Profiling Gingival Crevicular Fluid β-Catenin Level With 8-OHdG and Total Antioxidants Capacity During Healing of Periodontal Pockets: A 3-month Clinical Trial

Background: To profile gingival crevicular fluid (GCF) β-Catenin, 8-OHdG, and total antioxidants capacity (TAC) in periodontitis patients during healing of periodontal pockets following non-surgical periodontal therapy (NSPT).Methods: Periodontitis patients (n = 21) will included in this clinical trial. Clinical periodontal parameters will recorded and GCF samples will collected from randomly selected 4-6 mm periodontal pockets at baseline (T0) as well as 4 weeks (T1) and 12 weeks (T2) after NSPT. GCF levels of β-catenin, 8-OHdG, and TAC will be assayed by enzyme linked immunosorbent assay (ELISA).

Keywords: periodontal diseases; periodontal debridement; dental polishing; periodontal pocket

Why is this important?

This research explained the effects of non surgical periodontal treatment for patients with periodontal disease on the epithelial tissue regeneration during the healing process.

Introduction Periodontitis is a destructive disease of tooth-supporting tissues which initiated in response to frank dysbiosis of dental biofilm complemented by aggravated immune response. The hallmark of periodontitis is pathologic deepening of gingival sulci which are transformed into periodontal pockets when the front of dental biofilm advances apically. In such situation, immune responses are initiated within gingival tissue in order to counterattack the pathobionts and its virulent products . Polymorph nuclear cells, mainly neutrophils, are among the principle immune system components responsible for this task via series of biological activities such as phagocytosis and netosis. Prolonged inflammation of periodontal lesions and continuous heavy recruitment of these inflammatory cells leads to the release of oxygen free radicals (ROS) in an escalating pace which overwhelm antioxidant capacity of the host . Overproduction of ROS trigger series of destructive events characterized by increased metabolites of lipid peroxidation, DNA damage and protein damage that led to tissues damage . This shift of oxidative-antioxidant balance towards oxidative stress leads to dramatic changes in the levels of oxidative stress-related molecules such as total antioxidant capacity (TAC) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) . These biomarkers can be clinically detected in oral biofluids such as saliva and GCF with a potential to discriminate between periodontal health and disease .

In healthy periodontium, the gingival sulcus is lined by sulcular epithelium which is continued with the junctional epithelium at sulcus base. The epithelial cells are adherent to each other by range of junctional attachment including desmosomes, adherens junctions, and tight junctions. The integrity of this coherent epithelial cells provides a physical barrier against the ingress of pathobionts to deeper tissues or systemic circulation. Adherens junctions is the predominant cellular junctions which structurally composed of E-cadherin that grouped in extracellular, transmembrane, and intracellular domains. β-catenin forms a complex with E-cadherin, mediating the attachment of intracellular domain of adherens junctions to the cytoskeleton . Persistent inflammation associated with excessive oxidative stress culminating in the tissue compromises epithelial barrier function . Damage of E-cadherin-β-catenin complex leads to the release of β-catenin and its subsequent nuclear translocation; thereby, activating Wnt signaling pathway which is responsible for compromising epithelial phenotype . This notion is supported by detecting elevated level of β-catenin in GCF samples collected from periodontitis patients associated with clinical attachment loss (CAL) in comparison to healthy controls .

The goal of periodontal therapy is to preserve and maintain natural dentition as well as improve oral and systemic health. Non-surgical periodontal therapy (NSPT) considered as gold standard decision to remove biofilm and reduce pathobionts load to restore symbiotic state of oral microbiome and tissue homeostasis . Demand of Successful periodontal therapy depends on improvement in clinical parameters in the term of bleeding on probing (BOP), probing pocket depth (PPD) and CAL . Therefore, this study aimed to track changes in the level GCF of β-catenin ,8-OHdG, and TAC and investigate the association between these biomarkers during the healing period.

Materials and methods Study design This study is a clinical trial involving periodontitis patients who will followed for up to 3 months. The study will conducted from June 2026 to September 2026. The participants will be selected from the pool of patients attending the clinics in the College of Dentistry, University of Babylon. The volunteered patients should sign consent form, which will be collected by the examiner.

Inclusion and exclusion criteria The patients should be ≥ 18 years, have no history of systemic disease, have unstable periodontitis at any stage and grade with at least one moderately deep periodontal pocket (4 - 6 mm) at a single-rooted tooth. The patients were excluded when they were smokers, pregnant or lactating, taking medications which might influence the healing such as corticosteroids and calcium channel blockers, having previous periodontal therapy within the last 3 months, or using antibiotics within 3 months prior to enrolment.

Sample size The required sample size for this study was estimated after considering a reduction in probing pocket depth (PPD) from 7.64 ±1.76 mm to 5.68 ±2.3 mm in shallow pockets [12]. By using G*Power software 3.1, it was estimated that a sample of 17 patients was enough for rejecting the null hypothesis at a power of 0.8 and probability margin of error of 0.05. An extra four patients were invited to participate in this study to avoid any dropout during the follow-up.

Clinical examination and procedure At baseline visit (T0), the selected patients will diagnosed with unstable periodontitis which will defined by the presence of interdental clinical attachment loss (CAL) at ≥ 2 non-adjacent teeth and PPD > 5 mm or 4 mm pockets with bleeding on probing (BOP). In absence of interdental CAL, patients having buccal or oral CAL ≥ 3 mm with PPD > 3 mm detectable at ≥ 2 teeth were also diagnosed as periodontitis [13]. After diagnosis, plaque Index (PI) will used to record presence/absence of dental plaque on 4 sites/tooth with the aid of disclosing agent (EMS®, Switzerland) [14]. Then, BOP, PPD and CAL will recorded at 6 sites/tooth using UNC-15 periodontal probe (PHOENIX®, UK) by a calibrated examiner.

GCF samples will collected from randomly selected sites with periodontal pockets of 4 -6 mm after recording the clinical parameters. Each site will dried using cotton and air blowing by triple syringe. A paper strip (Oraflow Inc.®, New York, USA) will inserted into the selected pocket until feeling slight resistance. After 30 seconds, this pre-weighted paper strip will removed and immediately immersed in an Eppendorf tube (CNWTC/OEM, Jiangsu, China) containing 200 µl phosphate buffer saline (PBS) [15]. Then, the tube containing the strip and PBS will weighted using an electronic balance (JOANLAB®, China). The GCF weight will estimated by subtracting the weight of the tube containing the strip and PBS after sampling from the weights of the dry strip and the tube with PBS before sampling the GCF. Then, the tubes containing GCF samples will saved at -20 °C until further analysis.

After GCF samples collection, patients will receive professional mechanical plaque removal (PMPR) using ultrasonic scaler by the same examiner. Finally, oral hygiene instructions will given to all patients including tooth brushing twice a day and interdental cleaning with an appropriate tool such as interdental brush and dental floss. One week later, root surface debridement (RSD) will performed for all sites with pockets with Gracey curettes (Sakura, YDM®, Japan). Then, all teeth will polished by rubber cups and prophylaxis paste. After 4 weeks (T1) and 12 weeks (T2), all clinical parameters will recorded and GCF samples will recollected as describe above by the same examiner.

Enzyme-linked immunosorbent assay The frozen GCF samples will thawed at room temperature and centrifuged at 4000 RPM for 5 min (Rotofix® 32A, Germany). The optical density will measured at 450 nm and the operator who performed the procedures will blinded to the tested groups. Concentration of 8-OHdG was quantified competitive inhibition enzyme immunoassay technique (USCN®, USA). Sensitivity values was 26.81 pg/ml, with a detection range of 74.07-6.000 pg./ml. ELISA-based sandwich technique will used for quantitative measurement of β-catenin kit (USCN®, USA) with sensitivity values was 5.9 pg./ml and detection range of15.6-1.000 pg/ml. Colorimetric assay facilitated by an ELISA TAC kit (Elabscience®, USA) sensitivity value was 0.62 U/ml with detection range 0.62-190.43 U/ml.

Statistical analysis Data will analyzed using Prism 9 for macOS (Version 9.1.1) and IBM SPSS Statistics (Version 27). Data will presented as frequency, percentage or means (SD). The normality of continuous data will tested using Shapiro-Wilk test. Comparisons among means of variables at different time points will carried out using Friedman test. Dunn's test was used for Post hoc comparison between means of variables at two time points with consideration of adjusting considered p value. Pearson test and linear regression will used to assess the association and possible interactions between the study variables. The significance level will set at p value < 0.05.