Study participants
Participants were recruited from the Department of Periodontology, West China Hospital of Stomatology, Sichuan University from 2018 to 2021. The study protocol was approved by the Ethics Committee of West China Hospital of Stomatology (Number: WCHSIRB-D-2017-092) and was conducted in accordance with the Declaration of Helsinki in 2013. Written informed consent was obtained from all study participants. All participants were systemically healthy, as confirmed by physical examination and a comprehensive blood examination. The inclusion criteria for the severe periodontitis group were as follows: (i) at least 20 natural teeth, including at least 12 premolars and molars (excluding third molars); and (ii) at least five sites with probing depth (PD) and clinical attachment loss/level (CAL) ≥ 6 mm at baseline in different quadrants. A full-mouth series of X-rays within the past 6 months were required to further assess the diagnosis of periodontitis. All patients with periodontitis recruited met the criteria for a diagnosis of severe periodontitis [26], and they also need to be diagnosed with Stage III/IV periodontitis according to the 2018 classification of periodontitis [1]. Healthy controls had no periodontal sites with attachment loss or PD > 3 mm, and a whole mouth BOP positive site count of < 10%.
The exclusion criteria were as follows: (i) pregnancy or lactation; (ii) known medical disorders that could affect local and systemic inflammatory status and the cytokine levels in oral fluids, such as diabetes mellitus, rheumatoid arthritis, and immunological disorders; (iii) treatment with any anti-inflammatory drugs or antibiotics in the previous three months; (iv) history of periodontal treatment in the previous six months; (v) past or current smokers; (vi) significant occlusal disharmony or a history of orthodontic treatment in the past 10 years; and (vii) an inability to provide consent.
Sample size calculation
Changes of GCF LXA4 levels in periodontitis after treatment were used to calculate the optimal sample size. Due to a lack of related information in the literature, a priori sample calculation could not be performed. Thus, a pilot study which included five periodontitis patients was performed, and the changes in LXA4 levels before and after treatment were recorded. Sample size was determined by a function of power and level of significance. A sample size of 20 in the periodontitis group was required to detect a significant difference of 10 pg/µl of LXA4 with 80% statistical power and a 5% level of significance. If the sample size is calculated based on the detection of changes of PD, only about 6 patients are needed to achieve 90% statistical power and a 5% level of significance. Therefore, we chose a sample size of 20 to meet the requirement for statistical testing of both PD and LXA4.
Clinical assessment
After demographic factors were recorded, all participants underwent a full-mouth periodontal examination by the same examiner (RM). This examiner was trained in a calibration process to reduce intra-examiner error [27]. Five periodontitis patients were chosen for calibration. PD was measured on two occasions, 24 h apart. For PD, the percentage of agreement within ± 1 mm between repeated measurements was at least 95%. PD was measured from the free gingival margin to the base of the pocket. CAL was calculated as PD plus gingival recession. PD and CAL were recorded to the nearest millimeter at six sites per tooth (mesio-buccal, mid-buccal, disto-buccal, disto-lingual, mid-lingual, and mesio-lingual), except for the third molars, with a UNC-15 probe (Hu-Friedy, Chicago, IL, USA ). BOP was recorded as present or absent after 30 s of PD measurements. The BOP percentage was calculated by dividing the number of bleeding sites by the total number of sites examined in each participant. The plaque score (PS) was recorded as the presence or absence of plaque [28]. Additionally, PD reduction (≥ 2 mm) and CAL gain (≥ 3 mm) are regarded as the primary treatment outcome measures.
Clinical intervention
At the initial visit, periodontal measurements of all participants were recorded, and their baseline GCF samples were collected at least two days later. Patients with severe periodontitis underwent oral hygiene instructions, full-mouth supragingival debridement, and quadrant-based SRP using both ultrasonic scalers (MiniPiezon, EMS Dental, Nyon, Switzerland) and hand instruments (Gracey Curette; Hu-Friedy, Chicago, IL, USA) under local anesthesia within one month. Follow-up examinations and GCF sample collections were conducted at 1, 3, and 6 months after treatment, with clinical measurements taken at each subsequent follow-up appointment. In addition, supportive periodontal therapy including oral hygiene instructions and supragingival debridement was provided at each subsequent visit.
GCF sampling
In both groups, two to four sites per individual were selected as the sampling sites and GCF samples were collected by a single calibrated investigator (RM). In the healthy group, GCF samples were collected from sites with no clinical inflammation. With regard to patients with severe periodontitis, GCF samples were collected from each individual at the deepest sites (PD and CAL ≥ 6 mm) of their mouth.
After isolation of the sampling sites with cotton rolls to prevent the contamination of saliva, large and visible supragingival plaques were removed with Gracey curettes without touching the marginal gingiva. The sampling site was gently air-dried, and a 30-second GCF sample was collected using a filter paper strip (3MM, Whatman, Kent, UK), which was gently inserted into the sulcus/pocket, 1–2 mm subgingivally [29]. Samples visibly contaminated with blood or saliva were discarded and then collected from another site. Every filter paper strip was placed in a sterile Eppendorf tube (Axygen, Corning Incorporated, NY, USA) that had been weighed before sampling, using an analytical balance (AE 240s, Mettler, Zurich, Switzerland) with a sensitivity of 0.01 mg, and reweighed immediately after sampling. The increase of the weights was used to calculate the volume of GCF, according to relationship between weight and volume of GCF reported [30, 31]. These samples were stored at -80 °C until use. Eventually, 64 GCF samples from periodontally healthy participants and 74 GCF samples from severe periodontitis participants were collected in total, which were all individually used for biochemical and microbiological analysis.
Analysis of LXA4 in GCF
GCF samples were analyzed by an enzyme-linked immunosorbent assay (ELISA). Before the quantification of LXA4 levels, 300 µl phosphate-buffered saline containing 0.25% bovine serum albumin was added to each Eppendorf tube, and then the samples were centrifuged for 60 min at 300 rpm (8 g) and then for 2 min at 12,000 rpm (13,201 g) at a temperature of 4 °C [29]. The supernatants were collected and LXA4 levels were estimated using a commercially available ELISA kit (Neogen Corp., Lansing, MI, USA ) in accordance with the manufacturer’s instructions.
The diluted GCF samples were placed in duplicate in two wells. The total amount (pg) of LXA4 per sample in a 30 s period was identified using standard curves according to the manufacturer’s guidelines and dilution factor [29], and then divided by the GCF volume to give the LXA4 concentration (pg/µl) in the original GCF sample. Two values were obtained for each sample and the average of these values was taken as the final figure.
Analysis of periodontal pathogens
The precipitate of the pretreatment GCF sample from patients with severe periodontitis at baseline and 6 months after treatment was used for bacterial DNA extraction. DNA was extracted using an DNA extraction kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions. The bacterial 16 S ribosomal DNA of P. gingivalis, A. actinomycetemcomitans, Treponema denticola (T. denticola), Prevotella intermedia (P. intermedia) and Tannerella forsythia (T. forsythia) were amplified by polymerase chain reaction (PCR) system (Bio-Rad, California, USA ), using primers as previously reported [32, 33]. Genomic DNA from the plasmid-generated standards or reference strains were used as the positive control and distilled water was used as the negative control. PCR products were separated by electrophoresis in 2% agarose gels.
Statistical analysis
Statistical analysis was performed using the statistical software program SPSS version 21.0 (IBM Corp., Armonk, NY, USA ) and MedCalc version 19.0, and p-value < 0.05 was considered statistically significant. Graphs were created using GraphPad Prism version 7.0 (GraphPad Software Inc., San Diego, CA, USA ). Results based on continuous data were presented as mean ± SD, and results based on categorical data were presented as numbers and percentages.
Normal distribution of the data at baseline was checked using the Shapiro-Wilk test. Since the data was found to be non-normally distributed, the variables were analyzed with non-parametric methods. First, when examining group differences in demographic and clinical characteristics at baseline, the Pearson’s chi-square (χ2) test was used for categorical variables, whereas the non-parametric Mann-Whitney U were applied to determine differences in continuous data. Second, comparisons of the clinical parameters and the LXA4 levels before and after treatment in the severe periodontitis group were analyzed using the Wilcoxon signed rank test. Third, given that different GCF sampling sites within a patient could be more correlated with each other than the sites between patients and independent variables like gender, age and oral hygiene could potentially have relevant effects on the outcomes, we used generalized estimating equation (GEE) models adjusting for these variables to examine the relationship between LXA4 levels and clinical parameters (PD reduction ≥ 2 mm or CAL gain ≥ 3 mm) in this longitudinal study. The discrimination of the predicting model was assessed with receiver operating characteristic (ROC) analyses and the area under the curve (AUC). The optimal criterion for each ROC analysis was identified according to Youden’s index. Additionally, GEE analysis was also used to identify relationships between LXA4 levels and specific periodontal pathogens. Note that two patients with severe periodontitis were unavailable for analysis at the 1-month and 3-month follow-up visits, respectively.
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