The goal of this clinical trial is to evaluate whether hyaluronic acid (HA) improves periodontal healing in people with periodontal disease who smoke. Moreover, the effects of HA on biomolecules and bacteria levels will be assessed during the follow up period.

The main questions it will answer are:

Will periodontal sites treated with HA gel after non-surgical periodontal treatment (NSPT) lead to better outcomes in clinical parameters compared to the sites treated with NSPT only in smokers?

Will adjunctive use of HA gel reduce oxidative stress markers and bacteria levels during follow-up?

Researchers will compare periodontal sites receiving NSPT with adjunctive HA gel application to sites receiving NSPT alone to determine whether HA provides additional clinical, biochemical and microbiological benefits.

Participants will:

• first receive full-mouth NSPT then, randomization will be performed in selected jaw to determine the test sites. These two interproximal test sites will additionally receive HA gel application.
• attend follow-up visits at 1, 3 and 6 months for clinical periodontal measurements and to provide gingival crevicular fluid samples (GCF) and subgingival samples.

The GCF samples will be evaluated for Metallothionein (MT) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels and subgingival samples for periodontal pathogens.

This study is a split-mouth study that investigates the clinical, biochemical and microbiological efficacy of Hyadent BG® gel application in periodontal pockets after non-surgical periodontal treatment (NSPT) in smoking patients.

The study will include 23 smokers with periodontitis who applied to the Department of Periodontology, Faculty of Dentistry, Marmara University. Eligible participants will receive detailed information regarding study procedures, and written informed consent will be obtained prior to enrollment.

Treatment Procedures

Two days before NSPT, radiographic and full mouth clinical evaluations will be carried out to determine which jaw will be included for the study. The intraoral photographs will be taken, and impressions of the related jaw will be taken for stent fabrication.

One day before NSPT, clinical measurements of the related sites with the stent will be repeated. NSPT will be completed in three sessions within two weeks.

Before starting NSPT, baseline pooled gingival crevicular fluid (GCF) samples then pooled subgingival microbiological samples will be collected sequentially from the related sites of the selected jaw. Then oral hygiene instructions will be provided, followed by full-mouth supragingival and subgingival scaling using ultrasonic and hand instruments within two weeks.

At the last session of NSPT, test sites (non-adjacent sites with ≥ 6mm pocket depth) for adjunctive use of HA gel will be randomly determined by coin toss from the selected jaw, and HA will be applied subgingivally.

All patients will be followed up at 1-, 3-, and 6-month intervals.

Periodontal Clinical Measurements

At baseline and at 1-, 3-, and 6-month, full-mouth clinical measurements will be performed.

Periodontal clinical measurements will be recorded at six sites per tooth-mesiobuccal, midbuccal, distobuccal, mesiolingual, midlingual, and distolingual-using a Williams periodontal probe (Hu-Friedy).

Plaque Index (PI): Dental plaque accumulation will be assessed after air drying the teeth using a periodontal probe and visual inspection, according to the PI system developed by Silness and Löe.

Gingival Index (GI): The gingival inflammatory status will be assessed using the GI developed by Löe and Silness.

Probing Depth (PD): PD will be measured as the distance from the free gingival margin to the base of the periodontal pocket by inserting the periodontal probe to the pocket base.

Clinical Attachment Level (CAL): CAL will be recorded by measuring the distance from the cemento-enamel junction to the base of the pocket.

Bleeding on Probing (BOP) (%): BOP will be assessed 25-30 seconds after pocket probing. Sites showing bleeding will be recorded as positive (+), while those without bleeding will be recorded as negative (-).

Collection of GCF and Subgingival Microbiological Samples

At baseline, both GCF and pooled subgingival microbiological samples will be collected after clinical measurements from test and control sites, but at follow-up visits (1st, 3rd, and 6th months) prior to clinical measurements. Before sampling, supragingival plaque will be carefully removed, then the test (n=2) and control (n=2) sites will be isolated from saliva using cotton rolls and dried.

First, GCF samples will be collected. Periopaper strips will be inserted 1-2 mm into the periodontal pocket and left in place for 30 seconds at the selected sites. GCF volume will be measured.

Following GCF sampling, subgingival microbiological sampling will be carried out by inserting sterile paper points (#30) to the base of the periodontal pockets and keeping them in place for 10 seconds.

All GCF samples and the subgingival microbiological samples will be stored at -80°C until analysis day.

GCF samples will be analyzed by using commercially available Enzyme-Linked Immunosorbent Assay kits for metallothionein (MT) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels.

Quantification of A. actinomycetemcomitans, F. nucleatum, P. gingivalis, T. forsythia, and P. intermedia in subgingival microbiological samples will be performed using Real-Time Polymerase Chain Reaction.

Statistical Analysis

Statistical analyses will be performed using IBM SPSS Statistics for Windows, Version 20.0 (IBM Corp., Armonk, NY, USA). A confidence level of 95% will be adopted for all analyses, and a p-value of less than 0.05 will be considered statistically significant.

Due to the split-mouth study design, test and control sites within the same individual will be treated as paired samples.

Descriptive statistics for clinical parameters (PI, GI, BOP, PD, and CAL) and biochemical markers (MT and 8-OHdG) will be expressed as mean ± standard deviation, median, and minimum-maximum values.

The normality of data distribution will be assessed using the Shapiro-Wilk test. For normally distributed variables, within-group comparisons over time (baseline, 1 month, 3 months and 6 months) will be analyzed using one-way repeated measures analysis of variance (ANOVA). When a statistically significant difference is detected, pairwise comparisons will be performed using the Tukey post hoc test.

Comparisons between test and control sites will be conducted using the paired samples t-test.

For variables that do not follow a normal distribution, within-group comparisons over time will be analyzed using the Friedman two-way analysis of variance. When significant differences are observed, pairwise comparisons will be performed using the Wilcoxon signed-rank test.

Between-group comparisons for non-normally distributed variables will be performed using the Mann-Whitney U test.

Correlations between clinical, biochemical, and microbiological variables will be evaluated using Spearman's correlation analysis. Variables found to be significant will be further analyzed using multiple linear regression to assess their independent effects.

Inclusion Criteria:

1. Individuals who sign the informed consent form
2. Participants who are systemically healthy
3. Participants who smoke (for >5 years and ≥10 cigarettes per day)
4. Participants diagnosed with Stage III or Stage IV periodontitis
5. Participants with at least 20 natural teeth present in the mouth (excluding third molars)
6. Participants with two non-adjacent interproximal sites in each quadrant of either jaw, presenting probing depth (PD) and clinical attachment level (CAL) ≥6 mm, and with no caries, restorations, or furcation involvement

Exclusion Criteria:

1. Individuals who do not sign the informed consent form
2. Individuals having any systemic disease
3. Participants who have used antibiotics within the last 3 months
4. Participants who have received any periodontal treatment within 6 months prior to the study
5. Participants who are pregnant or lactating