Enamel microstructure—a truly three-dimensional structure

Journal of Human Evolution 45 (2003) 81–90 News and Views Enamel microstructure—a truly three-dimensional structure Gabriele A. Macho a*, Yong Jiang a, Iain R. Spears b a Hominid Palaeontology Research Group, Department of Human Anatomy and Cell Biology, The University of Liverpool, Liverpool L69 3GE, England, UK b Sport and Exercise Subject Group, School of Social Sciences and Law, University of Teesside, Middlesbrough TS1 3BA, England, UK Keywords: Computer modeling; 3D Enamel microstructure; Hominoids; Tooth development; Biophysical processes Introduction Paleoanthropological studies often center on teeth, not only because these elements are commonly preserved in the fossil record, but because they apparently contain a wealth of information with regard to development, phylogeny, and function. However, despite a plethora of studies, some fundamental problems are still unresolved. For example, while it is recognized that the 3dimensional arrangement of enamel prisms may hold important information with regard to phylogeny (von Koenigswald and Sander, 1997) and function (Rensberger, 2000), many paleoanthropological studies have thus far relied on investigating enamel microanatomy as a 2dimensional structure (e.g., Dean et al., 2001). This is mainly due to difficulties in visualizing and quantifying the 3-D structure of prisms. In order to overcome these limitations a computer model was developed (Jiang et al., 2003) which attempted to simulate the effects of biophysical processes governing enamel formation in modern humans (adapted from Osborn, 1970). Here we extend our model and present preliminary data on inter* Corresponding author. Tel.: +44-(0)151-794-5466; fax: +44-(0)151-794-5517 E-mail address: gama1@liverpool.ac.uk (G.A. Macho). specific variation in prism arrangement among primates. Furthermore, during our work to recreate the 3D microstructure of prismatic enamel it became increasingly clear that there are not only limitations with previous dental growth studies, but that these studies are based on fundamentally different concepts regarding evolutionary processes from those assumed in our approach. These limitations and differences will be highlighted also. Material and methods As part of a larger study we have developed a graphical computer model (C++, OpenGL) that allows the recreation of the 3-dimensional arrangements of prisms from broken surfaces (Jiang et al., 2003). The mathematical algorithms underlying these models are based on biophysical processes governing enamel formation in modern humans and, presumably, dogs (Osborn, 1970). Specifically, the model relies on the fact that layers of prisms differentiate successively apicocervically, that the advancing enamel front retains its integrity throughout enamel formation, and that cell movement is more constrained closer to the dentino-enamel junction (DEJ) than it is towards the outer enamel surface (OES). These assumptions suffice to create the enamel microstructure of 0047-2484/03/$ - see front matter  2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0047-2484(03)00083-6 82 G.A. Macho et al. / Journal of Human Evolution 45 (2003) 81–90 modern human teeth (Jiang et al., 2003). It should be noted that thus far no provision has been made to account for changes in prism diameter from the DEJ to the OES. In order to test the usefulness of our approach (i.e., computer modeling) for the creation of various enamel microstructures, transverse and longitudinal breaks in molars (and 1 premolar) were analyzed for 12 individuals of 6 species (2 Gorilla gorilla, 3 Pan troglodytes, 3 Pongo pygmaeus, 1 Hylobates sp., 2 Symphalangus syndactylus, 1 Papio sp.). The material consisted of flakes of enamel broken off dried skulls in our collections. In some instances, these breaks affected the integrity of the DEJ, and a few microns close to the DEJ are therefore missing. The material was broken in a transverse and longitudinal plane as apically as possible, using small chisels and hammers. The rationale underlying this procedure is the fact that enamel tends to break along, rather than across, prisms (Boyde, 1989; Lin and Douglas, 1994; Rasmussen et al., 1976) and, given the complex nature of prism decussation, this method was deemed better suited to visualize and/or determine the actual prism paths. All transverse breaks were along the buccal or lingual wall of the tooth, and were never below mid-crown level, although one Pongo molar fractured relatively low. The broken surfaces were cleaned and etched in 5% HCl for 20 seconds, and viewed with SEM (ISS) using backscattered mode (BSE). SEM images were then imported into our program and displayed and, after appropriate scaling, individual prisms were traced digitally. This was done first for transverse breaks [pattern of prism deviation in the x-z plane (Fig. 1)], and subsequently for the longitudinal breaks [patterns of prism deviation in the y-z plane (Fig. 1)] of the same specimen, thus creating the 3-dimensional pathway of a prism. From the longitudinal break we subsequently determined cycle length (i.e., the number of layers between same-phased prisms) and the angle between the c-axis of the prism and the DEJ (Fig. 1). Due to prism decussation, clean breaks could not always be obtained and bundles of prisms were broken across their long-axis. In order to account for this fact, cutting planes were introduced in our models for verification purposes; this allowed the comparison between the SEM images and the models created. Results Models could be created for all specimens analyzed here (Figs. 2, 3) and the pathways of prisms as viewed in the transverse (x-z) and longitudinal (y-z) planes are shown in Fig. 3. All species exhibit deviations in the transverse plane, while deviations in the longitudinal plane are markedly different. Notably, modern humans exhibit substantially less deviation in this plane than the other primates (Fig. 3). Common to all primates analyzed here, the regularity with which the prisms go into and out of the plane in longitudinal view suggests rhythmic phase changes in prism undulation apicocervically. These observations suffice that small pieces of enamel could be created for each individual (Fig. 2) and the co-ordinates of prism pathways saved for further analyses (Fig. 3). Despite the similarities in enamel structures and the small sample sizes, distinct patterns among species are found. It would appear that even closely-related species (e.g., the hominoids) differ substantially in their enamel microstructures when analyzed in the three dimensions (Fig. 3). For example, while gorillas and chimpanzees are similar in showing relatively little prism deviation in terms of amplitude and frequency in the x-z plane they differ fundamentally in the y-z plane. Prisms along the z-axis in longitudinal breaks take a sinusoidal pathway in chimpanzees, whereas they do not in gorillas. In this regard, the prism path of gorillas appears unique. Pongo shows the greatest prism undulations, not only with regard to frequency and amplitude of deviation in the transverse plane, but also in the longitudinal plane. While all primates studied thus far, except Papio and modern humans, show slight undulations horizontally as seen in Fig. 2Cb, this situation is most pronounced in orangutans. This extreme decussation in both planes results in Pongo having the largest ratio of projected prism length as measured from the transverse break, and the actual prism length (Fig. 4). Conversely, gibbons show the simplest structures with very little prism G.A. Macho et al. / Journal of Human Evolution 45 (2003) 81–90 83 Fig. 1. Planes and axes of reference used in this study. In (A) the planes of fractures are indicated, whereby (a) shows the intact tooth, (b) the longitudinal break and (c) the transverse break. The z-direction is set perpendicular to the DEJ (B, C). The orthogonally aligned y- and x-directions are aligned approximately along the long-axis of the tooth and transverse to the long-axis of the tooth, respectively. In addition, in a 3-dimensional view (D) of a simplified model, frontal (x-y), transverse (x-z) and longitudinal (y-z) planes are also shown. DEJ = dentino-enamel junction; OES = outer enamel surface. deviation; nonetheless decussation planes do occur. Finally, the microstructure observed in Papio is noteworthy as it shows high frequency curves in the inner part of the enamel combined with additional sinusoidal curves in the x-z and y-z planes in this area (Fig. 2Aa). Discussion Tooth size, shape and microanatomy are commonly considered to contain important information with regard to phylogeny and function (e.g., von Koenigswald and Sander, 1997), 84 G.A. Macho et al. / Journal of Human Evolution 45 (2003) 81–90 Fig. 2. SEM pictures of transverse breaks are shown for (A) Papio (a), (B) Pan (a) and (C) Pongo (a and b at greater magnification). Next to each SEM picture is the reconstructed enamel piece (Ab, Bb, Cc) and a simplified view of one cycle in transverse view (Ac, Bc, Cd). G.A. Macho et al. / Journal of Human Evolution 45 (2003) 81–90 85 Fig. 3. The tracked enamel prism pathways shown in the longitudinal (left) and transverse (right) planes for (a) Homo (b) Pan, (c) Gorilla, (d) Pongo, (e) Hylobates, (f) Papio. Data for Homo are taken from Jiang et al. (2003). whereby evolutionary changes in dental features are attributed to changes in ecology and dietary adaptations (e.g., Evans and Sanson, 2003; von Koenisgwald et al., 1987). While overall tooth size, shape and enamel thickness have been the subject of many anthropological studies, an assessment of the microanatomy has been wanting. This is in large part due to the difficulties in visualizing and quantifying the complex 3-dimensional arrangement of prisms. To this end, we have developed a computer model that, despite its limitations, makes it possible to recreate small enamel pieces from broken surfaces. Although sample sizes are relatively small, our approach (a) further confirms the functional significance of prism decussation and (b) contributes towards an understanding of the processes governing the development of prism decussation. (a) Functional significance of prism decussation Prism decussation is generally considered to have evolved in large-bodied, thick enameled 86 G.A. Macho et al. / Journal of Human Evolution 45 (2003) 81–90 Fig. 4. The percentage differences between the straight prism length in transverse view and the ‘true’ prism length (taking into account the 3-dimensional deviations) are plotted on the left axis, while the ‘true’ prism length is shown on the right axis. species as a crack-stopping mechanism (von Koenigswald et al., 1987). Despite its possible importance for evolutionary inquiry, an assessment of prism decussation from longitudinal sections has long been considered problematic for technical reasons (e.g., Boyde, 1989; Osborn, 1968; Risnes, 1986) and these will not be reiterated here. It should also be noted that the start of prisms at the DEJ is often offset in relation to the plane of the outer straight prisms (Fig. 3); this constitutes another crack-stopping mechanism (Jiang et al., 2003). Any longitudinal crack, or cut, through the long-axis of the tooth would therefore travel across the undulating prism path; this would complicate an assessment of prism decussation from longitudinal sections even further. Despite these difficulties, Dean (1998) concluded that it is possible to determine prism decussation from longitudinal sections; this study forms the basis for calculations of cuspal formation times of his and his col- laborators’ subsequent work (e.g., Dean et al., 2001). Based on analyses of ground sections, Dean (1998) proposed that whereas Pongo exhibits little decussation, prism decussation in Pan is similar to that in modern humans. The opposite seems to be the case. Specifically, Pongo was found to have the most extreme decussation in the present study (Figs. 2–4). Given the large body size of orangutans, as well as their dietary and functional adaptations (Lucas et al., 1994), such findings appear more reasonable. Even when the curvature in the y-z plane for Pongo is disregarded, the percentage difference is greater than it is in Pan and Gorilla, and only marginally smaller than in Homo1. As all tooth fragments were fractured at 1 It should be noted that reanalyses of Fig. 2 given in Risnes (1986) using different methods (i.e., string, calculations, our model) shows that the 15% difference obtained by Risnes with a map distance tracer is grossly overestimated. G.A. Macho et al. / Journal of Human Evolution 45 (2003) 81–90 comparable levels (i.e., mid-crown) the results are unlikely to be an artefact. Consequently, previous propositions about decussation based on histological analyses of ground sections need to be reappraised both from a methodological and biological point of view (e.g., Dean, 1998; Schwartz and Dean, 2001). The fact that enamel decussation is extreme in three planes in Pongo, but only in one plane in Homo, suggests that these teeth are particularly strong under multi-directional loads. While having a similar ratio between projected and true prism length, humans exhibit virtually no crack-stopping mechanism in the transverse plane. Indeed, when preparing the specimens by propagating cracks across control planes, the enamel of Homo was noticeably easier to fracture than that of Pongo (Fig. 2C), despite the fact that the latter came from dried specimens. Pongo thus seems highly derived in its microstructure compared to the other primates studied here. In addition, it is noteworthy that both Pongo and Papio exhibit vertical decussation planes (Fig. 2). However, the morphologies of these decussation planes differ between these primates, and also from those reported for the rhinoceros (Rensberger and von Koenigswald, 1980). Although the functional consequences of these different designs need to be explored further, our findings clearly support proposals that there exists a general relationship between size, function and decussation. (b) Processes underlying enamel formation For each species studied here small pieces of enamel could be recreated using our computer model (Jiang et al., 2003). Given that the mathematical algorithms underlying these models are based on assumed biophysical processes governing modern human tooth formation (Osborn, 1970), it is reasonable to conclude that primate, and probably mammalian, enamel formation follows the same rules. The imbalance of forces due to cell adhesion of the advancing enamel front, on the one hand, and the pressure exerted by the secreting ameloblasts, on the other, may be sufficient to cause the undulating trajectories of prisms. To test this proposition further, a forward dynamics 87 micro-model is currently being developed to explore the possible kinematic/kinetic relationships. However, fundamental to these calculations are the inertial characteristics of the ameloblasts, which in turn are dependent on secretion rates and diameters of the prisms. A dilemma has arisen in this regard between what we believe to be logical considerations based on a 3-dimensional model, as well as our previous work, and some of the existing literature on enamel development. Dental developmental studies that report “secretion rates” are considered particularly valuable for palaeoanthropological inquiry (GuatelliSteinberg, 2002). However, these investigations have largely failed to produce insights into evolutionary processes other than those that can be explained by changes in body and brain size alone (Macho, 2001). The life span of ameloblasts, and consequently tooth crown formation, are apparently highly correlated with brain/body size among primates. As tooth size and shape is generally considered the target of selection (e.g., Evans and Sanson, 2003), it is reasonable to infer that modulation of secretion rates may be a mechanism by which this can be achieved. Certainly, this would be the case when we attempt to simulate the process of formation. Unfortunately, while the temporal constraints on tooth formation times are acknowledged by Dean and co-workers (2001), the importance of differences in secretion rates are not related to other aspects of size, shape and development. Furthermore, not only do teeth vary in size and shape, but prism diameter also differs between species and increases from the DEJ towards the OES in order to accommodate the larger outer radius. Despite such considerations no attempts have been made to scale for these differences in previous growth studies (e.g., Dean et al., 2001; Schwartz and Dean, 2001). Hence, the linear, one-dimensional “secretion rates” are inappropriate for the description of the volume of material secreted (i.e., of the 3-dimensional prism structure), cell activity and/or growth rates. Interpretation of the results of growth studies within a coherent evolutionary and developmental framework is therefore hampered. Finally, inspection of our 3-dimensional models (Fig. 2) and the data derived from them (Fig. 3) 88 G.A. Macho et al. / Journal of Human Evolution 45 (2003) 81–90 Fig. 5. The prism path of the P4 for Pongo shown in Fig. 3 is reproduced. In (A) the linear secretion rate from the dentino-enamel junction (DEJ) towards the outer enamel surface (OES) is either unchanged (a), gradually increased (b), or decreased (c). A hypothetical stria of Retzius (dark line) is then superimposed. In all instances, S-shaped striae of Retzius are the result of marked prism undulations in the y-z plane. (B) depicts the proposal of Dean and Shellis (1998) that prism width (height) decreases from the DEJ toward the OES thus resulting in S-shaped striae (and change in prism direction) farther cervically. The bundle of dark prisms is that shown in (A) and empirically derived from SEM images. Prism curvature straightens out farther cuspally. If this were the case, and without increase in prism width or interprismatic matrix, radial and/or longitudinal weaknesses or cracks would result. indicate that prism undulations may determine the appearance of growth markers such as striae of Retzius. As a case in point, it is evident that the pronounced sinusoidal curvature of prisms in the longitudinal plane in orangutans would result in S-shaped striae of Retzius irrespective of changes in secretion rates and/or prism diameters (contra Dean and Shellis, 1998) (Fig. 5). Owing to the larger outer radius of the tooth surface when compared to that of the DEJ, it is implausible that prism width would decrease towards the outer enamel surface as suggested by Dean and Shellis (1998), at least not without concomitant increase in interprismatic matrix, which was not observed either. If it did, circumferential (or longitudinal) openings in Pongo teeth would be the result. In light of these considerations, and bearing in mind that all species in the current study exhibit some decussation in the z-y plane, it is also unsurprising that other species occasionally show S-shaped striae of Retzius (Dean and Shellis, 1998); they would occur when the ameloblasts move in a sinusoidal way in the z-y plane from the DEJ towards the OES (Fig. 3). It is not necessary to invoke unique developmental processes in explaining S-shaped striae, when inspection of prism pathways/decussation would suffice. This may seem a minor point of emphasis, but the evolutionary and biological implications could be fundamental. Given current thinking in evolutionary biology, it seems more plausible to assume that selection acts on the phenotype, i.e., the tooth and its microanatomy, rather than the processes underlying its formation. Owing to its functional significance, enamel decussation is generally considered to be under selection pressure (von Koenigswald and Sander, 1997; von Koenigswald et al., 1987; Rensberger, 2000). If, on the other hand, selective G.A. Macho et al. / Journal of Human Evolution 45 (2003) 81–90 pressures acted on secretion rates and prism width, as implied by Dean and Shellis (1998), it would be interesting to know what these selection pressures were. The overwhelming evidence for evolutionary adaptation (e.g., Rose and Lauder, 1996) would also need to be explained. Conclusions As part of a larger study to determine the functional importance of enamel decussation and the dietary adaptations of extinct species, we have developed a computer model which is based on mathematical algorithms derived from biophysical processes. The success of our model implies that primate enamel formation is governed by comparable processes. Despite these commonalities there are considerable systematic differences in microstructure among even closely related species, suggesting that enamel microstructure could become a valuable feature in phylogenetic and/or functional inquiry. Our preliminary data suggest that enamel decussation may indeed be an adaptation to functional demands (e.g., a crackstopping mechanism in large-bodied, thickenameled species, such as the orangutan, which are capable of high bite forces). In addition, our study has noted limitations with regard to the methodology, findings and interpretations of previous growth studies. While we acknowledge that differences in results with regards to tooth formation times may not matter too much in broad interspecies comparisons (Macho, 2001), several aspects of dental growth studies are problematic for heuristic reasons; such investigations give the impression that the results are empirically derived, the histological methods are well-proven, and the sectioning of fossil teeth is a worthwhile pursuit. Based on our work, none of these seems to be the case. Perhaps even more fundamental, dental growth studies appear to imply that growth processes themselves, rather than the phenotype, are the target of selection. Here we adhere to traditional thinking in evolutionary biology, which allows us to integrate our findings within a coherent framework consistent with evolutionary theory, functional adaptation, as well as developmental and biophysical processes. 89 Acknowledgements This work was supported by The Leverhulme Trust (F/00 025/A). We thank Prof. Jim Gallagher for comments on the manuscript. References Boyde, A., 1989. Enamel. In: Berkovitz, B.K.B., Boyde, A., Frank, R.M., Ho °hling, H.J., Moxham, B.J., Nalbandian, J., Tonge, D.H. (Eds.), Teeth. Springer Verlag, Berlin, pp. 309–463. Dean, M.C., 1998. A comparative study of cross striation spacings in cuspal enamel and of four methods of estimating the time taken to grow molar cuspal enamel in Pan, Pongo and Homo. J. Hum. Evol. 35, 449–462. Dean, M.C., Shellis, R.P., 1998. Observations on stria morphology in the lateral enamel of Pongo, Hylobates and Proconsul teeth. J. Hum. Evol. 35, 401–410. Dean, C., Leakey, M.G., Reid, D., Schrenk, F., Schwartz, G.T., Stringer, C., Walker, A., 2001. Growth processes in teeth distinguish modern humans from Homo erectus an earlier hominins. Nature 414, 628–631. Evans, A.R., Sanson, G.D., 2003. The tooth of perfection: functional and spatial constraints on mammalian tooth shape. Biol. J. Linn. Soc. 78, 173–191. Guatelli-Steinberg, D., 2002. Book Reviews. Am. J. Phys. Anthrop. 117, 277–279. Jiang, Y., Spears, I.R., Macho, G.A., 2003. An investigation into fractured surfaces of enamel of modern human teeth: a combined SEM and computer visualization study. Archs. Oral Biol. 48.44, 449–457. von Koenigswald, W., Sander, P.M., 1997. Tooth Enamel Microstructure. A.A. Balkema, Rotterdam. von Koenigswald, W., Rensberger, J.M., Pfretzschner, H.U., 1987. Changes in the tooth enamel of early Paleocene mammals allowing increased diet diversity. Nature 328, 150–152. Lin, C.P., Douglas, W.H., 1994. Structure-property relations and crack resistance at the bovine dentin-enamel junction. J. Dent. Res. 73, 1072–1078. Lucas, P.W., Peters, C.R., Arrandale, S.R., 1994. Seedbreaking forces exerted by orang-utans with their teeth in captivity and a new technique for estimating forces produced in the wild. Am. J. Phys. Anthrop. 94, 365–378. Macho, G.A., 2001. Primate molar crown formation times and life history evolution revisited. Am. J. Primatol. 55, 189–201. Osborn, J.W., 1968. Evaluation of previous assessments of prism directions in human enamel. J. Dent. Res. 47, 217–223. Osborn, J.W., 1970. The mechanism of ameloblast movement: a hypothesis. Calc. Tiss. Res. 5, 344–359. 90 G.A. Macho et al. / Journal of Human Evolution 45 (2003) 81–90 Rasmussen, S.T., Patchin, R.E., Scott, D.B., Heuer, A.H., 1976. Fracture properties of human enamel and dentin. J. Dent. Res. 55, 54–64. Rensberger, J.M., 2000. Pathways to functional differentiation in mammalian enamel. In: Teaford, M.E., Smith, M.M., Furgerson, M.W.J (Eds.), Development, Function and Evolution of Teeth. Cambridge University Press, Cambridge, pp. 252–269. Rensberger, J.M., von Koenigswald, W., 1980. Functional and phylogenetic interpretation of enamel microstructure in rhinoceroses. Paleobiology 6, 477–495. Risnes, S., 1986. Enamel apposition rate and the prism periodicity in human teeth. Scand. J. Dent. Res. 94, 394–404. Rose, M.R., Lauder, G.V. (Eds.), 1996. Adaptation. Academic Press, San Diego. Schwartz, G.T., Dean, C., 2001. Ontogeny of canine dimorphism in extant hominoids. Am. J. Phys. Anthrop. 115, 269–283.