Clinical Conditions and Prevalence of Periodontopathogens in Smokers and Non-smokers After Periodontal Therapy

Sixty patients (30 smokers (SM) and 30 non-smokers (NS) will be selected to participate of this study. All subjects will be recruited from the Department of Periodontology, School of Dentistry, Fluminense Federal University, Nova Friburgo, Rio de Janeiro State, Brazil. The study protocol was approved (protocol number: CAAE - 0070.0.258.000-10) by the Ethics Committee of the School of Medicine, Fluminense Federal University. Prior to participation, the purpose and procedures will be fully explained to all patients, who consequently gave written informed consent in accordance with the Helsinki Declaration. Medical and dental histories will be taken and patients will receive clinical evaluation at prescreening visits.

Clinical examination, microbiological collects and periodontal therapy Previous clinical examination, information about years of consumption of cigars and quantity of daily consumption will be recorded to select smokers and non- smokers subjects for the study. An experienced periodontist will perform clinical periodontal parameters in teeth for the protocol procedure. Each selected tooth selected will be measured for: Plaque Index (PI), Bleeding On Probe (BOP), Pocket Probing Depth (PPD), Gingival Recession (GR), Clinical Attachment Level (CAL) using a periodontal probe PCP15 (PCP-UNC15, Hu-Friedy, Chicago, IL), six sites (mesio-buccal, mediobuccal, disto-buccal, mesio-lingual, medio-lingual, disto-lingual) will be recorded. Sites with probing depth (PPD) > 5mm will be selected for microbiological analysis. After clinical measurements, the supragingival biofilm will be removed with sterile gauze. Gingival crevicular samples will be taken from 4 sites with the deepest PPD (>5mm) in each patient using a sterile paper point from the deepest pocket for 30s. Pooled biofilms from each site will be separated in two microtubes containing Tris -EDTA buffer (10 mM Tris-Hcl, 0.1 mM EDTA, pH 7.5) and were stored at -20°C. The samples will be analyzed microbiologically by Polymerase Chain Reaction - PCR. Periodontal treatment will be consisted of scaling and planning root, 4 times in first month and after that once a month until complete 1 year. Patients will be clinically evaluated and microbiological collects will be made at baseline, 3 months, 6 months and 1 year after periodontal treatment.

Microbiological evaluation - PCR assays DNA will be extracted and quantified in a spectrophotometer at 260 nm (Genesys 10UV, Rochester, NY, USA), in order to obtain a standard concentration of 100 ng/mL and stored at -20 °C for subsequent PCR reactions. Briefly, samples will be submitted to a lise solution (extraction buffer and proteinase K) and then purified using chloroform:isoamil-alcohol, followed by DNA precipitation with isopropanol and 70% ethanol. The DNA will be resuspended in TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 7.5, with 10 μg/mL RNAse). Microbial molecular identification will be carried out by PCR with specific primers for Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, Campylobacter rectus, Candida albicans, Candida glabrata, Candida tropicalis and Candida dublinienses. PCR amplification will be performed with a GeneAmp PCR system 2400 (Perkin-Elmer - Applied Biosystems) for TGradient 96 (Biometra, Germany) under thermal conditions specific for each pair of primers. The PCR products will be separated by electrophoresis in 2% agarose gels and Tris-borate-EDTA running buffer (pH 8.0). The molecular mass ladder (100 bp DNA ladder, Gibco, Grand Island, NY, USA) will be included for running in the agarose gel. The DNA will be stained with 0.1µl of Sybr Safe/mL (Invitrogen, CA, USA) and visualized under UV illumination (Pharmacia LKB-MacroVue, San Gabriel, CA, USA). Photographs of the images will be taken (Image Mater - LISCAP, VDS, Pharmacia Biotech Piscataway, NJ, USA) and analyzed.