Interaction Between Hypertension and Periodontitis

Forty-two participants were included in the study. The study groups consisted of 20 healthy individuals without any previously diagnosed systemic disease (10 periodontally healthy and 10 with periodontitis) and 22 patients with hypertension (13 periodontally healthy and 9 with periodontitis). The patient population was randomly selected among the patients who referred to Periodontology Clinic. Hypertensive patients with at least 5 years of follow up were included in the hypertension group. Hypertension patients treated with antihypertensive drugs known to cause gingival overgrowth were excluded from the study.

Other exclusion criteria were diabetes mellitus, high cholesterol levels, CVD and drugs usage other than antihypertensive drugs. A calibrated examiner performed a comprehensive periodontal examination included plaque index (Silness \& Loe, 1964) (PI), gingival index (Loe \& Silness, 1963) (GI), probing depth (PD), clinical attachment level (CAL) and bleeding on probing (BOP). The measurements were made at six 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., USA) To perform the intra-examiner calibration, PD and CAL were measured twice at six sites of each tooth within 24 hours. Intra-examiner agreement was at least 90% for both PD and CAL within 1 mm. Examiner who recorded the clinical indexes was blinded to the systemical status of the patients and the periodontologists who were responsible for patients periodontal treatment were not blinded because of the anesthesia applied to hypertension patients and the precautions needed to be taken.

During the study period, patients were instructed not to use any local/systemic antibiotics and antimicrobial agents.Patients diagnosed with periodontitis had Stage II periodontitis in a generalized pattern with probing depths of 3-4mm. The diagnosis was performed based on criteria that maximum probing depth ≤5 mm, mostly horizontal loss and >30% of teeth involved. Healthy periodontium was identified as the presence of BOP≤10% and PD≤3 mm.The deepest four pockets of non-adjacent single rooted teeth were selected for GCF sampling. The participants were distributed into four groups; healthy subjects with healthy periodontal condition (control), healthy subjects with periodontitis (CP), hypertension patients with periodontitis (CP + HP) and hypertension patients with healthy periodontal condition (HP). The study was approved by the local ethics committee (291208/11) and all patients signed an informed consent form. Exclusion criteria were smoking, pregnancy, established secondary hypertension diagnosis, other systemic diseases and previous periodontal and/or antibiotic treatment within the last six months. All patients with periodontitis received phase I periodontal therapy including oral hygiene instruction (OHI) and scaling/root planning (SRP) without any antimicrobial therapy. GCF and saliva samples were obtained and clinical measurements were performed at baseline (before treatment (BT) and four weeks after the phase I periodontal therapy (AT). The timeline for the phase I therapy (within 2 weeks of baseline examination 4 days). Primary biochemical outcome measures included salivary and GCF IL-6 and CRP, and primary periodontal outcome measures were PI, GI, PD, CAL and BOP. Secondary outcome measure was GCF volume.

GCF and saliva 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 contamination.

Standardized paper strips (Periopaper, Pro Flow, Amityville, NY, USA) were inserted into the sulcus until slight resistance was felt and left in place for 30 seconds. Strips contaminated by bleeding or exudates were discarded. GCF volumes were determined as described previously. To determine the amount of GCF, an electronic balance was used to weigh the paper strips before and immediately after collection. The mass (mg) of the fluid on each strip was converted to a volume in millimeters by assuming that the density of GCF was 1. Strips were placed into coded micro centrifuge tubes and stored at -800C until processing. Before biochemical analysis, paper strips were placed in 0,1 % bovine serum albumin/phosphate buffered saline solution in Eppendorf tubes, and the fluid from the paper strip was eluted by centrifugation for 6 minutes at 5,000 · g at 4 C. Following centrifugation, the strips were removed.The participants were asked to stop eating and drinking 2 hours before each sampling. They were instructed to rinse their mouth for 30 seconds with 10 mL of water and to rest for 2 minutes before unstimulated saliva was collected by spitting. Approximately 1 mL of unstimulated whole saliva was collected into Eppendorf tubes. Samples were centrifuged for 10 minutes at 15.000 × g at 4°C to remove any particulate matter. The supernatants were stored at -20°C until analysed.

GCF and saliva enzyme-linked immunosorbent assay (ELISA) analysis for IL-6 and CRPThe levels of IL-6 and CRP in GCF and saliva were measured using a sandwich ELISA kit (Biosource, Invitrogen Corporation, Carlsbad, California 92008). The ELISA procedures were carried out according to the manufacturer's instructions. Results were expressed as pg/mL. Total amounts were also calculated by multiplying concentrations and CRP volumes.