This prospective clinical study was reported in accordance with the Consolidated Standards Statement on Research Reporting Standards (CONSORT, 2010) and was carried out with the ethical rules of the Declaration of Helsinki. The study was approved by the Ethical Committee of the Kirikkale University and was conducted with a single-blind, randomised design. The required minimum sample size was determined using G*Power v.3.1.9.2 (University of Dusseldorf, Germany) with a 5% type-1 alpha error, 0.40 effect size, and 90% power in accordance with Mehrvarzfar et al. [13]. The minimum sample size required for each study group was 12. Considering the estimated 20% loss resulting from inaccurate or absent data, the final total sample size was established at 120 participants, with 15 participants per group.

Patient enrollment

Adult patients (aged 18–60) who were referred to the Dental Faculty of Kirikkale University for root canal treatment were included in this study. One hundred and twenty single-rooted anterior and premolar teeth that underwent endodontic treatment from July 2023 to November 2023 were selected.

Inclusion criteria

The inclusion criteria were good oral hygiene and not reporting pain before the appointment. Pulpal diagnosis was applied during clinical examination and confirmed upon accessing the teeth according to the AAE consensus. This was an AIP caused by extremely deep carious lesions, which are radiographically detectable and penetrate the entire thickness of the dentin. Pulp exposure would only occur during the removal of the caries, as there is no clinical or radiographic pathology or symptoms present (vital cases). Pulpal diagnosis was performed via clinical examination and radiographs for necrotic pulp with CAP (nonvital cases).

Exclusion criteria

The exclusion criteria were pregnancy, autoimmune diseases, uncontrolled diabetes, smoking, moderate and advanced periodontal disease (with 5 mm or greater probing depth), patients requiring endodontic treatment for more than one tooth, unrestorable coronal destruction, incomplete root formation, systemic or allergic sensitivity to local anaesthetics or NSAIDs, patients with ASA II and analgesic or antibiotic intake within 7 days before the beginning of treatment.

Informed consent

All participants received information about the aim of the study and the treatment protocol. Informed consent was obtained from patients who wanted to participate in the study.

Diagnosis and data record

The vitality was diagnostically examined using both electric pulp testing and thermal tests with the help of a secondary investigator who was not involved in the study. The same investigator recorded gender, age, preoperative pain and periapical index (PAI) scores and contact information.

Randomisation, blinding, and allocation

The participants were blinded and unaware of the specific treatment protocol. They were then randomly assigned to the four groups at a 1:1 ratio using a computer algorithm program (http://randomizer.org). After randomisation, patients were assigned consecutive numbers from 1 to 4 in the order of enrolment. The root canal sealers were coded (1: GuttaFlow Bioseal, 2: MTA-Bioseal, 3: NeoSealer Flo, 4: AH Plus). Just before the obturation protocol, a secondary investigator notified the clinician about the assigned root canal sealer. The tooth was obturated with this root canal sealer. The main components of the sealers are listed in Table 1.

Table 1 Sealer formulation, lot number and composition in accordance with the manufacturerTreatment protocols

This study was designed with two different pulp status: vital and nonvital pulp. All the teeth that met the inclusion criteria were treated in a single visit by a specialist (S.D.B.) with over five years of clinical experience in the Department of Endodontics. The treatment of the patients who were excluded from the study was conducted by a clinician who was not involved in the research.

In AIP patients, the presence or absence of bleeding in the pulp chamber served as verification. If the bleeding in the pulp could not be stopped by applying pressure to the pulp exposed with a sterile cotton pellet for 5 min, RCT was performed. However, if bleeding could be prevented, vital pulp therapy was applied, and patients were excluded from the study. In necrotic pulp with CAP patients, PAI was used as previously described [14, 15]. According to this index, periapical lesions with scores of 3 and 4 (less than 1 cm in diameter) were included in the study to achieve better standardisation. In patients with CAP, the change in bone structure with a noticeable radiolucent area was defined as a score of 3, and a well-defined radiolucent area was defined as a score of 4. The periapical lesion diameter was measured on digital radiographs using a software tool. When there was doubt about the score, a higher score was given. Only one tooth was treated in each patient. Following rubber dam placement and disinfection of the crown, a sterile bur was used to create an access cavity under an operating microscope (OPMI Pico; Carl Zeiss, Gottingen, Germany).

A local anaesthetic solution (containing 40 mg of articaine hydrochloride and 0.006 mg of epinephrine) was administered to all patients. Mandibular anaesthesia was used for the mandibular premolars. Buccal infiltration anaesthesia was used for other teeth. Supplementary anaesthesia was not required for any of the patients. The working length was established using a 15 K-file (Dentsply Maillefer, Ballaigues, Switzerland) and an apex locator (Root ZX Mini, Morita Corp., Kyoto, Japan), which was set 0.5 mm short of the 0.0 signal of the device. Initial instrumentation was performed up to a size of 25 K files, and then a Reciproc R25 (size 25, 0.08 taper) file (VDW, Munich, Germany) was used. If needed, the root canal was enlarged with Reciproc R40 (size 40, 0.06 taper) file (VDW). A total of 10 mL of 5% sodium hypochlorite was used to rinse the root canal during instrumentation with a 30-gauge side-vented needle. The needle tip was inserted 1.5 mm shorter than the working length, and moderate pressure was applied to avoid irrigant extrusion through the apex. The final irrigation mixture included 3 mL of 17% EDTA (Werax, Izmir, Turkiye), 2 mL of distilled water, 3 mL of 5% NaOCl (Werax), and 2 mL of distilled water respectively. Ultrasonic activation (Mini Piezon, EMS, Nyon, Switzerland) was performed by inserting it 2 mm shorter than the working length, and it was activated in 3 cycles of 20 s for both the NaOCl and EDTA solutions during the final irrigation stage. The root canals were subsequently dried with paper points (Sure-Endo, Seoul, Korea).

During obturation, a single tapered gutta-percha cone was adapted to the root canal, and its position was confirmed by periapical radiography. The gutta-percha cone used was either the Reciproc R25 (VDW) or Reciproc R40 (VDW). All the root canal sealers were applied using the same technique. The technique outlined by Castelo-Baz et al. [16] was subsequently employed to apply sealer with appropriate paper point cones (1 mm shorter than the working length) in the following manner: First, the sealer was administered to the root canal with the initial paper point and then uniformly distributed with a second paper point before any excess sealer with a third paper point was removed. Once the sealer was applied, Reciproc R25 or Reciproc R40 was adjusted to fit the root canal. The excess gutta-percha cone was subsequently removed, and the pulp chamber was cleaned. The coronal access cavities were restored with a composite resin material (Charisma Smart; Kulzer GmbH, Hanau, Germany).

Postobturation pain assessment

Pain was assessed using a visual analogue scale (VAS) card at 6 h, 24 h, 48 h, 72 h, 7 d and 30 d postoperatively. All the participants were asked to rate their perceived pain on the scale, with 0 indicating no pain and 10 indicating the highest possible pain. Pain levels were classified as none (0), mild (1 to 3), moderate (4 to 7), or severe (8 to 10). Patients were informed that they might experience mild postobturation pain and were instructed to take 400 mg of ibuprofen only when they experienced severe pain, as previously suggested [17]. The patients recorded the number of pain relief tablets taken on the back of the VAS score card, and the patients were advised to contact their healthcare providers if they needed to make additional appointments for pain management. Patients were recalled for clinical observation at 7 d, and VAS scores were recorded at the same time. Patients submitted their last VAS score for 30 d via their phones, and data were recorded.

Statistical analysis

Pain scores were statistically analysed with IBM SPSS Statistics version 22.0 (IBM, Armonk, NY, USA). The Mann‒Whitney U and Kruskal‒Wallis H tests, along with post hoc multiple comparison tests, were used to investigate the differences between groups. The Friedman test was used to compare changes in pain scores over time. Spearman’s correlation test was also used. A p value < 0.05 was considered significant.