In the present study, after the first cycle of radiation morphological changes were observed in the enamel compared to the non-irradiated sample. In re-irradiated specimens, with the escalation of radiation dose, the changes became even more evident [27,29,31].
Analysis of the area and intensity of the phosphate peaks, demonstrate that RT is affect the mineral and crystalline structure. Analyzing hydroxyapatite peaks (582nm) and carbonate (1070nm), as well as the carbonate/phosphate ratio, the results suggest the occurrence of the hydroxyl substitution process of hydroxyapatite (Ca10(PO4)6OH2) by carbonate, with a consequent increase in the solubility of dental enamel and dissolution of enamel crystals, favoring the demineralization process. Also altering the crystal dimensions, surface texture and stability of the hydroxyapatite, causing damage to this tissue that is even more severe with the re-irradiation process. Similarly, studies suggest that are loss of protein post radiation [14,46,48] and mineral [46,48]. It is important to clinical consideration about the use of fluoride to prevent RRC post-radiotherapy and mainly post re-irradiation therapy [48].
There is a tendency for the carbonate/phosphate ratio (1070/960) to increase with increasing radiation doses in all regions, regardless of the region analyzed, re-irradiation is not more damaging to one region than the other, but rather affects the tooth structure as a whole. Others studies suggest that this damages are even higher in cement-enamel junction [14,46].
It was possible to observe degradation of the interprismatic region and destruction of the enamel prisms and hydroxyapatite crystals, in SEM images. In dentin, indicated obliteration and degradation of the intertubular, peritubular and intratubular structure, presence of cracks and degradation of the collagen fiber [24,30,49]. Grötz et al. (1997) [27] attributed the obliteration of dentinal tubules, to the degeneration of the odontoblastic processes, being the result of direct radiogenic damage to the cells. The dentin acts as support for the enamel, and if its structure is compromised, it is possible that this support becomes less efficient, contributing to the occurrence of enamel fractures and cracks [46].
In order to observe the behavior of some elements were used EDS. In the irradiated enamel, after the 60Gy dose, a slight increase in the oxygen and a small decrease in the phosphorus and calcium were observed. These variations became even greater and more evident when analyzing the re-irradiated specimens. The decrease in the amount of calcium and phosphorus observed may be due to changes in the solubility of enamel after irradiation. This tissue would lose ions to the environment, differently of other studies [50–52], who verified a decrease in subsurface demineralization of enamel after irradiation, and attributed this event to a decrease in solubility. Another explanation, it is apatite crystals in dental enamel incorporate sodium, carbonate and magnesium during their formation and irradiation, it would probably cause punctual defects within the apatite and thus, ions could be removed from the crystal surfaces inside the enamel pores [52].
In addition, a study demonstrated greater expression of matrix metalloproteinase 20 (MMP-20), in the dentin-enamel junction of irradiated teeth. The proteolytic activity of MMP-20 may be responsible for the degradation of the structure of non-collagenous proteins, such as amelogenin and ameloblastin, located in the organic matrix of enamel, leading to delamination of this tissue and contributing to the etiology of RRC [35–37,53–56].
The interaction between radiation and water is high in dentin [57]. The radiolysis process releases H + and OH- ions into the environment, which can interact with other ions and produce new compounds. This fact explains the decrease in C ions and the lower values of Ca/P weight after exposure to radiation. These ions could induce the formation of a secondary non-apatite calcium phosphate phase, which would make the tissue more susceptible to degradation [58], even for long period of time [57], a fact that would justify the increased risk of developing RRC and/or fractures in the dentin structure
Velo (2018) [59] observed the incorporation of magnesium (Mg) after irradiation with 55 and 70Gy. Mg as a substituent component inhibits crystal growth and strongly influences the lattice parameters, which may have made the apatite amorphous. This change favors the occurrence of a less well-structured crystal arrangement, increasing the permeability and susceptibility of this substrate to cracks, contributing to the obliteration of dentinal tubules [60]. These structural defects can make dentin dry and friable [24], in addition to xerostomia that can occur [61], these changes would act synergistically as factors for the occurrence of RRC.
The clinical extrapolation of the results must be cautious, because, despite being reproduce, as much as possible, it is not possible to fully mimic what actually occurs in the living organism [21,33,62]. However, in vitro studies allow the standardization not only of samples, but also of experimental conditions, which would be extremely difficult to achieve in in vivo studies, both due to the nature of the study and the clinical and emotional conditions of the patients. Thus, research conducted with this study model is necessary and contributes to the understanding of the effects of RT on dental tissues, and can contribute to clinical repercussions on patients.
In this way, through the results obtained, this study contributes to the evaluation, quantification and understanding of the direct effects of RT on dental tissues and suggest that the toxicity caused by radiation to dental enamel is even greater and more complex in cases of re-irradiation, being of it is fundamentally important to expand knowledge about the effects caused by additional doses used in cases of retreatment, often above the tolerance doses of irradiated tissues.