The three root canal sealers included in this study were AHplus, ADseal, and Dia-Proseal. The chemical composition of the tested materials is shown in Table 1.

The pH was measured according to the criteria used in a previously published study.8 The sealer samples with 1 mm thickness and 5 mm diameter were prepared and allowed to set for 1 day (n = 3). After setting, each specimen was immersed in glass tubes containing 10 mL deionized water. Then, the pH was measured with a pH meter (Orion 3 star, Thermo Scientific, Singapore) previously calibrated with pH 7.0 and 4.0 solutions after 1, 2, 3, 7, 10, and 14 days.

The dimensional change was measured using the method recommended by ISO 6876/2012. Each sealer was put into a cylindrical silicon mold with a diameter of 6 mm and a height of 12 mm (n = 5). After setting, the mold was measured for length (M₁) with a micrometer caliper (accuracy of 10 µm). The samples were then stored in distilled water at 37℃. After 7, 14, and 21 days, the length (M₂) was measured. The changes in length were measured 3 times, and mean values were recorded as the dimensional change using the following formula: Dimensional change = (M₂ - M₁) / M₁ × 100.

The solubility was measured using the method recommended by ISO 6876/2012. Paraffin wax mold in 1.5 mm thickness and inner diameter of 20 mm was used for each sealer (n = 5). The molds were filled with the sealers and the assembly was placed in an incubator (37℃, > 95% relative humidity) for a period of time 100% longer than the setting time. After the sealers were removed from the mold, they were weighed 3 times each in analytical balance (HM-200, A&D Engineering Inc., Bradford, MA, USA). The mean weight was recorded as W₁. Then, the specimens were immersed in tubes containing 10 mL of distilled water for 7 days. After this period, the specimens were dried with absorbent paper and placed in a dessicator, and its weight was recorded as W2. The solubility of the sealer was calculated using following formula: Solubility = (W₁ - W₂) / W₁ × 100.

The flow was assessed using the method recommended by ISO 6876/2012. A volume of 0.5 mL sealer was put on a glass plate (n = 3). After 180 ± 5 seconds, the second glass plate was placed centrally on top of the sealer to make a total mass on the plate of 120 g. Ten minutes after mixing the sealer, the load was removed and the average of the major and minor diameters of the compressed sealer was measured by a digital caliper. The mean of 3 measurements for each sample was taken as the flow of the sealer.

The radiopacity was evaluated using the method recommended by ISO 6876/2012. Cylindrical samples from each material were fabricated by pouring the manipulated sealers into metallic rings with 10 mm internal diameter and 1 mm thickness (n = 3). The filled rings were kept at 37℃ until cements were set completely. The specimens were placed on occlusal x-ray film (Kodak Insight, Rochester, NY, USA) with an aluminum step-wedge graduated from 1 to 10 mm (in 1 mm increments). A Kodak-2200 X-ray machine (Kodak) operating at 70 kV, 10 mA, 18 pulses/second and with a focus-sensor distance of 30 cm was used. The radiographs were digitized and analyzed using a densitometer (GS-800, Bio-Rad, Hercules, CA, USA).

The tested material was placed into a silicon mold (1 mm thickness and 10 mm diameter). After setting, the cement was removed from the mold and stored in 10 mL of minimal essential medium-α (MEM-α, HyClone Laboratories, Logan, UT, USA) containing 10% fetal bovine serum (FBS, HyClone Laboratories) for 3 days.

L929 cell line was purchased from Korean Cell Line Bank, Seoul, Korea. The cells were seeded in 24-well plates (2 × 10⁴ cells/well) and pre-incubated in growth medium for 24 hours (n = 4). After overnight attachment, cells were treated with the prepared extracts of sealers for 1, 3, 7, and 14 days. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Briefly, 200 µL of MTT solution (0.5 mg/mL in phosphate buffered saline) (Amresco, Solon, OH, USA) was added to each well, and the wells were incubated for 2 hours. Subsequently, 200 µL of dimethyl sulfoxide (Amresco) was added to each well. Reduced MTT was then measured spectrophotometrically at 540 nm in a dual-beam microtiter plate reader (SPECTROstar Nano, BMG Labtech, Ortenberg, Germany).

Thirty-four freshly extracted mandibular premolars with straight and single root canal were used. After access openings, a size 15 file was inserted into the root canal. Working length was determined by placing a size 15 K-file into the canal until it became visible at the apical foramen and then decreasing the file length by 1 mm. The root canals were shaped with ProTaper rotary files up to an F3. During the preparation, the root canal was irrigated with 5 mL 3.0% sodium hypochlorite (NaOCl). After instrumentation, 17% ethylenediaminetetraacetic acid (EDTA) was applied for 1 minute to remove the smear layer. The root canals were dried with paper points. Samples were randomly allocated into three groups (Dia-Proseal, AHplus, and ADseal, n = 10) while setting positive (n = 2) and negative (n = 2) controls. All teeth were obturated to its working length by vertical compaction of 0.06 taper guttapercha cones and sealers. The access cavity was sealed with composite resin, and the samples were stored for 24 hours in 100% relative humidity at 37℃. The root surfaces were coated with two layers of nail polish except for the apical 3 mm. Then, the samples were stored in 1% methylene blue dye at 37℃ for 2 weeks. After removal from the dye, the samples were washed with distilled water. The root of each tooth was sectioned longitudinally using a diamond disc, and each half was analyzed under a stereomicroscope (Leica MZ16FA, Leica, Wetzlar, Germany). The amount of leakage was measured from the working length to the most coronal part of the root canal to which the dye had penetrated. Two independent measurements were made for each tooth. The microleakage experimental procedures were approved by the Institutional Review Board of the Chonbuk National University Hospital (IRB No.: 2014-10-001).

Statistical analysis was performed by one-way ANOVA followed by Tukey test (p = 0.05)

Dia-Proseal showed significantly higher pH value compared to other sealers (p < 0.05, Figure 1a). ADseal showed significantly higher dimensional change compared to AHplus and Dia-Proseal (p < 0.05, Figure 1b). The solubility values of AHplus and Dia-Proseal were similar, whereas ADseal showed the lowest solubility value (p < 0.05, Figure 1c). The flow values of sealer in increasing order were AHplus, Dia-Proseal, and ADseal, and there was statistical significance (p < 0.05, Figure 1d). The radiopacity of AHplus was higher than ADseal and Dia-Proseal (p < 0.05, Figure 2).

The cell viability of the tested materials were similar throughout the experimental period (Figure 3).

There was no significant difference in microleakage value among the tested samples (Figure 4).