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Micromechanical characterization of prismless enamel in the tuatara, Sphenodon punctatus
journal of the mechanical behavior of biomedical materials 39 (2014) 210 –217
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Short Communication
Micromechanical characterization of prismless enamel in the tuatara, Sphenodon punctatus Ezgi D. Yilmaza, Sabine Bechtlea, Hu¨seyin O¨zcobana, Jules A. Kieserb, Michael V. Swainc,d, Gerold A. Schneidera,n a
Institute of Advanced Ceramics, Hamburg University of Technology, Hamburg, Germany Sir John Walsh Research Institute, University of Otago, Dunedin, New Zealand c Prosthetic Department, Freiburg University, D 29106 Freiburg, Germany d Biomaterials, Faculty of Dentistry, University of Sydney, NSW 2006, Australia b
ar t ic l e in f o
abs tra ct
Article history:
Dental enamel – a naturally occurring biocomposite of mineral and protein – has evolved from
Received 16 June 2014
a simple prismless to an advanced prismatic structure over millions of years. Exploring the
Received in revised form
mechanical function of its structural features with differing characteristics is of great
23 July 2014
importance for evolutionary developmental studies as well as for material scientists seeking
Accepted 27 July 2014
to model the mechanical performance of biological materials. In this study, mechanical
Available online 4 August 2014
properties of prismless tuatara Sphenodon punctatus enamel were characterized. Using micro-
Keywords:
cantilever bending samples the fracture strength and elastic modulus were found to be
Biological materials
640787 MPa and 4276 GPa, respectively in the orientation parallel to the crystallite long axis,
Prismless enamel
which decreased in the orthogonal direction. The intrinsic fracture toughness of tuatara
Strength
enamel ranged from 0.21 MPa m1/2 and 0.32 MPa m1/2. These values correspond to the lower
Fracture toughness
limit of the range of values observed in prismatic enamel at the hierarchical level 1.
1.
Introduction
Dental enamel is the most highly mineralized tissue of vertebrates and investigations of its diversification has become an important tool to elucidate the evolutionary relationships among different species whose dietary habits, and hence the mechanical demands on their dental tissues, are closely related to their chemical composition and microstructural architecture (Koenigswald, 2000; Rensberger, 2000). For example, in lower vertebrates (fish, amphibians and
& 2014 Published by Elsevier Ltd.
reptiles) the physiological task of the dentition is usually limited to the capturing, piercing and holding of the prey with no contact between the opposing jaws (Line and Novaes, 2005; Kieser et al., 2011). Accordingly, they have a relative simple enamel type in contrast to most mammals, whose microstructural features exhibit a high level of complexity and diversity, which have evolved as a result of adaptations in response to an increase in stress levels imposed on teeth due to the formation of dental occlusion via chewing and grinding (Rensberger, 2000; Line and Novaes, 2005).
n Correspondence to: Hamburg University of Technology, Institute of Advanced Ceramics, Denickestraße 15, 21073 Hamburg, Germany. Tel.: þ49 40 42878 3037; fax: þ49 40 42878 2647. E-mail address: [email protected] (G.A. Schneider).
http://dx.doi.org/10.1016/j.jmbbm.2014.07.024 1751-6161/& 2014 Published by Elsevier Ltd.
journal of the mechanical behavior of biomedical materials 39 (2014) 210 –217
Although dental enamel of many animals consists predominantly of inorganic crystallites of hydroxyapatite (HAP) combined with a small amount of organic matter and water, it is evident that some interspecies differences in composition and crystallite geometry exist (Maas and Dumont, 1999). However, the main categorization of enamel structures is based on the arrangement and orientation of the crystallites. In basal species, enamel formed into parallel arranged crystallites (prismless enamel); whereas, in more advanced species HAP crystallites arranged into rod-shaped cylindrical bundles of crystallites known as “prisms” (prismatic enamel) delimited by organic sheaths and interprismatic crystallites. Considering the fact that enamel contributes to tooth stability essentially by resisting wear and fracture, evolutionary changes in enamel morphology should have enhanced its mechanical
211
properties as has been often hypothesized (Koenigswald, 2000; Rensberger, 2000; Line and Novaes, 2005; Maas and Dumont, 1999; Sander, 2000) but not experimentally verified. The latter requires detailed comparison of prismless and prismatic enamel types. Although extensive research has been carried out on the mechanical characterization of the mammalian prismatic enamel (Bechtle et al., 2010; Ang et al., 2010; Bechtle et al., 2012; He and Swain, 2008), very little is known about the mechanical properties of prismless enamel (Kieser et al., 2011; Enax et al., 2013). The thickness of prismless enamel ( 100 to 200 mm) has been the limiting factor for macroscopic characterization until recent years. Through the development of smallscale mechanical characterization techniques (Bechtle et al., 2012, 2011; Chan et al., 2011, 2009; Yilmaz et al., 2013) it is now possible to isolate and characterize sub-micron scale specimens
Fig. 1 – The lower jaw of the tuatara Sphenodon punctatus used in this study (a), and a longitudinal cross-section of a tooth (b) is illustrated. Tuatara enamel composed of nano-scaled HAP crystallites extending at almost right angles from dentin-enamel junction (the interface between enamel and dentin). The crystallites have a parallel arrangement in the outer part of the tuatara enamel and they form columnar units at the inner core (c). High numbers of enamel tufts having a width of 1 lm are also present in the tuatara enamel (d). These are known to be protein-rich crack like defects. A higher magnification image of the tuft region (e) visualizes thin films and ligaments connecting and wrapping the crystallites. These are believed to be the organic remnants.
212
journal of the mechanical behavior of biomedical materials 39 (2014) 210 –217
by means of focused ion beam (FIB) machining. By using this technique the mechanical characterization of prismless enamel of tuatara Sphenodon punctatus is undertaken in this study. Tuatara S. punctatus, often referred to as a living fossil (originated around 240–250 mya), is a small reptile restricted to New Zealand (Kieser et al., 2011). The dentition of the tuatara is characterized by a single row of conical shaped teeth in the lower jaw (Fig. 1a) fitting perfectly between the two upper rows when the mouth is closed (Kieser et al., 2011). Tuatara enamel has an increasing thickness along the long axis of the tooth reaching 100 mm at the tip of the tooth. Tufts (protein-rich crack like defects) originating from the dentin-enamel junction (DEJ) and dentine tubules exist in the inner part of the tuatara enamel as shown in the longitudinal cross-section in Fig. 1b. HAP crystallites extend from the DEJ at almost right angles to the outer enamel surface (OES). At the outer enamel layer, crystallites align relatively parallel to each other, whereas diverging crystallites from a central axis that develop in a fanshaped pattern can be seen in the inner layer close to the DEJ (Fig. 1c). These are known as columnar units, which are differentiated by lines of changing crystallite orientations (Sander, 2000). Tuatara enamel is reported to be richer in protein remnants compared to mammalian enamel (Kieser et al., 2011). This can be observed in Fig. 1d showing a tuft region at higher magnification. The crystallites appear to be wrapped and connected by very thin organic protein/peptide layers (Kieser et al., 2011). Although enamel appears to experience essentially compressive forces, it was shown by simulation studies that compressive chewing loads in a vertical direction also produce horizontal tensile stresses within ellipsoidal cusps (Rensberger, 2000) and are considered critical for cracking and fracture in enamel. In this regard, the mechanical behavior under tension was of interest in this work. However, due to the size of the enamel of tuatara and difficulties in preparation of tensile specimens for typical experimental setups, micro-cantilever bending experiments were conducted, where fracture was induced in the tensile stress region of the cantilever beams. By means of notched and unnotched micro-cantilever bending tests, mechanical properties including; fracture strength, elastic modulus and, fracture toughness are analyzed.
2.
Materials and methods
The tuatara teeth sample consisted of the lower jaw of a single individual from the collection of the Sir John Walsh Research Institute, University of Otago, Dunedin, New Zealand. Three teeth were resected from the jaw using the water cooled Buehler Isomet 4000 precision saw and a very thin (150 μm) diamond saw blade. Due to their small size ( 2 mm long, 1 mm in diameter), whole teeth were glued on electron microscope sample stubs using a mounting resin. The fixed teeth were ground down using 500 grit SiC paper until a cross section was exposed as shown in Fig. 1b. The teeth were then prepared using 1200 SiC grit paper and then polished with 1 mm diamond suspension. One of the prepared teeth was etched for 20 s using 36% hydrochloric acid to enhance the HAP crystallites visibility for scanning electron
microscope investigations. The two non-etched samples were used for focused ion beam (FIB) sample preparation. Finally, specimens were sputter-coated with a thin gold-palladium layer (some nanometers thick). Notched and unnotched triangular cantilever beams were prepared with a Cross-Beam Zeiss Auriga Canion FIB system. A detailed description of the milling procedure and parameters used is in our previous publications (Bechtle et al., 2012, 2011). Both notched and unnotched cantilever beams consisted of two orientation groups, namely parallel and perpendicular to the crystallite long axis. Please note that the unnotched parallel cantilever beams were milled from the tooth sample 1, whereas the unnotched perpendicular and notched cantilever beams were milled from the tooth sample 2. The beams fabricated in the same tooth were located in approximately the same region (middle-distal with respect to the distance to cusp and 10 to 20 mm with respect to the distance to DEJ) to prevent local structural and chemical variations that is commonly observed in mammalian enamel for instance. Moreover, milling sites were chosen at locations that are free of any tufts and tubules. Representative samples and the geometrical details are demonstrated in Fig. 2. Tables 1 and 2 list the dimensions of all samples tested in this study. L denotes the loaded cantilever length that is the distance between the attached end of the cantilever and the loading point for unnotched samples and the distance between the notch and the loading point for notched samples. W is the cantilever height, B is the cantilever width. For the notched samples, the notch depth is designated by, a, and the distance of the notch from cantilever fixed end by d. FIB prepared cantilever beams were tested using an MTS nanoindenter system equipped with a Berkovich tip. The nanoindenter tip was then positioned close to the cantilever's free end using the optical system. Samples were loaded at a loading rate of 0.2 mN s 1 up to max. displacements of 3 mm. Thereafter the samples were unloaded. The load on the sample, P, and the tip displacement, s, were monitored continuously. To calculate the actual cantilever displacement, sn, the sharp indenter tip penetration into the cantilever has to be subtracted from the total indenter displacement as described previously (Bechtle et al., 2012). To do this, nanoindentation tests were performed within the bulk tuatara enamel adjacent to where the FIB beams were obtained. Zeiss Supra 55VP scanning electron microscope (3 kV, 10 6 mbar) was used for microstructural investigations after mechanical testing. For the unnotched samples, bending stress and associated strain, fracture strength and elastic modulus were calculated from the load–displacement data monitored using the following equations (Dubbel et al., 2007) for triangular shaped cantilevers: σ¼
12 P L B W2
ð1Þ
sn H L2
ð2Þ
and ε¼
where σ is the bending stress and, ε is the bending strain on the outer surface of the beam. The elastic modulus E was estimated for all samples within the linear-elastic regions
journal of the mechanical behavior of biomedical materials 39 (2014) 210 –217
213
Fig. 2 – Scanning electron micrographs show the positions from which unnotched parallel (a) and unnotched perpendicular (b) tuatara micro-cantilevers were obtained. The white arrows indicate the crystallite orientation in the beams with respect to the long axis. The notched samples were produced also in parallel and perpendicular orientations. A schematic representation is shown in (d) (reprinted from Ref. Bechtle et al. (2011) with permission from the publisher) to elucidate sample geometry of the notched micro-cantilever beams as illustrated.
Table 1 – Dimensions of the FIB prepared unnotched tuatara cantilevers. Sample
Unnotched Unnotched Unnotched Unnotched Unnotched
parallel 1 parallel 2 parallel 3 perpendicular 1 perpendicular 2
L, Loaded cantilever length [mm]
W, Height [mm]
B, Width [mm]
16.76 17.73 14.92 19.70 13.19
5.84 7.74 6.04 7.94 7.46
5.44 6.43 5.50 7.46 6.38
Table 2 – Dimensions of the FIB prepared notched tuatara cantilevers. Sample
L, Loaded cantilever length [mm]
W, Height [mm]
B, Width [mm]
d, Distance of notch from cantilever fixed end [mm]
a, Notch depth [mm]
Notched parallel Notched perpendicular
17 22.3
6.19 5.02
5.16 4.57
2.32 6.543
0.92 0.39
214
journal of the mechanical behavior of biomedical materials 39 (2014) 210 –217
from the slope of the stress-strains curves, namely E¼
Δσ Δε
ð3Þ
For the notched samples, the formula for the fracture toughness Kc (critical stress intensity factor) calculation is given by pffiffiffiffiffiffi KIc ¼ FI; ∇ σ πa ð4Þ where the subscript I indicates the mode I fracture (tension normal to the crack plane). FI; ∇ is the geometric function parameter for notched triangular cantilevers. Stress intensity factors for rectangular bending bars are well known from fracture mechanics handbooks; however, there is a lack of such information for bars with triangular cross-sections (Bechtle et al., 2011). In order to compute the fracture toughness for the triangular cantilevers tested, a series of finite element computations was performed for the specific notch and sample geometry, and notch position of our specimens as explained in our previous study (Bechtle
et al., 2011). The geometric function was found to depend upon variations a/W and d/W ratios. For d/WZ0.5 and 0ra/ Wr0.15 the geometric function FI; ∇ for a triangular cantilever tested in bending notched at distance d from the cantilever fixed end can be approximated by Bechtle et al. (2011). a FI;∇ ffiFI;rect 1 þ 0:87 W
ð5Þ
with the geometric function FI,rect for the rectangular bar from Fett and Munz (1997). FI;rect ¼
1:1215 3=2
ð1αÞ
5 5 1 3 α þ α2 þ 5α2 ð1αÞ6 þ expð 6:1342α=ð1αÞÞ 8 12 8 8
ð6Þ where α¼a/W. The effect of parameter d/W in the region d/ W Z0.5 was less than 1% for FI; ∇ and can be neglected. It should be mentioned here that the d/W of the notched parallel sample is 0.37 but it is considered that this discrepancy does not play a significant role.
Fig. 3 – The load–displacement curves resulting from bending tests of the unnotched (a) and notched (c) tuatara microcantilevers are illustrated. The post-test SEM image of the unnotched sample “parallel 1” is shown in (b), where a major crack is apparent at the fixed end of the cantilever. The post-test investigation of the “parallel notched” sample (d) revealed an uneven fracture surface (underneath the FIB-induced notch) with some pores and cavity like features in the retained part after fracture.
journal of the mechanical behavior of biomedical materials 39 (2014) 210 –217
215
Table 3 – Mechanical properties of tuatara micro-cantilevers. Sample
σ, Strength [MPa]
ε, Strain [%]
E, Elastic modulus [GPa]
KIc, Fracture toughness [MPa m1/2]
Unnotched parallel 1 Unnotched parallel 2 Unnotched parallel 3 Mean7s.d. Unnotched perpendicular 1 Unnotched perpendicular 2 Mean7s.d. Notched parallel Notched perpendicular
581 725 566 624787 260 327 293747 – –
1.16 1.89 1.4 1.4870.37 0.7 1.36 1.0370.47 – —
49 38 40 4276 37 24 3079 – –
– – – – – – – 0.32 0.21
3.
Results
The load–displacement curves of the parallel and perpendicular unnotched samples are shown in Fig. 3a. All samples deformed in a linear elastic fashion until a sudden fracture took place. Table 3 summarizes the mechanical properties obtained from the unnotched micro-cantilevers. In the direction parallel to the crystallite long axis the average fracture strength values under bending was found to be 624787 MPa and 293747 MPa in the perpendicular direction. The average elastic modulus was respectively 4276 GPa and 3079 GPa while the strain at failure was 1.4870.37% and 1.0370.47% parallel to the crystallites and in the direction perpendicular to the crystallites. A representative post-test SEM micrograph of a fractured unnotched sample “parallel 1” is shown in Fig. 3b. A single large crack, which initiated at the fixed end of the cantilever (where the bending stress was the highest), is apparent. The crack surface that developed exposes the fractured crystallites. The load–displacement curves of the parallel und perpendicular notched samples are given in Fig. 3c. With initiation of crack propagation unstable fracture occurred. The fracture toughness KIc of the parallel and perpendicular notched samples are estimated as 0.32 MPa m1/2 and 0.21 MPa m1/2 respectively. The post-test SEM micrograph of the parallel notched sample is illustrated in Fig. 3d. The individual crystallites cannot be recognized (probably due to the resolution of the image) but an uneven fracture surface (underneath the FIBinduced notch) with some holes and cavities is seen.
4.
Discussion
To begin with, it is important to mention that due to the long operating time and complexity of the experimental set-up conducted in this study (which is the only way to examine the required material properties from these tissues having an extremely small volume), only a limited number of samples could be tested. The values obtained here from tuatara enamel do not allow us to determine an absolute conclusion about the mechanical properties of the prismless enamel due to the low statistical power. However, given the fact that the mechanical data of the enamel from basal species are largely lacking in the literature, we consider that even these small numbers of samples provide valuable information.
The microscale bending strength of mammalian (bovine and human) enamel was reported in the previous studies as 540– 1420 MPa in parallel and 380–560 MPa in perpendicular directions (Bechtle et al., 2012; Chan et al., 2009, 2010). And the microscale bending modulus of mammalian enamel was reported as 42–60 GPa in parallel and as 22–33 GPa in perpendicular direction (Bechtle et al., 2012; Chan et al., 2009, 2010). The strength and elastic modulus values estimated here for tuatara enamel are close to the lower limits of the previously reported values in general and this can be explained by the lower degree of mineralization in basal prismless enamel. A mineral volume range of 87–96% has been measured for mammalian (human and bovine) enamel (Elliott, 1997), whereas there is only one report referring the chemical composition of tuatara enamel. Kieser et al. demonstrated by Raman spectroscopy that tuatara enamel, tuatara dentin and human dentin share similar chemical compositions (Kieser et al., 2011). Given the fact that mineral content of human dentin is 50 vol% (Bertassoni and Swain, 2012), the mineral content of tuatara enamel can be considered as 50 vol%. As with most of the biological materials, tuatara enamel is found to be anisotropic. Almost two-fold lower bending strength values are obtained from the tuatara perpendicular in comparison to the tuatara parallel micro-cantilevers. This is most probably due to the load transfer from the matrix to the HAP fibers when loaded in parallel direction. However, the difference between the mean elastic modulus values of tuatara parallel and tuatara perpendicular sample groups was statistically not significant. This is astonishing and not expected if the Voight and Reuss models are applied (Hull and Clyne, 1996) to the parallel and perpendicular oriented micro-cantilevers. It reflects the fact that HAP crystallites are not perfectly aligned parallel and perpendicular to the loading direction. Moreover, another possibility is that the mineral content of the tooth sample 2 (where the perpendicular unnotched samples were milled) could be slightly higher than the tooth sample 1 (where the parallel unnotched samples were milled) reducing the expected variation in stiffness due to anisotropy. Based on our findings, the fracture toughness values obtained in tuatara enamel appears to be slightly lower than the fracture toughness values of bovine enamel 0.4–0.9 MPa m1/2 estimated using the same testing method with identical sample dimensions (Bechtle et al., 2011). The drawback of the FIB method is dehydration and exposing cutting edges of the sample to gallium ions, which can generate large amounts of heat. In order to
216
journal of the mechanical behavior of biomedical materials 39 (2014) 210 –217
assess whether this methodology alters the mechanical properties in human enamel or not, Chan et al. tested the hardness and elastic modulus of enamel samples before and after the treatment and measured no difference (Chan et al., 2009). However, this may not be valid for the fracture toughness which is reported to be influenced by the degree of hydration (Baldassarri et al., 2008). Water is known to be a plasticizer and additionally modify the protein structure and influence its deformability (Nalla et al., 2005). As a consequence, the fracture toughness estimated here could have been underestimated. Please note that to be able to conclude that the fracture toughness of prismless enamel of reptiles are less than prismatic enamel of mammals, larger numbers of samples from the former group should be tested in the future. Analytical models (Hull and Clyne, 1996; Ji and Gao, 2010) for predicting the elastic constants of simple composite structures cannot be implemented for tuatara enamel due to the structural mismatch. It is very difficult to estimate whether the crystallites have a straight or curvy outline from the SEM micrographs shown in Fig. 1. In addition, structures like tufts and tubules interrupt the continuity of the crystallites. Characterization of the hard particle length and aspect ratio is essential for mechanical modeling since it determines the load transfer in the composites. Even though the length of the crystallites in tuatara enamel extending from DEJ and running to the boundaries of the columnar units (see Fig. 1c) appears between 3 and 5 mm, SEM images is not very reliable in this regard since the appearance of the crystallites depends on the sectioning angle and sample preparation.
5.
Conclusion
In order to understand the reasons for evolutionary development in enamel morphology from simple to high order hierarchical structure, the mechanical properties of different enamel types should be compared. Although extensive research has been carried out on highly developed mammalian (human and bovine) enamel, only a few studies have attempted to investigate basal reptilian enamel. To fill this gap in the existing literature, the basic mechanical properties of tuatara S. punctatus enamel, which possess only one level of hierarchy, is investigated in thus study. In summary, by characterizing at the same hierarchical level the tuatara enamel appeared to be slightly weaker than mammalian enamel, but the difference was not pronounced. This suggests that the greater mechanical advantage of advanced prismatic mammalian enamel over basal prismless enamel may lie in the macroscopic fracture behavior where the toughening mechanisms operating at higher order hierarchical levels can contribute.
Acknowledgment The authors wish to express gratitude to German Research Foundation (Grant no. SCHN 372/18-1) for financial support. The authors gratefully acknowledge the technical support of Daniel Laipple and Uwe Lorenz (Helmholtz-Zentrum Geesthact) for the FIB system.
r e f e r e n c e s
Ang, S.F., Bortel, E.L., Swain, M.V., Klocke, A., Schneider, G.A., 2010. Size-dependent elastic/inelastic behavior of enamel over millimeter and nanometer length scales. Biomaterials 31, 1955–1963, http://dx.doi.org/10.1016/j.biomaterials. 2009.11.045. Baldassarri, M., Margolis, H., Beniash, E., 2008. Compositional determinants of mechanical properties of enamel. J. Dent. Res. 87, 645–649, http://dx.doi.org/10.1177/154405910808700711. Bechtle, S., Habelitz, S., Klocke, A., Fett, T., Schneider, G.A., 2010. The fracture behaviour of dental enamel. Biomaterials 31, 375–384, http://dx.doi.org/10.1016/j.biomaterials.2009.09.050. ¨ zcoban, H., Yilmaz, E.D., Fett, T., Rizzi, G., Lilleodden, Bechtle, S., O E.T., Huber, N., Schreyer, A., Swain, M.V., Schneider, G.A., 2011. A method to determine site-specific, anisotropic fracture toughness in biological materials. Scr. Mater. 66, 515–518, http://dx.doi.org/10.1016/j.scriptamat.2011.12.027. ¨ zcoban, H., Lilleodden, E.T., Huber, N., Schreyer, A., Bechtle, S., O Swain, M.V., Schneider, G.A., 2012. Hierarchical flexural strength of enamel: transition from brittle to damage-tolerant behaviour. J. R. Soc. Interface 9, 1265–1274, http://dx.doi.org/ 10.1098/rsif.2011.0498. Bertassoni, L.E., Swain, M.V., 2012. Influence of hydration on nanoindentation induced energy expenditure of dentin. J. Biomech. 45, 1679–1683, http://dx.doi.org/10.1016/j. jbiomech.2012.03.021. Chan, Y., Ngan, A., King, N., 2009. Use of focused ion beam milling for investigating the mechanical properties of biological tissues: a study of human primary molars. J. Mech. Behav. Biomed. Mater. 2, 375–383, http://dx.doi.org/10.1016/j. jmbbm.2009.01.006. Chan, Y., Ngan, A., King, N., 2010. Degraded prism sheaths in the transition region of hypomineralized teeth. J. Dent. 38, 237–244, http://dx.doi.org/10.1016/j.jdent.2009.11.003. Chan, Y., Ngan, A., King, N., 2011. Nano-scale structure and mechanical properties of the human dentine–enamel junction. J. Mech. Behav. Biomed. Mater. 4, 785–795, http://dx. doi.org/10.1016/j.jmbbm.2010.09.003. Dubbel, H., Grote, K.H., Feldhusen, J., 2007. Dubbel: Taschenbuch fu¨r den Maschinenbau, 22nd ed. Springer, Berlinhttp://dx.doi. org/10.1007/978-3-540-68191-5. Elliott, J.C., 1997. Structure, crystal chemistry and density of enamel apatites. Ciba Found. Symp. 205, 54–67, http://dx.doi. org/10.1002/9780470515303.ch5. Enax, J., Fabritius, H., Rack, A., Prymak, O., Raabe, D., Epple, M., 2013. Characterization of crocodile teeth: correlation of composition, microstructure, and hardness. J. Struct. Biol. 184, 155–163, http://dx.doi.org/10.1016/j.jsb.2013.09.018. Fett, T., Munz, D., 1997. Stress Intensity Factors and Weight Functions. Computational Mechanics Publications, Southampton, UK. He, L.H., Swain, M.V., 2008. Understanding the mechanical behaviour of human enamel from its structural and compositional characteristics. J. Mech. Behav. Biomed. Mater. 1, 18–29, http://dx.doi.org/10.1016/j.jmbbm.2007.05.001. Hull, D., Clyne, T.W., 1996. An Introduction to Composite Materials, 2nd ed. Cambridge University Press, Cambridge163. Ji, B., Gao, H., 2010. Mechanical principles of biological nanocomposites. Annu. Rev. Mater. 40, 77–100, http://dx.doi. org/10.1146/annurev-matsci-070909-104424. Kieser, J.A., He, L., Dean, M.C., Jones, M.E., Duncan, W.J., Swain, M. V., Nelson, N.J., 2011. Structure and compositional characteristics of caniniform dental enamel in the tuatara Sphenodon punctatus (Lepidosauria: Rhynchocephalia). N. Z. Dent. J. 107, 44–50.
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von Koenigswald, W., 2000. Two different strategies in enamel differentiation: Marsupialia versus Eutheria. In: Teaford, M.F., Smith, M.M., Ferguson, M.W.J. (Eds.), Development, Function and Evolution of Teeth. Cambridge University Press, Cambridge, New York, pp. 107–118, http://dx.doi.org/10.1017/ CBO9780511542626.020. Line, S.R., Novaes, P.D., 2005. The development and evolution of mammalian enamel: structure and functional aspects. Braz. J. Morphol. Sci. 22, 67–72. Maas, M.C., Dumont, E.R., 1999. Built to last: the structure, function, and evolution of primate dental enamel. Evol. Anthropol. 8, 133–152, http://dx.doi.org/10.1002/(SICI)15206505(1999)8:4o133::AID-EVAN443.0.CO;2-F. Nalla, R., Balooch, M., Ager, J., Kruzic, J., Kinney, J., Ritchie, R., 2005. Effects of polar solvents on the fracture resistance of dentin: role of water hydration. Acta Biomater. 1, 31–43, http://dx.doi.org/10.1016/j.actbio.2004.08.002.
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Available online at www.sciencedirect.com
www.elsevier.com/locate/jmbbm
Short Communication
Micromechanical characterization of prismless enamel in the tuatara, Sphenodon punctatus Ezgi D. Yilmaza, Sabine Bechtlea, Hu¨seyin O¨zcobana, Jules A. Kieserb, Michael V. Swainc,d, Gerold A. Schneidera,n a
Institute of Advanced Ceramics, Hamburg University of Technology, Hamburg, Germany Sir John Walsh Research Institute, University of Otago, Dunedin, New Zealand c Prosthetic Department, Freiburg University, D 29106 Freiburg, Germany d Biomaterials, Faculty of Dentistry, University of Sydney, NSW 2006, Australia b
ar t ic l e in f o
abs tra ct
Article history:
Dental enamel – a naturally occurring biocomposite of mineral and protein – has evolved from
Received 16 June 2014
a simple prismless to an advanced prismatic structure over millions of years. Exploring the
Received in revised form
mechanical function of its structural features with differing characteristics is of great
23 July 2014
importance for evolutionary developmental studies as well as for material scientists seeking
Accepted 27 July 2014
to model the mechanical performance of biological materials. In this study, mechanical
Available online 4 August 2014
properties of prismless tuatara Sphenodon punctatus enamel were characterized. Using micro-
Keywords:
cantilever bending samples the fracture strength and elastic modulus were found to be
Biological materials
640787 MPa and 4276 GPa, respectively in the orientation parallel to the crystallite long axis,
Prismless enamel
which decreased in the orthogonal direction. The intrinsic fracture toughness of tuatara
Strength
enamel ranged from 0.21 MPa m1/2 and 0.32 MPa m1/2. These values correspond to the lower
Fracture toughness
limit of the range of values observed in prismatic enamel at the hierarchical level 1.
1.
Introduction
Dental enamel is the most highly mineralized tissue of vertebrates and investigations of its diversification has become an important tool to elucidate the evolutionary relationships among different species whose dietary habits, and hence the mechanical demands on their dental tissues, are closely related to their chemical composition and microstructural architecture (Koenigswald, 2000; Rensberger, 2000). For example, in lower vertebrates (fish, amphibians and
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reptiles) the physiological task of the dentition is usually limited to the capturing, piercing and holding of the prey with no contact between the opposing jaws (Line and Novaes, 2005; Kieser et al., 2011). Accordingly, they have a relative simple enamel type in contrast to most mammals, whose microstructural features exhibit a high level of complexity and diversity, which have evolved as a result of adaptations in response to an increase in stress levels imposed on teeth due to the formation of dental occlusion via chewing and grinding (Rensberger, 2000; Line and Novaes, 2005).
n Correspondence to: Hamburg University of Technology, Institute of Advanced Ceramics, Denickestraße 15, 21073 Hamburg, Germany. Tel.: þ49 40 42878 3037; fax: þ49 40 42878 2647. E-mail address: [email protected] (G.A. Schneider).
http://dx.doi.org/10.1016/j.jmbbm.2014.07.024 1751-6161/& 2014 Published by Elsevier Ltd.
journal of the mechanical behavior of biomedical materials 39 (2014) 210 –217
Although dental enamel of many animals consists predominantly of inorganic crystallites of hydroxyapatite (HAP) combined with a small amount of organic matter and water, it is evident that some interspecies differences in composition and crystallite geometry exist (Maas and Dumont, 1999). However, the main categorization of enamel structures is based on the arrangement and orientation of the crystallites. In basal species, enamel formed into parallel arranged crystallites (prismless enamel); whereas, in more advanced species HAP crystallites arranged into rod-shaped cylindrical bundles of crystallites known as “prisms” (prismatic enamel) delimited by organic sheaths and interprismatic crystallites. Considering the fact that enamel contributes to tooth stability essentially by resisting wear and fracture, evolutionary changes in enamel morphology should have enhanced its mechanical
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properties as has been often hypothesized (Koenigswald, 2000; Rensberger, 2000; Line and Novaes, 2005; Maas and Dumont, 1999; Sander, 2000) but not experimentally verified. The latter requires detailed comparison of prismless and prismatic enamel types. Although extensive research has been carried out on the mechanical characterization of the mammalian prismatic enamel (Bechtle et al., 2010; Ang et al., 2010; Bechtle et al., 2012; He and Swain, 2008), very little is known about the mechanical properties of prismless enamel (Kieser et al., 2011; Enax et al., 2013). The thickness of prismless enamel ( 100 to 200 mm) has been the limiting factor for macroscopic characterization until recent years. Through the development of smallscale mechanical characterization techniques (Bechtle et al., 2012, 2011; Chan et al., 2011, 2009; Yilmaz et al., 2013) it is now possible to isolate and characterize sub-micron scale specimens
Fig. 1 – The lower jaw of the tuatara Sphenodon punctatus used in this study (a), and a longitudinal cross-section of a tooth (b) is illustrated. Tuatara enamel composed of nano-scaled HAP crystallites extending at almost right angles from dentin-enamel junction (the interface between enamel and dentin). The crystallites have a parallel arrangement in the outer part of the tuatara enamel and they form columnar units at the inner core (c). High numbers of enamel tufts having a width of 1 lm are also present in the tuatara enamel (d). These are known to be protein-rich crack like defects. A higher magnification image of the tuft region (e) visualizes thin films and ligaments connecting and wrapping the crystallites. These are believed to be the organic remnants.
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by means of focused ion beam (FIB) machining. By using this technique the mechanical characterization of prismless enamel of tuatara Sphenodon punctatus is undertaken in this study. Tuatara S. punctatus, often referred to as a living fossil (originated around 240–250 mya), is a small reptile restricted to New Zealand (Kieser et al., 2011). The dentition of the tuatara is characterized by a single row of conical shaped teeth in the lower jaw (Fig. 1a) fitting perfectly between the two upper rows when the mouth is closed (Kieser et al., 2011). Tuatara enamel has an increasing thickness along the long axis of the tooth reaching 100 mm at the tip of the tooth. Tufts (protein-rich crack like defects) originating from the dentin-enamel junction (DEJ) and dentine tubules exist in the inner part of the tuatara enamel as shown in the longitudinal cross-section in Fig. 1b. HAP crystallites extend from the DEJ at almost right angles to the outer enamel surface (OES). At the outer enamel layer, crystallites align relatively parallel to each other, whereas diverging crystallites from a central axis that develop in a fanshaped pattern can be seen in the inner layer close to the DEJ (Fig. 1c). These are known as columnar units, which are differentiated by lines of changing crystallite orientations (Sander, 2000). Tuatara enamel is reported to be richer in protein remnants compared to mammalian enamel (Kieser et al., 2011). This can be observed in Fig. 1d showing a tuft region at higher magnification. The crystallites appear to be wrapped and connected by very thin organic protein/peptide layers (Kieser et al., 2011). Although enamel appears to experience essentially compressive forces, it was shown by simulation studies that compressive chewing loads in a vertical direction also produce horizontal tensile stresses within ellipsoidal cusps (Rensberger, 2000) and are considered critical for cracking and fracture in enamel. In this regard, the mechanical behavior under tension was of interest in this work. However, due to the size of the enamel of tuatara and difficulties in preparation of tensile specimens for typical experimental setups, micro-cantilever bending experiments were conducted, where fracture was induced in the tensile stress region of the cantilever beams. By means of notched and unnotched micro-cantilever bending tests, mechanical properties including; fracture strength, elastic modulus and, fracture toughness are analyzed.
2.
Materials and methods
The tuatara teeth sample consisted of the lower jaw of a single individual from the collection of the Sir John Walsh Research Institute, University of Otago, Dunedin, New Zealand. Three teeth were resected from the jaw using the water cooled Buehler Isomet 4000 precision saw and a very thin (150 μm) diamond saw blade. Due to their small size ( 2 mm long, 1 mm in diameter), whole teeth were glued on electron microscope sample stubs using a mounting resin. The fixed teeth were ground down using 500 grit SiC paper until a cross section was exposed as shown in Fig. 1b. The teeth were then prepared using 1200 SiC grit paper and then polished with 1 mm diamond suspension. One of the prepared teeth was etched for 20 s using 36% hydrochloric acid to enhance the HAP crystallites visibility for scanning electron
microscope investigations. The two non-etched samples were used for focused ion beam (FIB) sample preparation. Finally, specimens were sputter-coated with a thin gold-palladium layer (some nanometers thick). Notched and unnotched triangular cantilever beams were prepared with a Cross-Beam Zeiss Auriga Canion FIB system. A detailed description of the milling procedure and parameters used is in our previous publications (Bechtle et al., 2012, 2011). Both notched and unnotched cantilever beams consisted of two orientation groups, namely parallel and perpendicular to the crystallite long axis. Please note that the unnotched parallel cantilever beams were milled from the tooth sample 1, whereas the unnotched perpendicular and notched cantilever beams were milled from the tooth sample 2. The beams fabricated in the same tooth were located in approximately the same region (middle-distal with respect to the distance to cusp and 10 to 20 mm with respect to the distance to DEJ) to prevent local structural and chemical variations that is commonly observed in mammalian enamel for instance. Moreover, milling sites were chosen at locations that are free of any tufts and tubules. Representative samples and the geometrical details are demonstrated in Fig. 2. Tables 1 and 2 list the dimensions of all samples tested in this study. L denotes the loaded cantilever length that is the distance between the attached end of the cantilever and the loading point for unnotched samples and the distance between the notch and the loading point for notched samples. W is the cantilever height, B is the cantilever width. For the notched samples, the notch depth is designated by, a, and the distance of the notch from cantilever fixed end by d. FIB prepared cantilever beams were tested using an MTS nanoindenter system equipped with a Berkovich tip. The nanoindenter tip was then positioned close to the cantilever's free end using the optical system. Samples were loaded at a loading rate of 0.2 mN s 1 up to max. displacements of 3 mm. Thereafter the samples were unloaded. The load on the sample, P, and the tip displacement, s, were monitored continuously. To calculate the actual cantilever displacement, sn, the sharp indenter tip penetration into the cantilever has to be subtracted from the total indenter displacement as described previously (Bechtle et al., 2012). To do this, nanoindentation tests were performed within the bulk tuatara enamel adjacent to where the FIB beams were obtained. Zeiss Supra 55VP scanning electron microscope (3 kV, 10 6 mbar) was used for microstructural investigations after mechanical testing. For the unnotched samples, bending stress and associated strain, fracture strength and elastic modulus were calculated from the load–displacement data monitored using the following equations (Dubbel et al., 2007) for triangular shaped cantilevers: σ¼
12 P L B W2
ð1Þ
sn H L2
ð2Þ
and ε¼
where σ is the bending stress and, ε is the bending strain on the outer surface of the beam. The elastic modulus E was estimated for all samples within the linear-elastic regions
journal of the mechanical behavior of biomedical materials 39 (2014) 210 –217
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Fig. 2 – Scanning electron micrographs show the positions from which unnotched parallel (a) and unnotched perpendicular (b) tuatara micro-cantilevers were obtained. The white arrows indicate the crystallite orientation in the beams with respect to the long axis. The notched samples were produced also in parallel and perpendicular orientations. A schematic representation is shown in (d) (reprinted from Ref. Bechtle et al. (2011) with permission from the publisher) to elucidate sample geometry of the notched micro-cantilever beams as illustrated.
Table 1 – Dimensions of the FIB prepared unnotched tuatara cantilevers. Sample
Unnotched Unnotched Unnotched Unnotched Unnotched
parallel 1 parallel 2 parallel 3 perpendicular 1 perpendicular 2
L, Loaded cantilever length [mm]
W, Height [mm]
B, Width [mm]
16.76 17.73 14.92 19.70 13.19
5.84 7.74 6.04 7.94 7.46
5.44 6.43 5.50 7.46 6.38
Table 2 – Dimensions of the FIB prepared notched tuatara cantilevers. Sample
L, Loaded cantilever length [mm]
W, Height [mm]
B, Width [mm]
d, Distance of notch from cantilever fixed end [mm]
a, Notch depth [mm]
Notched parallel Notched perpendicular
17 22.3
6.19 5.02
5.16 4.57
2.32 6.543
0.92 0.39
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journal of the mechanical behavior of biomedical materials 39 (2014) 210 –217
from the slope of the stress-strains curves, namely E¼
Δσ Δε
ð3Þ
For the notched samples, the formula for the fracture toughness Kc (critical stress intensity factor) calculation is given by pffiffiffiffiffiffi KIc ¼ FI; ∇ σ πa ð4Þ where the subscript I indicates the mode I fracture (tension normal to the crack plane). FI; ∇ is the geometric function parameter for notched triangular cantilevers. Stress intensity factors for rectangular bending bars are well known from fracture mechanics handbooks; however, there is a lack of such information for bars with triangular cross-sections (Bechtle et al., 2011). In order to compute the fracture toughness for the triangular cantilevers tested, a series of finite element computations was performed for the specific notch and sample geometry, and notch position of our specimens as explained in our previous study (Bechtle
et al., 2011). The geometric function was found to depend upon variations a/W and d/W ratios. For d/WZ0.5 and 0ra/ Wr0.15 the geometric function FI; ∇ for a triangular cantilever tested in bending notched at distance d from the cantilever fixed end can be approximated by Bechtle et al. (2011). a FI;∇ ffiFI;rect 1 þ 0:87 W
ð5Þ
with the geometric function FI,rect for the rectangular bar from Fett and Munz (1997). FI;rect ¼
1:1215 3=2
ð1αÞ
5 5 1 3 α þ α2 þ 5α2 ð1αÞ6 þ expð 6:1342α=ð1αÞÞ 8 12 8 8
ð6Þ where α¼a/W. The effect of parameter d/W in the region d/ W Z0.5 was less than 1% for FI; ∇ and can be neglected. It should be mentioned here that the d/W of the notched parallel sample is 0.37 but it is considered that this discrepancy does not play a significant role.
Fig. 3 – The load–displacement curves resulting from bending tests of the unnotched (a) and notched (c) tuatara microcantilevers are illustrated. The post-test SEM image of the unnotched sample “parallel 1” is shown in (b), where a major crack is apparent at the fixed end of the cantilever. The post-test investigation of the “parallel notched” sample (d) revealed an uneven fracture surface (underneath the FIB-induced notch) with some pores and cavity like features in the retained part after fracture.
journal of the mechanical behavior of biomedical materials 39 (2014) 210 –217
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Table 3 – Mechanical properties of tuatara micro-cantilevers. Sample
σ, Strength [MPa]
ε, Strain [%]
E, Elastic modulus [GPa]
KIc, Fracture toughness [MPa m1/2]
Unnotched parallel 1 Unnotched parallel 2 Unnotched parallel 3 Mean7s.d. Unnotched perpendicular 1 Unnotched perpendicular 2 Mean7s.d. Notched parallel Notched perpendicular
581 725 566 624787 260 327 293747 – –
1.16 1.89 1.4 1.4870.37 0.7 1.36 1.0370.47 – —
49 38 40 4276 37 24 3079 – –
– – – – – – – 0.32 0.21
3.
Results
The load–displacement curves of the parallel and perpendicular unnotched samples are shown in Fig. 3a. All samples deformed in a linear elastic fashion until a sudden fracture took place. Table 3 summarizes the mechanical properties obtained from the unnotched micro-cantilevers. In the direction parallel to the crystallite long axis the average fracture strength values under bending was found to be 624787 MPa and 293747 MPa in the perpendicular direction. The average elastic modulus was respectively 4276 GPa and 3079 GPa while the strain at failure was 1.4870.37% and 1.0370.47% parallel to the crystallites and in the direction perpendicular to the crystallites. A representative post-test SEM micrograph of a fractured unnotched sample “parallel 1” is shown in Fig. 3b. A single large crack, which initiated at the fixed end of the cantilever (where the bending stress was the highest), is apparent. The crack surface that developed exposes the fractured crystallites. The load–displacement curves of the parallel und perpendicular notched samples are given in Fig. 3c. With initiation of crack propagation unstable fracture occurred. The fracture toughness KIc of the parallel and perpendicular notched samples are estimated as 0.32 MPa m1/2 and 0.21 MPa m1/2 respectively. The post-test SEM micrograph of the parallel notched sample is illustrated in Fig. 3d. The individual crystallites cannot be recognized (probably due to the resolution of the image) but an uneven fracture surface (underneath the FIBinduced notch) with some holes and cavities is seen.
4.
Discussion
To begin with, it is important to mention that due to the long operating time and complexity of the experimental set-up conducted in this study (which is the only way to examine the required material properties from these tissues having an extremely small volume), only a limited number of samples could be tested. The values obtained here from tuatara enamel do not allow us to determine an absolute conclusion about the mechanical properties of the prismless enamel due to the low statistical power. However, given the fact that the mechanical data of the enamel from basal species are largely lacking in the literature, we consider that even these small numbers of samples provide valuable information.
The microscale bending strength of mammalian (bovine and human) enamel was reported in the previous studies as 540– 1420 MPa in parallel and 380–560 MPa in perpendicular directions (Bechtle et al., 2012; Chan et al., 2009, 2010). And the microscale bending modulus of mammalian enamel was reported as 42–60 GPa in parallel and as 22–33 GPa in perpendicular direction (Bechtle et al., 2012; Chan et al., 2009, 2010). The strength and elastic modulus values estimated here for tuatara enamel are close to the lower limits of the previously reported values in general and this can be explained by the lower degree of mineralization in basal prismless enamel. A mineral volume range of 87–96% has been measured for mammalian (human and bovine) enamel (Elliott, 1997), whereas there is only one report referring the chemical composition of tuatara enamel. Kieser et al. demonstrated by Raman spectroscopy that tuatara enamel, tuatara dentin and human dentin share similar chemical compositions (Kieser et al., 2011). Given the fact that mineral content of human dentin is 50 vol% (Bertassoni and Swain, 2012), the mineral content of tuatara enamel can be considered as 50 vol%. As with most of the biological materials, tuatara enamel is found to be anisotropic. Almost two-fold lower bending strength values are obtained from the tuatara perpendicular in comparison to the tuatara parallel micro-cantilevers. This is most probably due to the load transfer from the matrix to the HAP fibers when loaded in parallel direction. However, the difference between the mean elastic modulus values of tuatara parallel and tuatara perpendicular sample groups was statistically not significant. This is astonishing and not expected if the Voight and Reuss models are applied (Hull and Clyne, 1996) to the parallel and perpendicular oriented micro-cantilevers. It reflects the fact that HAP crystallites are not perfectly aligned parallel and perpendicular to the loading direction. Moreover, another possibility is that the mineral content of the tooth sample 2 (where the perpendicular unnotched samples were milled) could be slightly higher than the tooth sample 1 (where the parallel unnotched samples were milled) reducing the expected variation in stiffness due to anisotropy. Based on our findings, the fracture toughness values obtained in tuatara enamel appears to be slightly lower than the fracture toughness values of bovine enamel 0.4–0.9 MPa m1/2 estimated using the same testing method with identical sample dimensions (Bechtle et al., 2011). The drawback of the FIB method is dehydration and exposing cutting edges of the sample to gallium ions, which can generate large amounts of heat. In order to
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assess whether this methodology alters the mechanical properties in human enamel or not, Chan et al. tested the hardness and elastic modulus of enamel samples before and after the treatment and measured no difference (Chan et al., 2009). However, this may not be valid for the fracture toughness which is reported to be influenced by the degree of hydration (Baldassarri et al., 2008). Water is known to be a plasticizer and additionally modify the protein structure and influence its deformability (Nalla et al., 2005). As a consequence, the fracture toughness estimated here could have been underestimated. Please note that to be able to conclude that the fracture toughness of prismless enamel of reptiles are less than prismatic enamel of mammals, larger numbers of samples from the former group should be tested in the future. Analytical models (Hull and Clyne, 1996; Ji and Gao, 2010) for predicting the elastic constants of simple composite structures cannot be implemented for tuatara enamel due to the structural mismatch. It is very difficult to estimate whether the crystallites have a straight or curvy outline from the SEM micrographs shown in Fig. 1. In addition, structures like tufts and tubules interrupt the continuity of the crystallites. Characterization of the hard particle length and aspect ratio is essential for mechanical modeling since it determines the load transfer in the composites. Even though the length of the crystallites in tuatara enamel extending from DEJ and running to the boundaries of the columnar units (see Fig. 1c) appears between 3 and 5 mm, SEM images is not very reliable in this regard since the appearance of the crystallites depends on the sectioning angle and sample preparation.
5.
Conclusion
In order to understand the reasons for evolutionary development in enamel morphology from simple to high order hierarchical structure, the mechanical properties of different enamel types should be compared. Although extensive research has been carried out on highly developed mammalian (human and bovine) enamel, only a few studies have attempted to investigate basal reptilian enamel. To fill this gap in the existing literature, the basic mechanical properties of tuatara S. punctatus enamel, which possess only one level of hierarchy, is investigated in thus study. In summary, by characterizing at the same hierarchical level the tuatara enamel appeared to be slightly weaker than mammalian enamel, but the difference was not pronounced. This suggests that the greater mechanical advantage of advanced prismatic mammalian enamel over basal prismless enamel may lie in the macroscopic fracture behavior where the toughening mechanisms operating at higher order hierarchical levels can contribute.
Acknowledgment The authors wish to express gratitude to German Research Foundation (Grant no. SCHN 372/18-1) for financial support. The authors gratefully acknowledge the technical support of Daniel Laipple and Uwe Lorenz (Helmholtz-Zentrum Geesthact) for the FIB system.
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