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 0.80.16 Samples were analyzed at the Biochemistry Department.
This study was conducted in accordance with the Declaration of Helsinki and 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 took antibiotics or anti-inflammatory drugs in the last 30 days.
All clinical examinations were performed by a single experienced clinician (GD). The examiner was calibrated prior to perform of the study (intra-examiner kappa>0.8)
The gingival conditions of the patients were evaluated by Silness-Loe plaque index (PI)17 and Loe gingival index (GI)18. Decayed-missing-filled teeth (DMFT/dmft) and decayed-missing-filled teeth surfaces (DMFS/dmfs) were recorded. The extraction of primary teeth due to physiological root resorption was not recorded as a missing tooth. The teeth were scored using International Caries Detection and Assessment System (ICDAS) II19 and PUFA/pufa index (Exposed pulp, Ulceration, Fistula, Absess) (Table I) 20. The diagnosis was based on clinical and radiographic features only.
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.
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 analysis.
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. 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.
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 with Shapiro-Wilk test. The parameters between the groups were compared with One-way ANOVA, Tukey post hoc and Kruskal-Wallis tests. Multiple regression analysis was used to explain YKL-40 level. Sperman rank correlation test was used to verify the correlations between the parameters.