Batool.doc J Bagh College Dentistry Vol. 27(1), March 2015 Salivary level of Basic Sciences 189 Salivary level of RANKL and OPG in chronic periodontitis Batool Hassan Al-Ghurabi, B.Sc., M.Sc., Ph.D. (1) Sara Mohammed Mohssen, B.Sc., M.Sc. (2) ABSTRACT Background: Periodontal diseases are initiated by microbial plaque, which accumulates in the sulcular region and induces an inflammatory response. The Receptor activator of nuclear factor-kappa B ligand / osteoprotegerin (RANKL/OPG) axis is involved in the regulation of bone metabolism in periodontitis, in which an increase in receptor activator of nuclear factor-kappa B ligand or a decrease in osteoprotegerin can tip the balance in favor of osteoclastogenesis and the resorption of alveolar bone that is the hallmark of periodontitis. This study was performed to investigate the role of salivary levels of RANKL and OPG in pathogenesis of chronic periodontitis. Subjects and Methods: Fifty five subjects with chronic periodontitis with ages range from 24-64 years and 25 apparently healthy volunteers their ages and sexes were matched with the patients were participated in this study. Periodontal parameters used in this study were plaque index, gingival index, probing pocket depth, clinical attachment level and bleeding on probing. Saliva samples were collected from all patients and controls. Enzyme- linked immunosorbent assay was carried out for estimation the salivary level of RANKL and OPG in studied groups. Results: The present data revealed that the median salivary levels of RANKL was elevated in patient as compared with control group (P<0.001), whereas the salivary levels of OPG doesn’t show any significant differences between the study groups (p>0.05). In contrast the ratio of RANKL/OPG ratio was significantly higher in patients when compared with the ratio in control group. Furthermore, negative significant correlation was noticed between RANKL and OPG. Regarding correlation between salivary (RANKL and OPG) and clinical periodontal parameters, RANKL levels was showed significant positive correlation with each of probing pocket depth and clinical attachment level. Otherwise no association between OPG levels and clinical parameters of periodontitis was found. Moreover; the ratio RANKL/OPG was showed significant positive correlation with each of gingival index, probing pocket depth and clinical attachment level. Conclusion: This study demonstrates that salivary levels of RANKL and OPG play a crucial role in pathogenesis of periodontitis, and the relative RANKL/ OPG ratio appears to be indicative of disease occurrence. Keywords: chronic periodontitis, RANKL, OPG. (J Bagh Coll Dentistry 2015; 27(1):189-194). INTRODUCTION Periodontitis has a multifactorial etiology, and the characteristic tissue destruction is mediated mainly by the aberrant immune response of the host to periodontopathic bacteria. The role of oral microflora in the etiology of various inflammatory periodontal diseases has been well established, and specificity may vary between bacterial etiologies and different forms of periodontal disease (1,2). The chronic form of periodontitis, termed chronic periodontitis, is the most prevalent disease type (3). While headways have been made in various areas concerning the molecular pathogenesis of periodontitis, one area which of increasing importance is the involvement of the (RANKL/OPG) axis in bone remodeling and bone loss in periodontitis. RANKL and its receptor “Receptor Activator of Nuclear Factor-kappa B” (RANK) have been recognized as key factors regulating osteoclast formation (4). RANKL a membrane-bound or soluble protein belonging to the tumor necrosis factor (TNF) superfamily that is primarily produced in osteobl- (1)Assistant Professor. Department of Basic Sciences, College of Dentistry, University of Baghdad. (2)Department of Basic Sciences, College of Dentistry, University of Baghdad. astic lineages and activated T cells. RANKL stimulates osteoclast differentiation and activation, and inhibits osteoclast apoptosis. Binding of RANKL to RANK expressed on the surfaces of osteoclasts and their precursors, promotes osteoclast differentiation and activation (5). OPG is a soluble TNF receptor–like molecule, is the inhibitor of osteoclast differentiation. It binds to RANKL and blocks RANKL from interacting with RANK and neutralizes its activity by inhibiting the cell-to-cell signaling between osteoblast/bone stromal cells and osteoclast precursor cells, resulting in the inhibition of osteoclast formation. RANKL and OPG are crucial molecules that act as positive and negative regulators, respectively, in osteoclastogenesis and bone resorption. Under normal physiologic conditions, there is a balance between bone resorption and bone formation. Upregulation of RANKL has been seen in inflamed periodontal tissues, indicating that RANKL strongly participates in the processes of periodontal tissue destruction (6). Therefore this study was performed to investigate the role of salivary levels of RANKL and OPG in pathogenesis of chronic periodontitis. J Bagh College Dentistry Vol. 27(1), March 2015 Salivary level of Basic Sciences 190 SUBJECTS AND METHODS Fifty five patients with chronic periodontitis (38 male and 17 female) were enrolled in this study, their age range from 24 to 64 years. They were from attendants seeking treatment in the department of periodontics, College of Dentistry, Baghdad University from Novmber 2012 to Janraury 2013. Diagnosis was made by specialized dentists in the College. Oral examination was performed at four surfaces of each tooth except 3rd molar according to the following criteria: Assessment of dental plaque by (PI) of Silness and Loe (7). Assessment of gingival condition by (GI) (8). Probing pocket depth (PPD), clinical attachment level (CAL) and bleeding on probing (BOP). All the cases had received no treatment with no complain of chronic or systemic diseases. Apparently healthy periodontium volunteers their ages and sexes were matched to patients consisted of 25 individuals who were considered as control (19 males and 6 females). The saliva obtained from patients and healthy controls were analyzed for RANKL and OPG by using commercially available ELISA and performed as recommended in leaflet with kits, (Human RANKL and OPG ELISA Kit/ Cusabio/China). Statistical analysis It was assessed using P (Mann-Whitney-test), P (Bonferroni-test). Correlation between the different parameters was calculated by the spearman test and p values of P<0.01and P<0.05 were considered significant. RESULTS In the present study the mean age of patients was 40.15 ± 10.53 years, and there was male’s predominance among patients, about (69.1%) of CP patients were males, while only (30.9%) were females. Furthermore, (34.5%) of patients had positive family history of chronic periodontitis, while (65.5%) showed negative family history as clearly shown in table (1). The differences in clinical periodontal parameters in patients and healthy controls are summarized in table (2). Table (3) revealed a highly significant elevation in level of RANKL among patients (56.8 pg/ml) in comparison to that of healthy control (2.21 pg/ml), (p<0.001). While there is no significant differences (p>0.05) in median level of OPG between patients and healthy control groups (15 ng/ml; 17.99 ng/ml) respectively, as shown in table (4). Determination the ratio of RANKL / OPG in current study revealed, that there was high difference between two study groups. The median RANKL / OPG ratio in chronic periodontitis patients (3.61) was significantly higher (P<0.001) in comparison to the ratio in healthy control (0.12), according to tables (5). The results of correlation between RANKL and OPG are clearly shown in table (6). An anticipated, salivary RANKL level was showed significant negative correlation with OPG (r=- 0.331, p=0.024). On the other hand the current study found that RANKL/OPG ratio was positively correlated with RANKL(r=0.724, p=0.001), whereas negatively correlated OPG (r=- 0.300, p=0.006). Regarding the correlation of RANKL and OPG with clinical periodontal parameters, RANKL level was showed significant positive correlation with each of PPD (r=0.387, P=0.004) and CAL (r=0.267, P=0.049), as observed in table (7). In contrast, there is no association between OPG levels and other clinical parameters of periodontitis was found (p>0.05), table (8). Moreover; the current study found that the RANKL/OPG ratio was positively correlated with each of GI, PPD and CAL (r= 0.250, P=0.024; r=0.409, p<0.001; r=0.334, P=0.002) respectively, as shown in table (9). Table 1: Demographic Characteristic in CP Patients and Healthy Control Study groups P-value CP Patients n=55 Healthy control n=25 Age and Sex Age (years) Range (24-64) (20-51) Mean ± SD 40.15±10.53 37.38±9.10 0.254NS Gender type Female 17 (30.9%) 6 (24%) 0.128NS Male 38 (69.1%) 19 (76%) 0.672NS NS=Not significant (p>0.05). ** = Highly significant difference (p≤0.001). J Bagh College Dentistry Vol. 27(1), March 2015 Salivary level of Basic Sciences 191 Table 2: Clinical Periodontal Parameters in CP Patients and Healthy Control. Study groups P-value CP Patients n=55 Healthy control n=25 Clinical periodontal Parameters (Mean ± SD) Plaque index 1.50±0.62 0.85±0.37 <0.001** Gingival Index 1.26±0.49 0.74±0.29 <0.001** Probing Pocket Depth (mm) 2.10±0.66 1.17±0.62 <0.001** Clinical Attachment Loss 3.08±3.74 0.00±0.00 <0.001** Bleeding on Probing (BOP) 28.85±30.57 6.12±8.08 <0.001** ** = Highly significant difference (p≤0.001). Table 3: Differences in saliva concentration of RANKL between patients and healthy controls CP patients (n=55) Healthy control (n=25) P (Mann-Whitney) Saliva RANKL Range (6.21-284.34) (0-10.18) Median 56.8 2.21 <0.001** Inter-quartile range (15.99-85.4) (1.2-6.27) Mean Rank 53.15 15.29 Table 4: Differences in saliva concentration of OPG between patients and healthy controls CP patients (n=55) Healthy control (n=25) P (Mann-Whitney) Saliva OPG Range (2.3-57) (4.57-28.3) Median 15 17.99 0.070NS Inter-quartile range (10.25-19.3) (12.68-20.91) Mean Rank 37.84 47.69 Table 5: Differences in ratio of RANKL\OPG between patients and healthy controls CP patients Healthy control P (Mann-Whitney) (n=55) (n=25) Salivary ratio RANKL/OPG Range (0.3-34.24) (0-1.54) Median 3.61 0.12 <0.001** Inter-quartile range (1.24-9.04) (0.06-0.43) Mean Rank 52.9 15.83 Table 6: Correlation between RANKL and OPG among patients Variables RANKL OPG RANKL/OPG RANKL 1 r=-0.331 p=0.024* r=0.724 p=0.001** OPG r=-0.331 p=0.024* 1 r=-0.300 p=0.006* RANKL/OPG r=0.724 p=0.001** r=-0.300 p=0.006* 1 Table 7: Correlation between saliva level RANKL and clinical periodontal parameters in patients RANKL Correlation P- value Plaque index 0.119 0.388NS Gingival Index 0.047 0.733 NS Probing Pocket Depth (mm) 0.387 0.004* Clinical Attachment Loss 0.267 0.049* Bleeding on Probing (BOP) -0.108 0.432 NS J Bagh College Dentistry Vol. 27(1), March 2015 Salivary level of Basic Sciences 192 Table 8: Correlation between saliva level OPG and clinical periodontal parameters in patients OPG Correlation P- value Plaque index 0.077 0.574 NS Gingival Index -0.072 0.600 NS Probing Pocket Depth (mm) 0.019 0.892 NS Clinical Attachment Loss 0.015 0.913 NS Bleeding on Probing (BOP) 0.072 0.600 NS Table 9: Correlation between serum level RANKL\OPG and clinical periodontal parameters in patients RANKL/OPG ratio Correlation P value Plaque index 0.209 0.062NS Gingival Index 0.250 0.024* Probing Pocket Depth (mm) 0.409 <0.001** Clinical Attachment Loss 0.334 0.002* Bleeding on Probing (BOP) 0.214 0.055 NS DISCUSSION Inflammation and bone loss are hallmarks of periodontal disease. Accumulated evidence demonstrates that periodontitis involves bacterially derived factors and antigens that stimulate a local inflammatory reaction and activation of the innate immune system. Proinflammatory molecules and cytokine networks play essential roles in this process. IL-1 and TNF-α seem to be primary molecules that, in turn, influence cells in the lesion. Antigen- stimulated lymphocytes (B and T cells) also seem to be important. Eventually, a cascade of events leads to osteoclastogenesis and subsequent bone loss via the RANK–RANKL–OPG axis. This axis and its regulation are not unique to periodontitis but rather are critical for pathologic lesions involving chronic inflammation (5). The current results denoted a predominance of CP among males than a female which is comparable with other Iraqi study conducted by Fadil and Al- Ghurabi in 2012 (9), whereas, disagree with the result reported by Ali and colleagues (10) who observed that periodontitis in males less than as in females. The large prevalence of periodontitis among males may be due to that male less interest in oral hygiene than males. Positive family history was observed in 34.4% of patients with CP. This high rate of a positive family history in the present study raises the possibility of a genetic basis for CP transmission. However; this percentage which is in agreement with a local study reported by Ali et al., (10) who found 32% of patient had positive family history, and disagree with broad studies (11, 12). The findings of the present study indicate that the RANKL levels in saliva of chronic periodontitis patients were increased compared to controls, whereas the OPG levels were unchanged. These results are in agreement with other studies (13, 14, 15), who found an increase in soluble RANKL concentrations without a corresponding change in OPG levels in individuals with chronic periodontitis compared to healthy controls. Conversely, a reciprocal relationship was also found, in which RANKL protein expression was higher and OPG levels were lower in diseased gingival tissues compared to healthy controls (16). In consistent with our results Wara and colleagues, pointed out to that although the exact levels of RANKL and OPG varied from study to study, the trend was generally the same; the RANKL/OPG ratio was higher in periodontitis patients than in healthy controls (15). These findings correspond well with the critical role of RANKL in driving osteoclastogenesis and bone loss in periodontitis (17). Similarly, Corrti and colleagues showed that study on RANKL versus OPG concentrations in gingival tissue extracts clearly demonstrated a trend toward a higher RANKL/OPG ratio in individuals with periodontitis than in healthy controls. In a cross-sectional study, Bostanci et al. (18) quantified the RANKL and OPG levels in GCF from 21 healthy subjects, 22 gingivitis, 28 CP, 25 generalized aggressive periodontitis and 11 chronic periodontitis immunosuppressed patients, detecting that RANKL levels increased and OPG decreased in periodontitis patients compared with either gingivitis or healthy individuals, and concluded that RANKL/OPG ratio may predict disease occurrence. J Bagh College Dentistry Vol. 27(1), March 2015 Salivary level of Basic Sciences 193 The higher RANKL/ OPG ratio could explain in part the amount of bone loss in periodontitis patients, and this either be related to lower levels of OPG or higher levels of RANKL in periodontitis patients compared with healthy control. However, an increased RANKL/OPG ratio also may be associated with the clinical severity of periodontitis. The current study found that the RANKL/OPG ratio was positively correlated with each of GI, PPD and CAL, this result was consistent with that reported by Fatemeh et al., (19) in which there was statistically significant correlation between the concentration of RANKL and CAL in chronic periodontitis patients, there was also negative correlation between OPG concentration and CAL in those patients. Meanwhile, Bostanci et al., stated that although periodontitis is associated with an increased RANKL/OPG ratio compared to healthy controls, the ratio may not necessarily distinguish between mild, moderate, and severe forms. Nevertheless, results show that RANKL contributes to alveolar bone loss in periodontitis and tooth loss, other findings have suggested that the increased RANKL/OPG ratio may serve as a biomarker that denotes the occurrence of periodontitis, but may not necessarily predict ongoing disease activity (14). Microbial stimulation with Aggrigatibacter in chronic periodontitis induced RANKL expression on the surface of CD4 + cells and this may explain the elevation of RANKL/OPG ratio in periodontitis (20). In conclusion this study demonstrates that salivary levels of RANKL and OPG play a crucial role in pathogenesis of periodontitis, and the relative RANKL/ OPG ratio appears to be indicative of disease occurrence. REFERENCES 1. Craig RG, Yip JK, Mijares DQ, LeGeros RZ. J Clin Periodontol 2003; 30: 1075-83. 2. Carenza A. Clinical periodontology. 10th ed. Philadelphia: WB Saunders Company; 2009. 3. Cazalis J, Tanabe S, Gagnon G, Sorsa T, Grenier D. Tetracyclines and chemically modified tetracycline-3 (CMT-3) modulate cytokine secretion by lipopolysaccharide-stimulated whole blood. Inflammation 2009; 32: 130-7. 4. Garlet GP, Cardoso CR, Sliva TA, et al. Cytokine pattern determines the progression of experimental periodontal disease induced by Actinobacillus actinomycetemcomitans through the modulation of MMPs, RANKL, and their physiological inhibitors, Oral Microbiol Immunol 2006; 21: 12-20. 5. Cochran DL. Inflammation and bone loss in periodontal disease. J Periodontol 2008; 79: 1569–76. 6. Kirkwood KL, Cirelli JA, Rogers EJ, Giannobile WV. Novel host response therapeutic approaches to treat periodontal diseases. Periodontol 2000 2007; 43: 294- 315. 7. Silness J, Löe H. Periodontal disease in pregnancy ΙΙ. Acta Odontol Scand 1964; 24: 747-59. 8. Löe H. The gingival index, the plaque index and the retention index system. J Periodontol 1976; 38: 610- 6. 9. Fadil Z, Al-Ghurabi B. Study the role of pro-and anti- inflammatory cytokines in Iraqi chronic periodontitis patients. J Bagh Coll Dentistry 2012; 24(1): 164-69. 10. Ali AO, Saleem SS, Algobory SH. Reliability of family history report among relatives of aggressive periodontitis patients. J Bagh Coll Dentistry 2008; 20(1): 152-160. 11. Hart TC, Marazita ML, McCanna KM, Schenkein HA, Diehl SR. Reevaluation of the chromosome 4q andidate region for early onset periodontitis. Human Genetics 1993; 91: 416–22. 12. Llorente MA, Griffiths GS. Periodontal status among relatives of aggressive periodontitis patients and reliability of family history report. J Clin Periodontol 2006; 33: 121–5. 13. Kawai T, Akira, S. Pathogen recognition with Tolllike receptors. Curr Opin Immunol 2005; 17: 338–44 14. Lu HK, Chen YL, Chang HC, Li CL, Kuo MY. Identification of the osteoprotegerin/receptor activator of nuclear factor-kappa B ligand system in gingival crevicular fluid and tissue of patients with chronic periodontitis. J Periodontal Res 2006; 41: 354- 60. 15. Wara-aswapati N, Surarit R, Chayasadom, Boch JA, Pitiphat W. RANKL upregulation associated with periodontitis and Porphyromonas gingivalis. J Periodontol 2007; 78: 1062-1069. 16. Crotti T, Smith MD, Hirsch R, et al. Receptor activator NF kappaB ligand (RANKL) and osteoprotegerin (OPG) protein expression in periodontitis. J Periodontal Res 2003; 38: 380-87. 17. Taubman MA, Valverde P, Han X, Kawai T. Immune response: the key to bone resorption in periodontal disease. J Periodontol 2005; 76: 2033- 41. 18. Bostanci N, Ilgenli T, Emingil G, Afacan B, Han B, Toz H, Atilla G, Hughes FJ, Belibasakis GN. Gingival crevicular fluid levels of RANKL and OPG in periodontal diseases: implications of their relative ratio. J Clin Periodontol 2007; 34: 370-76. 19. Fatemeh S, Mandana S, Shilan R, Malihe N. Receptor activator of nuclear factor kappa B ligand and osteoprotegerin levels in gingival crevicular fluid. Dent Res J 2012; 9(6): 752-7. 20. Teng YT, Nguyen H, Gao X, Kong YY, Gorczynski RM, Singh B, Ellen RP, Penninger JM. Functional human T-cell immunity and osteoprotegerin ligand control alveolar bone destruction in periodontal infection. J Clin Invest 2000; 106: 59-67. J Bagh College Dentistry Vol. 27(1), March 2015 Salivary level of Basic Sciences 194 الخالصة یساھم في OPGو RANKL ان محور. تنشا امراض ما حول اللثة عن طریق الصحیفة الجرثومیة التي تتراكم في منطقة التلمیة والتي تحث االستجابة االلتھابیة :الخلفیة العلمیة و التي ھي لتي فیھا الزیادة في الرانكل والنقصان في االوبجي یمكن ان یرجح التوازن باتجاه عملیة نقض العظم وارتشاف العظم النسغيعملیة تنظیم ایض العظم في مرض النساغ وا .المزمنةفي التسبب في اللثة OPGو RANKLتم إجراء ھذه الدراسة لبحث دور مستویات اللعابیة من .السمة الممیزة لمرض النساغ طابقة مع تم اعمارھم و اجناسھم صحاء كانتاالتطوعین ن المم 25عاما و 64-24 بین أعمارھمبمرض النساغ المزمن تتراوح مریضا مصاباخمسون خمسة و: العمل وطرق المرضى فقدان و اللثة و بوجیعمق التھابات اللثة ومؤشر الصحیفة الجرثومیة ومؤشر ھي المستخدمة في ھذه الدراسةما حول االسنان معلمات وكانت . في ھذه الدراسة شاركوا المرضى المستوى اللعابي من لتقدیر المرتبط الممتز المناعیة فحص مقایسة االنظیم وأجري. السیطرةوالمرضى مجموعتيتم جمع عینات من اللعاب من . اثناء الفحصنزیف الواالنسجة الرابطة RANKL وOPG مجموعتي الدراسة في. مستویات اللعابیة ال، في حین أن )P >0.001(مقارنة مع مجموعة التحكم ىضارتفع في مجموعة المرقد RANKLمستویات اللعابیة من ال متوسط كشفت البیانات الحالیة أن: النتائج أعلى بكثیر لدى المرضى بالمقارنة مع النسبة في RANKL / OPGفي المقابل كانت نسبة ) . P <0.05(تظھر أي فروق ذات داللة إحصائیة بین مجموعات الدراسة لم OPGمن ومعلمات اللثة ) OPGو RANKL(من اللعابیة المستویات رتباط بینباالوفیما یتعلق . OPGو RANKLوعالوة على ذلك، لوحظ ارتباط كبیر سلبي بین . ضابطةالمجموعة ال والمعلمات السریریة OPGمستویات بین یالحظ وجود عالقةلم و. فقدان االنسجة الرابطةب ووارتباط إیجابي كبیر مع كل من عمق الجی RANKLالسریریة، وقد أظھر مستویات .وفقدان االنسجة الرابطةب وعمق الجی التھاب اللثة،ارتباط إیجابي كبیر مع كل من مؤشر RANKL / OPGقد أظھرت نسبة وعالوة على ذلك، . اللتھاب اللثة تكون مؤشرا على قد RANKL / OPGنسبة ان اللثة، و امراض تسبب فيتلعب دورا حاسما في ال OPGو RANKLتوضح ھذه الدراسة أن مستویات اللعابیة من : االستنتاجات .حدوث المرض RANKL ،OPGالتھاب اللثة المزمن، : مفتاحیةكلمات ال