Effect of therapeutic doses of radiotherapy on 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, the Ca/P ratio, and 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 exhibited a
decrease in P wt % (p=0.016), an increase in the Ca/P ratio (p=0.000), and a reduction in the integrated
area of the phosphate band (p=0.046). An increase in the Ca/P ratio (p=0.000) and a reduction in the areas
of the carbonate and phosphate bands were found in the RTb/RTc treatments. Conclusions RT application
at a therapeutic dose reduced the organic content of the deciduous enamel. Clinical Relevance Preventive
measures should be included in the patient treatment protocol because of RT-induced chemical changes to
the deciduous enamel.
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, 56], and these changes significantly
impede the quality of life of these patients [6, 29]. Radiation caries also develop rapidly [20, 27] in a
distinctive manner, unlike typical decay, with an initial shear fracture of enamel that sometimes results in
partial to total enamel delamination, followed by a subsequent decay of the exposed underlying dentin
[18, 21, 22, 58]. Brown-black tooth surface discoloration is also sometimes associated with teeth exposed
to radiotherapy. Notably, post-radiation dental lesions differ considerably from decay in non-irradiated
patients in clinical appearance, pattern of development and progression [21, 22]. Typical dental decay
occurs in pits, fissures and proximal areas between teeth. In contrast, post-radiation dental lesions tend to
occur at cervical (junction between crown and root), cuspal and incisal areas [58].
Radiation-induced hyposalivation is one of the most important etiological factors for the
development of caries [8, 21, 51, 58], 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, 17, 21], saliva composition [10], intensity of radiation dose on the tooth [4, 58] and poor
hygiene [22, 24, 29], should be considered. All of these factors characterise radiation decay as a
multifactorial disease [28, 29].
Scientific evidence indicates that teeth undergoing RT are not more susceptible to caries
development [17, 19, 22- 24]. However, damage to the mineralised tissue and changes in the biophysical
properties of the tooth, such as the resistance and morphology of the dentinoenamel junction [32, 33, 39],
are described in the literature. Nevertheless, controversies on the deleterious effects of RT on dental
enamel remain [17, 19, 39].
Information on 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 [37, 41, 48] and
micro-energy-dispersive X-ray fluorescence (µ-EDXRF) [5, 42, 47, 49] were applied in several areas, but
these types of analyses have not been used to study the effects of RT on the structure of deciduous
enamel. Raman spectroscopy is a non-destructive technique that detects changes in the structure and
composition of mineral and organic components of enamel [30, 40, 41, 48, 53, 55].
Complementing the information obtained from Raman Spectroscopy, µ-EDXRF may
qualitatively and quantitatively analyse the components of the structure of the enamel apatite to provide
information on the chemical interactions between the enamel and the RT.
Several investigations on the deleterious effects of RT on dental elements were performed [4, 8, 10, 17,
20-22, 32, 33, 56, 58], 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 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.
Materials and methods
Sample preparation
The 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 stored in deionised water [13, 17]. De-ionised water (DI water) is
water with the ions removed. Tap water generally contains ions from the soil (Na+, Ca 2+), the pipe (Fe2+,
Cu2+) and other sources. Water is generally de-ionised using an ion exchange process. The ions in water
will often interfere in solutions and sample storage during chemistry experiments, such as the present
study, when the samples are demineralised using chemical solutions. The ions in water can switch places
with other ions that you may be interested during your experimental analysis of the mineral structure. The
dissolution of samples in water and testing the results are a common technique, and contaminants in the
water will interfere with the results and storage media. DI water is not necessarily pure water based on the
usual de-ionisation process. Therefore, DI water was also filtered through 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 corono-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). The films were removed after the nail polish dried, which resulted in a 2
mm × 3 mm surface window.
Sample treatment
The 20 samples were randomly divided into two groups of 10 samples per group (Fig. 1).
Radiotherapy Group (RT) - The samples were evaluated before RT (RTb), after RT (RTa) and after pH
cycling (RTc).
Normal Group (N) - These samples were evaluated before (N) and after pH cycling (Nc).
Radiotherapy parameters
RT of the samples was performed at the Radiotherapy Center of the Integrated Oncology Clinics (Clínicas
Oncológicas Integradas - Grupo COI) in Rio de Janeiro, Brazil. RT planning was performed using
computed tomography of the samples to simulate the clinical patterns of a juvenile patient with head and
neck cancer. The samples received 54 Gy in the form of 2 Gy in 27 daily fractions, 5 days 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 (DI water),
which simulated the 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 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 DI water to minimise possible ion exchange [17].
Water forms free radicals of hydrogen and hydrogen peroxide with the absorption of radiation. These
radicals cause denaturation of the organic components of teeth, which changes the integrity and
mechanical properties of the enamel [1]. This configuration simulates the water content of saliva.
Caries-like lesion formation (pH cycling process)
All samples were submitted to the process of superficial induction of caries lesions formation
using the pH cycling model of ten from Cate and Duijsters [54] as modified by Mendes and Nicolau [34].
Samples in this experimental model were submitted to alternate solutions of demineralisation and
remineralisation for 7 uninterrupted days at room temperature without agitation. The specimens were
placed individually in plastic containers containing 8 ml of a demineralisation solution (DE) composed of
CaCl2 (2.2 mM), NaH2PO4 (2.2 mM), acetic acid (0.05 M) pH 4.8 adjusted with KOH (1 M), per litre of
solution for 8 hours followed by 16 hours in 8 ml of a remineralisation solution (RE) composed of CaCl2
(1.5 mM), NaH2PO4 (0.9 mM) and KCl (0.15 M) pH 7.0 adjusted with KOH (1 M) per litre of solution to
simulate daily periods of 8 hours of remineralisation and demineralisation and 8 hours of night time
remineralisation. Daily solution changes were performed and maintained at room temperature. The
solutions were prepared using DI water.
Micro energy-dispersive X-ray fluorescence
A semi-quantitative elemental analysis of calcium (Ca) and phosphorus (P) was performed using a μEDX spectrometer (μ-EDX 1300, Shimadzu, Kyoto, Japan) equipped with a rhodium X-ray tube and a Si
(Li) semiconductor detector cooled by liquid nitrogen (N 2). 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, Ca 10(PO4)6(OH)2,
99.999% purity, Lot 10818HA/SIGMA 2008) as a reference.
Measurements were collected under basic parameters for X-ray emissions that were
characteristic of Ca and P elements, and O2 and H elements were used for equilibrium and chemical
balance. A total of 150 spectra (3 points per sample) were collected in the μ-EDXRF analyses. The mean
of the three points was calculated, and 50 spectra were used for statistical analyses. Measurements were
performed using 15 kV and 100 sec per point.
FT-Raman spectroscopy analysis
The enamel slabs were analysed using FT-Raman Spectroscopy to evaluate treatment-induced changes in
inorganic and organic content. An FT-Raman spectrometer (RFS 100/S – Bruker, Karlsruhe, Germany)
with a germanium detector cooled by liquid N2 was used to collect the data. 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 three spectra were collected for each specimen
with 100 scans for a total of 150 spectra.
Enamel spectra were baseline corrected and normalised using the 960 cm-1 band for qualitative
and semi-quantitative spectral analyses [27, 33]. Changes in organic and inorganic enamel components
were analysed using the areas of the Raman bands centred 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 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 analysed 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 statistical analyses.
Results
The RT and N groups were evaluated at distinct time points. The 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 in the normal group were evaluated before (N) and
after pH cycling (Nc).
µ-EDXRF analysis
No significant changes were found in calcium or phosphorus weight percentages (wt %) at RTa (Table 1
and Fig. 2A, B) or in the Ca/P ratio (Fig. 2C). A 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 of the RTb and RTc
revealed a significant increase in the Ca/P ratio (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 demonstrated
that the calcium, phosphorus, and oxygen wt % were not modified after pH cycling (Table 1).
Longitudinal analyses of differences between experimental time points were performed via RTb/RTc and
N/Nc comparative analysis, but no significant differences were found in calcium, phosphorus, and oxygen
wt % (Table 2 and Figs. 2A-D).
FT-Raman spectroscopy analysis
There was a significant reduction of the organic content at RTa (p = 0.000) (Table 3 and Fig. 3A). The
phosphate area decreased at RTc (p = 0.046) compared with RTa (after RT) (Table 3 and Fig. 3B). The
phosphate (p = 0.035) and carbonate areas decreased (p = 0.004) between RTc and RTb (Table 3 and Fig.
3B,C). Comparisons of the band areas of Nc and RTc did not reveal significant changes in 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.
The physical and chemical changes in the dental enamel caused by RT in patients with head and
neck cancer remain controversial [17, 19, 22, 23, 39]. It is difficult to establish an exact parallel among
the various studies due to the different methods and doses of radiotherapy [19, 22, 26], methodologies
used (in vitro, in situ, or in vivo) [14, 39], and demineralisation conditions [19].
An evaluation of the organic balance using μ-EDXRF demonstrated a relationship between the
organic and inorganic components. 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 was no
significant difference compared to the radiotherapy group (RT) (Table 1 and Fig. 2D). Longitudinal
analyses of differences in the averages of the elemental weight of oxygen revealed similar observations
(Table 2). However, FT-Raman assessments demonstrated a significant reduction of organic content in
the samples submitted to RT (RTa) (Table 3 and Fig. 3A), which may be 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, which may react with and damage organic
components [13, 33]. 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.
Our findings demonstrated that RT affected collagen in the mineralised structure of dental tissue (Table 3
and Fig. 3A). Other studies demonstrated that pulp collagen [52] and dentin collagen [13, 39] were also
affected, which may cause a reduction in the anchor between the enamel and dentin and increase the
possibility of enamel fracture in incisal and occlusal surfaces [11, 32], primarily during mastication. The
gap formed in the DEJ causes denaturation of the organic matrix and a greater weakening of the enamel
[39]. The degeneration process of odontoblasts and obliteration of dentinal tubules are due to the
radiotherapy damage, which leads to changes in metabolism and vascularisation [14]. Radiation also
reduces dentin microhardness [21, 27]. This change can result in enamel ablation along the DEJ with
crack formation in the cervical region, incisal or occlusal [44] and GAP formation in the DEJ region,
which combined with the masticatory stress, can cause bacterial colonisation [21] and a higher risk of
caries, which rises with poor oral hygiene [25].
The organic matrix is present in tooth enamel at very low concentrations (1%) [28], but it plays
an important role. This matrix is composed of small peptides and amino acids that are distributed
throughout the mature tissue, and it presumably represents the remains of the initial developmental matrix
that is perhaps retained via links with hydroxyapatite crystals. The organic matrix provides the template
for enamel mineralisation, and it continues to be the means of transport for substances into the interior.
The organic matrix plays a major role in the control of ionic diffusion into this tissue, and it prevents,
facilitates, or manages enamel demineralisation [30]. Damage to the organic matter and the interprismatic
substances of enamel also contribute to RT by causing chemical reactions with water molecules [1],
which alters the diffusion properties [17]. Water is present in a small proportion of the enamel, but it
plays an important role in enamel function because dehydration affects the mechanical properties of the
enamel structure [13, 35].
One factor that could contribute to this difference in organic content between the µ-EDXRF
and FT-Raman analyses is the different penetration depths, as shown in a previous study [37]. This
difference is explained by the operating principles of the two techniques. Raman spectra provide analyses
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 at a penetration depth of only a few microns. 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 [37].
There are 10 calcium (Ca) ions per unit of hydroxyapatite. Therefore, the calcium activity is
raised to the tenth power in the solubility product equation [45], 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 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 enamel undergoing RT and after RT and pH cycling (Table 1 and Fig. 2A). Our
results are consistent with Kielbassa et al. [26], who observed that enamel that has undergone RT is not
more susceptible to demineralisation compared with enamel that did not undergo RT. These authors
suggested that RT caused changes in the ultrastructure of enamel without clinically impacting the
beginning of demineralisation [22]. However, we must consider that the free radicals found in enamel
apatite submitted to RT may 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 creates empty spaces between the molecules that cause
irreversible changes in the tooth structure [43] and significant micromorphometrical differences in enamel
[14]. These alterations make teeth more vulnerable to acid attack [21, 25] and cause changes in their
biomechanical properties [1, 13, 25, 46
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, pH cycling caused a significant
reduction in 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 pH cycling affected the enamel apatite that had undergone
RT, which caused some structural damage to the enamel from the phosphate component.
Micromorphometrical differences were also observed during the dental enamel demineralisation
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 makes it more unstable and
susceptible to damage [5, 36].
The 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 mineralisation, i.e., a higher value indicates that
the tooth structure is more mineralised with Ca. The minimum and maximum ranges for the Ca/P ratio of
hydroxyapatite in human dental structures lie between 1.3 for intratubular dentin and 2.3 for enamel [2].
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 demonstrates that despite the pH cycling in teeth that underwent RT, this
difference was sufficient to alter the inorganic P component (Table 1). This finding is consistent with
other studies that reported no differences in enamel solubility and the depth of caries lesions in teeth
undergoing RT [17, 23].
The FT-Raman Spectroscopy evaluation in this study demonstrated that the relative area of
carbonate band decreased significantly in 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 the exclusive application of RT because the group that received only RT (RTa) exhibited
no significant reduction. There is a positive correlation between carbonate and enamel solubility [50]. The
micro-spaces that are formed as a result of the loss of carbonate and organic matrix can prevent
demineralisation and ion dissolution. These results suggest that teeth that underwent RT and pH cycling
tended to have an initial loss of carbonate, which is an element that provides greater solubility but is most
likely the first element lost, and corroborates the results of Jansma et al. [17]. However, no significant
difference in the carbonate area was observed between healthy teeth subjected to pH cycling (Nc) and RT
and pH cycling (RTc) (Table 3).
Notably, caries and radiation caries are multifactorial diseases [9], in which the sum of several
factors may be responsible for damage to the tooth structure. Comparisons of the RTc and Nc groups
using μ-EDXRF analysis revealed no significant changes in the Ca, P, or oxygen weight percentages or
the Ca/P ratio (Table 1 and Figs. 2A-D), which was confirmed in previous in vitro studies [17, 31]. FTRaman spectroscopy analyses also demonstrated no differences in the band values of the organic content,
phosphate, and carbonate between the RTc and Nc groups (Table 3 and Figs. 3A-C). In vitro studies do
not adequately reproduce clinical conditions, but in situ and in vivo studies have limitations because the
effects of radiation differ between individuals (e.g., differences in salivary flow, composition of oral
microbiota, diet, etc.) and due to the fragility of these patients. Therefore, the study of radiation caries
development is very difficult, primarily because other factors may be associated with its development [17,
22, 23, 29, 51, 57, 58].
Radiation caries is a frequent severe disease that develops rapidly, and it is difficult to control.
This condition causes cosmetic problems, altered eating habits, pain and changes the quality of life of
cancer patients [16]. The use of preventive protocols [26] after radiotherapy treatment and a multiprofessional 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].. The use of preventive protocols [27] after radiotherapy
treatment and multi-professional monitoring for preventive and curative treatments will allow these
patients to live better with the consequences: taste loss, hyposalivation, radiation caries and trismus of
radiotherapy treatment [27]. These sequelae for radiotherapy for head and neck cancer become
increasingly important, and have a tremendous effect on quality of life. Recent studies demonstrated that
the intensity of the radiation dose is an important factor in the development of radiation decay [32, 58].
Teeth undergoing radiation higher than 60 Gy exhibit changes in their mineral structure and collagen in
dentin and enamel, and there is a reduction in hardness and tensile strength and an increased possibility of
fracture that reaches amputation of the crown [58]. The few mineral and organic changes in teeth
submitted to RT found in the present study demonstrates the need for further studies to better understand
the pathophysiology of radiation caries and establish the best means to prevent and treat oral
complications in patients who undergo RT.
Conclusion
The µ-EDXRF assessment revealed phosphorus ion reduction and an increase in the Ca/P ratio in samples
subjected to RT and pH cycling. The FT-Raman spectroscopy results demonstrated that therapeutic doses
of RT exclusively reduced the organic content. pH cycling reduced the phosphate content. RT with pH
cycling reduced the carbonate and phosphate contents compared with those of healthy enamel. Radiation
damaged 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 involving human participants were performed in accordance with the
ethical standards of the institutional and/or national research committee and 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
RTb versus RTc
-3.25 (6.19)
0.436
N versus Nc
-2.11 (6.83)
RTb versus RTc
0.83 (2.21)
N versus Nc
0.88 (2.60)
RTb versus RTc
2.44 (8.36)
N versus Nc
0.40 (9.69)
Calcium
0.853
Phosphorus
Oxygen
Non-parametric Mann-Whitney test.
0.631
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
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
comparision
Paired Student’s t test and Student’s t test.