This clinical study aims to investigate the oxidative stress-related effects of ozone irrigation in patients with asymptomatic apical periodontitis undergoing root canal treatment. A total of 60 patients who meet the inclusion criteria will be randomly assigned into two groups using an online randomization tool: an ozone irrigation group and a saline irrigation (control) group. The primary objective is to assess the biochemical effects of ozone therapy on oxidative stress markers in periapical interstitial fluid, including but not limited to TAS, TOS, MDA, and 8-OHdG, by using ELISA kits.

After routine canal preparation and initial irrigation protocol (NaOCl-EDTA-NaOCl), baseline periapical fluid samples will be collected using standardized sterile paper points. Following this, experimental irrigation will be applied: ozone water in the study group and saline solution in the control group. Second periapical fluid samples will be collected immediately after this procedure. Subsequently, calcium hydroxide dressing will be placed into the canals, and the patients will be recalled after one week. At the second visit, after removal of the intracanal medicament, a third sample will be collected from each patient.

Ethical approval was obtained from the Non-Interventional Clinical Research Ethics Committee of Atatürk University Faculty of Medicine. The results of this study are expected to contribute to the understanding of ozone therapy as a biocompatible and effective disinfection agent in endodontic procedures and to provide insights into its role in modulating oxidative stress within the periapical environment.

This randomized controlled clinical trial investigates the effect of ozone irrigation on oxidative stress levels in teeth diagnosed with asymptomatic apical periodontitis. Apical periodontitis, characterized by inflammation of periapical tissues in response to persistent root canal infection, often progresses silently and can affect long-term endodontic success. In recent years, ozone therapy has gained attention due to its strong antimicrobial, anti-inflammatory, and tissue-regenerative properties. This study evaluates whether ozone irrigation during endodontic treatment can modulate oxidative stress biomarkers more effectively than traditional saline irrigation.

Study Objective

The primary aim is to compare oxidative stress marker levels (TAS, TOS, MDA, and 8-OHdG) in periapical fluid samples following ozone versus saline irrigation

. Study Design and Participants

A total of 60 patients aged between 18-65, each diagnosed with a single-rooted tooth exhibiting asymptomatic apical periodontitis, were included based on strict inclusion/exclusion criteria. Patients with systemic disease, recent antibiotic use, or periodontal involvement were excluded. Randomization was conducted via [www.randomizer.org\ [blocked]](http://www.randomizer.org) to allocate participants into two groups (n=30 each):

Group A: Ozone irrigation group Group B: Saline (control) irrigation group

Each patient provided written informed consent, and the study received ethical approval from the Atatürk University Non-Interventional Clinical Research Ethics Committee (Decision No: B.30.2.ATA.0.01.00/557, Date: 27.09.2024).

Endodontic Procedure and Sampling Phases

After local anesthesia (2% articaine with 1:100,000 epinephrine) and rubber dam isolation, standardized root canal preparation was performed using rotary NiTi systems. During shaping, 2.5% NaOCl was used for initial irrigation. Final irrigation protocol common to both groups involved:

• 5 mL of 2.5% NaOCl
• 5 mL of 17% EDTA
• 5 mL of 2.5% NaOCl again

All irrigations were performed using 30-gauge side-vented needles.

Periapical Fluid Sampling Points

Sampling was done using sterile paper points inserted 2 mm beyond the apex, each held for 1 minute, and repeated with 3 paper points per canal. Each sample was stored in PBS-filled, coded Eppendorf tubes at -80°C.

Sample 1 (Baseline):

Collected immediately after final irrigation.

Sample 2 (After group-specific irrigation):

Group A received 5 mL of ozone water irrigation. Group B received 5 mL of sterile saline irrigation.

Samples were collected again using the same protocol.

Sample 3 (Post-medication):

Calcium hydroxide was placed as intracanal medicament. Patients were recalled after 7 days, Ca(OH)₂ was removed, and a third sample was obtained.

Laboratory Analysis

All samples were stored at -80°C until biochemical analysis. The oxidative stress markers were quantified using commercial ELISA kits according to manufacturer protocols.

Outcome Measures

Change in oxidative stress levels (TAS, TOS, MDA, 8-OHdG) at three time points.

Significance of the Study

This is the first known randomized clinical trial to assess the influence of ozone irrigation on oxidative stress biomarkers in periapical fluid. Given the potential of oxidative stress to affect tissue healing and inflammation, understanding the biochemical impact of ozone could offer novel insight into improving endodontic outcomes, especially in cases where apical healing is critical.

Inclusion Criteria:

• 1. Patients must be between 18 and 65 years of age 2. Single-rooted, asymptomatic apical periodontitis with necrotic teeth 3. Teeth with periapical lesion size classified as score 3 or 4 according to the Orstavik classification 4. Periapical Assessment Index (PAI) of 2 or higher 5. Patients with ASA I status

Exclusion Criteria

1. Teeth with multiple roots
2. Patients with ASA II or higher
3. Pregnant women or those suspected of being pregnant
4. Patients with a history of allergies
5. Teeth with excessive material loss where a rubber dam cannot be applied
6. Teeth with root fractures or an open apex
7. Teeth that have previously undergone root canal treatment
8. Presence of canal curvature greater than Schilder 25°,
9. Patients with generalized periodontitis,
10. Presence of a periodontal pocket greater than 3mm in the relevant tooth

This study uses a single-blind design in which the participants are unaware of the intervention type (ozone or saline irrigation) they receive. The operator performing the root canal therapy is aware of the group assignment due to the nature of the intervention procedure. Outcome assessments are performed by the same clinician and are thus not blinded.