Ninety for orthodontical reasons extracted caries free premolars were covered with varnish leaving a 3 × 3 mm window and divided into 6 groups of 15 teeth in each group. They were kept in a demineralizing gel (hydroxyethylcellulose) at pH 4.8 for 50 days. After demineralization the lower half of the window was also covered with varnish serving as positive control. The teeth of each group were then incubated in different toothpaste slurries, control medium or remineralization solution for 48 hours which simulates 2 years of tooth brushing 2 times for 2 minutes per day [23
]. The remineralization solution contained potassium chloride (KCl 1 mmol/l), sodium acetat (0,2 mol/l), calcium chloride (CaCl2
150 mmol/l) and potassium hydrogen phosphate (KH2
90 mmol/l). Incubation media are summarized in Table .
After treatment with slurries 30 teeth were embedded in Technovit 9100 (Kulzer, Weinheim, Germany) and serial sections through the lesions with a thickness of 80 μm were cut using a saw microtome (Leica 1600, Bensheim, Germany). All sections were investigated with polarization light microscopy (PLM) and the birefringence of the lesions was categorized according to their morphological appearance, and to each category a numerical index number was assigned as follows: no lesion (1), single porosities (2), interrupted lesion band (3), inhomogeneous lesion (4) and completely homogeneous lesion (5). The numerical values were statistically compared using the nonparametric Mann-Whitney test.
Three sections of each lesion were then coated with carbon and examined with a scanning electron microscope (Philips XL 30 FEG, Eindhooven, The Netherlands) at 20 kV using the backscattered electron detector. In each experimental and control window of the different teeth 3 spot measurements (spot size 2 nm) were carried out on the enamel surface, within the body of the lesion and on surrounding sound enamel, resulting in a total number of 9 measuring points per window. Element content in weight % of Ca, P, C, and F was measured with energy dispersive X-ray analysis (EDX) with a S-UTW detector (EDAX INC, Mahwah, NJ, USA). The count rate of the EDX detector was between 1800 and 2000 counts per second with a dead time of 30 %. Measuring time was 30 s (live seconds) with a resolution of 135.8 eV and an amplification time of 100 μs. Line scans through the lesions were made at 256 points with a dwell time of 1000 ms and amplification time of 100 ms. The values of the spot measurements were statistically evaluated using the nonparametric ANOVA test for repeated measurements. As 3 calculations were made on the same set of data, the Bonferroni correction for p = 0.05 was p = 0.016.
On ten teeth of each group incubated with slurries (n = 60), enamel etch samples were conducted on control windows and on experimental windows by etching an isolated round surface of the teeth for 1 min with 4 μl of 0.5 % perchloric acid [25
]. The enamel samples were analyzed for fluoride using the ion-specific Orion-9609 electrode. The resistance to acids of dental enamel was measured in terms of the amount of dissolved phosphorus resulting from the etching process. Phosphorus was determined photometrical by using flow injection analysis. The results were evaluated with Student's paired t-test.
This study was conducted with the approval of the Ethical Committee of the University of Witten/Herdecke.