Effects of gamma radiation on hard dental tissues of albino rats: investigation by light microscopy
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- El-Faramawy, N., Ameen, R., El-Haddad, K. et al. Radiat Environ Biophys (2013) 52: 375. doi:10.1007/s00411-013-0469-6
The present work aims at studying the effect of gamma radiation on the hard dental tissues. Eighty adult male albino rats with weights of about 250 g were used. The rats were irradiated at 0.2, 0.5, 1.0, 2.0, 4.0 and 6.0 Gy whole-body gamma doses. The effects on hard dental tissue samples were investigated after 48 h in histological and ground sections using light microscopy. Areas of acid phosphatase activity were detected using tartrate-resistant acid phosphatase (TRAP) stains. Observation of histological sections revealed disturbance in predentin thickness and odontoblastic layer as the irradiation dose increased. In cementum, widened cementocytes lacunae were occasionally detected even with low irradiated doses. On the other hand, relatively homogenous enamel was detected with darkened areas in enamel surface at doses over than 0.5 Gy. TRAP-positive cells were detected on the surface of the dentin of irradiated groups as well as cementum surface. Minimal detectable changes were observed in ground sections.
KeywordsGamma radiationDental tissuesLight microscopeDental histology
During the last few decades, there have been substantial improvements in cancer treatment optimization. Radiotherapy is currently an important part of several cancer treatments including head and neck cancer. It utilizes ionizing radiation to destroy neoplastic cells and thus to reduce or eliminate the malignant lesion. The head and neck region is a complex area composed of several dissimilar structures, such as mucosal linings, skin coverings, subcutaneous connective tissue, salivary gland tissue, teeth, bone and cartilage, that differ in their response to radiation (Cooper et al. 1995; Taylor and Miller 1999).
Several oral sequelae of radiotherapy of the head and neck region have been reported. Trismus may develop as a result of radiotherapy if masticatory muscles and/or the temporo-mandibular joints (TMJ) are included in the field of radiation (Goldstein et al. 1999). Mucositis is the most frequent and often most distressing radiotherapy complication (Scully and Epstein 1996), followed by alteration in taste sensation (Spielman 1998). Osteoradionecrosis is a very unfortunate complication of head and neck radiotherapy that may result in the loss of a significant volume of bone, mostly mandibular. The salivary glands show, in contrast to the other tissues, both early and late response to radiation. Vissink et al. (2003) studied the damage of the salivary glands as the most frequently occurring inconvenient side effect of head and neck radiation. Moreover, mucosal ulcers may result in severe pain for the patient, causing poor nutritional intake and poor oral care (Lalla et al. 2008; Clarkson et al. 2008).
Regarding the radiosensitivity of the specialized dental cells, Timothy et al. (1977) have early reported that ameloblasts are less sensitive to radiation damage than odontoblasts. In 1994, Shuichi et al. reported deleterious effects of gamma radiation on the development of tooth and jaws. In addition, Saad et al. (1991) have confirmed in an experiment on rat maxillary and mandibular incisors that radiation inhibits the normal odontogenesis.
Anneroth et al. (1985) have summarized the effects of radiation on the periodontium as: decreasing the vascularity of the periodontal membrane, rupturing, thickening and disorientation of Sharpey’s fibers and widening of the periodontal space after irradiation. Earlier reports revealed nonalterations in the irradiated pulps using light microscopy (e.g., Matson et al. 1978). However, Fawzi et al. (1985) have found that radiation at 4 Gy dose caused depression of the normal response of the albino rat dental pulp to the trauma and induced infection by pulpal exposure. Also, nuclear alterations in the irradiated pulp fibroblasts and odontoblasts have been reported by Vier-Pelisser et al. (2007).
The most threatening complication for the hard dental tissues is radiation-related caries. Radiation caries is a highly destructive form of dental caries which has a rapid onset and progression (Jansma et al. 1993). It has always been a matter of debate whether radiation caries is due to a direct or indirect effect of irradiation on teeth, or to both. Several investigators have reported that the development of radiation caries is not dependent on the presence of teeth in the field of irradiation. Rather, the determining factor is whether or not the main salivary glands are within the radiation field, which leads to salivary gland damage resulting in hyposalivation (e.g., Kielbassa et al. 2001).
Silva et al. (2009) studied the suggestion that direct radiogenic destruction of the teeth seems to be unessential to the microscopic progression of radiation-related caries using polarized light microscopy and scanning backscattered electron microscopy. This study has concluded that radiation-related caries seems to have the same morphological and demineralization pattern as ordinary caries.
Moreover, early dental restoration failure in teeth affected by radiation-related caries may have the same etiological factors as ordinary dental restoration failure and direct radiogenic damage to dentition would not be essential to early restoration failure in radiation-related caries (Silva et al. 2010).
According to many clinical studies, however, teeth in the field of irradiation seem to lead to a greater caries incidence (Grotz et al. 2001). When the oral cavity and salivary glands are exposed to high doses of radiation, the risk of rampant tooth decay with its sudden onset is a lifelong threat due to multifactorial causes (Kielbassa et al. 2006). The exact nature of this direct radiogenic damage is still to be elucidated.
Concerning the direct effect of radiation on the physical properties and histological structure of hard dental tissues, Jansma et al. (1990) have stated that the use of gamma radiation at a therapeutic level has no influence on its enamel permeability and thus on its organic component. It was reported that a medical gamma radiation dose of 25 kGy does not affect enamel surface microhardness (Lidiany et al. 2004). In contrast, Grötz et al. (2000) and El-Faramawy et al. (2011) have observed deep cracks histomorphologically in the irradiated enamel that reached the dentino–enamel junction (DEJ). In addition, radiation treatment significantly decreases the ultimate tensile strength of dentin and enamel, due to the harmful effects of radiation on the organic components of the different hard dental tissues (Soares et al. 2010). Many investigators have reported that irradiated teeth decalcify more readily than nonirradiated teeth. Enamel has been found less resistant to acid attack after irradiation (Grötz et al. 1998). Other researchers have reported a decrease in the enamel and dentin solubility after therapeutic radiation (e.g., Kielbassa et al. 1999, 2002).
In spite of a great number of relevant studies, the etiology of radiation-induced caries is still controversial. The contradictory findings in the literature lead to a need for more investigations. These investigations should aim to clarify the induced lesions in the hard tissue under the direct and indirect effects of radiation.
The aim of the present work is to evaluate the direct radiogenic effects of radiation on the hard dental tissues using light microscopic techniques.
Materials and methods
Eighty adult male albino rats (4 months old) with weights of about 250 g were used. The rats were supported by the animal house of Ain Shams University, Cairo, Egypt. The rats were housed in wire mesh cages under controlled temperature and dark–light cycle. Each cage contained five rats; the rats were fed bread and milk. Tap water was available ad libitum. The experimental procedure was conducted in compliance with ethical principles for animals’ research which have been reviewed and approved by the institutional guidelines of the Bio-ethical Committee of Ain Shams University.
For the irradiation process, a Cobalt-60 gamma source (Gammatron S-80, manufactured in 1976 by Siemens, Germany) with dose rate 63.1 mGy/min at 100 cm was used. A secondary standard dosimetry system composed of an electrometer (NPL–NE2560, UK) and an ionization chamber (NE2561, UK) was used to determine air kerma. Air kerma (Kair) was determined in accordance with the International Atomic Energy Agency (IAEA). The expanded uncertainty associated with Kair determination was about 0.9 % at 95 % level of confidence (coverage factor = 2).
Rats were separated into six groups; each group consisted of five rats. Each group was irradiated once in a cage to a definite dose of total body irradiation. To examine the background effects, two additional groups were left without irradiation as a control group; one was kept away from the building of radiation facility, and the other was moved with the irradiated groups but kept outside the irradiation cell. The irradiation process was achieved at the Radiation Department, National Institute of Standards, Cairo, Egypt. The irradiation process was done at a distance of 95 cm between the source and the surface of the cage. The field size setup was 17 × 43 cm covering the cage dimensions. Groups were irradiated to doses of 0.2, 0.5, 1.0, 2.0, 4.0 and 6.0 Gy. Care was taken to prevent self-shielding due to rat over crowdedness inside the cage (by designing the cage size and dimensions to be tightly fitted over the rats), and hence, more homogeneous irradiation was guaranteed.
The rats were killed by cervical dislocation 48 h after irradiation, and the molar areas of the mandibles were dissected to two halves. One half of molar area of each rat was identified. The ten identified halves of molar areas of each group were fixed immediately in 10 % formalin solution for at least 1 week. Five halves were prepared as decalcified sections and the other five halves prepared as ground sections.
Preparation of decalcified sections
To study the histological effects of radiation on the organic fractions of dentin and cementum, decalcified sections from the first five halves have been prepared through the following procedure (Bancropt et al. 1996): The samples were rinsed well to remove blood and adhering tissues before they were placed in 10 % buffered formalin for 10 days. Then the samples were decalcified by being placed in 12 % ethylene diamine tetra-acetic acid (EDTA) solution which was being changed every 48 h for 2 weeks. The decalcification process was tested by piercing the samples with a sharp probe. Then samples were rinsed well to remove all residues of the acid. The specimens were dehydrated by transferring them through different concentrations of alcohol increasing gradually: (50, 60, 80, 90, 96 % and absolute alcohol). Specimens were then transferred to xylol to be cleared from alcohol. The specimens were infiltrated in paraffin wax and were embedded in the center of paraffin wax blocks. The embedded specimens were sectioned by microtome (4–6 micron thick). These sections were transferred in different concentrations of alcohol decreasing gradually: 96 and 70 %, and then distilled water. The resulting sections were immersed in hematoxylin and eosin (H&E) and tartrate-resistant acid phosphatase (TRAP) special stains. These sections were examined by light microscope.
TRAP special stain was used for the demonstration of acid phosphatase activity (Tsukamoto-Tanaka et al. 2006). This stain requires 4 buffer solutions.
Staining procedure of TRAP
Two Coplin jars of Buffer I Solution (sodium acetate anhydrous 9.2 g/l, tartaric acid 11.4 g/l and glacial acetic acid 2.8 ml; pH = 4.7–5.0) were preheated to 37 °C. Then, one-half milliliter of Buffer II Solution (20 g/l naphthol phosphate in ethylene glycol monoethyl) was added to one Coplin jar, and the slides were added and incubated at 37 °C. One milliliter of Buffer III Solution (sodium nitrite 50 g/l) and one milliliter of Buffer IV Solution (2 g/l pararosaniline chloride) were mixed. This mixed solution was added to the other Coplin jar contains Buffer I Solution. The slides were rinsed, stained with counterstain (hematoxylin) and put in ammonia water. Afterward, the slides were dehydrated through alcohol, cleared in xylene and mounted.
The areas of TRAP activity were represented by orange to brown color, while areas of no acid phosphatase activity were violet (if they were hematoxyphilic).
Preparation of ground sections
To study the histological effects of radiation on the inorganic fractions of enamel, dentin and cementum, ground sections from the second five halves were prepared through the following procedure: Samples were cut longitudinally in a mesiodistal plane with thickness of 0.07 mm by a carborundum wheel in a low-speed hand piece under wet conditions. Due to size limitations of rat teeth, the molar areas with teeth intact were used. The finished ground sections were rinsed well with water. The sections then were cleared in clove oil and mounted with plasticizer, distyrene and xylene (PDX) mounting medium on a glass slide (Ho et al. 2004).
These sections were examined by light microscopy (Olympus model: BX60F5- Olympus optical co. Ltd—Japan) at different magnifications (40, 100, 200 and 400) according to the examined structure and the measurement requirements. The measurements of the predentin thickness in 3 different regions in pulp chamber were done by image analysis software UTHSCSA IT3 (UTHSCSA Image Tool, version 3, Texas). This software was developed in the Department of Dental Diagnostic Science at the University of Texas Health Science Center, San Antonio, Texas.
Histological effects of radiation on dentin
H&E stain results
Similar results are observed in the specimens irradiated with 0.2 Gy (Fig. 1b). In contrast, the predentin thickness became uneven in many areas from specimens irradiated at doses from 0.5 up to 6 Gy (Fig. 1c, d). In some regions the predentins are absent and the dentinal tubules are widely opened in the dentin–pulp border (Fig. 1e, f).
TRAP stain results
Ground section results
Histological effects of radiation on cementum
H&E stain results
Dispersed bay-like resorption concavities are observed on the surface of the cellular cementum for samples irradiated at doses higher than 0.5 Gy (Fig. 6e, f). Occasionally, multinucleated cells are observed adjacent to the cementum surface concavities, identical to the cementoclasts appearing in the Howship’s lacunae (Fig. 6f). These observable resorption areas exhibit evidence for the presence of cementoclasts (identical cells to osteoclasts).
TRAP stain results
Ground section results
Histological effects of radiation on enamel (ground section results)
The present study was designed to evaluate the radiogenic effect on hard dental tissues by in vivo irradiation of rats in order to simulate the effect of radiation on living patients receiving radiotherapy treatment. Six different doses (0.2, 0.5, 1, 2, 4 and 6 Gy) were applied to the animals under investigation. This offered an excellent opportunity to assess whether the increase in the dose would affect the degree of changes in the different dental tissues.
According to Tchanque-Fossuo et al. (2011), a dose of 7 Gy to rat mandibles approximates the bioequivalent dose which the mandibles receive in the clinical regimen of head and neck cancer radiotherapy patients. They stated also that the conventional therapy for head and neck cancer involves the use of radiation with absorbed doses up to 70 Gy delivered via a fraction size of 1.8–2 Gy. So, 7 Gy dose to rat is approximately equivalent to 70 Gy dose to human. Therefore, the maximum dose of our study (6 Gy) is approximately equivalent to 60 Gy given to human, while 0.2 Gy rat dose is equivalent to 2 Gy (which represents the daily fraction dose) for human.
In the present study, the predentin thickness was uneven in many areas of specimens exposed to radiation dose 0.5 up to 6 Gy, whereas in other regions the predentin was absent. This means that there is a disturbance in the secretory process of dentin. This disturbance could be attributed to the affection of odontoblasts themselves with irradiation exposure as a direct result of radiogenic cell damage with hampered vascularization of the adjacent pulp tissues (Grötz et al. 1998). These changes were reported before by Vier-Pelisser et al. (2007) where a large number of pulp odontoblasts displayed nuclear alterations with radiation exposure. These alterations in the nuclei may explain the disturbance in the secretory potential of odontoblasts to predentin.
In addition, the spaces between odontoblasts exhibit different sizes for all irradiated groups. These observations can be interpreted in terms of what is reported on hyaline degeneration occurring in the head and neck tissues following irradiation. The direct cellular damage following radiation may be a result of the destruction of DNA, proteins, enzymes or other macromolecules (Anneroth et al. 1985; Grotz et al. 2001; Greenberger and Epperly 2009).
In the present study there was a widening of the dentinal tubules at their pulpal end, substantiating the data given by El-Faramawy et al. (2011). This is also in accordance with Rabinowitch (1972) who suggested a coincidental association between internal resorption of dentin and radioactive material and/or traumatic occlusion. To detect the resorptive activity in the pulpal end of dentin, TRAP (tartrate-resistant acid phosphatase) special stain was selected as a specific histological marker of cells with acid phosphatase enzyme activity. This enzyme is associated with many resorptive cells (Laura et al. 2006).
In the present study, examination of both decalcified and ground sections by light microscope revealed that the dentin bulk was not noticeably affected by radiation doses used in this study, which is in agreement with the results of Kielbassa et al. (1999) and Al-Nawas et al. (2000). The minute effects of radiation on dentin might be explained by enamel acting as a biological shield which absorbs major parts of the energy of gamma radiation. It was reported that the energy of the radiation decreases as it passes through a material, due to absorption and scattering (Richard et al. 2007).
The current results support the belief that the irradiation process is not the reason for dead tracts. It is worthy to take into consideration the fact that the ground sections display the inorganic part of the tissue and that minimal changes between the control and irradiated groups were detected in the current study. We conclude that the given radiation doses have minimal or close to none effects on the mineral content of dentin. The irradiation process is more harmful to organic components as discussed before by Moscovich et al. (1999) and Soares et al. (2010).
For all irradiation doses, a separation of Sharpey’s fibers was observed. The current results are matching with those obtained by many researchers (e.g., Anneroth et al. 1985) who detected rupturing, thickening and disorientation of Sharpey’s fibers and widening of the periodontal space with irradiation. Most probably, the radiation induces changes in the cellularity and vascularity of periodontium that lead to trophic defects, impaired immune defense capability and a reduced healing/remodeling potential of these tissues. And, it turns to direct radiogenic damage of the periodontal ligament. The combination of these factors contributes to increasing risk of periodontal involvement observed after irradiation therapy. Similar results are seen by Epstein et al. (1998).
In addition, gap areas in the cemento–dentinal junction were observed, which coincides with Anneroth et al. (1985). The cemento–dentinal junction is regarded as a wide zone containing large quantities of proteins including collagen, based on the fact that the integrity of the cement–dentinal junction is dependent to some extent on the intermingling between the collagen fibers of dentin and cementum (Yamamoto et al. 2000 and Nanci and Ten 2007). So, the separation between cementum and dentin can be explained by radiogenic effects on collagen fibers in the irradiated samples (Ingo et al. 2005).
The predominance of the resorptive cells and resorption sites related to cementum after irradiation might be linked to an enhanced radioresistance of resorptive cells (such as osteoclasts and odontoclasts) compared to synthetic cells such as cementoblasts and osteoblasts. Consequently, a relative increase in the lytic activity exists (Baker 1982). These resorption sites were also clearly observed in the scanning electron micrographs of irradiated samples in our earlier work (El-Faramawy et al. 2011).
In the present study, there were ill-defined enamel rods in the groups irradiated with doses higher than 0.5 Gy. The relatively homogenous enamel structure agrees with reports by Grötz et al. (1998). Darkened areas concentrated on the surface enamel might be attributed to the fact that surface is the area of enamel most affected due to the gamma radiation.
The affection of enamel surface was reported by Jham and Freire (2006) who stated that smooth enamel surfaces, normally resistant to decay, are affected and that caries development and progression are accelerated. Also, the irradiated enamel appeared less resistant to acid attack after irradiation (Grötz et al. 1998). In addition, diffuse brown discoloration of the smooth surface of enamel and cervical and incisal caries, which are very unusual sorts of lesion, were widely detected in irradiated teeth (Silva et al. 2009).
The scarcity of differences between irradiated and nonirradiated enamel in the ground sections might be due to the fact that radiation mainly affects the organic components of enamel (Soares et al. 2010) which were disintegrated during grinding. Expanded researches are needed to evaluate the definite effect of radiation on the inorganic (mineral) components of enamel and the chemical changes in enamel following radiation.
Our observed effects of radiation on enamel surface and the dentin reparative activity represented in odontoblasts may support the hypothesis that the direct effect of radiation on the hard dental tissues predisposes for radiation-related caries. However, more detailed chemical analysis of enamel surface as well as studies on the reversibility of these effects is needed to clarify this hypothesis.