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Heat Treatment Strengthens Human Dentin
1 Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan; Correspondence: * corresponding author, mikarin{at}dent.osaka-u.ac.jp
The flexural strength of Type I collagen, the major organic component of human dentin, increases with heat. We hypothesized that human dentin can be strengthened by heating, which may help prevent fracture of non-vital teeth after restoration. Beam-shaped dentin specimens were obtained from the crowns of human third molars. The dentinal tubular orientations were arranged to run parallel or perpendicular to loading surfaces. The flexural and microtensile strengths of dentin in the parallel specimens were 2- to 2.4-fold greater after being heated between 110°C and 140°C for 1 hr. The stress intensity factors at fracture also increased after specimens were heated. The x-ray diffraction analyses suggested that shrinking of the lateral packing of the collagen triple-helices from 14 Å to 11 Å was the probable cause of the strengthening of heated dentin. We conclude that heat treatment strengthens human dentin.
Key Words: dentin • flexural strength • heat • collagen
Arecent seminal study demonstrated that tooth fracture was the most frequent reason for tooth loss in persons involved in a long-term plaque control program (Axelsson et al., 2004). Altogether, 62% of extractions were performed because of tooth fracture, compared with only 7% and 5% due to caries and periodontal disease, respectively. This research suggested that tooth fracture is a critical issue that needs to be addressed urgently. A better understanding of the mechanical properties of human dentin in various environmental conditions could significantly help in preventing tooth fracture and thus be of potentially great assistance to clinicians in their vital front-line battle. Microstructure, especially tubular orientation, appears to play an important role in the strength of dentin. It is now acknowledged that there is an anisotropy of the ultimate tensile strength of dentin under static loadings (Carvalho et al., 2001; Lertchirakarn et al., 2001; Inoue et al., 2003). Such characteristics of anisotropy were also recently confirmed in dentin under fatigue loadings, and it has been suggested that collagen fibrils in dentin are responsible for such anisotropy (Nalla et al., 2003; Arola and Reprogel, 2006). In contrast, the effects of hydration and dehydration on the mechanical properties of dentin and its microstructure have not yet been fully elucidated. Several studies have claimed that dehydrated dentin has a toughness lower than that of hydrated dentin (Jameson et al., 1993; Kahler et al., 2003; Kruzic et al., 2003; Bajaj et al., 2006). However, it has been reported that some mechanical properties are not affected by dehydration (Huang et al., 1992; Sedgley and Messer, 1992). In addition, from a clinical viewpoint, the higher incidence of fracture in pulpless teeth has been attributed to dehydration, but this is still controversial (Papa et al., 1994; Kahler et al., 2003). It has been demonstrated that the strength of reconstituted tendon collagen can be improved by varying the time and temperature of dehydrothermal cross-linking (Wang et al., 1994). The optimum time for cross-linking appears to be 5 days at 100°C. The improved ultimate tensile strength probably derived from preventing interfibrillar slippage and limiting the number of water molecules that inhibit formation of hydrogen and electrostatic bonding between collagen molecules. Furthermore, it has been reported that the profile of collagen cross-linking varies, depending on the anatomical location in dentin; such differences may partly explain the site-specific tensile strength (Miguez et al., 2004). In human bone, which has a composition comparable with that of dentin, the toughening role of collagen in bone mechanics has been reported, and the fragility of aging bones may relate to changes in the collagen network (Zioupos et al., 1999). Another study indicated that the collagen network played an important role in the toughness of human bone, and the toughness and strength of bone decreased with increasing collagen denaturation (Wang et al., 2001). Therefore, it is reasonable to investigate the mechanical properties of human dentin in relation to the profile of its collagen in different environments. The purpose of this study was to investigate the effects of dehydration and heating on the flexural and tensile strengths of dentin by focusing on dentinal tubular orientation and the collagen arrangement. The null hypothesis tested was that neither dehydration nor heating affected the mechanical properties of human dentin.
Preparation of a Specimen Human third molars free of caries were stored in Hanks’ balanced salt solution (HBSS) at 4°C and used within 3 mos of extraction. Protocols were approved by the ethics committee of Osaka University, and all individuals gave informed consent. Beam-shaped dentin specimens—measuring approximately 1.7 x 0.9 x 8.0 mm for flexure testing and stress intensity analysis, 1.0 x 0.5 x 8.0 mm for microtensile testing, and 0.5 x 0.3 x 8.0 mm for x-ray diffraction study—were obtained from coronal central portions of the molars (Fig. 1a  ). Dentinal tubule orientations in the specimens were arranged to run parallel or perpendicular to loading surfaces (Fig. 1b). The tubular orientations were screened by microscopic inspection prior to the testing. The specimens were subjected to the following environmental treatments: wet-soaked in HBSS; dry-dried in a desiccator (0140-A2, Sanplatech Co., Osaka, Japan) for 1 wk at 23°C with 30% humidity; and heat-heated in an oven at 50, 70, 110, 140, or 200°C for 1 hr. Each experimental group in the flexural and microtensile testing treatment had 10 beam-shaped specimens, and there were 5 and 3 specimens, respectively, for the stress intensity analysis and the x-ray diffraction study.
Flexural and Tensile Testing Specimens subjected to flexural fracture testing were secured in a custom-made metallic holder with a cantilever beam geometry (Fig. 1c  ), and a flexural load was applied by means of a universal testing machine (Autograph AG-IS, Shimadzu Co., Kyoto, Japan) with a crosshead speed of 0.1 mm/min. Fracture stress (s, MPa) was calculated by the following equation:
where M (N) is the load at fracture, L (m) is the valid length in the cantilever geometry, and b (m) and d (m) are the width and thickness of a specimen, respectively. The Young’s modulus (E, GPa) was also calculated by the following equation:
where P (N) is the load at yield point, L (m) is the valid length, d (m) is the displacement of a specimen at the yield point, and I (m4) is the moment of inertia given by:
Microtensile testing was conducted with a beam-shaped specimen trimmed to provide a cross-sectional area of 0.25 mm2 (Fig. 1d The mechanical properties of specimens taken from different environments were compared by ANOVA and Scheffé’s F test at a 95% level of confidence. Fractured surfaces of all the specimens in the flexural and tensile testing groups were examined by scanning electron microscopy (SEM) (JSM9-840A, JEOL, Tokyo, Japan) at magnifications from X200 to X5000. Surfaces were sputter-coated with a gold-palladium alloy prior to being observed.
Stress Intensity Analysis
where s (MPa) is the fracture stress, a (m) is the total depth of the pre-notch and pre-crack, b (m) is the thickness of a specimen, and the function F(a/b) is given by:
The total depth of the pre-notch (a) was measured by optical microscopy.
X-ray Diffraction Study
The mechanical properties of human dentin under different dehydration conditions focused on tubular orientations (Table ). In the parallel specimens, flexural and tensile strengths were significantly increased by heating within the range of 50 to 140°C. The perpendicular specimens also showed a tendency for their mechanical strength to increase with heating, but the differences were not statistically significant. The Young’s moduli were stable regardless of dehydration or heating. The stress intensity factors were increased by heating. This was true for both parallel and perpendicular specimens, although the parallel ones showed greater strength.
From the SEM observations, it appeared that, in the wet condition, the peritubular regions were smooth and the intertubular dentin was rough, in both the parallel and perpendicular specimens (Figs. 2a, 2c ). In contrast, in the heated dentin, the entire surfaces were rough (Figs. 2b, 2d). In the perpendicular specimens, many dentinal tubules were visible in the dentin in the wet condition (Fig. 2c) compared with those in the heated condition (Fig. 2d).
The x-ray diffraction study demonstrated that the distance between the axes of the collagen triple-helices, which is acknowledged to be 14 Å in the wet condition (Fraser and MacRae, 1973), shrank to 11 Å after the specimens were heated at 110°C for 1 hr (Figs. 3b, 3c ).
Dentin is a natural composite material in which the collagenous matrix is enveloped by accumulated hydroxyapatite crystals. From the perspective of composite materials, the biomechanical properties of dentin depend on the quality and the spatial arrangement of its constituents. In the present study, the toughness of dentin was improved by heating. The strengthening effect of Type I collagen by heating at 110°C was previously reported to occur as a result of dehydrothermal cross-linking (Wang et al., 1994). According to the present results from the x-ray diffraction study, the distance between collagen triple-helices, which is acknowledged to be 14 Å in the wet condition, shrank to 11 Å after heating for 1 hr at 110°C. This shrinkage of the collagen networks is the most probable reason for the toughening of human dentin by heating. In contrast to the improvement of other mechanical properties, the elastic moduli were unchanged under all conditions of dehydration. The theoretical model of human bone (Kotha and Guzelsu, 2007) shows that an increase in the elastic modulus of the organic matrix leads to increases in the elastic modulus of bone tissue. However, in the present study, no change in the elastic modulus of human dentin was found by heating. Another study (Wang et al., 2001) also reported results similar to those of the present study in human bone after heating. This may be because the elastic moduli of the mineral and organic components were not changed, even after heating. Fractographic observations of human dentin with fatigue fractures showed distinct microcracks in the peritubular cuff, as well as separation between the peritubular and intertubular dentin (Bajaj et al., 2006). This study speculated that the dentinal tubules might serve as effective stress-raisers, and hence as crack-initiation sites. This possibility was supported by the frequent occurrence of microcracks in the peritubular dentin surrounding the tubules on the fracture surfaces found under conditions of higher stress associated with final overload failure (Imbeni et al., 2003). In the present study, the rougher fracture surfaces in the heated specimens indicated that they required higher fracture energy compared with those in the wet specimens. This was clearly observed particularly in the perpendicular specimens, where cracks propagated into dentinal tubules surrounded by peritubular dentin. If one hypothesizes that the boundary between peritubular and intertubular dentin was the precise location markedly strengthened by heating, it is logical to suggest that the perpendicular specimens required significantly higher fracture energy after heating than did the parallel specimens, where cracks were deflected from dentinal tubules. Conducting nano-indentation studies focused on microstructure before and after heating would help to address this issue. Although several studies demonstrated that dehydration decreased the toughness of dentin (Jameson et al., 1993; Kahler et al., 2003; Kruzic et al., 2003; Bajaj et al., 2006), in the present study the mechanical properties of human dentin were unaffected by drying. These differences can be explained by differences in the materials used and the methods of dehydration. Other studies used different resources, such as bovine dentin and elephant tusks. In addition, degrees of dehydration of dentin may not be identical in studies with different dehydration protocols, or among specimens with a different state of calcified collagen. Such inconsistencies may explain the differences in the behavior of dehydrated dentin. The effect of heating within the range of 37 to 200°C on the mechanical properties of human bone were investigated (Wang et al., 2001), and the properties decreased when heating exceeded 160°C, where more than 50% of collagen was denatured. In the present study, decreases in the mechanical properties were also detected in human dentin heated at 200°C. However, no toughening by heating was found in bone, such as we found in dentin, even though collagen shrinkage was observed in decalcified human bone at 60°C (Zioupos et al., 1999). This difference in behavior with heating between human bone and dentin may be due to differences in their microstructure, although bone and dentin have a morphology similar to that of a dendritic canalicular system (Lu et al., 2007). Compact bone includes tubular structures, Haversian canals, but those microstructures are not identical to those of dentinal tubules. Haversian canals are not surrounded by hyper calcified regions such as peritubular dentin. The probable explanation could be that Haversian canals in bone are not identical to dentinal tubules, and this affects the difference in the response when the bone has been heated. Further investigations may help to identify the fatigue fracture behavior of human dentin during heating. In addition, detailed quantitative and qualitative analyses of the collagen cross-linking after heat treatment are also required. In summary, the results of this study indicate that the mechanical properties of human dentin increase by heating, probably because of the shrinkage of lateral separation between collagen molecules.
This study was supported by Grants-in-Aid for Scientific Research (No. 19390482) from the JSPS, and by the 21st century COE at Osaka University Graduate School of Dentistry. Received for publication March 15, 2007. Revision received March 14, 2008. Accepted for publication April 18, 2008.
Journal of Dental Research, Vol. 87, No. 8,
762-766 (2008)
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ABSTRACT
INTRODUCTION
m) was calculated by the following equation (
