the periodontium, caused by dental biofilm and modulated by intrinsic and acquired factors.[1] Consistent with current knowledge on pathophysiology, three forms of periodontitis can be identified necrotizing periodontitis, periodontitis as a manifestation of systemic disease, and the “chronic” or “aggressive” forms of periodontitis were grouped under a single category: “periodontitis.”[2] In all forms, with the progression of the disease, proinflammatory events of innate and adaptive immunity, and imbalance in bone metabolism, results in the loss of tooth attachment.[3]

Bone resorption is mainly regulated by the receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG), through the positive or negative stimulation of the receptor activator of NF-κB (RANK) in osteoclasts. RANKL is expressed in the membranes of osteoblasts and mesenchymal stem cells.[4] The precursors of osteoclasts present the receptor RANK, which interacts with RANKL through intercellular contact, inducing their differentiation into osteoclasts. OPG, also expressed by osteoblasts, is a soluble decoy receptor that inhibits the osteoclastogenesis through binding to RANKL, which inhibits RANKL-RANK interactions and therefore, inhibiting bone resorption. RANKL and OPG expression is regulated for many systemic and local factors, including bacterial products.[5] Clinical studies have shown that RANKL is increased and OPG decrease in periodontitis compared to healthy sites[6] and may be related to periodontitis-related bone resorption.

The new classification for periodontitis is based on a multidimensional staging and grading system. Staging is largely dependent on the severity of disease at presentation as well as on the complexity of disease management, while grading provides supplemental information about biological features of the disease. This distinction was made to emphasize the need for a more comprehensive maintenance and surveillance of the successfully treated patient with periodontitis. A patient with gingivitis can revert to a state of health, but a periodontitis patient remains a periodontitis patient for life, even following successful therapy, and requires lifelong maintenance to prevent recurrence of disease.[2] The periodontal maintenance program should be based on each individual needs. In general, it can prevent periodontitis recurrence, but it is not clear if it contributes to reducing a loss of attachment as compared to healthy individuals.[7] Since the loss of alveolar bone tissue is one of the main and most damaging consequences of periodontitis, understanding the exact mechanism that regulates human bone metabolism has been a subject of great interest. However, there is still a lack of data available in the literature on the effects that a maintenance program might induce in the main molecular markers of bone resorption in patients treated of periodontitis. In this context, this preliminary study aimed to compare the RANKL/OPG ratio in healthy individuals and patients treated of periodontitis included in a long-term maintenance program.

  Materials and Methods Top

The clinical study procedures were conducted according to the Fluminense Federal University Research Ethical Board recommendations (Registration Number CMM/HUAP 060/2010). Informed consent was obtained from all the patients. Patients were told that they had the right to withdraw from the study at any time.

Participant selection

Thirty Brazilian participants were randomly recruited for the study from the patient pool at the Brazilian Institute of Periodontology, Rio de Janeiro, Brazil, during 1 year. According to the clinical and radiographic characteristics of the periodontium sites, patients were divided into two groups. Patients treated of periodontitis and undergoing periodontal maintenance, recalled regularly 3–6 months' intervals for at least 2 years, were included in test group, including active sites (n = 15), and nonactive sites of the disease (n = 15). A control group was characterized by healthy periodontium individuals (n = 15). Patients were included in the present study if they were free of medical complications, aged above 35 years and were not taking medications affecting periodontal status. Patients were excluded if presenting oral diseases other than caries and periodontal disease, any history of systemic or local disease under influence of the immune system, diabetes mellitus, and HIV infection. They were also excluded if under immunosuppressive chemotherapy, smoking, current pregnancy or lactation, chronic usage of anti-inflammatory drugs until 3 months before this study and osteoporosis diagnosed by bone densitometry or tomography.

Diagnosis of healthy and diseased sites

All periodontal regions were clinically and radiographically evaluated by two examiners. Patients from control group presented healthy periodontium, showing no clinical signs of periodontal disease (probing depth [PD] ≤3 mm and no evidence of bleeding on probing [BOP]), and no radiographic periodontal bone loss in any region. The diagnosis of nonactive or active sites of periodontitis, in the test group, was established by clinical parameters such as PD, clinical attachment loss (CAL), and BOP which were recorded at six sites (mesiobuccal, buccal, distobuccal, mesiolingual/palatal, palatal/lingual, and distopalatal/lingual). Nonactive sites exhibited no progression of the clinical parameters from the last maintenance appointment to the date of data collection. However, the active sites exhibited BOP, PD ≥4 mm, and progression of CAL.

Gingival crevicular fluid sampling and elution

Patients were asked to gargle with a glass of cold, sterile water to cleanse the periodontal regions of loosely adherent debris. Subgingival samples were collected from all patients in duplicate at the moment of clinical and radiographic analysis. In the control group, samples were obtained from superior premolar mesiobuccal gingival sulcus. After clinical evaluation, considering PD, BOP, and radiographic analysis in test group, samples from gingival sulcus were fulfilled in sites with horizontal bone loss characterized “active” (PD ≥4 mm and with BOP) as well as in a site considered “inactive” (PD ≤3 mm and no BOP) before periodontal therapy procedures were performed in the past – intrapatient control.

The area was gently air-dried and carefully isolated by cotton rolls and a saliva ejector to avoid saliva contamination. Samples from GCFs were collected using a filter paper absorption technique. Paper strips (TANARI® #30, Tanariman Industrial Ltda, Manacapuru, AM, Brazil) were inserted into the gingival crevice until mild resistance [Figure 1], and it was felt and left in place for 30 s. The strips were then eluted in 100 μl of phosphate-buffered saline added with 1% bovine serum albumin and a proteases inhibitor cocktail (P2714-1 BTL Sigma-Aldrich, Saint Louis, Missouri, USA). All samples were centrifuged for 10 min at 3000 g to promote blood, plaque, and cellular elements sedimentation. The samples were stored at −80°C for subsequent assays for the detection of RANKL/OPG.
Figure 1: Gingival fluid collection in control (a) and test group (b)

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Receptor activator of nuclear factor-κB ligand/osteoprotegerin determination

The RANKL/OPG ratio was determined on samples by the detection of each cytokine through a bead-based immunoassay, using the Luminex xMAP technology, with a specific detection kit (Milliplex Human Bone Panel I, Millipore, USA). Briefly, 25 microliters of each sample were incubated for 16 h with xMAP antibody-coupled beads specific for RANKL or OPG, on a 96-well plate with an adapted filter-membrane bottom, provided with the commercial kit. After the capture of the analytes present on each sample, the beads were isolated using a vacuum filtration unit (Millipore Vacuum Manifold, Millipore, USA). Biotinylated antibodies and streptavidin R-phycoerythrin conjugate were added sequentially to bind each analyte, and the mixture was analyzed with a Luminex S200 flow luminometer system. In this system, each bead is analyzed with two lasers, where the first detect the bead color and thereby, the identity of the analyte, and the second detect the R-phycoerythrin signal, reflecting the analyte level on the bead. The system was set for the reading of at least 100 beads before the calculation of the mean fluorescence. Standards containing increasing concentrations of both RANKL and OPG were also evaluated in the same plate, providing a concentration curve which enabled the conversion of mean fluorescence into cytokine concentration on the biological samples, in picograms per milliliter.

Statistical analysis

Individual patient's data, including clinical, radiographic, and OPG/RANKL characteristics, were transcribed into a statistical software program for PC (STATA 11.0 – Data Analysis and Statistical Software – StataCorp LP, Texas, USA). The Shapiro–Wilk test showed that PD values were normally distributed. Clinically, differences in PD among active, inactive, and healthy sites were assessed by analysis of variance. Bonferroni test showed a significant difference between each group. Differences in OPG and RANKL levels in GCF between control and test groups were assessed by Kruskal–Wallis nonparametric test, considering the P < 0.05 in Shapiro–Wilk test (nonnormal distribution). Mann–Whitney–Wilcoxon test evaluated the difference between groups. Results were considered statistically significant if a P < 0.05.

  Results Top

The demographic data and the participant's characteristics are shown in [Table 1]. From the total of 45 patients evaluated, 26 (57.8%) were female and 19 (42.3%) were male, with a mean age of 38.8 ± 4.5 in the test group and 59.4 ± 12.2 in the control group. The PD did not present a difference between the active and nonactive site from the test group (3.8 ± 1.3). However, this parameter was higher than the control group (2.2 ± 0.8). The BOP occurred on 100% of the active sites and was completely absent in inactive sites and control group.
Table 1: Clinical and demographic characteristics of control and test subjects

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According to the Bonferroni Test, the parameter PD reached statistical significance between active and nonactive sites in the same subject of the test group (P = 0.0001) and between active sites and control sites (healthy individuals) (P = 0.002). However, nonactive sites did not differentiate from control sites, considering PD (P = 0.209).

The median values of the total amounts and concentrations of RANKL and OPG in GCF in the active and nonactive sites in patients who were treated of periodontitis and healthy sites are shown in [Table 2]. Individuals (test group) exhibited greater total amounts of OPG than healthy sites, and the difference between test and healthy sites reached statistical significance (P < 0.05). In contrast, the control group showed significantly greater amounts of RANKL compared to nonactive (P = 0.04) and active (P = 0.01) sites. The intrapatient evaluation showed no significant difference in RANKL levels. Furthermore, the RANKL/OPG ratio was significantly higher (P = 0.0048) in the healthy group compared with the maintenance group (active sites), but without a significative difference from the nonactive sites [Figure 2]. Nonactive and active sites in patients treated of periodontitis showed no significant difference in the RANKL/OPG ratio (P > 0.05).
Table 2: Osteoprotegerin and receptor activator of NF-êB ligand levels in crevicular gingival fluid from control and test sites

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Figure 2: Receptor activator of nuclear factor-κB ligand/osteoprotegerin ratio (median ± interquartile) from the gingival crevicular fluid from healthy participants (n = 15), or patients presenting active (bleeding, n = 15) and inactive (notbleeding, n = 15) sites of periodontitis, after at least 2 years of supportive periodontal therapy. A line indicates a significant difference between groups (P < 0.05)

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  Discussion Top

Many studies in the literature have shown that patients with periodontitis have increased the release of RANKL in gingival fluids.[8] The RANKL/OPG ratio regulates osteoclastogenesis so when RANKL is increased, and when OPG decreased it shows bone loss.[9] RANKL is a member of the tumor necrosis factors (TNF) superfamily, produced by diverse cells such as osteoblasts, fibroblasts, chondrocytes, bone marrow stromal cells, malignant cells, and T lymphocytes. When it interacts with RANK, a receptor in the membranes of precursors of osteoclasts, it activates its maturation, by activating the expression of several specific genes. OPG, on the other hand, is a member of the TNF receptor superfamily, secreted mostly osteoblasts, as well as by periodontal tissues, gingival epithelium, and gingival fibroblasts.[10] This protein act as a dummy receptor binding competitively to RANKL, impairing the contact of osteoblasts with precursors and the mature multinucleated of osteoclasts, and therefore, impending osteoclastogenesis. The expression levels of RANKL and OPG can be influenced by some substances and cytokines, including those involved in the periodontal disease, such as interleukin-1 (IL-1), IL-6, and TNF-α.[10]

According to diverse studies, RANKL expression is directly linked to the severity of periodontal disease.[11],[12],[13] An important work by Mogi et al.[13] has shown that the gingival crevicular fluid (GCF) of patients with periodontitis presented increased RANKL and decreased OPG. Furthermore, the RANKL/OPG ratio in the GCF was significantly higher in that group, compared to healthy individuals. The present study compared the levels of RANKL and OPG in patients treated of periodontitis undergoing a long-term periodontal maintenance program with those from the GCF of healthy participants. Our results evidenced no intra-patient difference but identified a significant mean increase in OPG and a decrease in RANKL (P < 0.05) in the patients undergoing maintenance, when compared to healthy participants.

According to the American Academy of Periodontology, maintenance includes a periodical dental recall examination, periodontal reevaluation, and risk assessment, the removal of bacterial plaque and calculus, and retreatment of recurrent sites, including mechanical debridement of any plaque or calculus deposits.[7] In the present work, the clinical measures adopted for the maintenance seemed to exert effective control over the periodontopathogenic agents that, together with possible contributing factors, promoted the concomitant increase in RANKL and decrease in OPG. Thus, the follow-up of these patients in the long-term shows an important clinical measure to be adopted with the objective of preserving the periodontal support structures.

The RANKL/OPG ratios of the test and control groups were also compared in this study. There was a statistically significant difference between the RANKL/OPG ratios of the test group (“active” site) and the control group. Interestingly, the ratio was elevated in three members of the control group. After an investigation, some clinical data called attention to these individuals, such as Class II restorations and dental crowding, possible bacterial plaque retainers, which could justify a higher RANKL/OPG ratio caused by subclinical gingivitis.

The literature did not show a pattern of uniformity in the RANKL/OPG ratio values in the different periodontal conditions, but most studies reported values ranging from 0.45 to 1.0 for healthy individuals.[11],[14],[15],[16] In this research, despite a significant difference between the RANKL/OPG ratio of active sites of patients in maintenance and the control group, their median are within the range of healthy individuals.[17] This preliminary result points to the importance of inclusion of patients treated of periodontitis in a maintenance program because even with the presence of clinical signs of active disease, the RANKL/OPG values were compatible with health. Therefore, the periodicity in which the patients are submitted to maintenance appointments is again necessary for the control of osteoclastogenesis, since patients included in this treatment regimen presented a RANKL/OPG ratio equivalent to that presented by healthy individuals. It is important to notice that the present results were obtained through a multiplex bead-based assay (XMap Technology, LuminexCorp, Austin, TX, USA), with increased sensitivity when compared to ELISA but, more importantly, able to detect both molecules simultaneously in the same sample, reducing the risk of bias of comparing quantifications obtained in independent assays.

Thus, our results suggest the maintenance program contributes to the management of factors involved in periodontitis. As patients still presented higher levels of RANKL when compared to the control group, which demonstrate how necessary is the patients treated of periodontitis to have a periodical follow-up with a specialist aiming to decrease the risk of increase RANK associated to the prolonged presence of bacterial lipopolysaccharides and maintain the tooth support tissues as stable as possible. However, while the mechanisms behind bone resorption process in periodontal disease are still under investigation, including the understanding of the contribution of other physiological and pathological factors, the use of RANKL and OPG as markers or even therapeutic targets for bone resorption in periodontal disease still demands further investigations.

  Conclusions Top

Patients with active sites in the maintainance program have a significantly lower RANK./OPG ratio than healthy individuals, demonstrating its importance for patients' periodontal health.


The authors would like to acknowledge the financial support from CAPES, FAPERJ, and CNPq.

Financial support and sponsorship

This study was supported by CAPES, FAPERJ, CNPq.

Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Figure 1], [Figure 2]

  [Table 1], [Table 2]


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