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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES BANGALORE, KARNATAKA. ANNEXURE II PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION 1. NAME OF THE CANDIDATE AND ADDRESS ( IN BLOCK LETTERS) Dr. VAZHAKKAT POOJA RAJACHANDRAN POST GRADUATE STUDENT DEPARTMENT OF PERIODONTICS, THE OXFORD DENTAL COLLEGE AND HOSPITAL, HOSUR ROAD, BOMMANAHALLI, BANGALORE – 560068 2. NAME OF THE INSTITUTION THE OXFORD DENTAL COLLEGE AND HOSPITAL, BANGALORE 3. COURSE OF STUDY AND SUBJECT MASTER OF DENTAL SURGERY, PERIODONTICS 4. DATE OF ADMISSION OF COURSE 03 /06/2013 5. TITLE OF THE TOPIC COMPARATIVE EVALUATION OF THE EFFECT OF PERIODONTAL GINGIVAL NON-SURGICAL THERAPY CREVICULAR ADRENOMEDULLIN ON FLUID LEVELS IN CHRONIC PERIODONTITIS PATIENTS WHO ARE SMOKERS AND NON- SMOKERS – A CLINICOBIOCHEMICAL STUDY. 6. BRIEF RESUME OF THE INTENDED WORK 6.1 NEED FOR THE STUDY Human gingival epithelium is constantly exposed to multiple assaults by microbes that live harmoniously in the oral niche1. Periodontal diseases are initiated by breakdown of the bacteria - host equilibrium that results in inflammatory destruction of tooth supporting tissues1. Gingival epithelium is the first line of host defence, represented by its barrier function and antimicrobial properties1. Oral keratinocyte innate immune effector molecules include the antimicrobial peptides like defensin, cathelicidin, adrenomedullin and calprotectin2. Adrenomedullin, a 52 amino acid peptide has antimicrobial properties against both Gram positive and Gram negative bacteria, local bactericidal effect, potent microvascular vasodilatory properties, supply inflammatory cells and mediators to inflamed periodontal tissues3. Production of adrenomedullin is up-regulated by oxidative stress (particularly in smokers), pro-inflammatory cytokines, angiotensin II, hypoxia, hyperglycemia, natriuretic peptide, aldosterone, etc4 Therefore, the aim of the present study is to determine the effect of non-surgical periodontal therapy on gingival crevicular fluid adrenomedullin levels in chronic periodontitis patients who are smokers and non-smokers. 6.2 REVIEW OF LITERATURE 1. A study was conducted to investigate the levels of gingival crevicular fluid adrenomedullin and human neutrophil peptides1-3 (HNP1-3) in patients with different periodontal diseases. This study demonstrated that periodontitis groups had a significantly higher total amount of GCF adrenomedullin compared to the gingivitis and healthy control groups after adjusting for age and gender. Additionally, GCF adrenomedullin levels were positively correlated with clinical periodontal parameters of sampling sites. The total amount of gingival crevicular fluid HNP1-3 level was not different among the study groups, and there was no correlation between the total amount of gingival crevicular fluid HNP1-3 level and clinical periodontal parameters5. 2. The aim of the study was to compare GCF adrenomedullin levels of periodontally healthy individuals (GROUP I), individuals with chronic periodontitis (GROUP II), periodontally healthy individuals with diabetes mellitus type 2(GROUP III) and individuals with chronic periodontitis and diabetes mellitus type 2(GROUP IV). This study concluded that groups with diabetes mellitus type 2(GROUP I and GROUP II) had significantly higher periodontal clinical indices than nondiabetes mellitus groups (GROUP I and GROUP II). The group of individuals with chronic periodontitis and diabetes mellitus type 2 had a significantly higher total GCF adrenomedullin level compared with other groups. A higher total GCF adrenomedullin level was found in diabetes mellitus type 2 groups (GROUP I and GROUP II) compared with nondiabetes mellitus groups (GROUP I and GROUP II)6. 3. A study was carried out to investigate whether adrenomedullin (ADM), a multifunctional peptide with key roles in host antimicrobial defence and inflammation, was present and quantifiable in human gingival crevicular fluid (GCF) and to study its relationship with periodontal health and disease. This study concluded that measurable adrenomedullin-like immunoreactivity (ADM-LI) was present in all the GCF samples collected and ADM-LI was significantly higher in periodontitis sites than in control healthy sites3. 4. A study was performed to elucidate the significance of oxidative stress in the modulation of endothelial functions. They examined the effects of H2O2 on the expression of two endothelium-derived vasoactive peptides, endothelin(ET) and adrenomedullin(AM), and their interaction. It was concluded that hydrogen peroxide decreased ET secretion and suppressed ET-1 mRNA expression. Antioxidants prevented hydrogen peroxide induced decrease of ET secretion. Hydrogen peroxide increased AM secretion and AM mRNA expression with augmentation of cAMP production. Neutralizing AM monoclonal antibody attenuated H2O2 - induced suppression of ET secretion and exogenously administered AM and cAMP suppressed ET secretion7. 5. A study was conducted to assess the effect of non-surgical periodontal therapy on the expression frequencies of human beta-defensin (HBD)-1, 2, and 3 in the gingival crevicular fluid (GCF) of patients affected by localized aggressive periodontitis. In the patient group ,the expression frequencies of HBD-1,-2,and-3 mRNA at baseline were 30% ,85% ,and 35%, respectively ,which changed after periodontal therapy to 80%,45%,and 85%, respectively. In the healthy control subjects, the expression frequencies were 95%, 40%, and 95% for HBD-1, 2, and3, respectively, which were different from those of diseased patients at baseline. This study concluded that the appropriate expression of HBD peptides in health and disease may contribute to the maintenance of periodontal homeostasis, possibly through its antimicrobial effects and the promotion of adaptive immune responses8. 6. A study was carried out to determine whether Adrenomedullin (AM) exerts its inflammatory role in keratinocytes through the activation of NF-κB and IL-6 secretion. This study concluded that AM induces degradation of inhibitor IκB and as a result, NF-κB is translocated to the nucleus of HaCaT cells (skin epithelial cells) where it will induce expression of genes such as IL-69. 6.3 AIMS AND OBJECTIVES OF THE STUDY 1. To evaluate GCF adrenomedullin level before and after non-surgical periodontal therapy in chronic periodontitis patients. 2. To compare GCF adrenomedullin level before and after non-surgical periodontal therapy in chronic periodontitis patients. 3. To evaluate GCF adrenomedullin level before and after non-surgical periodontal therapy in chronic periodontitis patients who are smokers. 4. To compare GCF adrenomedullin level before and after non-surgical periodontal therapy in chronic periodontitis patients who are smokers. 7. MATERIAL AND METHODS 7.1 SOURCE OF DATA A total of 40 patients reporting to the Outpatient Department of Periodontics, The Oxford Dental College and Hospital, Bangalore will be selected. 7.2 METHOD OF COLLECTION OF DATA A total of 40 patients will be selected for the study based on inclusion and exclusion criteria. Patients will be explained in detail about the study procedure, and a written consent will be obtained from the patient before including him or her in the study. INCLUSION CRITERIA GROUP I ( CHRONIC PERIODONTITIS PATIENTS) 1. Patients with probing pocket depth of ≥ 5mm 2. Clinical attachment loss of ≥ 3 mm 3. Radiographic evidence of bone loss ≤ 50% GROUP II ( CHRONIC PERIODONTITIS PATIENTS WHO ARE SMOKERS) 1. Patients with probing pocket depth of ≥ 5mm 2. Clinical attachment loss of ≥ 3 mm 3. Radiographic evidence of bone loss ≤ 50% 4. Current smokers will be selected i.e. persons who have ever smoked 100 cigarettes and who currently smoke every day or some days. (National Health Interview Survey, NHIS, 2011, Centres for Disease Control and Prevention, U.S. Department of Health and Human Services)10 EXCLUSION CRITERIA 1. Patients with any systemic disorders or medically compromised patients 2. Pregnant or lactating mothers 3. Patients who have received medications that would alter the level of adrenomedullin in the last 6 months. (Anti-inflammatory drugs, Antibiotics, Corticosteroids) 4. Patients who have undergone any form of periodontal therapy within last 6 months STUDY DESIGN The patients will be divided into following groups based on inclusion and exclusion criteria: GROUP I : 20 samples : Chronic Periodontitis patients GROUP II : 20 samples : GROUP III: 20samples : GROUP IV : 20 samples : Chronic Periodontitis patients who are smokers GROUP I after non-surgical periodontal therapy (3 months recall) GROUP II after non-surgical periodontal therapy (3 months recall) The following clinical parameters will be recorded at baseline (at first visit) and 3 months after non- surgical periodontal therapy : Gingival index: (Loe and Silness,1963)11. Plaque index: (Silness and Loe,1964)12. Bleeding on probing (Ainamo and Bay, 1975)13. Probing pocket depth Clinical attachment level Radiographic assessment using OPG Probing pocket depth and clinical attachment measurements will be measured using FLORIDA PROBE and standardized using acrylic stent. Non- surgical periodontal therapy which includes scaling and root planing will be performed. Patients will be put on oral hygiene maintenance which includes oral hygiene instructions and recall every month for 3 months. At recall visits, reinforcement of oral hygiene instructions and if required scaling will be carried out. Patients requiring periodontal surgical therapy will be refrained from treatment for 3 months after non-surgical periodontal therapy as per Helsinki declaration for the purpose of the study. METHOD OF COLLECTION OF SAMPLE Collection of gingival crevicular fluid GCF sample collection will be done one day after recording clinical parameters to avoid stimulation of the sample and its contamination with blood. The tooth to be sampled will then be dried with warm air for 10 seconds. Each site selected for GCF sampling will be isolated with cotton rolls and a saliva ejector will be used to decrease the risk of salivary contamination. GCF sample collection (4µl) will be done by extrasulcular method using calibrated microcapillary pipettes of length 125mm and bore size of 0.01mm. GCF samples will be collected from sites with deepest probing pocket depth before non-surgical periodontal therapy in group I and II and after non-surgical periodontal therapy in group III and IV .The collected GCF samples will be stored in a freezer (temperature maintained at - 80°C) till the assay procedure is carried out. The collected GCF samples will be subjected to ELISA (CUSABIO) for estimating the levels of ADRENOMEDULLIN. STUDY DURATION: 1 year. STATISTICAL ANALYSIS: Analysis of variance, Paired t test. 7.3 DOES THIS STUDY REQUIRE ANY INVESTIGATION OR INTERVENTIONS TO BE CONDUCTED ON PATIENTS OR OTHER BEINGS? Yes, GCF samples will be collected from patients for quantitative analysis of adrenomedullin level which will later be subjected to statistical analysis. 7.4 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION? Yes, The ethical clearance certificate has been attached. 8. LIST OF REFERENCES : 1. Lu Q, Samaranayake LP, Darveau RP, Jin L. Expression of human beta- defensin-3 in gingival epithelia. J Periodont Res 2005;40:474-81. 2. Hiroshima Y, Bando M, Kataoka M, Inagaki Y, Herzberg CM, Ross FK et al. Regulation of antimicrobial peptide expression in human gingival keratinocytes by interleukin-1α. Arch Oral Biol 2011;56(8):761-7. 3. Lundy FT, O’Hare MMT, McKibben BM, Fulton CR, Briggs JE, Linden GJ. Radioimmunoassay quantification of adrenomedullin in human gingival crevicular fluid. Arch Oral Biol 2006;51(4):334-8. 4. Ali SM, Mohammed SMH. Adrenomedullin as a novel promising therapeutic approach. International Journal of Science and Technology (IJST) 2013 Oct;2(10):747-53. 5. Turkoglu O, Emingil G, Kutukculer N, Atilla G. Evaluation of gingival crevicular fluid adrenomedullin and human neutrophils peptide 1-3 levels of patients with different periodontal diseases. J Periodontol 2010 Feb;81(2):284-91. 6. Ertugrul AS, Dilkilitas A, Sahin H, Alpaslan NZ, Bozoglan A. Gingival crevicular fluid adrenomedullin level in individuals with and without diabetes mellitus type 2. J Periodont Res 2013;48:342-9. 7. Saito T, Itoh H, Chun T, Fukunaga Y, Yamashita J, Doi K et al. Coordinate regulation of endothelin and adrenomedullin secretion by oxidative stress in endothelial cells. Am J Physiol Heart Circ Physiol 2001;281:H1364-H1371. 8. Ebrahem MA, Expression of human beta defensins (HBDs) 1,2 and 3 in gingival crevicular fluid of patients affected by aggressive periodontitis. Saudi Dent J 2013;25(2):75-82. 9. Pleguezuelos O, Pavli EN, Crowther G, Kapas S. Adrenomedullin signals through NF-κB in epithelial cells. FEBS Letters 2004;577(1-2):249-54. 10. CDC. 2011 National Health Interview Survey (NHIS) public use data release. Hyattsville, MD: US Department of Health and Human Services, CDC, National Center for Health Statistics; 2011. 11. Loe H, Silness J. Periodontal disease in pregnancy. I. Prevalence and severity. Acta Odontol Scand 1963;21:533-51. 12. Silness J, Loe H. Periodontal disease in pregnancy. II. Correlation between oral hygiene and periodontal conditions. Acta Odontol Scand 1964;22:121-35. 13. Ainamo J, Bay I. Problems and proposal for recording gingivitis and plaque. Int Dent Journal 1975;25:229-35. 9. SIGNATURE OF THE CANDIDATE 10. REMARKS OF THE GUIDE 11. NAME AND DESIGNATION 11.1 GUIDE Dr. SHOBHA K.S READER DEPARTMENT OF PERIODONTICS THE OXFORD DENTAL COLLEGE AND HOSPITAL, BANGALORE 560068. SIGNATURE 11.2 HEAD OF THE DEPARTMENT SIGNATURE 12. REMARKS OF THE PRINCIPAL SIGNATURE Dr. ANIRBAN CHATTERJEE DEPARTMENT OF PERIODONTICS THE OXFORD DENTAL COLLEGE AND HOSPITAL, BANGALORE 560068.