Possible Association Between Behçet's Disease and Periodontal Diseases

The study population consisted of 40 patients with BD (24 female and 16 male) who received treatment at Gazi University Faculty of Medicine, Department of Rheumatology, Ankara. Forty-seven systemically healthy gingivitis or periodontitis patients without BD (26 female, 21 male) from Gazi University Faculty of Dentistry, Department of Periodontology were included as controls. Periodontitis was diagnosed with a severity of Stage II, pocket depth 3-4 mm and Stage III, pocket depth ≥5 mm, a generalized pattern according to the criteria by the criteria 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions. Gingivitis was diagnosed as according to a BOP score ≥ 10%, without attachment loss and radiographic bone loss. The participants were divided into 4 groups; systemically healthy patients with gingivitis (HG), systemically healthy patients with periodontitis (HP), BD patients with gingivitis (BG), and BD patients with periodontitis (BP). This study was approved by the human subject's ethics board of Gazi University Clinical Research Ethics Committee (22122014/579) and was conducted in accordance with the Helsinki Declaration of 1975, as revised in 2013. All patients signed an informed consent form. Exclusion criteria were smoking, pregnancy, lactation, other systemically diseases, and previous periodontal and/or antibiotic treatment within the last 6 months.

All examinations were conducted by a single experienced dental examiner (FO). GCF samples and clinical measurements were obtained. The clinical examination included plaque index (PI), gingival index (GI), clinical attachment level (CAL), probing pocket depth (PPD), and bleeding on probing (BOP). The measurements were performed at 6 sites per tooth (mesio-buccal, mid-buccal, disto-buccal, mesio-lingual, mid-lingual and disto-lingual) using a periodontal probe (Williams periodontal probe, Nordent Manufacturing, Elk Grove Village,IL). GCF was obtained before recording clinical measurements. The deepest 4 pockets of non-adjacent single rooted teeth were selected for GCF sampling.

Diagnosis of BD Patients were diagnosed according to the International Criteria for Behçet's Disease (ICBD). ICBD is based on ocular lesions, genital ulcers, oral aphthous ulcers, skin manifestations, vascular involvement and positive pathergy test. Systemic involvement associated with BD, current symptoms and the use of any systemic medications were recorded.

Saliva sampling and processing Saliva sampling was performed before the periodontal examination and GCF collection. For each patient, unstimulated whole mixed saliva was collected using a modified method. Participants were asked to rinse her/his mouth with water for a minute, 10 min before the sampling. The saliva samples were collected into a sterile plastic container for 5 min. Samples were centrifuged for 10 min at 15,000 x g and the supernatant was frozen at -20 until analyses.

GCF sampling and processing The GCF sampling site was gently air dried and supragingival plaque was removed. The area was carefully isolated with cotton rolls in order to prevent from contamination. Standardized paper strips (Periopaper, OraFlow, Amityville, NY, USA) were inserted into the sulcus until slight resistance was felt and left in place for 30 seconds. Strips contaminated by bleeding were discarded. GCF volumes were determined by using a calibrated Periotron 8000® . The readings from the Periotron 8000® were converted into microliter (μL) by referencing to the standard curve with MILCONVERT.exe program ( MILCONVERT.exe software version 2.52, Oraflow, Amityville, NY, USA). Strips were placed into coded micro centrifuge tubes and stored at -80C until processing. Before biochemical analyses, paper strips were placed in 300 μL of phosphate buffered saline solution containing 0.5% bovine serum albumin in Eppendorf tubes, and GCF was eluted from the strips by centrifugation for 6 min at 5000 x g at 4C.

Biochemical analyses NO level of samples were determined using colorimetric nitric oxide assay kit (Oxford Biomedical Research, Oxford, MI, USA) (Product number: NB98). In aqueous solution, NO rapidly degrades to nitrate and nitrite. This kit employs the NADH-dependent enzyme reductase (NaR) for conversion of nitrate to nitrite prior to quantification of nitrate using Griess reagent-thus providing for accurate determination of total NO production. This colorimetric nitric oxide assay kit can be used to accurately measure as little as 1 pmol/μL (1μM) NO produced in aqueous solutions. The completed reaction is read at 540 nm. NO concentration was expressed as μM.

TNF-ɑ Picokine ELISA (human, Catalog Number: EK0525) and IL-1β Picokine ELISA (human, Catalog Number: EK0392) kits are sandwich ELISA kits for quantitative detection of human TNF-ɑ and human IL-1β. Detection range for TNF-α Picokine ELISA kit was 7.8-500 pg/mL and for IL-1β 3.9-250 pg/mL. Sensitivities for TNF-ɑ and IL-1β kits were \<1 pg/mL and \<0,15 pg/mL, respectively. Analyses and microplate readings were performed using an ELISA autoanalyzer (ChemWell 2900, Awareness Technology, Palm City, FL, USA).

TNF-ɑ (human) and IL-1β (human) Picokine ELISA kits (Boster Biological Technology Co., Pleasanton, Ltd., CA, USA) were based on similar standard sandwich ELISA technology. A monoclonal antibody (MoAb) from mouse specific TNF-ɑ or IL-1β has been precoated onto 96 well plates. Following the addition of standards and samples to the wells, a biotinylated detection polyclonal antibody from goat specific TNF-ɑ or IL-1β was added and then washed. Avidin-Biotin-Peroxidase Complex was added and unbound conjugates were washed away. Horseradish peroxidase (HRP) substrate 3,3',5,5'-tetramethylbenzidine (TMB) was used to visualize HRP enzymatic reaction. The blue color product changed into yellow after adding stop solution. The density of the yellow color which absorbs at 450 nm is proportional to the human TNF-ɑ or IL-1β amount of sample captured in plate. Results were expressed as pg/mL.