ORIGINAL RESEARCH Year : 2023  |  Volume : 14  |  Issue : 2  |  Page : 49-51

Assessment of Salivary Levels of the RANKL and RANK in Patients with Healthy Gingiva on Reduced Periodontium Versus Periodontitis: An Analytical Cross-Sectional Study

Shurooq Abdulkareem Muhssin1, Hadeel Mazin Akram2
1 Department of Periodontology, Collage of Dentistry, University Of Baghdad, Baghdad; Al Mazraa Primary Health Care Sector, Al-Karkh Health Directorate of Baghdad, Iraq
2 Collage of Dentistry, University of Baghdad, Baghdad, Iraq

Date of Submission26-Jan-2023Date of Decision03-May-2023Date of Acceptance12-May-2023Date of Web Publication28-Jun-2023

Correspondence Address:
Shurooq Abdulkareem Muhssin
Al Mazraa Primary Health Care Sector, Al-Karkh Health Directorate of Baghdad, Baghdad
Iraq

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/denthyp.denthyp_17_23

Introduction: We aimed to compare the changes in the two salivary biomarkers, RANKL and RANK, among patients with healthy gingiva on reduced periodontium versus generalized periodontitis stages II and III. Methods: Study subjects were divided into three groups: (1) healthy periodontium (control group) (n = 15), (2) generalized periodontitis stages II and III (n = 30), and (3) healthy gingiva on reduced periodontium (n = 30). Salivary levels of RANKL and RANK were assessed using an enzyme-linked immunosorbent assay. Data analysis was done by the one-way ANOVA and the Tukey post hoc test using R software. Results: There was a statistically significant difference among the three study groups regarding salivary levels of the RANKL (P < 0.001) and RANK (P < 0.001). A Post hoc test showed that the difference between salivary levels of the RANKL (P = 0.50) and RANK (P = 0.86) among periodontitis groups and healthy gingiva in the reduced periodontium group was not statistically significant. Conclusion: High salivary levels of RANKL and RANK in comparison with healthy gingiva are not necessarily associated with the active phase of periodontal disease and progressive bone destruction.

Keywords: ELISA assay, osteoclasts, osteoimmunology, periodontal disease, periodontitis, RANK, RANKL, salivary biomarkers

How to cite this article:
Muhssin SA, Akram HM. Assessment of Salivary Levels of the RANKL and RANK in Patients with Healthy Gingiva on Reduced Periodontium Versus Periodontitis: An Analytical Cross-Sectional Study. Dent Hypotheses 2023;14:49-51
How to cite this URL:
Muhssin SA, Akram HM. Assessment of Salivary Levels of the RANKL and RANK in Patients with Healthy Gingiva on Reduced Periodontium Versus Periodontitis: An Analytical Cross-Sectional Study. Dent Hypotheses [serial online] 2023 [cited 2023 Jun 29];14:49-51. Available from: http://www.dentalhypotheses.com/text.asp?2023/14/2/49/379887   Introduction 

Clinically healthy gingiva should be able to be observed in an unharmed periodontium, which means that there is no clinical attachment loss or bone loss, and in a reduced periodontium in either a non-periodontitis patient (such as a patient who has some recession of the gingiva in some way or who has undergone surgery of crown lengthening) or who has had periodontitis in the past but is currently periodontally stable.[1]

The well-known definition of gingival recession is the apical displacement of the gingival edge below the cementoenamel junction (CEJ). Mechanical trauma and the inflammatory periodontal disease have been identified as the two primary causes of gingival recession.[2]

Alveolar bone loss is a significant contributor to tissue deterioration in periodontal disease. The process is influenced by a number of molecular events, such as receptor activator of nuclear factor kappa-B ligand (RANKL). A cytokine called RANKL governs the activation, proliferation, and differentiation of osteoclasts, which leads to the loss of alveolar bone. It also functions as a ligand for the RANK receptor. When the ligand activates the cell surface receptor activator of nuclear factor kappa-B, the cells transform into fully differentiated and activated osteoclasts. RANK is an important molecule that facilitates the interaction of osteoblasts and osteoclasts during bone remodeling.[3],[4] A recent review stated that further studies are needed to establish a clear link between these proteins and bone resorption related to periodontitis.[5]

However, we aimed to compare the changes in the two salivary biomarkers, RANKL and RANK, among patients with healthy gingiva on reduced periodontium versus generalized periodontitis stage II and III.

  Materials and methods 

The ethics committee at the University of Baghdad’s, College of Dentistry (Project number: 522622, Reference number: 522) approved the protocol of this analytical cross sectional study on April 17, 2022. G*Power (http://www.gpower.hhu.de/) is used for sample size calculation. Considering a power of 0.90, an alpha error probability of 0.05, and an effect size of 0.42, a sample of 75 subjects is required. Systemically healthy subjects with a minimum of 20 teeth, and no recent use of antibiotics or anti-inflammatory medications were recruited. The study groups included: (1) Control group (healthy periodontium) (n = 15); (2) generalized periodontitis stages II and III (n = 30); and (3) healthy gingiva on reduced periodontium (n = 30). Staging was carried out according to the 2017 world workshop on the classification of periodontal and peri-implant diseases and conditions.[1]

Patients in all these groups were instructed to stop eating or drinking for 1 to 2 hours before saliva sample collection, to clean their mouths only with tap water, and avoid any oral hygiene activities.[6] Unstimulated samples of pure saliva were collected between 9 a.m. and 12 p.m. and blindly labeled with their subject numbers. Samples were spun at 3000 rpm for 10 minutes (Thermo Scientific, Pico 17 centrifuge, MA, USA). Salivary clear supernatants separated from the cellular debris and were stored in a clean Eppendorf tube (Eppendorf, Hamburg, Germany) and frozen at −20 °C.[7],[8] Using the ELISA method, salivary levels of RANKL (Elabscience Biotechnology Co., Ltd., Houston, USA) and RANK (YL Biont, Shanghai, China) were biochemically assessed using the GloMax Discover Microplate Reader (Promega Corporation, Madison, USA). Data analysis was done by one-way ANOVA and the Tukey post hoc test using R software (R Foundation for Statistical Computing, Vienna, Austria).

  Results 

Seventy-five participants were included in this study (mean age  =  38, range 27–51, 51% male) (a flow diagram is available via https://doi.org/10.6084/m9.figshare.22796132.v1). There was a statistically significant difference among the three study groups regarding salivary levels of the RANKL (P < 0.001) and RANK (P < 0.001). A Post hoc test showed that the difference between salivary levels of the RANKL (P = 0.50) and RANK (P = 0.86) among periodontitis groups and healthy gingiva in the reduced periodontium group was not statistically significant [Figure 1].

Figure 1 Box and whisker plot showed descriptive data collected from ELISA test (pg/mL) related to the salivary levels of the RANKL and RANK.

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  Discussion 

It is well-known that the osteoclastic bone destruction that happens throughout periodontitis is reliant on the receptor activation of RANKL. Periodontal ligament cells, osteoblasts, osteocytes, and gingival epithelial cells have been reported to have the ability to encourage osteoclastogenesis by generating RANKL, which links the antibacterial immune reaction to bone demolition. The proliferation and differentiation of undifferentiated cells into the osteoclast phenotype were reported to be stimulated by a greater amount of RANKL, which causes bone resorption. RANKL is responsible for promoting osteoclast development, osteoclast adhesion to bone tissue, osteoclast activation, and osteoclast maintenance.[9]

To our knowledge, this is the first study to assess salivary levels of the two biomarkers, RANKL and RANK, among patients with healthy gingiva on reduced periodontium versus generalized periodontitis stages II and III. Results of this study showed salivary levels of the RANKL and RANK among periodontitis groups and healthy gingiva in the reduced periodontium group were comparable. On the other hand, significantly high salivary levels of these biomarkers in comparison with healthy gingiva are not necessarily associated with the active phase of periodontal disease and progressive bone destruction.

Similarly, Teodorescu et al. showed a significantly higher salivary level of RANKL among patients suffering from periodontitis in comparison with healthy individuals.[10] Bostanci et al. demonstrated a significantly higher level of RANKL in gingival crevicular fluid among chronic periodontitis, generalized aggressive periodontitis, and chronic periodontitis subjects under immunosuppressant therapy in comparsion with healthy and gingivitis groups.[11] Chairatnathrongporn et al. demonstrated that salivary RANK and C gene expression had a positive significant correlation with clinical periodontal conditions among type 1 diabetes mellitus patients.[12] Al-Ghurabi et al. reported a significantly higher salivary level of RANKL among subjects with chronic periodontitis in comparison with healthy volunteers.[13] A recent systematic review and meta-analysis by Theodoridis et al., which involved four articles, showed the RANKL level in peri-implant crevicular fluid did not show a significant difference between peri-implantitis and healthy group.[14]

However, this study’s limitations were the small sample size. Readers must note the inherent restrictions of cross-sectional studies. The relationship between the outcome and the exposure cannot be evaluated directly because they are analyzed simultaneously. More clinical studies with adequate sample size and a long-term flow-up period are needed to assess dynamic of salivary levels of the two biomarkers, RANKL and RANK among healthy subjects and patients suffering from gingivitis and periodontitis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 

  References 
1.Chapple ILC, Mealey BL, Van Dyke TE et al. Periodontal health and gingival diseases and conditions on an intact and a reduced periodontium: consensus report of workgroup 1 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions. J Periodontol 2018;89(1):S74–S84.  
    2.Hassan MN, Aziz MA. Gingival recession and periodontal therapy. J Baghdad Coll Dent 2019;31(1):31–6.  
    3.Shazam H, Shaikh F, Hussain Z, Shazam H, Shaikh F, Hussain Z. Bone turnover markers in chronic periodontitis: a literature review. Cureus. 2020;12(1):e6699.  
    4.Chen B, Wu W, Sun W, Zhang Q, Yan F, Xiao Y. RANKL Expression in periodontal disease: where does RANKL Come from? Biomed Res Int. 2014;2014:731039.  
    5.Asquino N, Vigil G, Pereira-Prado V et al. Bone resorption in periodontal disease: the role of RANK, RANKL and OPG. A literature review. Odontoestomatologia. 2022;24(40).  
    6.Al-Musawi M, Ali O. Assessment of salivary interleukin-1β levels in patients with gingivitis and periodontitis: an analytical cross-sectional study. Dent Hypotheses 2023;14(1):3–6.  
    7.Ali BT, Mahmood MS. Assessment of salivary total antioxidants capacity levels of patients with chronic periodontitis in comparison to Healthy Control. J Baghdad Coll Dent 2018;30(1):58–62.  
    8.Surgery M, Nanakaly HT. Effect of periodontal therapy on serum and salivary interleukin-2 levels in chronic periodontitis. J Baghdad Coll Dent 2016;28(2):73–8.  
    9.Tsukasaki M. RANKL and osteoimmunology in periodontitis. J Bone Miner Metab. 2021;39(1):82–90.  
    10.Teodorescu AC, Martu I, Teslaru S et al. Assessment of Salivary Levels of RANKL and OPG in Aggressive versus Chronic Periodontitis. J Immunol Res. 2019; 2019.  
    11.Bostanci N, Ilgenli T, Emingil G et al. Gingival crevicular fluid levels of RANKL and OPG in periodontal diseases: implications of their relative ratio. J Clin Periodontol. 2007;34(5):370–376.  
    12.Chairatnathrongporn R, Tansriratanawong K, Santiprabhob J, Boriboonhirunsarn C, Promsudthi A. salivary gene expression of RANK, RANKL, and OPG in Type 1 diabetes mellitus and periodontal disease patients. J Int Soc Prev Community Dent. 2022;12(6):603–11.  
    13.Al-Ghurabi BH, Mohssen SM. Salivary level of RANKL and OPG in chronic periodontitis. J Baghdad Coll Dent 2015;27(1):189–94.  
    14.Theodoridis C, Doulkeridou C, Menexes G, Vouros I. Comparison of RANKL and OPG levels in peri-implant crevicular fluid between healthy and diseased peri-implant tissues. A systematic review and meta-analysis. Clin Oral Investig. 2022;26(1):823–36.  
    
  [Figure 1]