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Journal of Human Evolution 62 (2012) 424e428

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Journal of Human Evolution

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Tracking cellular-level enamel growth and structure in 4D with synchrotronimaging

Paul Tafforeau a,*, John P. Zermeno b, Tanya M. Smith b

a European Synchrotron Radiation Facility, 38043 Grenoble, FrancebDepartment of Human Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA

a r t i c l e i n f o

Article history:Received 31 May 2011Accepted 6 January 2012Available online 3 February 2012

Keywords:Enamel microstructureDental morphologyEnamel prismsTooth developmentDecussation

* Corresponding author.E-mail address: paul.tafforeau@esrf.fr (P. Tafforeau

0047-2484/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.jhevol.2012.01.001

other words, while enamel is more resistant to abrasion whenprisms are perpendicular to the occlusal surface (Rensberger and

model is based on 2D observations of cut or fractured teeth, and hasyet to be empirically validated.

Enamel microstructure

Resolving the three-dimensional (3D) organization of complexbiological microanatomy is of fundamental importance for under-standing tissue development and function. Dental hard tissues,including enamel and dentine, have been of particular interestsince the development of light microscopy (reviewed in Boyde,1989; Smith, 2008). A special property of these tissues is thatthey permanently record their own spatiotemporal development,and do not remodel in response to use. In mammalian enamel,continuous “fossilized tracks” known as prisms are formed byenamel-secreting cells (ameloblasts) during tooth growth (Fig. 1A).As enamel is secreted and mineralized, hydroxyapatite crystallitesare bound together to form these long thin prisms, which runcontinuously from the enamel-dentine junction (EDJ) to the enamelsurface (Fig. 1B). Prisms are separated from each other (or frominterprismatic enamel) by discontinuities among peripheralbundles of crystallites, known as prism sheaths (Fig. 1C).

In rodents and most mammals with first molar breadthsexceeding 4 mm, enamel microstructure has a complex 3D orga-nization, with alternating undulations of grouped prisms, known asdecussation (crossing) (Fig. 2A) (see reviews in Boyde, 1969;Koenigswald and Sanders, 1997). Prism decussation is believed toserve as structural reinforcement during mastication (Pfretzschner,1988; Rensberger, 1997; Lucas et al., 2008a,b; Chai et al., 2009). In

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Koenigswald, 1980; Koenigswald et al., 2011), cracks may propa-gate more easily between prisms that run parallel to each other(Koenigswald et al., 1987; Lucas et al., 2008a; Chai et al., 2009). Thusenamel microstructure may reflect biomechanical adaptations tophysical constraints and/or material properties of dietary items(Lucas et al., 2008a,b; Chai et al., 2009). Macho and colleagues haverecently proposed a model to explain the 3D organization ofhominoid enamel and to infer aspects of hominin dietary ecology(Macho et al., 2003; Macho and Shimizu, 2009, 2010). However, this

Remarkably, enamel prisms permanently record their dailyformation, represented by circadian features termed cross-striations and laminations (Fig. 2B) (Boyde, 1989; Bromage, 1991;Smith, 2006; Tafforeau et al., 2007). Identification of this internalbiological rhythm permits quantification of secretion rates, toothformation times, and age at death in juveniles (reviewed in Smith,2008; Antoine et al., 2009). This, in turn, facilitates the study ofprimate growth and development with greater precision thantraditional skeletal techniques, and it has been of particular interestfor reconstructing the evolution of hominin developmentalpatterns (e.g., Bromage and Dean, 1985; Dean et al., 2001; Smithet al., 2010). However, conventional assessments of develop-mental time, as well as functional design, often rely on the criticalassumption that enamel prisms run from the EDJ to the toothsurface with predictable 3D geometry and tight cellular cohesion(Risnes, 1986; Macho et al., 2003). Given the potential for signifi-cant variation in these characteristics, the establishment of cuspalenamel formation time has not proven easy, with a number ofdifferent methods having been devised for its calculation (Dean,1998; Smith, 2008). Attempts to document prism orientationhave relied on electron microscope imaging of sectioned or frac-tured surfaces (e.g., Boyde, 1969; Macho et al., 2003), confocalimaging of subsurface features (Radlanski et al., 2001; Dean, 2004),and/or serial sectioning and extrapolation between slices(Helmcke, 1967; Gantt, 1977; Hanaizumi et al., 1996, 1998;Radlanski et al., 2001). Precise 3D imaging of enamel microstruc-ture within intact teeth has been a methodological impossibility,limiting the extent to which its functional design and develop-mental constraints may be fully understood.

Figure 1. Micrographs of primate enamel showing enamel prisms. A) Scanning elec-tron micrograph of naturally fractured developing chimpanzee enamel. Enamel prismsrun from left to right; the exposed ends on the right show the impressions left by theenamel-forming cells (Tomes’ process pits of ameloblasts), which were removed withplasma ashing. Scale bar is equal to 0.1 mm. Image courtesy of Lawrence Martin. B)Transmitted light micrograph of a histological thin section of a human third molar.Enamel prisms (slightly curved vertical lines) run from the enamel-dentine junction(dark boundary above the conical dentine horn tip) to the enamel surface (upperboundary); a distance of more than 2.5 mm. Scale bar is equal to 0.5 mm. C) Scanningelectron micrograph of fossil primate enamel showing circular prisms, surrounded byprisms shealths (crystallite discontinuities) and interprismatic enamel (prepared byphosphoric acid etching as in Boyde (1989). Scale bar is equal to 0.005 mm.

Figure 2. Scanning electron micrographs of primate enamel showing prism structureand circadian features. A) Backscattered electron image of a chimpanzee upper molarcusp showing decussation. Prisms run in approximately vertical twisting courses fromthe enamel-dentine junction (dark boundary in the lower left) to the enamel surface(upper boundary). Image courtesy of Lawrence Martin. Scale bar is equal to 0.1 mm. B)Secondary electron image of a naturally fractured fossil ape tooth showing layers ofnearly vertical enamel prisms cross-cut by fine daily cross-striations (alternating lightand dark lines). Scale bar is equal to 0.1 mm.

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Synchrotron virtual imaging

This study builds upon a preliminary report on synchrotronimaging of primate dental tissues (Tafforeau and Smith, 2008) byemploying substantial improvements in acquisition techniques,processing protocols for data reconstruction and optimization, and3D segmentation tools. Here we reveal the fundamental units ofenamel microstructure in living and fossil primate teeth bydetailing a protocol for isolating enamel prisms, and present thefirst segmented cohorts of prisms through the full thickness ofdental enamel.

Data were collected on beamline ID19 of the EuropeanSynchrotron Radiation Facility (ESRF) in Grenoble, France usinglocal propagation phase contrast X-ray synchrotron micro-tomography (Tafforeau et al., 2006, 2007; Tafforeau and Smith,2008). Results from two primate teeth are presented here: a chim-panzee (Pan troglodytes) M2 developing crown (detailed inTafforeau and Smith, 2008), and a Neanderthal (Homo nean-derthalensis) M1 that reached crown formation a fewmonths beforedeath (Engis 2 juvenile detailed in Smith et al., 2010). Phase contrastscans were performed at an isotropic voxel size of 0.678 mm witha beam monochromatized using a multilayer mirror, propagationdistance of 150 mm, and energy of 40 keV for the chimpanzee and

Figure 3. Synchrotron image of segmented prisms coursing the entire enamel thickness of a developing chimpanzee molar. Enamel prisms in the upper lateral enamel run from theenamel-dentine junction (EDJ) (lower right) to the tooth surface (upper left) (animated in Videos S1 and S2). Individual continuous prisms are represented by unique colors. Notethe difference in the degree of prism decussation in mesial and lateral perspectives, represented by the direct and shadowed perspectives, respectively. The shadow projection of theprisms on the background enamel virtual section was obtained by using a virtual light source from the top right corner and projecting the prism path with a 60� vertical angle fromthe virtual slice, perpendicular to the general prism direction. Cross-sectional boxes illustrate seven enamel prisms that are tightly packed near the EDJ (lower box) and relativelydispersed just below the enamel surface (upper box). Daily lines (cross-striations) can be seen as fine, paired light and dark bands on enamel prisms in the left side of the virtualhistological slice (background); the total amount of time represented by these prisms is approximately 222 days. Scale bar is equal to 0.25 mm.

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52 keV for the fossil sample.We used a CCD FreLoN (Charge CoupledDevice Fast Readout Low noise) camera mounted on a microscoperevolver system with a 20 mm thick europium doped GGG (Gado-linium Gallium Garnet) scintillator to capture 1500 equally-spacedprojections over 180� in continuous rotation mode to improveresults in local tomography (Lak et al., 2008).

Radiographs were processed using tools developed at the ESRFfor enamel processing (including corrections for flatfield, darkfield,ring artifacts, small movements, and low frequencies normalization)(Tafforeau, 2004; Lyckegaard et al., 2011). Volumes were recon-structed using a filtered back-projection algorithm (PyHST, ESRF)adapted for local tomography, and thefinal sliceswere converted into

Figure 4. Synchrotron image of a Neanderthal molar showing a group of enamelprisms in two orientations. Enamel prisms in the mid-lateral enamel run from theenamel-dentine junction (EDJ) (left scalloped vertical line) to the tooth surface (right).The upper panel shows a mesial section (cusp tip is toward the top, cervix towards thebottom), and the lower panel shows a transverse plane perpendicular to the upperpanel. Prism shadows show cohort orientations from a 45� perspective, illustrating theirregular geometry. Scale is equal to 0.2 mm.

P. Tafforeau et al. / Journal of Human Evolution 62 (2012) 424e428 427

16-bit TIFF stacks for 3D processing. Processed image stacks wereimported into VG Studio MAX 2.0 (Volume Graphics, Heidelberg,Germany) formanualprismsegmentation.A3Ddrawtoolwasusedtoselect and add spherical portions of the volume to a region of interest(ROI). Individualprismswere segmented intouniqueROIsbyscrollingthrough the slices and adding spherical portions in a point-and-clickmethod.Segmentationwasdoneperpendicular to theprismlongaxis,and orthogonal views were used concurrently to ensure the 3Dintegrity of the prism. A cohort of prisms was chosen near the EDJ toensure the identity of the center prism; these were subsequently re-segmented, and intervening prisms were located and followedbetween cohort prisms to confirm the accuracy of prism identifica-tion. For a small sectionnear theEDJ, prismswerenot clearenough forindividual segmentation; this section is indicatedwithauniform lightgreen in Fig. 3 and Supplementary Online Videos S1 & S2.

Supplementary videos related to this article can be found atdoi:10.1016/j.jhevol.2012.01.001.

Visualization of enamel microstructure

Herewe report the first 3D segmentation of enamel prisms fromthe EDJ to the enamel surface of a developing chimpanzee molar(Fig. 3, Video S1). When observed in 3D after virtual extraction,prisms show surprisingly straight paths in a mesial perspective(direct view of the 3D rendering in Fig. 3), while a more lateralperspective reveals an irregular wave-like deviation from innerenamel to the tooth surface (rendered as the projected shadow onthe background enamel microstructure in Fig. 3). Prisms were alsosegmented in the lateral enamel of a crown-complete Neanderthalmolar (Fig. 4), although it wasmore difficult to follow a single prismthrough the full enamel thickness in this case. In both taxa, prismpaths were observed to deviate from a straight path preferentiallyin one plane. It was also possible to reconstruct the temporalprogression of the enamel-forming front from the perspective ofthe enamel secretory cells (Video S2).

The two samples illustrated here were specifically chosen fromdozens of submicron scans of primate teeth. We find that inherentvariability in the clarity ofmicrostructure is a serious limiting factor.In many cases it is possible to follow prisms for several hundredmicrons, but poor clarity in the inner enamel often prohibits fullsegmentation. It appears that this variation is due, in part, to themineralization level. In mature modern teeth, it is difficult tovirtually resolve enamel microstructure, while developing enameloften clearly reveals prismatic structures. This is due to the fact thatphase contrast imaging is based on heterogeneities in tissuedensities. In fully mature enamel, density differences betweenprisms and prism sheaths may be reduced relative to developingenamel, which prohibits effective edge-detection with propagationphase contrast at resolutions around 1 mm. This may also explainvariation within teeth, as enamel microstructure is often clearlyvisible in the outer and middle regions, becoming gradually lessvisible toward the EDJ (inner enamel). In developing teeth, enamelis more mineralized near the EDJ than near the surface (Suga, 1983;Tafforeau et al., 2007; Smith and Tafforeau, 2008: Figure 6, p. 223).Prism diameters and prism sheaths also tend to be larger near thesurface than near the EDJ (Video S2; also see Dean, 2004: Fig. 1, p.634; reviewed in Risnes, 1998; Smith, 2004). Additional study isneeded to understand the relative effects of mineralization andstructural variation on microstructure visibility.

This transformative approach enables reconstruction of thespatiotemporal (four-dimensional) development of the hardesttissue in the body at submicron resolution. Our results demon-strate that when comparing segmented (virtually isolated)prisms with conventional theoretical models (typically spiralingrods or undulating sinusoid curves: Risnes, 1986; Macho et al.,

2003), enamel prisms do not conform to simple geometricshapes. These results imply that 3D correction factors proposedfor the deviation of enamel prisms, such as the widely-cited 15%correction attributed to Risnes (reviewed in Macho et al., 2003;Smith, 2008), are prone to overestimation of the true prismlength. The cohort of prisms in the chimpanzee developing molarwas approximately 7% longer than the straight-line distance(local enamel thickness), and true prism lengths in the Nean-derthal were approximately 3% longer than the enamel thickness.However, the degree of prism decussation appears to be variablewithin and among hominoid teeth (also see Dean, 1998; Machoet al., 2003), making it unlikely than any single value will bean appropriate correction for estimating cuspal formation timewithin and among hominoid taxa. Moreover, our results suggesta reconsideration of Macho et al. (2003)’s model of 3D prismmorphology; neighboring secretory cells that form cohorts ofenamel prisms near the EDJ do not necessarily retain tightcellular cohesion during secretion, as the same group of prismsmay be spatially diffuse near the enamel surface. While weconcur that enamel prism paths appear to deviate with variablegeometry in different planes, we do not find support for theregular geometric shapes reported by Macho et al. (2003), nor dowe find cohorts of aligned prisms in the inner or middle enamel(as in their Fig. 1 schematic, p. 83). Dietary inferences for fossilhominins based on these models (Macho and Shimizu, 2009)should be reconsidered in light of these findings.

This approach may also shed light on the biomechanicalproperties of mammalian teeth. Dental adaptations are likely re-flected at different organizational scales (Koenigswald and

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Clemens, 1992), which may range from the complete thickness tothe fine structure of prismatic enamel (Rensberger andKoenigswald, 1980; Fortelius, 1981; Boyde and Fortelius, 1986;Koenigswald, 1988; Rensberger, 1997; Lucas et al., 2008a,b;Constantino et al., 2011). Lucas et al. (2008b) have postulated anadaptive model of mammalian dental morphology that integratesenamel thickness, enamel distribution, and the degree of prismdecussation (also see related discussions in Chai et al., 2009). Byusing the approach detailed above it is now possible to determinethe degree and extent of prism decussation non-destructively, andto assess how accurate these models are for reconstructingprimate (and ultimately hominin) dietary ecology. These advancesalso have broader implications for understanding craniofacialevolution, functional morphology, skeletal biomineralization, andthe design of biomaterials, including dental restoratives. Given theabundance of tooth crowns in the mammalian fossil record, thismay also represent an ideal approach to study the evolution ofcellular developmental dynamics.

Acknowledgments

This study was funded by the European Synchrotron RadiationFacility, Harvard University, the HMS Milton Fund, and the MaxPlanck Society. Lawrence Martin and Michel Toussaint areacknowledged for the samples, and Donald Reid, Daniel Lieberman,Fernando Ramirez-Rozzi, and two anonymous reviewers com-mented on the text.

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