Abstract

The aim of this study was to evaluate the influence of radiation therapy on root canal sealer push-out bond strength (BS) to dentin and the sealer/dentin interface after different final irrigation solutions (NaOCl, EDTA, and chitosan). Sixty-four maxillary canines were distributed into two groups (n=30): non-irradiated and irradiated with 60 Gy. Canals were prepared with Reciproc-R50 and subdivided (n=10) for final irrigation (NaOCl, EDTA, chitosan) and filled. Three dentin slices were obtained from each root third. The first slice of each third was selected for BS evaluation, and the failure mode was determined by stereomicroscopy. SEM analysis of the sealer-dentin interface was performed in the remaining slices. Two-way ANOVA and Tukey's tests (α=0.05) were used. Lower BS (P<0.0001) was obtained after irradiation (2.07±0.79 MPa), regardless of the final irrigation solution used. The NaOCl group (P<0.001) had the lowest BS in the irradiated (1.68±0.72) and non-irradiated (2.39±0.89) groups, whereas the EDTA (irradiated: 2.14±0.77 and non-irradiated: 3.92±1.54) and chitosan (irradiated: 2.37±0.73 and non-irradiated: 3.51±1.47) groups demonstrated a higher BS (P<0.05). The highest values were observed in the coronal third (3.17±1.38) when compared to the middle (2.74±1.36) and apical ones (2.09±0.97)(P<0.0001). There were more cohesive failures and more gaps in irradiated specimens, regardless of the final solution. The present study showed that radiation was associated with a decrease in BS, regardless of the final solution used, whereas chitosan increased BS in teeth subjected to radiation therapy.

Keywords
Radiotherapy; Dental Pulp Cavity; Chitosan

Introduction

Head and neck cancer ranks seventh among the most common neoplasms worldwide, with an annual incidence of approximately 640,000 new cases.¹1. Rettig EM, D’Souza G. Epidemiology of head and neck cancer. Surg Oncol Clin N Am. 2015 Jul;24(3):379-96. https://doi.org/10.1016/j.soc.2015.03.001
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Treatment for head and neck cancer depends on factors such as the type of cancer, staging, and location,⁵5. Bessell A, Glenny AM, Furness S, Clarkson JE, Oliver R, Conway DI, et al. Interventions for the treatment of oral and oropharyngeal cancers: surgical treatment. Cochrane Database Syst Rev. 2011 Sep;7(9):CD006205. https://doi.org/10.1002/14651858.CD006205.pub3
https://doi.org/10.1002/14651858.CD00620... and can be accomplished by surgery, radiation therapy, chemotherapy, or a combination thereof.⁵5. Bessell A, Glenny AM, Furness S, Clarkson JE, Oliver R, Conway DI, et al. Interventions for the treatment of oral and oropharyngeal cancers: surgical treatment. Cochrane Database Syst Rev. 2011 Sep;7(9):CD006205. https://doi.org/10.1002/14651858.CD006205.pub3
https://doi.org/10.1002/14651858.CD00620... Radiation therapy acts directly on the DNA by inhibiting cell division, or indirectly, by producing free radicals and resulting in cellular necrosis.⁵5. Bessell A, Glenny AM, Furness S, Clarkson JE, Oliver R, Conway DI, et al. Interventions for the treatment of oral and oropharyngeal cancers: surgical treatment. Cochrane Database Syst Rev. 2011 Sep;7(9):CD006205. https://doi.org/10.1002/14651858.CD006205.pub3
https://doi.org/10.1002/14651858.CD00620... Conventional radiation fractionation is the most commonly regimen used to minimize the side effects of radiation therapy on healthy tissues, favoring their repair.⁶6. Fletcher GH. Regaud lecture perspectives on the history of radiotherapy. Radiother Oncol. 1988 Aug;12(4):iii-v. https://doi.org/10.1016/0167-8140(88)90014-X
https://doi.org/10.1016/0167-8140(88)900... ^,77. Alam MS, Perween R, Siddiqui SA. Accelerated versus conventional radiation fractionation in early stage carcinoma larynx. Indian J Cancer. 2016 Jul-Sep;53(3):402-7. https://doi.org/10.4103/0019-509X.200677
https://doi.org/10.4103/0019-509X.200677... However, adjacent tissues are rarely preserved during head and neck radiation therapy,⁸8. Parahyba CJ, Ynoe Moraes F, Ramos PA, Haddad CM, Silva JL, Fregnani ER. Radiation dose distribution in the teeth, maxilla, and mandible of patients with oropharyngeal and nasopharyngeal tumors who were treated with intensity-modulated radiotherapy. Head Neck. 2016 Nov;38(11):1621-7. https://doi.org/10.1002/hed.24479
https://doi.org/10.1002/hed.24479... and this may vary according to the patient's age and to the dose and location of the ionizing radiation.⁵5. Bessell A, Glenny AM, Furness S, Clarkson JE, Oliver R, Conway DI, et al. Interventions for the treatment of oral and oropharyngeal cancers: surgical treatment. Cochrane Database Syst Rev. 2011 Sep;7(9):CD006205. https://doi.org/10.1002/14651858.CD006205.pub3
https://doi.org/10.1002/14651858.CD00620... ^,99. Khaw A, Logan R, Keefe D, Bartold M. Radiation-induced oral mucositis and periodontitis - proposal for an inter-relationship. Oral Dis. 2014 Apr;20(3):e7-18. https://doi.org/10.1111/odi.12199
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Some complications can be acute, such as pain and soft tissue sensitivity, qualitative and quantitative changes in saliva, loss of taste, fungal infections, and mucositis,¹⁰10. Gruber S, Dörr W. Tissue reactions to ionizing radiation-Oral mucosa. Mutat Res. 2016 Oct - Dec;770 Pt B:292-8. https://doi.org/10.1016/j.mrrev.2016.07.007
https://doi.org/10.1016/j.mrrev.2016.07.... whereas others develop later, such as xerostomia, mandibular osteoradionecrosis, muscular atrophy, trismus, radiation caries, changes in the bacterial microflora, and ultrastructural alterations in dentin.⁹9. Khaw A, Logan R, Keefe D, Bartold M. Radiation-induced oral mucositis and periodontitis - proposal for an inter-relationship. Oral Dis. 2014 Apr;20(3):e7-18. https://doi.org/10.1111/odi.12199
https://doi.org/10.1111/odi.12199... ^,1010. Gruber S, Dörr W. Tissue reactions to ionizing radiation-Oral mucosa. Mutat Res. 2016 Oct - Dec;770 Pt B:292-8. https://doi.org/10.1016/j.mrrev.2016.07.007
https://doi.org/10.1016/j.mrrev.2016.07.... ^,1111. Martins CV, Leoni GB, Oliveira HF, Arid J, Queiroz AM, Silva LAB et al. Influence of radiotherapy on the bond strength of different sealers to the root dentin. Int Endod J. 2016;49:1065-72. https://doi.org/10.1111/iej.12556
https://doi.org/10.1111/iej.12556...

Structural changes in enamel and dentin¹²12. Jervoe P. X-ray diffraction investigation on the effect of experimental and in situ radiation on mature human teeth. A preliminary report. Acta Odontol Scand. 1970 Nov;28(5):623-31. https://doi.org/10.3109/00016357009058587
https://doi.org/10.3109/0001635700905858... and direct damages to collagen,¹³13. Kochueva MV, Ignatieva NY, Zakharkina OL, Kamensky VA, Snopova IB, Kulabukhova KS et al.. Collagen structural changes in early radiation-induced damage. Biomed Invest. 2012;4:24-8.^,1414. Gonçalves LM, Palma-Dibb RG, Paula-Silva FW, Oliveira HF, Nelson-Filho P, Silva LA et al. Radiation therapy alters microhardness and microstructure of enamel and dentin of permanent human teeth. J Dent. 2014 Aug;42(8):986-92. https://doi.org/10.1016/j.jdent.2014.05.011
https://doi.org/10.1016/j.jdent.2014.05.... as well as reduction of dentin microhardness,¹⁴14. Gonçalves LM, Palma-Dibb RG, Paula-Silva FW, Oliveira HF, Nelson-Filho P, Silva LA et al. Radiation therapy alters microhardness and microstructure of enamel and dentin of permanent human teeth. J Dent. 2014 Aug;42(8):986-92. https://doi.org/10.1016/j.jdent.2014.05.011
https://doi.org/10.1016/j.jdent.2014.05.... favor the development and progression of radiation caries, which may lead to pulpal changes⁹9. Khaw A, Logan R, Keefe D, Bartold M. Radiation-induced oral mucositis and periodontitis - proposal for an inter-relationship. Oral Dis. 2014 Apr;20(3):e7-18. https://doi.org/10.1111/odi.12199
https://doi.org/10.1111/odi.12199... ^,1111. Martins CV, Leoni GB, Oliveira HF, Arid J, Queiroz AM, Silva LAB et al. Influence of radiotherapy on the bond strength of different sealers to the root dentin. Int Endod J. 2016;49:1065-72. https://doi.org/10.1111/iej.12556
https://doi.org/10.1111/iej.12556... ^,1515. Beech N, Robinson S, Porceddu S, Batstone M. Dental management of patients irradiated for head and neck cancer. Aust Dent J. 2014 Mar;59(1):20-8. https://doi.org/10.1111/adj.12134
https://doi.org/10.1111/adj.12134... and result in the need for endodontic treatment.¹¹11. Martins CV, Leoni GB, Oliveira HF, Arid J, Queiroz AM, Silva LAB et al. Influence of radiotherapy on the bond strength of different sealers to the root dentin. Int Endod J. 2016;49:1065-72. https://doi.org/10.1111/iej.12556
https://doi.org/10.1111/iej.12556...

According to Martins et al.,¹¹11. Martins CV, Leoni GB, Oliveira HF, Arid J, Queiroz AM, Silva LAB et al. Influence of radiotherapy on the bond strength of different sealers to the root dentin. Int Endod J. 2016;49:1065-72. https://doi.org/10.1111/iej.12556
https://doi.org/10.1111/iej.12556... radiation therapy performed before endodontic treatment reduces bond strength of the filling material to root dentin, regardless of the sealer type used, once it damages the dentin collagen fiber network.¹¹11. Martins CV, Leoni GB, Oliveira HF, Arid J, Queiroz AM, Silva LAB et al. Influence of radiotherapy on the bond strength of different sealers to the root dentin. Int Endod J. 2016;49:1065-72. https://doi.org/10.1111/iej.12556
https://doi.org/10.1111/iej.12556... ^,1313. Kochueva MV, Ignatieva NY, Zakharkina OL, Kamensky VA, Snopova IB, Kulabukhova KS et al.. Collagen structural changes in early radiation-induced damage. Biomed Invest. 2012;4:24-8.^,1414. Gonçalves LM, Palma-Dibb RG, Paula-Silva FW, Oliveira HF, Nelson-Filho P, Silva LA et al. Radiation therapy alters microhardness and microstructure of enamel and dentin of permanent human teeth. J Dent. 2014 Aug;42(8):986-92. https://doi.org/10.1016/j.jdent.2014.05.011
https://doi.org/10.1016/j.jdent.2014.05....

Recently, chitosan has been proposed as a final irrigation solution for the treatment of dentin surface and removal of the smear layer,¹⁶16. Silva PV, Guedes DF, Pécora JD, Cruz-Filho AM. Time-dependent effects of chitosan on dentin structures. Braz Dent J. 2012;23(4):357-61. https://doi.org/10.1590/S0103-64402012000400008
https://doi.org/10.1590/S0103-6440201200... ^,1717. Silva PV, Guedes DF, Nakadi FV, Pécora JD, Cruz-Filho AM. Chitosan: a new solution for removal of smear layer after root canal instrumentation. Int Endod J. 2013 Apr;46(4):332-8. https://doi.org/10.1111/j.1365-2591.2012.02119.x
https://doi.org/10.1111/j.1365-2591.2012... with the increase in collagen fiber degradation by collagenase,¹⁸18. Taravel MN, Domard A. Relation between the physicochemical characteristics of collagen and its interactions with chitosan: I. Biomaterials. 1993 Oct;14(12):930-8. https://doi.org/10.1016/0142-9612(93)90135-O
https://doi.org/10.1016/0142-9612(93)901... which may improve the long-term stability of exposed fibers inside the root canals during endodontic treatment.¹⁸18. Taravel MN, Domard A. Relation between the physicochemical characteristics of collagen and its interactions with chitosan: I. Biomaterials. 1993 Oct;14(12):930-8. https://doi.org/10.1016/0142-9612(93)90135-O
https://doi.org/10.1016/0142-9612(93)901... Therefore, considering radiation-induced changes in the collagen fiber network in the intertubular, peritubular, and intratubular dentin¹¹11. Martins CV, Leoni GB, Oliveira HF, Arid J, Queiroz AM, Silva LAB et al. Influence of radiotherapy on the bond strength of different sealers to the root dentin. Int Endod J. 2016;49:1065-72. https://doi.org/10.1111/iej.12556
https://doi.org/10.1111/iej.12556... and the ability of chitosan to stabilize the collagen structure, it is important to evaluate the behavior of different final irrigation solutions before root canal filling of teeth undergoing radiation therapy.

The aim of this study was to assess the push-out bond strength of epoxy resin-based sealer to intraradicular dentin after final irrigation with different solutions (NaOCl, EDTA, and chitosan), as well as the sealer/dentin interface in teeth subjected to fractionated radiation.

Methodology

Sample selection

After approval by the Research Ethics Committee of the Dental School of Ribeirão Preto, University of São Paulo, Brazil, protocol no. 53666016.0.0000.5419, 60 straight, single-rooted human maxillary canines with complete rhizogenesis, absence of calcifications and internal resorption, no previous endodontic treatment, as well as absence of metallic restorations,¹¹11. Martins CV, Leoni GB, Oliveira HF, Arid J, Queiroz AM, Silva LAB et al. Influence of radiotherapy on the bond strength of different sealers to the root dentin. Int Endod J. 2016;49:1065-72. https://doi.org/10.1111/iej.12556
https://doi.org/10.1111/iej.12556... with root length of at least 17 mm were selected from the endodontic laboratory collection. The teeth were kept in individual and labeled plastic vials with artificial saliva (pH 7.0, 37°C) and renewed daily.

Irradiation protocol

The teeth were randomly distributed into two groups (n = 30): one group without irradiation, and the other one was subjected to fractionated radiation with 6 MV X-rays. The teeth prepared to receive radiation were kept inside plastic vials with distilled water to obtain a uniform radiation dose (approximately 2.85 Gy/min).¹¹11. Martins CV, Leoni GB, Oliveira HF, Arid J, Queiroz AM, Silva LAB et al. Influence of radiotherapy on the bond strength of different sealers to the root dentin. Int Endod J. 2016;49:1065-72. https://doi.org/10.1111/iej.12556
https://doi.org/10.1111/iej.12556... ^,1414. Gonçalves LM, Palma-Dibb RG, Paula-Silva FW, Oliveira HF, Nelson-Filho P, Silva LA et al. Radiation therapy alters microhardness and microstructure of enamel and dentin of permanent human teeth. J Dent. 2014 Aug;42(8):986-92. https://doi.org/10.1016/j.jdent.2014.05.011
https://doi.org/10.1016/j.jdent.2014.05.... Treatment was done using a computer-assisted linear accelerator with 6 MV X-rays (RS 2000, RAD Source Technologies, Inc., Suwanee, USA) with energy of 200 kVp and 25 mA and a 0.3-mm standard copper filter. The cumulative radiation dose of 60 Gy was divided into 30 fractions (2 Gy/fraction) and delivered in 5 consecutive days/week, for 6 weeks. Between the irradiation cycles, the samples were stored in artificial saliva as previously described.

Root canal preparation and filling

Conventional endodontic cavities were made and the root canal was irrigated with 1% NaOCl (2 mL). The coronal thirds were serially prepared up to a 45.06 taper size with an LA Axxess bur (SybronEndo Corporation, Orange, USA). The working length was determined at 1.0 mm of the apical foramen.

Reciproc R50 (taper size 50.05) files were used in a reciprocating motion by the VDW Silver motor (VDW GmbH, Munich, Germany). The root canals were prepared using an in-and-out pecking motion (3 mm in amplitude and light apical pressure) and cleaned after three pecking motions, following the manufacturer's recommendations. The irrigation was performed with 2 mL of 1% NaOCl using a plastic syringe and a 30 G needle during each step.

After biomechanical preparation, the teeth were redistributed into three subgroups (n=10) according to the final irrigation solution used (5 mL): Subgroup A – 1% NaOCl (control), Subgroup B – 17% EDTA, and Subgroup C – 0.2% chitosan, for 5 min. Root canals were then flushed using 2 mL of distilled water and R50 absorbent paper cones (Dentsply Maillefer, Baillagues, Switzerland) were used for drying. After that, the canals were filled using the lateral condensation technique (R50 and R8 accessory cones) with AH Plus sealer (Dentsply, De Trey, Konstanz, Germany). Endodontic access was temporarily restored with a self-setting filling material (Coltosol, Coltène\Whaledent, S.A., Alstatten Switzerland) and kept at 95% humidity and 37 °C for a period of three times longer than the AH Plus sealer setting time (8 h).

Bond strength and SEM analysis

The roots were sectioned perpendicularly into 1-mm-thick slices with a water-cooled low-speed diamond disc (Isomet 1000; Buehler, Lake Forest, USA), obtaining three slices from each root third.

The first slice was used for the push-out test in a universal testing machine (Instron 2519-106; Instron, Canton, USA) at a crosshead speed of 0.5 mm/min⁻¹. The apical surface was placed towards the tip, making sure forces were applied from the apical to the coronal direction. Four-millimeter-long shafts with tip diameters of 0.4 mm, 0.6 mm, and 1.0 mm were used, respectively, for the apical, middle, and coronal root slices. A constant load was applied until displacement of the root filling material. The bond strength was determined in MPa by dividing the force needed to displace the filling material by the lateral area. The lateral area formula of tapered inverted cone (SL) was used to calculate the bonding area of the root canal filling material: SL = π(R + r)h, where π = 3.14, R is the mean radius of the coronal area of the slice in mm, r is the mean radius of the apical area of the slice in mm, and h is the height/thickness of the filling material.

Before the test, the height (h) of the slices was measured through a digital caliper (Digimess, Shiko Precision Gaging Ltd, China) and the perimeter and radius (major and minor) were measured by a stereo microscope (Leica, M165C, Leica Microsystems, Germany) using Las software v4.4 (Leica, M165C, Leica Microsystems, Germany) for the calculation of the adhered lateral area.

After the bond strength test, the slices were analyzed at 25× magnification to verify the failure mode, which was divided into five groups, as follows: (a) adhesive failure in the dentin – filling material dislodged from the dentin; (b) adhesive failure in the filling material – gutta percha dislodged from the sealer; (c) mixed, when both adhesive failures occurred; (d) dentin cohesive failure – dentin fractured; and (e) sealer cohesive failure – sealer fractured.

The second slice of each third was used for analysis of sealer penetrability into dentinal tubules according to the surface decalcification protocol (6 mol L⁻¹ of hydrochloric acid), followed by deproteinization in 2.5% NaOCl. For the evaluation of gaps, the third slice of each sample was dehydrated in an ascending ethanol series. Both analyses were performed on the coronal face of the slices (coronal, middle, and apical thirds). All slices were covered with gold-palladium and examined under a scanning electron microscope (EvoMa10, Carl Zeiss, Munich, Germany) at 75×, 100× and 500× magnifications.¹¹11. Martins CV, Leoni GB, Oliveira HF, Arid J, Queiroz AM, Silva LAB et al. Influence of radiotherapy on the bond strength of different sealers to the root dentin. Int Endod J. 2016;49:1065-72. https://doi.org/10.1111/iej.12556
https://doi.org/10.1111/iej.12556...

Statistical analysis

The data were subjected to normality tests (Shapiro-Wilk) and homogeneity of variance (Levene's test). Bond strength data were analyzed by two-way ANOVA with a split-plot design and post-hoc Tukey's test (α = 0.05) using the SAS 9.1 software (SAS, Cary, USA).

Results

Bond strength data are shown in Table 1.

There was a significant difference in irradiation (p < 0.0001), final irrigation solution (p < 0.0001), and root third (p < 0.0001). The interactions between irradiation and final irrigation solution influenced bond strength (p = 0.0444). However, there was no statistically significant difference in irradiation and root third (p = 0.0900), final irrigation solution and root third (p = 0.8413), and irradiation, final irrigation solution, and root third (p = 0.5660).

Irradiated teeth had lower bond strength values than non-irradiated teeth (p < 0.0001) (Table 1). Considering the interaction between irradiation and the final irrigation solution, Tukey's test (Table 2) showed that irradiation reduced bond strength in the final irrigation with NaOCl (p = 0.0191), EDTA (p < 0.0001), and chitosan (p = 0.0003). Regarding non-irradiated teeth, treatment with EDTA and chitosan showed the highest bond strength, with no statistical difference between them (p = 0.1758), and the NaOCl-treated teeth presented the lowest bond strength values (p < 0.05). For the irradiated teeth, the final irrigation with chitosan demonstrated higher bond strength than with NaOCl (p = 0.0211), and EDTA treatment presented similar results to the groups treated with chitosan (p = 0.4391) and NaOCl (p = 0.1115).

Regarding final irrigation protocols, Tukey's test showed that the use of EDTA and chitosan resulted in higher bond strength compared to the NaOCl group (p < 0.001), and that there was no statistical difference when compared to EDTA and chitosan (p = 0.9080) (Table 3).

The failure mode is presented in Table 4. For NaOCl, cohesive failures in the dentin increased after radiation at the cervical and middle thirds. For EDTA, there was an increase in adhesive failures in the dentin at all thirds after irradiation. For chitosan, there was an increase in adhesive failures in the filling material and a decrease in adhesive failures in the dentin at all thirds after irradiation.

The SEM analysis showed that, irrespective of the final irrigation solution used, irradiated specimens had a larger number of gaps at the dentin-filling material interface when compared to non-irradiated ones. Non-irradiated specimens treated with EDTA and chitosan showed greater homogeneity and adaptation of the filling material to the root dentin (Figure 1) when compared to non-irradiated teeth treated with NaOCl in all of the analyzed regions.

Moreover, the non-irradiated specimen treated with EDTA and chitosan showed few areas of short and interrupted resin tags, irregularly distributed and parallel to the peritubular dentin (Figure 2) when compared to irradiated specimens, which did not show resin tag formation. Regarding NaOCl-irrigated specimens, the presence of sealer tags was rarely observed, and when present, they were smaller, less numerous, and even more irregularly disposed compared to the EDTA and chitosan groups and were observed only in non-irradiated teeth.

Discussion

Although current protocols recommend fractionation of radiation for the repair of adjacent healthy tissues,⁶6. Fletcher GH. Regaud lecture perspectives on the history of radiotherapy. Radiother Oncol. 1988 Aug;12(4):iii-v. https://doi.org/10.1016/0167-8140(88)90014-X
https://doi.org/10.1016/0167-8140(88)900... ^,77. Alam MS, Perween R, Siddiqui SA. Accelerated versus conventional radiation fractionation in early stage carcinoma larynx. Indian J Cancer. 2016 Jul-Sep;53(3):402-7. https://doi.org/10.4103/0019-509X.200677
https://doi.org/10.4103/0019-509X.200677... acute and late adverse events have been commonly observed following radiation therapy.⁸8. Parahyba CJ, Ynoe Moraes F, Ramos PA, Haddad CM, Silva JL, Fregnani ER. Radiation dose distribution in the teeth, maxilla, and mandible of patients with oropharyngeal and nasopharyngeal tumors who were treated with intensity-modulated radiotherapy. Head Neck. 2016 Nov;38(11):1621-7. https://doi.org/10.1002/hed.24479
https://doi.org/10.1002/hed.24479... ^,99. Khaw A, Logan R, Keefe D, Bartold M. Radiation-induced oral mucositis and periodontitis - proposal for an inter-relationship. Oral Dis. 2014 Apr;20(3):e7-18. https://doi.org/10.1111/odi.12199
https://doi.org/10.1111/odi.12199... ^,1313. Kochueva MV, Ignatieva NY, Zakharkina OL, Kamensky VA, Snopova IB, Kulabukhova KS et al.. Collagen structural changes in early radiation-induced damage. Biomed Invest. 2012;4:24-8.

Among late adverse effects, radiation caries has been shown to be highly prevalent due to the increased survival of these patients and changes in the dentin ultrastructure, causing severe destruction of enamel and dentin, with pulp alterations in most cases, determining the need for endodontic treatment.¹⁹19. Shenoy VK, Shenoy KK, Rodrigues S, Shetty P. Management of oral health in patients irradiated for head and neck cancer: a review. Kathmandu Univ Med J (KUMJ). 2007 Jan-Mar;5(1):117-20. Therefore, it is important to evaluate different protocols for the endodontic treatment of teeth that have been subjected to radiation therapy.

The present study aimed to simulate the radiation doses used to treat cancer patients subjected to fractionated doses of 2 Gy for 5 consecutive days with 30 cycles/6 weeks, totaling 60 Gy.⁶6. Fletcher GH. Regaud lecture perspectives on the history of radiotherapy. Radiother Oncol. 1988 Aug;12(4):iii-v. https://doi.org/10.1016/0167-8140(88)90014-X
https://doi.org/10.1016/0167-8140(88)900... ^,77. Alam MS, Perween R, Siddiqui SA. Accelerated versus conventional radiation fractionation in early stage carcinoma larynx. Indian J Cancer. 2016 Jul-Sep;53(3):402-7. https://doi.org/10.4103/0019-509X.200677
https://doi.org/10.4103/0019-509X.200677... This protocol has been used to study the changes radiation therapy produces in the dental structure.¹¹11. Martins CV, Leoni GB, Oliveira HF, Arid J, Queiroz AM, Silva LAB et al. Influence of radiotherapy on the bond strength of different sealers to the root dentin. Int Endod J. 2016;49:1065-72. https://doi.org/10.1111/iej.12556
https://doi.org/10.1111/iej.12556... ^,2020. Aggarwal V. An in vitro evaluation of effect of ionizing radiotherapy on push-out strength of fiber posts under cyclic loading. J Endod. 2009 May;35(5):695-8. https://doi.org/10.1016/j.joen.2009.01.010
https://doi.org/10.1016/j.joen.2009.01.0... ^,2121. Soares CJ, Castro CG, Neiva NA, Soares PV, Santos-Filho PC, Naves LZ et al. Effect of gamma irradiation on ultimate tensile strength of enamel and dentin. J Dent Res. 2010 Feb;89(2):159-64. https://doi.org/10.1177/0022034509351251
https://doi.org/10.1177/0022034509351251...

Another important factor was the use of artificial saliva for tooth storage, which, although not exactly reproducing exactly the qualities of natural saliva, mainly in cases of irradiated patients with altered salivary flow, secretion, and composition, is still the option that most closely resembles the clinical conditions of natural saliva in patients not subjected to radiation therapy.¹¹11. Martins CV, Leoni GB, Oliveira HF, Arid J, Queiroz AM, Silva LAB et al. Influence of radiotherapy on the bond strength of different sealers to the root dentin. Int Endod J. 2016;49:1065-72. https://doi.org/10.1111/iej.12556
https://doi.org/10.1111/iej.12556... Note that the samples were kept in distilled water during irradiation, since immersion in artificial saliva could hamper the homogeneous distribution of irradiation due to its viscosity and high ion concentration.¹¹11. Martins CV, Leoni GB, Oliveira HF, Arid J, Queiroz AM, Silva LAB et al. Influence of radiotherapy on the bond strength of different sealers to the root dentin. Int Endod J. 2016;49:1065-72. https://doi.org/10.1111/iej.12556
https://doi.org/10.1111/iej.12556... ^,1414. Gonçalves LM, Palma-Dibb RG, Paula-Silva FW, Oliveira HF, Nelson-Filho P, Silva LA et al. Radiation therapy alters microhardness and microstructure of enamel and dentin of permanent human teeth. J Dent. 2014 Aug;42(8):986-92. https://doi.org/10.1016/j.jdent.2014.05.011
https://doi.org/10.1016/j.jdent.2014.05.... Furthermore, water is a major constituent of human tissue, thus the use of distilled water during radiation therapy can physically and chemically simulate the surrounding soft tissues by formation of free radicals.¹¹11. Martins CV, Leoni GB, Oliveira HF, Arid J, Queiroz AM, Silva LAB et al. Influence of radiotherapy on the bond strength of different sealers to the root dentin. Int Endod J. 2016;49:1065-72. https://doi.org/10.1111/iej.12556
https://doi.org/10.1111/iej.12556...

Teeth with amalgam restorations were excluded since, when in contact with irradiation, these restorations can increase the amount of secondary radiation,¹¹11. Martins CV, Leoni GB, Oliveira HF, Arid J, Queiroz AM, Silva LAB et al. Influence of radiotherapy on the bond strength of different sealers to the root dentin. Int Endod J. 2016;49:1065-72. https://doi.org/10.1111/iej.12556
https://doi.org/10.1111/iej.12556... ^,2222. Katsura K, Utsunomiya S, Abe E, Sakai H, Kushima N, Tanabe S et al. A study on a dental device for the prevention of mucosal dose enhancement caused by backscatter radiation from dental alloy during external beam radiotherapy. J Radiat Res (Tokyo). 2016 Nov;57(6):709-13. https://doi.org/10.1093/jrr/rrw092
https://doi.org/10.1093/jrr/rrw092... which could hamper the control of variables and the standardization of results.¹¹11. Martins CV, Leoni GB, Oliveira HF, Arid J, Queiroz AM, Silva LAB et al. Influence of radiotherapy on the bond strength of different sealers to the root dentin. Int Endod J. 2016;49:1065-72. https://doi.org/10.1111/iej.12556
https://doi.org/10.1111/iej.12556...

In this study, regardless of the final irrigation solution and root third, irradiated teeth had lower bond strength than non-irradiated ones, which is probably associated with changes in the dentin ultrastructure such as obliteration of dentinal tubules,¹⁴14. Gonçalves LM, Palma-Dibb RG, Paula-Silva FW, Oliveira HF, Nelson-Filho P, Silva LA et al. Radiation therapy alters microhardness and microstructure of enamel and dentin of permanent human teeth. J Dent. 2014 Aug;42(8):986-92. https://doi.org/10.1016/j.jdent.2014.05.011
https://doi.org/10.1016/j.jdent.2014.05.... ^,2323. Grötz KA, Duschner H, Kutzner J, Thelen M, Wagner W. [New evidence for the etiology of so-called radiation caries. Proof for directed radiogenic damage od the enamel-dentin junction]. Strahlenther Onkol. 1997 Dec;173(12):668-76. German. alterations in the intertubular, peritubular, and intratubular dentin,¹⁴14. Gonçalves LM, Palma-Dibb RG, Paula-Silva FW, Oliveira HF, Nelson-Filho P, Silva LA et al. Radiation therapy alters microhardness and microstructure of enamel and dentin of permanent human teeth. J Dent. 2014 Aug;42(8):986-92. https://doi.org/10.1016/j.jdent.2014.05.011
https://doi.org/10.1016/j.jdent.2014.05.... and with the fragmentation of the network of collagen fibers of the dentinal tissue and its deproteinization.⁹9. Khaw A, Logan R, Keefe D, Bartold M. Radiation-induced oral mucositis and periodontitis - proposal for an inter-relationship. Oral Dis. 2014 Apr;20(3):e7-18. https://doi.org/10.1111/odi.12199
https://doi.org/10.1111/odi.12199... ^,1313. Kochueva MV, Ignatieva NY, Zakharkina OL, Kamensky VA, Snopova IB, Kulabukhova KS et al.. Collagen structural changes in early radiation-induced damage. Biomed Invest. 2012;4:24-8.^,1414. Gonçalves LM, Palma-Dibb RG, Paula-Silva FW, Oliveira HF, Nelson-Filho P, Silva LA et al. Radiation therapy alters microhardness and microstructure of enamel and dentin of permanent human teeth. J Dent. 2014 Aug;42(8):986-92. https://doi.org/10.1016/j.jdent.2014.05.011
https://doi.org/10.1016/j.jdent.2014.05.... ^,2424. Liang X, Zhang JY, Cheng IK, Li JY. Effect of high energy X-ray irradiation on the nano-mechanical properties of human enamel and dentine. Braz Oral Res. 2016;30(1):1-9. https://doi.org/10.1590/1807-3107BOR-2016.vol30.0009
https://doi.org/10.1590/1807-3107BOR-201... It is suggested that head and neck radiation therapy may lead to alterations in the amide III group present in the collagen structure and result in disorganization of the secondary structure of the protein unit that forms the collagen fibers,²⁵25. Bet MR, Goissis G, Lacerda CA. Characterization of polyanionic collagen prepared by selective hydrolysis of asparagine and glutamine carboxyamide side chains. Biomacromolecules. 2001;2(4):1074-9. https://doi.org/10.1021/bm0001188
https://doi.org/10.1021/bm0001188... modifying the natural arrangement between mineral and organic contents of dentin, changing its physical and mechanical properties.

In irradiated teeth, final irrigation with 0.2% chitosan showed higher bond strength compared to teeth irrigated with 1% NaOCl and 17% EDTA, which were statistically similar. Although chitosan has a similar chelating action to that of EDTA¹⁶16. Silva PV, Guedes DF, Pécora JD, Cruz-Filho AM. Time-dependent effects of chitosan on dentin structures. Braz Dent J. 2012;23(4):357-61. https://doi.org/10.1590/S0103-64402012000400008
https://doi.org/10.1590/S0103-6440201200... , teeth undergoing radiation therapy with deproteinization and defragmentation of the network of collagen fibers,²⁶26. Del Carpio-Perochena A, Bramante CM, Duarte MA, Moura MR, Aouada FA, Kishen A. Chelating and antibacterial properties of chitosan nanoparticles on dentin. Restor Dent Endod. 2015 Aug;40(3):195-201. https://doi.org/10.5395/rde.2015.40.3.195
https://doi.org/10.5395/rde.2015.40.3.19... chitosan probably increased the resistance of collagen fibers to degradation by collagenase,²⁷27. Shrestha A, Friedman S, Kishen A. Photodynamically crosslinked and chitosan-incorporated dentin collagen. J Dent Res. 2011 Nov;90(11):1346-51. https://doi.org/10.1177/0022034511421928
https://doi.org/10.1177/0022034511421928... besides stabilizing the structure of collagen already compromised by irradiation.¹⁸18. Taravel MN, Domard A. Relation between the physicochemical characteristics of collagen and its interactions with chitosan: I. Biomaterials. 1993 Oct;14(12):930-8. https://doi.org/10.1016/0142-9612(93)90135-O
https://doi.org/10.1016/0142-9612(93)901...

However, it is known that the use of 1% NaOCl alone does not promote smear layer removal, preventing the contact between endodontic sealers and intertubular dentin, which may justify the decreased bond strength found in this study.²⁸28. Scelza MF, Pierro V, Scelza P, Pereira M. Effect of three different time periods of irrigation with EDTA-T, EDTA, and citric acid on smear layer removal. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2004 Oct;98(4):499-503. https://doi.org/10.1016/j.tripleo.2004.03.027
https://doi.org/10.1016/j.tripleo.2004.0...

Dentin is characterized as a heterogeneous substrate due to its constitution, which has about 70% of inorganic material, 20% of organic material, and 10% of water,²⁹29. Pashley DH, Carvalho RM. Dentine permeability and dentine adhesion. J Dent. 1997 Sep;25(5):355-72. https://doi.org/10.1016/S0300-5712(96)00057-7
https://doi.org/10.1016/S0300-5712(96)00... ^,3030. Haller B. Recent developments in dentin bonding. Am J Dent. 2000 Feb;13(1):44-50. wherein the organic material consists mainly of collagen fibers³¹31. ten Cate JM. Remineralization of caries lesions extending into dentin. J Dent Res. 2001 May;80(5):1407-11. https://doi.org/10.1177/00220345010800050401
https://doi.org/10.1177/0022034501080005... . According to Soares et al.,²¹21. Soares CJ, Castro CG, Neiva NA, Soares PV, Santos-Filho PC, Naves LZ et al. Effect of gamma irradiation on ultimate tensile strength of enamel and dentin. J Dent Res. 2010 Feb;89(2):159-64. https://doi.org/10.1177/0022034509351251
https://doi.org/10.1177/0022034509351251... due to the fragmentation of the collagen fiber network, radiation has been shown to be more damaging to organic than to inorganic components, which may add to the explanation of the bond strength reduction of the AH Plus sealer after irradiation, regardless of the final irrigation solution used. The literature shows that hybridization is the primary process during the adhesion of hydrophobic resin materials to dentin and, in this process, most of the adhesion is promoted by micromechanical fixation of the collagen matrix inside of the intertubular dentin.³²32. Pashley EL, Tao L, Mackert JR, Pashley DH. Comparison of in vivo vs. in vitro bonding of composite resin to the dentin of canine teeth. J Dent Res. 1988 Feb;67(2):467-70. https://doi.org/10.1177/00220345880670020601
https://doi.org/10.1177/0022034588067002... ^,3333. Schwartz RS. Adhesive dentistry and endodontics. Part 2: bonding in the root canal system-the promise and the problems: a review. J Endod. 2006 Dec;32(12):1125-34. https://doi.org/10.1016/j.joen.2006.08.003
https://doi.org/10.1016/j.joen.2006.08.0...

Qualitative SEM analysis also evidenced the presence of gaps at the dentin-filling material interface more expressively in specimens treated with 17% EDTA and 0.2% chitosan after irradiation, corroborating the decrease of bond strength and an increase in the prevalence of adhesive failures in dentin and in the filling material, respectively. The interface integrity is crucial for the sealing, minimizing the chances of recontamination, which could induce to failure of endodontic treatment.³⁴34. Tabassum S, Khan FR. Failure of endodontic treatment: the usual suspects. Eur J Dent. 2016 Jan-Mar;10(1):144-7. https://doi.org/10.4103/1305-7456.175682
https://doi.org/10.4103/1305-7456.175682... During the curing of resin materials, shrinkage stress increases and the root canal sealer tends to dislodge the sealer-dentin interface, forming gaps.³⁵35. Bergmans L, Moisiadis P, De Munck J, Van Meerbeek B, Lambrechts P. Effect of polymerization shrinkage on the sealing capacity of resin fillers for endodontic use. J Adhes Dent. 2005;7(4):321-9.

Besides the influence of irradiation on the reduction of resin tag formation, probably due to obliteration of dentinal tubules¹⁴14. Gonçalves LM, Palma-Dibb RG, Paula-Silva FW, Oliveira HF, Nelson-Filho P, Silva LA et al. Radiation therapy alters microhardness and microstructure of enamel and dentin of permanent human teeth. J Dent. 2014 Aug;42(8):986-92. https://doi.org/10.1016/j.jdent.2014.05.011
https://doi.org/10.1016/j.jdent.2014.05.... ^,2323. Grötz KA, Duschner H, Kutzner J, Thelen M, Wagner W. [New evidence for the etiology of so-called radiation caries. Proof for directed radiogenic damage od the enamel-dentin junction]. Strahlenther Onkol. 1997 Dec;173(12):668-76. German. and to alterations in the intertubular, peritubular, and intratubular dentin at 60 Gy cumulative doses, short, asymmetric and low-density resin tags were observed in SEM when 17% EDTA and 0.2% chitosan were used.

Cohesive failure was also observed, independently of the final irrigation solution and root third, which may have occurred due to the association of the action of biomechanical preparation system, as reciprocating counterclockwise and clockwise motion can create areas of tension that can generate dentinal cracks,³⁶ and dentin ultrastructure alterations in irradiated teeth such as deproteinization, microhardness reduction, alterations in its crystal structure, fragmentation of collagen fibers, and obliteration of dentinal tubules.¹²12. Jervoe P. X-ray diffraction investigation on the effect of experimental and in situ radiation on mature human teeth. A preliminary report. Acta Odontol Scand. 1970 Nov;28(5):623-31. https://doi.org/10.3109/00016357009058587
https://doi.org/10.3109/0001635700905858... ^,1313. Kochueva MV, Ignatieva NY, Zakharkina OL, Kamensky VA, Snopova IB, Kulabukhova KS et al.. Collagen structural changes in early radiation-induced damage. Biomed Invest. 2012;4:24-8.^,1414. Gonçalves LM, Palma-Dibb RG, Paula-Silva FW, Oliveira HF, Nelson-Filho P, Silva LA et al. Radiation therapy alters microhardness and microstructure of enamel and dentin of permanent human teeth. J Dent. 2014 Aug;42(8):986-92. https://doi.org/10.1016/j.jdent.2014.05.011
https://doi.org/10.1016/j.jdent.2014.05....

Considering that radiation therapy is associated with high caries prevalence, which may heighten the need for endodontic treatment, and that it is a commonly used treatment for head and neck cancer, the results presented herein open perspectives for new studies, such as demonstrating whether the use of filling materials is reproducible in different filling techniques, investigating more suitable techniques and materials, and developing new dentin surface treatment protocols to mitigate the harmful effects of irradiation on bond strength, consequently contributing for a better understanding and endodontic treatment plan for a more successful treatment of patients undergoing radiation therapy.

Conclusion

Radiation of teeth before root canal filling was correlated with lower push-out bond strength, regardless of the final irrigation solution used. In addition, the chitosan solution increased the bond strength of teeth subjected to radiation therapy when compared to teeth treated with NaOCl and EDTA.

Acknowledgement

This work was supported by FAPESP – São Paulo Research Foundation (2015/14880/8).

References

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