Effect of therapeutic doses of radiotherapy oin the organic and inorganic contents of the deciduous enamel: an in vitro study

Abstract

Objectives This study evaluated the effects of radiotherapy on the composition of deciduous teeth enamel, using micro energy-dispersive X-ray fluorescence and Fourier transform Raman spectroscopy before and after a pH-cycling process. Materials and MethodsTen deciduous molars were sectioned and divided into two groups (n=10). The radiotherapy group (RT) was irradiated with 54 Gy at 2 Gy/day, 5 days per a week for 5 weeks and 2 days, and the normal group (N) was not irradiated. The RT group was evaluated before radiotherapy (RTb), after radiotherapy (RTa), and after radiotherapy and pH cycling (RTc). The normal group was evaluated before (N) and after pH cycling (Nc). The weight percentage (wt %) of calcium (Ca), phosphorus (P), and organic content, and the Ca/P ratio, ands well as the integrated area of the Raman bands relative to the organic, carbonate, and phosphate contents were also evaluated. ResultsThe exclusive use of RT reduced the organic content of enamel (p=0.000). The RTc group exhibitshowed a decrease in P wt % (p=0.016), an increase in in the Ca/P ratio (p=0.000), and a reduction in the integrated area of the phosphate band (p=0.046). Among the RTb/RTc treatments,Aan increase in the Ca/P ratio (p=0.000) and a reduction in the areas of the both carbonate and phosphate bands were found in the RTb/RTc treatments. Conclusions The RT applicationedat ain therapeutic dose reduced the organic content of the deciduous enamel. Clinical Relevance Due to chemical changes caused by RT on the deciduous enamel,Ppreventive measures should be included ion the patient treatment protocol because of RT-induced chemical changes to the deciduous enameltreatment.

Keywords: Radiotherapy, Deciduous enamel, Energy-Dispersive X-ray Spectroscopy, Fourier transform Raman spectroscopy, Head and neck cancer.

Introduction

Caries, erosion, and damage to dental hard tissues are among the frequently observed late clinical changes in patients who undergo radiotherapy in the head and neck region [7, 565], and these changes, which significantly impede the quality of life of these patients [6, 298]. Radiation caries also develop rapidly [1209, 276] in a distinctive manner, unlike typical decay, with an initial shear fracture of enamel that, sometimes resultsing in partial to total enamel delamination, followed by a subsequent decay of the exposed underlying dentin [187, 210, 221, 587]. Brown-black tooth surface discoloration is also sometimes associated with teeth exposed to radiotherapy. Notably,It is important to note that post-radiation dental lesions differ considerably from decay in non-irradiated patients in clinical appearance, pattern of development and progression from decay in non-irradiated patients [210, 221]. Typical dental decay occurs in pits, fissures and proximal areas between teeth. In contrast, post-radiation dental lesions tend to occurs at cervical (junction between crown and root), cuspal and incisal areas [58].

RAlthough radiation-induced hyposalivation is considered one of the most important etiological factors for the development of caries [8, 210, 510, 587], but other factors, such as a reduction in the protective properties of saliva, salivary pH reduction, quantitative and qualitative changes in the bacterial flora [8], dietary changes [8, 176, 212], saliva composition [10], intensity of radiation dose on the tooth [4, 587] and poor hygiene [221, 243, 298], should be considered. All of these factors characterizse radiation decay as a multifactorial disease[286, 298].

Scientific evidence indicates that teeth undergoing RT are not more susceptible to caries development [176, 198, 221- 243]. However, damage to the mineralizsed tissue and changes in the biophysical properties of the tooth, such as the resistance and morphology of the dentinoenamel junction [321, 332, 398],arehave been described in the literature. Nevertheless, controversies onremain regarding the deleterious effects of RT on dental enamel remain [176, 198, 398].

Information onabout the organic and inorganic composition of dental enamel is necessary to obtain a better understanding of the effects of RT on dental hard tissues. Raman spectroscopy [376, 401, 487] and micro- energy-dispersive X-ray fluorescence (µ-EDXRF) [5, 421, 476, 498] werehave been applied in several areas; however, but these types of analyses have not yet been used to study the effects of RT on the structure of deciduous enamel. Raman spectroscopy is a non-destructive technique that can detects changes in the structure and composition of mineral and organic components of enamel [3029, 4039, 410, 487, 532, 554].

Complementing the information obtained from Raman Spectroscopy, micro energy-dispersive X-ray fluorescence (µ-EDXRF)[Editor1]maycanbe used to qualitatively and quantitatively analyzse the components of the structure of the enamel apatite to, thus provideing information onabout the chemical interactions between the enamel and the RT.

SAlthough several investigations on have been performed regarding the deleterious effects of RT on dental elements were performed [4, 8, 10, 176, 2019-212, 321, 332, 565, 587], but studies on the molecular structure, and organic and inorganic composition of tooth enamel are required to determine the pathophysiology of radiation caries.

We tested theThe null hypothesis tested here was that if the therapeutic dose of radiation does not alter the composition and molecular structure of deciduous enamel, then this will not cause damage to the organic and inorganic contentsofin the deciduous tooth enamel.The aim of this study was to used micro energy-dispersive X-ray fluorescence(µ-EDXRF) and FT-Raman to evaluate in vitro whether RT interferes with the composition and molecular structure of deciduous tooth enamel both before and after a pH cycling.

Materials and methods

Sample preparation

This study was approved by Tthe Ethics and Research Committee of the Cruzeiro do Sul University (Universidade Cruzeiro do Sul), São Paulo, Brazil approved this study, under Protocol Nº 058/2010. Ten deciduous, caries-free, extracted, or exfoliated first and second molar teeth were cleaned using a rubber cup (Viking, KG - Sorensen, Barureri, SP, Brazil) and water and, then they were stored in deionizsed water[13, 176].De-ionizsed [Editor2]water (also called DI water) is water withthat has the ions removed. Tap water generis usually containsfull of ions from the soil (Na+, Ca 2+), from the pipe (Fe2+, Cu2+) and other sources. Water is generusually de-ionizsed by using an ion exchange process. Often during the chemistry experiments as this one, when we demineralized the samples using chemical solutions, Tthe ions in water will oftenhave interference in solutions and also in the storage of sample storage during chemistry experiments, such as the present study, when the samples are demineralised using chemical solutions. The ions in watery can switch places with other ions that you may be interested during your experimentaling and analyszisng ofn in the mineral structure. The dDissolution ofving samples in water and doing testings on the resultsareis a common technique, and contaminants in the water will interfere with themake the whole test give wrong results and all storage mediaum and all storage media was stored for future studies. DIe-ionized water is no't necessariely pure water based ongiven the usual de-ionizsation process.;Therefore,henceDI waterfor this study it was also filtered throughin biological filters in this study. Artificial saliva was not used in the present study because it does not have exactly the same characteristics as the natural saliva, especially in patients who underwent radiotherapy in the head and neck, because these patients have alterations of salivary flow and saliva compositio[15]. Longitudinal hemisectioning was performed in a (cCorono-root direction) using a low-speed micromotor (LB100 Beltec, Araraquara, SP, Brazil) and carborundum disk (Dentaurum, Pforzhein, Germany) under cooling (running water) to obtain two samples of each dental element with an up to 2 mm thickness of tooth enamel. A 2 mm × 3 mm rectangle of laboratory film (Parafilm M Barrier Film, West Chester, PA, USA) was cut and placed in the middle third of each sample. The surfaces were covered with two layers of red nail polish (Revlon, New York, NY, USA). After the nail polish dried,Tthe films were removed after the nail polish dried, which resulteding in a 2 mm × 3 mm surface window.

Sample treatment

The 20 samples were randomly divided into two groups ofwith 10 samples perin each group (Fig. 1).

Radiotherapy Group (RT)-The samples were first evaluated before RT (RTb), after RT (RTa) and after ; they then underwent RT and were evaluated again after RT (RTa). Finally, the samples were subjected to pH cycling and evaluated again (RTc).

Normal Group (N) -These samples were first evaluated before (N) and afterthen submitted topH cycling and evaluated again (Nc).

Radiotherapy parameters

RT of the samples was performed at the Radiotherapy Center of the Integrated Oncology Clinics (Clínicas Oncológicas Integradas - Grupo COI), located in Rio de Janeiro, Brazil. RT planning was performed using computed tomography of the samples to, simulateing the clinical patterns of a juvenile patient with a head and neck cancer. The samples received 54 Gy in the form of 2 Gy in 27 daily fractions, 5 days per weekly for 5 weeks and 2 days. A 6 MV photon energy dose was delivered through a direct field on the surface of each tooth using a linear accelerator (ONCOR Expression model, Siemens, Erlangen, Bayern, Germany). The effect of a photon beam of this energy produced a build-up region of approximately 1.5 cm (DIdeionized water), which simulated theing 1.5 cm of tissue above the tooth. Thereafter, each tooth was irradiated with a total dose of 54 Gy at an energy level of 6 MV. The samples were placed on two wax plates, with 10 samples on each plate positioned 0.5 cm apart. The plates were then placed in 5.0 cm of solid water to account for backscatter. A 10 × 10 field was used at a distance of 100 cm. The wax plates were fixed in a plastic container that was held in place with a lead ring to prevent displacement. All samples received the dose at the same time and remained immersed in 2.0 cm of DIdeionized water to minimizse possible ion exchange [176]. WOnce water forms free radicals of hydrogen and hydrogen peroxideexhibits severe chemical reactions with the absorption of radiation, it forms free radicals of hydrogen and hydrogen peroxide. These radicals in turn cause denaturation of the organic components of teeth, which causing changes in the integrity and mechanical properties of the enamel and, consequently, in its mechanical properties [1]. This configuration simulates the water content of saliva.

Caries-like lesion formation (pH -cycling process)

All the samples were submmitted to the process of superficial induction of caries lesions formation using the [pH cycling model of ten from Cate and Duijsters [534] as modified by Mendes and Nicolau [334]. Samples iIn this experimental model, the sample wereas submitted to alternate solutions of demineraliszation and remineraliszation, for 7 uninterrupted days at, in room temperature and without agitation. The specimens were placedut individually in plastic containerpots containing 8 ml of a demineraliszation solution (DE) composed ofbyCaCl2 (2.,2[Editor3]2m mM),; NaH2PO4 (2.,22m mM),; acetic acid (0.,055M M) pH 4.,8 adjusted with KOH (11M M), per litere of solution for 8 hours followed by 16 hours and then in 8 ml of a remineraliszation solution (RE) composed ofby CaCl2 (1.,55m mM),;NaH2PO4 (0.,99m mM) ande KCl (0.,155M M) pH 7.,0 adjusted with KOH (11M M), per litere of solution for16 hours, in order to simulate similar daily periods of 8 hours ofeach to remineraliszation and demineraliszation and 8 hours oftonight time remineraliszation corresponding night time. Daily solution changes were performed and maintained atin room temperature. The solutions were prepared usingwithDIdeionizated water.

Micro energy-dispersive X-ray fluorescence (μ-EDXRF)

A semi-quantitative elemental analysis of calcium (Ca) and phosphorus (P) was performed using a μ-EDX spectrometer (μ-EDX 1300, Shimadzu, Kyoto, Japan) equipped with a rRhodium X-ray tube and a Si (Li) semiconductor detector cooled by liquid nitrogen (N2). The tension in the tube was set at 15 kV, with an automatic adjustment of the incident beam diameter to 50 microns. The equipment was adjusted using a certified commercial reagent of stoichiometric hydroxyapatite (Aldrich synthetic, Ca10(PO4)6(OH)2, 99.999% purity, Lot 10818HA/SIGMA 2008) as a reference.

MThe measurements were collected under basic parameters for the X-ray emissions that were characteristic of the Ca and P elements, and the O2 and H elements were used for equilibrium and chemical balance. AIn total of, 150 spectra (3 points per sample) were collected in the μ-EDXRF analyses. The mean of each of the three points was calculated, and 50 spectra were used for statistical analyseis. MThe measurements were performed usingwith 15 kV and 100 sec per point.

FT-Raman spectroscopy analysis

The enamel slabs were analyzsed usingby FT-Raman Spectroscopy to evaluate treatment-induced changes in the inorganic and organic content caused by the treatments. AnThe FT-Raman spectrometer (RFS 100/S – Bruker, Karlsruhe, Germany) with a germanium detector cooled by liquid N2was used to collect the data. SThe samples were excited by an air-cooled Nd:YAG laser ( = 1064.1 nm). The power of the Nd:YAG laser incident on the sample was 400 mW. The spectral resolution was set to 4 cm-1, and for each specimen, three spectra were collected for each specimen with 100 scans for a, total ofing 150 spectra.

For the qualitative and semi-quantitative spectral analysis, the Eenamel spectra were baseline corrected and then normaliszed using the 960 cm-1 band for qualitative and semi-quantitative spectral analyses [276, 332]. Changes in the organic and inorganic enamel components were analyszed using the areas of the Raman bands centered at 430 cm-1 (ν2 PO43-) (p1), 1071 cm-1 (ν1 CO32-) (p2), and 2942 cm-1 (CH bonds of collagen) (p3) relative to the 961 cm-1 (ν1 PO43-) (p4) [42]. The integrated areas of the bands were calculated using the Microcal Origin 8.0 software (Microcal Software, Northampton, MA, USA).

Statistical Analysis

A power test was initially performed for sample verification (n): for n = 10, Z alpha = 0.05 and Z Beta = 0.20, with a test power of = 0.80. The arithmetic means of the three points of each sample were calculated and analyszed by group for each element. Paired Student’s t tests, Student’s t test, and nonparametric Mann-Whitney test were used. A significance level of 5% probability was adopted (p ≤ 0.05), and IBM SPSS Statistical Software version 17.0 (New York, USA) was used to perform the statistical analyses.

Results

The radiotherapy group (RT) and Nthe normal groups (N) were evaluated at distinct time points. In the RT group,Tthe effect of radiotherapy treatment on the deciduous tooth enamel in the RT group was evaluated at three time points: before RT (RTb), after RT (RTa), and after RT and pH cycling (RTc). Samples iIn the normal group, the samples were evaluated before (N) and after pH cycling (Nc).

µ-EDXRF analysis

After the radiotherapy (RTa), Nno significant changes were found in the calcium orand phosphorus weight percentages (wt %) at RTa (Table 1 and Fig. 2A, B) or in the Ca/P ratio (Fig. 2C). After the radiotherapy and pH cycling (RTc), Aa significant reduction in phosphorus wt % (p = 0.016) and an increase in the Ca/P ratio (p = 0.000) occurred at RTc (Table 1). Comparison ofg the RTb and RTc revealed, a significant increase in the Ca/P ratio was found (p = 0.000) (Table 1 and Fig. 2C). The pH cycling in the normal group (Nc) resulted in an increase in the Ca/P ratio compared with the normal group without pH cycling (N) (p = 0.002) (Table 1 and Fig. 2C). Comparisons between RTc and Nc groups demonstratshowed that the calcium, phosphorus, and oxygen wt % were not modified after pH cycling (Table 1). Longitudinal analyseis of the differences between the experimental time points wereas performed via RTb/RTc and N/Nc comparative analysis. However, but no significant statistical differences were found in calcium, phosphorus, and oxygen wt % (Table 2 and Figs. 2A-D).

FT-Raman spectroscopy analysis

After RT (RTa) Tthere was a significant reduction of the organic content at RTa (p = 0.000) (Table 3 and Fig. 3A). After RT and challenge (RTc) Tthe phosphate area decreased at RTc (p = 0.046) when compared with the RTa (after RT) (Table 3 and Fig. 3B). When comparing the group submitted to RT and challenge (RTc) with the group before RT (RTb), Tthe phosphate (p = 0.035) and carbonate areas decreased (p = 0.004) between RTc and RTb (Table 3 and Fig. 3B,C). CoComparisons ofamong the band areas of the groups Nc and RTc did not revealshow significant changes in the collagen, carbonate, and phosphate contents (Table 3 and Fig. 3A-C).

Discussion

We tested the null hypothesis that if the therapeutic dose of radiation does not alter the composition and molecular structure of deciduous enamel, then this will not cause damage to the organic and inorganic contents of deciduous tooth enamel. This study used µ-EDXRF and FT-Raman to evaluate in vitro whether RT interferes with the composition and molecular structure of deciduous tooth enamel before and after pH cycling. The choice to work with deciduous teeth is related to the large number of children with cancer. Understanding the damage caused by RT, at molecular and compositional level, we can establish preventive measures and provide a better quality of life for these children. In this study the use of human deciduous teeth was due to their chemical and structural similarity to young permanent teeth, proven to be more susceptible to caries [50], allowing a wider range of our results. i

The physical and chemical changes in the dental enamel caused by RT in patients with head and neck cancer remain controversial [176, 198, 221, 232, 398]. It is difficult to establish an exact parallel among the various studies due to the different methods and doses of radiotherapy [198, 221, 265], methodologies used (in vitro, in situ, or in vivo) [14, 398], and demineraliszation conditions [198].

An evaluation of the organic balance using μ-EDXRF demonstratshowed a relationship between the organic and inorganic components. TAlthough the means of the organic components were lower in the group that underwent RT (RTa) compared with the group receiving RT and pH cycling (RTc), but there were was no statistically significant differences compared with to the radiotherapy group (RT) (Table 1 and Fig. 2D). Similar observations were made using the Llongitudinal analyseis of the differences in the averages of the elemental weight of oxygen revealed similar observations (Table 2). However, the assessment by FT-Raman assessments demonstratshowed a significant reduction of organic content in the samples submitted to RT (RTa) (Table 3 and Fig. 3A), which may bewas possibly due to the constant inorganic content of enamel when the stability in the stoichiometry of the crystalline structure was maintained (Table 1). It is likely that alterations in the interprismatic region, which concentrates water, resulted from free radicals and reactive oxygen species accumulation,whichthat may react with and damage organic components [13, 332]. However, theses studies were conducted in vitro, which presents limitations to reproducing exact clinical situations. Factors, such as changes in the oral microflora, hyposalivation, and diet, could not be considered.