## 2 Materials and Methods  
A total of 110 adult incisors (both upper and lower) with a single canal system were extracted for periodontal concerns. The teeth were thoroughly cleaned using ultrasonic technology to remove debris and then placed in a solution containing 0.1% thymol for 3 h. Immediately after extraction, the incisors were decorated by cutting them 1 mm above the junction between the enamel and cement. Then, an opening was made to access the inside of the tooth. The tooth length was determined using a number 10 ISO hand K-file (Dentsply/Sirona, Switzerland). After that, the tooth was prepared using the TruNatomy system (Dentsply/Sirona, Switzerland) until it reached a foraminal diameter of either 0.25 or 0.35, depending on the specific case.  
The teeth were flushed with 20 mL sodium hypochlorite 5.25% (NaOCl) and 5 mL ethylenediaminetetraacetic acid 17% (EDTA), and then dried using sterile paper points (Dentsply/Sirona, Switzerland) and then sealed with Ah Plus(r) Bioceramic Sealer (Dentsply, Konstanz, Germany) and TruNatomy Conform Fit gutta-percha points (Dentsply/Sirona, Switzerland) using the hydraulic technique. The roots were placed into plastic containers and embedded into alginate ([PERSON], Melville, NY, USA). Afterwards, the roots were stored in an incubator (Memmert IN 75 Incubator, Buechenbach, Germany) at 100% humidity and a temperature of 37 \({}^{\circ}\)C for 2 weeks. Subsequently, the teeth were placed into blocks made of auto-polymerizing methyl methacrylate acrylic resin.  
The roots were horizontally severed at the apical region, specifically 5 mm away from the apex, the samples were taken from the apical and cervical regions. The slices were cut with a microtome (IsoMet Low Speed Precision Cutter, Buehler, Dusseldorf, Germany) at 1 mm thickness, and two slices were cut from each tooth. After that, the samples were dried and sputter-coated with gold. Finally, the surfaces were analyzed with a scanning electron microscope at \(\times\)100\(\sim\)\(\times\)25,000 magnification. Scanning electron microscopy (SEM) was carried out using the Quanta Inspect F50 (FEI Company, Eindhoven, The Netherlands) at a 10 kV and 20 kV acceleration electron voltage. A total of 220 slices from 110 teeth were analyzed using SEM-EDX.  
This study utilized a series of SEM images taken at different levels of magnification (ranging from 100\(\times\) to 25,000\(\times\)) to examine the detailed structure and the interface between dentin and the sealer. The X-ray energy dispersive spectrometer (EDX) (Thermo Fisher, Eindhoven, The Netherlands) was used in order to determine the elemental distribution at the interface between the dentin and the sealer as well as the elemental distribution of intermediary dentin located at a minimum distance of 50 microns from the dentin in direct contact with the sealer. Measurements were taken in four zones from each specimen, with two from the intermediate dentin level (Area 1) and two from the dentin-sealer interface (Area 2). The results were assessed comprehensively, both qualitatively using SEM pictures and elemental mapping, and quantitatively using element weight (wt%) and atomic (at%) percentages from the EDX study. The particle size was determined using ImageJ 1.52d software ([PERSON], National Institutes of Health, USA) at the intermediate dentin level and at the dentin-sealer interface. The study examined the data using descriptive analysis, calculated the distribution frequencies in percentages, and conducted chi-squared tests to compare proportions. Significance was attributed to \(p\)-values less than 0.05. The statistical analyses were conducted using the DATAtab software accessed on 9 September 2024, version (2024) [22].