The association between receptor activator of nuclear factor kappa-β ligand and clinical attachment level among waterpipe smoker.

BACKGROUND: Higher levels of receptor activator of nuclear factor kappa-β ligand, i.e., RANKL, were reported among periodontitis patients compared with their healthy peers. This study aimed to measure the salivary level of RANKL among waterpipe smokers and to find the possible correlation between salivary level of RANKL and clinical periodontal parameters.
MATERIALS AND METHODS: This study is a cross-sectional study involving 89 (49 waterpipe smokers and 40 nonsmokers) participants. The whole unstimulated saliva samples were collected for quantitative determination of RNAKL using a human RANKL enzyme-linked immunosorbent assay kit. Plaque index, gingival index, bleeding on probing, periodontal pocket depth (PPD), and clinical attachment level (CAL) were recorded for each participant. Mann-Whitney U-test was used to detect any difference between groups at P < 0.05. Spearman correlation coefficient test was used to test the association between variables. Linear regression was used to investigate the cause-and-effect relationship between the associated variables.
RESULTS: In a comparison, RANKL, PPD, and CAL showed statistical differences (P < 0.05) between waterpipe smokers and nonsmokers. RANKL was found to be positively associated with CAL, and approximately 62.8% of CAL in this study sample could be explained by RANKL. The regression equation was CAL = 1.744 + 0.004 (RANKL).
CONCLUSIONS: The salivary level of RANKL was positively related to CAL in this study sample.

INTRODUCTION

Smoking is the major risk factor for the development and progression of periodontal diseases.[1] Heavy smokers were reported to have more attachment and alveolar bone loss compared to nonsmokers with less response to periodontal treatment.[2] Many smoking tobacco products are available in the markets including cigarettes, cigars, cigarillos, and waterpipe. Waterpipe tobacco smoking is a common practice in Eastern Mediterranean countries, Middle East, and parts of Asia.[3] Waterpipe device consists of bowel, hose, body, valve, and water jar.[4] A negative pressure is formed above the water each time the smoke is inhaled from the hose, and air passes over the charcoal and tobacco, transmits through the body of the waterpipe, and reaches the jar bubbling through water. Upon smoking, water in the jar lowers the temperature and moisturizes the aerosol of smoke. This leads to the misbelief of considering waterpipe smoking is safer than cigarette and cigar smoking.[5] Waterpipe smoke contains many toxic heavy metals such as cadmium, cobalt, nickel, led, cobalt, chromium, and arsenic. These toxic constituents were reported to be identified in significantly higher levels in waterpipe smoke comparing to cigarette smoke.[6] The volume of waterpipe smoke production (500,000 mm) is much higher than that of cigarette (500–600 mm). Nicotine blood level of daily waterpipe smokers is the same as in person who smokes ten cigarettes a day. Polycyclic aromatic hydrocarbons in the waterpipe smoke are about twenty times that of cigarette smoke.[7] These findings support the more harmful effect of waterpipe smoking comparing to cigarette.

Periodontal clinical parameters and radiographic assessment of alveolar bone loss are informative to evaluate the periodontal disease severity. On the other hand, they provide few useful determinants of disease activity.[8] Salivary biomarkers may help in this concept. Determining the biomarkers levels in saliva may offer an easy and safe approach for disease prediction and detection.[9]

Receptor activator of nuclear factor kappa-β ligand (RANKL) is a cytokine which belongs to the tumor necrosis factor (TNF) family. It is a polypeptide of 314 amino acids encoded by the gene TNF superfamily 11 (TNFSF11). It is secreted freely by the inflammatory cells or expressed in the form of membrane-bound protein.[1011] RANKL has a role in alveolar bone resorption.[1213] Higher levels of RANKL were reported among periodontitis patients compared with their healthy peers. The levels of RANKL were found to be positively associated with the increase in severity of periodontitis and related to the load of periodontal pathogenic Porphyromonas gingivalis within periodontal tissue.[14]

After reviewing the literature, this study is the first to measure the salivary level of RANKL among waterpipe smokers and to find the possible correlation between salivary level of RANKL and clinical periodontal parameters.

MATERIALS AND METHODS

This study was a cross-sectional study. A sample of 49 waterpipe smokers and 40 nonsmokers (aged 30–40 years) was collected from the recruitment of patients in the Department of Periodontics, at teaching hospital, College of Dentistry, University of Baghdad. The collection was started in February 2017 until April 2017. The sample size was determined after conducting a pilot study involving 10 participants and was calculated using G*Power software (version 3.1.9.2) at probability power of 0.8 and 0.05 type I error probability. The ethical approval to conduct the study and collect patients was obtained from the College of Dentistry, University of Baghdad committee (13s/1277). The participants selected for this study were male waterpipe smokers and with no history of any systemic disease. Any patient examined who had undergone periodontal treatment or on a course of anti-inflammatory or antimicrobial therapy during the last 3 months or a cigarette smoker was excluded. All selected participants voluntarily enrolled in the study after a well explanation about the aim of the study. Written informed consents were obtained from all participants.

The whole unstimulated saliva samples were collected at 9–11 am. At the time of collection, participants gargled thoroughly with water to remove any possible debris. Then, they were asked to roll saliva into test tubes (Eppendorf tube 5.0 mL) for 5 min. The tubes were kept in a small cooling box after collection. Then, the collected saliva was centrifuged at 2000 rpm for 20 min, and the clear supernatant saliva was withdrawn by micropipette into Eppendorf tubes and stored at −20°C until biochemical analysis of RANKL.[15]

After salivary sample collection, plaque index (PI),[16] gingival index (GI),[17] bleeding on probing (BOP), periodontal pocket depth (PPD), and clinical attachment level (CAL) were recorded for each participant using Williams probe (Hu-Friedy, Chicago, ILUSA). All measurements were recorded by a single examiner. A weak before clinical examination, the examiner alignment was carried out using Hefti AF and Preshaw PM protocol.[18] Intraexaminer calibration was achieved with kappa value >0.7. For PI and GI, four surfaces (mesial, buccal/labial, distal, and lingual/palatal) of all teeth except third molars were recorded. For BOP, the periodontal probe was inserted to the bottom of gingival crevice or periodontal pocket and was moved gently along the root surface. If bleeding occurred within 30 s, the site was given score (1) and score (0) was given for the nonbleeding site. While for PPD and CAL, the sites of measurement were midbuccal line, midpalatal/lingual line, and mesiobuccal and distobuccal line angles.

For quantitative determination of RANKL in the saliva samples, human RANKL enzyme-linked immunosorbent assay kit (Shanghai Yehua Biological Company, China [Cat. No YHB2552Hu]) was used. The procedure followed the manufacturer instructions of the kit. The concentration of RANKL in each sample was determined by comparing the average sample optical density record with the concentrations from the assay standard curve.

Data were analyzed using Statistical Package of Social Science software version 19 (IBM SPSS Statistics for Windows, Version 19.0. Armonk, NY: IBM Corp). Kolmogorov–Smirnov test was used to check the normality of data (results are not shown). Since abnormal distribution of the data was assumed, Mann–Whitney U-test was used to detect any difference between groups at P < 0.05. Spearman correlation coefficient test was used to test the association between variables. Linear regression was used to investigate the cause-and-effect relationship between the associated variables.

RESULTS

Among 49 waterpipe smokers and 40 nonsmokers in this study, 45 participants had periodontitis. The mean, SD, and median of RANKL and the clinical periodontal parameters of smokers and nonsmokers are shown in Table 1. In a comparison, RANKL, PPD, and CAL showed statistical differences (P < 0.05) between these two groups.

Table 1

Statistical description and comparison between waterpipe smokers and nonsmokers

RANKL was found to be positively associated with CAL as shown in Table 2. Table 3 shows the linear regression between CAL and RANKL. It was found that approximately 62.8% of CAL in this study sample can be explained by RANKL. In every one-unit increase in RANKL, there will be an increase in CAL by 0.004. The regression equation was estimated (CAL = 1.744 + 0.004 [RANKL]). The unstandardized residuals in the regression model were normally distributed indicating the validity of the regression model outcome.

Table 2

Spearman correlations between receptor activator of nuclear factor kappa-β ligand and periodontal parameters

Table 3

Linear regression analyses for the association between clinical attachment level (dependent variable) and receptor activator of nuclear factor kappa-β ligand (predictor)

DISCUSSION

In this study, there were no statistical differences in the means of PI, GP, and BOP parameters between waterpipe smokers and nonsmokers. These findings might be attributed to the fact that plaque accumulation and gingivitis are associated to the oral hygiene control performance of participants.[19] Thus, smoker and nonsmoker participants in this study might perform approximately the same level of mechanical plaque control measures. In contrast, waterpipe smokers showed deeper pockets and more attachment loss. This agrees with earlier reports of the role of smoking as a risk factor for the development and progression of periodontal diseases.[1]

The interesting finding in this study was the higher level of RANKL in saliva among waterpipe smokers. This result agrees with earlier studies[2021] and disagrees with others.[2223] It is important to remember that this study is the first study to measure RANKL level in saliva of waterpipe smokers. Earlier mentioned studies reported either the salivary level of RANKL among cigarette smokers or the RANKL level in gingival crevicular fluid among waterpipe smokers. As a limitation of this study, it was better to include cigarette smokers group to compare with waterpipe smokers, also the absence of quantitative measures of waterpipe smoking effect to standardize it with cigarette smokers, such as nicotine and carbon monoxide concentrations.

The level of RANKL in the saliva of waterpipe smokers was found to be positively related to the mean of CAL. This interesting result may help to develop a noninvasive diagnostic tool that provides accurate and predictive evaluation of periodontal disease status. The loss of attachment in 62.8% of the examined participants could be explained by the higher level of salivary RANKL among smokers. Earlier studies reported that higher level of RANKL induces bone resorption by stimulating the proliferation and differentiation of undifferentiated cells to form the osteoclast phenotype and thus increasing bone resorption.[2425]

CONCLUSIONS

The salivary level of RANKL is positively related, correlation coefficient = 0.724, to the loss of attachment among waterpipe smokers. The loss of attachment in 62.8% of the examined periodontitis participants could be explained by the elevated salivary levels of RANKL among waterpipe smokers. These findings emphasize on the detrimental effect of waterpipe smoking on periodontal attachment level.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest