The investigation received approval from the University Scientific Studies Review and Ethics Committee under decision number 2019/259. Participants were enrolled upon providing both written and verbal consent.
2.1. Subject Inclusion
The sample size determination employed a two-tailed hypothesis test, with a significance level of 5% and a power of 85%, maintaining an allocation ratio of 1:1. Based on these parameters, the estimated minimum sample size required was 17 patients per treatment group. In our study, we conducted research involving 78 teeth from 60 patients. Thorough anamnesis was conducted with all participants. Our inclusion criteria encompassed lower premolars, upper central and lateral incisors with a single root and canal, along with apical radiolucent lesions. Exclusion criteria comprised teeth exhibiting periodontal pockets exceeding 4 mm, mobility, nonrestorability, immaturity, or any indications of fracture or dental anomalies. Additionally, patients who declined participation, those with systemic illnesses, and chronic drug users were not included.
Vitality tests yielded negative results for all teeth. The treatments were administered consistently by the same operator to ensure uniformity. However, due to irregular attendance at follow-up appointments by six patients and one instance of tooth extraction, the study ultimately involved 70 teeth, as elaborated in (Table 1).
Table 1
Data of teeth included in the study
| Control | Methylene Blue | Curcumin | Erythrosine B |
Female/male | 10/7 | 9/9 | 9/8 | 9/9 |
Age (mean±standard deviation) | 32.82 ± 13.92 | 37.66 ± 15.32 | 31.64 ± 12.78 | 35.77 ± 13.43 |
Anterior/premolar | 9/8 | 9/9 | 9/8 | 9/9 |
Re-treatment | 2 | 3 | 3 | 3 |
2.2. Study Design
Participants were randomized into four groups. Periapical radiographs of the teeth were captured using a dental X-ray unit (Satalect X-Mind, France) set at 250 kV, 4 mA, and 0.12 seconds of irradiation time, employing a parallel technique guided by a cone indicator. For teeth with indistinct lesion sizes on periapical radiographs, three-dimensional images were acquired using cone beam computed tomography (CBCT) (Planmeca, Helsinki, Finland) with settings of 90 kVp, 12 mA, 14 seconds, and a 16x5 imaging field.
Following rubber dam isolation, access cavities were prepared using a diamond bur. The working length was determined using a 15 K-file (Dentsply Maillefer, Ballaigues, Switzerland) coupled with an apex locator (Root ZX, Morita, Japan), ensuring a 0.5 mm short of the "00" point. Radiographic confirmation of the working length was obtained.
Canal instrumentation up to a 20 K-file was carried out, followed by further preparation using the R25 Reciproc instrument (VDW, Munich, Germany) operated by a VDW Silver endodontic motor (VDW) at the 'Reciproc All' setting. After three pecking motions, irrigation with 5% Sodium hypochlorite (NaOCl) (Werax, İzmir, Türkiye) was conducted until the full canal length was reached. Mechanical instrumentation is completed with the R40 instrument.
Throughout the procedure, each canal received irrigation with 10 mL of 5% NaOCl at a flow rate of 0.1 ml/sec via a 30-G irrigation needle (Endo-Top Wola, Poland), positioned 1 mm short of the working length. Subsequently, canals were rinsed with distilled water and then irrigated with 5 mL of 17% EDTA (Kemiger, Ankara, Turkey) for 1 minute, allowing an additional 1-minute dwell time. Ultrasonic activation involved using 2 mL of NaOCl in total, administered three times for 20 seconds each, employing a Niti U-File #25 attached to the E2 tip of an ultrasonic device (Woodpecker, China) in endodontic mode, positioned 1 mm short of the working length. After each irrigation step, canals were rinsed with distilled water.
Following this stage, participants were allocated into four groups for the final irrigation protocol (Table 2).
Group 1: A curcumin solution was prepared by dissolving curcumin (Sigma-Aldrich, Steinheim, Germany) in 0.5% dimethyl sulfoxide (DMSO) (Aromel Medikal, Konya, Türkiye) to achieve a final concentration of 5.0 mg/mL. The photosensitizer was delivered into the canal using an irrigation needle until the pulp chamber was filled. After a 3-minute wait, ultrasonic activation was performed for 20 seconds. The solution was then refreshed, and the activation process was repeated two more times for a total of 60 seconds. The last refreshed photosensitizer was irradiated with a 200mW diode laser (SiroLaser Blue, Dentsply, Germany) at a wavelength of 450 nm, employing spiral movements with an EasyTip 200 µm Endo fiber optic tip for 20 seconds (Fig. 1). This process was repeated two more times. Finally, the photosensitizer was rinsed with distilled water.
Group 2: Erythrosine B (Sigma Aldrich, St. Louis, MO, USA) was diluted with phosphate-buffered saline solution to achieve a final concentration of 0.3 mg/mL. Ultrasonic activation was performed similarly to Group 1, but laser application utilized a wavelength of 660 nm and a power of 100 mW.
Group 3: Methylene blue (Sigma-Aldrich, Germany) at a concentration of 0.1 mg/mL was introduced into the canal as in the previous groups and activated by both ultrasonic and laser methods [13]. The parameters for this group were also set to a wavelength of 660 nm and a power of 100 mW.
Group 4: Canals were irrigated with 10 mL of 5% NaOCl at a flow rate of 0.1 mL/sec.
Table 2
photosensitizer concentrations and diode laser parameters
| Curcumin | Erythrosine B | Methylene Blue | Control |
Concentration | 5 mg/mL with DMSO | 0.3 mg/mL with PBS | 0.1mg/mL | 10ml %5 NaOCl |
Wavelength | 445 nm | 660 nm | 660 nm | - |
Power | 200 mW | 100 mW | 100 mW | - |
Irradiation time | 60 sec | 60 sec | 60 sec | - |
All canals were subsequently rinsed with distilled water. The final irrigation involved the use of 2 mL of 1% EDTA for 1 minute. A 40/04 master gutta-percha cone was placed and confirmed radiographically. Canals were dried using sterile paper points. The obturation of canals with the 40/04 gutta-percha cones was carried out using Endoplus (President/München/ Germany) and the lateral condensation technique. Following the removal of excess gutta-percha, access cavities were cleaned with alcohol and restored incrementally with resin-modified glass ionomer cement (Riva Light Cure HV, Australia) and composite resin (G-Premio Bond, Tokyo, Japan).
Participants were scheduled for follow-up appointments at 6, 12, and 24 hours, as well as at 1 week, during which they were asked to report any pain experienced. Pain levels were assessed using the Visual Analogue Scale (VAS), ranging from 0 (no pain) to 10 (most severe pain), and recorded accordingly. Subsequent follow-up appointments were scheduled at 1, 3, 6, and 9 months, and a final 1-year follow-up, during which radiographs were taken to monitor the healing of the lesions (Figs. 2, 3, 4, and 5).
2.3. Evaluation of lesions
The area of lesions in periapical radiographs, captured using the parallel technique, was calculated in mm² utilizing Image J 1.28 software (Fig. 6) (National Institute of Health, USA). Two blinded and experienced endodontists underwent calibration on 150 radiographs. The endodontists, assessing the radiographs in a subdued and quiet environment, engaged in discussions to resolve any discrepancies until reaching a consensus.
Statistical analysis was conducted using SPSS for Windows version 24.0 (IBM, New York, USA). The normality of the data distribution was assessed using the Shapiro-Wilk test, revealing a normal distribution. Consequently, the results were subjected to analysis using One-Way ANOVA followed by post-hoc Tukey HSD tests. Statistical significance was considered at p < 0.05.