Introduction

Head and neck cancer is the sixth most common malignancy worldwide, with approximately 650,000 new cases diagnosed annually [1,2]. For the treatment of head and neck cancer, surgical resection is often combined with radiotherapy and/or chemotherapy [3]. Radiotherapy is one of the most common treatment options for head and neck cancer usually resulting in good rates of tumor control [4]. Despite being a non-invasive treatment option due to the preservation of the tissue structure, some side effects in sites can develop up to 12 months after the completion of treatment [5,6]. These side effects may include mucositis, taste loss, hyposalivation, radiation-induced caries, periodontal disease, osteoradionecrosis, and trismus [6].

Radiation-induced caries has been considered one of the most common side effects of head and neck radiotherapy [7,8] and reported in approximately 29% to 37% of patients [9]. Clinically, these lesions begin in the labial surface at the cervical third of the teeth with superficial enamel changes and brownish pigmentation [8]. With the development of the lesion, areas of enamel demineralization and delamination occur, leading to a possible dental crown amputation [10,11].

Radiation-induced caries have a multifactorial etiology from a combination of direct damage and indirect effects. Hyposalivation impairs enamel remineralization and reduces saliva’s antimicrobial properties. Poor oral hygiene allows cariogenic bacteria to grow, while a carbohydrate-rich diet acts as a substrate for acid production, accelerating tooth demineralization. The direct ionizing radiation affects the hard tissues of the teeth [6,12].

The destruction of collagen found within the dental pulp of teeth located in the irradiation field appears to contribute to fibrosis and a reduction of vascularity within the dental pulp, especially by higher radiation doses (over 50Gy), impairing the metabolism of odontoblasts, which are responsible for dentin production and mineralization [12,13]. Several studies have reported changes in the mechanical and chemical properties of dental hard tissues caused by the direct effect of radiation, such as microhardness, elastic modulus, and chemical composition [14,15].

Collagen degradation in the organic matrix involves the chemical reaction known as decarboxylation, which is based on removing carboxyl groups (COOH). When collagen is exposed to ionizing radiation, decarboxylation of specific amino acid side chains takes place releasing carboxyl groups and simultaneously causing the loss of phosphate groups, directly associated with the collagen matrix. Additionally, during this process, new phosphate groups are formed, which can bridge with calcium ions, changing the structural integrity of collagen and contributing to its breakdown [16].

Studies investigating the properties of dental tissues using natural teeth with radiation-induced caries are very scarce. Most studies use sound-extracted teeth subjected to different radiotherapy protocols [17–21]. These simulation methods have limitations since they do not reproduce the oral environment, such as the presence of saliva, bone, and oral tissues, inherent patient factors, type of diet, and oral hygiene. Therefore, the outcomes of these types of research may not contribute to the complete understanding of the etiopathogenesis of radiation-induced caries and indicate more effective restorative treatments.

Understanding how dental tissue changes after radiation is important for establishing an effective treatment protocol that restores function, aesthetics, and phonetics and improves the quality of life of patients affected by radiation caries. Therefore, this exploratory study aimed to analyze the microstructure and changes in the chemical composition of radiation-induced carious dentin and irradiated dentin from patients who underwent head and neck radiotherapy compared to sound dentin. The null hypothesis posits that there would be no difference in the microstructural and chemical analyses of the substrates after scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR) analysis.

Materials and methods

Specimens’ preparation

Six carious teeth were used to obtain six fragments of radiation-induced carious dentin (CD) and six fragments of adjacent irradiated dentin (ID). For the control group (SD), six sound teeth were also sectioned to produce six fragments of sound dentin, matching the number of specimens obtained from the irradiated teeth. The specimens were sectioned using a diamond disk to obtain 3-mm thick specimens under constant water cooling in a precision cutting machine (IsoMet 1000, Buehler Ltd., Lake Bluff, IL), and then embedded in self-curing acrylic resin (Jet, Classico, São Paulo, Brazil) inside a 3-mm high plastic tube. The surfaces of all specimens were ground flat and polished using a metallographic polisher (Ecomet 30, Buehler Ltd., Lake Bluff, IL) with #600 silicon carbide sandpaper for 30 s, #1200 for 5 min, and #2000 for 10 min. Subsequently, the specimens were polished with 3-, 1-, and 0.25-µm diamond pastes (Metadi II, Buehler Ltd., Lake Bluff, IL) for 15 s each. The specimens were ultrasonically cleaned for 10 min with distilled water between each sandpaper and paste, and for 20 min after the completion of polishing procedure.

The specimens were prepared for the tests according to a previous study [23]. Initially, the dentin surfaces were etched with 10% phosphoric acid for 5 s and washed for 15 s. This was followed by 10% sodium hypochlorite for 10 min and an ultrasonic bath for 20 min. Specimens were then fixed by immersion in 2.5% glutaraldehyde for 24 h to preserve structural integrity. Dehydration was conducted through sequential immersion in graded alcohol concentrations, beginning with 25%, 50%, and 75% for 20 min each, followed by 95% for 30 min, and concluding with 100% for 1 h. Finally, the specimens were immersed in hexamethyldisilazane (Merck KGaA, Darmstadt, Germany) for 10 min and kept in a desiccator with silica to avoid humidity until testing. Fig 1 illustrates the study design along with the detailed steps involved in the specimens’ preparation and analysis.

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Fig 1. Study design and steps of specimens’ preparation and analyses.

https://doi.org/10.1371/journal.pone.0337062.g001

Results

SEM and EDS analyses

The dentin morphology of specimens from the evaluated groups analyzed by SEM is presented in Fig 2. Sound dentin (SD) exhibiting surface integrity (Fig 2A and 2B) of the intertubular (asterisk) and peritubular dentin (arrows) with the view of the odontoblastic process (Fig 2C) and absence of visible craters and microfractures. Adjacent irradiated dentin (ID) presented craters with dentin tubules partially obliterated (Fig 2D–2F). Radiation-induced carious dentin (CD) with the presence of craters (Fig 2G). Fig 2H and 2I illustrate that CD exhibits numerous craters indicative of dental degradation. However, the distinction between intertubular and peritubular dentin remains unclear.

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Fig 2. SEM representative images of specimens from the evaluated groups.

(A) Sound dentin (SD) exhibiting normal organization of dentin with open tubules (1000×). (B) Intertubular dentin (asterisk) and peritubular dentin (arrows) (10000×). (C) Odontoblast process inside a tubule (25000×). (D) Adjacent irradiated dentin (ID) shows irregularities and partially obliterated dentin tubules. (E) and (F) Peritubular dentin at the tubule internal walls showing signs of degradation (arrows) (10000× and 25000×). (G) Radiation-related carious dentin (CD) showing partially obliterated tubules (1000×). (H) and (I) Radiation-related carious dentin (CD) with numerous craters in the peritubular dentin (10000× and 25000×).

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Table 1 presents the weight percentage (wt%) of calcium (Ca), phosphorus (P), carbon (C), and oxygen (O) as determined by Energy Dispersive Spectroscopy (EDS). The results indicate that the atomic percentages of Ca and P decreased significantly when comparing samples labeled as SD and CD, with p-values of 0.027 and 0.039 respectively. Conversely, the weight percentage of C increased significantly between these two groups (p = 0.027). However, no statistically significant difference was observed in the O percentages (p = 0.066).

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Table 1. Weight percentage (wt%) ± standard deviation of calcium (Ca), phosphorus (P), carbon (C), and oxygen (O) of SD, ID and CD groups as determined by Energy Dispersive Spectroscopy (EDS).

https://doi.org/10.1371/journal.pone.0337062.t001

After the statistical analysis, the Ca/P/C ratio was calculated. The Ca/P/C ratio was 0.09 in SD, 0.06 in ID, and 0.03 in CD.

XRD analysis

Fig 3 shows the XRD spectra of the dentin surface of specimens from the SD, ID, and CD groups. Miller’s (hkl) indices shown in the graph were indexed according to the ICDD file number 01-072-7552 for Hydroxyapatite. Most of the identified peaks resulted in a decrease in ID and CD specimens compared to SD. We observed an absence of the 1-1-2 and 3-0-0 peaks in ID and CD specimens. Most of the other denoted peaks presented a reduction in intensity for ID and predominantly for CD.

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Fig 3. XRD patterns of the dentin specimens of the evaluated groups.

HAP was identified using the ICDD [01-072-7552] file.

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FTIR analysis

The representative spectra of hydroxyapatite in specimens from the evaluated groups are presented in Fig 4. The organic phase of dentin, mainly composed of type I collagen, is represented by specific spectral bands known as Amide I (1600–1700 cm ⁻ ¹), Amide II (1500–1600 cm ⁻ ¹), and Amide III (1200–1350 cm ⁻ ¹). The intensity of peaks was similar for SD and ID samples, while CD exhibited higher peak intensity, especially in the Amide I and II bands. The spectra of ID and CD showed peaks associated with phosphate vibrations (PO₄³), ν₁ 940–980 cm ⁻ ¹, ν₃ 1000–1100 cm ⁻ ¹, ν₄ 500–600 cm ⁻ ¹ bands. While the distinct bands attributed to carbonate vibrations (CO₃^2-) ν₂ 850–890 cm ⁻ ¹, ν₃ 1400–1500 cm ⁻ ¹ bands are less pronounced in these groups compared to SD.

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Fig 4. FTIR spectra of specimens from each group showing HAP vibration bands.

https://doi.org/10.1371/journal.pone.0337062.g004

Discussion

This study focused on the chemical characterization and structure analysis of sound, adjacent irradiated dentin, and radiation-induced carious dentin. According to the results, there were changes in the morphological structure and chemical composition of the adjacent irradiated dentin and radiation-induced carious dentin. Therefore, the null hypothesis that there would be no difference in the microstructural and chemical analyses between the substrates was rejected.

The demineralization patterns of radiation-induced caries appear like those of conventional caries, characterized by demineralized dentin, translucent zones, dentin dead tracts, and the deposition of reactionary and intratubular dentin [24]. The presence of the odontoblastic process in sound dentin images and its absence in adjacent irradiated dentin and radiation-induced carious dentin likely reflect the disruption of metabolic activity in odontoblasts, as these processes are essential for maintaining dentin-pulp homeostasis. This absence may indicate impaired nutrient transport and reduced cellular activity, directly correlating with altered pulp vitality and decreased odontoblastic metabolism due to radiation exposure [12,13]. Although some laboratory simulations of radiotherapy studies have observed similarities between sound dentin and radiation-induced carious dentin [25,26], our SEM images showed obliteration, and craters in the adjacent irradiated dentin and more numerous and deeper craters in radiation-induced carious dentin. Our findings suggest that ionized radiation therapy leads to demineralization and degradation of the dentin, compromising its structure mechanically and increasing susceptibility to further damage or decay. A previous study analyzing the effect of radiation therapy on the morphology of enamel and dentin of deciduous teeth in SEM also reported dentin with fissures, tubule obliteration, and fragmentation of collagen fibers observed especially after higher cumulative doses [27].

In laboratory studies on the effects of radiotherapy [18,21,28], extracted sound human teeth are used, and the oral conditions are simulated either with a physiological sodium chloride solution immersion [21], relative humidity [18], or even under dry conditions [28], and the interference of natural tissues is not reproduced at all. Therefore, in the present study teeth from patients who received radiotherapy and developed radiation-induced caries were used. One of the radiotherapy’s most important local effects is demineralization of dental tissues by changing both the organic and inorganic components [18,26]. This effect can be exacerbated by hyposalivation and xerostomia, which has been reported in almost 100% of the patients undergoing head and neck radiotherapy and interferes directly with the mineral balance and pH of the oral environment [29].

The significant decrease in Ca and P content in radiation-induced carious dentin has been associated with the effects of ionizing radiation on hydroxyapatite [28]. Besides that, our results also showed a decrease in Ca and P content in the adjacent irradiated dentin. The Ca/P/C ratio is associated with the mineral composition of the crystalline structure of the teeth, which directly affects the mechanical properties of dental hard tissues [26,30]. The increased carbon (C) content reflects changes in organic components, which may contribute to tissue breakdown. A recent study reported a non-significant difference in the Ca/P ratio between irradiated and non-irradiated root dentin. However, the samples were obtained by sectioning impacted third molars and the radiation was performed under dry conditions [31]. Indeed, interactions between water and radiation exposure have been reported, in which ions are released by water, inducing the formation of a distinct nanostructured phase, known as the secondary nanoapatitic calcium phosphate phase, which is primarily composed of nano-sized apatite crystals, leading to an increase in the degradation susceptibility of Hap [32,33].

Magnesium is important in Hap crystal growth, replacing calcium ions in the hydroxyapatite structure. This substitution helps stabilize the crystal lattice and influences Hap crystals’ size, shape, and overall development [34]. However, its level was below the detectable threshold in our study, so we did not include it in the results.

X-ray diffraction (XRD) was used to analyze dentin because it provides detailed information on its mineral composition, crystal structure, and crystallinity, assessing changes in dentin due to demineralization, aging, or treatments [35]. The interaction between the X-rays and the specimen resulted in a diffraction pattern of the inorganic composition of Hap. The most visible differences among the spectra of Fig 3 are the disappearance of the (1 1 2) and (3 0 0) peaks. These changes in the adjacent irradiated dentin indicate preferential damage or dissolution of crystal peaks, likely influenced by their stability or orientation within the dentin structure. Additionally, there was a clear difference in peak intensity among the three dentin substrates, particularly at (0 0 2), (2 1 0), (3 1 0), and (2 2 2) peaks, suggesting damage to Hap crystals and degradation in the adjacent irradiated dentin, and even more in the radiated-induced carious dentin. This reduction may result from a decrease in crystallite size, an increase in lattice strain, or a combination of both, potentially caused by direct radiation-induced damage or demineralization processes [27].

FTIR analysis explored the inorganic and organic parts of the dentin, including collagen. The crystallinity of dentin and the presence of collagen crosslinks are identifiable in FTIR spectra, where the degradation of dentin is characterized by lower crystallinity and higher collagen crosslinks [35]. Collagen cross-linking involves forming covalent or non-covalent bonds between collagen molecules, enhancing tissue strength and stability, which is important for resistance to degradation and dentin bonding durability [36], as well as hardness and resistance to mechanical stress [37]. The amide bands I, II, and III, characteristic of collagen’s triple helix structure, are primarily associated with the protein components of materials like collagen, a major organic component in dentin [38]. Changes in the amide bands, particularly Amide I and Amide II, can reflect modifications in the collagen structure [39]. Qualitative differences can be observed in the amide band regions of the spectra between the sound, irradiated, and carious dentin groups, with higher absorbance in amide for carious dentin.

The combined analysis of phosphate and carbonate vibrations provides a comprehensive understanding of how caries-induced degradation impacts dentin mineral content and structural integrity [33]. Changes in FTIR spectra intensity suggest a potential decrease in the relative quantity of carbonate incorporated within the dentin structure following irradiation and caries development. Carbonate groups are often substituted into hydroxyapatite, affecting its biological and mechanical properties and indicating changes in the mineral composition [38]. The apparent reduction in the intensity of carbonate bands aligns with the concept of radiation and caries-induced mineral loss and alteration in dentin composition. Phosphate groups are hydroxyapatite’s primary mineral component, forming dentin’s structural backbone. The presence and intensity of phosphate-specific vibrations are key indicators of the degree of mineralization [40]. In our results, the decrease in FTIR spectra intensity associated with phosphate groups (e.g., the region around ~1000−1100 cm-1) suggests alterations in the mineral content of the dentin. This outcome could be related to a demineralization process, where the mineral component of dentin is reduced.

The reduction in the intensity of peaks indicates a decrease in hydroxyapatite content, particularly in carious dentin specimens, which can suggest a weakened dentin structure caused by degradation. These findings are consistent with previous studies showing that radiation therapy causes significant alterations in tooth mineralization, compromising the dentin’s resistance to caries and fractures [18–20]. Due to the nature of our experimental design and the limited sample size, it was not possible to perform statistical analysis on the data. Spectral normalization would be a valuable step in allowing quantitative or comparative analysis of collagen degradation. Since it was not performed in the present study, making these statements could be inappropriate.

A previous study relates the significance of cross-linking in collagen fibers in dentin hardness and elastic modulus [41]. It explains that cross-linking involves the formation of covalent bonds between collagen molecules, with amides playing an important role. Enzymatically modified lysine and hydroxylysine residues form aldehydes, which react with neighboring residues to create stable cross-links, essential for a stable collagen network in dentin [42]. Additionally, hydrogen bonding within the protein backbone stabilizes collagen’s triple helix structure, indirectly supporting dentin resilience [42]. Changes in FTIR spectra intensity associated with phosphate groups indicate variations in hydroxyapatite concentration, reflecting demineralization, while alterations in amide bands signal collagen structure modifications, such as denaturation or degradation, due to factors like radiation or caries. Similarities in the spectral curves of irradiated dentin and non-irradiated dentin were reported previously, with an increase in the irradiated dentin curve primarily in the peaks of the bands, including phosphate [43]. The opposite was found in the results of the present study, with clear differences in the spectral bands for all tested substrates.

Radiation-induced caries is known for its destructive and quick progress when untreated [43]. Therefore, one of the limitations of this study was the difficulty in obtaining numerous and large fragments due to the fragility of the irradiated and carious dentin structure. Therefore, the results should be interpreted as exploratory, warranting further studies with larger samples. The reduction of sample size impaired the inclusion of spectral normalization which would enable a more robust and quantitative assessment of collagen and Hap degradation following irradiation and radiation-induced caries development. Future studies including spectral deconvolution, peak fitting, and specific collagen crosslink ratio calculations might provide more detailed information about the changes in mineral and organic dentin components submitted to irradiation and caries development.

Based on our results, it can be assumed that the adhesion of restorative materials to radiation-affected dentin surfaces is deeply compromised. Collagen degradation impairs the chemical and mechanical interaction of the resin monomers of adhesive materials with the dental structure, which is essential to the retention and integrity of dental restorations [44]. The management and restorative treatment of radiation-induced caries is still challenging for dental professionals [45]. Clinical trials investigating the longevity of restorative treatments in patients with radiation-induced caries reported failures such as marginal integrity, secondary caries, and retention, with different adhesive restorative materials [45–48]. It could be inferred that the restorative treatment is significantly influenced by the complex and still poorly understood nature of the substrate available for adhesion, which directly impacts the longevity of restorations [49]. Therefore, the current study was designed to contribute to a better understanding of the impact of head and neck radiation on dentin tooth structure in both radiation-induced carious dentin and adjacent areas. It is important to comprehend the characteristics of dental substrates affected by radiation to recommend the proper treatment aiming to increase the longevity of restorations, decrease repetitive restorative cycles, and improve the quality of life of patients after oncologic treatment.

Conclusion

The effect of radiation on the dentin was expressed chemically as a reduction in calcium (Ca) and phosphorus (P) levels in hydroxyapatite and microstructurally by the presence of tubule obliteration, and craters.

According to the XRD results the intensity of most peaks was decreased in irradiated dentin, and predominantly carious dentin, suggesting degradation of hydroxyapatite in these specimens.

FTIR findings showed a reduction of the intensity of carbonate bands leading to caries-induced mineral loss and alteration in dentin composition of both irradiated and carious dentin. The XRD and FTIR results suggest the degradation of collagen and Hap of the irradiated and carious dentin.

Supporting information