Can you interpret together the data and the results of the study “Serum vitamin D concentration is associated with antimicrobial peptide level in periodontal diseases” and especially concerning HBD-2? These are the data and results : 1. Materials and methods
“This study was approved by the Ondokuz Mayıs University Clinical Research Ethics Committee on 22/12/2016 (OMÜ KAEK 2016/38), and the study was conducted in accordance with the principles of the Helsinki Declaration. The individuals included in the study were given detailed information about the purpose and content of the study, and written informed consent was obtained from participants.
Patients who were referred to the biochemistry department of OMU medical school for 25(OH)D3 measurements from February to September 2017 invited to participate in our study. Serum 25(OH)D3 levels (ng/mL) were determined by liquid chromatography‐mass spectrometry (LC‐MS/MS; München, Germany). The patients were divided into two categories based on serum 25(OH)D3 as vitamin D sufficient and insufficient. The Institute of Medicine’s (IOM) guidelines’ definition of the Vitamin D status was adopted, whereby vitamin D insufficiency listed at < 20 ng/mL (≤ 50 nmol/L)and levels ≥ 20 ng/μL represented sufficiency status. Among these patients who fulfilled the following criteria were included in the study.
• Participants between 18–35 years of age
• Systemically healthy
• Not taking any medication regularly
• No periodontal treatment in the previous six months • Non-smokers
Within one week of determination of vitamin D status, patients were invited for a periodontal examination. Clinical and radiological ex- aminations of all volunteers were performed. The periodontal ex- amination included probing depth, clinical attachment level (CAL), plaque index (PI), gingival index (GI), and bleeding on probing. All measurements were performed by a single examiner. The examiner was calibrated against an expert periodontist. Calibration was validated if 90 % of the recordings could be reproduced within a 1 mm difference. Diagnosis and classification of periodontal diseases (CP and peri- odontal health) was done according to the 1999 American Academy of Periodontology classification (Armitage, 1999). Participants were classified as having CP if they satisfied the following criteria: radio- graphic evidence of alveolar bone loss with periodontal pocket depth ≥4 mm, CAL of > 3 mm in at least two interproximal sites, not on the same tooth. Participants were classified as a control group if they had no previous or existing evidence of periodontal diseases, no evidence of radiographic or clinical attachment loss. All control groups had bleeding on probing > 10 %, therefore defined as gingivitis (Trombelli, Farina, Silva, & Tatakis, 2018). Study groups: Based on serum 25(OH)D levels and periodontal status subjects were classified as: Group 1: Vitamin D sufficient controls (n:20 GCF, 14 tissue sample) Group 2: Vitamin D sufficient CP (n:20 GCF; 15 tissue sample) Group 3: Vitamin D deficient controls (n:20 GCF; 13 tissue sample) Group 4: Vitamin D deficient CP (n:20 GCF; 14 tissue sample)”,
2.GCF sample collection
“In the control group (gingivitis), GCF samples were collected from the vestibular aspects of maxillary central incisors. In the CP group, sample sites were selected from the teeth having deepest periodontal pockets PD (≥4 mm deep). One site from one tooth for each patient was used for sample collection. One strip was used per site. The GCF levels were presented as the total amount of AMPs recovered in 30 s (Lamster, Oshrain, & Gordon, 1986; Lamster, Oshrain, Fiorello, Celenti, & Gordon, 1988). No missing teeth were present for the control group. Samples were obtained the day after the periodontal diagnosis. Prior to the GCF sample collection, the supragingival plaque was removed with a sterile scaler without touching the gingival margin. The tooth was isolated with a cotton roll and saliva ejector and dried with sterile 2 × 2 gauze In the collection of GCF, 2 × 14 mm paper strips (Periopaper, Proflow, Inc., Amityville, NY, USA), standard in size and absorbency, were used. These special paper strips (periopaper; Oraflow Inc., NY) were placed in the orifice of the gingival margin and left for 30 s. Paper strips contaminated with saliva or blood were not included in the as- sessment. Volumetric analysis of the liquid collected in the paper strips was immediately measured with a pre-calibrated volume measuring device (Peritron® 8000; Pro Flow Inc., Amityville, NY, USA). The paper strips were placed in sterile Eppendorf tubes containing 100μL of phosphate‐buffered saline. The samples were centrifuged at 10000xg for 10 min (Shimadzu UV160A, SNo: 28006648, JAPAN). The super- natants obtained from the GCF were separated for study. The samples were mixed thoroughly stored at -80 °C for up to the working day.”,
3. Tissue sampling
"Gingival tissue samples were taken from the same patients used for GCFs sampling before any periodontal treatment applied. All necessary periodontal treatments of all participants were completed after then. In the chronic periodontitis patients, tissue samples of the deepest pocket were removed from the interproximal area under local anesthesia by internal bevel, sulcular, and interproximal incisions.
In the gingivitis group, gingival tissue samples were collected from a patient who was undergoing the gingivectomy procedure for an esthetic reason under local anesthesia by internal bevel, sulcular, and interproximal incisions. The samples were homogenized in the phosphate buffer solution (PBS, 10mM, pH 7.2) and sonicated (Fisher, Sonic Dismembrator; Mosel 300) for 1 min at 220 V at + 4 °C. They then stored in a freezer at -80 °C until processing. ",
4… Preparation of the tissue sample
“Homogenates brought to room temperature on the day of analysis. Then they were centrifuged at + 4°C at 14000rpm for 5min. The collected supernatants were used for biochemical analysis. Protein content in gingival tissue homogenates of each sample was determined by the Lowry method. (Lowry, Rosebrough, Farr, & Randall, 1951) Biochemical values were expressed as nanograms per mg protein.”,
5.ELISA assay
“GCF and tissue concentrations of LL-37 and hBD-2 were quantified ELISA technique (Sun-Red Bio Company, Shanghai, China; Cat No: 201- 12-2198 and 201-12-1937, respectively) in the Ondokuz Mayıs University Faculty of Medicine Medical Biochemistry Research Laboratory according to manufacturer’s instruction. The enzymatic re- actions were quantified in an automatic microplate photometer (Absorbance at 450 nm using Tecan Spectrafluor Plus, Tecan Trading AG). Sample concentrations were calculated according to the standard curve generated using standard values for each antimicrobial peptide. Kit sensitivity was 0.05 ng/mL and assay range was 0,1 ng/mL-10 ng/ mL. Upper and lower outliers were studied in duplicate.”,
6. Statistical analysis
“All computations were performed with R software 3.5.1. The data were examined for normal distribution using the Shapiro Wilk test. Data were presented as an arithmetic mean ± standard deviation for normally distributed variables, and the data which did not fit the normal distribution were presented as median (min-max). One-way analysis of variance (ANOVA) was used to compare the data with a normal distribution. Tukey HSD was used in case of homogeneity of variance from multiple comparison tests, and Tamhane’s T2 test was used in cases where variance was not homogeneous. The Kruskal Wallis test was used to compare the data that were not normally distributed. Post-hoc comparisons were carried out using the Mann-Whitney U test (p-value<0.05, adjusted for multiple comparisons using the Holm correction). The generalized additive model (GAM) analyses were used to examine the effects of diagnosis, Vitamin D status, and clinical variables on tissue and GCF AMP (hBD-2 and LL-37) levels as this al- lows the modeling of non-linear relationships. A backward variable selection was applied, including all variables that were significant at the 5% level. If an interaction term was included, the main effect was also included regardless of its significance. The GAMs were run using the “mgcv” package in R (Wood & Wood, 2015). The sample size calculation was performed based on the previous study (Ertugrul et al., 2014). A sample size of 15 per group would achieve a statistical power of 80 % to detect differences among the means versus the alternative of equal means using an F test with a 0,05 significance level.”.
1. Demographic and clinical parameters
“This case-control study was performed on 80 GCF samples and 58 gingival tissue samples collected in four groups. The majority of participants consisted of women (70 %). However, given the sex ratio of the participants, there was statistically no significant difference by gender among groups (p = 0.23). The mean age of the participants was 28.74 years. The difference among study groups was not significant (p = 0.34). No smokers included in this study. The periodontal para- meters, PI, GI, CAL, PD, and BOP, are displayed in Table 1. While there was statistically no significant difference in periodontal parameters between gingivitis groups of sufficient 25(OH)D3 and low 25(OH)D3 patients, all clinical measurements were higher in periodontitis groups compared to gingivitis groups (p < 0.001). Periodontitis group con- sisted of localized CP. PD of GCF sampling sites ranged between 4 mm–5 mm.”,
2. Antimicrobial peptide GCF levels
3.HBD-2 levels
“GCF hBD-2 levels are presented in Fig. 1. The median hBD-2 level in GCF was found to be highest in the periodontitis group with sufficient serum Vitamin D levels (2.58 ng/30 s; range: 2.39–2.86); this is fol- lowed by the periodontitis group with deficient serum vitamin D levels (2.34 ng/30 s; range: 1.92–2.54). The subsequent highest hBD-2 levels were the gingivitis group with sufficient serum vitamin D levels 1.67(0.90–1.87) and the gingivitis group with insufficient serum vi- tamin D levels 0.76(0.58–1.53). The difference was found to be statis- tically significant between (p < 0.001) and within groups (p < 0.05; adjusted with the Holm method). Vitamin D sufficient gingivitis pa- tients had significantly higher GCF hBD-2 levels than Vitamin D defi- cient gingivitis patients (p < 0.001); similarly, Vitamin D sufficient CP patients had significantly higher GCF hBD-2 levels than those of vi- tamin D deficient CP patients (p < 0.001). The GCF hBD-2 levels of CP patients were significantly higher than that of gingivitis patients both within the vitamin D sufficient (p < 0.001); and the vitamin D defi- cient group (p < 0.001).”,
4. LL-37 levels
“The GCF LL37 median values for vitamin D sufficient periodontitis patients were 24.33 ng/30 s (16.29–28.30) compared to 20.29 ng/30 s (11.29–26.88) for vitamin D deficient periodontitis group (p = 0.028). For the gingivitis group, the corresponding values were 17.92 (6.42–28.93) in vitamin D sufficient patients and 7.36 (6.18–10.37) in vitamin D deficient patients (p < 0.001; Fig. 1). Similar to the gingival tissue LL37 levels, there was a statistically significant difference be- tween the median values GCF LL37 levels of CP patients and gingivitis patients within the vitamin D deficient group (p = 0.028) and within Vitamin D sufficient group (p < 0.001; Fig. 1).”,
5. Antimicrobial peptide gingival tissue levels
6. HBD-2 levels
“There was strong evidence of a difference (p-value < 0.001) in the tissue hBD-2 concentrations between the mean ranks of periodontitis and gingivitis groups with different 25(OH)D levels (Fig. 2). The post- hoc paired comparison showed that there was evidence (p-value< 0.05) of the differences between all the groups. The median hBD-2 concentration of the gingivitis group having deficient vitamin D levels was 4.23 ng /mg protein (range: 2.37–5.87 ng/mg protein) compared to 7.38 ng/mg protein (range: 4.21–13.35 ng/mg) in the gingivitis group with sufficient 25(OH)D levels, while for the periodontitis group with deficient 25(OH)D levels the median hBD-2 concentration was 9.28 ng/ mg protein (6.29–10.81 ng/mg protein) and with sufficient 25(OH)D levels were 12.06 ng/mg protein (9.11–18.18 ng/mg protein). The hBD- 2 concentration in the gingivitis group with deficient levels of 25(OH)D was about half of those in gingivitis patients with sufficient levels of vitamin D (p < 0.001). Likewise, in the CP group, hBD-2 concentration was significantly higher in vitamin D sufficient patients compared to Vitamin D deficient patients; p < 0.0001. As expected, hBD-2 gingival tissue concentration was higher in chronic periodontitis groups com- pared to corresponding gingivitis groups (for both in deficient (p < 0.001) and sufficient serum 25(OH)D levels (p < 0.001)).”,
7. LL-37 levels
“The tissue level of LL37 was higher compared to those of hBD-2 (Fig. 2). Similar to hBD-2, the median values of gingival tissue LL37 concentration differed among groups (p < 0.001). The median value of LL37 of the gingivitis group having deficient vitamin D levels (19.89 ng/mg protein range: 11.22–27.63) was lower than all other groups. The highest median value of LL37 gingival tissue levels (53.91 ng/mg protein; 37.21–85.13) was obtained in chronic periodontitis patients with 25(OH)D levels≥20 ng/mL. Concerning gingivitis patients, significantly higher LL37 tissue levels were observed in vitamin D sufficient samples (24.39 ng/mg protein) when compared to Vitamin D deficient samples (19.89 ng/mg protein; p = 0.027). Like- wise, in the periodontitis group, higher LL37 tissue levels were detected in vitamin D sufficient samples (53.9 ng/mg protein) compared to vitamin D deficient specimens (24.93 ng/mg protein; p < 0.0001). Comparison of the tissue LL37 Levels with and without periodontitis revealed that CP patients had higher LL-37 levels than gingivitis patients both in vitamin D sufficient (p < 0.0001)and deficient group (p = 0.014).”,
8. Regression model
“GAM analysis showed that significant correlations of GCF hBD-2 and LL-37 level with 25(OH)D levels and periodontal destruction (Table 2). Inclusion of potential confounding factors such as age and gender in the regression model did not alter this association. Ad- ditionally, clinical variables (CD, CAL, PI, GI) were pre-screened by fitting GAM models to each variable in isolation in a backward fashion.
None of the clinical variables were identified as independent predictors of AMPs. Parameters included in GAM analysis were periodontal disease status, vitamin D levels, the interaction between Vitamin D levels and periodontal disease status, age, and gender. The final model explained 87 % of the variance of GCF hBD-2 (Adjusted r2 = 0.87, p = 0.03 (for all variables), GCV = 0.04); 52 % of variance of the GCF LL-37 (Adjusted r2 = 0.52, P < 0.0001, GCV = 0.09). Due to the low sample size, three parameters were included in the GAM analysis to identify predictors of local AMPs in gingival tissue samples. The final model consisting of periodontal disease status, vitamin D levels, interaction between Vitamin D levels and periodontal disease status explained 60 % of variance of the tissue hBD-2 (adjusted r2 = 0.60, P < 0.0001 (for all variables), GCV = 0.09); 63 % of variance of the tissue LL-37 (ad- justed r2 = 0.63, P < 0.0001 (for all variables), GCV = 0.06). The re- sults of the regression analysis are shown in Table 2.”,
9. Correlation
“Finally, to test for an association between GCF and tissue levels of both AMPs, a Spearman correlation coefficient was used. There was a positive correlation between GCF and tissue levels of hBD-2 (r = 0.82; < 0.0001) and LL-37 (r = 0.65; < 0.0001).”.