2.1. Patients Study
The study group comprises 189 adult European Caucasian subjects: 85 men and 104 women between 20 and 71. Patients were selected and recruited from the Department of Periodontology at the Medical University of Lublin, and the study began on January 31, 2015, and ended on March 1, 2017. After a full explanation of this study's aim, written informed consent forms were obtained from all participants by the Helsinki Declaration. Medical and dental histories of each patient were gathered. Patient inclusion criteria were as follows: no treatment of periodontitis for the last 6 months that could affect periodontal treatment outcomes; no use of antibiotics and/or anti-inflammatory drugs for 3 months before taking part in the study. Criteria for exclusion from the study were as follows: pregnancy or breastfeeding for women. cigarette smoking, hormonal disorders or taking hormonal agents, infections (such as HIV, hepatitis, and tuberculosis), systemic diseases (e.g., diabetes, osteoporosis, and immunological disorders, and patients with a history of oncological treatment).
The diagnosis of patients was based on clinical and radiographic criteria. The clinical examination included Löe and Silness gingival index (GI), probing pocket depth (PD), clinical attachment level (CAL), and bleeding on probing (BOP). Both PD and CAL measurements were performed using a WHO-621 periodontal probe. GI measurements were assessed at four surfaces per tooth/implant (mesial, distal, buccal, lingual, and palatal surface), while PD and CAL measurements were taken at six surfaces per tooth (stepping method) (mesiobuccal, mid-buccal, distobuccal, mesio-oral, mid-oral and disto-oral). GI, PD, and CAL are given as average values. One examiner assessed all clinical parameters. Based on the clinical data, patients were later divided into five clinical groups as follow: (1) a control group of periodontally healthy patients (group I), (2) patients with mild periodontitis (group II), (3) patients with moderate periodontitis (group III) (4) patients with severe periodontitis (group IV), (5) periodontally healthy subjects who received an implant treatment (implants with a new alternative hydrophilic surface SPI ELEMENT INICELL, Thommen Medical AG, Grenchen, Switzerland and Brånemark system implant, Nobel Biocare, Gothenburg, Sweden) (group V). In the study group of patients who have received dental implants, none showed any symptoms of peri-implantitis and had no prior history of periodontitis. The intraoral periapical radiographic method was used to evaluate each implant's bone condition and was obtained for each implant using the paralleling technique with a radiographic positioner. In the qualification, we also considered the type of prosthetic work performed on implants. In all patients from group V, the bone density grade was determined according to a scale of D1-D4 defined by Misch .
2.2. GCF/PISF Sampling and Processing
Before a GCF sampling, the supragingival plaque was carefully removed. The collection sites were isolated using cotton rolls and dried with air jets. The GCF samples were subsequently obtained from the mesiobuccal root using sterile Periopaper strips (Oraflow Inc., Plainview, NY, USA) that were overlaid and placed at the gingival crevice region until mild resistance was felt. The strips were left in place for 30 seconds to prevent any mechanical irritation. The strips contaminated with blood were discarded. Following the GCF collection, the strips were kept in sterile test tubes and stored in aliquots at -80°C until needed for analysis.
Clinical examinations in the group of patients with implants were performed after removal of the supra-constructions. PISF samples have been drawn, at least 18 months after the surgery, similarly using sterile Periopaper strips that were inserted into the gingival crevice until mild resistance was felt. For 30 seconds, the strips were left in place. After that, the paper points were transferred to a sterile test tube and then immediately stored in aliquots at a temperature of -80°C.
2.3. Cytokine measurements
For GCF/PISF extraction, paper strips were put in tubes containing 500 µL of phosphate-buffered saline (PBS) (pH 7.2) (Sigma Aldrich) and next gently shaken and incubated at room temperature for 1 hour. After that, the strips were pulled, and the fluids were analyzed. Commercially available enzyme-linked immunosorbent assays (ELISA) were used to measure the concentration of IL-1β (catalog no DLB50), CXCL8 (catalog no D8000C), and TNF-α (catalog no DTA00C) (Quantikine R&D Systems Inc., Minneapolis, MN, USA) on Thermo Scientific™ Multiskan™ FC Microplate Photometer. All ELISA procedures were performed according to the manufacturer's instruction. All measurements were done in triplicate. The GCF/PISF IL-1β, CXCL8, and TNF-α concentrations were equated to a standard calibration curve.
2.4. Statistical analysis
The statistical analysis for this study was conducted using Statistica 13.1 (Statsoft Inc., USA). Shapiro-Wilk test was used to analyze the normality of distribution, while the Mann-Whitney U test was performed to analyzed differences in the levels of IL-1β, CXCL8, and TNF-α in GCF, and differences in the levels of IL-1β, CXCL8, and TNF between G1 and G2 bone density groups, as well. Associations between studied proteins were tested using linear regression (adjusted for age and sex. The Spearman's rank correlation coefficient was used to test correlations between IL-1β, CXCL8, and TNF-α concentrations in G1 and G2 bone density groups. Statistical significance was set at p = 0.05.