## 2 Material and Methods
### Experimental Design  
The teeth were randomly divided into four experimental groups (n = 25) as follows:  
Group 1, canals were obtrutated using SC technique with TotalFill(r) BC Sealer.  
Group 2, canals were obtrutated using SC technique with TotalFill(r) BC Sealer mixed with AgNPs.  
Group 3, canals were obtrutated using CWC technique with TotalFill(r) HiFlow BC Sealer.  
Group 4, canals were obtrutated using CWC technique with TotalFill(r) HiFlow BC Sealer mixed with AgNPs.  
To prepare the modified sealer (AgNPs incorporated with calcium silicate-based sealer), 0.06 mg of nanoparticles was measured for every 1 g of sealer using a digital analytical balance, resulting in a nanoparticle concentration of 0.06% in the modified sealer in line with previous work [21]. Manual mixing was conducted with a metal spatula on a glass plate, then for 1 min using a vortex mixer. This was succeeded by ultrasonication using a probe sonicator (Sigma-Aldrich, St Louis, MO, USA) for 1 h to improve the dispersion of NPs. This was conducted at 25 \({}^{\circ}\)C and in the absence of light. Table 1 lists the experimental groups used in the study.  
In the SC technique, the BC sealer was injected by the intracanal tip, then the size-matched ISO size 30/0.06 taper TotalFill(r) BC Point (TotalFill(r) BC point, FKG, La Chaux-de-Fonds, Switzerland) was coated with the TotalFill(r) BC sealer followed by its slow insertion to the full working length. After that, the cone was cut off with activated system B pluggers (Sybron Endo, Orange, CA, USA) just below the orifice, and a quick-setting temporary filling Cavit G (3 M ESPE, Seefeld, Germany) was placed. In the CWC technique, we used TotalFill(r) HiFlow BC Sealer (FKG, La Chaux-de-Fonds, Switzerland) because, according to the manufacturer, it exhibits lower viscosity and increased radiopacity when used with the heated obturation procedure than TotalFill(r) BC Sealer. In this technique, the placement of the BC sealer and the BC point were the same as in the SC technique. However, the BC point was cut 3 mm from the working length using activated system B pluggers, then the root canal was backfilled with plasticized GP using TotalFill(r) BC pellets (TotalFill(r) BC pellets, FKG, La Chaux-de-Fonds, Switzerland), and the orifice was sealed with Cavit G. All treatment procedures were performed by a single operator. Specimens were stored at 37 \({}^{\circ}\)C in 100% humidity for 2 weeks to enable a complete set of the sealers. Afterwards, roots were horizontally sectioned using a 0.3 mm-thick, water-cooled slow-speed diamond blade (IsoMet 4000; Buehler, Lake Bluff, IL, USA) to obtain 1 mm-thick dentin slices from the middle third of each root.  
\begin{table}
\begin{tabular}{c c c} \hline
**Groups** & **Sealer Type** & **Filling Technique** \\ \hline Group 1 & TotalFill® BC Sealer & Single-cone technique \\ Group 2 & TotalFill® BC Sealer + AgNPs & Single-cone technique \\ Group 3 & TotalFill® HiFlow BC Sealer & Continuous wave condensation technique \\ Group 4 & TotalFill® HiFlow BC Sealer + AgNPs & Continuous wave condensation technique \\ \hline \end{tabular}
\end{table}
Table 1: Summary of the experimental groups.