Can you interpret together the data and the results of the study “Gingival Crevicular Fluid Levels of Human Beta-defensin 2 and 3 in Healthy and Diseased Sites of Individuals with and without Periodontitis” and especially concerning HBD-2? These are the data and results : 1.Study design and sampling strategy
“This cross-sectional study comprised participants from a previous study (Costa et al. 2018), totalling 40 individuals selected from the Periodontology Clinic at the Federal University of Minas Gerais. Sample size was calculated using statistical software (GPower 3.1 Statistical Power Analysis for Windows, Düsseldorf, Germany) consider- ing mean values from previous studies (Dommisch et al. 2005, Brancatisano et al. 2011, Ertugrul et al. 2013), an effect size of 0.30, a significance level of 0.05 and a study power of 0.80. Based on these parameters, a sample size of ~20 individuals per group was determined to be necessary. Sampling strategy was as previously described (Costa et al. 2018). Briefly, during the period of data collection (August 2012 to December 2014), approximately 360 individuals sought dental care in the university, were determined to be eligible and invited to participate in the study. According to their availability and accessibility during the dental care routine, individuals who met the inclusion criteria were added to the study groups until the required sample size was reached. Individuals were excluded if they were pregnant or lactating women, smokers and former smokers or if they have any systemic diseases. Exclusion was also applied to individuals using drugs that could influence periodontal health, having received periodontal treatment within the last 6 months or having used antibiotics and anti- inflammatory drugs within the last 3 months.”,
2. Periodontal clinical examination
“All participants underwent a full-mouth periodontal examination and the parameters of probing depth (PD), clinical attachment level (CAL), and bleeding on probing (BOP) were recorded at 4 sites (buccal, distal, lingual, mesial) for all teeth present except any third molars. Intraoral radiographs were taken to detect bone loss (Costa et al. 2018). One single trained examiner performed all examinations with a manual periodontal probe (PC- PUNC 15 Hu-Friedy®, Chicago, USA). Evaluations of PD and CAL parameters were performed and repeated within a 1-week interval for 10 individuals randomly selected during the recruitment of participants, in order to determine intra-examiner agreement. Analyses retrieved kappa values for PD and CAL greater than 90% and intra- class correlation coefficient greater than 89%. Those individuals were not included in the study. Participants were divided into 2 groups (n = 20): periodontally healthy individuals (H) and individuals with periodontitis §. Periodontally healthy individuals (H) should have a minimum of 20 natural teeth, PD ≤3 mm and no more than 20% of sites with BOP. It is important to stress that CGF samples were collected in these individuals from sites with no BOP. Individuals with periodontitis should have a minimum of 15 natural teeth and at least 4 teeth with 1 or more sites with PD ≥4 mm and CAL ≥3 mm in the same site. The diagnosis of periodontitis was based on clinical and radiographic parameters as previously established (American Academy of Periodontology Task Force Report on the Update to the 1999 Classification of Periodontal Diseases and Conditions, 2015). Diagnosis and definition of periodontitis were updated according to the new classification scheme for periodontal diseases and conditions (Tonetti et al. 2018) and participants’ periodontal condition was confirmed.”,
3. Gingival crevicular fluid sampling
“GCF sampling was carried out 1 week after the periodontal examination among all participants. GCF fluid samples were obtained from healthy sites (Hh) of each periodontally healthy individual and from healthy sites, sites with gingival inflammation but no attachment loss (Pg) and sites with gingival inflammation and attachment loss (Pp) of each individual with periodontitis. For each sample category, 4 representative sites were selected in each participant and analyzed together (pool samples) to provide a mean value per category/individual (Hh [n=20],Ph[n=20],Pg[n=20],Pp[n=20]).A total of 320 sites were sampled (80 in H individuals and 240 in P individuals).
Healthy sites (Hh and Ph) for GCF collection showed PD and CAL ≤3 mm without BOP and without alveolar bone loss. In P individuals, sites with gingival inflammation but no attachment loss (Pg) for GCF collection showed PD and CAL ≤3 mm and BOP; sites with gingival inflammation and attachment loss (Pp) showed PD ≥4 mm, CAL ≥3 mm and BOP. The 4 Pp sites were selected according to the deepest PD at different non-adjacent teeth, preferably 1 site in each quadrant. Clinical parameters of sampling Pp sites were: mean PD 6.01±2.03 mm and mean CAL 6.40±2.31 mm). The 4 Hh and Ph sites (healthy sites), as well as the 4 Pg sites (sites with gingival inflammation but no attachment loss), were also collected at different non- adjacent teeth, preferably 1 site in each quadrant (Costa et al. 2018).
Before GCF sampling, all selected sites were isolated with cotton rolls to avoid contamination with saliva, supragingival plaque was removed and the tooth was gently air-dried. Paper strips (Periopaper, PerioCol Collection Strip, Oraflow, Plainview, NY, USA) were inserted into the gingival sulcus/pocket until mild resistance was felt and left in position for 30 seconds. Care was taken to avoid mechanical injury. Paper strips contaminated with blood were discarded (Griffiths 2003, Wassall and Preshaw 2016). The total GCF volume collected by each strip was measured with a calibrated device (Periotron 6000, IDE Interstate, Amityville, NY, USA). Representative samples of the same site category (Hh, Ph, Pg, Pp) in the individual were placed into a dry Eppendorf tube (4 paper strips per tube) and frozen at −80°C until laboratory analysis.” 1. Quantification of hBD-2 and hBD-3
“GCF levels of hBD-2 and hBD-3 (pg/ml) were deter- mined using the Enzyme-linked Immunosorbent Assay (ELISA) sandwich technique according to manufactur- ers’ instructions (ELISA kits catalog #900-K172 and #900-K210, Peprotech, Rocky Hill, NJ, USA). Before the quantification of hBDs, the absorbed fluid was eluted from the paper strips by adding 600 μl (150 μl per strip) of phosphate- buffered saline (PBS; 10 mmol/L, pH 7.2) to each Eppendorf tube and shaking the tubes on an ELISA plate shaker for 30 minutes. Subsequently, the tubes were centrifuged at 13 000 g for 10 minutes at 4°C.
For the analysis, 96 wells were coated with 100 μl (0.25 mg/ml) of anti-hBD specific antibody for 12 hours. After 4 washes with PBS with 0.05% Tween 20 (PBST), wells were blocked for 1 hour at room tem- perature with 300 μl of blocking solution (1% bovine serum albumin in PBST). The plates were washed and 100 μl of sample or standard solution was added to the wells in duplicate; following this, the plates were incubated for 2 hours. After further washing, 100 μl of the detection antibody (0.5 μg/ml) was added to the wells and the plates were incubated for another 2 hours. After this period, the plates were washed and 100 μl of streptavidin-peroxidase (1:2000 in PBST) was added to the wells. Plates were incubated at room temperature for 30 minutes. Colorimetric reactions were produced with the use of o- phenylenediamine in the presence of 0.02% H2O2. After blocking the reaction with H2SO4 (2N), the absorbance was measured at 450 nm using an ELISA reader. The minimum and maximum detectable limits of hBD-2 were 16 - 2000 pg/ml and of hBD-3 was 63 - 4000 pg/mL. The levels of hBD-2 and hBD-3 in each sample were determined using the concentration values of standards included in the kit contents. The results were expressed in pg/ml.”,
2. Ethical considerations
“The present study was approved by the Research Ethics Committee of the Federal University of Minas Gerais – COEP/UFMG (CAAE #0529.0.203.0001-11). All participants were informed of the aims of the study and they signed an informed consent form before inclu- sion in the study. Legal rights of the participants were preserved at all times.”,
3. Statistical analysis
“Data normality was assessed using the Kolmogorov- Smirnov test. Descriptive analyses were presented for periodontal parameters and levels of hBDs. Descrip- tive and bivariate analyses, as well as the creation of 2 databases using site and individual values of hBD-2 and hBD-3, were performed using statistical software (Statistical Package for Social Sciences, IBM SPSS Sta- tistics for Macintosh version 20, IBM Corp., Armonk, NY, USA). These databases were then used to perform the multilevel analyses (Hierarchical Linear and Non- linear Modeling, HLM 6.06 Statistical Package, Skokie, IL, USA). The results were considered significant if p < 0.05.
For each defensin, data were hierarchically structured in 2 levels: periodontal sites (Level 1: healthy sites/sites with gingival inflammation but no attachment loss/sites with gingival inflammation and attachment loss) nested within individuals (Level 2: individuals with or without periodontitis). The multilevel structure of analyses in- cluded 80 mean site values (Level 1) from 40 individual values (Level 2). Multilevel linear modelling was used to assess the differences in hDB-2 and hBD-3 expression. During the first stage, a “null model” estimated the basic partition of data variability between the 2 levels before the inclusion of site and individual characteristics were taken into account. Level 1 variables were incorporated into the model. In the sequence, Level 2 variables were incorporated, with the calculation of p-values (Student’s t test). Only variables with p < 0.05 were retained in the final model. The intraclass correlation coefficient, i.e., the proportion of total variance attributed to the cluster level (individual characteristics), was also calculated.”,
4. Results
“The group of periodontally healthy individuals (H) was composed of 7 women and 13 men, mean age 39.6 ± 16.7 years (range 18 – 79). The group of subjects with periodontitis § was composed of 10 women and 10 men, mean age 44.4 ± 10.2 years (range 25 – 68). Full- mouth periodontal parameters of the study groups are shown in Table 1. Individuals with periodontitis presented a higher mean PD and CAL when compared to periodontally healthy individuals.
GCF levels of hBD-2 were [mean ± s.d.; median (Q3 – Q1)]: Hh = 18.03 ± 28.11 (4.80; 0.00 – 30.69); Ph = 33.43 ± 6.53 (33.29; 28.04 – 38.25); Pg = 29.60 ± 8.68 (27.56; 23.14 – 35.50); and Pp = 35.10 ± 20.04 (26.20; 23.20 – 42.54) (Figure 1). GCF levels of hBD- 3 were [mean ± s.d.; median (Q3 – Q1)]: Hh = 41.12 ± 120.71 (0.00; 0.00 – 0.00); Ph = 1121.74 ± 565.87 (978.44; 760.48 – 1268.12); Pg = 1424.45 ± 1309.38 (938.19; 806.75 – 1266.38); and Pp = 852.76 ± 1121.98 (613.63; 325.50 – 854.68) (Figure 2). It is important to note that 17 hBD-3 samples (17/20) and 6 hBD-2 samples (6/20) in the control group were under the detection limit for the ELISA test. These samples represented 14,38% of the total samples. They were imputed a value of 0 (zero) for statistical analyses purposes.
In the hierarchical modelling, the analysis of the null model showed a significant variance in the values of hBD-2 (p < 0.01) and a marginally significant variance in the values of hBD-3 (p = 0.096) by the individual level (Level 2 groupings), indicating a need for running HLM analyses (Table 2).
Results from the multilevel model showed that periodontitis was associated with higher levels of hBD-2 (coefficient 14.68; p = 0.023) and hBD-3 (coefficient 1091.86; p < 0.001). There were no effects of site phe- notype on the GCF levels of hBD-2 (p = 0.266 for Pg and p = 0.625 for Pp) (Table 3) and hBD-3 (p = 0.302 for Pg and p = 0.359 for Pp) (Table 4).” .