i will  provide you with data from two  studies. Can you interpret together the data and the results of the two  studies and especially concerning HBD-2 ? Here are the data from study 1:1. Study design
“The study protocol was approved by the Ethics Committee on Human Research of the School of Dentistry at Araraquara - UNESP (protocol number 01394712.5.0000.5416). The purpose and content of the study were explained to eligible subjects, and written informed consent was obtained from all participants for inclusion in the study. A total of 70 participants were recruited from June 2016 to March 2018 at the Periodontology Clinic, School of Dentistry at Araraquara - UNESP. The diagnosis of periodontitis was performed according to the classification of the 2017 World Workshop on the Classification of Periodontal and Peri-implant Diseases and Conditions 12 . All patients should present a minimum of 15 natural teeth. Twenty patients were included in the periodontally healthy control group. All subjects were free of systemic and periodontal diseases. They should present probing depth (PD) ≤ 3 mm, no bleeding on probing (BOP), no clinical attachment loss, plaque index (PI) and gingival index (GI) ≤ 15%. Fifty patients with periodontitis stage III or IV were included in periodontitis groups. In addition to the diagnostic criteria 12 , they should present at least five sites in non-adjacent teeth with PD ≥ 4 mm, BOP, and clinical attachment level (CAL) ≥ 4 mm, except for third molars. From the 50 patients, 25 had never smoked and 25 were smokers who should smoke at least 10 cigarettes/day for a minimum of 5 years.
None of the patients had a history of systemic diseases or immunological disorders. Subjects who had received antibiotics and anti-inflammatory drugs within the last 3 months or received periodontal treatment within the last 6 months were not included in the study. Patients using hormone replacement therapy, pregnant and lactating women and orthodontic patients were also excluded.
The sample size was calculated using statistical software (GPower 3.1 Statistical Power Analysis for Windows, Düsseldorf, Germany). Considering mean values of hBD expression and standard deviation from previous studies 13 , 14 , 15 , an effect size of 0.3 was obtained. Based on this effect size, a significance level of 0.05 and a study power of 0.80, a sample size of 20 individuals per group were determined to be necessary.”,
2.Clinical examination
“One calibrated examiner performed all clinical examinations. The intra-examiner reproducibility was assessed by measuring the PD in six teeth (16, 21, 24, 36, 41 and 44), according to the Ramfjord index, in a total of six patients (36 sites/patient) on two different occasions in a time interval of 7 days. Data were submitted to Kappa concordance analysis (k=0.91). The parameters PD, BOP and CAL were assessed at six sites per tooth (mesio-buccal, mid-buccal, disto-buccal, mesio-lingual, mid-lingual, disto-lingual), while PI and GI were registered as absence (negative site) or presence (positive site) of visible plaque and gingival bleeding at four sites per tooth (mesial, distal, buccal and lingual), excluding the third molars. Measurements were performed with a manual periodontal probe (UNC15; Hu-Friedy, Chicago, IL, USA).”, 3. Gingival crevicular fluid (GCF) sampling :
“GCF samples were collected from healthy (n=5) sites of each periodontally healthy patient and from healthy (n=5) and diseased (n=5) sites of each periodontitis patient. Sites at different non-adjacent teeth, preferably one site per quadrant, were selected for GCF collection in each subject, one day after the periodontal examination. Healthy sites had no gingival inflammation, PD ≤ 3 mm, no BOP or clinical attachment loss; and diseased sites had gingival inflammation, PD and CAL ≥ 4 mm with BOP. Diseased sites were selected according to the deepest probing depth in each quadrant. A single examiner performed GCF sampling. The sites to be sampled were isolated with cotton rolls, the supragingival plaque was removed with a sterile curette and the surfaces were gently air-dried. GCF was sampled by inserting absorbent paper strips (Periopaper®, Oraflow Inc., New York, USA) into the gingival sulcus or periodontal pocket for 30 seconds. Paper strips contaminated with blood and saliva were rejected. An electronic device Periotron 8000 determined the GCF volume absorbed into each strip (Periopaper, ProFlow, Inc., Amityville, NY, USA), which was calibrated based on a protocol described before 16 (calibration of the electronic device by polynomial regression). The GCF was obtained as a pooled sample of the same category (healthy or diseased site) in each subject. The papers strips were immediately placed into a dry sterile polypropylene tube (5 paper strips per tube) and kept at -80 °C until analysis. The readings from the Periotron 8000 were converted to an actual volume (μL) by reference to a standard curve.”,
4. Immunosorbent assay for hBD 1 and hBD 2 quantification:
“First, the absorbed GCF was eluted from the paper strips by adding 750 μL (150 μL per strip) of 1x Phosphate-Buffered Saline (PBS) to each tube. The tubes were left on ice in a horizontal shaker for 30 minutes and then centrifuged at 4 °C, 13.000 g for 10 minutes. The GCF levels of hBD 1 and 2 were measured using the sandwich ELISA technique according to the manufacturer’s guidelines (Peprotech, Rocky Hill, NJ, USA). The absorbance was measured by a spectrophotometer at 450 nm wavelength. The levels of AMPs in each sample were determined using the concentration values of standards included in the kit contents. The results were expressed in pg/mL.” and
5. Statistical analysis
“The statistical analysis was performed using the software GraphPad Prism 7 (San Diego, CA, USA). For clinical data, normal distribution was analyzed using the D’Agostino and Pearson test. For normally distributed data, it was used One-way ANOVA followed by Tukey for healthy sites and unpaired t test for diseased sites on inter-group analyses and paired t test for intra-group analyses. For non-normally distributed data, Kruskal-Wallis and Dunn were used for healthy sites and Mann-Whitney tests was used for diseased sites on inter-group analyses, while the Wilcoxon test was performed for intra-group comparison. A p-value of less than 0.05 was considered significant. The effect size was estimated for the primary outcome of the study (AMPs level in GCF). Since non-parametric tests were used for intra and inter-group comparisons, the eta squared and the correlation coefficient r were calculated.” 1. Demographics and periodontal parameters
“A significant age difference was found between periodontally healthy subjects (25.79 ± 3.95) and periodontitis patients (NS [40.32 ± 12]; S [43.88 ± 10.64]) (p<0.05). The distribution of the male and female participants had no significant difference between the study groups, periodontally healthy subjects (5 males/15 females) and periodontitis patients (NS - 9 males/16 females; S - 12 males/13 females) (p = 0.28). The periodontal clinical parameters are presented in table 1. Regarding the full mouth periodontal clinical parameters, PI, BOP, PD and CAL were significantly lower in periodontally healthy subjects than in periodontitis patients (p<0.0001). Only for GI, a significant difference was observed between periodontally healthy patients and smoker patients (p=0.0001). The sampled sites of periodontally healthy subjects presented significantly lower values of PD and CFV than healthy sampled sites of smokers and non-smokers with periodontitis (p<0.05), as well as lower CAL than healthy sampled sites of smokers (p<0.05) (Table 2). In patients with periodontitis, the intra-group comparison revealed lower levels of PD, CAL and CFV in the healthy sampled sites than in the diseased sites (p<0.0001) (Table 2).” ,
2. hBD 1 and hBD 2 quantification
“The GCF levels of hBD 1 and hBD 2 are presented in Figure 1. The hBD 1 levels were significantly higher in healthy sites of the patients without periodontal disease than in healthy sites of smokers and non-smokers with periodontitis (p<0.0001), with a mean difference of 31.62 (95% CI: 17.15 - 46.08) between SH and H and a mean difference of 31.20 between NSH and H (95% CI: 13.81 - 48.57). The lowest GCF hBD 2 level was observed in healthy sites of non-smokers (p=0.0003). A mean difference of 33.61 was observed between SH and H (95% CI: -3.41 - 70.63), and a mean difference of 6.56 was found between NSH and H (95% CI: -1.68 - 14.79). The magnitude of the effect observed for GCF levels of hBDs in healthy sites was large (eta squared= 0.497 and r= 0.70 for hBD 1; eta squared= 0.176 and r= 0.42 for hBD 2). The comparison between diseased sites of patients with periodontitis revealed significantly higher levels of hBD 2 and lower levels of hBD 1 in the GCF of smokers compared to non-smokers (p<0.05). The mean difference between SD and NSD was 74.67 (95% CI: -50.82 - 200.16) for hBD 2 and 6.89 (95% CI: 0.64 - 13.14) for hBD 1. The magnitude of the effect observed for GCF levels of hBDs in diseased sites of smokers and non-smokers was intermediate (hBD 2 eta squared= 0.073 and r= 0.27; hBD 1 eta squared= 0.081 and r= 0.28) 17 . In non-smokers, higher levels of hBD 1 and 2 were observed in diseased sites than in healthy sites (p<0.05). A mean difference of 4.34 for hBD 1 (95% CI: -6.74 - 15.41) and 118.95 for hBD 2 (95% CI: -3.14 - 241.04) was observed between NSD and NSH. The magnitude of the effect observed was intermediate for hBD 2 (eta squared= 0.097 and r= 0.31) and small for hBD 1 (eta squared= 0.021 and r= 0.14). However, in smokers, no significant difference in hBDs level was found between healthy and diseased sites (p>0.05). A mean difference of 2.13 for hBD 1 (95% CI: -2.71 - 6.96) and 10.67 for hBD 2 (95% CI: -36.24 - 57.58) was observed between SD and SH. The magnitude of the effect observed was small for hBD 2 (eta squared= 0.019 and r= 0.14) and intermediate for hBD 1 (eta squared= 0.108 and r= 0.33) .” and these are the data of study 2 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). Can you interpret together the data and the results of the two studies and especially concerning HBD-2 ?
