*2.2. Treatment and Study Groups*

Ten samples were kept un-instrumented as positive controls (group 1). A size #15 K patency file was used to determine the correct working length (WL). For fifty specimens, the k file was placed in the canal until it extrudeed the apex and then the file was pulled 1 mm shorter to the apex. Fifty specimens were then prepared using the crown-down technique by Profile rotary system up to #40/0.04 taper file (excluding positive control specimens-group 1). Between each instrumentation, constant irrigation was performed with 2.5% NaOCl (1 mL). After completion of the canal preparation, a final rinse was performed with 2.5% NaOCl (2 mL) for 1 min followed by 2 mL 17% EDTA for 1 min and 10 mL distilled water. Root canal surfaces were dried using paper points and were further divided into 5 groups (*n* = 10) based on instrumentation and obturation techniques.

Group 1 (Positive control): Samples were left un-instrumented and unfilled.

Group 2 (Negative control): Roots were instrumented and left unfilled.

Group 3: GP and AH Plus sealer using cold lateral compaction technique.

Group 4: GP and AH Plus sealer using Single Cone technique

Group 5: TotalFill GP and TotalFill BC sealer using cold lateral compaction technique Group 6: TotalFill GP and TotalFill BC sealer using Single Cone technique.

After completing obturation, periapical radiographs were exposed to assess the quality of root filling. Any root with inadequate obturation was excluded and replaced with a newly prepared specimen. The access cavity was closed using temporary filling (3M™ Cavit™). The specimens were kept at 37 ◦C and 100% humidity for 2 weeks to allow complete setting of the sealer.

#### *2.3. Specimen Testing*

To simulate the periodontal attachment, the specimen's root surface was coated with a thin-layer of polyvinylsiloxane (PVS) impression material up to 2 mm apical to the coronal

end of the root. All of the roots were mounted perpendicularly in a poly-vinyl ring filled with self-cure acrylic resin (Opti-Cryl, South Carolina, Columbia) exposing 2 mm from the root (cervical). Root fracture resistance was assessed using a universal testing machine (UTM) (Lloyds, LF, plus, Ametek Inc., Great Britain, UK). All of the samples were placed in the lower plate of the testing machine and in the upper part a custom-made metal spreader with a diameter of 0.8 mm was secured. The tip was oriented in the center of the canal orifice and force was applied vertically to the long axis of the root at a crosshead speed of 0.5 mm/min until root fracture. A drop in >25% of applied force was observed when the fractures occurred. The amount of load necessary for root fracture was recorded in Newtons (N).
