|Ahead of print publication
Demineralization of tooth enamel following radiation therapy; An in vitro microstructure and microhardness analysis
Jagadish Kudkuli1, Ashish Agrawal2, Om Prakash Gurjar3, Sunil Dutt Sharma4, PD Rekha1, Muhammed A. P. Manzoor1, Balwant Singh2, BS Rao1, Riaz Abdulla5
1 Yenepoya Research Centre, Yenepoya (Deemed to be University), Indore, India
2 Imaging Beamline (BL-4), BARC Beamline Section, Technical Physics Division, Indus-2, RRCAT, Indore, India
3 Department of Radiotherapy, Sri Aurobindo Institute of Medical Sciences, Indore, Madhya Pradesh, India
4 Radiological Physics and Advisory Division, Bhabha Atomic Research Centre; Department of Health Sciences, Homi Bhabha National Institute, Mumbai, Maharashtra, India
5 Depatment of Biomaterials & Research centre, Department of Oral pathology, Yenepoya Dental College, Yenepoya (Deemed to be University), Indore, India
|Date of Submission||03-Jan-2019|
|Date of Decision||23-May-2019|
|Date of Acceptance||22-Aug-2019|
|Date of Web Publication||16-May-2020|
Deparment of Biomaterials & Research centre Professor, Department of Oral pathology, Yenepoya Dental College, Yenepoya (Deemed to be University)
Source of Support: None, Conflict of Interest: None
Objective: The objective of this study is to evaluate the effects of radiotherapy doses on mineral density and percentage mineral volume of human permanent tooth enamel.
Materials and Methods: Synchrotron radiation Xray microcomputed tomography (SRμCT) and microhardness testing were carried out on 8 and 20 tooth samples, respectively. Enamel mineral density was derived from SRμCT technique using ImageJ software. Microhardness samples were subjected to Vickers indentations followed by calculation of microhardness and percentage mineral volume values using respective mathematical measures. Data were analyzed using paired t-test at a significance level of 5%. Qualitative analysis of the enamel microstructure was done with two-dimensional projection images and scanned electron micrographs using μCT and field emission scanning electron microscopy, respectively.
Results: Vickers microhardness and SRμCT techniques showed a decrease in microhardness and an increase in mineral density, respectively, in postirradiated samples. These changes were related to mineral density variation and alteration of hydroxyapatite crystal lattice in enamel surface. Enamel microstructure showed key features such as microporosities and loss of smooth homogeneous surface. These indicate tribological loss and delamination of enamel which might lead to radiation caries.
Conclusions: Tooth surface loss might be a major contributing factor for radiation caries in head-and-neck cancer patients prescribed to radiotherapy. Such direct effects of radiotherapy cause enamel abrasion, delamination, and damage to the dentinoenamel junction. Suitable measures should, therefore, be worked out to protect nontarget oral tissues such as teeth while delivering effective dosages to target regions.
Keywords: Head-and-neck cancer, microhardness, radiotherapy, SRμCT, tooth enamel
|How to cite this URL:|
Kudkuli J, Agrawal A, Gurjar OP, Sharma SD, Rekha P D, Manzoor MA, Singh B, Rao B S, Abdulla R. Demineralization of tooth enamel following radiation therapy; An in vitro microstructure and microhardness analysis. J Can Res Ther [Epub ahead of print] [cited 2020 Jun 2]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=284491
| > Introduction|| |
Oral squamous cell carcinoma is the sixth most common cancer reported globally, with an annual incidence of over 3,000,000 cases of which 62% arise in developing countries. Vital head-and-neck (H and N) anatomy and severe metastasis of affected squamous epithelial tissues are serious challenges for successful implementation of radiation which is a principal mode of H and N cancer treatment to improve the tumor control probability of target areas. However, the focus has become increasingly prominent to reduce normal tissue complication probability that is likely to occur on normal tissues surrounding the target. This has led to rapid advancements in technology and delivery methods of radiotherapy which has opened up preferences between60 Co gamma-rays and high-energy X-rays (typically 6 megavoltage [MV] X-rays from medical electron linear accelerators [LINACs]) among various other upgraded systems. Notwithstanding such developments, the severity of oral sequelae continues to persist, affecting quality of life (QOL) of H and N cancer patients. The high ionization potential of MV radiation and maximum fractions of radiation dose administered to oral tumor cells are the major factors for indirect side effects induced upon salivary gland damage such as mucositis, xerostomia, taste loss, trismus, and direct side effects such as progressive loss of periodontal ligament, osteoradionecrosis, and radiation caries.
Dental and oral health is adversely affected due to direct effects of radiation therapy, and teeth are one of the worst affected normal dental hard tissues due to radiation received during a therapeutic procedure. Although it has lesser electrons per gram as compared to soft tissue, the mass density of teeth/bone is much higher, and therefore, the electrons per unit volume are much higher than that in soft tissue. This is shown to cause more dose deposition in soft tissue–tooth interface region affecting surrounding muscles which supply nutrients to the teeth. Radiation-induced caries is seen as a major complication affecting long-term QOL of H and N cancer patients. It can lead to severe dentition breakdown and is one of the several long-term side effects encountered due to direct effects of radiotherapy. Tooth enamel delamination from direct exposure to therapeutic radiation coupled with radiation-induced xerostomia alters oral hygiene and induces tooth surface loss. Clinical studies suggest a three-tier dose response to categorize the direct effect of radiation on the tooth. Minimal tooth damage was observed below 30 Gy; at doses between 30 and 60 Gy, there were a 2–3 times increased risk of tooth damage likely related to salivary gland impact and a 10 times increased risk of tooth damage when the tooth-level dose is >60 Gy indicating radiation-induced damage to the tooth in addition to salivary gland damage.
Studies regarding evaluating dental erosion/demineralization are extensively carried out by profilometric, tomographic, and spectroscopic techniques which provides accurate means of quantitative analysis. Scanning electron microscopy (SEM) analysis is often restricted to qualitative domain unless supplemented with specific observer scoring criteria through features observed in scanned images and derives quantitative data when incorporated and validated effectively.,In vitro studies have shown deleterious effects on wear behavior and tribological properties of both enamel and dentin specimens exposed to simulated clinical doses of radiotherapy. LINAC-based X-ray photon irradiation was done on enamel–dentin sections that revealed a decrease in protein content in the enamel and dentin regions resulting in increased stiffness of enamel and dentin near the dentinoenamel junction (DEJ). Ultimate tensile testing of the enamel and dentin regions of tooth sections have shown that radiotherapy decreases the biomechanical properties of dentin where the organic composition is vital, while areas with an inorganic composition such as enamel are comparatively more resistant to radiotherapy. It was also shown that indentation patterns and microstructure of enamel and dentin are affected under radiation intervention, suggesting that enamel may become more brittle followingin vitro andin vivo radiations. Variation was also observed in wear and friction behavior of human tooth enamel exposed to60 CO gamma-radiation. Changes in microstructure and composition of enamel were assessed through nano-scratch tests, surface profilometer, and SEM analysis, and results of which were closely related to changes in the crystallography, chemical composition, and surface microhardness of the enamel. These mechano-morphological property changes in tooth enamel and dentin could potentially lead to biomechanical failure leading to enamel delamination and progressive teeth decay or radiation caries.
| > Materials and Methods|| |
Twenty freshly extracted human permanent premolar teeth, noncarious, without any attrition/abrasion, were examined and collected after obtaining ethical clearance from the institutional ethics committee. Patient consent was not required in this study since the patient information was delinked before collecting the samples from clinicians. Samples were collected henceforth and preserved in phosphate-buffered saline (PBS) maintained at constant pH of 7.4. Hemi-sections of the tooth were obtained in the buccolingual orientation with diamond disk diamond carbide burr attached to an electric micromotor. The sections included anatomical crown and 2 mm of the cementoenamel junction (CEJ) [Figure 1]. Irregularities on the sample surface were curated using straight fissure burr to obtain relatively uniform surface, and distal surfaces were polished thoroughly to get flat base suitable for indentation on the mesial enamel surface. While all 20 buccal hemi-section samples were subjected to Vickers indentation and SEM to obtain microhardness values and micrographs, respectively, 8 samples were used for synchrotron X-ray micro-computed tomography (SRμCT) experiment.
|Figure 1: Representation of premolar tooth sectioning. Buccal sections of premolar teeth were cut and used as samples in the experiment|
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To study the effect of radiation dose, tooth samples were irradiated using 6 MV X-rays from a medical electron LINAC. Prior to irradiation of the tooth samples, peak dose rate (i.e., dose rate at the depth of dose maximum, 1.5 cm) of the 6 MV X-ray beam was verified using ionization chamber (calibrated cylindrical chamber)-based measurement in a 30 cm × 30 cm × 30 cm water phantom (Source to surface distance (SSD)). From this measurement, dose per monitoring unit was calculated following the standard calculation approach. The tooth samples were placed on a thin perspex sheet, and this sheet was immersed into the water phantom in such a fashion that tooth samples were located at the depth of dose maximum (1.5 cm). While positioning the perspex plate inside the water phantom and adjusting the irradiation field, it was kept in mind that all the tooth samples should be well within the flat region of the radiation field. Enamel–dentin buccal sections were then irradiated using the 6 MV X-ray beam for radiation-absorbed dose in the range of 20–80 Gy.
Synchrotron X-ray micro-computed tomography (SR-μCT)
The experimental work was carried out at imaging beamline (BL-4) at Indus-2, synchrotron radiation (SR) source, Raja Ramanna Centre for Advanced Technology. The representation of optical and experimental hutch is shown in [Figure 2]. Synchrotron source being operated at 2.5 GeV energy and 150 mA current generates a polychromatic X-ray beam tangential to the bending magnet which is passed through a monochromator to form monochromatic synchrotron X-ray beams. This beam is transmitted through the sample and collected in a two-dimensional (2D) imaging area detector (fiber-optic-coupled Charge-coupled device (CCD) camera having Gadox scintillator) to form a projection image on it. SRμCT was carried out at 28 keV beam energy to obtain a total of 900 such projections for each sample while rotating it about its axis in the angular range of 180° through a 0.2° rotation step. Acquisition time for each projection was set to 1 s. Data obtained were preprocessed for noise removal, flat-field corrected and normalized before applying tomographic reconstruction of cross-sectional slice image using filtered backprojection. Three-dimensional (3D) images were prepared by stacking of the reconstructed slice image and applying appropriate volume rendering to highlight the desired features. Comparison of material density and micromorphology of samples was done using histogram analysis and qualitative examination of enamel regions before and after the intervention. Regions of interest measuring 0.01 cm × 0.01 cm were selected, and density histogram values were generated using ImageJ software.
|Figure 2: Schematic representation of optical and experimental hutches at X-ray synchrotron micro-computed tomography setup, beamline 4, Indus 2, Raja Ramanna Centre for Advanced Technology|
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The experiment was carried out using MicroMet auto-turret microhardness tester (Matzusawa Co. Ltd., Model MMT XA, Tohshima, Japan). Vickers microhardness test was carried out before and after irradiation by indentations made using Vickers diamond microindenter with 100 gf load for 15 s dwelling time. Diamond-shaped indentations were made at the central axis of the buccal hemi-sections approximately 3 mm from CEJ (vertical axis) and 1 mm from horizontal axis where the surface was relatively uniform. Average diagonal lengths were measured and Vickers microhardness number was derived using the following formula.
HV = 1.854F/L2 (1)
where HV is Vickers microhardness number, F is pressure in gf (gram force), and L is the diagonal length in μm.
Hardness number interconversion indices gave Knoop microhardness numbers for corresponding Vickers microhardness data which were converted to percentage mineral volume using the relation:
Mineral content (%) volume = 4.3 (√KHN)+1.3 (2)
where KHN = Knoop microhardness number.
Field emission scanning electron microscopy
Enamel surfaces of tooth hemi-sections were sputter-coated with gold (Au) and observed using field emission SEM (FE-SEM) (Carl Zeiss, Germany). Electron micrographs were recorded for surface microstructure comparison of pre- and postirradiated samples. The extra high-tension voltage level was set at 3 kV and magnification/resolution to ×1000/2 μm.
| > Results|| |
Mineral density measurement given here is analogous to pixel gray value variation observed in 2D slice projections of pre- and postirradiated enamel surface. SRμCT analysis of 180 image projections showed that average mineral density of enamel was significantly increased in radiation-exposed samples compared to controls. Furthermore, there was a steady rise in the rate of increase in the material density of enamel from 20 to 80 Gy [Figure 3]. The variation was found to be significant by a statistical method, paired t-test (P < 0.0001). Overall, enamel density was increasing with the radiation dose which indicates changes in the structural integrity of hydroxyapatite crystal lattice that encompasses around 97% of enamel composition. Since all the experiments were carried out using a monochromatic beam of the same energy, pixel gray values in the reconstructed tomographic slice images are directly proportional of the pixel-averaged linear attenuation coefficient of each pixel in the sample. The linear attenuation coefficient, in turn, depends on the atomic number and physical density of the materials. The variation observed here in image gray values due to radiation exposure is thus attributed to the changes in the atomic number and physical density of the samples. There were no marked changes in enamel microstructure observed through 2D slice projections of microtomography at around 15–20 μ resolution [Figure 4]. However, In control samples [Figure 5]a, electron micrographs showed normal integrated enamel morphology whereas, in radiation-exposed enamel surfaces, distinct changes in micromorphology features were observed. Micro porosities on enamel prism structures, loss of regular key whole patterns were noted after 20 Gy radiation exposure [Figure 5]b. Dissolution of prism cores [Figure 5]c and d] and loss of smooth homogeneous surface [Figure 5]e were observed after radiation exposure of 40Gy, 60Gy, and 80Gy respectively.
|Figure 3: Mineral density gray values of enamel slice projections for control and treatment groups. **Implies, treatment groups have a significantly higher mineral density (P < 0.0001) as compared to controls. Thirty projections each from control and different treatments were analyzed for mineral density parameter using ImageJ software tool. Error bars represent the Standard error. Sample size (n) for controls = 8, 20 Gy = 2, 40 Gy = 2, 60 Gy = 2, 80 Gy = 8|
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|Figure 4: Two-dimensional slice projections of buccal hemi-sections showing enamel and dentin regions in control and irradiated samples. (a, c, e and g) Nonirradiated tooth hemi-sections and (b) irradiated sample (20 Gy), (d) irradiated sample (40 Gy), (f) irradiated sample (60 Gy), and (h) irradiated sample (80 Gy)|
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|Figure 5: Electron micrographs of the enamel surface of the permanent premolar teeth. The images were obtained by scanning electron microscopy at ×1000/2 μm resolution. Scanning electron microscopy micrographs are shown for before radiation exposure (a) control and after exposure (b) 20 Gy, (c) 40 Gy, (d) 60 Gy, (e) 80 Gy samples. Sample size (N) for controls = 22, 20 Gy = 4, 40 Gy = 5, 60 Gy = 4, 80 Gy = 4|
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Vickers microhardness tests involved derivation of microhardness numbers from diagonal lengths of rhombus -shaped indentation, interconversion to Knoop microhardness number, and then, obtaining percentage mineral volume from the equation (2). Mean and standard deviations of percentage mineral volume are shown in [Figure 6]. Results revealed gradual loss of percentage mineral volume on enamel surface with an increase in radiation doses from 20 to 80 Gy. However, the loss was significant in 80 Gy exposed samples (P < 0.05) when compared to controls.
|Figure 6: Percentage mineral volume of teeth before and after treatment. *Implies, compared to control, 80 Gy treatment samples had a significant reduction in percentage mineral volume (P < 0.013). Average diagonal values generated from triplicate indentations were calculated for microhardness and mineral volume (%) parameters using hardness number interconversion indices and percentage mineral volume formula. Error bars represent the standard error. Sample size (n) for controls = 22, 20 Gy = 4, 40 Gy = 5, 60 Gy = 4, 80 Gy = 4|
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| > Discussion|| |
In this study, we have chosen two quantitative material characterization techniques, namely microhardness and SRμCT, to assess changes in enamel surface induced by different doses of radiation. While the former is relatively well standardized to characterize surface changes of dental tissues, no such analysis has been carried out so far using the SR-μCT technique. Mineral volume and mineral density parameters were derived by respective techniques to study the effect of different radiation doses on surface morphological properties of human permanent tooth enamel.
According to the strategy designed by treatment planning system which is specific to the type of diagnosis, radiotherapy doses are delivered in fractions using a planned regimen such as 2 Gy/day × 5 days/week cumulating the total radiation dose to about 60–70 Gy. Most of thein vitro studies have used fractionated doses to mimic this clinical situation. However, it is to be noted that delivering fractionated radiation treatment is based on&%#8220;5Rs” (repair, redistribution, reoxygenation, regeneration, and radiosensitivity) of radiobiology. Since thisin vitro study was to assess variations in the inorganic composition of enamel layer and would not be relevant to mechanisms involved in the 5Rs, it was preferred to deliver the cumulated dose in a single fraction in our experiments. This procedure is in agreement with similarin vitro studies where teeth were exposed to cumulated doses of radiotherapy and studied for microhardness and the atomic percentage of constituting elements in tooth enamel.,
Results may vary depending on the type of teeth, patient individuals, storage condition, etc., To maintain the uniformity of sample type, we chose premolar teeth and samples were moistened with PBS pH 7.2 during radiation exposure and used as a storage solution to simulate the oral environment of neutral pH and aqueous condition. Other studies have used artificial saliva, deionized water, and 0.9% saline as a storage solution for the samples along with antifungal supplements to prevent contamination., However, no contamination was observed in our test samples stored in PBS for 3 months of experimental period. Enamel is the most mineralized of all tissues and the hardest substance in invertebrates. It is dominated by the mineral phase, which is about 95 wt% (percentage by weight). Only about 1 wt% is an organic matrix and 4 wt% is water. The inorganic mineral phase is attributed to hydroxyapatite crystal matrix while organic and water contents lie underneath in DEJ, dentin, and pulp cavity. As a crucial component in dental tissue, mineral volume and density variations in enamel layer can greatly influence the stability of DEJ and teeth decay thereafter as a direct effect of radiotherapy.
In evaluating mechano-morphological properties of human tooth enamel under the effect of oral cancer radiotherapy treatment, this is the first study to have estimated mineral density and mineral volume parameters by the use of two of the nondestructive techniques, namely microhardness and SR-μCT. SR-μCT is a nondestructive 3D imaging technique that offers high flux for fast data acquisition times with high spatial resolution. Determination of mineral density profiles based on changes in X-ray attenuation is the principle of SRμCT analysis and is extensively used forin vitro dental research. In addition, synchrotron sources score higher compared to lab-based X-ray systems owing to the superior degree of both spatial and chromatic coherences which allow to analyze density parameters and demonstrate changes happening at micron resolution. 2D image slice projections obtained from SRμCT revealed an increase in density in enamel regions as shown in increased pixel gray values of enamel areas of teeth exposed to radiation doses. Mineral density usually chosen is that of hydroxyapatite crystal matrix, and XRD (X ray diffraction) peaks of enamel samples have indicated enlargement in crystal size due to radiation exposure. Material density is the physical property of a substance and gives the relationship between its mass and the volume. The inverse relationship that exists between density and volume of a substance is well correlated with our results which showed a simultaneous reduction in percentage mineral volume and increased density of enamel. Qualitative analysis of micromorphology features by 2D slice projections and FE-SEM signifies that SR-μCT did not offer required resolution to clearly identify micromorphology features unlike FE-SEM method which demonstrated microporosities on enamel prism structures, loss of regular key whole patterns [Figure 5]b, dissolution of prism cores [Figure 5]c and d], and loss of smooth homogeneous surface [Figure 5]e in radiation-exposed samples.
In some studies, sample preparation involved polishing the tooth surface to get a uniform area for making clear indentations for microhardness test. Microhardness values obtained from different areas such as middle enamel, outer enamel, and inner enamel were nonconcurrent because of uneven distribution of enamel layer with a cusp at the central axis of teeth having a maximum thickness of 2.5 mm and getting thinned out at the borders. Hence, we made triplicate indentations at the central axis of the enamel before and after radiation for calculating microhardness number and percentage mineral volume of the enamel regions. Microhardness numbers obtained by Vickers and Knoop microhardness indentations are related to percentage mineral volume of the surface according to the mathematical relation established and validated by Featherstone et al. Mineral volume values of enamel surfaces were derived using relation (i) and (ii). Percentage mineral volume of enamel was dependent on radiation doses showing gradual demineralization with an increase in radiation exposure from 20 to 80 Gy. There was a significant reduction in mineral volume at 80 Gy exposure (P < 0.013) compared to controls. Evaluation of microhardness provides knowledge about the susceptibility of teeth to the demineralization process induced by erosive agents such as bacterial acids, ingestion of acid diet, and therapeutic radiation exposure in cases of H and N cancers.
However, controversial outputs were drawn from this technique regarding the effect of radiation on microhardness properties of dental hard tissues. Some studies have shown that an increase in enamel microhardness was found after radiotherapy. They argued that an increase in enamel microhardness and elastic modulus was because of the lower organic content of irradiated enamel which alters Hunter–Schreger band patterns making enamel increasingly stiff and brittle and susceptible to enamel delamination due to radiation exposure., There are reports of decreased enamel hardness postradiation which can be explained by decarboxylation of the tissue due to radiation which can induce microcracks in hydroxyapatite minerals and form lumps of smaller crystallites., These observations are in agreement with our results where decreased enamel microhardness is related to percentage mineral volume loss and a simultaneous increase in mineral density which is due to variation in hydroxyapatite crystal matrix as mentioned earlier. Further studies are needed to incorporate these techniques on much larger sample sizes to clearly understand the effects of radiotherapy on patterns of changes occurring on dental hard tissues.
| > Conclusions|| |
In this study, we have reported variation in micromorphology and mineralization properties of tooth enamel due to exposure to therapeutic doses of megavoltage X-rays from a medical electron LINAC. Increase in the enamel density in irradiated enamel surface might have to do with tampering of the crystal lattice, hydroxyapatite, which forms the major part of enamel ultrastructure. Enamel density variation could be the initiation of direct impact of radiation on teeth, followed by tribological loss, demineralization, and subsequent incidence of radiation caries. Loss of tooth enamel might be a major contributing factor for radiation caries in H and N cancer patients, prescribed to radiotherapy, and doses above 40 Gy can cause serious loss of mineral volume of teeth. In conclusion, we emphasize protecting teeth and surrounding normal oral tissues from radiation-induced complications which would help in delivering an effective treatment to H and N cancer patients.
All procedures performed in studies involving human participant samples were in accordance with the ethical standards of the institutional research committee (Protocol No: 2017/073) and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
For this type of study, formal consent was not required since the patient information was delinked before collecting the samples from clinicians.
Financial support and sponsorship
This work was funded by the Board of Research in Nuclear Sciences (BRNS) research grant vide the Sanction No. DAE-BRNS 34 (1)/14/36/2014.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]