can you produce the risk of bias according to the Agency for Healthcare Research and Quality for the study “Serum vitamin D concentration is associated with antimicrobial peptide level in periodontal diseases” while i provide you with the following data from the study :                                                                                                                                                                                                                                                                                           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.".