Adjunctive Hyaluronic Acid Gel in Non-Surgical Periodontal Treatment of Patients With Diabetes Mellitus

This study is a randomized controlled split-mouth clinical trial that investigates the clinical, biochemical, and microbiological effects of adjunctive Hyadent BG® gel application into selected interproximal periodontal pocket sites after non-surgical periodontal treatment (NSPT) in patients with diabetes mellitus (DM) and stage III/IV periodontitis.

Periodontitis is a chronic inflammatory disease characterized by the progressive destruction of the tooth-supporting tissues. DM is an important modifying factor for periodontal disease progression and treatment response. Hyperglycemia-related alterations in host immune response, collagen metabolism, microvascular function, and wound healing may contribute to increased periodontal inflammation and delayed periodontal healing. Therefore, adjunctive local approaches that may enhance the response to NSPT are clinically relevant in this patient population.

Hyaluronic acid (HA) is a naturally occurring glycosaminoglycan found in periodontal tissues and the extracellular matrix. Because of its hydrophilic, viscoelastic, anti-inflammatory, and wound-healing properties, HA has been investigated as an adjunctive agent in periodontal therapy. In this study, HA gel will be applied locally into periodontal pockets after scaling and root planing to evaluate whether it provides additional benefit beyond NSPT alone.

The study will include a total of 23 patients with DM and Stage III/IV periodontitis who applied to the Department of Periodontology, Faculty of Dentistry, Marmara University. Eligible participants will receive detailed information regarding the study procedures, and written informed consent will be obtained prior to enrollment.

TREATMENT PROCEDURES

Before treatment, recent HbA1c and fasting plasma glucose (FPG) test results will be obtained. Two days before NSPT, radiographic and full-mouth clinical evaluations will be carried out to determine which jaw will be included in the study. 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 and 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 in two weeks time. At the last session of NSPT, test sites (non-adjacent sites with ≥6 mm pocket depth) for adjunctive use of HA gel will be randomly determined by a toss of a coin from the selected jaw, and HA will be applied subgingivally.

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

Periodontal Clinical Measurements

At baseline and at 1, 3, and 6 months, 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 kept in place for 10 seconds. All GCF and the subgingival microbiological samples will be stored at -80°C until the day of analysis. GCF samples will be analyzed for interleukin-34 (IL-34) and beta C-terminal telopeptide (β-CTX) levels using commercially available enzyme-linked immunosorbent assay kits. IL-34 will be assessed as a marker related to local inflammatory activity, and β-CTX will be assessed as a marker of type I collagen degradation.

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 (IL-34 and β-CTX) 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.

• Change of Bleeding on Probing (%) [Time frame: Baseline, 1, 3 and 6 months after NSPT]
• Change in Plaque Index [Time frame: Baseline, 1, 3 and 6 months after NSPT]
• Change in Gingival Index [Time frame: Baseline, 1, 3 and 6 months after NSPT]
• Change in GCF IL-34 Levels [Time frame: Baseline, 1, 3 and 6 months after NSPT]
• Change in GCF β-CTX Levels [Time frame: Baseline to 1, 3 and 6 months after NSPT]
• Change in level of Porphyromonas gingivalis [Time frame: Baseline, 1, 3 and 6 months after NSPT]
• Change in level of Fusobacterium nucleatum [Time frame: Baseline, 1, 3 and 6 months after NSPT]
• Change in level of Aggregatibacter actinomycetemcomitans [Time frame: Baseline, 1, 3 and 6 months after NSPT]
• Change in level of Tanerella forsythia [Time frame: Baseline, 1, 3 and 6 months after NSPT]
• Change in level of Prevotella intermedia [Time frame: Baseline, 1, 3 and 6 months after NSPT]