In this study, the changes that might be caused by radiation up to 60Gy in the extracted human primary and permanent teeth were investigated. To date, no consensus has been reached on this issue in the literature yet.
It is known that storage solutions are effective on the hardness of extracted teeth. In previous studies, extracted teeth were stored in Hanks' balanced salt solution, PBS (pH:7.4), NaCl (0.9%), normal saline, thymol (0.2%), artificial saliva, and distilled water. A 47% decrease was reported in the dentin hardness of teeth stored in the NaCl for 30 days.20 However, no significant change was determined in the hardness of the teeth stored in Hanks' balanced salt solution.21 Dry environment is also known to adversely affect the mechanical properties of dental specimens due to dehydration.22
In order to simulate the xerostomia/ hyposalivation caused by radiotherapy in patients, Reed et al.14 prefered to use a solution containing a small amount of storage medium instead of immersing dental specimens in PBS completely.
Marangoni-Lopes et al.23 stated that the Ca and P concentrations significantly increased in the artificial saliva in which the specimens were kept during the enamel and dentin irradiation because of the Ca and P loss from the enamel surfaces. Thus, this solution was used to keep the specimens only during radiotherapy.
In this study, the dental samples were stored in the distilled water. During radiotherapy procedures, the teeth were wrapped in a gauze patch impregnated with artificial saliva and placed in the center of a styrofoam container filled with rice. Rice was preferred for the homogeneity of radiation dose distribution in all regions.24 Rice grains were partially crushed to minimize the gaps between the grains. Furthermore, after each daily radiotherapy application, the teeth were put into artificial saliva and kept in the etuve at 37 0C in order to simulate the real oral environment.
It is reported that there will be little or no enamel detachment from the tooth when microhardness is measured with Vickers test rods placed perpendicularly to the cut tooth surface,13 and this affects the microhardness measurement. In our study, the measurement was performed with Vickers measurement rods which were perpendicular to the enamel cut surface.
The measurement points selected for the microhardness analysis were shaped in the light of previous studies.4,10 Because of the increasing amount of organic structure in the DEJ region, this region was stated to be affected more by radiotherapy than the other regions.4,10,12,14 It was reported that the enamel and dentin hardness values increased depending on the point of the measurement, and the hardness value when moved away from the DEJ region.25 In our study, the measurements were made from the enamel and dentin regions 50 µm away from the DEJ region.
Lu et al.12 and Gonçalves et al.4 determined a decrease in the enamel microhardness close to the DEJ region, while de Siqueria et al.10 determined first a decrease and then an increase. As for the dentin, while Gonçalves et al.4 found a decrease in the microhardness close to the DEJ region after radiation, de Siqueria et al.10 first found a decrease and then an increase. In this study, a statistically significant difference was not determined in both enamel and dentin close to the DEJ region in the primary teeth when compared with the nonirradiated teeth. However, the microhardness of the deep enamel in the permanent teeth decreased significantly after 50Gy as compared with 40Gy and decreased significantly after 60Gy as compared with 50Gy.
In the literature, some studies have reported no changes,26,27 some have reported increases,4,10 and some have reported decreases 12,13,16,17,23,28-30 in the overall enamel microhardness after radiotherapy. In this study, we found that in the permanent teeth, the microhardness in the surface/middle/deep enamel and the overall enamel decreased or increased significantly after the 20, 30, 50, and 60Gy doses as compared with the previous lower doses (Figure 3).
There were statistically significant differences in the microhardness of the middle/deep/overall enamel among the groups in the primary teeth, while there were statistically significant differences in the microhardness of the surface/middle/deep enamel and the overall enamel among the groups in the permanent teeth.
There are studies in the literature indicating a decrease in the overall dentin microhardness.4,10,15,23,31-34 It was explained that the reason of this decrease could be the high water content of dentin (10%), decreased vascularization, obliteration of dentinal tubules due to the slowing process of irradiated odontoblast cell metabolism, and the degeneration of collagen fibers due to the effect of free radicals released after irradiation.11 In this study, it was found that, in the permanent teeth, although there was a statistically significant difference among the groups in terms of the microhardness of the surface/middle/deep dentin and the overall dentin, there were no statistically significant differences in them after all the radiation doses when compared with the nonirradiated teeth. The microhardness in the middle dentin of the permanent teeth only increased significantly after 40Gy as compared with the increase after 30Gy. No statistical difference was encountered in the dentin microhardness of the primary teeth after radiotheraph. In addition, there was a statistically significant difference only in the microhardness of the deep dentine among the groups in the primary teeth. For this study, the permanent teeth were collected as a result of the surgical removal of the impacted third molars that had not been erupted in the mouth yet. We think that they may have been more affected by the radiation since the post-eruptive calcification or maturation of the enamel did not occur.
In the literature, there are studies examining the changes in the chemical structure of the teeth after radiation.
Velo et al.15 examined Ca, P, O, C, Mg, and Ca/P weight ratio in the irradiated root dentin by EDX. They reported decreases in O, C, Mg elements, and Ca/P weight ratio after radiotherapy. Cambi et al.11 examined phosphate, carbonate, and amide ratios in the dentin by Raman spectroscopy and reported that they decreased in the irradiated dentin. Reed et al.14 determined a decrease in the protein/mineral ratio and in the carbonate/phosphate ratio in the enamel region close to the DEJ when analyzed with Raman spectroscopy in the human teeth. They attributed the decrease in the protein/mineral ratio to the structural change of collagen in both enamel and dentin. Marangoni-Lopes et al.23 stated that radiotherapy caused a reduction in the mineral and organic contents of the enamel, and a growing increase followed by a reduction after the 0.03Gy dose in the organic contents of the dentin.
On the other hand, Lu et al.12 reported a slight increase in the protein/mineral ratio in the enamel and a decrease in the dentin when analyzed with Raman spectroscopy. They also examined the Ca/P ratio by an Electron Probe Micro-Analyzer and reported that Ca and P elements decreased and the Ca/P ratio increased. de Barros da Cunha et al.30 stated that radiation did not interfere with the enamel Ca and P content.
In this study, Na, K, Mg, P, and Ca elements and the Ca/P weight ratio in the analysis of the primary and permanent teeth's hard tissues were performed with ICP-OES. It was observed that there were statistically significant differences in all the elements investigated and the Ca/P weight ratio among the groups in both primary and permanent teeth. Irregular increases and decreases in Na and Mg elements and Ca/P weight ratio in both primary and permanent teeth were observed with every 10Gy radiation dose increased. However, at the end of the 6-week radiotherapy, the five elements of the primary and permanent teeth decreased when compared with the nonirradiated teeth.
In ICP-OES, elemental analyses of all the hard tissues were performed without distinguishing between enamel and dentin. The reason for the decrease in these elements after radiotherapy can be explained by the fact that they may be replaced by heavy metals or free radicals released. Free radicals are produced by the effect of ionizing radiation. As a result of this, oxidative stress can cause structural and functional modifications by damaging important biomolecules such as DNA, proteins and lipids. Oxidative stress caused by reactive oxygen species has been reported to be effective in the etiology of heavy metal toxicities.35,36 Heavy metals, which are important inducers of oxidative stress, are activated to act as catalysts.
Miculescu et al.37 stated that heavy elements accumulate faster than the major elements of teeth which is lost with aging. It can be thought that radiation may have revealed a similar effect of aging.
Previous studies have also reported that radiation doses have a greater effect on teeth as the doses are increased.4,8,10,12,15,38 In our study, the significant changes in elements started generally after 30Gy and these changes were observed after 40, 50, and 60Gy, too. In the microhardness assessment, the significant changes were observed only in the permanent teeth enamel after 40 and 60Gy.
Even though significant changes were observed in the inorganic structure of the teeth according to the results of the elemental analysis, these changes were slight in microhardness analyses. We estimate that this may be due to the fact that hyposalivation was not fully reflected in vitro conditions. Because the teeth were soaked in the distilled water, collagen fibers could have absorbed the water. So, the flexibility of the teeth may have increased.
This is the first study in which primary and permanent teeth are examined together. This gave us the opportunity to compare the responses of the primary and permanent teeth with the different doses of the radiation within the same study protocol. Furthermore, there is no study in the literature examining five different major elements of teeth, which is one of the most significant elements that make this study original. Moreover, very different findings in the previous studies indicate that there is still no clear data on the subject in the literature, and that similar studies are needed for the future.
In this study, we investigated the direct effect of the radiation on the dental hard tissue regardless of the best known side effect of radiation, which is hyposalivation. The obtained results confirmed the negative effect of the radiation on teeth. Thus, the null hypothesis was rejected since there were differences between the nonirradiated and irradiated teeth.
It is necessary to develop strategies to minimize the damage caused by radiation in the dental hard tissue for the patient's dental health. It should be remembered that patients undergoing radiotherapy are individuals at high risk of caries, and protective applications (such as oral hygiene education, application of caries prevention agents, and non-cariogenic diet recommendations) should be focused in these patients.
The limitations of this study can be listed as follows: (i) in vivo conditions are not fully met, (ii) only the 5 major elements of the tooth were examined, but heavy metals that are thought to increase in the teeth were not examined. On the other hand, the strength of the study is that the effect of radiation doses (from 10 to 60Gy) on the morphogical, mechanical and chemical properties of both primary and permanent teeth were examined in the same study protocol, which has never been studied before.