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 Methods Ten 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. Results

The 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 applicationed at 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) maycan be 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 contents ofin 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 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 results areis 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,hence DI 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 to pH 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 ofby CaCl2 (2.,22m 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 ofto night time remineraliszation corresponding night time. Daily solution

changes were performed and maintained atin room temperature. The solutions were prepared usingwith

DIdeionizated 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 N2 was 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 CO3

2-) (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.

OAccording to our findings demonstrated that, RT affecteds collagen inof the mineraliszed structure of

the dental tissue (Table 3 and Fig. 3A). Other studies demonstratedhave shown that the pulp collagen

[521] and the dentin collagen [13, 398] weare also affected, which may cause a reduction inof the anchor

between the enamel and dentin and, thereby increaseing the possibility of enamel fracture of the enamel

in the incisal and occlusal surfaces [11, 321], primarily during mastication. Furthermore, Tthe gap formed

in the DEJ causes denaturation of the organic matrix and therefore a greater weakening of the enamel

[398]. The degeneration process of odontoblasts and obliteration of dentinal tubules are due to the

radiotherapy damage, which leadsing to changes in metabolism and vasculariszation [14]. Radiation also

reduces dentinMeanwhile, the microhardness of dentin is also reduced by radiation [210, 276]. This

change can result in enamel ablation along the DEJ with crack formation in the cervical region, incisal or

occlusal [443] and GAP formation in the DEJ region, which combined with the masticatory stress, can

cause bacterial coloniszation [210], and a higher risk of caries, which rises with poor oral hygiene [254].

TAlthough the organic matrix is present in tooth enamel atin very low concentrations (1%)

[287], but it plays an important role. This matrix is cComposed of small peptides and amino acids that

areand distributed throughout the mature tissue, and it presumably represents the remains of the initial

developmental matrix that is, perhaps retained via links with the hydroxyapatite crystals. The organic

matrixIt provides the template for enamelthe mineraliszation, of the enamel and it continues to be the

means of transport for substances into the interior. The organic matrixBy playsing a major role in the

control of the ionic diffusion into this tissue, and itorganic matrix can prevents, facilitates, or manages

enamel demineraliszation [3029]. Damage to the organic matter and in the interprismatic substances of

enamel also contributed to RT by causing chemical reactions with water molecules [1], which alters

theresulting in changes in the diffusion properties [176]. Water isThough present in a small proportion

ofin the enamel, but itwater plays an important role in enamelthe function of the enamel because

dehydration affects the mechanical properties of the enamel structure [13, 354].

One factor that could contribute to this difference in organic contentthe results obtained

between the µ-EDXRF and FT-Raman analyseis, regarding to the organic content, is the different

penetration depths, as shown in a previous study [376]. This difference is explained by the operationg

principles of the two techniques. Raman spectra provide analyseis of bulk material because the laser

penetration depth is greater than 1.0 mm. µ-EDXRF analysis was performed with points that were 50 μm

in diameter atnd with a penetration depth of only a few μmicrons. The most important difference in

resolution between these techniques resides in the incident or excitation beam wavelength and energy. X-

rays are shorter and more energetic than the infrared lasers that are used in the Raman technique [376].

TBecause there are 10 calcium (Ca) ions per unit of hydroxyapatite. Therefore, the calcium

activity is raised to the tenth power in the equation for the solubility product equation [4544]. As a result,

and the solubility product of dental enamel is directly related to the strength of the enamel during pH

cycling, which is affected more by changes in Ca concentration than by changes in any other factor , both

in the tooth structure and in the external environment. Therefore, we can infer that mineral solubility is

linked to stoichiometric deviations in the components of hydroxyapatite. However, our study indicated no

significant changes in the weight percentage of Ca of when evaluating the enamel undergoing RT and

after RT and pH cycling (Table 1 and Fig. 2A). Our results are consistentaligned with those of Kielbassa

et al. [265], who observed that enamel that has undergone RT is not more susceptible to demineraliszation

compared with enamel that did not undergo RT. These authorsy suggested that the RT causeds changes in

the ultrastructure of enamel without presenting any clinically impacting in the beginning of

demineraliszation [221]. However, we must consider that the free radicals found in enamel apatite

submitted to RT maycan cause harmful chemical reactions after RT [12].

Another possibility is that the calcium phosphate found in tooth structure causes an extraordinary

loss of water molecules during RT, which createsallowing empty spaces between the molecules that cause

irreversible changes in the tooth structure [432] and significant micromorphometrical differences in

enamel [14]., These alterationswhich makes teeth more vulnerable to acid attack [210, 254] and causes

changes in their biomechanical properties [1, 13, 254, 465].

Exclusive treatment with RT did not change the phosphorus wt % (Table 1 and Fig. 2B) or the

phosphate band integrated area (Table 3 and Fig. 3B). However, the effect of the pH cycling caused a

significant reduction in both phosphorus (p = 0.016) and the phosphate area (p = 0.046) (Tables 1 and 3,

respectively). The reduction in mineral concentration is related to the low pH, which favours the

dissolution of hydroxyapatite [3]. Our results suggest that the pH cycling could affected the enamel

apatite that hads undergone RT, whichthus causeding some structural damage to the enamel from the

phosphate component. Micromorphometrical differences were also observshowed during the dental

enamel demineraliszation submitted to RT [14]. One possible explanation for this decrease is that the

phosphorus molecule that is present in the structure of hydroxyapatite is located more externally, which

makesing it more unstable and susceptible to damage [5, 365].

The rate of hydroxyapatite mineralization is determined by Tthe Ca and P ratio (Ca/P)

determines the rate of hydroxyapatite mineralisation. This ratio was calculated for stoichiometric

hydroxyapatite (1.67). However, the amount of hydroxyapatite found in hard biological tissue varies

according to the degree of tissue mineraliszation of the tissue, i.e., a higher value indicates that the tooth

structure is more mineraliszed with Ca. According to the literature, Tthe minimum and maximum ranges

for the Ca/P ratio of hydroxyapatite present in human dental structures lie between 1.3 for intratubular

dentin and 2.3 for enamel [2]. In this study, the RT and pH cycling resulted in a significant increase in the

Ca/P ratio (p = 0.000) in this study (Fig. 2C). This increase was due to a non-significant increase in Ca

and a significant decrease in the phosphorus weight percentage, which demonstratesing that despite the

pH cycling in the teeooth that underwent RT, this difference was sufficientjust enough to alterchange the

inorganic P component (Table 1). This finding is consistentwas in accordance with other studies that

reported no differences in enamel solubility and the depth of caries lesions in teeth undergoing RT [176,

232].

In this study, Tthe FT-Raman Spectroscopy evaluation in this study demonstrated that the

relative area of carbonate band decreased significantly in the group in which the samples that underwent

RT and pH cycling (RTc) compared with the group of teeth before receiving RT (RTb) (Fig. 3C). The

difference was most likely due to the pH cycling than in relation to the exclusive application of the RT

because the group that received only RT (RTa) exhibitshowed no significant reduction. There is a positive

correlation between the carbonate and enamel solubility [5049]. The micro-spaces that are formed as a

result of the loss of carbonate and organic matrix can prevent demineraliszation and ionthe dissolution of

ions. These results suggest that a teeooth that underwent RT and pH cycling tendeds to have an initial loss

of carbonate, which is an element that providgives greater solubility butand is most likely the first

element to be lost, and corroboratesing the resultsstudy of Jansma et al. [176]. However, no significant

difference in the carbonate area was observed betweenamong the healthy teeth subjected to the pH

cycling (Nc) andor the RT and pH cycling (RTc) (Table 3).

Notably,It is noteworthy that caries and radiation caries areis a multifactorial diseases [9], as is

radiation caries, in whichere the sum of several factors may be responsible for damage to the tooth

structure. In this study, when Ccomparisons ofg the RTc and Nc groups usingby μ-EDXRF analysis

revealed, no significant changes were observed in relation to the Ca, P, or oxygen weight percentages or

the Ca/P ratio (Table 1 and Figs. 2A-D), which wais confirmed in previousby other in vitro studies [176,

310]. The analysis by FT-Raman spectroscopy analyses also demonstratshowed no differences inamong

the band values of the organic content, phosphate, and carbonate, between the RTc and Nc groups (Table

3 and Figs. 3A-C). However, note that Iin vitro studies do not adequately reproduce clinical conditions,

butjust as in situ and in vivo studies have limitations because the effects of radiation differ between

individuals differ (i.e.g., differences in salivary flow, composition of oral microbiota, diet, etc.) and due

to the fragility of these patients. ThereforeHence, the study ofstudying radiation caries development is

very difficult, primarily because other factors may be associated with its development [167, 212, 223,

289, 501, 567, 578].

Radiation caries is a frequent severe disease that is severe, develops rapidly, frequent, and it is

difficult to control. This condition, and it causes cosmetic problems, altered eating habits, pain and

changes in the quality of life of cancer patients [156]. The use of preventive protocols [26] after

radiotherapy treatment and a multi-professional monitoring aiming preventive and curative treatments

will allow these patients to live better with the consequences: taste loss, hyposalivation, radiation caries,

trismus and osteoradionecrosis, acquired after radiotherapy treatment [27, 57].In addition, Tteeth with

great coronary destruction or pulpal infection may result in increase thed risk of developing

osteoradionecrosis [19]. The use of preventive protocols [276] after radiotherapy treatment and a multi-

professional monitoring foraiming preventive and curative treatments will allow these patients to live

better with the consequences: taste loss, hyposalivation, radiation caries and trismus ofacquired after

radiotherapy treatment [276]. These sequelae for radiotherapy for head and neck cancer become

increasingly important, and have a tremendous effect on quality of life. Recently, studies

demonstratedhave shown that the intensity of the radiation dose is an important factor to be considered in

the development ofing the radiation decay [ 321 ,, 587 ]. Teeth undergoing radiation higher than 60 Gy

exhibithave changes in their mineral structure and collagen in, both dentin and enamel, and there is a

reduction in hardness and , in tensile strength and an increased the possibility of fracture , that, reachesing

amputation of the crown [ 587 ]. The few mineral and organic changes in teeth submitted to RT found in

the present study demonstrateshows the need for further studies to better understand the pathophysiology

of radiation caries and to establish the best means to prevent and treat oral complications in patients who

undergowent RT.

Conclusion

The assessment by µ-EDXRF assessment revealshowed phosphorus ion reduction and an increase in the

Ca/P ratio inwhen samples were subjected to RT and pH cycling. The FT-Raman spectroscopy results

demonstratshowed that the RT, provided at therapeutic doses of RT, exclusively reduced the organic

content. The effect of the pH cycling enabled a reducedtion in the phosphate content. RT with pH cycling

resulted in a reducedtion in the carbonate and phosphate contents compared with those of healthy

enamel. Radiation damageds the organic content of the enamel. Other studies are needed to evaluate the

composition and molecular structure of enamel that has undergone RT, considering the influence of the

etiological factors of caries.

Compliance with Ethical Standards

Funding: This study was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP,

for the X-ray microfluorescence equipment (Grant no. 2005/50811-9) and FT-Raman spectroscopy

system (Grant no. 01/14384-8).

Conflicts of Interest: Author Elza Maria de Sá Ferreira declares that she has no conflict of interest. Author

Luís Eduardo Silva Soares declares that he has no conflict of interest. Author Héliton Spíndola Antunes

declares that he has no conflict of interest. Author Sofia Takeda Uemura declares that she has no conflict

of interest. Author Patrícia da Silva Barbosa declares that she has no conflict of interest. Author Hélio

Augusto Salmon Jr declares that he has no conflict of interest. Author Giselle Rodrigues de Sant’Anna

declares that she has no conflict of interest.

Ethical approval: All procedures performed in studies involving human participants were performed in

accordance with the ethical standards of the institutional and/or national research committee and with the

1964 Helsinki declaration and its later amendments or comparable ethical standards. This study was

approved by the Ethics and Research Committee of the Cruzeiro do Sul University (Universidade

Cruzeiro do Sul), São Paulo, Brazil, under Protocol Nº 058/2010.

Informed consent: Informed consent was obtained from all individual participants included in the study.

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Figure captions

Fig. 1 Description of the study design

Fig. 2 Mean and standard deviations (SD) of: (A) calcium, (B) phosphorus, (C) organic content weight

percentages (wt %), and (D) Ca/P molar ratio from enamel obtained by µ-EDXRF analysis for each group

and period of treatment: N - not irradiated, Nc - not irradiated after pH cycling, RTb - before

radiotherapy, RTa - after radiotherapy, and RTc - after radiotherapy and pH cycling

Fig. 3 Mean and standard deviations (SD) of the relative area of: (A) organic content band (2940cm -1 ),

(B) phosphate (960cm-1 ), and (C) carbonate (1070cm-1 ) bands obtained by FT-Raman spectroscopy for

each group and period of treatment: N - not irradiated, Nc - not irradiated after pH cycling, RTb - before

radiotherapy, RTa - after radiotherapy, and RTc - after radiotherapy and pH cycling

Table 1 Statistical comparisons of the average content of calcium (Ca), phosphorus (P),

and oxygen (O) weight percentages (wt %) in the enamel and the Ca/P weight ratios

obtained by x-ray fluorescence among stages RTb, RTa, RTc, N and Nc.

Groups comparision Calcium Phosphorus Oxygen Ca/P ratio

RTb versus RTa p=0.438 p=0.411 p=0.318 p=0.115

RTa versus RTc p=0.395 p=0.016 p=0.880 p=0.000

RTb versus RTc p=0.131 p=0.267 p=0.380 p=0.000

N versus Nc p=0.353 p=0.314 p=0.767 p=0.002

RTc versus Nc p=0.824 p=0.961 p=0.933 p=0.620

Paired Student’s t test and Student’s t test.

Table 2 Differences in means and weight percentages (wt %) of calcium, phosphorus,

and oxygen between stages RTb-RTc and N-Nc

Elements Groups comparision Means (SD) p value

CalciumRTb versus RTc -3.25 (6.19) 0.436

N versus Nc -2.11 (6.83)

PhosphorusRTb versus RTc 0.83 (2.21) 0.853

N versus Nc 0.88 (2.60)

OxygenRTb versus RTc 2.44 (8.36) 0.631

N versus Nc 0.40 (9.69)

Non-parametric Mann-Whitney test.

Table 3 Comparison of integrate area of Raman bands relative to the organic content

(2940/960 cm-1 ), carbonate (1070/960 cm-1 ) and phosphate (430/960 cm-1 ) among the

RTb, RTa, RTc, N, and Nc groups

Groups

comparision

Organic content Carbonate Phosphate

RTb versus RTa p=0.000 p=0.220 p=0.661

RTa versus RTc p=0.146 p=0.261 p=0.046

RTb versus RTc p=0.160 p=0.004 p=0.035

N versus Nc p=0.951 p=0.504 p=0.853

RTc versus Nc p=0.070 p=0.123 p=0.577

Paired Student’s t test and Student’s t test.