# An Evaluation of the Demineralizing Effects of Various Acidic Solutions
## 2 Materials and Methods  
Tooth collection was approved by the Ethical Committee of U.M.F. [PERSON] Cluj-Napoca (271/30 July 2019). The extracted teeth were mechanically cleaned with dental curettes and professional brushing (Depural(tm) Neo, Pentron, Orange, CA, USA). Chemical cleaning was performed by immersing the teeth for 60 min in a Chloramine T-Sin solution from Sintofarm (20 tablets were dissolved in 1 L of distilled water). The dental crowns were examined for enamel defects. In this study, 30 healthy molars and premolars with no caries on their vestibular and oral surfaces were included. The teeth were kept in a 0.9% saline (NaCl) solution at room temperature until they were used and in between the steps of the study. On the vestibular and oral surface of each tooth, a 6 mm wide circle was marked with a dental burr. This helped to localize the tip of the spectrophotometer and the DIAGNODent(tm) pen ([PERSON]) every time in the same position. The teeth were covered with acid-resistant nail varnish with the circles' surface being left out. Two silicone matrices were created for each tooth, covering half of the dental crown and root, except for the circle's surface. The base of the matrix was thin and straight. This helped to position the tooth for the X-ray so that the rays penetrated parallel to the marked circular zone of the enamel. Photographs of the teeth were taken in the initial situation, and their color was recorded. The baseline color was measured for all specimens and recorded using a spectrophotometer (VITA Easyshade(r) Advance 4.0, Vita Zahnfabrik, Bad Sackingen, Germany) according to the CIE (Commission Internationale de l'Eclairage) L*, a*, b* system. The values of L* represent lightness (white-black); a* and b* are the chromaticity coordinates (green-red and blue-yellow) [17]. Positive a* values indicate the red color range and negative values represent the green color range. Positive b* values show the yellow color range, while negative values indicate the blue color range. The teeth were measured three times (with recalibration after each tooth), and the mean values for L*, a* and b* were recorded. The VITA Easyshade(r) system measures the remission spectrum with a defined light, which passes perpendicular to the tooth (the measurement range is 400-700 nm). The color measurements were standardized by using the same black background, one operator and the same lighting conditions. The measurements were repeated after each demineralization cycle, and the total color change (\(\Delta\)E) was calculated using the following formula [18,19]:  
\[\Delta\text{E}_{\text{ab}} = \sqrt{\left(\text{L}_{2}-\text{L}_{1}\right)^{2}+\left(\text{a}_{2} -\text{a}_{1}\right)^{2}+\left(\text{b}_{2}-\text{b}_{1}\right)^{2}}\]  
The teeth were examined with a DIAGNODent(tm) pen ([PERSON], Brea, CA, USA) which uses laser fluorescence to detect caries within the tooth structure. When the incident laser light is dispersed on the enamel, a carious tooth structure will exhibit fluorescence directly related to the depth of the caries, resulting in higher reading values. Clean and healthy tooth structure reveals little or no fluorescence and will give low scale readings. The readings were performed using a probe with a flat tip, this being recommended for smooth surfaces. Measurement time was standardized to ten seconds [20]. The instrument was calibrated against a ceramic standard before each measurement session, and maximum readings were recorded. The values obtained can be classified to indicate the diagnosis [21,22]. This classification is presented in Table 1.  
The surface morphology was examined with a scanning electron microscope (SEM Inspect(tm) S, FEI, Hillsboro, OR, USA)--a type of electron microscope that produces images of a sample by scanning the surface with a focused beam of electrons. The electrons interact with the atoms of the enamel, producing various signals that present information about the surface topography and composition of the tooth [23].