Materials and methods

Study design and ethics declarations

This study employed a randomized, controlled clinical trial design, The study is approved by the Ethical Committee of Faculty of Dentistry Ain Shams University (FDASU) with Approval code: FDASU Rec IR052432. Also, the trial is registered at ClinicalTrials.gov with number NCT07138365 with initial release date 22/8/2025.

Sample size calculation

The sample size calculation was performed using data from a previous study [12]. with G*Power version 4.5.0, by adopting an alpha (α) level of (0.05), a beta (β) level of (0.2) (power = 80%) and an effect size (d) of (0.697). Based on this analysis, the predicted sample size was determined to be a total of 40 cases, with 10 participants assigned to each group. Noted that, the calculated sample size is a minimum estimation. If it is found to be minor, it can be safely increased.

Patients’ selection

Patients were selected from the normal group of people visiting the Dental Teaching Hospital at Ain Shams University. Participants required to be between the ages of 18 and 45, diagnosed with irreversible pulpitis and apical periodontitis, free of any underlying medical disorders, and free of analgesics for the week before the session.

According to radiographic analysis, eligible individuals had to have a mandibular single-rooted premolar identified. Furthermore, individuals reported > 7 VAS for both preoperative and percussion pain. Additionally, patients should have exhibited exaggerated prolonged lingering pain responses to cold application that was sustained for few minutes after removal of stimulus (Endo-Frost, Coltene-Whaledent, Switzerland).

Diagnosis was confirmed during access cavity preparation, evidenced by profuse pulpal bleeding. Exclusion criteria comprised pregnancy, anatomical variations in root canal morphology, and refusal to participate. Prior to enrollment, all patients were thoroughly informed about the study procedures and treatment protocol, and provided written informed consent.

Patients’ randomization, allocation, and preparation

Eligible patients were randomly assigned to one of three comparative groups using computer-generated randomization. The allocation sequence was concealed by an independent assistant, who prepared sealed black envelopes containing group assignments. Prior to treatment initiation, each patient selected an envelope at random from a box, thereby determining their group allocation. The study employed a double-blind design, ensuring that both the participants and the outcome assessor remained unaware of the assigned interventions throughout the trial.

All patients received an inferior alveolar nerve block using Artinibsa (Inibsa Dental 4% with 1:100,000 epinephrine). In cases requiring supplemental anesthesia, intra-pulpal injection was administered with a pressure syringe. Access cavities were prepared under copious irrigation, and teeth were isolated using a rubber dam. Upon pulp exposure, profuse bleeding within the chamber confirmed the diagnosis.

The initial glide path was established with a #10 K-file (Mani, Inc., Japan), followed by coronal flaring using an M-Pro orifice opener rotary file (19 mm length, 8% taper; Foshan Stardent Equipment Co. Ltd., Guangdong, China). Working length was determined to be 0.5 mm short of the full canal length using an electronic apex locator (Root ZX; J. Morita Corp., Tokyo, Japan), and verified with intraoral periapical radiography.

The initial baseline apical fluid (AF) sample (S1) was collected at this stage. A sterile size 20 paper point with a 2% taper (DiaDent Group, Seoul, Korea) was inserted 2 mm beyond the root apex and left in place for 30 s. It was then transferred to Eppendorf tubes containing 0.5 mL of phosphate-buffered saline (pH 7.4), which were subsequently stored at 10 °C [6]. Mechanical canal preparation was performed using the M-Pro rotary file system (Foshan Stardent Equipment Co., China), comprising instruments #20/4%, #25/6%, #35/4%, and #40/4%.

Patients’ classification and intervention

Group A Control group (n = 45)

The canals were irrigated with NaOCl at room temperature, followed by a final flush with saline at room temperature.

Broup B Intra-oral group (n = 45)

Intraoral cryotherapy was administered by placing a custom-made plastic pack (2.5 × 5 cm) filled with ice gel (DonJoy Orthopaedic Pty Ltd, Normanhurst, New South Wales, Australia) on the buccal vestibule directly over the treated tooth. Patients were instructed to retain the pack intraorally for 10 min, with the option to remove it for 2 min if they experienced excessive cold or a burning sensation [13]. Throughout the cryotherapy session, a thermocouple device was used to monitor and maintain the temperature at 10 °C. A designated member of the research team ensured strict adherence to the temperature protocol. If the temperature exceeded the target threshold, the gel pack was promptly replaced to restore the desired range. Each pack was applied for 10 min, with a minimum of three cycles completed over a 30-minute period, incorporating 2-minute breaks between applications. Following the completion of the three cryotherapy cycles, the second apical fluid sample (S2) was collected as previously described.

Group C Intra-canal cryotherapy with saline Group (n = 45)

The canals were irrigated with NaOCl at room temperature, followed by a final flush with cold saline [13]. The irrigation syringes in were kept in an ice box filled with cooling gel packs to maintain the liquid at a temperature of 2–4 °C. A thermocouple was used to confirm that the temperatures remained within the required range.

Group D Intra-canal cryotherapy with Hypochlorite group (n = 45)

The canals were irrigated with cold NaOCl and then rinsed with cold saline as a final flush [13]. The irrigation syringes in were kept in an ice box filled with cooling gel packs to maintain the liquid at a temperature of 2–4 °C. A thermocouple was used to confirm that the temperatures remained within the required range.

During canal instrumentation, irrigation was performed between rotary file sequences using a side-vented needle (30-gauge) positioned 2 mm short of the working length. Each interval was irrigated with 5 mL of 2.5% sodium hypochlorite (NaOCl). For the final flush, a saline solution was delivered over a 10-minute period using the same needle configuration to ensure thorough canal cleansing while maintaining consistent needle placement. Finally, the canals were dried with paper points corresponding to the master apical file, and the second apical fluid sample (S2) was collected.

Obturation and postoperative instructions

Following radiographic confirmation of the master cone, root canal obturation was performed during the same visit using the cold lateral condensation technique. Gutta-percha points and a resin-based sealer (Meta BioMed, Korea) were utilized. Prior to obturation, the canals were irrigated with 17% EDTA to eliminate the smear layer. After completion, the access cavity was sealed with glass ionomer cement (Meta BioMed, Korea).

A postoperative radiograph was taken to verify the accuracy of the obturation. Patients were given detailed postoperative instructions and provided with a numeric Visual Analog Scale (VAS) chart to assess pain levels, ranging from 0 (no pain) to 10 (worst imaginable pain).

Patients were contacted by phone at 6, 12, 24, 48, and 72 h after the procedure to record their pain scores. Each patient was prescribed an analgesic containing 50 mg of diclofenac potassium (Cataflam®), with instructions to take one tablet every eight hours for three days if experiencing persistent or intolerable pain. Patients were also asked to document the number of analgesic doses taken during the follow-up period.

Biochemical analysis

Substance P (SP) levels in samples S1 and S2 were measured using the Enzyme-Linked Immunosorbent Assay (ELISA). Split paper points were diluted in 600 mL of phosphate-buffered saline (PBS) at pH 7.4. The diluted samples were then centrifuged at 10,000 rpm for five minutes. SP concentrations were determined using a commercial ELISA kit (Catalog No. E-EL-0067, Elabscience Biotechnology), following the manufacturer’s protocol. The kit’s minimum detection threshold for SP was 3.9 pg/mL. Absorbance readings were obtained using a microplate reader (Spectra Max Plus 384, USA) at wavelengths between 420 and 450 nm. A standard curve was generated using known SP concentrations. The concentration of drug P in each sample was calculated based on this standard curve.

Statistical analysis

Statistical analysis was performed with SPSS 27®, Graph Pad Prism® and Microsoft Excel 2016. All data were explored for normality by using Shapiro Wilk Normality test Kolmogorov tests. Which revealed that all data deviated significantly from non-parametric distribution (P < 0.05), except age was normally distributed (P > 0.05). Comparison between different groups was performed by One Way ANOVA test in normal data. In non-parametric data, comparison was performed by using Kruskal Wallis test followed by Dunns test, comparison between different time points was performed by using Fridman’s test followed by Duns test, while comparison between before and after was performed by using Wilcoxon signed rank test. In categorical data (analgesics intake and sex) all comparisons were performed by using chi square test. The significant level was set to be at P ≤ 0.05.