- Email: [email protected]
Effects of different radiation doses on the microhardness, superficial morphology, and mineral components of human enamel
Accepted Manuscript Title: EFFECTS OF DIFFERENT RADIATION DOSES ON THE MICROHARDNESS, SUPERFICIAL MORPHOLOGY, AND MINERAL COMPONENTS OF HUMAN ENAMEL Authors: Sandra Ribeiro de Barros da Cunha, Felipe Paiva Fonseca, Pedro Augusto Minorin Mendes Ramos, Cec´ılia Maria Kalil Haddad, Eduardo Rodrigues Fregnani, Ana Cec´ılia Corrˆea Aranha PII: DOI: Reference:
S0003-9969(17)30117-6 http://dx.doi.org/doi:10.1016/j.archoralbio.2017.04.007 AOB 3851
To appear in:
Archives of Oral Biology
Received date: Revised date: Accepted date:
20-2-2017 5-4-2017 8-4-2017
Please cite this article as: de Barros da Cunha Sandra Ribeiro, Fonseca Felipe Paiva, Ramos Pedro Augusto Minorin Mendes, Haddad Cec´ılia Maria Kalil, Fregnani Eduardo Rodrigues, Aranha Ana Cec´ılia Corrˆea.EFFECTS OF DIFFERENT RADIATION DOSES ON THE MICROHARDNESS, SUPERFICIAL MORPHOLOGY, AND MINERAL COMPONENTS OF HUMAN ENAMEL.Archives of Oral Biology http://dx.doi.org/10.1016/j.archoralbio.2017.04.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
EFFECTS OF DIFFERENT RADIATION DOSES ON THE MICROHARDNESS, SUPERFICIAL MORPHOLOGY, AND MINERAL COMPONENTS OF HUMAN ENAMEL EFFECTS OF RADIATION ON HUMAN ENAMEL
Sandra Ribeiro de Barros da Cunha - D.D.S., Ph.D. student, Department of Restorative Dentistry, School of Dentistry, University of São Paulo, Av. Professor Lineu Prestes, 2227, São Paulo - SP, Brazil +55 11 30917645 [email protected] Felipe Paiva Fonseca - D.D.S., Ph.D. , Department of Oral Surgery and Pathology, School of Dentistry, Universidade Federal de Minas Gerais; Av. Antônio Carlos, 6627, Pampulha, Belo Horizonte-MG, Brazil. Pedro Augusto Minorin Mendes Ramos - B. Phys., Medical Physicist, Hospital Sírio Libanês, Rua Dona Adma Jafet, 91 - Bela Vista, São Paulo - SP, Brazil. Cecília Maria Kalil Haddad - B. Phys., Head of the Department of Medical Physicists, Hospital Sírio Libanês, Rua Dona Adma Jafet, 91 - Bela Vista, São Paulo - SP, Brazil Eduardo Rodrigues Fregnani - D.D.S., Ph.D., Head of the Department of Oral Medicine, Hospital Sírio Libanês, Rua Dona Adma Jafet, 91 - Bela Vista, São Paulo - SP, Brazil Ana Cecília Corrêa Aranha (Corresponding author) - D.D.S., Ph.D., Associate Professor of the Department of Restorative Dentistry, School of Dentistry, University of São Paulo Av. Professor Lineu Prestes, 2227, São Paulo - SP, Brazil. [email protected]
Highlights - After radiation, decrease in microhardness values was found in cervical enamel only. - Radiation caused no change in the Ca/P ratio of enamel. - Morphological changes were only found in cervical enamels irradiated with 70Gy.
ABSTRACT Objective: To evaluate the effects of three different radiotherapy doses (20, 40, and 70 Gy) on the microhardness, superficial morphology, and mineral content (based on Ca and P values) of three different depths of human enamel (cervical, middle, and occlusal). Design: Thirty-four third molars were cut, separated, and prepared. Microhardness samples (n=30) were embedded in acrylic resin and then polished, and depths were delimited. Microhardness tests were performed on cervical, middle, and occlusal enamel pre- and post-radiotherapy with a load of 50 g for 30 s. For the scanning electron microscopy (SEM) analysis (n = 4) and energy dispersive X-ray spectroscopy (EDS) (n=12), samples were fixed in a 3% glutaraldehyde solution, washed in 0.1 M cacodylate solution, and dehydrated in crescent concentrations of ethanol. Microhardness data were tested for significant differences using a two-way analysis of variance (ANOVA) and Tukey’s test (p<0.05), while SEM and EDS were evaluated qualitatively. Results: The results showed a decrease in microhardness values only in the cervical enamel, regardless of the radiation dose used; no morphological or mineral change was observed. Conclusion: Radiotherapy can affect the microhardness values of only cervical enamel without compromising the morphological or mineral (Ca and P) content at any depth.
KEYWORDS: Radiotherapy, Enamel, Microhardness, EDS, SEM, Head and Neck Cancer
INTRODUCTION Head and neck cancer represents a heterogeneous group of malignant tumors of the upper aerodigestive tract. This type of cancer is the seventh most common worldwide (Jemal et al., 2011). Oral cancer, which includes oral mucosa, hard and soft palate, gums, tongue, and floor of the mouth, is considered a worldwide health problem. Oral squamous cell carcinoma is the most common microscopic subtype, accounting for over 90% of all cases diagnosed. It is mainly associated with the simultaneous use of tobacco and alcohol, and in the year 2012 approximately 300,000 new cases and 145,000 deaths were estimated to have occurred worldwide from this type of cancer (Döbrossy, 2015). During the last decades, there have been improvements in head and neck cancer treatment, and radiotherapy is an important part (El-Faramawy et al., 2013). According to the World Health Organization (WHO), two-thirds of oncological patients will have radiotherapy as a treatment option. Radiotherapy is a local treatment that uses electromagnetic waves that carry energy and have no mass. The electromagnetic waves used in radiotherapy treatment are considered ionizing radiation, which means that the energy carried by these waves is higher than the energy that holds the electron in the electrosphere, creating free electrons. These free electrons can cause damage to the cell both directly and indirectly (Tauhata et al., 2014). Indirect damage is caused by the production of free radicals from the radiolysis of different molecules, such as water that produces H+ and
OH- ions, and the latter are highly unstable and reactive. These ions will bind to other molecules, which will be damaged and lose function, or will produce more free radicals. This radiation mechanism supports the consensus in the literature that radiotherapy of dental tissues has greater deleterious effects when higher concentrations of organic content are present in the tissue (Walker, 1975; Pioch, Golfels and Staehle, 1972; Soares et al., 2010). The standard treatment for squamous cell carcinoma in the head and neck regions is a final accumulated dose between 40 Gy and 70 Gy, which is fractionated in daily doses (Nutting et al., 2001; Seikaly et al., 2004; Jham and Silva-Freire, 2006; Kielbassa et al., 2006; Lieshout and Bots, 2014). Despite the advantage of preserving the tissue structure, radiotherapy in patients with head and neck cancer usually results in oral complications (Pioch, Golfels and Staehle, 1972; Soares et al., 2010; Jham and Silva-Freire, 2006). The literature reports changes in the salivary glands, decreased salivary flow, changes in salivary composition and oral microbiota, oral mucositis, muscular trismus, vascular alterations, and osteoradionecrosis from radiotherapy (Nutting et al., 2001; Kielbassa et al., 2006; Silva et al., 2010; Naves et al., 2012; Lieshout and Bots, 2014). In addition to these structural changes, there is also the possibility that radiotherapy may exert direct effects on the teeth, such as changes in the dentinenamel junction, crystalline structure, acid solubility of enamel, dentin elastic modulus, matrix metalloproteinases (MMPs), and enamel and dentin microhardness (Kielbassa et al., 2006; Silva et al., 2010; McGuire et al., 2014; Gonçalves et al., 2014; Reed et al., 2015). One of the most cited side effects of radiotherapy is radiation-related caries, which are a complex, multifactorial disease and an indirect effect of radiation
treatment that harms the tooth structure. It is a rapid, painless, and destructive form of tooth decay that can quickly lead to the amputation of crowns (Dreizen et al., 1977; Vissink et al., 2003). With the increasing life expectancy of the population worldwide, longevity of the teeth in the oral cavity, and increase in neoplastic diseases, it is essential that dentists be aware of changes in the oral cavity produced by the irradiation of head and neck cancer so that they can manage patients correctly both pre- and post-treatment. Thus, this study aimed to analyze superficial morphology, microhardness, and mineral content alterations of irradiated dental substrates at three different radiation doses.
MATERIAL AND METHODS Specimen preparation Thirty-four healthy human third molars were collected from the Tooth Bank of the School of Dentistry of the University of São Paulo, after approval from the Research Ethics Committee of the same institution (protocol 658.991). Teeth were cleaned and had their roots removed with a water-cooled diamond disc (Kg Sorensen, Barueri, São Paulo, Brazil). From each tooth, two sagittal slices were obtained with a low-speed diamond saw under water cooling (Isomet 1000, Buehler Ltd., Lake Bluff, IL, USA). Sagittal sections were prepared at the middle of the crown along the long axis of the tooth creating two slices: the mesial and distal slices. The mesial parts were used for microhardness and energy dispersive X-ray spectroscopy (EDS), and the three distal parts were used for the EDS control group. The remaining four teeth were cut along a transverse plane to obtain three samples for each depth of enamel and used for scanning electron microscopy (SEM) (Fluxogram 1).
Microhardness Microhardness refers to the testing of different materials hardness by applying small loads to it. The hardness of oral tissues is an essential aspect of their behavior, representing their resistance to masticatory loads, changes in the dentin-enamel junction, crystalline structure, acid solubility of enamel and dentin elastic modulus. For the microhardness test, samples were embedded in acrylic resin, and then a decreasing sequence of silicon carbide (SiC) paper (400, 600, 800, 1200, 2500, and 4000 grit) was used with the aid of a water-cooled polishing machine (Buehler Ltd., Lake Buff, IL, USA) to obtain a smooth and polished surface. The opposite surface of each specimen was also polished to obtain a parallel surface, which is essential for the correct measurement of microhardness. Between each polishing stage, samples were cleaned using distilled water in an ultrasonic cleaner (Digital Ultrasonic Cleaner CD4820, Kondortech, Sao Carlos, Brazil) for 8 minutes to remove any debris. The enamel depths were delineated with a permanent marker and ruler. Five indentations at each enamel depth were performed with a Knoop tip attached to a Microhardness Tester HMV-2000 (SHIMADZU Co., Tokyo, Japan) using a load of 50 g for 30 seconds. The indentations started 150 µm away from the dentin-enamel junction, and each indentation was spaced 100 µm apart. Microhardness values were measured
with
CAMS-WIN
software
(Newage
Testing
Instruments
Inc.,
Southampton, England). The microhardness test was performed before and after irradiation so that each sample could serve as its own control. After the first microhardness test analysis (control), the samples were randomly divided into three groups (n = 10) according to the radiation dose (Group 20
Gy, Group 40 Gy, and Group 70 Gy) and again submitted to the same microhardness analysis protocol. The results were separated according to depth. The quantitative analysis was performed by a two-way analysis of variance (ANOVA) (doses of 20, 40, and 70 Gy before and after radiotherapy) with one repeated measurement (time) and at a significance level of 5% (p ≥ 0,05).
Irradiation of the samples Samples were irradiated with a total and single application of 20, 40, or 70 Gy with X-rays from a linear accelerator (Mevatron MX2 6 mV; Siemens Healthcare, Erlangen, Germany) in the Department of Radiotherapy at Sírio-Libanês Hospital. This study used a 20-Gy dose in an attempt to simulate teeth that are situated in healthy tissues, but located near the target region, which can receive between 40 and 70-Gy. Samples were submerged in distilled water during radiotherapy. Moreover, all irradiations were done in the same container with the samples submerged in distilled water. An isocentric set-up was done, so that the geometric center of the container was aligned with the isocenter of the linear accelerator. This means that from whatever angle we point the gantry, the distance between the "radiation source" and the container's center would be 100cm. For a homogeneous radiation distribution for all samples, radiation was applied in two opposing fields parallel to the incident radiation.
Energy dispersive X-ray spectroscopy For the EDS test, 12 samples (n = 3) randomly selected from the microhardness analysis were used from the control and irradiated groups (20 Gy, 40
Gy, and 70 Gy). For the control group, the distal part of the tooth that was not used for SEM was selected. For samples used in the microhardness analysis, the acrylic resin was removed with the aid of a low-speed cutting machine (Labcut 1010 - Extec, Enfield, Connecticut, EUA). Samples were then fixed in 2.5% glutaraldehyde solution for 24 h at 4°C, washed in a solution of 0.1 M cacodylate, dehydrated in sequential ethanol baths for 10 min each (30%, 50%, 70%, 90%, 96%, and 100% for 20 min) and then transferred to a critical-point dryer (HMDS) for 30 min. Samples were mounted on aluminum stubs, properly identified, covered with a thin layer of carbon (MED 010, Balzers Union, Balzers, Liechtenstein), and observed by SEM to detect backscattered electrons (BSE) (LEO 435 VP, LEO Electron Microscopy Ltd., Cambridge, England). Quantifications were performed according to the depth used in the microhardness test. Three measurements of the enamel were performed at each depth (cervical, middle, and occlusal) and for each type of substrate (sound enamel and irradiated enamel). The duration in each area was 20 seconds, with the dead time in a 35% cobalt pattern.
Scanning electron microscopy For the SEM analysis, 12 samples were used from the distal parts of four different teeth. These samples were divided according to the depth of the tooth (occlusal, middle, and cervical) and then divided according to the radiation dose received (control, 20 Gy, 40 Gy, or 70 Gy). Samples were then fixed in 2.5% glutaraldehyde solution for 24 h at 4°C, washed in a solution of 0.1 M cacodylate, dehydrated in sequential baths of ethanol for 5 minutes each (30%, 50%, 70%, 90%, 96%, and 3 baths of 10 min each in 100%), and then transferred to a critical-point
dryer (HMDS) for 30 min. Samples were mounted on aluminum stubs, properly identified, covered with a thin layer of platinum, and observed using a Quanta 600 FEG SEM and the secondary electron detector (SE) (FEI, Hillsboro, OR, USA). Three images were obtained of each sample at three magnifications (1,000×, 5,000×, and 20,000×).
RESULTS Microhardness analysis The results of the ANOVA indicated a statistically significant difference only in microhardness of the cervical enamel for all irradiation doses and periods studied. The results of Tukey’s test and average hardness measurements indicated that in the cervical enamel, the average hardness was greater before radiation than after radiation (Table 1) (Figure 1).
Energy dispersive X-ray spectroscopy (EDX) Because Ca and P are the two main components of the enamel structure and are by far the most investigated elements in the majority of the studies, we detailed described the results for these two elements only. However, we further state that no significant difference was observed when investigating Na, Mg, Cl, and O values (data not shown). Hence, the results of the EDS test were analyzed descriptively, and indicated no significant difference in the Ca/P level for either substrate at any depth, independent of the irradiation dose and period (pre- and post-radiation) (Table 2) (Figure 1).
Scanning electron microscopy The images obtained show electron micrographs of the study groups based on the radiation dose (control, 20, 40, and 70 Gy) and region (occlusal, middle, and cervical regions). Three samples from each group were used to acquire the images and perform a qualitative analysis at three different magnifications (1,000×, 5,000×, and 20,000×) (Figure 1). Images of the control group indicated the presence of regular healthy enamel at all depths with the regular presence of prisms in the shape of a “door lock”, characteristic of enamel substrate as it can bee seen in the control group (Figure 2). Similar characteristics were also observed in all treated groups (Figure 3). However, at 20,000× magnification, a slight change in the enamel prisms was observed, which appeared shorter and rounded, and only in the 70 Gy group (Figure 4).
DISCUSSION Literature presents different ways to simulate in vitro radiotherapy with some of the authors using artificial saliva, distilled water or physiological saline solution (Gernhardt et al., 2001; Gonçalves et al., 2014; da Cunha et al., 2016). Since the main focus of this study is the direct effect of radiotherapy in dental tissue, distilled water was chose since it can provide an environment capable of radiolysis and no further interaction with the teeth. Radiotherapy consists of cumulative fractionated doses, about 2 Gy, that are delivered in daily sessions, with pauses at weekends, for up to 7 weeks. The standard treatment in patients with malignant tumors in the head and neck region results in a final accumulated dose of between 40 Gy and 70 Gy (Kielbassa et al., 2006; Naves et
al., 2012). It is well known that the effects of radiotherapy are cumulative and that the fractionation of radiation is based on "5Rs" (Repair, redistribution, reoxygenation, regeneration and radiosensitivity) (Harrington, Jankowska and Hingorani, 2007). Since there would not occur any of the 5Rs on the teeth in an in vitro study it was preferred to deliver the dose with a single fraction. In the present study, the enamel presented slight changes in microhardness, no change in mineral content, and a very slight morphological change within the radiotherapy protocols used. Only the cervical enamel showed decreased microhardness values after radiation at all doses used. This can be explained by the higher porosity and lower tissue thickness in this region, and thus its organic content may be more greatly influenced by the response to radiation. This is in agreement with Jansma et al., who concluded that the use of gamma radiation at therapeutic doses had no significant effect on chemical components, except in organic parts (Jansma et al., 1990). These results further support the necessity of evaluating changes in all regions of dental enamel, what is important to overcome limited reports previously described in literature that did not investigate all dental thirds. The EDX test confirmed that there was no modification regarding the concentrations of mineral content based on the values of Ca and P, regardless of the enamel location and dose of radiation, which is consistent with the literature (Walker et al., 1975; Jansma et al., 1990). In the clinic and in the literature, there is a consensus that radiation-related caries have a preference for the enamel’s cervical region. Thus, the changes found in the cervical third in this study are consistent with this clinical finding (Frank, Herdly and Philippe, 1965; Kielbassa et al., 2006; Soares et al., 2010).
For the enamel’s middle and occlusal thirds, this study found no significant change in microhardness values in the EDX or SEM analysis. Although several studies investigating the radiotherapy effects on human enamel have been published, literature is quite controversial regarding the hardness of irradiated enamel, possibly due to the high variability on methodological approaches used. Some authors are in agreement with our results. For example, Kielbassa et al., who studied the effects of radiotherapy on enamel microhardness and caries susceptibility in vitro and in situ, found no difference in microhardness values or caries between irradiated and nonirradiated enamel (Kielbassa et al., 1999; Kielbassa, Schenderh and Schulte-Mönting, 2000). Other previous publications, where structural changes were not found after radiotherapy, are in agreement with the results of the present study (Wiemann et al., 1972; Zach, 1976; Jansma et al., 2006). A more recent study showed that radiotherapy did not influence the bonding strength to enamel, irrespective of the adhesive or radiation dose used, showing that the chemical and prismatic structures of enamel probably were unaltered after radiotherapy (da Cunha et al., 2016). Other authors found an increase in enamel microhardness after radiotherapy. They explained the increase through changes in Hunter-Schreger band patterns making irradiated enamel more friable and susceptible to crack formation, and possible alterations in the organic matrix of the enamel decreasing the protein/mineral ratio and leading to an increase in microhardness and the elastic modulus because of the lower organic content of irradiated enamel (Gonçalves et al., 2014; Reed et al., 2015). It is important to highlight some differences in the methodology used by these authors if compared to the present study: Gonçalves et al. (2014) used a smaller sample (20 vs 34 teeth), did not evaluate different thirds of the teeth (only different
depths), used a different irradiation source, used a shorter distance between the source and the teeth, used a maximum cumulative dose of 60Gy and the authors used a score system to define the presence or absence of morphological changes. Reed et al. (2015) also used a much smaller sample size than ours (7 vs 34 teeth), did not evaluate different thirds of enamel, used a different radiation source and the distance between the teeth and the radiation source was not provided. The literature also contains studies in which ionizing radiation decreased enamel microhardness in all regions, and the authors explained that this occurs because of decarboxylation of the tissue. This means that the organic matrix interacts with apatite crystals, creates microcracks in hydroxyapatite minerals, and forms smaller crystallites, thus making the surface of the enamel tissue more rough (Kielbassa, Hellwig, Meyer-Lueckel, 2006; Lieshout and Bots, 2014; Qing et al., 2015; Liang et al., 2016). In the systematic review of Lieshout and Bots (2014) the authors clearly demonstrated the contradictory results described in literature regarding the
effects
of
radiation
in
the
enamel
and
dentin
components.
Kielbassa, Hellwig, Meyer-Lueckel (2006) observed significant differences in the demineralization of irradiated bovine incisors enamel, but not in their human molars sample. Qing et al. (2014) in their study used 13 teeth submitted to microhardness assay, the authors used a maximum of 60Gy irradiation and a different radiation source than ours, and they also immersed the samples in artificial saliva. The authors did not specify the exact anatomical location of the tests and the distance from the irradiation source to their teeth. Liang et al. (2016) obtained interesting results using a well designed study, but in contrast to ours they did not investigate different areas of enamel.
With the increase in the number of patients with head and neck cancer, and consequently, the increased use of radiotherapy, it is important to know the effects of ionizing radiation on the oral cavity and how its side effects can contribute to the evolution of radiation-related caries. It is known that interactions of the oral microflora change with decreased pH in the oral cavity, reduced salivary flow, and saliva composition, and these are important factors in the development of radiationrelated caries (Silva et al., 2010). In conclusion, the findings of this in vitro study demonstrate that radiotherapy was able to cause only slight morphological changes after 70Gy radiation and that microhardness decreased only in the cervical third of the enamel, but it did not interfere with enamel Ca and P content.
ACKNOWLEDGEMENTS The authors would like to express their gratitude to the School of Dentistry of the University of São Paulo and Sírio-Libanês Hospital for supporting this research.
CONFLICT OF INTEREST Disclosures: None
REFERENCES 1. da Cunha, S.R., Ramos, P.A., Haddad, C.M., da Silva, J.L., Fregnani, E.R. and Aranha, A.C. (2016). Effects of different radiation doses on the bond strengths of two different adhesive systems to enamel and dentin. Journal of Adhesive Dentistry, 18, 151-156.
2. Döbrossy, L. (2005). Epidemiology of head and neck cancer: magnitude of the problem. Cancer Metastasis Reviews, 24, 9-17. 3. Dreizen, S., Daly, T.E., Drane, J.B., Brown, L.R. (1977). Oral complications of cancer radiotherapy. Postgraduate Medical Journal , 61, 85-92. 4. El-Faramawy, N., Ameen, R., El-Haddad, K., El-Zainy, M. (2013). Effects of gamma radiation on hard dental tissues of albino rats: investigation by light microscopy. Radiation and Environmental Biophysics, 52, 375-387. 5. Frank, R.M., Herdly, J., Philippe, E. (1965). Acquired dental defects and salivary gland lesions after irradiation for carcinoma. Journal of the American Dental Association, 70, 868-883. 6. Gernhardt, C.R., Kielbassa, A.M., Hahn, P., Schaller, H.G. (2001). Tensile bond strengths of four different dentin adhesives on irradiated and nonirradiated human dentin in vitro. Journal of Oral Rehabilitation, 28, 814-820. 7. Gonçalves, L.M., Palma-Dibb, R.G., Paula-Silva, F.W., Oliveira, H.F., Nelson-Filho, P., Silva, L.A., Queiroz, A.M. (2014). Radiation therapy alters microhardness and microstructure of enamel and dentin of permanent human teeth. Journal of Dentistry, 42, 986-992. 8. Harrington, K., Jankowska, P., Hingorani, M. (2007). Molecular biology for the radiation oncologist: the 5Rs of radiobiology meet the hallmarks of cancer. Clinical Oncology, 19, 561-571. 9. Jansma, J., Buskes, J.A., Vissink, A., Mehta, D.M., Gravenmade, E.J. (1988). The effect of X-ray irradiation on the demineralization of bovine dental enamel. A constant composition study. Caries Research, 22, 199-203.
10. Jansma, J., Borggreven, J.M., Driessens, F.C., Gravenmade, E.J. (1990). Effect of X-ray irradiation on the permeability of bovine dental enamel. Caries Research, 24, 164-168. 11. Jemal, A., Bray, F., Center, M.M., Ferlay, J., Ward, E., Forman, D. (2011). Global cancer statistics. CA A Cancer Journal for Clinicians, 61, 69-90. Erratum in: CA A Cancer Journal for Clinicians, 61, 134. 12. Jham, B.C., da Silva-Freire, A.R. (2006). Oral complications of radiotherapy in the head and neck. Brazilian Journal of Otorhinolaryngology, 72, 704-708. 13. Kielbassa, A.M., Wrbas, K.T., Schulte-Mönting, J., Hellwig, E. (1999). Correlation of transversal microradiography and microhardness on in situinduced demineralization in irradiated and nonirradiated human dental enamel. Archives of Oral Biology, 44, 243-251. 14. Kielbassa,
A.M.,
Schendera,
A.,
Schulte-Mönting,
J.
(2000).
Microradiographic and microscopic studies on in situ induced initial caries in irradiated and nonirradiated dental enamel. Caries Research, 34, 41-47. 15. Kielbassa, A.M., Hinkelbein, W., Hellwig, E., Meyer-Lückel, H. (2006). Radiation-related damage to dentition. Lancet Oncology, 7, 326-335. 16. Kielbassa AM, Hellwig E, Meyer-Lueckel H. (2006). Effects of irradiation on in situ remineralization of human and bovine enamel demineralized in vitro. Caries Research, 40, 130-5. 17. Liang, X., Zhang, J.Y., Cheng, I.K., Li, J.Y. (2016). Effect of high energy Xray irradiation on the nano-mechanical properties of human enamel and dentine. Brazilian Oral Research, 30. 18. Lieshout, H.F., Bots, C.P. (2014). The effect of radiotherapy on dental hard tissue--a systematic review. Clinical Oral Investigation, 18, 17-24.
19. McGuire, J.D., Gorski, J.P., Dusevich, V., Wang, Y., Walker, M.P. (2014). Type IV collagen is a novel DEJ biomarker that is reduced by radiotherapy. Journal of Dental Research, 93, 1028-1034. 20. Naves, L.Z., Novais, V.R., Armstrong, S.R., Correr-Sobrinho, L., Soares, C.J. (2012). Effect of gamma radiation on bonding to human enamel and dentin. Supportive Care in Cancer, 20, 2873-2878. 21. Nutting, C.M., Morden, J.P., Harrington, K.J., Urbano, T.G., Bhide, S.A., Clark, C., Miles, E.A., Miah, A.B., Newbold, K., Tanay, M.A., Adab, F., Jefferies, S.J., Scrase, C., Yap, B.K., A’hern, R.P., Sydenham, M.A., Emson, M., Hall, E. (2001). PARSPORT Trial Management Group. Parotid-sparing intensity modulated versus conventional radiotherapy in head and neck cancer (PARSPORT): a phase 3 multicentre randomised controlled trial. The Lancet. Oncology ,12, 127-136. 22. Pioch, T., Golfels, D., Staehle, H.J. (1992). An experimental study of the stability of irradiated teeth in the region of the dentinoenamel junction. Endodontics and Dental Traumatology, 8, 241-244. 23. Qing, P., Huang, S., Gao, S., Qian, L., Yu, H. (2015). Effect of gamma irradiation on the wear behaviour of human tooth enamel. Scientific Reports, 5, 11568. 24. Reed, R., Xu, C., Liu, Y., Gorski, J.P., Wang, Y., Walker, M.P. (2015). Radiotherapy effect on nano-mechanical properties and chemical composition of enamel and dentine. Archives of Oral Biology, 60, 690-697. 25. Seikaly, H., Jha, N., Harris, J.R., Barnaby, P., Liu, R., Williams, D., McGaw, T., Rieger, J., Wolfaardt, J., Hanson, J. (2004). Long-term outcomes of
submandibular gland transfer for prevention of postradiation xerostomia. Archives of Otolaryngology and Head and Neck Surgery, 130, 956-961. 26. Silva, A.R., Alves, F.A., Berger, S.B., Giannini, M., Goes, M.F., Lopes, M.A. (2010). Radiation-related caries and early restoration failure in head and neck cancer patients. A polarized light microscopy and scanning electron microscopy study. Supportive Care in Cancer, 18, 83-87. 27. Soares, C.J., Castro, C.G., Neiva, N.A., Soares, P.V., Santos-Filho, P.C., Naves, L.Z., Pereira, P.N. (2010). Effect of gamma irradiation on ultimate tensile strength of enamel and dentin. Journal of Dental Research, 89, 159164. 28. Tauhata, L., Salati, I.P.A., Di Prinzio, R., Di Prinzio, A. (2014). Radioproteção e Dosimetria: Fundamentos – 10a rev. Rio de Janeiro, IRD/CNEN, 2014, 111-119. 29. Vissink, A., Jansma, J., Spijkervet, F.K., Burlage, F.R., Coppes, R.P. (2003). Oral sequelae of head and neck radiotherapy. Critical Reviews in Oral Biology and Medicine, 14, 199-212. 30. Walker, R. (1975). Direct effect of radiation on the solubility of human teeth in vitro. Journal of Dental Research , 54, 901. 31. Wiemann, M.R., Davis, M.K., Besic, F.C. (1972). Effects of x-radiation on enamel solubility. Journal of Dental Research, 51, 868. 32. Zach, G.A. (1976). X-ray diffraction and calcium-phosphorous analysis of irradiated human teeth. Journal of Dental Research, 55, 907-909.
Figures legends Figure 1. Graphical abstract of the main results obtained in this study. Radiotherapy caused significant microhardness changes only in the cervical third of the enamel no matter the dose used, but it did not interfere with its mineral content (based on Ca/P ratio) and caused morphological changes only when 70Gy was applied and limited to the cervical area of the irradiated teeth.
Figure 2. Electromicrographs of cervical region of Control Group with 1.000 (A), 5.000 (B) and 20.000x (C) magnificatgion.
Figure 3. Electromicrographs of cervical region of 20 Gy (A), 40 Gy (B) and 70 Gy (B) with 5.000x magnification.
Figure 4. Electromicrographs of cervical region of Control Group (A), 20 Gy (B), 40 Gy (C) and 70 Gy (D) with 20.000x magnification.
Table 1 – Microhardness (means and standard deviations – values in Knoops) for each region and dose.
Dose
20Gy 40Gy 70Gy
Cervical Enamel Mean(SD) Mean(SD) PrePostRadioteraphy Radioteraphy 298.04 294.36 (11.80) (6.68) 285 275.3 (19.45) (36) 291.98 283.68 (16.51) (21.64)
Medium Enamel Mean(SD) Mean(SD) PrePostRadioteraphy Radioteraphy 300.16 296.14 (11.19) (11.78) 291.96 293.04 (15.44) (17.53) 293.22 288.78 (13.24) (14.25)
Occlusal Enamel Mean(SD) Mean(SD) PrePostRadioteraphy Radioteraphy 281.82 281.76 (8.41) (11.12) 271.80 274.48 (20.21) (16.48) 289.82 279.90 (14.12) (9.10)
Table 2 - Mean concentrations by weight of the elements Ca and P, and Ca/P Index obtained with EDX for each region and dose (values in %mass).
Group and Region Control Cervical Medium Occlusal 20Gy Cervical Medium Occlusal 40Gy Cervical Medium Occlusal 70Gy Cervical Medium Occlusal
Ca 38.11 38.07 38.03 38.08 38.25 38.14 38.33 38.19 38.15 38.13 38.11 38.11
P 19.71 19.53 19.52 19.57 19.43 19.50 19.47 19.47 19.47 19.53 19.50 19.53
Ca/P index 1.93 1.95 1.95 1.95 1.97 1.96 1.97 1.97 1.96 1.95 1.95 1.95
S0003-9969(17)30117-6 http://dx.doi.org/doi:10.1016/j.archoralbio.2017.04.007 AOB 3851
To appear in:
Archives of Oral Biology
Received date: Revised date: Accepted date:
20-2-2017 5-4-2017 8-4-2017
Please cite this article as: de Barros da Cunha Sandra Ribeiro, Fonseca Felipe Paiva, Ramos Pedro Augusto Minorin Mendes, Haddad Cec´ılia Maria Kalil, Fregnani Eduardo Rodrigues, Aranha Ana Cec´ılia Corrˆea.EFFECTS OF DIFFERENT RADIATION DOSES ON THE MICROHARDNESS, SUPERFICIAL MORPHOLOGY, AND MINERAL COMPONENTS OF HUMAN ENAMEL.Archives of Oral Biology http://dx.doi.org/10.1016/j.archoralbio.2017.04.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
EFFECTS OF DIFFERENT RADIATION DOSES ON THE MICROHARDNESS, SUPERFICIAL MORPHOLOGY, AND MINERAL COMPONENTS OF HUMAN ENAMEL EFFECTS OF RADIATION ON HUMAN ENAMEL
Sandra Ribeiro de Barros da Cunha - D.D.S., Ph.D. student, Department of Restorative Dentistry, School of Dentistry, University of São Paulo, Av. Professor Lineu Prestes, 2227, São Paulo - SP, Brazil +55 11 30917645 [email protected] Felipe Paiva Fonseca - D.D.S., Ph.D. , Department of Oral Surgery and Pathology, School of Dentistry, Universidade Federal de Minas Gerais; Av. Antônio Carlos, 6627, Pampulha, Belo Horizonte-MG, Brazil. Pedro Augusto Minorin Mendes Ramos - B. Phys., Medical Physicist, Hospital Sírio Libanês, Rua Dona Adma Jafet, 91 - Bela Vista, São Paulo - SP, Brazil. Cecília Maria Kalil Haddad - B. Phys., Head of the Department of Medical Physicists, Hospital Sírio Libanês, Rua Dona Adma Jafet, 91 - Bela Vista, São Paulo - SP, Brazil Eduardo Rodrigues Fregnani - D.D.S., Ph.D., Head of the Department of Oral Medicine, Hospital Sírio Libanês, Rua Dona Adma Jafet, 91 - Bela Vista, São Paulo - SP, Brazil Ana Cecília Corrêa Aranha (Corresponding author) - D.D.S., Ph.D., Associate Professor of the Department of Restorative Dentistry, School of Dentistry, University of São Paulo Av. Professor Lineu Prestes, 2227, São Paulo - SP, Brazil. [email protected]
Highlights - After radiation, decrease in microhardness values was found in cervical enamel only. - Radiation caused no change in the Ca/P ratio of enamel. - Morphological changes were only found in cervical enamels irradiated with 70Gy.
ABSTRACT Objective: To evaluate the effects of three different radiotherapy doses (20, 40, and 70 Gy) on the microhardness, superficial morphology, and mineral content (based on Ca and P values) of three different depths of human enamel (cervical, middle, and occlusal). Design: Thirty-four third molars were cut, separated, and prepared. Microhardness samples (n=30) were embedded in acrylic resin and then polished, and depths were delimited. Microhardness tests were performed on cervical, middle, and occlusal enamel pre- and post-radiotherapy with a load of 50 g for 30 s. For the scanning electron microscopy (SEM) analysis (n = 4) and energy dispersive X-ray spectroscopy (EDS) (n=12), samples were fixed in a 3% glutaraldehyde solution, washed in 0.1 M cacodylate solution, and dehydrated in crescent concentrations of ethanol. Microhardness data were tested for significant differences using a two-way analysis of variance (ANOVA) and Tukey’s test (p<0.05), while SEM and EDS were evaluated qualitatively. Results: The results showed a decrease in microhardness values only in the cervical enamel, regardless of the radiation dose used; no morphological or mineral change was observed. Conclusion: Radiotherapy can affect the microhardness values of only cervical enamel without compromising the morphological or mineral (Ca and P) content at any depth.
KEYWORDS: Radiotherapy, Enamel, Microhardness, EDS, SEM, Head and Neck Cancer
INTRODUCTION Head and neck cancer represents a heterogeneous group of malignant tumors of the upper aerodigestive tract. This type of cancer is the seventh most common worldwide (Jemal et al., 2011). Oral cancer, which includes oral mucosa, hard and soft palate, gums, tongue, and floor of the mouth, is considered a worldwide health problem. Oral squamous cell carcinoma is the most common microscopic subtype, accounting for over 90% of all cases diagnosed. It is mainly associated with the simultaneous use of tobacco and alcohol, and in the year 2012 approximately 300,000 new cases and 145,000 deaths were estimated to have occurred worldwide from this type of cancer (Döbrossy, 2015). During the last decades, there have been improvements in head and neck cancer treatment, and radiotherapy is an important part (El-Faramawy et al., 2013). According to the World Health Organization (WHO), two-thirds of oncological patients will have radiotherapy as a treatment option. Radiotherapy is a local treatment that uses electromagnetic waves that carry energy and have no mass. The electromagnetic waves used in radiotherapy treatment are considered ionizing radiation, which means that the energy carried by these waves is higher than the energy that holds the electron in the electrosphere, creating free electrons. These free electrons can cause damage to the cell both directly and indirectly (Tauhata et al., 2014). Indirect damage is caused by the production of free radicals from the radiolysis of different molecules, such as water that produces H+ and
OH- ions, and the latter are highly unstable and reactive. These ions will bind to other molecules, which will be damaged and lose function, or will produce more free radicals. This radiation mechanism supports the consensus in the literature that radiotherapy of dental tissues has greater deleterious effects when higher concentrations of organic content are present in the tissue (Walker, 1975; Pioch, Golfels and Staehle, 1972; Soares et al., 2010). The standard treatment for squamous cell carcinoma in the head and neck regions is a final accumulated dose between 40 Gy and 70 Gy, which is fractionated in daily doses (Nutting et al., 2001; Seikaly et al., 2004; Jham and Silva-Freire, 2006; Kielbassa et al., 2006; Lieshout and Bots, 2014). Despite the advantage of preserving the tissue structure, radiotherapy in patients with head and neck cancer usually results in oral complications (Pioch, Golfels and Staehle, 1972; Soares et al., 2010; Jham and Silva-Freire, 2006). The literature reports changes in the salivary glands, decreased salivary flow, changes in salivary composition and oral microbiota, oral mucositis, muscular trismus, vascular alterations, and osteoradionecrosis from radiotherapy (Nutting et al., 2001; Kielbassa et al., 2006; Silva et al., 2010; Naves et al., 2012; Lieshout and Bots, 2014). In addition to these structural changes, there is also the possibility that radiotherapy may exert direct effects on the teeth, such as changes in the dentinenamel junction, crystalline structure, acid solubility of enamel, dentin elastic modulus, matrix metalloproteinases (MMPs), and enamel and dentin microhardness (Kielbassa et al., 2006; Silva et al., 2010; McGuire et al., 2014; Gonçalves et al., 2014; Reed et al., 2015). One of the most cited side effects of radiotherapy is radiation-related caries, which are a complex, multifactorial disease and an indirect effect of radiation
treatment that harms the tooth structure. It is a rapid, painless, and destructive form of tooth decay that can quickly lead to the amputation of crowns (Dreizen et al., 1977; Vissink et al., 2003). With the increasing life expectancy of the population worldwide, longevity of the teeth in the oral cavity, and increase in neoplastic diseases, it is essential that dentists be aware of changes in the oral cavity produced by the irradiation of head and neck cancer so that they can manage patients correctly both pre- and post-treatment. Thus, this study aimed to analyze superficial morphology, microhardness, and mineral content alterations of irradiated dental substrates at three different radiation doses.
MATERIAL AND METHODS Specimen preparation Thirty-four healthy human third molars were collected from the Tooth Bank of the School of Dentistry of the University of São Paulo, after approval from the Research Ethics Committee of the same institution (protocol 658.991). Teeth were cleaned and had their roots removed with a water-cooled diamond disc (Kg Sorensen, Barueri, São Paulo, Brazil). From each tooth, two sagittal slices were obtained with a low-speed diamond saw under water cooling (Isomet 1000, Buehler Ltd., Lake Bluff, IL, USA). Sagittal sections were prepared at the middle of the crown along the long axis of the tooth creating two slices: the mesial and distal slices. The mesial parts were used for microhardness and energy dispersive X-ray spectroscopy (EDS), and the three distal parts were used for the EDS control group. The remaining four teeth were cut along a transverse plane to obtain three samples for each depth of enamel and used for scanning electron microscopy (SEM) (Fluxogram 1).
Microhardness Microhardness refers to the testing of different materials hardness by applying small loads to it. The hardness of oral tissues is an essential aspect of their behavior, representing their resistance to masticatory loads, changes in the dentin-enamel junction, crystalline structure, acid solubility of enamel and dentin elastic modulus. For the microhardness test, samples were embedded in acrylic resin, and then a decreasing sequence of silicon carbide (SiC) paper (400, 600, 800, 1200, 2500, and 4000 grit) was used with the aid of a water-cooled polishing machine (Buehler Ltd., Lake Buff, IL, USA) to obtain a smooth and polished surface. The opposite surface of each specimen was also polished to obtain a parallel surface, which is essential for the correct measurement of microhardness. Between each polishing stage, samples were cleaned using distilled water in an ultrasonic cleaner (Digital Ultrasonic Cleaner CD4820, Kondortech, Sao Carlos, Brazil) for 8 minutes to remove any debris. The enamel depths were delineated with a permanent marker and ruler. Five indentations at each enamel depth were performed with a Knoop tip attached to a Microhardness Tester HMV-2000 (SHIMADZU Co., Tokyo, Japan) using a load of 50 g for 30 seconds. The indentations started 150 µm away from the dentin-enamel junction, and each indentation was spaced 100 µm apart. Microhardness values were measured
with
CAMS-WIN
software
(Newage
Testing
Instruments
Inc.,
Southampton, England). The microhardness test was performed before and after irradiation so that each sample could serve as its own control. After the first microhardness test analysis (control), the samples were randomly divided into three groups (n = 10) according to the radiation dose (Group 20
Gy, Group 40 Gy, and Group 70 Gy) and again submitted to the same microhardness analysis protocol. The results were separated according to depth. The quantitative analysis was performed by a two-way analysis of variance (ANOVA) (doses of 20, 40, and 70 Gy before and after radiotherapy) with one repeated measurement (time) and at a significance level of 5% (p ≥ 0,05).
Irradiation of the samples Samples were irradiated with a total and single application of 20, 40, or 70 Gy with X-rays from a linear accelerator (Mevatron MX2 6 mV; Siemens Healthcare, Erlangen, Germany) in the Department of Radiotherapy at Sírio-Libanês Hospital. This study used a 20-Gy dose in an attempt to simulate teeth that are situated in healthy tissues, but located near the target region, which can receive between 40 and 70-Gy. Samples were submerged in distilled water during radiotherapy. Moreover, all irradiations were done in the same container with the samples submerged in distilled water. An isocentric set-up was done, so that the geometric center of the container was aligned with the isocenter of the linear accelerator. This means that from whatever angle we point the gantry, the distance between the "radiation source" and the container's center would be 100cm. For a homogeneous radiation distribution for all samples, radiation was applied in two opposing fields parallel to the incident radiation.
Energy dispersive X-ray spectroscopy For the EDS test, 12 samples (n = 3) randomly selected from the microhardness analysis were used from the control and irradiated groups (20 Gy, 40
Gy, and 70 Gy). For the control group, the distal part of the tooth that was not used for SEM was selected. For samples used in the microhardness analysis, the acrylic resin was removed with the aid of a low-speed cutting machine (Labcut 1010 - Extec, Enfield, Connecticut, EUA). Samples were then fixed in 2.5% glutaraldehyde solution for 24 h at 4°C, washed in a solution of 0.1 M cacodylate, dehydrated in sequential ethanol baths for 10 min each (30%, 50%, 70%, 90%, 96%, and 100% for 20 min) and then transferred to a critical-point dryer (HMDS) for 30 min. Samples were mounted on aluminum stubs, properly identified, covered with a thin layer of carbon (MED 010, Balzers Union, Balzers, Liechtenstein), and observed by SEM to detect backscattered electrons (BSE) (LEO 435 VP, LEO Electron Microscopy Ltd., Cambridge, England). Quantifications were performed according to the depth used in the microhardness test. Three measurements of the enamel were performed at each depth (cervical, middle, and occlusal) and for each type of substrate (sound enamel and irradiated enamel). The duration in each area was 20 seconds, with the dead time in a 35% cobalt pattern.
Scanning electron microscopy For the SEM analysis, 12 samples were used from the distal parts of four different teeth. These samples were divided according to the depth of the tooth (occlusal, middle, and cervical) and then divided according to the radiation dose received (control, 20 Gy, 40 Gy, or 70 Gy). Samples were then fixed in 2.5% glutaraldehyde solution for 24 h at 4°C, washed in a solution of 0.1 M cacodylate, dehydrated in sequential baths of ethanol for 5 minutes each (30%, 50%, 70%, 90%, 96%, and 3 baths of 10 min each in 100%), and then transferred to a critical-point
dryer (HMDS) for 30 min. Samples were mounted on aluminum stubs, properly identified, covered with a thin layer of platinum, and observed using a Quanta 600 FEG SEM and the secondary electron detector (SE) (FEI, Hillsboro, OR, USA). Three images were obtained of each sample at three magnifications (1,000×, 5,000×, and 20,000×).
RESULTS Microhardness analysis The results of the ANOVA indicated a statistically significant difference only in microhardness of the cervical enamel for all irradiation doses and periods studied. The results of Tukey’s test and average hardness measurements indicated that in the cervical enamel, the average hardness was greater before radiation than after radiation (Table 1) (Figure 1).
Energy dispersive X-ray spectroscopy (EDX) Because Ca and P are the two main components of the enamel structure and are by far the most investigated elements in the majority of the studies, we detailed described the results for these two elements only. However, we further state that no significant difference was observed when investigating Na, Mg, Cl, and O values (data not shown). Hence, the results of the EDS test were analyzed descriptively, and indicated no significant difference in the Ca/P level for either substrate at any depth, independent of the irradiation dose and period (pre- and post-radiation) (Table 2) (Figure 1).
Scanning electron microscopy The images obtained show electron micrographs of the study groups based on the radiation dose (control, 20, 40, and 70 Gy) and region (occlusal, middle, and cervical regions). Three samples from each group were used to acquire the images and perform a qualitative analysis at three different magnifications (1,000×, 5,000×, and 20,000×) (Figure 1). Images of the control group indicated the presence of regular healthy enamel at all depths with the regular presence of prisms in the shape of a “door lock”, characteristic of enamel substrate as it can bee seen in the control group (Figure 2). Similar characteristics were also observed in all treated groups (Figure 3). However, at 20,000× magnification, a slight change in the enamel prisms was observed, which appeared shorter and rounded, and only in the 70 Gy group (Figure 4).
DISCUSSION Literature presents different ways to simulate in vitro radiotherapy with some of the authors using artificial saliva, distilled water or physiological saline solution (Gernhardt et al., 2001; Gonçalves et al., 2014; da Cunha et al., 2016). Since the main focus of this study is the direct effect of radiotherapy in dental tissue, distilled water was chose since it can provide an environment capable of radiolysis and no further interaction with the teeth. Radiotherapy consists of cumulative fractionated doses, about 2 Gy, that are delivered in daily sessions, with pauses at weekends, for up to 7 weeks. The standard treatment in patients with malignant tumors in the head and neck region results in a final accumulated dose of between 40 Gy and 70 Gy (Kielbassa et al., 2006; Naves et
al., 2012). It is well known that the effects of radiotherapy are cumulative and that the fractionation of radiation is based on "5Rs" (Repair, redistribution, reoxygenation, regeneration and radiosensitivity) (Harrington, Jankowska and Hingorani, 2007). Since there would not occur any of the 5Rs on the teeth in an in vitro study it was preferred to deliver the dose with a single fraction. In the present study, the enamel presented slight changes in microhardness, no change in mineral content, and a very slight morphological change within the radiotherapy protocols used. Only the cervical enamel showed decreased microhardness values after radiation at all doses used. This can be explained by the higher porosity and lower tissue thickness in this region, and thus its organic content may be more greatly influenced by the response to radiation. This is in agreement with Jansma et al., who concluded that the use of gamma radiation at therapeutic doses had no significant effect on chemical components, except in organic parts (Jansma et al., 1990). These results further support the necessity of evaluating changes in all regions of dental enamel, what is important to overcome limited reports previously described in literature that did not investigate all dental thirds. The EDX test confirmed that there was no modification regarding the concentrations of mineral content based on the values of Ca and P, regardless of the enamel location and dose of radiation, which is consistent with the literature (Walker et al., 1975; Jansma et al., 1990). In the clinic and in the literature, there is a consensus that radiation-related caries have a preference for the enamel’s cervical region. Thus, the changes found in the cervical third in this study are consistent with this clinical finding (Frank, Herdly and Philippe, 1965; Kielbassa et al., 2006; Soares et al., 2010).
For the enamel’s middle and occlusal thirds, this study found no significant change in microhardness values in the EDX or SEM analysis. Although several studies investigating the radiotherapy effects on human enamel have been published, literature is quite controversial regarding the hardness of irradiated enamel, possibly due to the high variability on methodological approaches used. Some authors are in agreement with our results. For example, Kielbassa et al., who studied the effects of radiotherapy on enamel microhardness and caries susceptibility in vitro and in situ, found no difference in microhardness values or caries between irradiated and nonirradiated enamel (Kielbassa et al., 1999; Kielbassa, Schenderh and Schulte-Mönting, 2000). Other previous publications, where structural changes were not found after radiotherapy, are in agreement with the results of the present study (Wiemann et al., 1972; Zach, 1976; Jansma et al., 2006). A more recent study showed that radiotherapy did not influence the bonding strength to enamel, irrespective of the adhesive or radiation dose used, showing that the chemical and prismatic structures of enamel probably were unaltered after radiotherapy (da Cunha et al., 2016). Other authors found an increase in enamel microhardness after radiotherapy. They explained the increase through changes in Hunter-Schreger band patterns making irradiated enamel more friable and susceptible to crack formation, and possible alterations in the organic matrix of the enamel decreasing the protein/mineral ratio and leading to an increase in microhardness and the elastic modulus because of the lower organic content of irradiated enamel (Gonçalves et al., 2014; Reed et al., 2015). It is important to highlight some differences in the methodology used by these authors if compared to the present study: Gonçalves et al. (2014) used a smaller sample (20 vs 34 teeth), did not evaluate different thirds of the teeth (only different
depths), used a different irradiation source, used a shorter distance between the source and the teeth, used a maximum cumulative dose of 60Gy and the authors used a score system to define the presence or absence of morphological changes. Reed et al. (2015) also used a much smaller sample size than ours (7 vs 34 teeth), did not evaluate different thirds of enamel, used a different radiation source and the distance between the teeth and the radiation source was not provided. The literature also contains studies in which ionizing radiation decreased enamel microhardness in all regions, and the authors explained that this occurs because of decarboxylation of the tissue. This means that the organic matrix interacts with apatite crystals, creates microcracks in hydroxyapatite minerals, and forms smaller crystallites, thus making the surface of the enamel tissue more rough (Kielbassa, Hellwig, Meyer-Lueckel, 2006; Lieshout and Bots, 2014; Qing et al., 2015; Liang et al., 2016). In the systematic review of Lieshout and Bots (2014) the authors clearly demonstrated the contradictory results described in literature regarding the
effects
of
radiation
in
the
enamel
and
dentin
components.
Kielbassa, Hellwig, Meyer-Lueckel (2006) observed significant differences in the demineralization of irradiated bovine incisors enamel, but not in their human molars sample. Qing et al. (2014) in their study used 13 teeth submitted to microhardness assay, the authors used a maximum of 60Gy irradiation and a different radiation source than ours, and they also immersed the samples in artificial saliva. The authors did not specify the exact anatomical location of the tests and the distance from the irradiation source to their teeth. Liang et al. (2016) obtained interesting results using a well designed study, but in contrast to ours they did not investigate different areas of enamel.
With the increase in the number of patients with head and neck cancer, and consequently, the increased use of radiotherapy, it is important to know the effects of ionizing radiation on the oral cavity and how its side effects can contribute to the evolution of radiation-related caries. It is known that interactions of the oral microflora change with decreased pH in the oral cavity, reduced salivary flow, and saliva composition, and these are important factors in the development of radiationrelated caries (Silva et al., 2010). In conclusion, the findings of this in vitro study demonstrate that radiotherapy was able to cause only slight morphological changes after 70Gy radiation and that microhardness decreased only in the cervical third of the enamel, but it did not interfere with enamel Ca and P content.
ACKNOWLEDGEMENTS The authors would like to express their gratitude to the School of Dentistry of the University of São Paulo and Sírio-Libanês Hospital for supporting this research.
CONFLICT OF INTEREST Disclosures: None
REFERENCES 1. da Cunha, S.R., Ramos, P.A., Haddad, C.M., da Silva, J.L., Fregnani, E.R. and Aranha, A.C. (2016). Effects of different radiation doses on the bond strengths of two different adhesive systems to enamel and dentin. Journal of Adhesive Dentistry, 18, 151-156.
2. Döbrossy, L. (2005). Epidemiology of head and neck cancer: magnitude of the problem. Cancer Metastasis Reviews, 24, 9-17. 3. Dreizen, S., Daly, T.E., Drane, J.B., Brown, L.R. (1977). Oral complications of cancer radiotherapy. Postgraduate Medical Journal , 61, 85-92. 4. El-Faramawy, N., Ameen, R., El-Haddad, K., El-Zainy, M. (2013). Effects of gamma radiation on hard dental tissues of albino rats: investigation by light microscopy. Radiation and Environmental Biophysics, 52, 375-387. 5. Frank, R.M., Herdly, J., Philippe, E. (1965). Acquired dental defects and salivary gland lesions after irradiation for carcinoma. Journal of the American Dental Association, 70, 868-883. 6. Gernhardt, C.R., Kielbassa, A.M., Hahn, P., Schaller, H.G. (2001). Tensile bond strengths of four different dentin adhesives on irradiated and nonirradiated human dentin in vitro. Journal of Oral Rehabilitation, 28, 814-820. 7. Gonçalves, L.M., Palma-Dibb, R.G., Paula-Silva, F.W., Oliveira, H.F., Nelson-Filho, P., Silva, L.A., Queiroz, A.M. (2014). Radiation therapy alters microhardness and microstructure of enamel and dentin of permanent human teeth. Journal of Dentistry, 42, 986-992. 8. Harrington, K., Jankowska, P., Hingorani, M. (2007). Molecular biology for the radiation oncologist: the 5Rs of radiobiology meet the hallmarks of cancer. Clinical Oncology, 19, 561-571. 9. Jansma, J., Buskes, J.A., Vissink, A., Mehta, D.M., Gravenmade, E.J. (1988). The effect of X-ray irradiation on the demineralization of bovine dental enamel. A constant composition study. Caries Research, 22, 199-203.
10. Jansma, J., Borggreven, J.M., Driessens, F.C., Gravenmade, E.J. (1990). Effect of X-ray irradiation on the permeability of bovine dental enamel. Caries Research, 24, 164-168. 11. Jemal, A., Bray, F., Center, M.M., Ferlay, J., Ward, E., Forman, D. (2011). Global cancer statistics. CA A Cancer Journal for Clinicians, 61, 69-90. Erratum in: CA A Cancer Journal for Clinicians, 61, 134. 12. Jham, B.C., da Silva-Freire, A.R. (2006). Oral complications of radiotherapy in the head and neck. Brazilian Journal of Otorhinolaryngology, 72, 704-708. 13. Kielbassa, A.M., Wrbas, K.T., Schulte-Mönting, J., Hellwig, E. (1999). Correlation of transversal microradiography and microhardness on in situinduced demineralization in irradiated and nonirradiated human dental enamel. Archives of Oral Biology, 44, 243-251. 14. Kielbassa,
A.M.,
Schendera,
A.,
Schulte-Mönting,
J.
(2000).
Microradiographic and microscopic studies on in situ induced initial caries in irradiated and nonirradiated dental enamel. Caries Research, 34, 41-47. 15. Kielbassa, A.M., Hinkelbein, W., Hellwig, E., Meyer-Lückel, H. (2006). Radiation-related damage to dentition. Lancet Oncology, 7, 326-335. 16. Kielbassa AM, Hellwig E, Meyer-Lueckel H. (2006). Effects of irradiation on in situ remineralization of human and bovine enamel demineralized in vitro. Caries Research, 40, 130-5. 17. Liang, X., Zhang, J.Y., Cheng, I.K., Li, J.Y. (2016). Effect of high energy Xray irradiation on the nano-mechanical properties of human enamel and dentine. Brazilian Oral Research, 30. 18. Lieshout, H.F., Bots, C.P. (2014). The effect of radiotherapy on dental hard tissue--a systematic review. Clinical Oral Investigation, 18, 17-24.
19. McGuire, J.D., Gorski, J.P., Dusevich, V., Wang, Y., Walker, M.P. (2014). Type IV collagen is a novel DEJ biomarker that is reduced by radiotherapy. Journal of Dental Research, 93, 1028-1034. 20. Naves, L.Z., Novais, V.R., Armstrong, S.R., Correr-Sobrinho, L., Soares, C.J. (2012). Effect of gamma radiation on bonding to human enamel and dentin. Supportive Care in Cancer, 20, 2873-2878. 21. Nutting, C.M., Morden, J.P., Harrington, K.J., Urbano, T.G., Bhide, S.A., Clark, C., Miles, E.A., Miah, A.B., Newbold, K., Tanay, M.A., Adab, F., Jefferies, S.J., Scrase, C., Yap, B.K., A’hern, R.P., Sydenham, M.A., Emson, M., Hall, E. (2001). PARSPORT Trial Management Group. Parotid-sparing intensity modulated versus conventional radiotherapy in head and neck cancer (PARSPORT): a phase 3 multicentre randomised controlled trial. The Lancet. Oncology ,12, 127-136. 22. Pioch, T., Golfels, D., Staehle, H.J. (1992). An experimental study of the stability of irradiated teeth in the region of the dentinoenamel junction. Endodontics and Dental Traumatology, 8, 241-244. 23. Qing, P., Huang, S., Gao, S., Qian, L., Yu, H. (2015). Effect of gamma irradiation on the wear behaviour of human tooth enamel. Scientific Reports, 5, 11568. 24. Reed, R., Xu, C., Liu, Y., Gorski, J.P., Wang, Y., Walker, M.P. (2015). Radiotherapy effect on nano-mechanical properties and chemical composition of enamel and dentine. Archives of Oral Biology, 60, 690-697. 25. Seikaly, H., Jha, N., Harris, J.R., Barnaby, P., Liu, R., Williams, D., McGaw, T., Rieger, J., Wolfaardt, J., Hanson, J. (2004). Long-term outcomes of
submandibular gland transfer for prevention of postradiation xerostomia. Archives of Otolaryngology and Head and Neck Surgery, 130, 956-961. 26. Silva, A.R., Alves, F.A., Berger, S.B., Giannini, M., Goes, M.F., Lopes, M.A. (2010). Radiation-related caries and early restoration failure in head and neck cancer patients. A polarized light microscopy and scanning electron microscopy study. Supportive Care in Cancer, 18, 83-87. 27. Soares, C.J., Castro, C.G., Neiva, N.A., Soares, P.V., Santos-Filho, P.C., Naves, L.Z., Pereira, P.N. (2010). Effect of gamma irradiation on ultimate tensile strength of enamel and dentin. Journal of Dental Research, 89, 159164. 28. Tauhata, L., Salati, I.P.A., Di Prinzio, R., Di Prinzio, A. (2014). Radioproteção e Dosimetria: Fundamentos – 10a rev. Rio de Janeiro, IRD/CNEN, 2014, 111-119. 29. Vissink, A., Jansma, J., Spijkervet, F.K., Burlage, F.R., Coppes, R.P. (2003). Oral sequelae of head and neck radiotherapy. Critical Reviews in Oral Biology and Medicine, 14, 199-212. 30. Walker, R. (1975). Direct effect of radiation on the solubility of human teeth in vitro. Journal of Dental Research , 54, 901. 31. Wiemann, M.R., Davis, M.K., Besic, F.C. (1972). Effects of x-radiation on enamel solubility. Journal of Dental Research, 51, 868. 32. Zach, G.A. (1976). X-ray diffraction and calcium-phosphorous analysis of irradiated human teeth. Journal of Dental Research, 55, 907-909.
Figures legends Figure 1. Graphical abstract of the main results obtained in this study. Radiotherapy caused significant microhardness changes only in the cervical third of the enamel no matter the dose used, but it did not interfere with its mineral content (based on Ca/P ratio) and caused morphological changes only when 70Gy was applied and limited to the cervical area of the irradiated teeth.
Figure 2. Electromicrographs of cervical region of Control Group with 1.000 (A), 5.000 (B) and 20.000x (C) magnificatgion.
Figure 3. Electromicrographs of cervical region of 20 Gy (A), 40 Gy (B) and 70 Gy (B) with 5.000x magnification.
Figure 4. Electromicrographs of cervical region of Control Group (A), 20 Gy (B), 40 Gy (C) and 70 Gy (D) with 20.000x magnification.
Table 1 – Microhardness (means and standard deviations – values in Knoops) for each region and dose.
Dose
20Gy 40Gy 70Gy
Cervical Enamel Mean(SD) Mean(SD) PrePostRadioteraphy Radioteraphy 298.04 294.36 (11.80) (6.68) 285 275.3 (19.45) (36) 291.98 283.68 (16.51) (21.64)
Medium Enamel Mean(SD) Mean(SD) PrePostRadioteraphy Radioteraphy 300.16 296.14 (11.19) (11.78) 291.96 293.04 (15.44) (17.53) 293.22 288.78 (13.24) (14.25)
Occlusal Enamel Mean(SD) Mean(SD) PrePostRadioteraphy Radioteraphy 281.82 281.76 (8.41) (11.12) 271.80 274.48 (20.21) (16.48) 289.82 279.90 (14.12) (9.10)
Table 2 - Mean concentrations by weight of the elements Ca and P, and Ca/P Index obtained with EDX for each region and dose (values in %mass).
Group and Region Control Cervical Medium Occlusal 20Gy Cervical Medium Occlusal 40Gy Cervical Medium Occlusal 70Gy Cervical Medium Occlusal
Ca 38.11 38.07 38.03 38.08 38.25 38.14 38.33 38.19 38.15 38.13 38.11 38.11
P 19.71 19.53 19.52 19.57 19.43 19.50 19.47 19.47 19.47 19.53 19.50 19.53
Ca/P index 1.93 1.95 1.95 1.95 1.97 1.96 1.97 1.97 1.96 1.95 1.95 1.95