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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 14  |  Issue : 2  |  Page : 83-88

Evaluation of receptor activator of nuclear factor-κB ligand and osteoprotegerin levels in saliva and gingival crevicular fluid in patients with chronic periodontitis


1 Department of Oral Medicine, Periodontology, Diagnosis and Radiology, Faculty of Medicine, Tanta University, Tanta, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt

Date of Submission27-Dec-2016
Date of Acceptance28-Mar-2017
Date of Web Publication30-May-2017

Correspondence Address:
Amr M Gabr
Elawkaf Towers 1, 6 October Street, Elmehalla Elkobra, Gharbia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/tdj.tdj_62_16

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  Abstract 


Background and objective:
Recent findings have suggested that osteoclastogenesis is directly regulated by receptor activator of nuclear factor-κB ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG).
Aim:
Evaluate and compare the level of RANKL and OPG and its ratio (RANKL/OPG ratio) in saliva and gingival crevicular fluid (GCF) of patients with chronic periodontitis and clinically healthy individuals.
Patients and methods:
Ninety patients were selected and divided into group 1: 45 patients with clinically healthy gingiva as a control group. Group 2: 45 patients with moderate to severe chronic periodontitis probing pocket depth, bleeding on probing, plaque index and attachment level were measured, unstimulated whole saliva and GCF samples were collected from four deepest sites in different quadrants, both analyzed by enzyme-linked immunosorbent assays.
Results:
All periodontal indices (pocket depth, bleeding on probing, plaque index and attachment level) were significantly higher in the test groups than in the control group, it was found that RANKL and RANKL/OPG ratio significantly increased while OPG level significantly decreased in GCF and saliva in the chronic periodontitis group compared with the controlled group.
Conclusion:
Both GCF and saliva collection are a noninvasive approaches as a diagnostic markers for RANKL, OPG and RANKL/OPG ratio.

Keywords: bone resorption, enzyme-linked immunosorbent assays, gingival crevicular fluid, osteoprotegerin, receptor activator of nuclear factor-κB ligand


How to cite this article:
Gabr AM, El-Guindy HM, Saudí HI, Morad MA. Evaluation of receptor activator of nuclear factor-κB ligand and osteoprotegerin levels in saliva and gingival crevicular fluid in patients with chronic periodontitis. Tanta Dent J 2017;14:83-8

How to cite this URL:
Gabr AM, El-Guindy HM, Saudí HI, Morad MA. Evaluation of receptor activator of nuclear factor-κB ligand and osteoprotegerin levels in saliva and gingival crevicular fluid in patients with chronic periodontitis. Tanta Dent J [serial online] 2017 [cited 2017 Oct 22];14:83-8. Available from: http://www.tmj.eg.net/text.asp?2017/14/2/83/207308




  Introduction Top


Bone is subjected to continuous remodeling to meet the functional adaptation needs. The remodeling process is characterized by coupling of resorption of the bone matrix by osteoclasts and its reformation by osteoblasts. Under physiological conditions, a dynamic balance is established between bone formation and resorption. But, if the balance switches towards enhanced bone resorption, then bone destructive pathology will occur [1].

Chronic periodontitis is defined as chronic destructive inflammatory condition of the anatomical structures which surround and support the teeth, namely gingiva, periodontal ligament and alveolar bone resulting in tissue injury including loss of connective tissue attachments and alveolar bone destruction [2].

Although clinical diagnosis of periodontitis based on visual and radiographic assessment in addition to measurement of pocket depth (PD), tissue attachment and bleeding on probing (BOP) in different locations in multiple teeth is well established in dental practice, these traditional diagnostic procedures give an indication of severity and, therefore, reflect historical disease activity but not current disease activity. Moreover, they neither identify susceptible individuals who might be at risk of future periodontitis nor the risk for disease progression [3],[4].

Oral fluid biomarkers related to periodontal disease can be associated with soft tissue inflammation, alveolar bone loss, bacterial products, antimicrobial proteins, etc., [5],[6]. The requirement for reliable biomarkers to distinguish progressive periodontitis from normal biological processes is considered fundamental for identifying periodontitis at an earlier or even preclinical stage, initiating preventative pretreatment and also for the conduct of epidemiological studies [7],[8].

Biomarkers have been defined as cellular, biochemical, molecular or genetic alterations by which a normal, abnormal or simply biologic process can be recognized or monitored [9]. The goal of biomarker research in periodontology is to develop a high impact diagnostic which has significant impact on clinical decision making, patient outcomes and healthcare economics [10].

There is emerging interest in a number of cytokines such as receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) which are regulators of bone cell activity and, as such, mediate bone loss characteristic of periodontitis [11],[12]. Increasing evidence has shown an important role of both the RANKL and OPG in human periodontitis [13],[14] that could potentially be used for diagnostic or therapeutic purposes [15].

RANKL, a member of the tumor necrosis factor ligand superfamily, was identified as a cell membrane-bound factor responsible for stimulation of osteoclast differentiation and bone resorption [16]. RANKL is produced as a membrane-bound or secreted ligand by osteoblasts, fibroblasts or activated T cells and B cells.

The action of RANKL can be blocked by its soluble decoy receptor OPG, which is a member of the tumor necrosis factor receptor superfamily, with structural homology to receptor activator of nuclear factor-κB (RANK) [16]. By binding to RANKL, OPG prevents its further interaction with RANK, and subsequently prevent all the downstream molecular events that lead to osteoclast differentiation and bone resorption. The production of RANKL and OPG by various cells types is controlled by systemic and local stimuli, including hormones, inflammatory mediators and bacterial products [17],[18].

OPG also inhibits the development of osteoclast [19]. The biological effects of OPG on bone cells include the inhibition of terminal stages of osteoclast differentiation, suppression of mature osteoclast activation, and induction of apoptosis [20],[21]. So, bone remodeling appears to be mainly controlled by the balance RANKL/OPG [22].

OPG is expressed by different cell types like osteoblasts, osteoclastic stromal cells, T cells, B cells, chondrocytes and follicular dendritic cells [20]. It is also found in organs (like the kidney, liver, heart, lung, spleen, thyroid, lymph nodes, thymus, brain and placenta) [23], in periodontal and dental tissues (like gingiva and periodontal ligament) [24], in the internal and the external enamel epithelium, as well as in the mesenchyme of the dental papilla during tooth development [25]. Moreover, a prominent expression of OPG in the cartilaginous primordial of developing maxilla, mandible and hyoid bone has been reported [23].

Increased RANKL or decreased OPG local expression can cause bone resorption at different sites of the human skeleton. On the other hand, decreased RANKL or increased OPG expression could result in enhanced bone formation, leading to osteoporotic conditions. The involvement of the RANKL/OPG system is well established in the pathogenesis of diseases of bone and mineral metabolism [26]. Among various biomarkers of bone destruction, the investigation of RANKL and OPG in biological analytes of relevance may deliver reliable information on the state of periodontal disease, but may not be able to predict future activity [12].

In an attempt to find biomarkers associated with periodontal disease and its progression, extensive studies have been done on gingival crevicular fluid (GCF), glandular saliva and whole saliva [5],[6].


  Patients and Methods Top


Study population

Ninety patients were selected from the Outpatient Clinic, Faculty of Dentistry, Tanta University for this case–control, cross-sectional study. Their ages ranged between 30 and 55 years old.

Inclusion criteria

All patients were physically healthy, nonsmokers (They had never smoked, or had stopped smoking >5 years previous to the date of examination.) [27] and free of any systemic diseases that may affect bone (like rheumatoid arthritis and systemic lupus erythematosus) and have minimum 20 teeth excluding third molars.

Exclusion criteria

Patients having any systemic illness that could affect the progression of periodontal disease (such as diabetes mellitus) and pregnant or lactating women were excluded.

Patients who had received any periodontal therapy and/or antibiotics, bisphosphonates, nonsteroidal anti-inflammatory therapies during the previous 6 months or had periapical pathology and orthodontic appliances were also excluded.

Patients with history of alcoholism, liver, kidney or salivary gland dysfunction were excluded from this study.

Study design

All patients were divided into two groups:

  1. Group 1: 45 patients with clinically healthy gingiva were recruited from the faculty as a control group.
  2. Group 2: 45 patients with moderate to severe chronic periodontitis based on the criteria defined by the American Academy of Periodontology [28].


Approval for this project was obtained from Tanta Faculty of Dentistry, Tanta University Research Ethics Committee. The purpose of the present study was explained to the patients and informed consents were obtained according to the guidelines on human research published by the Research Ethics Committee at Faculty of Dentistry, Tanta University.

Clinical assessment

Complete medical and dental histories were obtained. All patients received clinical periodontal examination assessed by William's periodontal probe according to Sexton et al.[29] including: probing PD, BOP, plaque index (PI) and attachment level (AL).

Saliva sampling

Unstimulated whole expectorated saliva (5 ml) was collected before clinical assessment from each patient [30]. Patients were asked to avoid oral hygiene measures (i.e., flossing, brushing, mouth rinses, eating, drinking, or gum chewing) for 1 h before collection. They were asked to swallow first and allow the saliva to drain passively for 5 min over the lower lip into sterile tube while seated in an upright position. Saliva samples were immediately transferred to a container at 4°C and samples were frozen and stored at −80°C until final analysis at Clinical Pathology, Faculty of Medicine, Tanta University within 6 months.

Gingival crevicular fluid sampling

Before GCF sampling, the PI was recorded. GCF samples were collected from four deepest sites (PD ≥5 mm and 30% bone loss) in different quadrants using paper strips. The selected sample sites were cleared of all detectable supragingival plaque, isolated with cotton rolls and a stream of air to prevent the samples from being contaminated by saliva. The paper strips was gently inserted into the periodontal pocket until mild resistance is felt or in any way not more than 1 mm and left in place for 30 s. To completely extract the sample from the paper, the fluid was eluted by the method of centrifugation with portions of buffers. Briefly, each strip was placed in a sterile Eppendorf tube and incubated in 200 ml of a PBS (50 mmol/l; pH 7.2) for 30 min; thereafter; each tube was centrifuged at 2000g for 10 min. After removal of the strips using centrifugal filtration, supernatants were stored at −80°C until analysis by enzyme-linked immunosorbent assays (ELISA). An identical method was used to obtain GCF samples from the control group [31].

Biomarker analysis

ELISA were used for the quantitative measurement of RANKL and OPG. RANKL was measured by using RANKL E0855 ELISA Kit (EIAab, Wuhan, China) and OPG was measured by using Human Osteoprotegerin ELISA E0108h (EIAab). The results of RANKL and OPG assays were expressed as ng/ml for concentrations. All of laboratory tests were performed in the Clinical Pathology Department, Faculty of Medicine, Tanta University.

Statistical analysis

The collected data were organized, tabulated and statistically analyzed using statistical package for the social sciences (SPSS, version 19; IBM, Illinois, Chicago, USA). The differences between mean values of the two studied groups were tested used using Student's t test. The level of significant was adopted at P valueless than or equal to 0.05.


  Results Top


Clinical findings

All periodontal indices were significantly higher in the test group than in the control group (P < 0.001).

The mean value of PD was 2.01 ± 0.61 mm in control group. In chronic periodontitis group the mean value of PD was 5.61 ± 0.70. There was a highly significant difference between the two groups as P valueless than 0.001.

It was found that the mean value of BOP was 0.61 ± 0.31 in the control group. In chronic periodontitis group the mean value of BOP was 2.39 ± 0.38. There was a highly significant difference between the two groups as P value less than 0.001.

The mean value of PI was 0.77 ± 0.46 in the control group. In chronic periodontitis group the mean value of PI was 2.29 ± 0.59. There was a highly significant difference between the two groups as P valueless than 0.001.

The mean value of AL was 0.26 ± 0.37 mm in the control group. In chronic periodontitis group the mean value of AL was 3.69 ± 0.78. There was a highly significant difference between the two groups as P value less than 0.001.

The differences in clinical periodontal parameters in chronic periodontitis patients and healthy controls are summarized in [Table 1].
Table 1: Clinical findings in chronic periodontitis patients and healthy controls

Click here to view


Immunoenzymatic results

The mean value of RANKL–GCF was 0.21 ± 0.02 in the control group. In chronic periodontitis group the mean value of RANKL–GCF was 0.33 ± 0.06. There was a highly significant difference between the two groups as P valueless than 0.001.

The mean value of OPG–GCF was 0.28 ± 0.04 in the control group. In chronic periodontitis group the mean value of OPG–GCF was 0.20 ± 0.03. There was a highly significant difference between the two groups as P valueless than 0.001.

The mean value of RANKL/OPG ratio–GCF was 0.77 ± 0.14 in the control group. In chronic periodontitis group the mean value of RANKL/OPG ratio–GCF was 1.65 ± 0.37. There was a highly significant difference between the two groups as P valueless than 0.001.

The mean value of RANKL–saliva was 0.21 ± 0.03 in the control group. In chronic periodontitis group the mean value of RANKL–GCF was 0.35 ± 0.08. There was a highly significant difference between the two groups as P value less than 0.001.

The mean value of OPG–saliva was 0.39 ± 0.09 in the control group. In chronic periodontitis group the mean value of OPG–saliva was 0.22 ± 0.03. There was a highly significant difference between the two groups as P value less than 0.001.

Determination of the ratio of RANKL/OPG revealed that there was high difference between the study groups. The mean value of ratio in chronic periodontitis patients 1.62 ± 0.43 was significantly higher P valueless than 0.001 in comparison with the ratio in healthy controls [Table 2].
Table 2: Summary of immunoenzymatic findings obtained from the study patients

Click here to view



  Discussion Top


To find a reliable molecular marker of periodontal tissue destruction with high sensitivity, specificity and utility [12] is considered a major challenge in clinical periodontics. Many different biomarkers associated with bone resorption, formation and turnover have been assessed in GCF and saliva [4]. The RANK/RANKL/OPG system plays a significant role in the production and activation of osteoclasts and in the regulation of bone resorption [32].

The present study was designed to evaluate and compare the level of RANKL and OPG and its ratio (RANKL/OPG ratio) in saliva and GCF of 45 patients with chronic periodontitis and 45 clinically healthy individuals as a controlled group.

The rationale for using of RANKL and OPG and its ratio (RANKL/OPG ratio) in the current study is based on that RANKL, its receptor RANK and the decoy receptor OPG are the three key molecules that regulate osteoclast recruitment and function. RANKL binding to its receptor RANK on osteoclast precursor cells elicits their differentiation and activation. On the other hand, OPG binding to RANKL interrupts RANKL–RANK ligation; consequently, OPG inhibits the ability of RANKL to induce osteoclastogenesis. RANKL is involved not only in physiological osteoclastogenesis, but also in pathological bone loss [33].

All patients were medically free, to avoid the possible impact of systemic disorders on the periodontal condition and their possible effect on the tested clinical parameters, since many systemic disorders have been implicated as risk factors or indicators for adverse periodontal conditions [34]. All patients are not current smokers as smoking is capable of reducing sign of inflammation.

According to the present study it was found that RANKL and RANKL/OPG ratio increased while OPG level decreased in GCF in the chronic periodontitis group compared with the controlled group.

This was in agreement to Bostanci et al.[35] who compared the levels of RANKL, OPG and their relative ratio in GCF of healthy and periodontal disease patients. RANKL and OPG concentrations in GCF were measured by ELISA. In their study, RANKL levels were low in health and gingivitis groups, but increased in all three forms of periodontitis. OPG levels were higher in health than all three periodontitis, or gingivitis groups. There were no differences in RANKL and OPG levels between chronic and generalized aggressive periodontitis groups, whereas these were lower in the immunosuppressed chronic periodontitis group. The RANKL/OPG ratio was significantly elevated in all three periodontitis forms, compared with health or gingivitis, and positively correlated to probing PD and AL. So, level of RANKL and OPG in GCF were oppositely regulated in periodontitis, but not gingivitis, resulting in an enhanced RANKL/OPG ratio. This ratio was similar in all three periodontitis groups and may therefore predict disease occurrence.

However the current results were in contradiction with findings ofIbrahim [36]that compared the levels of osteoclastogenesis-related factors sRANKL and OPG and their ratios in GCF from patients with chronic periodontitis, generalized aggressive periodontitis and controlled type 2 diabetes mellitus patients with chronic periodontitis. Clinical periodontal parameters were measured. Concentrations of sRANKL and OPG in GCF were analyzed by ELISA. Higher concentration levels of sRANKL, OPG, RANKL/OPG ratios in the three diseased periodontitis groups compared with the control group. Thus she concluded that GCF total amount of sRANKL, OPG were significantly increased in periodontal disease, supporting its role in the alveolar bone changes developed in this disease.

The reasons for these variations in the levels of OPG may be due to difference of study population, also diabetes mellitus can affect the bone remodeling process, environmental factors, and volume of bone present during the time of GCF collection or as a result of methodological variations.

In the present study it was found that salivary RANKL and RANKL/OPG ratio significantly increased while OPG level decreased in saliva in chronic periodontitis group compared with controlled group. It is not surprising that chronic periodontitis patients have increased salivary and GCF RANKL and RANKL/OPG levels than the control group since these proteins are known to play a major role in the tissue destruction in chronic periodontitis patients.

This was in agreement toTobon-Arroyave et al.[37] who conducted a study to determine the variations in salivary concentrations of sRANKL, OPG and its ratio, regarding the periodontal status of smoking and nonsmoking chronic periodontitis patients and healthy controls. sRANKL and OPG salivary levels were analyzed by ELISA and showed that sRANKL and sRANKL/OPG ratios were increased, whereas OPG was significantly decreased in chronic periodontitis compared with healthy controls.

However these results were contradicting the findings ofAl-Sabbagh et al.[38] who conducted a study to examined levels of salivary OPG, in patients within a case–control study. Unstimulated whole saliva of patients with moderate to severe chronic periodontitis and sex-matched and age-matched healthy control were measured using ELISA. They concluded that OPG levels were elevated 1.6-fold in chronic periodontitis, although not significantly, than in control group.

Also, Costa et al.[39] conduct a study to evaluate salivary concentrations of OPG in patients with periodontitis with type 2 diabetes by ELISA. The salivary OPG concentrations in the diabetes mellitus group were higher than in periodontitis and control groups. They concluded that salivary OPG concentrations were elevated regardless of periodontal inflammation in patients with diabetes. Therefore, periodontitis and diabetes are conditions that may interfere with protein expression and should be considered when using saliva for diagnosis.

In contrast to the above mentioned examinations, the OPG was significantly lowers in patients with chronic periodontitis compare with that in healthy individual. The reasons for these variations in the levels of OPG may be due to at least in part, to differences in the sampling method, sensitivity of the immunoassay and sample size.


  Conclusion Top


Based on the results of the current study, it could be concluded that:

  1. RANKL/OPG concentration ratio in GCF and saliva proved to be an important indicator of periodontitis, as it mirrors the relative expression ratio in the tissue.
  2. Both GCF and saliva collection are a noninvasive approaches as a diagnostic markers for RANKL, OPG and RANKL/OPG ratio. So, saliva can be used instead of GCF as its easier approach.
  3. GCF and saliva may have therapeutic value as an advantageous approach to periodontal disease management, targeting RANKL and OPG regulation by the host.


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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