# Impact of X-ray Irradiation on the Chemistry and Structure of Biogenic Hydroxyapatite: The Case of Human Enamel

Thibaud Coradin, Aline Percot, Lotfi Slimani, Mohamed Selmane, Nabila Masri, Stéphane Le Goff, Julia Bosco

► To cite this version:

Thibaud Coradin, Aline Percot, Lotfi Slimani, Mohamed Selmane, Nabila Masri, et al. Impact of X-ray Irradiation on the Chemistry and Structure of Biogenic Hydroxyapatite: The Case of Human Enamel. ChemistrySelect, 2025, 10 (22), pp.e00956. 10.1002/slct.202500956. hal-05107667

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# Impact of X-ray Irradiation on the Chemistry and Structure of Biogenic Hydroxyapatite: The Case of Human Enamel

Thibaud Coradin,*[a] Aline Percot,[b] Lotfi Slimani,[c] Mohamed Selmane,[d] Nabila Masri,[c] Stéphane Le Goff,[c] and Julia Bosco*[c, e]

The influence of X-ray irradiation on dental tissues has been widely studied in the context of radiotherapy for the treatment of head and neck cancer. An observation common to almost all previous reports is the decrease of enamel hardness after irradiation. However, the origin of this loss of mechanical resistance remains a matter of debate. Here, we have conducted a chemical and structural study of the effect of a single 70 Gy dose on human teeth mineralized tissues. Vickers hardness of both enamel and dentin was decreased after irradiation. Microcomputed tomography showed that only the hydroxyapatite density of enamel was modified. Scanning electron microscopy, X-ray

diffraction and Raman micro-spectroscopy indicated that the chemistry and crystallinity of the inorganic phase of enamel, and its organization at the micron scale, were not modified. However, Raman spectra highlighted a disorientation of hydroxyapatite nanocrystallites over the whole enamel thickness. The decrease of enamel hardness was correlated with the decrease of mineral density by assuming the presence of an additional irradiation-induced porosity. Our data suggest that X-ray irradiation can create local defects in the enamel structure, decreasing its mechanical stability. These results shed new light on the interactions between X-rays and hydroxyapatite-based materials.

## 1. Introduction

Surgical resection, radio-, and chemotherapy, used either alone or in combination, are the three most common modalities used in cancer treatment.[1] The aim of radiotherapy is to kill cancer cells by damaging their DNA, either by direct interaction or by generating radicals in the cell cytoplasm.[2] This can be achieved using an external radiation (using photons from X-rays or γ-rays source, electrons, neutrons, protons or heavy ions) or internal radioactive sources. Indeed, a major challenge is to achieve efficient treatment of the tumor while limiting impact on surrounding safe tissues.

Head and neck cancers affect the oral cavity as well as larynx, pharynx and sinuses.[3] A major complication of radiation

therapy in this region is an increased risk for developing caries, not only during or immediately after treatment, but over the patient lifetime.[4] For sites exposed to 30–60 Gy, the odds of developing caries damage are 12–200 times greater than for unexposed sites.[5] Caries result from the dissolution of enamel, the outermost tissue of tooth, a highly mineralized material containing > 90 w% carbonated hydroxyapatite, by the action of oral bacteria.[6] If mineral loss occurs throughout the enamel thickness, oral fluids get access to dentin, a porous material of composition close to bone (45 w% mineral, 30 w% organics, 25 w% water).[7] Further deeper lies the pulp, a non-mineralized, cellularized tissue, integrating nerves and blood vessels.[8] Hence, if the carious process reaches pulp, it not only causes pain but also allows oral fluids, including bacteria, to have access the blood circulation.

Formerly, it was assumed that radiotherapy-induced caries were caused by the destructive action of the radiation on the dental tissues themselves, making them more prone to dissolution.[9] However, they are nowadays mainly seen as a sequela of the reduced function of the salivary glands caused by the radiation, making the oral fluids more aggressive.[10] Yet many in vitro/ex vivo studies have suggested that irradiation *per se* can impact dental tissues, although data available in the literature are highly variable, when not apparently contradictory.[11] These discrepancies can have many origins, such as different irradiation conditions and sample preparation protocols (including full teeth versus tissue sections, wet versus dried samples). The variety, adequacy and sensitivity of the selected analytical techniques are also to be considered. Yet, a few observations are common to most studies. First, morphological modifications, such as microcracks, were reported in enamel, dentin and at the dentin-enamel junction (DEJ).[12–15] Second, hardness of both enamel and dentin were almost systematically shown to decrease after irradiation.[12,15,16] However, no clear correlation

[a] Dr. T. Coradin
Sorbonne Université, CNRS, Laboratoire de Chimie de la Matière Condensée
de Paris, Paris 75005, France
E-mail: thibaud.coradin@sorbonne-universite.fr

[b] Dr. A. Percot
Sorbonne Université, CNRS, De la Molécule aux Nano-objets: Réactivité, Interactions et Spectroscopies, Paris 75005, France

[c] Dr. L. Slimani, N. Masri, S. Le Goff, Dr. J. Bosco
Université Paris Cité and Sorbonne Paris Nord, Inserm, Santé Orale,
Montrouge 92120, France
E-mail: julia.bosco@u-paris.fr

[d] Dr. M. Selmane
Sorbonne Université, Fédération de Chimie et Matérieux de Paris-Centre,
Paris 75005, France

[e] Dr. J. Bosco
Service de Médecine Bucco-Dentaire, AP-HP, Hôpital Pitié-Salpétrière, Paris
75013, France

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© 2025 The Author(s). ChemistrySelect published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
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between these two types observations has been established so far.

The aim of the present study was to bring a new contribution to our understanding of the impact of X-ray irradiation on dental hydroxyapatite by combining techniques allowing to characterize chemical and structural modifications from the ionic to the macroscale. A correlation between hardness and mineral density was established and discussed in term of radiation-induced porosity.

## 2. Materials and Methods

### 2.1. Samples

Teeth were obtained from the dental departments of AP-HP, France. All experiments were conducted in accordance with the principles articulated in the Declaration of Helsinki. All teeth were collected with the informed oral consent of all patients, in accordance with the ethical guidelines set by French law and with specific authorization by Paris University Dental School (n°DC- 2009-927, Cellule Bioethique DGRI/A5, Paris, France). Non-carious molars extracted from nonirradiated individuals for orthodontic reasons were collected. The exclusion criteria were the presence of fissures, enamel hypoplasia, and white spots.

### 2.2. Irradiation Procedure

Twenty healthy freshly extracted human permanent molars were obtained and stored in Chloramine T at 4 °C for a period not exceeding 1 month. Teeth were randomly divided in two groups. Ten teeth that belong to the irradiated groups (group I) received a 70 Gy single dose irradiation (Faxitron X-ray machine). This dose corresponds to the cumulated dose of current head and neck cancer treatment (2 Gy/day, 5 days/week over 7 weeks).[3] During the radiation exposure, teeth were immersed in water. Irradiation was carried out in the UMS 028 (Sorbonne Université, INSERM) at Hôpital Pitié Salpétrière. The other teeth were not irradiated (group NI) and stored in Chloramine T at 4 °C until the end of the irradiation treatment.

Full teeth were used for µCT analysis (n = 10 per group) and then longitudinally sectioned into two slabs, for SEM analysis (n = 10 per group) and microhardness (n = 5 per group) and for XRD analysis (n = 10 per group) and Raman analysis (n = 3 per group).

### 2.3. Microhardness Measurements

The samples were cleaned and their pulpal side ground wet with 600- and 1200-grit silicon carbide papers. Then, the specimens were placed in an ultrasound bath in water for 5 min. Microhardness measurements were performed on a Vickers microhardness tester (Struers DuraScan-20; Ballerup, Denmark), with a 200-gf load applied for 15 s in enamel and a 100-gf load for 15 s in dentin. Six sites were chosen on the dental longitudinal section surface

and measured for each dental slab. Sites were set at 50 µm from the enamel surface (surface enamel, SE), one-half the thickness of buccal enamel (middle enamel, ME), 50 µm from the DEJ in enamel (deep enamel, DE), 50 µm from the DEJ in dentin (surface dentin, SD), one-half the thickness of dentin (middle dentin, MD) and 50 µm from the pulp chamber (deep dentin, DD). For each site, three indentations spaced 100 µm from each other in enamel and 150 µm in dentin were made by the same examiner. The dentin and enamel microhardness values were obtained as the average of the results for the three indentations.

### 2.4. Micro-X-ray Computed Tomography (µCT) Studies

The samples were imaged using an X-ray micro-CT device (Quantum FX Caliper; Life Sciences, Perkin Elmer, Waltham, MA) hosted by the PIV Platform, URP2496, Montrouge, France. The X-ray source was set at 90 kV and 160 µA. Full 3D high-resolution raw data were obtained by rotating both the X-ray source and the flat panel detector 360° around the sample (scanning time: 2 min). After acquiring 360° projections, 3D reconstructions were obtained using the RigakuSW (Caliper) software and tridimensional images were reconstructed into a DICOM image stack in 3D matrix 512 × 512 × 512 voxel. A 20 mm × 20 mm field of view was used to obtain an isotropic voxel size of 40 µm. Quantification of the dental hard tissue was performed with Analyze 14.0 software (Biomedical Imaging Resource; Mayo Clinic, Rochester, MN, USA). A low pass filter with a 3 × 3 × 3 kernel was applied to reduce noise and images were threshold to separate the different mineralized tissue (dentin and enamel). After segmentations, the tissue density was quantified within the Region of Interest (ROI). To convert CT intensity, in Hounsfield unit, of the mineralized tissue into mineral density, a set of phantoms, calibrated in mg of hydroxyapatite per cm³, was used.

### 2.5. Scanning Electron Microscopy (SEM) Imaging

The specimens were fixed in a glutaraldehyde solution in cacodylate buffer, cleaned for 10 min in an ultrasonic vat (Ultrasonic-Cleaner T-1449-D; Odontobrás Ind. e Com, Ribeirão Preto, Brazil) containing distilled and deionized water, dehydrated in aqueous solutions of increasing ethanol content (25%, 50%, 75%, 95%, and 100%), and immersed in hexamethyldisilazane (HMDS) for 10 min. Subsequently, they were fixed on stubs with a double-sided adhesive carbon tape and coated with a 20–30 nm gold layer in a vacuum-metallizing machine (SDC 050; Bal-Tec AG, Balzers, Germany). The specimens were then subjected to analysis using a scanning electron microscope (JSM-6400; JEOL Ltd., Tokyo, Japan) working at 15 kV.

### 2.6. X-ray Diffraction (XRD) Analyses

X-ray diffraction analyses were carried out using a BRUKER DISCOVER X-ray powder diffractometer equipped with a X-ray tube with a Copper anode (doublet K<sub>α1</sub> = 1.54056 Å and K<sub>α2</sub> = 1.54439 Å) and a 1D LynxEye detector (XE-T) from Bruker in
<figure><img src="image_5.png" /></figure>

Bragg-Brentano configuration (θ/2θ scan). The sample was irra-
diated in variable slit mode over a length of 4 mm across the
enamel area. Data acquisitions were made in the 10°–60° angu-
lar range with a step of 0.03° for 10 h. Data were analyzed using
the Fityk 1.3.1 software, allowing to obtain the position, intensity
and full-width at mid-height (FWMH) of main diffraction peaks.

## 2.7. Raman Spectroscopy Analyses

Raman analyses were performed following a single-blind proto-
col, using a Senterra Raman micro-spectrometer (Bruker Optics),
equipped with a 785 nm laser and a power at the sample
of ca. 10 mW. Data collection was controlled by the OPUS 7.5
software (Bruker Optics). Measurements were collected across
one spectral window (99–1540 cm⁻¹) at a spectral resolution of
about 3 cm⁻¹, and each analysis was the coaddiated of 2 spec-
tra accumulated at up to 4 s of exposure time for each. Analyses
were performed using a 50 × long working distance IR objec-
tive (Olympus, Tokyo, Japan), giving an analytical spot size of
approximately 12 µm in diameter. Analyses were performed on
polished sections. For mapping, the sample was moved by a
computer-controlled stage. Each surface was scanned by mov-
ing the sample by 25 µm steps. Maps were performed on a
2350 × 185 µm surface for the control sample and 1515 × 160 µm
surface for the irradiated sample. Chemical maps were generated
by integrating the area of bands centered at 1074, 1048, 595, 580,
455, and 436 cm⁻¹. The integration mode calculated the area of
the zone delimited by the x-axis after baseline correction and
two manually-defined threshold wavenumbers, spaced 7 cm⁻¹
from each other. The average spectra were calculated over the
entire surface, excluding dentin and the first 50 µm on the exter-
nal surface. Baseline subtractions, fitting, integration and map
generation were performed using the OPUS 8.7 software (Bruker
Optics).

## 2.8. Statistical Analyses

When relevant (n ≥ 5), data were submitted to analysis of vari-
ance (ANOVA), and means were compared using the Tukey test.
Analyses were performed by comparing nonirradiated and irra-
diated samples only. Stars represents statistically different levels:
\*p-value < 0.05; \*\*p-value < 0.01; and \*\*\*p-value < 0.001.

# 3. Results and Discussion

## 3.1. Effect of X-ray Irradiation on Enamel and Dentin
Hydroxyapatite Density

The impact of irradiation of full teeth samples was first investi-
gated by measuring the density of hydroxyapatite ρ in enamel
and dentin using micro-X-ray computed tomography (µCT)
(Figure 1a,b). For nonirradiated samples, ρ<sub>NI</sub> was 2.1 ± 0.2 g.cm<sup>-3</sup>
and 1.10 ± 0.05 g.cm<sup>-3</sup> in enamel and dentin, respectively.
After irradiation, ρ<sub>I</sub> was significantly decreased in enamel
(1.95 ± 0.03 g.cm<sup>-3</sup>) but not in dentin (1.07 ± 0.05 g.cm<sup>-3</sup>).

<figure><img src="image_6.png" /><figcaption>**Figure 1.** (a) Representative µCT images and (b) corresponding hydroxyapatite density for nonirradiated (NI, green) and irradiated (I, red) teeth in enamel (E) and dentin (D). *n* = 10 per group. Comparisons were made between NI and I samples in the same tooth area, \* *p* < 0.05.</figcaption></figure>

Only one comparable study using synchrotron radiation µCT
is available,[17] unexpectedly reporting increased mineral density
after irradiation. Importantly, the mineral density as obtained
from µCT is not the density of enamel (in g.cm⁻³) but the
density of the hydroxyapatite (HAP) phase within enamel (in
g of HAP.cm⁻³). It is calculated using reference samples made
of synthetic hydroxyapatite with varying porosity.[18] Thus, such
a change in mineral density can indicate either a modification
of the hydroxyapatite phase of enamel, or a change in enamel
porosity.

3.2. Effect of X-ray Irradiation on Enamel and Dentin Hardness

Vickers hardness (VH) measurements were then performed on
teeth sections (Figure 2a,b). Noticeably, whereas the imprint of
the measurement probe in enamel was neat for the nonirra-
diated samples, it was less regular for the irradiated samples,
a first indication that a change has occurred in the enamel
structure.[19] For nonirradiated samples, VH<sub>NI</sub> was 390 ± 10 for
superior (SE) and middle (ME) enamel as well close to the dentin-
enamel junction (DEJ). In the dentin area, VH<sub>NI</sub> was ca. 80 for
surface (SD) and middle (MD) dentin (80 ± 5) and slightly smaller
for deep (DD) dentin (70 ± 5). For irradiated samples, VH<sub>I</sub> was
significantly smaller in all enamel areas (310 ± 10) and in all
dentin zones, except for SD. The irradiation-induced decrease
of ca. 20% in microhardness of enamel was comparable to
previously-reported data.[13–15]

3.3. Effect of X-ray Irradiation on Enamel and Dentin
Morphology

In order to identify any structural defects at the micron scale,
teeth sections were imaged using Scanning Electron Microscopy
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>**Figure 2.** Hardness measurements of nonirradiated (NI) and irradiated (I) enamel and dentin. (a) Representative SEM images of indentation imprints (scale bar: 10 mm) and (b) corresponding Vickers hardness values measured on sections of nonirradiated (green) and irradiated (red) teeth in superior (SE), middle (ME) and deep enamel (DE), and for surface (SD), middle (MD) and deep (DD) dentin. *n* = 5 per group. Comparisons were made between NI and I samples in the same tooth area, \*p < 0.05, \*\*p < 0.01.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**Figure 3.** Representative SEM images of enamel, dentin-enamel junction (DEJ) and dentin for nonirradiated (NI) and irradiated (I) samples. E = enamel; D = dentin; yellow arrows: enlarged DEJ; red arrows: cracks near and in dentin microtubules. Scale bar: 10 µm.</figcaption></figure>

(SEM) (Figure 3). The structure of enamel was apparently not modified after irradiation, with a clear visualization of hydroxyapatite rods and inter-rods area. An enlarged dentin-enamel junction could be observed in some irradiated samples but this observation was not systematic and was also sometimes observed in nonirradiated samples. Considering the dentin tissue, some cracks on the borders and inside the microtubules were observed in some irradiated samples, but not in nonirradiated samples. These observations are in good agreement with the literature and were mostly attributed to the effect of irradiation on the organic matter, and especially the collagenous component of dentin.[12-15]

### 3.4. Effect of X-ray Irradiation on Enamel Crystallinity

Since significant variation in mineral density was measured for enamel only, further investigations of this tissue were performed. First, the crystallinity of the mineral phase was studied by X-ray diffraction (XRD) performed on teeth sections (Figure 4). Typical diffraction patterns for enamel from nonirradiated and irradiated teeth are shown in Figure 4a with attribution of the main peaks according to the JCPDS card N°09-432 file. Based on the literature,[20] the Full-Width at Mid-Height (FWMH) of the (002) peak at 2θ = 25.9° and of the (310) peak at 2θ = 39.8° were determined and compared before and after irradiation. As shown
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>**Figure 4.** (a) Representative XRD diffractograms of nonirradiated (NI, green) and irradiated (I, red) enamel and (b) corresponding FWMH of the (002) and (310) peaks. Comparisons were made between NI and I samples.</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>**Figure 5.** Average Raman spectra of enamel in nonirradiated (NI) and irradiated (I) samples (inset: detail of the 350–650 cm⁻¹ region).</figcaption></figure>

on Figure 4b, there was no significant difference in the FWMH of both peaks between the two series of teeth, suggesting the absence of significant modification of the crystallinity of enamel under irradiation. No comparable study was found in the literature. It must be pointed out that crystallinity reflects the size of the domains where ions adopt an ordered spatial organization, also called crystallites. It has been shown that these crystallites do not correspond to enamel rods but to smaller particles constituting these rods.[21] Thus, the absence of variation in crystallite size does not necessarily mean that the organization of the rods is not perturbed by irradiation.

### 3.5. Raman Spectroscopy Studies of Irradiated Enamel

In parallel, to investigate the chemical structure of hydroxyapatite crystals, Raman spectra of enamel sections of nonirradiated and irradiated teeth were also recorded. As shown on Figure 5, main bands characteristic of biogenic hydroxyapatite structures were observed in the average spectra of both types of samples.[22] The most intense band at 960 cm⁻¹ (ν₁) corresponds to the symmetric stretching of the P—O bonds. At lower wavenumbers, a triplet in the 560–630 cm⁻¹ (ν₄) and a doublet in the 400–460 cm⁻¹ (ν₂) regions are mainly corresponding to

O—P—O bending modes. At higher wavenumbers, a triplet in the 1020–1090 cm⁻¹ (ν₃) corresponds to asymmetric stretching of the P—O bonds. The symmetric stretch of the C—O bonds of carbonates (ν₁CO₃) also contributes to the 1070 cm⁻¹ peak. Noticeably, while all vibration bands were present at the same wavenumbers for the two samples, some differences in the relative intensity of peaks in the ν₂ and ν₄ doublets were observed (Figure 5, inset).

To confirm this observation, a series of spectra were recorded from the outer enamel layer to the DEJ for nonirradiated and irradiated samples. Their intensities were normalized using the ν₁ band and maps of the area of selected peaks were established (Figure 6). Variations of the 1074 cm⁻¹ peak contribution were clearly visualized, and similar for both NI and I samples. In the case of NI samples, maps for other peaks were all quite homogeneous, indicating a small range of variations over the enamel thickness (Figure 6a). In contrast, for I samples, there was a clear gradient in the area of all peaks (Figure 6b). In addition, two sets of bands seemed to show comparable variations: the 1048, 595, and 436 cm⁻¹ group and the 580 and 455 cm⁻¹ group.

A more detailed examination of spectra obtained at specific positions of the sample was performed. For NI samples, there was a continuous but small increase in the intensity of all peaks with increasing analysis depth (Figure 6c). A similar global increase in intensity was also observed for I samples, but three bands show more significant variations (Figure 6d). The band at 1048 cm⁻¹, that was present as a shoulder in outer enamel, reached an intensity close to that of the 1074 cm⁻¹ band near the DEJ. The band at 595 cm⁻¹, that was of lower intensity compared to the 580 cm⁻¹ in outer layer, became more intense near the DEJ. In parallel, the relative intensity of the 436 cm⁻¹ band grew faster than that of the 455 cm⁻¹ band throughout the depth of enamel.

Raman spectroscopy is particularly well-adapted to study hydroxyapatite-based materials, including teeth,[23,24] because this mineral phase presents a large set of middle-to-high intensity bands corresponding to vibrations of the phosphate group.[25] Here, we observed that the intensity and spatial variation of the 1070 cm⁻¹ band, that has major contribution from the vibration of the carbonate groups, were not modified by irradiation. This points out the absence of modification of the carbonatation level of hydroxyapatite. In contrast, a change in
<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /><figcaption>**Figure 6.** Optical images and associated maps of areas of selected Raman bands (1074, 1048, 595, 580, 455 and 436 cm⁻¹, from top to bottom) in enamel for (a) nonirradiated and (b) irradiated samples. The color bar scales the relative value of the band area at each wavenumber. Detailed Raman spectra recorded in positions 1–4 for (c) nonirradiated and (d) irradiated samples (inset: detail of the 550–650 cm⁻¹ region).</figcaption></figure>

the relative intensity of bands belonging to the same vibration mode was noticed, not only when nonirradiated and irradiated samples were compared, but also depending on enamel area. Even more intriguingly, these variations seemed to occur sim-

ilarly in two groups of bands belonging to different vibration modes. The fact that bands corresponding to the same vibration mode vary in relative intensity but not in position indicates that no chemical modification has occurred. Rather it suggests
a change in the orientation of the phosphate groups within the enamel structure.

As a matter of fact, it was previously demonstrated that the relative intensity of bands could vary with the relative orientation of hydroxyapatite nanocrystals and of the Raman excitation light.[26,27] It must also be noticed that no peaks appeared in the 1200–1500 cm⁻¹ region, that would have signed for higher protein contents, suggesting the absence of a significative demineralization process.

### 3.6. Bridging Mechanics, Density, and Local Disorder

At the macroscale, a 70 Gy X-ray irradiation led to a decrease in hardness and of hydroxyapatite density in almost all parts of the enamel and dentin. No significant change in enamel morphology, crystallinity and chemistry was found. However, Raman spectroscopy suggested a perturbation of hydroxyapatite nanocrystal orientation after irradiation. On this basis, the hypothesis can be raised that X-rays create additional porosity in the enamel structure, that would lead to a decrease in mineral density and mechanical stability, and results in a local disorganization of the mineral particles.

As a matter of fact, there exists a general relationship between hardness VH and porosity p for a given material (Equation 1).[28]

$$ \frac{VH}{VH_0} = e^{-b \cdot p} \qquad (1) $$

where VH₀ is the hardness of the nonporous solid and b is characteristic of a given material. For hydroxyapatite, a b value of 4.8 was found in the literature.[28]

Porosity p is related to the density of the considered porous solid ρ and the density of the nonporous material ρ₀ by (Equation 2).

$$ p = \left(1 - \frac{\rho}{\rho_0}\right) \qquad (2) $$

Associating Equations (1) and (2) for nonirradiated and irradiated enamel leads to Equation (3).

$$ \frac{VH_I}{VH_{NI}} = e^{-b \cdot (\rho_{NI} - \rho_I) / \rho_0} \qquad (3) $$

Using literature values relevant to nanoscale hydroxyapatite (ρ₀ = 3), it is possible to calculate.

$$ \left( \frac{VH_I}{VH_{NI}} \right)_{cal} = 1.2 \pm 0.2 $$

This value can be compared to the experimental one obtained from VH_I and VH_NI values from microhardness measurements.

$$ \left( \frac{VH_I}{VH_{NI}} \right)_{exp} = 1.25 \pm 0.05 $$

The similarity between these two values indicates that the variation of microhardness can be, at least partially, related to

the measured decrease in mineral density, if one assumes that such a decrease is due to an increase in porosity.[29]

These pores can originate from a loss of organic matrix or water. In fact, these two components account for 10% in volume of enamel.[30] In parallel, it has been demonstrated that X-ray-irradiation of biological tissues can lead to water vaporization, creating bubbles.[31] It can therefore be suggested that these bubbles can contribute to the additional porosity by creating cavities. Moreover, they can induce local orientational disorder in the mineral structure, in agreement with the Raman data. The fact that these pores were not visible by SEM imaging suggest that they occur at the nanoscale.

### 4. Conclusions

In this study, the major impact of X-ray irradiation on enamel was a reduction of the hardness and of the hydroxyapatite density, that could be correlated to an increase of porosity. Such pores induce a local disorganization of the hydroxyapatite nanocrystals without impacting their chemical composition nor crystallinity. Although single dose irradiation conditions used here differ from the standard multiple dose clinical procedure for head and neck cancers treatment, our data can contribute to a better understanding of the effect of X-rays on dental tissues in a more general context and can also offer additional insights on the influence of irradiation on hydroxyapatite-based biomaterials.

### Acknowledgements

The authors have nothing to report.

### Conflict of Interests

The authors declare no conflict of interest.

### Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

**Keywords:** Hydroxyapatite · Radiotherapy · Raman spectroscopy · Teeth

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Manuscript received: February 17, 2025