i will provide you the data from the following study “Gingival crevicular fluid levels of Interleukin-22 (IL-22) and human β Defensin-2 (hBD-2) in periodontal health and disease : A correlative study”. Can you interpret together the data and the results of the study, especially concerning HBD-2 ? These are the data of the study : 1. Materials and methods
"The study population consists of patients from the Department of Periodontology, Dr.D.Y.Patil Dental College and Hospital, Pimpri, Pune. The study protocol was designed in accordance with Helsinki declara- tion of 1975, as revised in 2013 and was approved by the university ethics committee (DYPV/EC/122/17).All participants willing to participate in our study signed an informed consent, were in good general heath, had no history of periodontal therapy, nor had they taken antibiotics/anti-inflammatory drugs within 6 months. Smokers, pregnant women, patients with underlying systemic disease were excluded from the study. Sample size estimation was based on data provided by Chen et al., 2014 for expected means of GCF hBD-2 levels as measured by ELISA in chronic periodontitis, gingivitis and healthy control subjects (chronic periodontitis = 266.91 pg/mL ± 311.81 pg/mL, patients with chronic gingivitis:487.80 ± 420.66 pg/mL and healthy controls was 99.20 ± 99.98 pg/mL).14 Using a sample size of 25 subjects per group the overall estimated effect size was determined as f = 0.52 (F-test ANOVA) at p = 0.05 and 80% power, which was in the large range (> 0.40) and thus deemed acceptable.15 Since there are no preliminary data for correlation of IL- 22 and hBD-2 in each these groups, we estimated the power of this sample to detect a significant large correlation effect (>0.5) and at n = 25, the sample had 80% power at p = 0.05 to determine a Pearson’s correlation coefficient of 0.53.” , can you produce the risk of bias according to the Agency for Healthcare Research and Quality for the study “Gingival crevicular fluid levels of Interleukin-22 (IL-22) and human β Defensin-2 (hBD-2) in periodontal health and disease : A correlative study” while i provide you with the following data from the study : 1. Materials and methods “The study population consists of patients from the Department of Periodontology, Dr.D.Y.Patil Dental College and Hospital, Pimpri, Pune. The study protocol was designed in accordance with Helsinki declara- tion of 1975, as revised in 2013 and was approved by the university ethics committee (DYPV/EC/122/17).All participants willing to parti- cipate in our study signed an informed consent, were in good general heath, had no history of periodontal therapy, nor had they taken anti- biotics/anti-inflammatory drugs within 6 months. Smokers, pregnant women, patients with underlying systemic disease were excluded from the study. Sample size estimation was based on data provided by Chen et al., 2014 for expected means of GCF hBD-2 levels as measured by ELISA in chronic periodontitis, gingivitis and healthy control subjects (chronic periodontitis = 266.91 pg/mL ± 311.81 pg/mL, patients with chronic gingivitis:487.80 ± 420.66 pg/mL and healthy controls was 99.20 ± 99.98 pg/mL).14 Using a sample size of 25 subjects per group the overall estimated effect size was determined as f = 0.52 (F-test ANOVA) at p = 0.05 and 80% power, which was in the large range (> 0.40) and thus deemed acceptable.15 Since there are no preliminary data for correlation of IL- 22 and hBD-2 in each these groups, we estimated the power of this sample to detect a significant large correlation effect (>0.5) and at n = 25, the sample had 80% power at p = 0.05 to determine a Pearson’s correlation coefficient of 0.53.”, 2. Clinical measures : “Clinical measurements made were plaque index (PI), gingival index (GI), bleeding on probing (BOP), probing depth (PD) and clinical attachment level (CAL). The measurements were taken at 6 sites per tooth and entered manually in the data sheet. Probing pocket depth and attachment level measurements were made to the nearest millimeter using an UNC 15 Probe. A total of 79 subjects were recruited in the study and were grouped according to their periodontal status. Group 1 (Healthy controls) included 27 subjects with a plaque index (PI) of < 1, gingival index (GI) of < 1, probing depth (PD) of ≤ 3 mm, and no loss of attachment. Group 2 (Gingivitis) included 25 subjects with a PI of ≥1, GI of ≥1PD ≤ 3 mm, clinical evidence of inflammation based on presence of BOP ≥ 50% of sites and no clinical attachment loss. Group 3 (Chronic periodontitis) had 27 subjects with a PI of ≥1, GI of ≥1, Probing depth (PD) ≥ 5 mm and clinical attachment level(CAL) ≥ 3 mm and presence of BOP ≥ 50% of sites with radiographic evidence of bone loss.” , 3. GCF collection :
“GCF collection was done a day after the periodontal examination. In healthy individuals, sites with PD ≤ 3 mm and no signs of inflamma- tion, in gingivitis patients, sites with bleeding on probing and in chronic periodontitis subjects, the sites with PD ≥ 5 mm were chosen for ac- quiring GCF. The area was isolated with gauze to avoid saliva con- tamination, after drying the area with air, supragingival plaque was removed with curette (Hu Friedy, USA), a calibrated volumetric mi- crocapillary pipette (Sigma Aldrich) was placed at the entrance of the gingival sulcus, gently touching the gingival margin. From each test site, 2 μl of GCF was collected. The collected GCF was then transferred to a sterile Eppendorf tube and stored at −70oc until the assay. Micropipettes with contaminants like blood or saliva were discarded.18 The GCF level of IL-22, hBD-2 was assayed using commercially available ELISA kit (E Lab Biosciences) [Catalog no: E-EL-H0106 (IL- 22), Catalog no: E-EL-H0996 (hBD-2]. The procedure was performed according to the instructions given in the kit.” ? 1. Quantification of hBD-2
“Briefly, to a human hBD-2 antibody precoated ELISA plate 100 μL of the standard or sample is added and incubated for 90 minutes at 37 °C and the free components are washed. Add 100 μL biotinylated detection antibody is added and incubated for 1 hour at 37 °C and washed. Then 100 μL of horse radish peroxide (HRP) conjugate is added and incubated for 30 minutes at 37 °C and washed. 90 μL of substrate reagent is added and incubated for 15 minutes at 37 °C followed by the addition of 50 μL stop solution. The optical density is measured with a spectrometry at a wave length of 450 nm immediately. The minimum detection limit for hBD-2 is 37.50 pg/mL. The levels of hBD-2 in each sample were determined using the concentration values of standards included in the kit contents. The results were expressed in pg/mL.”, 2. Statistical analysis
“The statistical analysis was done using the Statistical package for the Social science (SPSS) for windows, version 19; SPSS Inc. Chicago, IL, USA. All the periodontal parameters were described and reported as mean values ( ± SD). The distribution and comparison of clinical parameters and gingival crevicular fluid IL-22, hBD-2 levels in healthy controls, gingivitis and chronic periodontitis groups were assessed using one way ANOVA test. Post Hoc Bonferroni test was used for intergroup comparison of clinical parameters and gingival crevicular fluid (GCF) IL-22, hBD-2 levels in healthy controls, gingivitis and chronic periodontitis groups. Association among the IL-22 and hBD-2 and clinical parameters in all three groups were examined using Pearson’s correlation test. Probability value of less than 0.05 was considered statistically significant for all analyses.”, 3. Clinical analysis
“The healthy control group had 27 subjects with a mean age of 25.14 years and 25 gingivitis subjects with a mean age of 31.92 years and 27 patients with periodontitis with a mean age of 47.00 years. Periodontal parameters of the study groups are shown in Table 1. All clinical parameters were significantly higher in the chronic periodontitis group than the healthy and gingivitis groups (P < 0.05).”, 4. GCF hBD-2 levels
” The mean GCF levels of hBD-2(Table 1, Graph 1) and the mean difference between groups are represented (Table 2). The hBD−2 concentration was significantly higher in the chronic periodontitis group compared to gingivitis group (P = 0.003) and healthy control group (P < 0.05).However there were no statistical differences be- tween healthy and gingivitis groups in terms of total amount of hBD-2 in GCF. " , 5. Correlations "There was significant positive correlation between GCF total amount of IL-22 and GI (P < 0.01), PI (P < 0.01), PD (P < 0.00.01), CAL (P < 0.05) [Table 3, Graph 2(A, B, C, D)] in chronic periodontitis group (Group 3). Positive correlation between GCF total amount of hBD-2 and PI (P < 0.05) and CAL (P < 0.01) [Table 3, Graph 3(B, D)] was observed in chronic periodontitis group. Positive correlation was observed between IL-22 and hBD-2 levels in GCF of CP Group (P < 0.005) [Table 3, Graph 3(E)]. The correlation between clinical parameters and gingival crevicular fluid levels of hBD-2 and IL-22 in healthy controls and gingivitis group was statistically insignificant (data not shown).