## 2 Materials and Methods
### Sample Selection and Inclusion Criteria  
The procedures of this study were in accordance with the institutional and national ethical standards on human experimentation and the Declaration of Helsinki of 1975, as revised in 2000, and they were approved by the Ethics Committee of the AORN of Ospedali Gelli with N. 117 del 19 February 2020. The first operator selected 40 healthy permanent molars extracted because of over-retention or lack of space. Inclusion criteria were as follows: absence of both carious lesions and fractures and excessive abrasion, presence of restoration. Samples were collected from patients of both sexes (15 males and 10 females). The age of patients ranged from 20 to 50 years. All patients were treated in the dental clinic of the multidisciplinary department of medical-surgical and dental specialties of the University of Campania \"[PERSON]\" in Naples. The freshly extracted molars, after being washed with spring water, were immersed and kept in a hermetically sealed glass container containing a 0.1% thymol solution and stored at 4 \({}^{\circ}\)C.  
At the beginning of the search, each tooth was removed from the container and rinsed abundantly in distilled water for 2 min. The second examiner numbered each specimen by putting a sequential number with a pencil in correspondence with the root tooth and marking the axial enamel surfaces of each tooth crown (vestibular, palatal, mesial, and distal) with a letter (A, B, O, and X) on the corresponding root surface (Figure 1a). In this way, it was possible to obtain four groups for the 40 specimens to test 4 different therapeutic interventions designed to evaluate the protective effect, from acid liquid, on the corresponding enamel surface. The same staff member then delineated a space of approximately 2 mm \(\times\) 4 mm (2 mm in height and 4 mm in width) for each tooth by making of an incision performed by a 0.8 mm diameter diamond ball drill (FG) in correspondence with the enamel of each of the four axial surfaces; this window will represent the test area for the clinical trial. The third examiner, who was not a clinician, randomly assigned each area test of each tooth to a specific therapeutic program to be tested using a computerized random distribution program. The fourth examiner, who was not a clinician, performed a preliminary scanning of the spot areas of each sample using a SEM (Zeiss EVO of Carl Zeiss Microscopy, White Plains, NY, USA), before administering a therapeutic protocol. In this way, photographic areas of the four surfaces of enamel of interest at time zero (healthy enamel) were obtained for a single sample belonging to the four study groups and compared with the same photographic images taken after erosive treatment. The SEM analyses were carried out at the Advanced Materials Laboratory of the Department of Architecture and Industrial Design of the University of Campania \"[PERSON]\". The samples to be analyzed were taken from the preservative fluid, rinsed, placed on bibula paper, and left to dry overnight. At the time of analysis, each tooth was attached on a metal support (grounded) of SEM with graphite-based adhesive tape (Figure 1b). For all study samples, scans of the four spot surfaces were performed in high and low vacuum mode by an electron detector (backscatter) with high sensitivity at different magnifications to identify the structure and morphology of the enamel surface before each type of treatment. In an analysis of the SEM in no spot area of the enamel, an alteration of the gray scale was highlighted that could be traced back to any type of structural change in the enamel. The subdivision of the tooth crown into four areas allowed the identification and formation of four study groups for each tooth. The fifth clinical operator was used to administer, in correspondence of the area test, the various treatment protocols to oppose the demineralizing action of an acidic solution according to the following scheme:  
* Group A: no pretreatment was administered (control group).