EDX-Element Analysis of the In Vitro Effect of Fluoride Oral Hygiene Tablets on Artificial Caries Lesion Formation and Remineralization in Human Enamel.

Aim of this in-vitro-study was to assess the remineralization potential of a tooth cleaning tablet with different fluoride content quantitatively using EDX analysis.Twenty three caries free impacted third molars were examined; enamel surfaces were wax coated leaving two 3x4mm windows for exposure to demineralization/remineralization cycles. The teeth were randomly assigned to 4 groups of 5 control and 6 experimental teeth each. Demineralization by standardized HEC-gel, pH 4.7 at 37°C for 72h, was alternated by rinsing in remineralization solution, pH 7.0 at 37°C for 72h, total challenge time 432h. The negative control group N was treated during remineralization cycles with saline; positive control group P was treated with remineralization solution; experimental group D1 was exposed to remineralization solution containing Denttabs(®)-tablets with 1450 ppm F; experimental group D2 was exposed to remineralization solution and Denttabs(®)-tablets with 4350 ppm F. Each tooth was cut into serial sections and analyzed by scanning electron microscopy with EDX element analysis for assessment of the different zones of the lesions in 3 representative sections. Statistical analysis was based on the AVOVA test for repeated measurements and post hoc Bonferroni adjustment. The results showed a significantly higher Ca and P content in the body of the lesion in both fluoride treated groups compared to the controls. It can be concluded that higher concentrations of NaF may be more effective in remineralization of early advanced caries lesions.

RCT Entities:   Population Interventions Outcomes

INTRODUCTION

The use of class="Chemical">fluorides is the most effective method iclass="Chemical">n class="Chemical">n class="Disease">caries inhibition. In caries preventive protocols fluoride containing dentifrices are widely used. Dentifrices contain usually between 1200 and 1450 ppm fluoride. Their effect on remineralization of initial caries lesions depends upon the fluoride concentration and the fluoride formulation [1]. Recently it could be shown, that remineralization of caries lesions is enhanced by higher fluoride concentrations [2, 3]. A novel application of fluoride is the use of oral hygiene tablets. The advantage of the use of oral hygiene tablets is that they are rapidly dissolved in saliva, and that the fluoride bioavailability immediately after tooth brushing is higher compared to a conventional dentifrice [4-6]. A recent study reported that the effectiveness of the remineralization of initial artificial caries lesions depends upon the fluoride concentration in the tablets [7].

In vitro studies address mechanistic questions of enamel de- and remineralization with standardized methods. During the past decades pH cycling models for the determination of the efficiency of class="Chemical">fluoride products for remiclass="Chemical">neralizatioclass="Chemical">n of class="Chemical">n class="Disease">caries lesions have become state of the art [3, 8, 9]. Different methods to determine the effectiveness of fluoride products on artificial caries lesions have been used. Mineral loss and uptake has been measured by determining the Ca content in the de- and remineralizing solutions [10, 11]. Most widely microradiography of the lesions has been applied for determining the demineralization and remineralization effect [2, 12]. It is the advantage of polarized light microscopy to characterize the morphological features of remineralization effects in initial artificial caries lesions [7]. Serial sections through caries lesions can further be investigated using SEM imaging and EDX element analysis [1, 13-15] It was therefore the aim of this study to determine the quantitative effect on de- and remineralization of artificial caries lesion of different NaF concentrations in a newly developed oral hygiene tablet.

MATERIALS AND METHODOLOGY

Tissue

Twenty three completely impacted class="Species">human third molars were used iclass="Chemical">n agreemeclass="Chemical">nt with class="Chemical">n class="Gene">GLP instructions governing the use of human tissue. These teeth were selected because of the uniform enamel structure with no individual challenges due to the non-exposure to the oral cavity. Immediately after clinically indicated surgical removal they were thoroughly cleaned of organic debris and stored in saline containing 0.1% thymol. No further surface treatment of the enamel was applied. The teeth were than coated with wax leaving a 3x4 mm window on the buccal and lingual smooth surfaces and finally randomly assigned to 4 groups (Table ).

Demineralization/Remineralization, pH-Cycling

pH-cycling conditions were chosen to create advanced artificial enamel lesions on natural smooth surfaces. Each cycle was scheduled for 3 days and was repeated 6 times. The experimental period of pH-cycling lasted therefore 18 days. After demineralization and remineralization (except for the negative control group) the specimens were rinsed in distilled class="Chemical">water to remove excess treatmeclass="Chemical">nt gel solutioclass="Chemical">n. Demiclass="Chemical">neralizatioclass="Chemical">n gel coclass="Chemical">ntaiclass="Chemical">ned 1.5 mM class="Chemical">n class="Chemical">CaCl2, 0.9 mM KH2PO4, 150 mM KCl, 0.1 M sodium acetate buffer, 30 mM acetate in hydroxyehtylcellulose. The pH was adjusted to 4.7 and controlled before and after each 3 day cycle [16].

Remineralization solutions were comprised of 1.5 mM class="Chemical">CaCl2, 0.9 mM class="Chemical">n class="Chemical">KH2PO4, and 150 mM KCl at pH 7.0, again controlled before and after each 3 day cycle.

The n class="Chemical">wax coated teeth were fixed with metal wires aclass="Chemical">nd haclass="Chemical">ng iclass="Chemical">n the respective solutioclass="Chemical">ns aclass="Chemical">nd the volume of each solutioclass="Chemical">n was 100 ml. These solutioclass="Chemical">ns were coclass="Chemical">nstaclass="Chemical">ntly agitated usiclass="Chemical">ng a magclass="Chemical">netic stirrer. All cycles were executed uclass="Chemical">nder coclass="Chemical">nstaclass="Chemical">nt climatic coclass="Chemical">nditioclass="Chemical">ns at 370.

Treatment Groups

The experimental scheme has been previously described in detail by Gaengler et al., 2009 [7]. The negative control group class="Chemical">N of 5 teeth with10 lesioclass="Chemical">ns uclass="Chemical">nderweclass="Chemical">nt demiclass="Chemical">neralizatioclass="Chemical">n cycles oclass="Chemical">nly aclass="Chemical">nd was kept duriclass="Chemical">ng the remiclass="Chemical">neralizatioclass="Chemical">n cycles iclass="Chemical">n class="Chemical">n class="Chemical">saline. The positive control group P of 6 teeth with 12 lesions was remineralized in the specified solution without fluoride content. The 2 experimental groups (D1 and D2) of 6 teeth with 12 lesions each were exposed to remineralization solution containing Denttabs ® with 1450 ppm F- (D1) or Denttabs ® with 4350 ppm F- (D2). The oral hygiene tablets contain fluoride from NaF, and the other ingredients according to INCI are microcrystalline hydroxyethylcellulose, hydrated silica, sodium hydrogen carbonate, sodium laurylsulfate, ascorbic acid, magnesium stearate, aspartame and mint flavor (Innovative Zahnputzgesellschaft mbH, Berlin, Germany).

The tablets were suspended in the remineralization solution with a ratio of one tablet per 5 ml solution to simulate the maximal bioavailability of n class="Chemical">fluoride immediately after brushiclass="Chemical">ng [4].

Scanning Electron Microscopy

After removal of the class="Chemical">wax coaticlass="Chemical">ngs staclass="Chemical">ndardized micro photos of all lesioclass="Chemical">ns at 10x magclass="Chemical">nificatioclass="Chemical">n were takeclass="Chemical">n. Before further processiclass="Chemical">ng the roots were removed. All teeth were theclass="Chemical">n dehydrated iclass="Chemical">n graded class="Chemical">n class="Chemical">alcohol and embedded in Technovit 9100 (Kulzer, Weinheim, Germany). Serial ground sections were cut with a saw microtome (LEICA CM 1900, Leica, Wetzlar, Germany) with a thickness of 80 µm in corono-apical direction. Three ground sections per tooth representing two approximal lesions on the buccal and lingual sides were used for quantitative assessment of calcium, phosphorus, fluoride and carbon content. The results of the polarization light microscopic investigations of the sections have already been published [7]. Prior to electron microscopy the sections were coated with carbon. The sections were investigated with a Philips XL 30 FEG scanning electron microscope (Philips, Eindhoeven, Netherlands). Acceleration voltage was 20 kV using a backscattered electron detector (BSE). Element analysis was carried out with an energy dispersive x-ray analysis system (EDX) using a S-UTW detector (EDAX INC, Mahwah, NJ, USA). In each lesion within the superficial zone, the body of the lesion, and sound enamel five measuring points were selected. The measuring time was 60 seconds with a resolution of 135.8 eV and an amplification time of 100 live seconds. Line scans through whole caries lesions of the teeth were made at 510 points with a dwell time of 1000ms and an amplification time of 10 live seconds.

Statistical Analysis

The obtained data were processed with the Statistical Package for Social Sciences (SPSS 17.0, Chicago, III., USA). class="Chemical">Normal distributioclass="Chemical">n of the data was tested with the Leveclass="Chemical">ne test aclass="Chemical">nd for comparisoclass="Chemical">n of the data betweeclass="Chemical">n the differeclass="Chemical">nt groups. the Aclass="Chemical">n class="Chemical">NOVA test for multiple comparisons with post hoc adjustment after Bonferroni was used. The data within the groups were compared using the non parametric sign test for related samples. The level of significance after α-adjustment was determined at p<0.025.

RESULTS

Scanning electron microscopy of the lesions revealed remarkable differences in the lesion morphology. The negative control group class="Chemical">N showed iclass="Chemical">nhomogeclass="Chemical">neous large lamiclass="Chemical">nated lesioclass="Chemical">ns (Fig. ). The positive coclass="Chemical">ntrol P showed also large lesioclass="Chemical">ns, but less expressed (Fig. ). Iclass="Chemical">n experimeclass="Chemical">ntal group D1 the lesioclass="Chemical">ns were smaller aclass="Chemical">nd clearly less demiclass="Chemical">neralised. The body of the lesioclass="Chemical">n was almost abseclass="Chemical">nt (Fig. ). Iclass="Chemical">n experimeclass="Chemical">ntal group D2 the lesioclass="Chemical">ns were eveclass="Chemical">n smaller with a class="Chemical">narrow baclass="Chemical">nd as body of the lesioclass="Chemical">n (Fig. ). The liclass="Chemical">ne scaclass="Chemical">n through a lesioclass="Chemical">n of group class="Chemical">n class="Chemical">N demonstrated the demineralization of the body of the lesion and the non demineralized superficial zone (Fig. ).

In the superficial layer the class="Chemical">calcium aclass="Chemical">nd class="Chemical">n class="Chemical">phosphorus content was similar in the control group N and experimental group D1 and D2 (Tables and , Fig. ). The content of phosphorous and calcium in the body of the lesion was significantly higher in groups D1 and D2 compared to both control groups N and P. The numeric values are summarized in table 1. All measurements showed a rather wide range of variation (Fig. ).

Table 1

Element Content in Weight % in The Different Lesion Zones

Lesion Zone	Group	Value	C	F	P	Ca	Ca/P
Superficial layer	N	Median	12.19	0.15	18.65	34.24	1.84
		Mean	12.52	0.17	19	35.08	1.84
		SD	2.8	0.13	1.5	3.36	0.06
	P	Median	12.48	0.2	18.08	32.74	1.82
		Mean	12.95	0.21	18.06	32.67	1.81
		SD	2.89	0.15	1.11	2.54	0.07
	D1	Median	11.48	0.21	18.88	34.62	1.84
		Mean	11.89	0.31	19.03	35.01	1.84
		SD	2.38	0.27	1.37	3.19	0.06
	D2	Median	11.63	0.21	18.56	34.31	1.85
		Mean	11.98	0.23	18.56	34.51	1.86
		SD	2.26	0.18	1.21	2.74	0.06
Body of the lesion	N	Median	26.19	0.18	15.49	28.6	1.86
		Mean	26.65	0.2	15.62	29.15	1.86
		SD	4.3	0.14	1.51	3.61	0.07
	P	Median	27.68	0.15	14.81	27.15	1.83
		Mean	28.33	0.16	14.73	26.93	1.82
		SD	7.34	0.12	2.05	4.64	0.10
	D1	Median	20.3	0.33	16	31.48	1.86
		Mean	21.39	0.42	16.79	31.27	1.86
		SD	6.23	0.3	2.01	4.39	0.07
	D2	Median	21.5	0.29	16.31	30.57	1.89
		Mean	21.9	0.33	16.31	30.71	1.88
		SD	6.44	0.2	1.82	3.5	0.05

For the body of the lesion statistical analysis showed significant differences in the content of all measured elements between group class="Chemical">N aclass="Chemical">nd P aclass="Chemical">nd class="Chemical">n class="Gene">D1 and D2. In the superficial layer the Ca, P and C content was significantly higher in group D2 compared to group P. All statistical results are summarized in Table .

The Ca/P ratio was between 1.81 and 1.88 reflecting not the ratio of chemically pure class="Chemical">hydroxyapatite, which is 1.66. However, a statistically sigclass="Chemical">nificaclass="Chemical">nt differeclass="Chemical">nce iclass="Chemical">n the Ca/P ratio was fouclass="Chemical">nd betweeclass="Chemical">n the superficial layer of class="Chemical">n class="Chemical">N-P, P-D1, P-D2 D1-D2 and in the body of the lesion between N-P, P-D21, PD2 and D1-D2 (Tables and ).

Comparison of the element content within the groups showed, that for C, Ca and P the element contend was significantly different (p < 0.001) between the superficial layer, the body of the lesion and sound enamel. The class="Chemical">fluoride coclass="Chemical">nteclass="Chemical">nt was sigclass="Chemical">nificaclass="Chemical">ntly higher iclass="Chemical">n the superficial layer of D1 compared to the coclass="Chemical">ntrol group class="Chemical">n class="Chemical">N. In the body of the lesion the fluoride content was significantly higher in D1 and D2 compared to control N, and P and to D2 (Table. ). In both D1 and D2 groups the fluoride content in the body of the lesion was higher than in the superficial layer.

DISCUSSION

The predominant effect of topically applied class="Chemical">fluoride is to iclass="Chemical">nhibit demiclass="Chemical">neralizatioclass="Chemical">n aclass="Chemical">nd iclass="Chemical">ncrease remiclass="Chemical">neralizatioclass="Chemical">n. class="Chemical">n class="Chemical">Newer studies showed that the efficiency of higher concentrations of fluoride in oral hygiene products for remineralization of advanced subsurface caries lesions is even better [2, 3, 7]. De- and remineralizing processes are yet not fully understood. This investigation showed that the mineral content within the surface layer was not different between the demineralized control group and the remineralised NaF groups. On the other hand the mineral content in the body of the lesion increased significantly compared to the demineralized control group. This indicates that fluoride enhances the remineralization of the body of the lesion. This is also supported by the increased fluoride content of the body of the lesion.

The pH cycling model, applied in this investigation, resulted in well defined advanced class="Disease">caries-like lesioclass="Chemical">ns, which have beeclass="Chemical">n described earlier [7]. Iclass="Chemical">n agreemeclass="Chemical">nt with the data already reported, the quaclass="Chemical">ntitative elemeclass="Chemical">nt aclass="Chemical">nalysis democlass="Chemical">nstrated a similar remiclass="Chemical">neralizatioclass="Chemical">n poteclass="Chemical">ntial of the 4350 ppm class="Chemical">n class="Chemical">fluoride concentration and the 1450 ppm fluoride concentration. This seems to be contradictory with previous studies, which demonstrated that higher fluoride concentrations enhance the remineralization of advanced caries lesions [2, 3, 12]. However, Yamazaki et al. (2007) [12] observed, that already low concentrations of fluoride prevented demineralization of sound enamel, and higher concentrations enhanced remineralization of artificial caries-like lesions. The presented results clearly demonstrate, that the remineralization effect is due to fluoride, because the content of Ca and P in D1 and D2 is higher in the body of the lesion compared to the positive control group.

Long term bioavailability of class="Chemical">fluoride is esseclass="Chemical">ntial for remiclass="Chemical">neralizatioclass="Chemical">n of class="Chemical">n class="Disease">caries lesions. In-vivo bioavailability of fluoride is dependent upon several factors which are fluoride formulation, fluoride concentration and salivary composition [5, 6]. After fluoride administration the fluoride concentration in dental plaque increases. [6, 17].

Inhibition of demineralization and/or enhancement of remineralization depend upon the availability of free class="Chemical">fluoride ioclass="Chemical">ns. It has beeclass="Chemical">n discussed that class="Chemical">n class="Chemical">fluoride may form CaF2 fluoride depots which slowly release fluoride ions [18, 19]. However, it could be shown that shortly after the application of a sodium fluoride mouth rinse CaF2 – like deposits were not formed in plaque [20].

The complex class="Chemical">fluoride kiclass="Chemical">netics iclass="Chemical">n saliva aclass="Chemical">nd plaque fluid are iclass="Chemical">nflueclass="Chemical">nced by both sources, the class="Chemical">n class="Chemical">fluoride secretion via salivary glands and, mainly, by alimentary uptake or topical application of fluoride containing oral hygiene products. The pathobiological conditions on biofilm coated tooth surfaces are not entirely reproduced by the in-vitro cycling remineralization model. However, the results clearly demonstrate the elevated bioavailability of fluoride contributes to enhanced remineralization of advanced caries-like enamel lesions.

The SEM images of subsurface lesions confirm the dynamic remineralization process with laminations at the forefront of mineral deposits and with decreasing pore volume. This is further supported by the significant increase of class="Chemical">calcium aclass="Chemical">nd class="Chemical">n class="Chemical">phosphorus exclusively in the body of the caries lesion if fluoride was available.

Table 2

Statistical Differences of the Element Content Within the Different Lesion Zones.

		p Value
Lesion Zone	Groups	C	F	P	Ca	Ca/P
Superficial layer	N/P	p = 0.864	p = 0.435	p < 0.001	p < 0.001	p<0.0001
	N/D1	p = 0.208	p < 0.001	p = 1.000	p = 1.000	p=1.000
	N/D2	p = 0.445	p = 0.064	p = 0.017	p = 0.554	p=0.230
	P/D1	p = 0.001	p < 0.001	p < 0.001	p < 0.001	p < 0.001
	P/D2	p = 0.003	p = 1.000	p = 0.002	p < 0.001	p < 0.001
	D1/D2	p = 1.000	p < 0.001	p = 0.004	p = 0.638	p=0.018
Body of the lesion	N/P	p = 0.125	p = 1.000	p < 0.001	p < 0.001	p<0.001
	N/D1	p < 0.001	p < 0.001	p < 0.001	p < 0.001	p=1.000
	N/D2	p < 0.001	p < 0.001	p = 0.011	p = 0.006	p= 0.230
	P/D1	p < 0.001	p < 0.001	p < 0.001	p < 0.001	p < 0.001
	P/D2	p < 0.001	p < 0.001	p < 0.001	p < 0.001	p < 0.001
	D1/D2	p = 1.000	p < 0.001	p = 0.092	p = 1.000	p= 0.018

Statistically significant differences are marked in red.