Ethical approval was obtained from the Clinical Research Ethics Committee of the Faculty of Medicine of Gaziosmanpasa University (project number: 15-KAEK-229). One hundred and five human maxillary incisors were used in this study. Mesiodistal and bucco-palatal direction radiographs were taken from the teeth to confirm the presence of a single canal. The teeth were decoronated using straight diamond burs (Komet, Gebr. Brasseler GmbH & Co. KG, Germany) in a conventional high-speed handpiece under water cooling so that each root had a standardized length of 15 mm. The WL was determined to be 1 mm short of the apex using a #10 K file (VDW GmbH, Munich, Germany). The WL was established at 14 mm. Roots were prepared with Reciproc rotary files up to size R40 at WL (VDW GmbH, Germany) and irrigation was performed with 10 mL 2.5% NaOCl. Next, roots were placed in Eppendorf tubes (Labosel, İstanbul, Turkey) filled with a silicone material (Zetaplus soft; Zhermack Clinical, Badia Polesine, Italy). After removal of the roots from the impression material, a longitudinal groove was prepared on the buccal and lingual surface using a narrow diamond bur without cutting the canal wall. A spatula was used to split longitudinally. A number 1S cavitron tip (Aceton, Merignac, France) was modified and inserted into an ultrasonic handpiece (Newtron P5; Satelec, Acteongroup, France) to create artificial standardized grooves. Two of the three standard grooves were created in the coronal and apical part of one segment, and another in the middle part of a second segment. The dimensions of grooves were 0.2 mm in width, 3 mm in length, and 0.5 mm in depth (Figure 1). The dimension of grooves was checked under a stereomicroscope (Zeiss Stemi 2000-C, Carl Zeiss MicroImaging, Göttingen, Germany) at 20X magnification. The root halves and grooves were irrigated with 5 mL of 17% EDTA for 1 min. and 5 mL 2.5% NaOCl for 1 min. while being activated with a toothbrush to remove debris and the smear layer. The standardized grooves were filled with CH paste (Ammdent, Punjab, India), and the root halves were reassembled. The root canals were fully filled with CH paste using a Lentulo spiral. Two radiographs (mesiodistal and bucco-palatal direction) were taken to confirm complete filling of the canals with CH paste. Roots were remounted and placed into Eppendorf tubes. Then samples were divided randomly into 7 groups, each containing 15 teeth. Seven different color stickers were pasted on the caps of the Eppendorf tubes to indicate each of the 7 groups. The access cavities were sealed with temporary filling material (Cavit, 3M ESPE, Seefeld, Germany) and stored in 37°C at 100% relative humidity for 2 weeks.