## 2 Materials and Methods  
For analysis, a number of 174 freshly extracted premolars for orthodontic purposes were collected. After extraction, teeth were kept at room temperature in 10% formalin solution for a period of 7 days, which was subsequently replaced by saline solution (NaCl 0.9%) and was changed daily for 10 more days. After storage, teeth were examined and inspected. Teeth that showed signs of enamel degradation, caries, tartar, or fillings on the vestibular surface, as well as fluorosis or hypoplasia, were excluded from the study. On the subsequent stage of the study, 147 premolars with intact vestibular surfaces were analyzed. Teeth from the study group underwent periodontal ligament removal using a GraceyTM 5-6 curette and then were polished with Depural NeoTM Spofa Dental, Jicin, Czech Republic, and stored back in the saline solution. Later in the study, an S-3400N electron microscope, Hitachi, Chiyoda, Tokyo, Japan, was used to analyze the enamel surface pretreatment (Figure 1). Data were recorded and used as a benchmark later in the study for comparison with the images obtained post treatment. The samples did not require prior preparation because the microscope has a vacuum function that allows the observation of nonconductive specimens at a working distance of 9 mm, without the need to apply any conductive layer. The acceleration voltage of the microscope was operated at 15 kV. The only intervention on the enamel surface was to draw an indicative perimeter of the future base of the bracket for an easier investigation of the surface. For this purpose, we used a round disk of 0.10 mm diameter. After the preliminary analysis using SEM, the study group was divided into 2 subgroups as follows:  
* Study sub-group 1 consisted of 72 premolars, in which we applied metal Gemini(tm) brackets (3M Unitec(tm), USA) with the following chemical composition: Ni 83.98%, Si 6.46%, Fe 5.90%, and Cr 3.52%;
* Study subgroup 2 consisted of 75 premolars, in which we applied ceramic brackets Forestadent(tm), Germany (Al2O3). The enamel's surface was dried for 10 s using the air spray, and 37% phosphoric acid (Unitek Etching Gel System from 3M Unitek) was applied for 30 s. The acid was removed by a jet of water applied for 10 s, followed by a jet of air from the unit's spray for 10 s.To promote adhesion, a primer Transbond Xtm from 3M Unitek was used; the primer was applied with a micro brush by circular movements on the previously prepared surface then it was photo-polymerized for 20 s by using a high-power led lamp Elipar S10(tm), 3M ESPE, with the tip of the lamp curing tube placed 1 mm away from the vestibular surface of the tooth. The bracket's base was loaded with the adhesive paste Transbond tm from 3M Unitek, then placed very similar to and following the positioning bonding rules described by [PERSON] et al. (2010) in the center of the vestibular surface. After correct positioning, brackets were pressed to 300 g using an orthodontic dynamometer. Excess composite resin was removed prior to light curing with a dental probe. The polymerization was performed mesially and distally from the bracket for 10 s on each side. After 24 h, the brackets were removed using straight take-off pliers and acting directly on the brackets' bases. The adhesive material was removed with a high-speed multi-laminated tungsten carbide burr. The cleaning process was continued until the adhesive material was no longer visible to the visual inspection and the enamel became smooth, without roughness detectable on examination with the dental probe. After the brackets were removed, the teeth were stored in saline solution at room temperature until the second analysis at SEM, when the samples were analyzed at the same parameters as before treatment, and possible irregularities and cracks in the enamel surface were monitored. In the next step of the study a quantitative analysis, the energy-dispersive X-ray spectroscopy (EDX) was also performed on the entire surface of the brackets' soles to detect any traces of enamel.