1http://dx.doi.org/10.20396/bjos.v18i0.8657265 Volume 18 2019 e191638 Original Article 1 Department of Restorative Dentistry, Faculty of Nova Esperança , PB, Brazil. 2 Department of Clinic Dentistry, Federal University of Pernambuco, PE, Brazil. 3 Department of Restorative Dentistry, Federal University of Paraíba, PB, Brazil. Corresponding author: Renally Bezerra Wanderley Lima Faculty of Nova Esperança Av. Frei Galvão, 12 58067-698, João Pessoa, PB, Brazil Phone: 55 19-982156352 E-mail: renallywanderley@gmail.com https://orcid.org/0000-0003-4477-7850 Conflict of Interest: The authors declare that they have no conflict of interest. Received: April 26, 2019 Accepted: June 30 2019 Impact of radiotherapy on the bond strength of different adhesive systems to human dentin: one-year in vitro evaluation Renally Bezerra Wanderley Lima1,*, Maria Luiza Pontual2, Raquel Venâncio Fernandes Dantas3, Sônia Saeger Meireles3, Ana Karina Maciel Andrade3, Rosângela Marques Duarte3 Aim: The aim of this study is to evaluate the effect of radiotherapy on the bond strength of resin-based composite restorations to dentin, performed either 24 h or 1 year before or after radiation. Methods: Ninety-six posterior teeth were randomly distributed into the following groups: IB (n = 16), irradiated teeth were restored 1 year after x-ray application; NB (n = 16), not irradiated teeth were stored for 1 year and then restored. IA (n = 32), teeth were restored and irradiated at 24 h or 1 year after the restoration. NA (n = 32), teeth were restored, not irradiated, and tested as IA. Eight samples from each group were randomly assigned to either the three-step or two-step etch-and rinse adhesive system procedure. The irradiated specimens were subjected to 60 Gy of x-ray radiation fractionally. The restored teeth were vertically sectioned, and 1-mm2 resin–dentin sticks were obtained and submitted to the microtensile bond strength test. The bond strength data were analyzed by two- way analysis of variance (ANOVA) followed by Tukey’s test (p < 0.05). Failure modes were examined by optical microscopy and scanning electron microscopy. Results: The IB group showed lower bond strength values compared to the NB group. The bond strength values between the adhesive systems were not statistically different. Conclusion: The application of radiation dose decreased the bond strength of the adhesive restorations to dentin when the bonding procedure was conducted 1 year after in vitro radiotherapy. Keywords: Dentin. Tensile strength. Radiotherapy. Head and neck neoplasms. https://orcid.org/0000-0003-4477-7850 2 Lima et al. Introduction Radiotherapy is the most common modality of treatment for malignant tumors in the head and neck regions. This treatment uses high-energy x-rays at doses of 40–60 Gy, and even low doses may cause changes in normal tissue located within the irradiation field, drastically diminishing the quality of life of irradiated patients1. Regarding radiation-induced damage to the orofacial tissues, severe alterations in these tissues have been documented, such as mucositis, candidosis, hyposaliva- tion, radiation caries, dysgeusia, trismus, and osteoradionecrosis2. Radiation caries is one of the principal consequences of radiotherapy that results in severe destruc- tion of human dentition3,4. According to some studies, the development of radiation caries is related to indirect or direct radiogenic damage to dental hard tissues. The indirect effects include sali- vary changes, hyposalivation, changes in the oral microbiota, limitations in performing adequate oral hygiene, and adoption of a soft diet due to swallowing difficulties4-7. With respect to the direct effects, there is a direct alteration in biological molecules, which appear to have a negative effect on the dentinoenamel junction, enamel, den- tin, and pulp components of teeth, increasing the severity of dentition breakdown7-13. Morphological alterations of the dentin structure such as degeneration of the odon- toblast processes and obliteration of the dentin tubules also have been observed14. In addition, previous studies have indicated the presence of x-rays-induced damage to collagen present in the main peptide chains of dentin15,16. The protocol for dental restorations in oral cancer patients is still under controversial discussion. Hence, the choice of the best restorative material for dental restorations in patients undergoing radiotherapy seems to be based on the clinical experience of the professional6. Recent studies have recommended the use of adhesive restorative techniques for the treatment of irradiated patients6,17-21. In these studies, irradiation treatment did not affect the bond strength of the adhesive restorations to the den- tin and enamel structure, when the restoration was carried out before radiotherapy. On the other hand, when the restoration protocol was performed after the application of radiation, lower bond strength values for irradiated teeth were observed compared to teeth that had not been irradiated19,21. However, there is limited information regard- ing the direct effects of x-rays on the bond strength of resin-based composite resto- rations to enamel and dentin as well as whether the restorative procedure should be carried out before or after irradiation. As we wanted to develop a restorative protocol based on adhesive materials for irra- diated patients and to determine whether the restorative procedure should be car- ried out before or after irradiation, the aim of this study was to evaluate the effect of radiotherapy on the bond strength of resin-based composite restorations to dentin, using a three-step etch-and-rinse and a two-step etch-and-rinse adhesive, performed either 24 h or 1 year before or 1 year after the radiation treatment. The null hypotheses tested in this study were as follows: 1) The bond strength of the resin-based compos- ite restorations to dentin would not be affected by radiotherapy either before or after restoration placement. 2) There would be no difference in the dentin bond strength 3 Lima et al. between the three-step etch-and-rinse and two-step etch-and-rinse adhesive systems for all study conditions. Materials and methods Ninety-six intact, non-carious, unrestored posterior human maxillary/mandibular molars extracted over the course of three months, were obtained under a protocol approved (0394/11) by the institutional review board of Federal University of Paraiba, Brazil. The teeth were stored in 0.2% thymol solution. Sample preparation All tooth roots were embedded using self-curing acrylic resin. The teeth were ran- domly distributed into the following groups: IB (n = 16), irradiated teeth were restored 1 year after x-ray application; NB (n = 16), not irradiated teeth were stored for 1 year and then restored; IA (n = 32), teeth were restored and irradiated at 24 h or 1 year after the restoration. NA (n = 32), teeth were restored, not irradiated, and tested as IA. Eight teeth from each subgroup were randomly assigned to one of two adhesive system protocols: three-step etch-and-rinse adhesive (Adper™Scotchbond MP Plus, 3M/ESPE, St. Paul, MN, USA - SC) and two-step etch-and-rinse adhesive (Adper™ Sin- gle Bond 2, 3M/ESPE, St. Paul, MN - SB). Compositions and batch numbers of each material are shown in Table 1. For restorative treatment after or before the application of x-rays, the occlusal enamel surface was removed using a diamond disc mounted in a low-speed laboratory cut- ting machine (Labcut 1010, Extec, Enfield, CT, USA) under cooling conditions. The superficial dentin was exposed and finished with 600-grit silicon carbide abrasive paper for 60 s in polishing machine (Politriz ERIOS – 27000, São Paulo, SP, Brazil), Table 1. Composition and batch numbers of materials used. Product (Batch number) Manufacturer Composition Manufacture’s instructions Adper™Scotchbond MP Plus Lot. N133000 3M/ESPE, St. Paul, MN, USA Primer: water, HEMA. Adhesive: Bis-GMA, HEMA, dimethacrylates, polyalkenoic acid copolymer, photoinitiator. Apply the phosphoric acid 37 % for 15 s, following rinse for 30 s. Apply 1 coat of primer and gently air dry for 5 s. Apply two consecutive coats of adhesive and applying a gentle stream of air for 10 s. Light-cured for 10 s. Adper™ Single Bond 2 Lot. N 30077 3M/ESPE, St. Paul, MN, USA Bis-GMA, HEMA, dimethacrylates, polyalkenoic acid copolymer, photoinitiator, water, ethanol Apply the phosphoric acid 37 % for 15 s, following rinse for 30 s. After apply two consecutive coats of adhesive and applying a gentle stream of air for 10 s. Light-cured for 10 s. 37% Phosphoric acid etching Gel FGM, Joinville, SC, BR Cobalt Aluminate Blue Spinel Apply the phosphoric acid 37 % for 15 s, following rinse for 30 s. Optilux Plus GNATUS GNATUS, Ribeirão Preto, SP, BR LED light cure Light-cured using a irradiance ³400 mW/cm2 4 Lima et al. and a flat dentin surface was obtained. The adhesives were applied according to the manufacturers’ instructions (Table 1). After the adhesion process, the resin block was built up using three layers of Z350 composite resin (3M/ESPE, St. Paul, MN, USA), resulting in a height of 4.5 mm. Each layer was light cured for 40 s with an intensity of 400 mW/cm2 (Optilux Plus GNATUS, Ribeirão Preto, São Paulo, Brazil). Radiotherapy The simulated radiotherapy was performed using a Primus K Linear Accelerator (Siemens Healthineers, USA) with an energy of 6 MeV, a source–surface distance of 100 cm, and a field size of 18 cm × 23 cm. The specimens were placed in individual containers, which were filled with distilled water above the resin blocks, in order to provide dose homogeneity. Radiation was applied perpendicular to the surface of the specimen, and a total dose of 60 Gy, in fractions, was delivered. All groups of speci- mens were stored in distilled water, changed daily, at 37 °C. Microtensile bond strength test For the bond strength test, the restored teeth were sectioned longitudinally in the mesio-distal and buccal-lingual directions across the bonded interface, using a slow-speed diamond saw (Isomet, 1000 Buehler Ltd., Lake Bluff, IL, USA) to obtain 15–30 resin–dentin sticks with a cross-sectional area of approximately 1 mm2 (±0.1 mm2). The resin–dentin bonded sticks were fixed to a testing jig with cyanoac- rylate glue (Super Bond Gel, Loctite Brazil Ltd.) and subjected to the tensile load at a crosshead speed of 0.5 mm/min until failure (Shimatzu, Kyoto, Japan). The microten- sile bond strength was expressed in MPa and derived by dividing the imposed force (N) at the time of fracture by the bond area (mm2). Failure mode analysis The fractured surfaces of all specimens were observed by using an optical micro- scope (XJM-400, KOZO, Nanjing, China) at a magnification of 100×. The fracture mode was classified as follows: (I) cohesive failure in the adhesive, (II) cohesive failure in the dentin, (III) cohesive failure in the hybrid layer, or (IV) mixed failure, cohesive failure in the adhesive and cohesive failure in the hybrid layer. Representative fractured sur- faces of each tested group exhibiting the most frequently observed failure mode were analyzed by scanning electron microscopy (JSM-5600, JEOL, Tokyo, Japan) operating at 15 kV and a working distance of 15 mm. Statistical analysis Bond strength data were submitted to two-way (adhesive system vs. moment of radiation) analysis of variance (ANOVA) followed by Tukey’s test at a significance level of p < 0.05. RESULTS ANOVA revealed that the material, radiation, and all possible interactions between the factors resulted in statistically significant differences (p < 0.005) for the two storage times (24 h and 1 year). There was no statistical difference in the bond strength values https://www.google.com.br/url?sa=t&rct=j&q=&esrc=s&source=web&cd=8&cad=rja&uact=8&ved=0ahUKEwiN9f3r65TRAhWJipAKHaBpCa4QFggyMAc&url=https%2525252525253A%2525252525252F%2525252525252Fusa.healthcare.siemens.com%2525252525252Fradiation-oncology%2525252525252Fupgrades-and-options-for-your-linac%2525252525252Ftreatment-data-management&usg=AFQjCNF6IIa6ZJeJaSHrx0q_eE166VaKEQ 5 Lima et al. between the control group (not irradiated) and irradiated group restored before radio- therapy, using both adhesive systems, after short- and long-term storage (24 h and 1 year) (Tables 2 and 3). When the adhesive restorations were carried out one year after radiation application, the irradiated group presented lower bond strength values compared to the control group (Table 4). There was no statistical difference in the bond strength values between the adhesive systems used for all study conditions. No pretesting failures were recorded for any group. Table 5 and 6 show the mode of failure. The predominance of cohesive failure in the adhesive was detected for the control and irradiated groups restored 24 h and 1 year before radiotherapy. On the other hand, mixed failures (cohesive failure in the adhe- sive and cohesive failure in the hybrid layer) predominated when the specimens were restored 1 year after radiotherapy (Fig. 3). Table 2. Means values of bond strength (MPa) and standard deviation of adhesive restorations performed before the radiation in 24 hours storage. Treatment group Adhesive system Single Bond Scotch Bond Irradiated 39,64 ± 4,9 Aa 38,28 ± 7,9 Aa Control (no irradiated) 41,67 ± 7,6 Aa 41,43 ± 10,06 Aa Groups identified with different upper case letter superscripts (analysis in rows) and lower case letters (analysis in columns) represent statistical significant differences (p<0.05). Table 3. Means values of bond strength (MPa) and standard deviation of adhesive restorations performed before the radiation in 1year storage. Treatment group Adhesive system Single Bond Scotch Bond Irradiated 39,65 ±5,44 Aa 38,15 ± 9,19 Aa Control (no irradiate) 40,67 ±5,67 Aa 40,34 ± 7,90 Aa Groups identified with different upper case letter superscripts (analysis in rows) and lower case letters (analysis in columns) represent statistical significant differences (p<0.05). Table 4. Means values of bond strength (MPa) and standard deviation of adhesive restorations performed after the radiation in 1year storage. Treatment group Adhesive systems Single Bond Scotch Bond Irradiated 35,39 ± 7,47 Ab 32,68 ± 7,45 Ab Control (no irradiated) 41,33 ±5,09 Aa 41,01 ± 7,63 Aa Groups identified with different upper case letter superscripts (analysis in rows) and lower case letters (analysis in columns) represent statistically significant differences (p<0.05). 6 Lima et al. DISCUSSION According to the results of this study, the bond strength of the adhesive systems tested to dentin decreased when the restorations were performed 1 year after radia- tion application. Moreover, no difference in the dentin bond strength between those adhesive systems was observed for all study conditions. Consequently, both hypoth- eses of this study were rejected. Therefore, the restorative treatment for oral cancer patients who have dental cavities should be carried out before initiating head and neck radiotherapy. because irradiation doses may modify human tissues structure, as enamel and dentin, impairing the formation of an adequate bond between adhesive treatment and tooth structure. The ionizing radiation used as a treatment for oral cancer patients has a short wave- length and a high energy, which may induce micro-morphological alterations in den- tin and enamel7,8,14,22,23. The results of this study showed that radiotherapy caused a (1) (3)(2) 15 kV x500 50 µm h a h 15 kV x500 50 µm15 kV x500 50 µm a Figure 1. SEM photo illustrating the mode of type I cohesive fracture in the adhesive (1). Type IV, fracture at the base of the hybrid layer (a) with filled dental tubules and resin and cohesive fracture in the hybrid layer (h) (2) and presence of filled (a) and unfilled (b) dental tubules (3). Table 5. Percentage (%) of specimens according to the fracture mode of restorations performed before radiation. Groups Failure mode (%) SB SC I II III IV I II III IV Control group/24h 68 7 5 20 50 10 30 10 Radiation group/24 h 60 5 10 25 65 5 10 20 Control group/ 1 year 60 4 18 18 55 7 18 20 Radiation group/ 1year 55 2 15 28 40 12 20 28 Table 6. Percentage (%) of specimens according to the fracture mode of restorations performed after radiation. Groups Failure mode (%) SB SC I II III IV I II III IV Control group/1 year 20 5 20 55 20 8 28 44 Radiation group/1 year 21 12 25 42 23 16 20 41 7 Lima et al. significant detrimental effect on the bond strength of adhesive systems to dentin when the adhesive restorations were carried out 1 year after radiation application to the teeth, using two- or three-step adhesives. This fact may be explained by a direct effect of high-dose radiation (60 Gy) on the dentin structure. It is well known that radiation reacts with water, forming hydrogen and hydrogen peroxide free radicals24. Dentin contains a considerable amount of water in its composition; therefore, x-rays act through the formation of free radicals, which may have a negative effect on the secondary and tertiary structures of dentin proteins, causing the loss of collagen fiber hydration and leaving the tissue dry and friable7,24. As a result of this process, some micro-morphological alterations in the dentin structure can occur, including collagen fiber fragmentation7,15 and obliteration of dentin tubules, which is preceded by degeneration of odontoblast processes14. This damage may impair formation of the hybrid layer, producing a permeable adhesive interface between the adhesive system and the irradiated dentin. Moreover, alteration of the structural organization of collagen may occur because some chemical bonds are broken by free radicals during radiation and reorganization of the chemical components may happen, thus altering the structure15. In this study, a self-adhesive system was not assessed and further investigation is required to evaluate if that adhesive system will produce a strong and durable adhesive interface with the irradiated dentin, since self-adhesive systems containing an acidic monomer, as MDP, is capable to bond chemically to dentin and enamel structure25. The results of this in vitro study corroborate with previous findings, which showed the lowest bond strength values to dentin when the adhesive restoration was carried out after radiotherapy19,21,26. However, in these studies, the adhesive procedure was performed immediately or 24 h after radiation therapy. In clinical conditions, dental bonding procedures are not performed immediately after finishing radiotherapy. Thus, in our study, the restorative procedure was carried out 1 year after the application of radiation to the teeth. This is the first study to evaluate the long-lasting effect of radi- ation on the dentin structure and its consequences on the bonding effectiveness of adhesive systems to irradiated dentin. A previous report has hypothesized that in vivo high-dose radiotherapy causes induction and activation of enzymes that degrade col- lagens over a period of months or years27. The effect of radiation (60 Gy) on the den- tin microstructure could be observed by the mode of failure for the irradiated group restored after radiotherapy (Table 6). The predominant fracture modes were mixed failures (cohesive failure in the adhesive and cohesive failure in the hybrid layer), which showed failure of the formation of a stable and strong hybrid layer (Figure 1). Regarding the group in which the restorative procedure was carried out before the radiation application, there were no significant differences for the bond strength val- ues between the irradiated and nonirradiated restored teeth after storage for 24 h or 1 year. This finding is in accordance with others studie.6,19,28 which allege that when hybridization is obtained prior to irradiation, the alterations in the substrate might not be great enough to affect the behavior of the pre-existing hybrid layer and to compro- mise the bonding effectiveness between dentin and the adhesive materials. For all groups in which the teeth were restored before radiation, the predominant failure mode was cohesive failure in the adhesive (Table 5). It is possible that radiation applied after 8 Lima et al. the restorative procedure did not influence the bond quality of the adhesive material to dentin; consequently, adequate bond strength values could be obtained. As observed in the present investigation, the application of x-rays had a harmful effect on the bond strength to human dentin when the adhesive restorations were placed 1 year after radiotherapy. Nevertheless, this scenario could be different in clinical prac- tice considering the dry mouth of patients, increased viscosity, decreased salivary pH, dietary changes, and deficiencies in oral hygiene during and after radiotherapy. There- fore, future research should be conducted to simulate intraoral conditions in order to validate the findings of this study. Within the limitations of the current study, it was concluded that the application of x-rays decreased the bond strength of the tested adhesive restorations to dentin when the bonding procedure was conducted 1 year after in vitro radiotherapy. In addition, the different adhesive systems used in this study (two- or three-step etch-and-rinse procedure) showed similar bond effectiveness to dentin, regardless of the time period after the restoration was introduced. Acknowledgements The work was supported Department of Restorative Dentistry, Federal University of Paraíba, State of Paraiba, Brazil. REFERENCES 1. Al-Nawas B, Al-Nawas K, Kunkel M, Grötz KA. Quantifying radioxerostomia: salivary flow rate, examiner’s score, and quality of life questionnaire. Strahlenther Onkol. 2006 Jun;182(6):336-41. 2. Sulaiman F, Huryn JM, Zlotolow IM. 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