Periodontally healthy subjects should require gingival removal during periodontal aesthetic surgery for the correction of gingival discrepancies and asymmetries. Exclusion criteria were pregnancy, lactation, current smoking, and smoking within the past five years, periodontal or/and antibiotic

therapies in the previous six months, use of mouthrinses containing antimicrobials in the preceding two months, systemic condition that could affect the progression of periodontal disease (e.g. diabetes, immunological disorders) and long-term administration of anti-inflammatory check details and immunosuppressive medications. Clinical examination.  All clinical examinations were performed by one examiner (VRS) who was calibrated, as previously described [16]. The intra-examiner variability was 0.21 mm for PD and 0.22 mm for CAL. The clinical parameters, registered dichotomously [i.e. BoP], were

calculated by the Kappa-Light test and the intra-examiner agreement was >0.85. The following parameters were assessed at six sites of all teeth, excluding third molars, using a manual periodontal probe (UNC15, Hu-Friedy, Chicago, IL, USA): plaque index (PI), BoP (presence/absence), suppuration (SUP, presence/absence), marginal bleeding (MB, presence/absence), PD (mm) and CAL (mm). Experimental groups.  Based on their periodontal status, the subjects were see more divided into one of the following groups: (1)

 Periodontally healthy (n = 15; control): Subjects with no sites with CAL >3 mm and <20% of sites presenting BoP and/or MB. Saliva sampling.  isometheptene The saliva samples were obtained around 8:00 a.m. Volunteers were instructed not to brush their teeth during the preceding 12 h and not to drink or eat anything for 1 h before sampling to avoid contamination with non-salivary components. Approximately 500 μl of saliva was transferred to 1.5 ml tubes in which 10 μl of 250 mm EDTA had been added. Samples were placed on ice and processed within 1 h after collection. Saliva samples were clarified by centrifugation at 13,000 g at 4 °C for 10 min, and the supernatants were collected and frozen at −70 °C until laboratory analysis. Total concentration of protein in saliva was determined by the method of Bradford to check for variations in salivary flow (Sigma-Aldrich, St Louis, MO, USA). Gingival biopsies sampling.  For the chronic periodontitis group, the gingival biopsies were collected from teeth indicated for exodontia due to advanced periodontitis in order to obtain representative areas of the periodontal inflammation. If the patient had two or more teeth with these characteristics, biopsy only one tooth with the worst diagnosis was included.