Does post-bleaching fluoridation affect the further demineralization of bleached enamel? An in vitro study

Bleaching has been accepted as one method of treating discolored teeth. Recently, novel in-office bleaching products that use high concentrations of hydrogen peroxide (HP) have made in-office treatments easier. However, the effects of these products on enamel are still an open issue and need to be clarified. When vital teeth are bleached, as a result of the direct contact between the bleaching agent and the outer enamel surface, the enamel surface of the tooth crown can be affected by high levels of HP in bleaching agents, causing structural and morphological changes. There are many studies on the reduction in microhardness as well as the loss of calcium from bleached enamel [1–4]. Furthermore, some changes in bleached enamel were described as slightly erosive defects promoted by the bleaching agent [4–7].

The positive effect of highly concentrated fluoride products related to the inhibition of demineralization and erosion is well documented [8]. Different topical fluoride applications such as sodium fluoride (NaF), acidulated phosphate fluoride, and stannous fluoride are widely used in promoting enamel remineralization. However, unlike the commonly used agents, it has been suggested in the literature that titanium tetrafluoride (TiF₄), may have a greater effect on enamel remineralization. Furthermore, the use of fluoride products after bleaching procedures has also been shown to be beneficial [2, 9, 10]. As topical fluoride is applied following bleaching, mineral loss is significantly reduced, microhardness is restored, and the resistance of enamel to demineralization is increased [4, 10, 11].

Fluoride has been confirmed to remineralize lesions by increasing resistance to acid attack by forming a calcium fluoride layer to inhibit demineralization [9]. In addition, the formation of a glaze layer has been shown when enamel surfaces were treated with low pH TiF₄[12, 13]. However, there are no data available in the literature on the preventive effect of these applications on further demineralization. Thus, the aim of this present study was to examine the effects of two different post-bleaching fluoridation agents (NaF and TiF₄) on the Ca²⁺ loss from enamel after an acidic challenge. The null hypotheses tested were: (1) no difference will be observed in Ca²⁺ release between the fluoridated and non-fluoridated groups of bleached enamel surfaces, after being subjected to acidic attack; thus, these fluoride agents will not affect the susceptibility of bleached enamel to further demineralization and (2) no differences in Ca²⁺ release will be noted between NaF- and TiF₄-treated enamel surfaces after being subjected to further demineralization following bleaching with 38% HP.

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.

The Ca²⁺ concentrations of the samples were measured at the end of the 4th, 8th^, 12th, and 16th days (Table 2, Figure 1). The loss of Ca²⁺ in the control, 38% HP, 38% HP + NaF, and 38% HP + TiF₄ groups were evaluated cumulatively every 4 days, and at the end of the 16th day, 15.07 ± 1.81 μg/mL, 22.44 ± 2.52 μg/mL, 13.67 ± 1.86 μg/mL, and 9.12 ± 2.40 μg/mL were obtained in total, respectively (Figure 2).

	N		4th day	8th day	12th day	16th day	Total
Control group	10	Mean	3,59	3,2	3,72	4,55	15,07
		Std dev	0,54	0,59	1,23	0,89	1,81
		Min.	3,01	2,46	2,46	2,74	12,6
		Max.	4,93	4,38	6,58	5,75	18,36
38% HP	10	Mean	5,75	5,18	5,56	5,95	22,44
		Std dev	1,6	1,52	0,71	0,37	2,52
		Min.	3,83	3,01	4,66	5,48	18,36
		Max.	7,95	8,5	6,85	6,58	27,68
38% HP + NaF	10	Mean	2,05	3,15	3,94	4,52	13,67
		Std dev	0,83	0,58	1,01	0,83	1,86
		Min.	0,82	2,19	2,46	3,29	10,68
		Max.	3,01	4,11	5,75	6,03	15,61
38% HP + TiF4	10	Mean	1,15	1,94	2,66	3,37	9,12
		Std dev	0,92	1,54	0,63	0,48	2,4
		Min.	0	0	1,64	2,46	4,93
		Max.	2,46	4,11	3,56	3,83	11,5

The loss of Ca²⁺ in each of the test groups was compared with that of the control group using the Friedman test. A statistically significant difference was observed among the groups after 4, 8, 12 and 16 days and in total (p < 0.05). The Wilcoxon test was used to identify possible statistically significant differences between the groups.

High concentrations of HP that promote enamel surface alterations soften the superficial layer of the enamel surface, increase surface porosity, and release more Ca²⁺ than low concentrations of HP and carbamide peroxide (CP) [4, 15, 17]. Thus, 38% HP has been recruited for the present study to investigate the Ca²⁺ released from the bleached enamel surfaces after an acidic challenge. With this designated high concentration, it was aimed to observe the maximum Ca²⁺ release after a further demineralization process.

Alterations in the inorganic component of hydroxyapatite might be an indicator of the changes in Ca²⁺ levels of enamel. Rotstein et al.[1] demonstrated that most bleaching agents may cause various changes in the levels of calcium, phosphorus, and potassium in dental hard tissues; whereas Tezel et al.[4] demonstrated that 35% and 38% HP caused Ca²⁺ loss from the enamel surfaces. In the present study, Ca²⁺ released from the enamel of specimens treated with 38% HP was significantly higher than the untreated control group (p < 0.05). Based on the Ca²⁺ values, this result suggests that high concentrations of bleaching agents cause surface alterations after an acidic challenge.

The application of highly concentrated fluoride favors the formation of a CaF₂-like layer [18]. This deposit is later dissolved, allowing fluoride to diffuse into the underlying enamel, saliva, or a plaque layer covering the tooth. It is assumed that some of the fluoride supports the remineralization of enamel. The results of a previous study confirmed that phosphates and proteins from saliva coated the calcium-fluoride layer on the enamel as a pH-controlling reservoir. This layer acted to decrease demineralization and promote remineralization [19].

Al-Qunaian et al.[20] investigated the effects of whitening agents on caries susceptibility of human enamel and reported that no significant differences in caries susceptibility were observed between the untreated control specimens and those specimens treated with 10% CP, 20% CP with fluoride, and 35% HP. There were no significant differences between the treated and controlled specimens for teeth treated with 10% CP or 35% HP. However, specimens treated with whitening gel containing 20% CP with fluoride had significantly reduced caries susceptibility when compared with their untreated controls. It was claimed that this effect could be related to fluoride incorporation in 20% CP gels containing fluoride, and the results were in agreement with laboratory studies that fluoride enhanced enamel remineralization.

In the present study, fluoride agents were applied to the bleached enamel and then subjected to further demineralization. When the test groups that were bleached with 38% HP were compared, the decrease in Ca²⁺ loss of the 1.5% TiF₄-treated group was detected to be the lowest (Table 2). Regarding this result, it can be assumed that TiF₄ may be effective in protecting the bleached enamel surface against acid attack. Interestingly, no Ca²⁺ release was detected from three specimens of TiF₄ group during the first 4 days, and there was also no Ca²⁺ release from two specimens during the second 4-day interval (Table 2). We assume that this effect might be the result of the glaze formation after topical TiF₄ application. It is known that formation of a glaze layer takes less than 10 s after the application of TiF₄[21]. The ability of TiF₄ to strongly protect enamel against the action of acid is a synergistic effect of glaze formation and increased enamel fluoride content. The high fluoride content and great reduction in solubility found in TiF₄-treated enamel suggests that a fluoride reaction with the enamel is involved [22]. In a previous study, Tezel et al.[16] reported that TiF₄ was found to be more effective than Duraphat (NaF, 2.26% F) or Elmex (amine fluoride, 1.25% F) in preventing artificial enamel lesion formation. Attin et al.[23] reported that fluoridation was effective in increasing the resistance of enamel against demineralization by erosive substances. Similarly, the findings of this present study demonstrated that the resistance of bleached enamel against acid attack increased after 1.5% TiF₄ treatment.

The comparison of the Ca²⁺ losses from the test groups that were bleached with 38% HP revealed that the decrease in Ca²⁺ losses of the 2.1% NaF-treated group was also lower, indicating that NaF could also protect enamel surfaces against acid attack. When the NaF-treated group was compared with the control group, it was seen that the amount of Ca²⁺ lost from the NaF group was significantly different during the first 4 days (p < 0.05) (Table 3). However, when the NaF-treated group was compared with the 38% HP group, the difference was statistically significant during the whole test period (16 days) (p < 0.05).

However, when the effect of NaF treatment against acid attack was compared with TiF₄ treatment, it was observed that its influence was not as strong as TiF₄ (Table 3, Figure 1). Tveit et al.[24] assumed that complexes were formed between TiF₄ and hydroxyapatite, based on a strong binding of the titanium compound and the oxygen atom of the phosphate group. Mundorff et al.[22] suggested that TiF₄ acted with enamel both chemically, by decreasing enamel solubility, and physically, owing to the formation of a protective glaze on the enamel surface. van Rijkom et al.[25] compared the erosion-inhibiting effect of topical fluoride treatment based on the deposition of CaF₂-like material using 1% NaF and 4% TiF₄. It was concluded that the reduction of Ca²⁺ loss was more stable for TiF₄ than for the NaF group, and the reduction appeared to be smaller with longer acid exposure times.

Generally, fluoride uptake of demineralized enamel is higher when compared with sound enamel [26]. It is assumed that the applied fluoride can easily penetrate through the porous structure of demineralized enamel and that can create a higher number of possible retention sites [27, 28]. According to the results of one study [29], the bleached and fluoridated enamel was more resistant against erosive attacks than the bleached/unfluoridated and unbleached/unfluoridated enamel. In the present study, at the end of the test period, the total amount of calcium released from the bleached/fluoridated specimens was lower than the control group (unbleached/unfluoridated) and the difference was significant in TiF₄-treated specimens (p < 0.05) (Table 3). This result is noteworthy in that bleached and fluoridated teeth may be more resistant to acid attack than sound teeth. These results would need to be investigated with further studies.

In the last decade, there has been a growing interest in demineralization and remineralization studies because of the demand for minimally invasive treatment techniques. When scanning through the literature, we encountered a number of different techniques applied in these types of studies. In vitro demineralization using acid buffers and in vitro demineralization/remineralization using a pH-cyling model are the most frequently used techniques that possess both advantages and disadvantages. It is important to choose the simplest and most practically appropriate model. Similar to the previous studies, we preferred using AAS for in vitro demineralization to observe the impact of fluoride agents on the Ca²⁺ loss following further demineralization. This method is a very sensitive but reliable method for calcium analysis, which avoids the interaction of other solutes [30, 31]. It can be used with confidence to quantify erosion of both enamel and dentine, and their chemical analyses of mineral release [32–34].

Based on the results of the present study, the null hypothesis that states there would be no difference between the amount of Ca²⁺ released from the enamel surfaces that were treated with NaF and TiF₄ after an acidic challenge was rejected. It was also shown that topical fluoride application decreased the amount of Ca²⁺ released from the 38% HP-treated enamel surfaces after further demineralization. TiF had a significantly more pronounced effect than NaF in protecting enamel surfaces against acidic attack after bleaching with 38% HP.

It may be concluded that post-bleaching fluoride application may be beneficial in reducing the risk of demineralization caused by acid attack after bleaching processes and to remineralize the bleached enamel surfaces. In addition, TiF₄-treated samples released less Ca²⁺ than NaF-treated samples, which indicates that TiF₄ may be more effective than NaF in preventing damage from acid attack.