This interventional study was conducted from September 2020 to December 2021, and the patients were recruited from the outpatient Department of Periodontology, VSPM Dental College and Research Centre, Nagpur, India in accordance with the Helsinki Declaration of 1975 as revised in 2013. Clinical Trial Registry–India (CTRI), the primary register of the WHO International Clinical Trials Registry Platform, was used to register this clinical trial. Registration number CTRI/2020/03/024346 was assigned for this trial. After objectives and the procedure of the trial were explained, a written informed consent was obtained from the patients willing to participate. The study was also approved by Institutional Ethics Committee of our institute.
Referring to the study by Veyisoglu G. et al. (2019)[7], the authors reported mean levels of SEMA-4D, PAD-2 and MMP-8 in GCF in control, gingivitis and chronic periodontitis (CP) patients. These data were used to estimate the effect size. Based on the means for SEMA-4D across three patient groups, the effect size was 0.4843, while for PAD-2 was 0.3735 and for MMP 8 was 0.5539. The proposed study also has three groups namely: Periodontally healthy, stage III periodontitis non-smokers and stage III periodontitis smokers. An effect size of 0.45 was considered appropriate, which resulted into a sample size of 51 (17 per group) patients to provide the desired effect with 80% power and 95% confidence level. Hence, 60 patients aged between 25 to 60 years was the finalized sample size for this trial with all the participants having ≥ 20 teeth present in the oral cavity.
The periodontal diagnosis was based on the new classification for periodontal and peri-implant diseases and conditions given in 2017[19, 20]. Thus 20 patients in each group were allotted with Group 1 comprising of periodontally healthy patients, Group 2 and Group 3 comprising of untreated Stage III periodontitis non-smoker and smoker patients respectively (Fig. 1). The allotted patients had to fulfill the following criteria based on the group of allotment as
Inclusion Criteria:
Group I
Periodontally healthy patients with no signs of periodontal disease and no history of smoking.
Group II
Non-smoker patients with stage III periodontitis as assessed by clinical findings of PPD ≥ 5 mm and CAL ≥ 4 mm. (≥ 30% of teeth affected) and with radiographic evidence of bone loss and with no history of smoking.
Group III
Smoker patients with stage III periodontitis who were current smokers with untreated stage III periodontitis, as assessed by clinical findings of PPD ≥ 5 mm and CAL ≥ 4 mm. (≥ 30% of teeth affected) and with radiographic evidence of bone loss and with history of smoking at least 10 cigarettes per day for the last 3 years.
Exclusion Criteria:
Patients with any systemic disease, pregnant and/or lactating females, use of antibiotics within the last 6 months and anti-inflammatory drugs within the last 3 months, use of steroids or any immunosuppressive therapy, those who had symptoms of acute illness or had undergone any type of periodontal therapy or oral prophylaxis in past 6 months were excluded from the study.
Clinical Examination:
For systematic recording of the findings in each of the study participant, case history was recorded and the clinical and radiographic examinations were carried out. The smokers’ status was determined by verbal questioning and smokers were enrolled if they regularly smoked 10 cigarettes per day while the non smokers were selected only if they did not smoke cigarettes in their entire lifetime. A single examiner (AI) used a manual periodontal probe to do an intraoral examination on 10 patients prior to the study†. PPD, CAL, Plaque Index (PI)[21], Gingival Index (GI)[22] and Papillary Bleeding Index (PBI)[23] score were the clinical parameters examined. The measurements were taken at six different locations around each tooth and then rounded up to the nearest 0.5 mm. Readings were repeated by the same examiner (AI) two hours after the first measurement in order to perform intra observer reproducibility analysis. Intra-class correlation (ICC) coefficient with a two-way mixed effects model was obtained for each periodontal parameter. The ICC ranged from 0.92 to 0.99 in the groups (P < 0.0001), indicating excellent intra observer reliability.
Gingival Crevicular Fluid Collection:
The selected site was air dried and isolated with sterile cotton rolls. The supragingival plaque was removed gently with a sterile curette without touching the gingival margin. The site showing the greatest signs of inflammation and CAL was selected for sampling. The microcapillary pipette‡ was placed at the entrance of the gingival sulcus, and GCF was collected. Sites that did not express any GCF or were contaminated with blood and saliva were excluded from the study. Approximately 10 µL of GCF was collected from the patients with stage III periodontitis and 4–5 µL from patients with healthy periodontium[24]. Collected GCF samples were immediately transferred to airtight plastic vials (Eppendorf tubes) and stored at − 20°C until final assay. GCF was assayed for the laboratory markers SEMA-4D, PAD-2 and MMP-8 at baseline and at three months using commercially available enzyme-linked immunosorbent assay (ELISA Kit)§. Samples were analyzed according to the instruction manual at the Department of Biochemistry, NKP Salve Institute of Medical Sciences, Nagpur, India. Briefly GCF samples were diluted with dilution buffer in the kit and the amount of SEMA-4D, PAD-2, MMP-8 were determined. All samples have been run in duplication.
Non-surgical Periodontal Therapy (Nspt)
All patients underwent NSPT, which included scaling and root planing (SRP) performed by a single operator (RK), along with oral hygiene instructions. On recall visits every month upto 3 months, supragingival plaque was removed, and oral hygiene instructions were reinforced and clinical parameters recorded.
Statistical analysis:
The descriptive statistics for the levels of periodontal parameters were obtained in terms of mean and standard deviation across study groups. The comparison of means was carried out using one-way analysis of variance (ANOVA). Paired t-test was applied to assess the difference from baseline to 3 months, unpaired t-test was applied to assess the difference between two groups. One-way ANOVA test assessed the difference between three groups at baseline and 3 months which was followed by post-hoc Tukey test. All the data analyses were performed using SPSS softwareǁ and the statistical significance will be tested at 5% level.
Footnotes
† PCPUNC 15; Hu Friedy, Chicago, IL, USA.
‡ Labo Glass Scientific Supply, Haryana, India.
§ Kinesis Dx ELISA Kits, Los Angeles, USA
ǁ SPSS ver. 20.0 (IBM corp. USA)