Effect of fluoride application during radiotherapy on enamel demineralization

Abstract Radiation-related caries are one the most undesired reactions manifested during or after head and neck radiotherapy. Fluoride application is an important strategy to reduce demineralization and enhance remineralizaton. Objective: To evaluate the effect of the topical application of fluoride during irradiation on dental enamel demineralization. Material and Methods: Thirty molars were randomly divided into three groups: Non-irradiated (NI), Irradiated (I), Irradiated with fluoride (IF). Each group was subdivided according to the presence or absence of pH-cycling (n=5). In the irradiated groups, the teeth received 70 Gy. The enamel's chemical composition was measured using Fourier Transform Infrared Spectrometry (organic matrix/mineral ratio - M/M and relative carbonate content - RCC). Vickers microhardness (VHN) and elastic modulus (E) were evaluated at three depths (surface, middle and deep enamel). Scanning electron microscopy (SEM) was used to assess the enamel's morphology. Results: The FTIR analysis (M/M and RCC) showed significant differences for irradiation, pH-cycling and the interaction between factors (p<0.001). Without pH-cycling, IF had the lowest organic matrix/mineral ratio and relative carbonate content. With pH-cycling, the organic matrix/mineral ratio increased and the relative carbonate content decreased, except for IF. VHN was influenced only by pH-cycling (p<0.001), which generated higher VHN values. ANOVA detected significant differences in E for irradiation (p<0.001), pH-cycling (p<0.001) and for the interaction between irradiation and pH-cycling (p<0.001). Increased E was found for group I without pH-cycling. With pH-cycling, groups I and IF were similar, and showed higher values than NI. The SEM images showed no morphological changes without pH-cycling. With pH-cycling, fluoride helped to maintain the outer enamel's morphology. Conclusions: Fluoride reduced mineral loss and maintained the outer morphology of irradiated and cycled enamel. However, it was not as effective in preserving the mechanical properties of enamel. Radiotherapy altered the enamel's elastic modulus and its chemical composition.


Introduction
Of the several therapeutic modalities, radiotherapy is one of the most widely used in the treatment of head and neck cancer 1 . Patients undergoing radiotherapy often develop early and late oral radiation-induced complications such as mucositis, hyposalivation and subsequent xerostomia, taste loss, osteoradionecrosis and radiation-related caries. 1 Radiation-related caries are a lifelong risk, and not only during or shortly after the treatment. 2 Dental caries have a rapid onset and progression, and may be accompanied by enamel delamination, leading to crown amputation. [2][3][4] The occurrence and severity of radiation caries are related to the changes in the quality and quantity of saliva, changes in the oral microbiota, difficulty in promoting oral hygiene, and increasingly cariogenic diet. [3][4][5] In synergy, there are direct effects on dental tissue, including changes in the crystalline structure, dentinoenamel junction (DEJ), acid solubility of the enamel, enamel and dentin microhardness, and tensile strength. 6,7 An important strategy to prevent dental caries is to reduce demineralization and enhance remineralization. 8,9 There are several fluoridecontaining products on the market for professional application and their anticariogenic effect will depend on the product formed in the enamel and its retention on the surface of the enamel over time. 8 There is no universal protocol for the treatment of radiationrelated caries, however, the importance of fluoride is well recognized. 10 The daily topical application of 1% neutral sodium fluoride gel with custom-made fluoride carriers has been shown to reduce post radiationrelated caries. 11   Uberlândia, MG, Brazil) was applied on the enamel 4 minutes before the radiation procedure, and was maintained throughout the entire irradiation process, totalizing 5 minutes of contact. 13 After this, the specimens were rinsed to remove the fluoride gel, and subsequently stored in deionized water that was changed weekly.

Specimens' preparation
The roots of all teeth were removed 1 mm below the cementoenamel junction, by using a water-cooled diamond saw (Isomet, series 15HC diamond, Buehler Ltd.; Lake Bluff, IL, USA) and a precision saw (Isomet 1000, Buehler; Lake Bluff, IL, USA). On five teeth in each group that was submitted to pH-cycling, an area

Dynamic microindentation test
The specimens' preparation and the experimental protocol were performed as previously described by Soares, et al. 16

FTIR analysis
The spectra for groups NI, I and IF with and without pH-cycling are shown in Figure 2 a Table 4.
Three-way ANOVA revealed significant effect for the irradiation factor (p<0.001), pH-cycling (p<0.001) and for the interaction between irradiation and pHcycling (p<0.001). However, no significant difference was found for enamel depths (p=0.293) and for the interactions between irradiation and enamel depths (p=0.850), pH-cycling and enamel depths (p=0.619), and between irradiation factor, pH-cycling and enamel depths (p=0.721). When comparing the groups without pH-cycling, group I had the highest E values, followed by groups IF and NI. When comparing the groups with pH-cycling, the E values of groups I and IF were similar and higher than those of group NI.
The E values for group I remained constant with or without pH-cycling.

SEM analysis
The enamel of all groups featured well-organized prisms, surrounded by interprismatic regions ( Figure   3A-C). The enamel exposed to pH-cycling resulted in discontinuity of the buccal contour that was more

Discussion
The null hypothesis tested in this study was rejected. The radiation from the linear accelerator altered the chemical composition and elastic modulus of the enamel, and pH-cycling altered its chemical composition and mechanical properties, whereas fluoride attenuated demineralization. Depending on the localization of the malignancy, the salivary glands, oral mucosa and jaws inevitably have to be included in the radiotherapy field 1,2 , causing damage to oral function, and interfering in the patient's quality of life. 5 Radiation-related caries are an atypical pattern of dental caries due to the combination of both hyposalivation and the direct effects on the hard dental tissue. [3][4][5] Under some conditions, the teeth may be completely lost within short periods 2 . There is still no consensus about how the dental hard tissues changed and how the changes may contribute to rapid deterioration of the teeth. 5,19 The literature shows several variables in the form of simulating in vitro radiotherapy. Third molars from 18-25 year-old patients were used to control variability, since it is known that patient age can interfere in the elastic modulus and hardness of the enamel. 20 It has been previously reported that physiological saline solution, artificial saliva or distilled water were used as storage solution. 12,19 In this study, deionized water was used in attempt to simulate the oral conditions of a patient whose head and neck were irradiated soon after the start of irradiation 21   The mechanical properties of the enamel were assessed using a dynamic indentation test.
Radiotherapy increased the VHN values, in spite of not being statistically significant, and the E values. An important aspect of radiotherapy is radiolysis, wherein radiation interacts with water. Despite enamel being mostly composed of mineral phase, it still has 4-6% of organic constituents, such as protein, peptides and water. 22 When radiolysis occurs, reactive unstable free radicals H + and OH − are released and then it can interact with other ions to produce new compounds. 23 This might explain the increase in amide I, phosphate and carbonate content after exposure to radiation, contributing to the altered mechanical properties.
Moreover, the degradation of the water may have led to a dehydrated and more hypermineralized enamel tissue, making it susceptible to the formation of cracks 24 . All these changes made the enamel more friable and could be the mechanism responsible for its delamination, frequently observed after radiotherapy. 25 One of the studied factors was enamel depth. Although In the same way, previous studies have noted the importance of fluoride in the protection of these highrisk patients from caries in combination with good oral hygiene. 7,11,[29][30][31] Kielbassa, et al. 7 (1997)  This finding is opposite to those of previous studies, which have shown no differences between irradiated and nonirradiated enamel lesions 2,3 probably because these authors used in situ experiments, with participants in good general health with normal salivary flow. There are some differences in the clinical oral environment of irradiated and non-irradiated patients, especially with regard to salivary flow. It should be considered that remineralization occurs only with resting salivary flow. 3 Thus, in patients with irreversible damages to the salivary glands or low resting flow rate, remineralization is impaired, which appears to promote an increase in the incidence of radiation-related caries.
No micromorphological alterations in the enamel were revealed after irradiation. However, when the tooth was subjected to pH-cycling, the surfaces evaluated showed aspects of demineralization, seen as discontinuity of the outer morphology of the enamel ( Figure 3G-H). The enamel irradiated with fluoride showed the lowest degree of demineralization ( Figure   3G-H). Since fluoride helps to decrease the solubility of enamel when exposed to acid challenges, 36 this process was expected to occur in a less pronounced way than in enamel that was not exposed to fluoride.
Clinical studies are needed to confirm the results of the in vitro studies and to create protocols with the purpose of minimizing or counteracting radiationrelated caries and damages to the dental hard tissue.
Nevertheless, the use of fluoride during radiation therapy is an applicable approach for patients with head and neck cancer, and a possible strategy for this procedure is the use of fluoride in trays.

Conclusions
The findings of this in vitro study demonstrated that the application of 1% neutral fluoride gel during the irradiation procedure maintained the morphological and chemical integrity of the irradiated and pH-cycled enamel. However, topical fluoride was not as effective in preserving its mechanical properties.