Ethical Approval This double-blind and randomized controlled clinical study was carried out on 55 patients with Type 2 DM and periodontitis who applied to the Periodontology Clinic of the Faculty of Dentistry of Recep Tayyip Erdoğan University between 2021/February and 2022/January and met the study criteria. Ethical approval was given by the Clinical Research Ethics Committee of the Faculty of Medicine of the same university within the framework of the Declaration of Helsinki (2008) (2022/03). The possible risks and benefits of the treatment to be applied to the participants were mentioned and their verbal and written consents were obtained.
Working Groups According to the WHO, in individuals in the young age category, "stage 3/4 periodontitis" was determined according to the current periodontal disease and condition classification as the diagnostic criterion for periodontitis. Accordingly, probing pocket depth (PPD) should be at least 6mm and bone loss should be at least 3mm. Our diagnostic criteria for Type 2 DM was fasting plasma glucose (FPG) level greater than 126 mg/dl and glycosylated hemoglobin level (HbA1c) greater than 6.5%. Those who smoke, use antibiotics, anti-inflammatory and antioxidant drugs in the last 6 months, work night shifts, are in pregnancy and lactation, have cancer and autoimmune diseases, have kidney disease, have received any periodontal treatment in the last 6 months obese patients were also excluded from the study. In Europe, obesity is defined as a body-mass index (BMI) of more than 30 kg/m2 and a waist circumference of more than 88 cm in women and 102 cm in men.
Treatment Protocol According to the above criteria, patients included in the study were randomly divided into control (fmSRP) and treatment \[fmSRP-melatonin (fmSRP-mel)\] groups by a computer program by an independent researcher (HA). The investigator (YSG), who provided the sample and conducted the clinical procedures, was blind to the study groups. fmSRP is the approach of completing supragingival and subgingival debridement procedures within 24 hours with periodontal curettes, scalers and ultrasonic instruments. Additional melatonin administration was made in the form of tablets containing 6mg of melatonin daily for 30 days from the day following the SRP. Patients were instructed to take the tablets just before bedtime. No additional application was made in the control group. Within the scope of the non-surgical periodontal treatment program, all patients were trained on brushing teeth with the modified bass technique and the use of appropriate interface cleaning tools. These trainings were repeated in the maintenance sessions. Participants were also instructed not to use any mouthwash.
Sample Collection and Clinical Periodontal Measurements Clinical periodontal measurements with gingival crevicular fluid(GCF) and serum samples were performed at baseline (T0) and at 3th (T1) and 6th months (T2) following fmSRP. In all three periods, sampling was done one day before clinical periodontal measurements.
GCF samples were taken from the interproximal regions of the two teeth with the most radiographic bone loss in each of the four quadrants in the morning. Initially, proper isolation was achieved by means of cotton rolls. The area was slightly dried for 10 seconds. Sterile paper strips (Periopaper, Proflow, Inc., Amityviile, NY) were advanced until minimal resistance was felt in the sulcus. Waited 30 seconds. Paper strips contaminated with blood or saliva were not included. The GCF volume in each paper strip was measured using the periotron device (Periotron 8010 Harco Electronics, Winnipeg, Canada) by electrical compedance method and recorded as µl. Immediately afterwards, 8 paper strips obtained from each patient were transferred to an eppendorf tube containing 250 µl of phosphate buffer solution. It was stored in a -80°C freezer (Thermo Fisher Scientific, Waltham, MA, USA) until the day of biochemical analysis.
Immediately following the GCF samples, 16 cc venous blood samples were taken from the left arm antecubital fossa region of the patients. After the blood was centrifuged at 3000 rpm at 2-8oC for 15 minutes, serum samples were obtained (Elektro-mag, Istanbul, Turkey). The samples were taken into Eppendorf tubes and kept in a -80°C freezer (Thermo Fisher Scientific, Waltham, MA, USA) until the day of analysis.
Plaque index (PI), gingival index (GI), bleeding on probing (BOP), PPD and clinical attachment level (CAL) measurements were performed for clinical periodontal status assessments. PPD was measured as the distance between the deepest point of the sulcus and the gingival margin, and CAL as the distance between the deepest point of the sulcus and the enamel-cementum junction. Scoring for each tooth was made from 6 different regions for PPD and CAL, and from 4 different regions for other indices. Williams periodontal probe (Hu-Friedy, Chicago, IL, USA) was used for measurements.
Biochemical Analysis Eppendorf tubes, which were removed from the freezer 24 hours before the experiment, were allowed to thaw gradually, first at -20oC and then at +4oC. The samples and reagents were brought to room temperature (18-25oC) just before the analysis. RANKL (Catalog no: E-EL-H5813), OPG (Catalog no: E-EL-H1341) and MMP-8 (Catalog no: E-EL-H1450) analyzes in GCF samples, IL-1ß (Catalog no: E- EL-H0149) analyzes were performed on serum samples using the enzyme-linked immunosorbent assay (ELISA) method and according to the instructions of the kit manufacturer (Elabscience, Houston, Texas, USA). Results were calculated as ng/ml (MMP-8) and pg/ml (IL-1β, RANKL and OPG).
Statistical Analysis Windows 17.0 for SPSS program was used for statistical analysis. (SPSS Inc., Chicago, USA) The conformity of the data to the normal distribution was evaluated with the Shapiro Wilk test. The Mann Whitney U test was used for intergroup comparisons, and the Friedman, Wilxocon tests and Bonferroni correction were used for the analysis of time-dependent within-group changes. Chi-square test was applied for comparison between groups in terms of genders. A p\<0.05 level was considered statistically significant.
