RESEARCH REPORTS

Biomaterials & Bioengineering

C.J. Soares1*, C.G. Castro1, N.A. Neiva1, P.V. Soares1, P.C.F. Santos-Filho1, L.Z. Naves1, and P.N.R. Pereira2
1 Biomechanics Group, School of Dentistry, Federal University of Uberlndia, Dept. of Operative Dentistry and Dental Materials, Av. Par 1720, Bloco 2B, Sala 2B-24, Campus Umuarama, Uberlndia, MG 38400-902, Brazil; and 2Department of Operative Dentistry, University of North Carolina, Chapel Hill, NC, USA; *corresponding author, carlosjsoares@umuarama.ufu.br

Effect of Gamma Irradiation on Ultimate Tensile Strength of Enamel and Dentin

INTRODUCTION
ndividuals diagnosed with head and neck cancer may be treated surgically (Hong et al., 2001) by radiotherapy (Lyons, 2006; Lazarus et al., 2007), by chemotherapy (Lyons, 2006; Lazarus et al., 2007), or by a combination of these treatments. Despite the advantage of preserving tissue structure, radiotherapy causes adverse changes in the oral cavity (Spetch, 2002). Several studies (Silverman, 1999; Craddock, 2006) have investigated the causes of radiation caries; however, little is known about the structural alterations occurring in the dentin and enamel after irradiation (Frnzel et al., 2006). Enamel is composed of 1-2% organic material and 3-4% water by weight, and 92-96% of inorganic matter organized in prisms, whose orientation determines enamels anisotropic behavior and influences its mechanical properties (Urabe et al., 2000; Giannini et al., 2004). Dentin is a hydrated biological composite composed of 70% inorganic material, 18% organic matrix, and 12% water (wt%), with various properties and structural components, depending on location (Mjr, 1972), which confer directional properties (anisotropy) on the tissue and have profound effects on its tensile strength (Carvalho et al., 2001; Giannini et al., 2004). It has been observed (Frnzel et al., 2006) that, after irradiation, the mechanical properties of teeth are altered. While several studies have measured the ultimate tensile strength (UTS) of root and coronal dentin or of enamel (Lertchirakarn et al., 2001; Giannini et al., 2004; Miguez et al., 2004), no systematic study has been carried out analyzing the effect of gamma irradiation therapy on the UTS of enamel and dentin in relation to prism orientation, dentin tubule orientation, and location. The null hypothesis to be tested in this study is that gamma irradiation therapy has no effect on the UTS of dental tissues, despite tubule or prism orientation and dentin location.

J Dent Res 89(2):159-164, 2010

ABSTRACT
The effect of gamma irradiation therapy on the ultimate tensile strength (UTS) of enamel and dentin in relation to prism orientation, dentin tubule orientation, and location is unknown. It was hypothesized that tubule and prism orientation, location, and irradiation have an effect on the UTS of dental structures. Forty human third molars were used, half of which were subjected to 60 Gy of gamma irradiation, in daily increments of 2 Gy. The specimens were evaluated by microtensile testing. Results showed that irradiation treatment significantly decreased the UTS of coronal and radicular dentin and of enamel, regardless of tubule or prism orientation. With or without irradiation, enamel was significantly stronger when tested parallel to its prismatic orientation. Coronal and radicular dentin of non-irradiated specimens presented significantly higher UTS when tested perpendicularly to tubule orientation. However, when the teeth were irradiated, the influence of tubule orientation disappeared, demonstrating that irradiation is more harmful to organic components.

KEY WORDS: ultimate tensile strength, irradiation, tubule orientation, prism orientation; dentin, enamel.

MATERIALS & METHODS

Forty sound human third molars free of caries, from individuals 18 to 23 yrs old, were collected with informed donor consent. The teeth were obtained under a protocol (248/05) analyzed and approved by the Ethical Committee of the Federal University of Uberlndia, MG, Brazil. They were stored in a solution of 0.2% thymol for no longer than 1 mo after extraction, then cleaned of gross debris and placed in distilled water for 24 hrs before the beginning of the experiment. The teeth were divided into 2 groups: an irradiated group (Ir) (number of specimens, N = 15) and a non-irradiated group (NIr) (N = 15). The Ir group received 60 Gy of gamma radiation in a 60Cobalt irradiation unit (Theratron Phoenix 60Cobalt Radiotherapy Treatment Unit - Theratronics International, Ltd., Atomic Energy of Canada, Ltd., AECL Medical, Ottawa, ON, Canada), with exposure to daily increments of 2 Gy, 5 days a wk. The dose was defined on the radiotherapy unit panel that self-measures the radiation level emitted. Both groups of samples were stored in distilled water changed daily.

DOI: 10.1177/0022034509351251 Received July 17, 2008; Last revision April 15, 2009; Accepted July 23, 2009

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and the cross-sectioned area at the site of fracture was measured with a digital caliper (S235, Sylvac, Crissier, Switzerland) to the nearest 0.01 mm. Mean tensile strength values were expressed in MPa, and data were analyzed by two-way ANOVA with tensile strength as the dependent variable, and radiotherapy and orientation of the dental structure as factors, followed by the Tukey test (significance, p < 0.05). The specimens were allowed to air-dry overnight, sputtercoated with gold (MED 010, Balzers, Balzers, Leichtenstein), and examined by scanning electron microscopy (LEO 435 VP, Carl Zeiss, Jena, Germany). Representative areas of tested sites were photographed at 2500-3000X magnification.

RESULTS
Figure 1. Schematic representation of specimen preparation. (A) Enamel parallel to the prism orientation. (B) Enamel stressed perpendicular to enamel prisms. (C) Coronal dentin stressed perpendicular to tubule orientation. (D) Coronal dentin tested parallel to tubule orientation. (E) Orientation for testing radicular dentin parallel to the long axis of the tubules. (F) Radicular dentin stressed perpendicular to tubule orientation.

Influence of Tubule and Prism Orientation and Irradiation on UTS of Enamel and Dentin
Two-way ANOVA revealed that there were significant differences between the UTS of the dental structures (p = 0.001) and the irradiation (p = 0.003), but not for both factors (p = 0.154). The Table shows the means of UTS for all groups. The direction of the tubules and prisms in the specimens tested, either parallel or perpendicular, was confirmed by lowmagnification scanning electron microscopy. The presence of irradiation significantly decreased the UTS of coronal and radicular dentin and of enamel, regardless of tubule or prism orientation. Enamel was significantly stronger when tested parallel to its prismatic orientation, regardless of the presence of irradiation. Non-irradiated dentin specimens, coronal and radicular, presented significantly higher UTS when tested perpendicularly to tubule orientation. When the dentin substrate was subjected to irradiation, tubule orientation had no influence on UTS.

The intact occlusal enamel surface was etched with 37% phosphoric acid (Dentsply Caulk, Milford, DE, USA) for 30 sec, rinsed, and bonded with Adper Single Bond 2 (3M-ESPE, St. Paul, MN, USA). A resin composite block (5 mm high) was built up in 3 layers with TPH Spectrum resin composite (Dentsply Caulk), to form an extension of the crown to facilitate further slicing and testing (Giannini et al., 2004). Each increment was light-polymerized for 20 sec, and the specimens were stored in distilled water at 37C for 24 hrs. Roots and crowns were then serially sectioned vertically in a buccal-lingual direction (Fig. 1) to obtain several slices of approximately 1.0 mm thick by means of a low-speed diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) under water cooling. Six slices 1.0 0.16 mm in thickness were obtained for each root and crown portion and were further trimmed to an hourglass shape that produced a cross-sectional testing area of 1.0 0.1 mm2. The samples of each tooth were randomly assigned to one of 6 groups (number of slices per group = 20). The slices were trimmed to an hourglass shape with a superfine diamond bur #1090 (KG Sorensen, Barueri, So Paulo, Brazil) under airwater irrigation, reducing enamel to a parallel positioning of prisms (Fig. 1A), enamel transverse to the orientation of prisms (Fig. 1B), coronal dentin transverse to the orientation of tubules (Fig. 1C), coronal dentin parallel to the orientation of tubules (Fig. 1D), root dentin parallel to the orientation of tubules (Fig. 1E), and coronal dentin transverse to the orientation of tubules (Fig. 1F). Each specimen was fixed to the grips of a microtensile testing device with cyanoacrylate glue (Loctite Super Bonder, Henkel Loctite Corporation, Munich, Germany) and subjected to tensile testing at 0.5 mm/min in a testing machine (EMIC 2000 DL, So Jos dos Pinhais, PR, Brazil) until failure. After fracture, the specimen was removed from the testing apparatus,

Appearance of Fracture Surfaces

The fractured site of non-irradiated enamel tested transversely to its prismatic orientation showed a preferential plane of fracture (Fig. 2A), with less oblique fractures of the prisms. The fractured site of non-irradiated enamel tested parallel to its prismatic orientation (Fig. 2B) presented a cone-shaped structures. Since fracture did not occur in a single plane, these structures appeared to be protruding from the surface. Fractured irradiated enamel tested transversely to its prismatic orientation showed predominantly oblique fracture orientation (Fig. 3A), tending to cross to the inter-prismatic region. The fracture of non-irradiated enamel tested transversely to its prismatic orientation presented a fractured surface with a melted appearance (Fig. 3A). The surfaces of irradiated samples stressed parallel to prisms showed more irregular surfaces involving greater interprismatic change (Fig. 3B), demonstrating alteration of interprismatic areas. Non-irradiated crown dentin (Fig. 2C) fractured transversely to tubules showed regular planes similar to non-irradiated root dentin (Fig. 2E). In these samples, intertubular dentin was smooth, showing collagen fibrils oriented transversely to the tubule direction (Figs. 2C, 2E). The plane of fracture passed

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Table. Ultimate Tensile Strength of Human Dentin in Crown vs. Root and Enamel Influenced by Gamma Irradiation Ultimate Tensile Strength Values (MPa) Parallel Orientation Tooth Structure Enamel Crown dentin Root dentin Non-irradiated 40.1 12.4 54.2 19.5 49.8 13.9
Aa Ab Ab

Perpendicular Orientation Irradiated Non-irradiated

Ba Ba Ba

Irradiated 10.9 3.9 Bb 51.3 16.9 Ba 43.7 15.4 Ba

29.4 7.9 39.8 13.4 30.2 12.0

17.6 5.7 86.5 20.2 64.7 14.7

Ab Aa Aa

Values are adjusted means SD. Different superscript letters indicate a statistically significant difference by the Tukey test (p < 0.05). Capital letters represent comparison of irradiation factors for each structural orientation. Lower-case letters represent structural orientation comparison (parallel or perpendicular) for each tooth structure in the same irradiation pattern (non-irradiated or irradiated). Number of slices = 20 for all groups.

approximately through the central axis of tubules and tangentially through others (Figs. 2C, 2E). Crown dentin fractured parallel to tubule orientation (Fig. 2D) was more irregular than root dentin (Fig. 2F), with peritubular dentin protruding from the surface as a distinct structure (Fig. 2D). Samples of irradiated crown dentin fractured transversely to tubule orientation showed the same plane of fracture, but with greater micro-cracks present in peritubular dentin (Figs. 3C, 3E). Irradiated dentin fractured parallel to tubule orientation showed more inhomogeneous fracture planes (Figs. 3D, 3F), with great numbers of micro-cracks in peritubular dentin which was less distinctly discerned (Fig. 3D).

DISCUSSION
The null hypothesis was rejected, since gamma radiation influenced the UTS of enamel in relation to prism and dentin tubule orientation. Dentin presented different character- Figure 2. Fracture pattern in sites of non-irradiated samples of stressed perpendicular enamel (A). Enamel tested parallel to prism orientation (B). Coronal dentin stressed perpendicular to tubule orientation (C). Coronal dentin istics after radiotherapy tested parallel to tubule orientation (D). Radicular dentin tested perpendicular to tubule orientation (E). Radicular treatment when compared dentin tested parallel to tubule orientation (F). Original magnification: 2500-3000X. with non-irradiated dentin, while the prism orientation maintained the in-fluence on UTS mean values in found that the UTS of dental structures varied widely accordirradiated ena-mel. While the structured biological hard tising to intra-tooth location (Staninec et al., 2002; Giannini sues presented anisotropic characteristics, other studies have et al., 2004). It has been observed (Giannini et al., 2004) that

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significantly higher UTS for enamel when tested under these conditions (Giannini et al., 2004). Several factors might influence the results of the microtensile test. In this study, all sample surfaces were fixed with glue for the testing (Soares et al., 2008). We stored the samples in thymol, because it has been shown (Giannini et al., 2004) that this procedure does not affect strength measurements. The cross-head speed was defined at 0.5 mm/min, because it is known to produce adequate stress/ strain distribution during load application. The protocol used for radiation therapy was the same as that normally used for the treatment of patients with head and neck cancer; the incremental fraction dose was important to simulate the same dose that patients receive daily. During radiation, teeth were stored in distilled water; studies should be conducted to clarify the effects of storage media during this period. Since the protein phase, Figure 3. Fracture pattern in sites of irradiated samples of stressed perpendicular enamel (A). Enamel tested parallel to prism orientation (B). Coronal dentin stressed perpendicular to tubule orientation (C). Coronal dentin which is concentrated in tested parallel to tubule orientation (D). Radicular dentin tested perpendicular to tubule orientation (E). Radicular the interprismatic region, is dentin tested parallel to tubule orientation (F). Parallel and perpendicular fractured surface of enamel presented more influenced by radioplasticized and cone-shaped characteristics (A,B). At higher magnification, typical microcracks along intertutherapy (Baker, 1982), the bular dentin stressed parallel (D) and perpendicular (E) can be noted. Original magnification: 2500-3000X. UTS was less in the perpendicular prismatic plane than when enamel was tested transversely to the orientation of in the parallel plane. We identified a greater degree of irreguprisms, fractures occurred preferentially along the interprislarity in the fracture pattern of irradiated than non-irradimatic substance, while, when it was tested parallel to its prisated enamel. This can possibly be explained by a matic orientation, fractures occurred obliquely to its long disarrangement of the crystalline portion of enamel (Jansma axis, as occurred in the present study. These observations et al., 1988), which represents the restructuring of the chemiclearly confirm the brittle and anisotropic behavior of enamel cal bonds into the mineral components that alter the crystal(Rasmussen et al., 1976; Hassan et al., 1981; Xu et al., 1998). line organization and alterations of protein interprismatic The weaker interprismatic substance was aligned perpendiculinks (Baker, 1982), which resulted in higher prism cleavage lar to the load, so the tension rapidly propagated across the independent of stress orientation. specimen, causing it to fail under a lower load. The enamel Previous results indicated that the UTS of dentin varies signififailure occurred by separation of prisms along the interpriscantly and is dependent on intra-tooth location (Wang, 2005) and matic substance; only a few prisms were obliquely cleaved depth (Konishi et al., 2002; Giannini et al., 2004). The present (Giannini et al., 2004). On enamel stressed parallel to its prisstudy yielded higher UTS values when tensile force was directed matic orientation, the stress concentrated on the stronger perpendicular to tubule orientation. It has been reported (Miguez prismatic units, and the fracture required all prisms to be et al., 2004) that the majority of the fibrils run either perpencleaved before catastrophic failure occurred, resulting in dicular or oblique to the tubule direction, so tensile force applied to

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collagen fibrils longitudinally results in higher UTS values than that applied perpendicularly. The results are consistent that fiber orientations of crown and root dentin, which involve the tubules perpendicularly in the long axis. The mechanical properties of dentin are defined partly by its organic components (Inoue et al., 2003; Miguez et al., 2004), so when dentin is stressed perpendicular to tubule orientation, this substrate is really stressed parallel to fiber orientation, which, since the fibers are stretched, results in increased load to failure. In 1975, Walker affirmed that therapeutic irradiation did not significantly affect the susceptibility of teeth to fracture in vivo but possibly may increase tooth fragility caused by protein degradation. It was considered that there are two important factors that promote the destruction of enamel and dentin: radiation-associated destruction and radioxerostomia (Frnzel et al., 2006). The interaction of the organic matrix with apatite crystals results from the electrostatic binding of collagen carboxylate sidechains and surface mineral phosphate groups via calcium ions. The loss of acidic phosphate groups through the decarboxylation side-chain, promoted by radiation, precedes the formation of new calcium ion bridge phosphate groups. Moreover, the mineralorganic interaction is reduced, and the development of carbon dioxide may induce microcracks in the hydroxyapatite mineral, resulting in a roughened surface (Hubner et al., 2005; Frnzel et al., 2006). While the mineral concentration is higher in peritubular dentin, the microcracks are concentrated in this area, reducing UTS values in coronal and root dentin. Additionally, the denaturation of the organic matrix caused by radiolysis would reduce the physical anchorage between enamel and dentin and the inner stability of dentin, since it contains collagen fibers as well (Pioch et al., 1992). This tended to be more evident in this study, because, initially, the orientation of dentin tubules influenced UTS, but when the teeth were irradiated, the effects of tubule orientation on UTS disappeared, demonstrating that irradiation was more damaging to organic components, mainly the collagen fibers. Radioxerostomia also accompanies tooth destruction (Frnzel et al., 2006). To avoid this, it is necessary that one use a method that can reduce tissue damage, such as a radio-protector (Hey et al., 2009). The irradiation effect on cross-linked collagen, and over the total integration or stability of collagen, in the testing of a new product that has been used to establish collagen structure (Salz et al., 2005; Bedran-Russo et al., 2007), should be tested in association with irradiation therapy. The stabilization of dentin collagen with biocompatible cross-linking agents may be of clinical importance to improve the UTS of dentin (Salz et al., 2005; Bedran-Russo et al., 2007), and it is necessary to study different methods using chlorhexidine (Carrilho et al., 2007) or other natural cross-linking agents (Bedran-Russo et al., 2007) and mechanisms to control the demineralization that occurs in enamel with the use of fluoride solutions (Ccahuana-Vsquez et al., 2007; Salar et al., 2007). Sealants applied to dentin and enamel (Gernhardt et al., 2007) should be tested during and between irradiation sessions, in an attempt to reduce this irradiation interference and increase the quality of life of persons undergoing irradiation therapy for head and neck cancer. Within the limitations of an in situ study, it can be concluded that irradiation

therapy significantly decreases the UTS of coronal and radicular dentin and enamel, regardless of tubule or prism orientation. However, this study also showed that the damage is potentially greatest for organic components.

ACKNOWLEDGMENTS
The authors are indebted to Dr. E.W. Kitajima (NAP/MEPAESALQ/USP) for SEM equipment support and to the Cancer Hospital of Uberlndia for irradiation equipment support. This study was supported by grants from FAPEMIG.

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