One hundred and twenty recently extracted human maxillary premolars were utilized in this study. The sample size was estimated by G*Power (version 3.0.10, Kiel University, Germany). Assuming type I statistical error of 5% and two-tailed statistical test, this study was designed to have a power of 90% based on previous study [18]. The calculated sample size was 10 teeth per subgroup.
The teeth were selected according to the following criteria: intact buccal enamel surface, no extraction damage or pitting and cracks and free of caries, dental fluorosis, and other hypomineralized lesions. Investigating the buccal surface was done with the aid of eye loupes ×6 magnification (Univet, Italy). All teeth were washed with tap water and stored in 0.1% thymol solution. The teeth were randomly divided into two main equal groups. Group I (n = 60) was left without bleaching and group II (n = 60) was subjected to bleaching with 30% carbamide peroxide gel (Vivastyle, Ivoclar Vivadent AG, Schaan/Liechtenstein, USA), according to manufacturer’s instructions. At first, the enamel surfaces were cleaned with non-fluoridated pumice and rubber cup connected to low-speed hand piece for 10 s. The carfaces for 30 min and repeated for 3 days. After bleaching, the teeth were rinsed with tap water. Finally, fluoride (Fluor protector, Ivoclar Vivadent AG, Schaan/Liechtenstein, USA) was applied for 10 min.
Metal brackets (NANDA, Ortho Organizers, CA, USA) were bonded to the buccal surfaces of all teeth. The buccal enamel area subjected to etching and bonding procedures was equal and standard in all teeth. The enamel was etched for 30 s with 37% phosphoric acid gel (Super Etch, SDI Limited, Bayswater, Australia), rinsed with water for 10 s and dried with air for 5 s. Transbond XT primer (3M Unitek, CA, USA) was applied to the etched enamel. Transbond XT adhesive (3M Unitek) was placed on the bracket base. The brackets were positioned on the correct position with firm pressure and excess composite was removed. Finally, the adhesive was light cured for 20 s.
Then, the teeth in either group were subdivided into equal six subgroups (A, B, C, D, E, and F) according to the materials utilized for prevention of enamel decalcification. All the materials were used according to manufacturer’s instructions.
Subgroup A: no material was applied (control).
Subgroup B: 5% NaF varnish (Profluorid varnish, VOCO GmbH, Cuxhaven, Germany) was applied on the labial surfaces around the brackets. Then, the teeth were leaved to dry for 5 min.
Subgroup C: 5% NaF-ACP varnish (Enamel Pro Varnish, Premier dental, PA, USA). The varnish was applied as in group B.
Subgroup D: fluoride-releasing, light-cured resin coat (Ortho-Choice Ortho-Coat, Pulpdent, Watertown, MA, USA) was applied. The coat was light-cured for 20 seconds.
Subgroup E: CPP-ACP (GC Tooth Mousse, GC Corp, Tokyo, Japan) was painted and left for 3 min undisturbed. Then, it was removed by cotton roll and allowed to dry for extra 2 min. Finally, the remaining residue was washed with tap water. These procedures were repeated daily.
Subgroup F: the same procedures were done as in subgroup E except CPP-ACFP (GC MI Paste plus, GC Corp, Tokyo, Japan) was applied instead of casein phosphopeptide amorphous calcium phosphate complex.
All teeth were cycled in a demineralization solution and artificial saliva for 15 days [18]. The teeth were immersed in the demineralizing solution (calcium nitrate 0.4723 g, potassium dihydrogen phosphate 0.2722 g, acetic acid 4.5083 g) for 8 h/day [19]. Then, the teeth were rinsed with tap water and putted in artificial saliva solution (sodium azide 0.75 g, potassium monohydrogen phosphate 0.804 g, sodium chloride 1.02 g, calcium chloride 0.166 g, magnesium chloride 0.059 g) for 30 min [20]. Each tooth was cleaned by using electronic dental brush (Oral-B, Braun GmbH, Germany) and Crest cavity protection toothpaste (1450 ppm F) for 2 s. Again, all teeth were rinsed with water and placed in the artificial saliva for approximately 15 h until the cycle was repeated.
Laser fluorescence device (Kavo DIAGNOdent pen, Kavo, Germany) was used to measure the level of mineralization of the enamel surfaces around the brackets. Measurements were taken before application of the materials utilized for prevention of enamel decalcification and after cycling in the demineralization solution and artificial saliva. At first, the laser device was calibrated against the device own ceramic standard to ensure an accurate reading. Then, measurements were obtained on the buccal surfaces 1 mm away and opposite the center of the mesial, distal, gingival, and occlusal borders of the brackets. The laser device gives readings from 0 to 99. Low readings indicate high mineral content of the enamel and hence clean healthy tooth structure. On the other hand, higher readings indicate greater demineralization.
The data were collected and analyzed using Statistical Package for the Social Sciences (SPSS version 17.0). Kruskal-Wallis test was used to evaluate if there was a significant difference in the changes between all subgroups. Mann-Whitney U test was utilized to compare the changes between each two subgroups. Wilcoxon signed-rank test was used to compare between the pre and post measurements in each studied subgroups. The statistical tests were based on a type 1 error value of 5% (α = 0.05) and on a power of 0.90 sample size.
