Netrin 1 and Its Receptor Unc5b as Markers of Periodontal Disease

A total of 60 individuals, comprising 20 patients with Periodontitis (P Group), 20 patients with gingivitis (G Group), and 20 periodontally healthy controls (H Group) were involved in the study. The patients were diagnosed according to the new classification scheme. Stage and extent of periodontitis were also determined.

The clinical periodontal criteria for the diagnosis of the groups were as follows: the periodontitis group had interdental clinical attachment loss (CAL) ≥5 mm at site of greatest loss and periodontal probing depth (PD) ≥ 6 mm in one or more sites of the 2 non-adjacent teeth in at least two quadrants of the mouth. Accordingly, Stage III generalized periodontitis patients were included in the study. Patients were diagnosed as gingivitis with bleeding on probing ≥ 50 %, PD ≤3 mm, and no radiographic bone loss or CAL. Periodontally healthy individuals had no recorded history of periodontal problems, with PD ≤3 mm, no radiographic bone loss, good oral hygiene, no gingival inflammation.

Inclusion criteria were as follows: (1) aged >18 years, (2) having at least 16 natural teeth (excluding third molar),(3) nonsmokers with no history of smoking, (4) not having any diagnosed medical illness or drug intake that could affect the periodontal condition. The patients who had (1) taken antibiotics, nonsteroidal antiinflammatory or any other drugs within the past 3 months,(2) received nonsurgical or surgical periodontal treatment, (3) a restorative and endodontic therapy requirement, (4) a removable partial denture and/or having orthodontic therapy were excluded from the study. Current pregnancy or lactation and having serum C reactive protein (CRP) > 3mg/L were also the exclusion criteria.

All individuals were examined at baseline and four weeks after non-surgical periodontal treatment including, whole mouth probing depth (PD), CAL, presence of bleeding on probing (BOP), papillar bleeding index (PBI), gingival index (GI) and plaque index (PI) except the third molars. PD and CAL were measured at six sites per tooth using a manual periodontal probe.

The non-surgical periodontal treatment for periodontitis group included supra- and subgingival scaling, root planing and oral hygiene instructions. Subgingival scaling and root planing were performed under local anesthesia in two sessions within the 48-72 h. Gingivitis group had supra-and subgingival scaling, polishing and oral hygiene instructions. The treatment of gingivitis and periodontitis patients were performed by a periodontist (BM) using hand and ultrasonic instruments. All measurements were performed by the same calibrated examiner (SG).

Saliva sampling

Whole unstimulated saliva samples were collected. Each participant was asked first to rinse the mouth completely with tap water for 1 minutes, wait for 10 minutes, and then expectorate into sterile polypropylene tube for 5 minutes.

Gingival crevicular fluid (GCF) sampling

GCF samples were obtained from two nonadjacent interproximal sites in one single- rooted and one multi-rooted teeth by standardized filter paper strips. GCF was sampled from two sites with GI < 1, PD ≤ 3 mm and without BOP in the healthy group; two sites with GI ≥ 2, PD ≤ 3 mm and positive BOP (visible signs of inflammation) in the gingivitis group; and two deepest pocket (≥ 5 mm) with GI ≥ 2 and positive BOP in periodontitis group.

Serum sampling

Serum samples were taken following saliva and GCF sampling before the periodontal treatment. Six milliliters of venous blood were obtained by a standard venipuncture method and the serum was separated from blood by centrifugation at 1,500 g for 20 minutes.

Measurement of Netrin 1 and Unc5b levels in GCF, Saliva and Serum Samples

Netrin 1 and Unc5b levels in GCF, saliva and serum samples were measured by the enzyme-linked immunosorbent assay (ELISA) using commercial kits according to the manufacturer's guidelines.

Statistical Analysis

All data analyses were performed using a statistical software package. Comparisons of clinical and biochemical parameters between the study groups were performed using the Kruskal- Wallis with Mann Whitney U test with Bonferroni correction method. The intragroup comparisons (at baseline and first month) were performed using Wilcoxon test for paired samples. Associations among levels of the GCF, saliva and serum biomarkers and clinical parameters were also examined using the Spearman rank correlation analysis.