In this study, changes that might be caused by radiation up to 60 Gy in extracted human primary and permanent teeth were investigated. To date, no consensus has been reached on the 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 NaCl for 30 days.19 However, no significant change was determined in the hardness of teeth stored in Hanks’ balanced salt solution.20 Dry environment is also known to adversely affect the mechanical properties of dental specimens due to dehydration.21
In order to simulate xerostomia/ hyposalivation formed due to 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.
In this study, dental samples were stored in 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.22 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.
When forming control and experimental groups, it should be taken into consideration that each tooth has different chemical and physical properties. In many studies, teeth in the control group and irradiated teeth are different.11,12,14,15,23 In addition, there are studies comparing the same teeth before and after radiation.4,10,13 In this study, the teeth undergoing ICP-OES analysis were subjected to irreversible processes (such as being crushed into powder). Due to this reason, it is not possible to conduct repetitive analyses. In this study, the other halves of the irradiated teeth were used as the control group. For microhardness measurement, symmetrical points were selected on the parts assigned as the control and experimental groups.
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 microhardness measurement. In our study, the measurement was performed with Vickers measurement rods which was perpendicular to the enamel cut surface.
The measurement points selected for 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.4,10,12,14 It was reported that the enamel and dentin hardness values depended on the point of measurement, and the hardness value increased when moving away from the DEJ region.24 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. In this study, statistically significant decreases were determined in the enamel close to the DEJ region in permanent teeth at 30 and 60 Gy.
While Gonçalves et al.4 found a decrease in the dentin microhardness close to the DEJ region after radiation, de Siqueria et al.10 first found a decrease and then an increase. In our study, there were significant decreases in this region in both primary and permanent teeth at 30 Gy.
In the literature, some studies have reported no changes 25,26, some have reported increases 4,10 and some have reported decreases 12,13,16,17,27,28 in the overall enamel microhardness after radiotherapy.
There are studies in the literature indicating a decrease in the overall dentin microhardness.4,10,15,23,29−31 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 of irradiated odontoblast cell metabolism, and degeneration of collagen fibers due to the effect of free radicals released after irradiation.11 In our study, a decrease was also observed in the overall dentin microhardness in primary and permanent teeth.
Velo et al.15 examined Ca, P, O, C, Mg, and Ca/P weight ratio in the irradiated human tooth 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 human dental 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 tooth. They attributed the decrease in the protein/mineral ratio to the structural change of collagen in both enamel and 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 decreased and the Ca/P ratio increased.
In this study, it was observed that after radiotherapy there were decreases in Na, K, Mg, P, and Ca elements and the Ca/P weight ratio in the analysis of primary and permanent teeth's hard tissues performed with ICP-OES. These decreases are more evident at doses of 30 and 60 Gy. Although primary and permanent teeth exhibited slightly different responses to radiotherapy, they often had similar responses (Fig. 3).
In ICP-OES, elemental analysis of all the hard tissues was 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.32,33 Heavy metals, which are important inducers of oxidative stress, are activated to act as catalysts.
Miculescu et al.34 stated that heavy elements accumulate faster than tooth major elements 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.4,8,10,12,15,35 In our study, more changes were observed in the morphological, mechanical, and chemical properties of teeth at 60 Gy in comparison with low doses.
Even though the significant changes were observed in the inorganic structure of the teeth according to the results of the elemental analysis, these changes were not very significant 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 primary and permanent teeth to different doses of radiation within the same study protocol. Furthermore, there is no study in the literature examining five different major elements of the tooth, which is one of the significant elements that make this study original.
In this study, we investigated the direct effect of radiation on dental hard tissue independently of the best known side effect of radiation, which is hyposalivation. The obtained results confirmed the negative effect of radiation on teeth.
It is necessary to develop strategies to minimize the damage caused by radiation in dental hard tissue for the patient’s dental health.
Li et al.23 reported that the use of fluoride, CPP-ACP, resin infiltration, and their paired combinations would prevent radiation-induced destruction of dentin.
Bekes et al.35 applied the paired combination of desensitizer and fluoride to the teeth prior to radiotherapy, kept the teeth in 10% sucrose during meals and reported that the depth of caries lesions caused by radiation at the end of a 5-week period was shallower than the depth of irradiated teeth without any protective application.
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.