Nano-scale structure and mechanical properties of the human dentine–enamel junction

J O U R N A L O F T H E M E C H A N I C A L B E H AV I O R O F B I O M E D I C A L M AT E R I A L S

4 (2011) 785–795

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/jmbbm

Research paper

Nano-scale structure and mechanical properties of the human dentine–enamel junction Y.L. Chan a,∗ , A.H.W. Ngan a , N.M. King b a Department of Mechanical Engineering, Faculty of Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong b Paediatric Dentistry and Orthodontics, Faculty of Dentistry, University of Hong Kong, Pokfulam Road, Hong Kong

A R T I C L E

I N F O

A B S T R A C T

Article history:

Despite being an interface between two mechanically mismatched phases of the soft

Received 12 March 2010

dentine and hard enamel, the dentine–enamel junction (DEJ) in a human tooth is in general

Received in revised form

capable of withstanding a long working life of repeated dynamic loading. The current

29 May 2010

poor understanding of the structure and properties of the DEJ has presented a major

Accepted 13 September 2010

obstacle to designing better therapeutic protocols for complications concerning the DEJ.

Published online 18 September 2010

In this investigation, it was discovered that the DEJ is a thin, but gradual interface with

Keywords:

in dentine enter into the enamel side of the DEJ and terminate in a region in which the

Transmission electron microscope

hydroxyapatite crystals begin to show enamel characteristics. Using focused ion beam

Dentine–enamel junction

machining, micro-beams were fabricated from regions within 50 µm of the DEJ and were

Biomineralization

subjected to bend tests. In spite of the similarity in the flexural strength of the DEJ and

Nanomechanical testing

enamel, fractographs revealed cracks in the DEJ that propagated along structures with

characteristics transiting from those of dentine to those of enamel. The collagen fibres

dentine characteristics. To the best of our knowledge, this is the first report on the testing of the mechanical properties of the DEJ. c 2010 Elsevier Ltd. All rights reserved. ⃝

1.

Introduction

The crown of the human tooth is composed of two calcified tissues, namely, enamel and dentine. Enamel, on the outer surface, is the hardest tissue in the human body, comprising ∼95 vol% of apatite crystals and ∼5 vol% of water and organic materials arranged in ∼5 µm keyholeshaped structures known as prisms. It protects the underlying dentine and acts as the cutting and grinding surface during mastication. Dentine is made up of ∼50 vol% apatite crystals, 20 vol% water, and 30 vol% organic matrix. It is distributed throughout the crown and root and so forms the bulk of ∗ Corresponding author. Tel.: +852 2859 7900; fax: +852 2858 5415. E-mail address: [email protected] (Y.L. Chan). c 2010 Elsevier Ltd. All rights reserved. 1751-6161/$ - see front matter ⃝ doi:10.1016/j.jmbbm.2010.09.003

tooth and has the function of absorbing and distributing stresses within the tooth. The structural and compositional dissimilarities between these two mineralised tissues induce marked differences in their mechanical behaviour—enamel is much stiffer (Young’s modulus E ∼ 80 GPa) and harder (hardness H ∼ 4 GPa) than dentine (E ∼ 20 GPa, H ∼ 1 GPa) (Angker and Swain, 2006). Sharp interfaces between such dissimilar materials are usually subjected to concentrated stresses which often cause delamination. Yet, surprisingly, a tight and durable junction known as the dentine–enamel junction (DEJ) exists between the two calcified tissues in the crown of the tooth and persists throughout the millions of cycles of mastication forces during the working life of

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a tooth, with only rare cases of mechanical failures. The life-long success of the bond between enamel and dentine stands in stark contrast to the adhesion of dental restorative materials applied to the oral mineralized tissues of the tooth which dislodge relatively easily. These restorations typically fail either by gross structural failure which results in dislodgement of the applied materials, or by microleakage at the restoration-tissue interface, leading to bacterial invasion and subsequent secondary caries or pulpal death (White et al., 2005). An accurate understanding of the structure–function relationship governing the DEJ would have significant clinical relevance and may permit the creation of improved interfaces between restorations and the odontogenic mineralized tissues. The DEJ has been described as a complex interface with at least three levels of microstructure: the 25–100 µm scallops with their convexities directed towards the dentine and concavities towards the enamel; the 2–5 µm micro-scallops housed within each scallop; and a finer nano-level structure within each micro-scallop (Marshall Jr. et al., 2001; Marshall et al., 2003). Although it has been proposed that the crack resistance of the DEJ may arise from gradual, rather than abrupt, changes in the structure and mechanical properties (White et al., 2000), optical microscopy and scanning electron microscopy (SEM) examinations have indicated a clear demarcation between enamel and dentine, such that the DEJ appears as a narrow, distinct interface (Hayashi, 1992; Lin et al., 1993). Despite the DEJ being renowned for its crack resistance, only a limited number of studies have investigated the fracture properties (Dong and Ruse, 2003; Fong et al., 2000; Habelitz et al., 2001; Imbeni et al., 2005; Marshall Jr. et al., 2001; Stock et al., 2008; White et al., 2000; Zaslansky et al., 2006; Zhang et al., 2007). None of the investigators reported the occurrence of a fracture along the DEJ due to their high fracture toughness, and this cast doubts on the accuracy of the measured values in representing the related fracture properties of the DEJ. The remarkable mechanical success of the DEJ has been attributed to structural characteristics such as the large surface area due to the scalloping structure, the collagen fibre reinforcement, and the intimate contact between the adjacent enamel and dentine crystals (Imbeni et al., 2005; Marshall et al., 2003). However, the contribution of these factors to the mechanical integrity of the DEJ has not been systematically studied, and interestingly, most of the known features of the DEJ were actually identified by microscopy and their roles in fracture resistance were inferred from the observed microstructure. Furthermore, as three different levels of microstructure are present in the DEJ, techniques such as traditional tensile testing would only be able to produce values which represent the collective effects from the three different levels. In this study, a micro-cantilever technique will be used to investigate the submicron-scale structures of the DEJ, so as to gain an insight into the structure–function relationship at this level of hierarchy. The microstructure and fracture properties of the DEJ will be compared to those of the enamel and dentine immediately next to the biological junction, so as to identify the possible toughening mechanisms at the nanolevel isolated from the influence of the scallops and microscallops.

2.

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Materials and methods

Four extracted non-carious human permanent molars were used in the present experiments. Prior to processing, the teeth were stored in Hank’s balanced salt solution (HBSS) at 4 ◦ C so as to prevent demineralization. The growth of microorganisms in the medium was prevented by the addition of sodium azide. The teeth were sectioned, using an EXAKT 300CP diamond saw, into 1 mm thick slices which were then mounted into matching holes drilled in brass blocks to facilitate subsequent polishing (Tang and Ngan, 2004). The mounted specimens were then polished with P 4000 silicon carbide paper and subsequently with 0.05 µm alumina suspensions to achieve a surface roughness of ∼10 nm, as measured by atomic force microscopy (AFM). The samples were then cleaned ultrasonically for 10 s in HBSS to remove any visible smear layer. During the entire preparation process, the samples were kept in HBSS except during grinding and polishing so as to maintain hydration of the samples. Since the functional width of the DEJ has been reported to vary from 1 to 100 µm, this investigation began with identifying the region where the transition in mechanical properties took place in the samples. A series of nanoindentations were conducted along a line starting from and ending at locations approximately 50 µm on either side of the DEJ. The indents were spaced 10 µm apart and the whole procedure was repeated twice near the same DEJ portion to check for consistency. A nanohardness tester (CSM Instruments SA, Switzerland), equipped with a Berkovich tip, was employed for making the nano-indentation measurements immediately after surface preparation. A large drop of HBSS was placed on the specimen surface and by surface tension effects, the space between the entire sample block and the nano-indenter tip was filled by HBSS; thus, the sample was kept hydrated during nanoindentation. The maximum loading of 2 mN was achieved at a rate of 24 mN/min followed by a holding time of 10 s before unloading to 0.5 mN at a rate of 24 mN/min which was then held for 60 s before final unloading to 0 mN. Effects on the measurement due to thermal drift, viscoelasticity of the sample and meniscus forces from the fluid were corrected using the protocols described previously in Feng and Ngan (2002) and Ngan et al. (2005). After identification of a region, the size of which, was manageable by the FIB milling technique, two types of microspecimens were made for bend tests: (i) micro-cantilevers and (ii) micro-beams which have both ends attached to the main tooth structure. These micro-specimens were milled out from the samples in an FEI Quanta 200 3D Dual Beam focusedion-beam (FIB) system, using a milling scheme similar to that employed in an earlier study (Chan et al., 2009). To the best of our technical skills, direct irradiation of the sample with gallium ions was kept to only those areas which are designated for sputter removal throughout the milling process such that damage to the remaining tissues involved in further experiments were kept to minimal. The first step was to make a cut by milling with gallium ions at 30 kV and a current of 3 nA after tilting the sample to 45◦ with respect to the ion direction. The procedure was then repeated on the other side after a simple 180◦ rotation about

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the specimen normal. This process created a micro-beam with a triangular cross-section and a width of 3–5 µm. For the micro-cantilevers, an additional step was to cut one free end of the beam to achieve a cantilever structure. The width and depth of the cross-section of the cantilevers were then measured up to a resolution of 0.1 µm using the SEM mode for calculation of the flexural strength and elastic modulus in the micro-cantilever bending experiments described below. Some micro-cantilevers and beams were made entirely within the enamel, or the dentine phase at least 10 µm from the DEJ. In the micro-cantilevers containing the DEJ, the DEJ was close to the fixed end of the cantilevers so that this would be subjected to the maximum stress under bending. For the micro-beams which contained the DEJ, the DEJ was at the mid-span of the 20 µm long beam where the load was applied and hence, under this configuration, it was also subjected to the maximum stress during bending. Schematic illustrations of the typical layout of the micro-cantilevers and micro-beams are shown in Fig. 1. A Hysitron nanoindenter mounted on a Thermomicroscopes atomic force microscope (AFM) and equipped with a Berkovich tip suitable for experiments in liquid conditions was employed to apply a downward point force on either the midpoint of the both-endfixed micro-beams, or the free end of the micro-cantilevers. Prior to the experiments, the view field of the optical microscope attached to the nano-indenter was carefully aligned with the position of the Berkovich tip, by making an array of indents on an aluminium block and carefully positioning the nano-indentor transducer until the centre of the indent array coincided with the centre of the view field of the microscope. Our combined nano-indenter/AFM system enabled AFM scanning using the Berkovich nano-indenter tip immediately before nano-indentation so that the subsequent nano-indentation could be made at a point extremely close to the centre of the recorded AFM image. After the Berkovich tip had been correctly positioned, a downward load was applied at a constant rate of 20 µN/s until the beam broke. Some of the micro-cantilevers/beams were rehydrated in an enclosed humid environment for 30 min before the mechanical testing was conducted. A minimum of eight micro-specimens were used to test dentine, enamel and the DEJ in each of the configurations. The elastic modulus, E, of the cantilever was calculated as E=

SL3 , 3I

(1)

where L is the length between the loading point and the fixed end of the cantilever, S is the slope of the load–deflection curve, I = wd3 /36 is the second moment of area of the cantilever’s triangular cross-section, w is the width and d is the height of the cantilever’s triangular cross-section. The flexural strength, σcant , of the cantilever was calculated as σcant =

PLd , 3I

(2)

where P is the failure load, and I, L and d have the same meanings as above. While the above samples were used for mechanical characterization, the adjacent sections immediately next to them were used for microstructural characterization in SEM. They were etched with 10% phosphoric acid for 10 s before

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Fig. 1 – Schematic illustration of the typical layout of the micro-cantilevers and micro-beams.

Fig. 2 – Hardness and elastic modulus profiles across the DEJ as measured using nano-indentation.

fixation with 10% phosphate buffered formalin for 24 h. Etching preferentially removes some of the apatite crystals to expose the organic matrix and exaggerate the microstructural features in the enamel. Dehydration was then conducted in increasing concentrations of alcohol, followed by critical-point drying using CO2 . To achieve electron conductivity, a layer of gold was sputter-coated onto the samples prior to examination in a Leo 1530 SEM. Further fine details on the apatite crystals and their spatial arrangement with the organic matrix at the DEJ were revealed by TEM. The TEM specimens, measuring 10 µm × 20 µm × 0.1 µm, were produced using the protocol described by Chan et al. (2010) from the non-etched enamel that had been mechanically characterized.

3.

Results

3.1.

Functional width of the DEJ

The elastic modulus and hardness data from the nanoindentation tests conducted across the DEJ are shown in Fig. 2. Enamel was found to have an elastic modulus of ∼95 ± 15 GPa and a hardness of ∼7 ± 2 GPa, whereas dentine had an elastic modulus of ∼19 ± 2 GPa and hardness of ∼1 ± 0.1 GPa. A sharp change in mechanical properties was observed across the DEJ as revealed under the present experiment with a spatial resolution of ∼10 µm.

3.2.

Microstructure of the DEJ

Fig. 3 shows a low-magnification SEM micrograph of the region near the DEJ. In the top half of the micrograph, the

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Fig. 3 – Scanning electron micrograph of an acid etched human permanent molar near the DEJ.

phosphoric acid dissolved away most of the apatite crystals in the dentine and so the remaining collagen network was revealed, which was rather uniform in structure throughout the whole region. Enamel occupies the lower half of the micrograph, where traces of prisms about 5 µm in width run approximately perpendicular to the DEJ. The estimated boundaries of the DEJ, judged according to the transition in the observed microstructure, are marked by dotted lines in Fig. 3, and the DEJ appears as a region with a thickness of approximately 5 µm. Such a microscopic estimate of the width of the DEJ is more refined than, and is not inconsistent with, the functional evaluation from the nano-indentation results presented in Fig. 2. Fig. 4(a) and (b) show a pair of bright-field (BF) and darkfield (DF) TEM micrographs of the same region near the DEJ. In the BF shown in Fig. 4(a), the fibrous-like bright contrast in the dentine phase is due to the network of collagen I fibres (∼100 nm thick) which absorbs less energy from the incident electrons, while the particle-like (sometimes elongated) dark contrast in the enamel phase is due to apatite crystals of 50–100 nm in size. These crystalline phases appear as bright contrast in the DF in Fig. 4(b) because they Bragg-diffracted electrons which were collected by the objective aperture. Fig. 4(a) and (b) reveal a ∼0.5 µm wide transition region running across the middle of the micrographs with contrast characteristics intermediate between those of dentine and enamel, i.e. both collagen and crystalline contrast are evident. This transitional region, the approximate boundaries of which are marked by dotted lines in Fig. 4(a) and (b), indicate the location of the DEJ; it is noteworthy that the DEJ is a transitional region with a hybrid structure of enamel and dentine. Such a transitional change in structure is further illustrated in Fig. 4(c), which shows a series of eight selected area diffraction patterns (SADP) taken across the DEJ. SADP 1–3 are in the dentine region and they appear identical, with continuous diffraction rings implying a dense distribution of fine crystalline phases in the structure. SADP 6–8 are in the enamel region and they consist of discrete diffraction spots, implying a distribution of coarse crystals. SADP 4–5 are in the DEJ region, and they consist of both continuous rings and discrete diffraction spots, i.e. the structure is a hybrid of dentine and enamel. Fig. 4(d) summarizes the BF, DF and SADP of the dentine, DEJ and enamel regions. The

DF micrographs in Fig. 4(d) show that the crystals in dentine are equi-axed and are much smaller (∼10 nm) in size than the usually elongated crystals in enamel (∼50–100 nm in length). This explains the continuous diffraction rings in the SADP of dentine and the spotty SADP of enamel. Again, the DEJ structure is apparently an intermediary of enamel and dentine.

3.3.

Mechanical behaviour of the DEJ

Fig. 5 shows the flexural strength data measured from the micro-cantilever tests under dehydrated and rehydrated conditions. In the dehydrated state, the flexural strength of enamel at 0.85 ± 0.20 GPa was the highest among the three types of structures, while dentine exhibited the lowest flexural strength of 0.48 ± 0.16 GPa. The flexural strength of the DEJ was 0.78 ± 0.20 GPa which was slightly lower than enamel but much higher than dentine. In the rehydrated state, the flexural strength of enamel and the DEJ were not significantly changed at 0.87 ± 0.29 GPa and 0.74 ± 0.24 GPa respectively, while for dentine this value decreased to 0.31 ± 0.11 GPa. Fig. 6 shows the elastic modulus of dentine measured from the micro-cantilever tests. Following rehydration, the elastic modulus of dentine suffered a significant decrease from 22 ± 2 GPa to 18 ± 1 GPa which illustrates the effect of the current rehydration protocol on the mechanical behaviour of dentine. Distinctive differences were observed between the fractured surfaces of enamel and the DEJ despite their similarity in the measured flexural strength (Fig. 7(a) and (b)). On the fractured surface of enamel are exposed longitudinal debris of the prisms (see the lower region of the fractured surface in Fig. 7(a)), while the fractured surface of the DEJ is much more fibrous with no visible prismatic structure exposed. For dentine, some of the cantilevers (e.g. Fig. 7(d)) did not fracture at their fixed ends, but at a location of the micro-cantilever where a dentinal tubule ran through (Fig. 7(c) vs. (d)). The tubule obviously weakened the local structure and raised the local stress to cause fracture there, instead of at the fixed end of the cantilever where the bending stress should be the highest if the cantilever was a solid bar. Fig. 8 shows the both-end-fixed micro-beams after the bend tests. Under the dehydrated condition, a major crack

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a

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c

b

Fig. 4 – The transmission electron micrographs of the DEJ along with the adjacent enamel and dentine in a human molar tooth under (a) bright-field (BF) condition, and (b) dark-field (DF) condition, (c) selected area diffraction patterns (SADP) from dentine across the DEJ to the enamel (scale bars indicate 5 nm−1 ), (d) typical BF, DF and SADP of dentine, the DEJ and enamel (scale bars in images indicate 0.2 µm, those in SADP indicate 5 nm−1 ).

appears to have propagated from the bottom of the DEJcontaining micro-beams across to their upper surfaces at mid-span where the DEJ is situated (Fig. 8(a)). In contrast, after rehydration (Fig. 8(b)), no discernible cracks along the DEJ could be observed after testing. The rehydrated dentine micro-beams also did not exhibit cracking after the bend tests as opposed to the dehydrated dentine micro-beams (Fig. 8(c) and (d)). However, by contrast to both dentine and DEJ, enamel exhibited cracking under both hydrated and dehydrated conditions (Fig. 8(e) and (f)). Fig. 9 shows typical load–displacement graphs for the micro-beams during bend tests with the rehydrated data in each condition shifted to the right by 30 nm for easier comparison. Generally, in the dehydrated state, the microbeams of the DEJ, dentine, and enamel exhibited linear responses until they failed by catastrophic crack propagation, and no yielding was observed (Fig. 9). After rehydration, enamel still exhibited catastrophic failure with no clear sign of yielding (Fig. 9(c)), but gradual yielding was observed in dentine and some of the DEJ cantilevers (Fig. 9(a) and (b)).

In cases when yielding occurred, the flexural strength was calculated using the data point just prior to the start of yielding. The yielding mechanisms in rehydrated dentine and the DEJ seem to have prevented cracks from propagating in a catastrophic manner.

4.

Discussion

4.1.

The width of the DEJ

Although there have been reports on mechanical testing across the DEJ using a variety of techniques such as microindentation, nano-indentation and AFM indentation (Fong et al., 2000; Marshall Jr. et al., 2001; White et al., 2005), the width of the region within which the transition in mechanical properties takes place remains controversial. An earlier micro-indentation investigation revealed a 100 µm wide region adjacent to the DEJ within which the mechanical properties gradually changed (White et al., 2000). However, another study using nano-indentation estimated the width

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Fig. 4 – (continued) of this transitional zone to be drastically smaller at only 10 µm (Fong et al., 2000), while another study using AFM indentation concluded a further reduced width to 1 µm (Habelitz et al., 2001). The variation in the width of the transitional zone has been attributed to the differences in the size of the indenter tips and the 3D nature of the DEJ (Habelitz et al., 2001). The assessed width of the DEJ from the present TEM examination shown in Fig. 4(a)–(d) is approximately 0.5–1 µm, but from the SEM examination (Fig. 3) it is about 5 µm. Both estimates are more refined than, but are not inconsistent with, the ∼10 µm revealed from the nano-indentation data in Fig. 2. The measurement of the functional width of the DEJ by nanoindentation is limited by the fact that the indent size is finite and successive indents should not be too closed to one another to avoid overlap of the plastic deformation zones. On the other hand, the SEM microstructure as seen from Fig. 3 is likely to be affected by the quality of the etching process and so the width of the DEJ estimated from this micrograph could only be considered as an upper bound value. By contrast, the TEM micrographs in Fig. 4 reveal the actual crystalline distribution in the tooth microstructure, and the estimated value of 0.5–1 µm in this case should be very accurate.

4.2.

Microstructural features at the DEJ

Previous SEM examinations (Lin et al., 1993; Marshall et al., 2003; White et al., 2000) have suggested that collagen fibres from dentine penetrate perpendicularly into enamel, thus

spanning the DEJ, and that this provides reinforcement for the junction. The present SEM and TEM examinations also revealed a similar phenomenon (Figs. 3 and 4). It is well known that the formation of calcified tissues involves a four-step process commencing with heterogeneous nucleation of inorganic calcium phosphate on an organic extra-cellular matrix, which is produced by the cells present in the tissues. The organic matrix of the calcified tissues, therefore, determines the crystals that form subsequently. High-resolution TEM revealed that the foetal DEJ had amorphous zones between dentine and enamel (BodierHoullé et al., 2000). Based on this, it was postulated that a progressive, inorganic phase transformation from amorphous to crystalline state occurs after calcium and phosphate ions have precipitated into the organic matrix in dentine and enamel, and that the continuity at the DEJ is brought about by the subsequent growth of the apatite crystals from enamel and dentine (Bodier-Houllé et al., 2000). Proteins which play a role in the nucleation of calcium phosphates in enamel and dentine include amelogenin and collagen I, respectively. Collagen I, from dentine, was observed to penetrate across the DEJ into enamel (Lin et al., 1993) and amelogenins were also found in the mantle dentine (Nanci et al., 1994). As the specimens in the present investigation were prepared from mature permanent teeth, it would be logical to infer that the crystals observed at the DEJ are reflective of both enamel and dentine since the relevant proteins from both tissues are present at the DEJ during the developmental stage.

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Fig. 5 – Flexural strengths of enamel, the DEJ and dentine under dehydrated and rehydrated conditions as measured with the micro-cantilever technique.

4.3.

Fracture behaviour of the DEJ

Only a few studies (Dong and Ruse, 2003; Giannini et al., 2004; Imbeni et al., 2005; White et al., 2005) have been dedicated to measuring the fracture properties of the DEJ. By drastically decreasing the specimen size, the present technique overcame two important problems in characterizing the stress required for delamination of the DEJ. First, as the width of the specimens was smaller than the micro-scallops of the DEJ, the recorded data should be reflective of the intrinsic properties of the interface, rather than being influenced by the effects of the micro-scallops. Second, during conventional testing of the DEJ, fracture normally occurs at the adjacent enamel, or dentine, rather than at the DEJ itself. By confining the tested volume to be close to the DEJ and subjecting the DEJ to the maximum tensile stress, the present experimental setup ensured that the recorded data are representative of the DEJ rather than the adjacent tissues. The stress required for cleavage of the DEJ was found to be slightly lower than that of enamel, but much higher than that of dentine. The similarity between the tensile strength of the DEJ and enamel has been reported previously (Giannini et al., 2004), and this was attributed to the complexity of the structure of the DEJ and its ability to modify crack propagation. The present fractographs shown in Fig. 7 indicate a marked difference in the morphology of the fractured surfaces of enamel and DEJ micro-cantilevers, despite their similarities in the measured flexural strength. In enamel, the cracks in the beams tended to propagate in defined directions as seen in Fig. 7(a), and the rod-like crystals are exposed on the fractured surface. It was observed from Fig. 4(c) that the SADP of enamel was obviously textured and the apatite crystals should therefore be aligned along particular directions. Such a configuration has been reported to have the effect of directing the propagation of cracks to preferred directions in enamel (Dong and Ruse, 2003). Conversely, no such elongated crystals were exposed on the fractured surface of the DEJ cantilevers (Fig. 7(b)) and the fractured surface assumes a morphology that more closely resembles dentine (Fig. 7(c)). The present data have shown that the flexural strength of dentine is much lower than that of both enamel and the DEJ. Although the major factor in influencing the fracture

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Fig. 6 – Elastic modulus of dehydrated and rehydrated dentine as measured with the micro-cantilever technique.

behaviour of dentine has been suggested to be its collagen fibre arrangement (Nalla et al., 2003b), the tensile strength of dentine also depends on the cross-sectional area of the dentinal tubules present and this has been proposed to be the main reason for the variation in dentine strength across a tooth (Giannini et al., 2004). In the present study, some of the dentine micro-cantilevers did not fracture at their fixed end and dentinal tubules were exposed on the fractured surfaces (Fig. 7(d)). The average diameter of dentinal tubules was ∼1 µm while the average depth of the cross-section of the cantilevers was ∼2.5 µm. Thus, the presence of a tubule would significantly raise the local stress on the intertubular dentine in these micro-specimens, and so fracture can occur at these locations, i.e. the dentinal tubules are regions of weakness.

4.4.

Effect of hydration on fracture behaviour

The micro-beam geometry used in this study represents an interesting alternative to the micro-cantilever geometry which was also used. In the micro-beam geometry, a crack is initiated at the lower surface of the beam at mid-span, and it propagates upwards against an increasing beam width, due to the triangular shape of the beam’s cross-section. The crack therefore propagates with decreasing stress intensity factor, and so it may eventually be arrested before reaching the upper surface. By contrast, in the micro-cantilever geometry, the crack is initiated on the upper, wider surface at the beam’s fixed end and it propagates downwards along a decreasing width, or increasing stress intensity factor. Therefore, once a crack is initiated in a micro-cantilever, it is less likely to be arrested. Thus, post-crack-arrest yielding may be seen in a both-end-fixed micro-beam, but not in a micro-cantilever. Due to its upward propagating nature, a crack that has emerged on the upper surface of the beam can be identified as a through-thickness crack simply by its presence at the upper surface of the micro-beam. Post-mortem SEM examination of the micro-beams showed that cracks were initiated at the recorded stress, but spontaneous crack propagation to the upper surfaces of the beams could only be observed in enamel under both hydrated and dehydrated conditions (Fig. 8(e) and (f)). In dentine and the DEJ, under the hydrated condition, the cracks initiated were arrested and the micro-beams exhibited softening after the first failure load (Fig. 9(a) and (b)) without noticeable crack

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Fig. 7 – Scanning electron micrograph of the fractured surface of (a) an enamel cantilever, (b) a DEJ cantilever, (c) a dentine cantilever, and (d) another dentine cantilever which broke at around the mid-span.

Fig. 8 – Scanning electron micrographs of (a) a dehydrated DEJ micro-beam in lower magnification, and middle sections of (b) a rehydrated DEJ micro-beam, (c) a dehydrated dentine micro-beam, (d) a rehydrated dentine micro-beam, (e) a dehydrated enamel micro-beam, and (f) a rehydrated enamel micro-beam, all after the bending experiments.

propagation to the back side (Fig. 8(b) and (d)). However, under dry conditions, both dentine and the DEJ were more or less linearly elastic, followed by catastrophic crack propagation and failure (Fig. 9(a) and (c)), with the crack propagating through to the upper surface of the micro-beams (Fig. 8(a)

and (d)). Collagen is a major constituent of dentine and its mechanical properties are known to be tightly convolved with its degree of hydration (Balooch et al., 1998). In fact, the fracture toughness of dentine was observed to significantly increase with the degree of hydration due to crack inhibition

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4.5.

Rehydrated Dentine Dehydrated Dentine

Fig. 9 – Typical load–displacement graphs of (a) DEJ cantilevers, (b) dentine cantilevers, and (c) enamel cantilevers.

mechanisms attributed to the collagen content (Nalla et al., 2005). In this study, the load–displacement graphs of dentine exhibited gradual deviation from linearity on increasing load only under hydrated conditions and the dentine microbeams failed rather abruptly when they were dehydrated. This suggests that cracks propagate more readily in the dehydrated state than in the rehydrated state. The same phenomenon was also observed in the DEJ, where the effect of dehydration was even more pronounced. Failure of the DEJ under dehydrated conditions is similar to that of enamel where the crack propagated catastrophically across the micro-beam as shown in Fig. 8(a), but under hydrated conditions, crack propagation was arrested and the upper surface of the micro-beam remained intact.

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Possible toughening mechanism in the DEJ

From the TEM images, the DEJ was observed to be a hybrid region exhibiting characteristics of both enamel and dentine. Due to the interdigitation of collagen fibres and the gradual transition of crystal sizes across from dentine to enamel, the DEJ would be expected to have properties intermediate of dentine and enamel. The presence of collagen in the DEJ would probably activate a similar crack arresting mechanism as observed in dentine, when the DEJ is in a hydrated state. Cracks form more easily in dentine than in enamel or DEJ, but the ability of collagen to slow down crack propagation would be expected to more effectively prevent the catastrophic failure of dentine than enamel. In order to make such deductions on the role of hydration on the fracture properties of dentine, the rehydration technique that was used must be verified. It has been proven that the elastic modulus of dentine varies with the level of hydration, showing a typical ∼20% increase from the completely hydrated state to the completely dehydrated state (Guidoni et al., 2006; Kinney et al., 2003). The elastic modulus of the dehydrated dentine cantilevers was 23.8 ± 3.4 GPa while dentine cantilevers rehydrated with the current protocol exhibited a value of 17.3 ± 4.9 GPa. This 27% drop in elastic modulus demonstrates that the rehydration protocol provided enough moisture to restore the hydrated mechanical behaviour of collagen. Major toughening mechanisms suggested for dentine include crack deflection, crack bridging, uncracked ligament bridging and micro-cracking (Nalla et al., 2003a). By analyzing the crack path and fracture toughness of dentine with respect to collagen arrangement, crack bridging by collagen fibres has been reported to make the largest contribution to the high fracture toughness of dentine (Nalla et al., 2003a). The structure and mechanics of collagen depends heavily on the presence of water and the degree of mineralization (Balooch et al., 2008). Water molecules are known to form hydrogen bonded bridges within and between the peptide chains, and a highly ordered inner hydration layer of water molecules forms hydrogen bonds along the peptide chains to maintain the spacing within the collagen fibrils (Bella et al., 1995, 1994). The loss of water from collagen fibrils has been postulated to cause the fibrils to become more tightly packed, and, in turn, increase their rigidity due to the larger inter-fibril attractive forces (Wenger et al., 2007). Such changes in the structure of collagen have been demonstrated to cause failure of the collagen cross-bridging mechanism effective for toughening dentine, and to induce unstable brittle failure in the odontogenic tissues (Jameson et al., 1993). The presence of collagen at the DEJ together with the similarity in its fracture behaviour to dentine suggests that a similar toughening mechanism may be operating for the integrity of this biological junction. However, in contrary to most existing data, the fracture resistance of the DEJ was not shown to be superior to its adjacent tissues. The experiments in this study were designed specifically to investigate a small region immediately adjacent to the DEJ such that many failure mechanisms at the higher levels of hierarchy were isolated from the experiment. While the fracture properties of the DEJ have usually been studied by propagating a crack from

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the enamel towards the DEJ (Imbeni et al., 2005; Lin and Douglas, 1994; Zhang et al., 2007), this study is the first to use a micro-cantilever setup to directly apply a tensile bending stress across the DEJ to delaminate it. The stress state in such a configuration should give much more direct information on the delamination strength of the DEJ, as opposed to the very complex stress states in earlier studies. The mechanisms in the immediate adjacent tissues which could hinder crack propagation across the DEJ in other investigations would be rendered irrelevant in such a situation. The present TEM and mechanical testing investigations revealed that the microstructures and mechanical properties of DEJ, although intermediate between those of enamel and dentine, are rather similar to those of enamel (Figs. 4 and 5). This suggests that additional mechanisms at other hierarchical levels are required for the significant improvement in fracture resistance of the DEJ commonly observed in comparison to enamel.

5.

Conclusions

Based on the apatite crystal size and presence of collagen as seen in the TEM, a 0.5–1 µm thick layer with distinct difference in morphology from bulk enamel and dentine was identified as the DEJ. The flexural strength of this layer was slightly lower than the adjacent enamel, but much higher than dentine. Upon rehydration, the layer demonstrated a crack arresting capability similar to dentine, without significant increase in its flexural strength. At the nano-level distant from the influence of the scalloped structures of the DEJ, the low delamination probability of the DEJ is therefore postulated to be due to the collagen from dentine crossing over to enamel.

Acknowledgements This investigation was supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, PR China (Project No. HKU7156/08E), as well as a grant from University Grants Council (SEG HKU06). AHWN is grateful to support from the Croucher Foundation in the form of a Senior Research Fellowship. REFERENCES

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