## 2 Materials and Methods  
### Preparation of Specimens  
The study was approved by the Ethics Committee of Tel-Aviv University (No: 230.17, 23 January 2018), and all protocols were conducted in accordance with the relevant regulations and guidelines.  
Fifty-five freshly extracted human permanent molars (for periodontal reasons) were used in this study. Teeth with previous endodontic treatment, visible sign of root resorption, caries, root fractures, or immature apices, were excluded from the study. Teeth with fused roots or with a single root were also excluded. Once cleaned of debris and soft tissue remnants, teeth were stored in phosphate-buffered saline.  
For use, the teeth were decorated to expose the pulp chamber cavities, and 5 mm of the apical segment of the roots was amputated using a high-speed diamond disc (IPR Diamond Disc; Dentsply Int./Maillefer, Ballagues, Switzerland) with water cooling. Pulpal remnants were removed from pulp chamber and root canals using a barbed broache (Barbed Broache; Dentsply Int./Maillefer, Ballagues, Switzerland). The canal orifices and apical foramen of each root were filled with resin composite (TE; Ivoclar Vivadent AG, Schaan, Liechtenstein, German). Perforations were created in the center of the pulpal floor by using a round #14 diamond bur while the tooth was visualized under an optical microscope (OPMI pico Dental Surgical Microscope, Carl Zeiss Meditec, Dublin, CA, USA) at 6 \(\times\) magnification [19].  
The specimens were randomly divided into the following experimental groups:  
1. Group 1 (\(n=10\)): The perforation defects were repaired using MTA-Angelus (Angelus, Londrina, PR, Brazil), mixed according to the manufacturer's recommendations. A metal spatula was used to mix a 1:1 ratio of MTA Angelus with distilled water on a sterilized glass slab. The mixture was homogeneous and had a consistency similar to wet sand.
2. Group 2 (\(n=10\)): The perforation defects were repaired using Endocem MTA (Maruchi, Wonju, Korea) cement, mixed according to the manufacturer's recommendations. A metal spatula was used to mix 300 mg Endocem MTA powder with 0.12 mL liquid on a sterilized glass slab.
3. Group 3 (\(n=10\)): The perforation defects were repaired using Biodentine (Septodont, Saint-Maur-des-Fosses, France), mixed according to the manufacturer's recommendations. The closed capsule was gently tapped on a hard surface to dispense the powder. Five drops of liquid were added into the capsule and mixed in a triturator for 30 s.
4. Group 4 (\(n=5\)) (positive control): The created perforations were left without repair material.
5. Group 5 (\(n=5\)) (negative control): The teeth were left without perforation and repair material, but the external tooth surface was covered with two layers of nail varnish.
6. Group 6 (\(n=5\)) (negative control MTA Angelus): The teeth were the same as group 1 without bacterial contamination.
7. Group 7 (\(n=5\)) (negative control Endocem MTA): The teeth were the same as group 2 without bacterial contamination.
8. Group 8 (\(n=5\)) (negative control Biodentine): The teeth were the same as group 3 without bacterial contamination.  
All teeth were left at 37 \({}^{\circ}\)C and 100% humidity for 24 h. All specimens were prepared by a single operator.