For groups G8F and G11F, the fluoride treatment was performed before the laser irradiation using an acidulated phosphate fluoride (APF) gel (DFL Ltd., Rio de Janeiro, Brazil) containing 1.23% of fluoride, pH 3.5, applied for 4 min. After application, samples were washed with distilled water and dried with absorbent paper. A pulsed CO2 laser emitting at 10.6 μm wavelength (UM-L30, Union Medical Engineering Co., USA) was used. The focal

distance was Inhibitor Library order adjusted in order to result in a beam diameter of 2.5 mm at the irradiation position and the other irradiation parameters were determined in a pilot study, ensuring that no visible ablation or carbonization was caused. A complete description is given in Table 1. Before the experiments began and after every 5 irradiations, the energy emitted was controlled with an energy meter (Coherent FieldMaster GS + Detector LM45; Coherent, USA). To standardize the irradiation conditions, the mirror arm of the laser was fixed onto a laboratory apparatus support and the samples were fixed onto an XY micropositioner mounted on a linear motorized stage (Newport Klinger MT160-250 Linear Stage, New York, USA). The motor was moved at a speed of 7.5 mm/s and two lines of irradiation were enough to irradiate the entire exposed area. For each irradiation line

3 pulses were overlapped per spot.21 After the irradiations all the samples were individually placed in plastic tubes (Falcon Tubes™, BD, Franklin Lakes, USA) and subjected to the following pH-cycling Natural Product Library model22 for 9 days (8 + 1 day remineralization bath) Casein kinase 1 at 37 °C: 1. 4 h in 50 ml demineralization bath (1.4 mM de calcium nitrate, 0.91 mM sodium dihydrogen phosphate, 0.05 M acetate buffer, 0.06 μg F/ml, pH 5.0). The proportions of the de- and remineralization solutions per area of exposed enamel were 6.25 and 3.12 ml/mm2 respectively. The plastic tubes containing the samples were maintained at 37 °C and under a constant agitation of 200 rpm throughout the entire cycling period. After

completion of cycling procedure, before and between the further investigations, the specimens were stored on wet cotton fabric at room temperature and at a constant relative humidity of 100%. After the end of the pH-cycling procedures, the samples were removed from the plastic tubes and the amount of calcium and phosphorous released into the two solutions (de- and remineralization) was measured with an inductively coupled plasma optical emission spectrometer (ICP-OES; Spectro Flame M 120, Spectro Analytical Instruments GmbH and Co. KG, Kleve, Germany). Before the elements were determined, calibration was performed with calcium and phosphorous standard solutions (Merck KGaA, Darmstadt, Germany).