The manuscript of this laboratory study has been written according to the Preferred Reporting Items for Laboratory Studies in Endodontology (PRILE) 2021 guidelines [22] (Fig. 1).

Fig. 1The alternative text for this image may have been generated using AI.

Flowchart of the study design according to PRILE guidelines

Ethics committee approval

This study was approved by the Bolu Abant İzzet Baysal University Non-Interventional Research Ethics Committee (date: 26/08/2025; approval number: 2025/342).

Sample selection, preparation and placement of intracanal medicaments

Relevant studies were reviewed to guide the sample size calculation, and the highest required number derived from the statistical methods aligned with the primary hypotheses was selected. Using G*Power 3.1.9.2 (Heinrich-Heine-University Düsseldorf, Germany) [23], the standardized effect size was determined to be 0.5125 based on a comparable study [24] with a 95% confidence level (α = 0.05) and a theoretical power of 0.80, the minimum sample size required for each group was calculated to be 11. Carious-free, single-rooted human mandibular premolars with closed apex were collected. All teeth were examined under a stereomicroscope (Olympus SZ61, Tokyo, Japan) to exclude specimens with fractures or anatomical anomalies. After removing periodontal tissue remnants from the root surfaces of 110 eligible teeth using a periodontal curette, the samples were stored in a 0.1% thymol solution at 4 °C until use.

Immature tooth models were prepared following the methodology described in a previous study [25]. The apical 3 mm of each root was resected, and the root length was standardized to 14 ± 1 mm. Therefore, considering that an apical diameter of 1.1 mm is critical for RET, root canals were enlarged using Gates-Glidden drills (#2–#4; Proud, London, United Kingdom) until they reached a diameter of 1.1 ± 0.1 mm [26]. Saline irrigation with SNI was performed between each instrument. Subsequently, each canal was irrigated with 20 mL of 1.5% sodium hypochlorite (NaOCl) for 5 min, followed by 20 mL of saline and dried with paper points in accordance with the AAE and ESE recommendations [1, 4]. To simulate clinical conditions during irrigation, the apical root ends were sealed with modeling wax, and suction aspiration was applied.

The teeth were randomly allocated into two main groups according to the intracanal medicament used. All medicaments were prepared according to the manufacturer’s instructions and delivered into the canals using a Lentulo spiral (VDW, Munich, Germany). Before placing the intracanal medicament, the modeling wax was briefly removed from the root apex to ensure that the canal lumen was completely filled. The medicaments were prepared as follows:

DAP (n = 55): Equal proportions of metronidazole (Flagyl, Sanofi, Istanbul, Turkey) and ciprofloxacin (Cipro, Biofarma, Istanbul, Turkey) were mixed with distilled water to obtain a final concentration of 1–5 mg/mL.

CH (n = 55): Calcium hydroxide powder (Kalsin, Aktu Tic., Izmir, Turkey) was mixed with distilled water at a 1:1 ratio.

Following confirmation of adequate medicament placement, the modeling wax was repositioned, and the access cavities were sealed with a temporary restorative material (Cavitimi, Imicryl Dental, Turkey). All specimens were stored at 37 °C and 95% relative humidity for three weeks to simulate clinical RET procedures.

Specimen allocation and randomization

All procedures were performed under standardized conditions by two operators (M.I.A. and A.Y.). After temporary sealing of the access cavities, specimens in the CH and DAP groups were randomly assigned to the irrigation activation subgroups using a simple randomization protocol. The allocation sequence was generated using a random number generator (Microsoft Excel, RAND function, Version 16.0) by an independent endodontist who was not involved in the experimental procedures or outcome assessment. Each specimen was assigned a unique numerical code, and all subsequent procedures were conducted using coded samples. Outcome assessment was performed on coded stereomicroscopic images, and the operators remained blinded to group allocation during image analysis.

Removal of intracanal medicaments

After a 3-week incubation period, all teeth treated with DAP or CH were randomly assigned to five subgroups (n = 11) according to the irrigation activation method. Each specimen was irrigated with 20 mL of 17% EDTA, followed by saline, as recommended by the ESE guidelines [1]. EDTA enhances the release of dentin-derived growth factors that promote stem cell differentiation, whereas a final saline rinse is advised to minimize the potential adverse effects of irrigants on target cells [1]. To simulate the clinical irrigation dynamics, the apices were sealed with modeling wax throughout the irrigation and retained until sectioning. Activation protocols were performed strictly according to the manufacturer’s instructions, as described below.

SNI

A 30-gauge side-vented closed-end needle (Endo-Top; Cerkamed, Stalowa Wola, Poland) was positioned 1 mm short of the working length and moved vertically with gentle movements. This needle design was selected to minimize the risk of irrigant extrusion in the simulated immature open-apex model, in accordance with current RET clinical recommendations that advocate irrigation systems designed to reduce extrusion (e.g., a closed-end, side-vented needle) and positioning the needle approximately 1 mm short of the root end [4]. This approach is also consistent with the ESE position statement, which recommends the use of a side-vented needle during irrigation in RET procedures [1]. Each 5 mL of EDTA and saline was delivered for 20 s.

PIPS F

The Fotona Light Walker Er:YAG laser (LightWalker AT; Fotona, Ljubljana, Slovenia) was set in Super Short Pulse (SSP) mode with water and air off, according to the manufacturer’s instructions, at 20 mJ, 15 Hz, 0.3 W. A PIPS (Fotona, Ljubljana) flat fiber tip (400/9 mm) (Fig. 2a) was placed in the reservoir area. Four activation cycles of 20 s each were performed using the solutions in the order and volume specified in the SNI. Each 5 mL of solution was activated for 20 s.

Fig. 2The alternative text for this image may have been generated using AI.

Laser fiber tips with different geometric designs used in the study (a PIPS F, b PIPS R, c SWEEPS F and d SWEEPS R)

PIPS R

Using identical laser parameters, the PIPS radial fiber tip (600/9 mm) (Fig. 2b) was placed in the reservoir, and activation was performed in the same sequence as that for PIPS-F.

SWEEPS F

The SWEEPS protocol was applied using the LightWalker Er:YAG laser (Fotona) in automatic SWEEPS mode (20 mJ, 15 Hz, 0.3 W; water/air off). A flat SWEEPS fiber tip (300/9 mm) (Fig. 2c) was coronally placed, and four cycles of 20-s activation were performed for each solution.

SWEEPS R

Using the same laser settings, the SWEEPS radial fiber tip (600/9 mm) (Fig. 2d) was applied, and activation was completed according to the protocol described for SWEEPS-F.

Evaluation of remaining intracanal medicaments

Following irrigation activation the modeling wax was removed, and each tooth was longitudinally sectioned using a diamond disc without invading the canal space of the tooth. Once an adequate groove was created, an enamel chisel was placed into the notch, and gentle pressure was applied to separate the two halves [27]. Each specimen was examined under a stereomicroscope (Olympus SZ61) at 20 × magnification, and images were captured using a digital camera (Olympus DP12; Olympus, Tokyo, Japan). Images were analyzed using ImageJ software (ImageJ 1.47 V; National Institutes of Health, USA). The outline of the root canal lumen was first delineated and designated as %100. The area containing residual intracanal medicament was measured, and the values were converted into percentage of remnants. This in vitro study employed a factorial design including two intracanal medicaments and five irrigation activation methods. The primary outcome was the percentage area of residual medicament quantified by image analysis. To improve interpretability and statistical sensitivity, analyses were performed using percentage data. The obtained percentage data were statistically analyzed. The reliability of repeated measurements was assessed using the intraclass correlation coefficient (ICC) based on the percentage values obtained from ImageJ analysis. The ICC value was 0.984, indicating excellent agreement between the measurements.

Statistical analysis

The data were analyzed using the R program (v4.5.0; R Foundation for Statistical Computing, Vienna, Austria). The conformity of the data to a normal distribution was examined with the Shapiro–Wilk test. For the comparison of values that did not conform to the distribution according to group and activation method factors, ART Anova was used with the ARTool package, and multiple comparisons were made using Holm correction. The results of the analysis were presented as median (minimum: maximum) for quantitative data. The significance level was set at p < 0.05.