Does Post-bleaching Fluoridation Affect the Further Demineralization of Bleached Enamel? An In Vitro Study

Hande Kemaloğlu; Hüseyin Tezel; Zeynep Ergücü


BMC Oral Health. 2014;14(113) 

In This Article


This study was approved by the Ege University, Faculty of Medicine, Research Ethics Committee (19/10/2012) and written informed consent was received from participants.

Sample Preparation

Ten maxillary premolars extracted for orthodontic purposes at Ege University, Faculty of Dentistry were selected for this in vitro study. All participants gave written consent prior to the extraction process. The extracted teeth were rinsed in tap water, and cleaned of plaque and debris with a dental hand piece and brush. The buccal, palatal, and occlusal surfaces were checked under a stereomicroscope, and teeth with enamel defects or cracks were rejected. Ten selected teeth were stored in 0.9% NaCl and 0.1% thymol for 1 week at 4°C to eliminate the reproduction of microorganisms, and then rinsed with distilled water. Each tooth was sectioned bucco-palatally into two halves with a diamond disc. These halves were then sectioned longitudinally into two parts, so that four specimens were obtained from each tooth. These specimens were later randomly assigned to one of the four groups, on the condition that each part of every tooth would be in one of the four different groups (Table 1). Then, the teeth were covered with wax except for the enamel surface.

Bleaching Procedure

All specimens in three of the test groups were treated with a commercial in-office bleaching agent of 38% HP (Opalescence Xtra Boost; Ultradent, South Jordan, UT, USA) according to the manufacturer's instructions. The untreated specimens in the fourth group were used as a control group and kept in artificial saliva (0.7 mmol/L CaCl2, 0.2 mmol/L MgCl2, 4.0 mmol/L KH2PO4, 30.0 mmol/L KCl, 20.0 mmol/L HEPES; pH 7.0) during the test period.[14,15]

A thick layer (~1 mm) of 38% HP (pH ≅ 7) was applied to the enamel surfaces of the specimens in the test groups (Table 1). To achieve optimum effectiveness, the bleaching gel was stirred/agitated every 5 min and refreshed every 15 min. The total time of application was 45 min per day. This procedure was repeated every other day for 3 days. After removing the whitening gel, the teeth were rinsed, dried, and kept in artificial saliva until the next procedure.

Post-fluoridation Process

Two out of the three test groups were treated with two different fluoride agents with approximately the same concentrations; 1.5% TiF4 (Aldrich Chem. Co, Milwaukee, WI, USA) (pH = 1.2, 9200 ppm) and 2.1% NaF (Merck, Switzerland) (pH = 1.2, 9500 ppm). They were applied for 60 s using a pipette while the third test group was left untreated and kept in artificial saliva during the test period after the bleaching process.

Demineralization Process

Immediately after the application of the bleaching and fluoride agents for the prescribed time, the specimens were rinsed with a water spray and dried with blasts of air. The enamel was then covered with standard "o"-shaped wax so as to expose a standard round window area (6.83 mm[2]) and acetic acid buffered with 0.34 M sodium acetate (pH = 4) was used as a demineralization buffer. A calcium monohydrate salt [Ca (H2PO4)2H2O)] was dissolved to obtain 10 mmol/L Ca2+ and 20 mmol/L PO43- in the solution.[16]

Each specimen was treated with 50 mL of solution in polyethylene test tubes. The specimens were demineralized in four consecutive periods over 4 days. At the end of the 4th day, each specimen was taken out of the test tube and placed in a new tube, which contained fresh buffer solution. The previous solutions were kept in their tubes to be tested afterwards for their Ca2+ concentration using an atomic absorption spectrophotometer (AAS), as performed in previous studies.[4,16]

Calcium analysis was undertaken with the AAS using 0.1 mL of each demineralization solution, which was diluted with 4.9 mL of distilled water. To prevent the interaction of magnesium and phosphate ions, 50,000 mg/L of lanthanum chlorine (LaCl2) was added to each test tube to make up 10% LaCl2 in each buffer solution. The same procedure was applied to blank (buffer) and standard solutions of calcium. The amount of calcium released from tooth to buffer was calculated by measuring the difference in Ca2+. The calcium concentration in the samples was detected with an AAS (Varian Spectra-10 plus AA; Varian, Melbourne, Australia) (wavelength: 422.7 nm; slit 0.5 nm). The calcium released to the buffer after the 4th, 8th, 12th, and 16th days were compared using Friedman and Wilcoxon tests.