## 2 Materials and Methods  
In this in vitro study, we used a total of 120 mandibular and maxillary premolars extracted for orthodontic purposes. The donor patients approved the use of their extracted teeth in the study by signing a written consent. The criteria for including teeth in the study were:  
* The crown integrity, namely the absence of any structural or developmental crown defects, decays, restorations or cracks caused by the extraction procedures [12; 13];
* The teeth not having undergone any chemical treatment [9]. The extracted teeth were washed in water then placed in a 0.1% thymol solution. Later, they were stored for a week in distilled water that was changed once a day. They were removed from distilled water on the day of testing and were gently cleaned with Depural Neo from Spofa Dental, a slightly abrasive fluoride-free paste that is generally used for professional teeth polishing [14]. We wanted to use only the premolar crowns for easier handling, so we separated the premolar roots and crowns at the anatomical cement-enamel junction using low-speed diamond disks and water cooling [15]. All the premolar crowns were mounted in Duracrol (a self-curing methacrylate resin produced by Spofa Dental, which now is part of Danaher Corporation, California) blocks with only their buccal surfaces exposed (Figure 1) in order to allow the brackets' bonding and testing [15]; the hard self-curing resin kept the crowns of the teeth immobile during the brackets' debonding. The cubes were prepared by putting the acrylic resin in plastic cubes previously insulated with Vaseline before starting the setting. The size of the cubes was 3.3 cm/3.3 cm/2 cm. Just before the setting, the crowns of the premolars were submerged so that only the vestibular surface remained exposed. We made sure that the vestibular surfaces were not contaminated with Duracrol [16]. For all the specimens in the study, we used Discovery brackets from DENTAURUM GmbH & Co., Ispringer, Germany. Thus, prepared teeth were randomly divided into six groups (20 samples for each group) according to the bonding system and enamel preparation:  
Group 1: Transbond Plus Color Change together with Transbond Plus Self-Etching Primer (3M). The buccal surfaces of teeth in this group were treated with Transbond Plus  
Figure 1: Sample preparation: (**a**) Duracrol cube; (**b**) before testing; and (**c**) position during the testing process. Self-Etching Primer; the primer was rubbed onto the buccal surfaces for 10 s using the disposable supplied. Then, the moisture-free spray was used for 10 s in order to deliver air to the primer. We applied Transbond Plus Color Change paste to the brackets' bases which then were pressed evenly and placed on the teeth, respecting the positioning bonding rules by localizing the center of the buccal surfaces [17]. Excessive bonding material around the bracket base was then gently removed by using a sealer, and the adhesive was light-cured with a Demetron LC lamp (SDS Kerr, USA) which had a light intensity of 800 mW/cm\({}^{2}\). The specimens were light-cured from occlusal, gingival, mesial and distal aspects for 10 s each, for a total of 40 s [18; 19]. Group 2: Transbond Plus Color Change together with Transbond Plus Self-Etching Primer (3M) moistened with distilled water. We first prepared the buccal surfaces of the teeth in this group as we did with the specimens from Group 1; the difference was that after the application of the primer and the use of the air spray, we moistened the entire buccal surfaces of the teeth in this group with water. The water was applied by using first a dental syringe and then using a microbrush. Then, we applied the bonding material Transbond Plus Color Change in the same way as we did in Group 1. Group 3: Transbond Plus Color Change together with Transbond Plus Self-Etching Primer (3M) moistened with saliva.