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# Protection of wear resistance behaviour of enamel against electron beam irradiation

## Abstract

### Introduction

Irradiation is known to cause oxidation process among the tissue-altering the properties of teeth leading to tissue necrosis and caries formation. Hence protection of the oral cavity is a major concern to deal with therapy side effects.

### Aim

Evaluation of wear resistance property of Enamel against electron beam radiation and analysing the radio protective effects of natural organic compounds.

### Materials and methods

Total of 36 healthy extracted human molar teeth were collected, four samples were used as control, and remaining 32 were divided into four groups (N = 8 each): radiation control group and three groups treated with organic compounds during radiation treatment. The enamel samples were tested for FTIR spectroscopy, XRD analysis, SEM and EDAX analysis before and after 70 Gy radiation treatment.

### Results and discussion

The particle size of radiation control samples had increased showing decrease in its crystallinity index. Calcium to Phosphorous ratio had also decreased along with structural changes as observed by SEM analysis. But groups treated with organic compounds has maintained tooth integrity in comparable to control groups after radiation treatment.

### Conclusion

Virgin coconut oil, vitamin E oil and curcumin has potential radioprotective action against radiation in protecting tissue properties. Hence, with further advanced research, these natural substances should emerge as a topical applicator during radiotherapy to oral cancer patients.

## Introduction

Oral cancer is one of the commonest cancer’s rankings sixth around the world.1 Ninety percent of the cases are epidermoid squamous cell carcinomas (ESCC) in the lining of the oral cavity.2 Alcohol, smoking and tobacco consumption are considered as risk factors elevating the levels of malignancy.3 While, human papilloma virus and chronic infection in the oral cavity might be the other causes for cancers in non- smokers and drinkers.4 The treatment of cancer involves a series of process including drugs, chemotherapy, surgery followed by radiotherapy or a combination of these therapies. However, the aggressive mode of treatment against the oncological condition has unavoidable effects on normal cells within the vicinity of exposure.5 It mainly affects salivary glands leading to xerostomia, muscular atropy, dental caries, fungal and bacterial infections and many more.6 Radiation caries is the late indirect effect observed in the patients causing irreversible damage on the dental tissues.7 Evidence suggest that demand for dental treatment is around 58–98% among oral cancer patient8 Also, dental treatments are highly recommended in prior to prevent post radiation effects, especially to extract teeth with poor prognosis having high risk of infection.8 Because, there are high possibility to develop caries or osteonecrosis in irradiated teeth due to softening of the tissue, plaque formation9 and changes in dental tissue properties decreasing surface microhardness (SMH),7,10 ultimate tensile strength11 and fracture resistance.12,13

## Materials and methods

### Tooth collection and specimen preparation

The healthy extracted human molar teeth were collected from the Department of Oral and Maxillofacial Surgery, A B Shetty Memorial Institute of Dental Sciences, Mangaluru. The consent waiver form was obtained from the institute to collect these extracted teeth samples. The experimental procedures were approved by the Human Ethics Committee of Nitte University, and the methods were carried out in accordance with the Declaration of Helsinki (2008). A total of 36 human teeth were selected for this study. They were maintained in artificial saliva (0.375 g/l calcium chloride, 0.125 g/l Magnesium chloride, 1.2 g/l Potassium chloride, 0.85 g/l Sodium Chloride, 2.5 g/l Sodium Hydrogen Phosphate, 1 g/l sorbine acid, 5 g/l carboxymethylcellulose sodium and 43 g/l sorbitol solution (70%, noncrystalline)) until the initiation of the experiment. Four samples were used as control (without radiation treatment), remaining 32 samples were divided into four groups (N = 8 each) based on the topical application used for protection during irradiation treatment: (1) radiation control group in distilled water, (2) test group in coconut oil, (3) test group in vitamin E oil and (4) test group in turmeric.

Out of eight samples from each group, two are used for Scanning Electron Microscopy and EDAX analysis and rest of the six samples are used for FTIR and XRD analysis.

The enamel portions of the teeth specimens (control and test sample groups) were drilled using airotar under running water to avoid the dehydration caused by local overheating. The powdered enamel samples were dried and used for the Fourier Transmission Infra-Red (FTIR) spectroscopy and X-Ray Diffraction (XRD) analysis.

The powdered teeth specimens (n = 6/group) were immersed in the solutions for Group 1,2 and 3 and layered with a turmeric in vaseline combination (polymer form) for group 4 in polypropylene Eppendorf tubes (Tarsons). FTIR and XRD testing was done at 0 Gy initially before starting the radiation treatment. Following the testing, the samples were again immersed in the solutions till they received 70 Gy of electron beam radiation using Linear Accelerator (Dual-energy photons, five energies of electrons and CT simulator) with daily exposure of 7 Gy fraction/day for ten days at Nitte Leela Narayan Shetty Memorial Cancer Institute. When they reached 35, 42, 49, 56, 63 and 70 Gy radiation, the samples were washed and dried to conduct FTIR and XRD testing at various radiation intervals. The total dosage of radiation and the course of therapy were followed as per the module for oral cancer patients to simulate the clinical situation.20 All the sample tubes were immersed in water to distribute the radiation dosage equally throughout the medium. After irradiation, the specimens were thoroughly rinsed with deionized water and then tested.

### Test period

The samples were tested for their change in the properties at various radiation intervals. Four tooth samples were used as control and tested without radiation treatment. The remaining six samples were tested for FTIR and XRD after receiving 0, 35, 42, 49,56,63 and 70 Gy Electron beam radiation dose. The samples were further processed and evaluated

### FTIR analysis

FTIR spectrometric analysis of the enamel was performed using a spectrometer (IRPRestige-21, Shimadzu, Japan) under the supervision of expert supervisor at Mangalore University. The spectra were recorded in the range of 400–4000 cm−1 at a 4 cm−1 resolution. The samples were positioned against the diamond crystal of the FTIR unit and pressed with a force gauge at a constant pressure to facilitate contact. The spectra of the enamel before and after irradiation at intervals were obtained. Data were recorded and analyzed with Origin Pro 8.0 SRO software (Northampton, USA). The band between 500 and 885 cm−1 represents CO32−v2, while the band between 885 and 1090 cm−1 provided information about PO43− v1, v3. After normalization, the ratio of the integrated areas of the CO32− v2 peak to the PO4 3−v1, v3 peak (C: M value) were measured.21

### X-Ray diffraction

The crystalline nature of the enamel specimens was evaluated before and after irradiation treatment at various study interval using X’pert XRD (X’pert PRO, Panalytical, Netherlands) with CuKα radiation source at 35 kV/25 mA. Data were obtained in the 2θ range of 10°–70°. Both, crystallinity and grain size were calculated. The crystal sizes were calculated using Scherrer’s formula22,23 as follows: D = 0.89 λ /βcosθ, where λ represents the wavelength (CuKα), θ is the peak diffraction angle that satisfies Bragg’s law for the hkl plane and β is the width of the diffraction profile. The CIXRD index of XRD patterns was measured using several peaks located at the proximity to the highest peak in the graph as suggested by Pearson et al.19 The reflections chosen with reference standard hydroxyapatite were (211), (202) and (300) located between 30 and 35° of the 2- theta angle. The height of the highest peak corresponding to (211) was measured from the baseline fit and heights of the other peaks were from the top to valley separating it from the corresponding peak.

The CIXRD value is given by:

$$\left( {{\mathrm{CI}}} \right){\mathrm{XRD}} = \frac{{{\mathrm{a}}\left( {{\mathrm{300}}} \right) + {\mathrm{b}}\left( {{\mathrm{202}}} \right)}}{{{\mathrm{h(211)}}}}$$

### SEM analysis

The eight samples were marked at a point on its enamel surface using a diamond marker pen. Morphological changes observed at these marked regions at various intervals using SEM (Carl Zeiss- Fesem, Oxford Instruments, EDS) to reveal the deformation and fracture patterns. SEM edax were performed to analyze the compositional change at the region on the enamel at different irradiation intervals. The results of compositional change for Calcium, Phosphate, Oxygen and sodium expressed in terms of percentage of atomic mass and weight.

### Statistical analysis

Data were analysed using Statistical Package for the Social Sciences (SPSS) version 16.0 software (SPSS Inc, Chicago, IL, USA). A paired t-test was used to compare crystallinity, crystal size, C: M ratio and Compositional change before and after the irradiation treatments at various intervals. A p- value less than 0.05 (p < 0.05) was considered statistically significant.

## Results

### XRD results

X-ray diffraction (XRD) spectra of the enamel before and after irradiation are shown in Figs. 1 and 2. The hkl Miller indices assigned correspond to the standard hydroxyapatite diffraction pattern (JCPDS 86–0740). The test enamel XRD patterns were compared with that of reference hydroxyapatite, and the peaks were found to be consistent. Meanwhile, the powder XRD analysis indicated that irradiation had decreased the crystallinity of enamel and enlarged the crystal size (Table 1). However, the protective mechanism was exhibited by the natural agents retaining the crystallinity of the enamel. Virgin coconut oil appeared as the best natural product among the studied materials followed by curcumin and vitamin E oil. Table 2 shows the particle size and crystallinity Index as measured from the Figs. 1 and 2 using squerrer equation and millers’ indices. It has been noted that the particle size of the radiated sample maintained in distilled water had increased showing a decrease in its crystallinity index. The CI XRD values of samples maintained in protective medium showed better crystalline Index than compared to radiation control group.

### FTIR analysis

The representative Fourier transform infrared spectroscopy (FTIR) spectra were measured for its absorbance in all the enamel test groups. The integrated area of CO2− v2 and PO3−v1, v3 peaks were measured. The radiation control group showed a significant decrease in peak size and area in the enamel components after 70GY irradiation treatment when compared to the non-radiated group. The decrease in PO43− v1, v3 integrated area was higher than the CO 2− v2 area. The results of the paired t- test revealed a significant increase in the carbonate: mineral ratio (C: M) after irradiation in the control groups and a significant decrease in the ratio among the protective media groups (Table 3). The test groups did not show a major difference at all the radiation interval periods maintaining insignificant stable mineral ratio thereby improving area retention at all the experimental periods. (Figs. 3 and 4)

## Discussion

On the other hand, the organic compound treated samples depicted significantly lower C: M ratio compared to that of the radiation control group. Also, there exists a positive correlation between microhardness and the mineral content of the tooth enamel.34,35 Hence the reduction in mineral content of enamel after irradiation is the cause for the decrease in micro hardness property of the enamel.36 But the test group compounds were successful in maintaining the nearly stable mineral content ratio during and after radiation therapy.

## Conclusion

The study findings have provided evidence to justify that radiation treatment up to 70 Gy is causing damage to the enamel tissue property where it loses its mechanical strength, undergoes compositional change along with structural variations as studied by XRD, FTIR and SEM EDAX analysis. The topical applications with organic compounds from natural source during radiation treatment have preserved the tissue integrity of teeth. This widens the scope for future research to develop a natural protective compound to be applied during radiotherapy.

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## Acknowledgements

We would like to thank the Board of Research in Nuclear Sciences (BRNS) for funding this research Project. (34(1)/14/31/2014-BRNS). We also thank Nitte (Deemed to be University) for providing platform and infrastructure to continue research work in the Institution.

## Author information

Authors

### Contributions

MNH designed the experiments and received the grants for this project. NDH provided technical support for sample collection. PG performed the measurements. MNH and PG co-wrote the paper. All authors have commented on the discussion of the manuscript.

### Corresponding author

Correspondence to Mithra N. Hegde.

## Ethics declarations

### Competing interests

The authors declare no competing interests.

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Hegde, M.N., Gatti, P. & Hegde, N.D. Protection of wear resistance behaviour of enamel against electron beam irradiation. BDJ Open 5, 11 (2019). https://doi.org/10.1038/s41405-019-0021-0