Patient selection
This study was performed in the Faculty of Dentistry, Department of Pedodontics, at Inönü University. According to the previous power analysis clarifying the changes of YKL-40 in saliva, the estimated number of participants was 15 per group, with an alpha level of 0.05 and a power of 0.80.13 Samples were analyzed at the Biochemistry Department.
This study was conducted in accordance with the Declaration of Helsinki after obtaining ethical approval. It was approved by a Clinical Research Ethics Committee in Turkey: Inönü University School of Medicine (ethic number: 2017/67). Written informed consent was obtained from the parents before the examination.
A total of 85 subjects, aged 6-15 (9.15 ± 2.16 years; 39 male, 46 female) participated in this study. The participants were divided into three groups: Group I (control, n=25, mean age=7.72 ± 1.34), Group II (n=30, with dental caries, mean age=9.40 ± 2.13) and Group III (n=30, with advanced dental caries, mean age=10.10 ± 2.19). The children were excluded from the study if they had any systemic and/or periodontal diseases, and if they had taken antibiotics or anti-inflammatory drugs in the last 30 days.
Calibration of the examiners
All clinical examinations were performed by an experienced clinician (GD). On the other hand, biochemical analysis was performed by another experienced researcher (EL).
The examiners were trained and calibrated prior to the study. Test-retest reliability was assessed using the intraclass correlation coefficient (ICC) calculated by the kappa coefficient. 15 randomly selected patients and their radiographs were re-examined one week after the initial examination by the examiner (GD). Spearman correlation coefficients were used to calculate kappa values between the two time periods. The results of the examiner for both clinical and radiological examinations were same figure 0.97 (κ > 0.75 was evaluated as a good agreement) for 15 random samples.
The examiner (GD) was calibrated to measure clinical parameters (DMFT/dmft, ICDAS II, and PUFA/pufa) and radiological parameters (periapical x-ray evaluation). One of the examiners (GD) was trained in the measurement of both GI and PI, and saliva collection and the other (EL) was trained in YKL-40 extraction from saliva samples and the measurement of its amount (ng/mL).
Clinical measurements
The gingival conditions of the patients were evaluated by Silness-Loe plaque index (PI)14 and Loe gingival index (GI)15. Decayed-missing-filled teeth (DMFT/dmft) and decayed-missing-filled teeth surfaces (DMFS/dmfs) were recorded. The extraction of the primary teeth due to the physiological root resorption was not recorded as a missing tooth. The teeth were scored using International Caries Detection and Assessment System (ICDAS) II16 and PUFA/pufa index (Exposed pulp, Ulceration, Fistula, Absess) (Table I) 17. The diagnosis was based on two factors: clinical and radiographic features.
Group I; control. ICDAS II code=0, DMFT/dmft=0
Group II; shallow caries; caries in dentin and cavitation was not exaggerated. ICDAS II code=1-4
Grop III; deep caries; caries lesion was close to the dentin-pulp interface, the dentin thickness was less than 1mm, with or without pulpal exposure. ICDAS II code=5-6. Group III had at least one tooth with ICDAS II code-5 or 6. The caries lesions extending into the pulp tissue were classified according to the PUFA/pufa index.
Radiological examination
The radiographic examination was made to confirm caries lesion depth and whether a pulpal involvement of caries lesions, especially in groups II and III.
Saliva collection
All the saliva samples were obtained in the morning and the participants were asked to avoid eating or drinking 1 h before the collection of samples. All the unstimulated saliva samples were collected by the spitting method and transferred into a 2-ml polypropylene tube. All the saliva samples were homogenized on a Vortex mixer (1 min) and centrifuged (10,000× g, 10 min) to remove cellular debris. The resultant supernatants of the samples were stored at −80 °C for further analyses.
YKL-40 assay
The level of YKL-40 in saliva was measured by ELISA (R&D Systems, Minneapolis, MN) and the analysis was performed according to the manufacturer’s instructions using human recombinant standards in Biochemistry Laboratory. All samples were run in duplicate and the results were averaged for the analysis. The results were reported in pg/mL. The detection limit was 3.5 pg/mL for YKL-40. The samples with YKL-40 levels below the limits of the assay’s detectability were scored 0. The results recorded in pg/mL were converted to ng/mL.
Statistical analyzes
Data analysis was performed using the statistical package SPSS 21 (SPSS Inc., Chicago Illinois, USA). The results were expressed as means ± standard deviations. The data were firstly analyzed for the normal distribution using Shapiro-Wilk test. The parameters between the groups were compared with one another using One-way ANOVA, Tukey post hoc and Kruskal-Wallis tests. Multiple regression analysis was used to determine the explanatory power of the variables causing the increasing of YKL-40 level in saliva. Sperman rank correlation test was used to verify the correlations between the parameters.