Wednesday, April 29, 2015

Accessing Developmental Information of Fossil Hominin Teeth Using New Synchrotron Microtomography-Based Visualization Techniques of Dental Surfaces and Interfaces

This is a great paper on cutting edge non destructive fossil hominin dental synchrotron imaging.  Life histories, including age at weaning, inter birth spacing and age at death can be determined from the study of hominin teeth.  Early hominin fossil teeth are very rare and not available for destructive testing.  Due to their hardness, hominin teeth are often the only hominin fossils that survive over hundreds of thousands of years.  The detailed morphology of teeth is information rich in terms of understanding life histories.  For these reasons, non-destructive testing procedures for early hominin teeth show great promise toward understanding the life histories of early hominins and human evolution.  (This research was funded by NSF Grant BCS 1126470, Harvard University, and the European Synchrotron Radiation Facility (Ec697).)

Adeline Le CabecNancy Tang and Paul Tafforeau
April 22, 2015
(Link) open access PLOS One paper
(Link) related movie


Quantification of dental long-period growth lines (Retzius lines in enamel and Andresen lines in dentine) and matching of stress patterns (internal accentuated lines and hypoplasias) are used in determining crown formation time and age at death in juvenile fossil hominins.  They yield the chronology employed for inferences of life history. Synchrotron virtual histology has been demonstrated as a non-destructive alternative to conventional invasive approaches. Nevertheless, fossil teeth are sometimes poorly preserved or physically inaccessible, preventing observation of the external expression of incremental lines (perikymata and periradicular bands). Here we present a new approach combining synchrotron virtual histology and high quality three-dimensional rendering of dental surfaces and internal interfaces.  We illustrate this approach with seventeen permanent fossil hominin teeth. The outer enamel surface and enamel-dentine junction (EDJ) were segmented by capturing the phase contrast fringes at the structural interfaces. Three-dimensional models were rendered with Phong’s algorithm, and a combination of directional colored lights to enhance surface topography and the pattern of subtle variations in tissue density. The process reveals perikymata and linear enamel hypoplasias on the entire crown surface, including unerupted teeth. Using this method, highly detailed stress patterns at the EDJ allow precise matching of teeth within an individual’s dentition when virtual histology is not sufficient.  We highlight that taphonomical altered enamel can in particular cases yield artificial subdivisions of perikymata when imaged using X-ray microtomography with insufficient resolution.  This may complicate assessments of developmental time, although this can be circumvented by a careful analysis of external and internal structures in parallel. We further present new crown formation times for two unerupted canines from South African Australopiths, which were found to form over a rather surprisingly long time (> 4.5 years). This approach provides tools for maximizing the recovery of developmental information in teeth, especially in the most difficult cases.


Scholars have long recognized the wealth of information preserved in dental hard tissues. Tooth microstructure has been used to study developmental defects, tooth formation times, and age at death (reviewed in [13]). Further, a strong correlation between dental development and important life history events has been suggested in human and non-human primates (reviewed in [47]). Life history can be described as series of developmental milestones in an individual’s life including birth, the duration of breast-feeding or weaning, as well as more complex aspects such as inter-birth intervals and lifespan; these events happen at different times and over different durations following species.

In order to determine growth periods and/or age at death of juveniles using a direct measurement independent of modern human or great ape standards, long-period incremental growth lines that course through the enamel (Retzius lines) and manifest on crown surfaces (perikymata) are the most commonly counted developmental features. Their periodicity however needs to be determined for calculating the crown formation time. This is achieved by counting the daily prism cross-striations between two successive long-period lines. This process is conventionally done by analyzing slices through the main cusp axis of the tooth under a microscope, and requires physical sectioning of the tooth. Other methods rely solely on the counts of cross-striations for determining age at death [810], but these approaches are extremely difficult to apply on fossils due to the high variability in the visibility of their enamel microstructure. In either case, these techniques are destructive, and are thus only rarely applied to valuable fossil specimens.

Retzius [11] was one of the first to observe long-period incremental growth lines in enamel on thin sections of vertebrate teeth, increments which have since been widely defined and described, especially in primates [1216]. Their etiology is still poorly understood [15,1721], although several explanatory hypotheses have been proposed to account for this growth disturbance: systemic origin [8,13,22], shift in the synchronization of different cellular biological rhythms [17] or even gastric disturbance (indigestion) caused by periodic feast days [23]. Structural variants of regular Retzius lines have been described, such as staircase-type Retzius lines [18] and S-shaped Retzius lines [24] while other lines running parallel to the developing front have been documented, e.g., irregular striae [19], pathological Wilson bands [2527], and chevron lines [28]. When Retzius lines reach the outer enamel surface (OES), they manifest as continuous wave-like structures around the circumference of the crown [1215,17,29]. First named perikymata by Preiswerk [30], these features are separated by grooves or imbrication lines following Pickerill’s description [31]. As early as 1854, Kölliker was one of the first to draw attention to the continuity between perikymata and Retzius lines which has then been confirmed by others [12,15,17,2933]. Newman and Poole [17] and others [34,35], reviewed in [3638] established the parallel between incremental growth features in enamel (Retzius lines, perikymata, cross-striations) and those existing in dentine (Andresen lines, periradicular bands, von Ebner lines). Dean [37] reports that periradicular bands are difficult to see because they are often packed close together, are shallower than perikymata, and lie beneath cementum, although they have been employed for reconstructions of developmental time [39]. Stress events often manifest as circumferential bands known as linear enamel hypoplasias on crown surfaces, in addition to irregular accentuated lines within the enamel (reviewed in [4042]).

Determination of the timing of these defects may provide insight into stress, and may facilitate matching synchronously-developing teeth within a dentition (e.g., [41,4346]). However, quantifying microscopic incremental features and documenting stress timing is often a serious challenge, since the clarity of Retzius lines within teeth is variable, and precise quantification of perikymata and hypoplasias is complicated by variation in their expression and the curvature of tooth surfaces [20,42]. Growth lines in teeth are conventionally observed from naturally-fractured teeth and histological (thin) sections under transmitted light microscopy. Nonetheless, perikymata counting from well-preserved tooth surfaces has the advantage of being non-destructive [47] and can be performed under a stereomicroscope or using scanning electron microscopy (SEM) with some preparation of the specimen (e.g., high resolution casts and sputter coating) [41], (reviewed in [48,49]]. Attempts for developing semi-automatic counting techniques of perikymata on isolated teeth with well-preserved outer enamel surfaces have so far demonstrated only limited success [50,51].

Histological assessments of incremental features often rely on physically-sectioned teeth, which limits material available for study, or on high-resolution impressions of tooth surfaces, which require accessible tooth germs or erupted teeth with well-preserved lateral surfaces. However, over the last decade, developments of propagation phase contrast X-ray synchrotron microtomography (PPC-SR-μCT) have permitted virtual histology, or non-destructive imaging of the internal aspects of dental tissues (e.g., [6,5254]). In addition to being non-destructive, PPC-SR-μCT data may be used to produce high resolution 3D models of OES, as well as virtual section planes of various orientations and thicknesses, which improve the visibility of growth lines (e.g., [6]). Propagation phase contrast scans reveal the interface between two materials as a double fringe (adjacent black and white halves, with the white being on the side of the denser material), which yields sharper surfaces than absorption scans. Importantly, the real physical interface between two materials is at the exact junction between these white and black fringes.

In brief, materials are characterized by their index of refraction (n) which is a combination of attenuation (β or μ) and phase shifts (δ), as n = 1- δ + iβ. Pure absorption occurs when the distance ‘D’ between the scanned object and the detector equals zero. In the case where transverse coherence of the beam is sufficient, such as in third generation synchrotron sources, when D increases but remains in the near field of the Fresnel diffraction region, phase dominates over absorption and the phase shifts resulting from the different densities of the matter become visible in the so-called ‘edge-detection regime’ [55,56]. In conventional CT, D remains small enough so that the phase is generally not detectable, except for small objects imaged at a resolution close to 1 μm, with average energies typically lower than 20 keV (see Figure 2 and the corresponding text in [57], and Figure 1 in [58]). The fringes related to the phase shifts represent one of the main advantages of synchrotron virtual histology for studies of dental development, as the phase contrast sensitivity to small density differences is orders of magnitude stronger than that of absorption (e.g., [52,59]). This approach facilitates the non-destructive observation of incremental growth lines in teeth, and yields exceptional microscopic clarity of surfaces and interfaces due to the strong phase fringes associated with these structures.

Here we describe and validate a new application of 3D virtual histology that enhances the identification and quantification of long-period growth lines on the OES, and stress pattern on both the OES and the enamel-dentine junction (EDJ). This is an alternative approach to conventional methods to determine tooth crown formation times and developmental defects, especially in the case of teeth with altered surfaces. This 2D-3D approach has been used to determine the age at death in juvenile dentitions that cannot otherwise be studied. This is the case for unerupted teeth that are not observable with other techniques, specimens inaccessible using classical histology due to conservation issues or 2D synchrotron paleohistology due to poor preservation of internal structures. The techniques presented in this paper have been developed during a broad comparative study involving Plio-Pleistocene juvenile hominins [60,61] and of the MH1 Au. sediba holotype [6264]. In the latter case, this combined 2D-3D approach has yielded age at death and overall dental development characterization despite poorly preserved external and internal structures. The goal of this paper is neither to challenge nor to solve potential methodological problems of previously published values of long-period line counts performed on teeth with good surface quality that are presently taken as references for comparison with our own results, but rather to propose new approaches to investigate specimens that would be inaccessible with other techniques.
The combination of 2D virtual histology and 3D high quality rendering of dental surfaces and interfaces facilitates detailed studies of fossil dentitions by enabling the use of any single fragmentary piece of information in a global approach. We present two case-studies of the lower canines of MLD2 (still enclosed in its crypt) and StW151, and we calculate their crown formation times. By maximizing the amount of information obtainable from rare and precious fossil specimens, this approach will allow us to better understand the evolution of human life history.

Images (they're fantastic!):

Fig 1. 3D Phong rendering and colored light system. Principle of the 3D Phong rendering illustrated with the URC of MLD11-30. Illumination of the 3D model simultaneously by two light sources (LS1 and LS2), each composed of three components (three first columns): ambient, diffuse and specular. Each of the three rows (left half of the picture) shows the individual effect of each component. The combination of all light components is presented in the fourth column. LS1 (middle row) employs a white hue and is oriented in a perpendicular direction to the computer screen (viewer’s perspective—labial side of the crown). Light source 2 has a low white ambient (5 in VGStudio MAX 2.2), an orange diffuse light of moderate intensity (35) and a pale blue specular component with a tenfold higher intensity than the orange light (about 200). Combined with LS1, LS2 is oriented from the top (for taking a first set of images during the rotation of the tooth when mounting a multiple view plate, see S1 Fig), and then from the bottom (second set of images, same conditions) to light the 3D model with a low angle incidence to make topographical and densitometric details more visible. Both sets of images were then combined in Adobe Photoshop to enhance and sharpen topographic details with a mask of high frequency reinforcement. This operation involved taking the top-light image and subtracting structures smaller than 20 pixels that were also present in the bottom-light image (low frequencies), resulting in the combination of unique details from each direction in the final 3D model (far right).

Fig 2. Topography and fine variation in density at the EDJ. Renderings of the EDJ of the ULC of StW151, with two white lights sources (default in VGStudio MAX 2.2). (A) The tooth rendered with ‘ScatterHQ’, which reveals only subtle density variation (gray values) at the EDJ. (B) The EDJ rendered with ‘Phong 3D’ and the ‘Normalize gradient’ commands; this renders only the topographical details of the EDJ surface, and omits shades related to density variation.

Fig 3. 3D rendering of the unerupted LLC of MLD2 showing perikymata. The unerupted MLD 2 LLC in its alveolar crypt, which filled with matrix during fossilization. Retzius lines could not be revealed in the virtual histological data, in spite of changing thickness and orientation of the virtual 2D slice, the two thick lines are likely parts of ring artifacts (A). Despite continuous contact between the OES and the sediment filling the crypt, and the noisy nature of the fringes at the OES, the enamel surface could be successfully segmented and rendered (B), revealing countable perikymata almost all the way from the cusp tip to the cervix. Linear enamel hypoplasias are also apparent encircling the tooth crown.

Fig 4. Enamel hypoplasia matching in the MLD11-30 URI2 and URC. Matching of the URI2 and URC of MLD11-30 based on linear enamel hypoplasias.  On the left-hand side, both teeth are shown in natural proportions: the incisor (left) is smaller than the canine (right). The transformation on the far right was created by enlarging the incisor to be the same size as the canine, so that its hypoplasia and perikymata pattern matches the canine. S2 Movie shows the procedure for matching.

Fig 5. Retzius lines and perikymata doubling in the LLI1 of KB 5223.  Virtual histological slices (grayscale images) showing subdivisions of Retzius lines (white arrows) in the LLI1 of KB5223 (labial view), and their corresponding expression as subdivisions of perikymata (white arrows) on the outer enamel surface. The dotted lines show the fidelity of 2D – 3D matching through horizontal alignment. The position of the labiolingual 2D section is indicated on the 3D model by the green stripe. See S1 Supporting Information (Section II) for a discussion about this phenomenon related to taphonomical alteration (local demineralization).

Fig 6. Complementarity of 2D and 3D developmental information illustrated for StW151 LLC and MLD11-30 URC.  When calculating the crown formation time of StW151 LLC, we use Retzius lines on a 2D virtual slice in the cervical area, since the 3D model does not show clearly identifiable perikymata in that region (A). The cervix of the MLD11-30 LLC (B) yields the largest variation for both inter- and intra-observer counts (S2 Supporting Information, Tab “Average deciles”). This is due to the presence of unequal subdivisions of perikymata and a poor visibility of the perikymata at the very bottom of the cervix. The exact alignment between the 2D slice (its thickness explains that the alignment appears not exact with the 3D, although it is) and the 3D model of the OES is shown by pink lines at the cervix and at a hypoplasia for StW151, and at the bottom edge of a fracture for MLD11-30.

Fig 7. EDJ matching in multiple teeth of a single individual.  The 3D models of the EDJs of the LRC (A) and LRM1 (B) of KNM-KP34725 are matched in (C) by superimposing a portion of each EDJ (red frames and arrows) following the stress pattern as a barcode on the EDJ and root surface (colored arrowheads). This is done with Abode Photoshop by rotation, translation, isometric scaling, perspective and skewing of the fragment of EDJ of the canine onto the fragment of molar that is taken as a reference. (The two apical thirds of the roots of the molar were out of the field of view during scanning, thus the roots appear to be cut in an abrupt manner.) 

Fig 8. Unwrapped external surface of the StW151 ULM1.  Virtual unwrapping of the outer surface of the StW151 ULM1 obtained from the concatenation of a single pixel-wide frames saved during the complete rotation of the tooth around its long axis. On the left side of the unwrapped surface, the tooth is viewed from the buccal side, and the mesio-buccal and disto-buccal roots can be seen in the front, while the lingual root is visible in the back. The lingual view, at the right of the unwrapped tooth, shows the lingual root in the front, and the two buccal roots in the back. Since not all points are at the same distance from the center of rotation of the tooth, some parts of the tooth can be distorted: the furcation area of the buccal roots is stretched in the middle of the unrolled tooth. Perikymata, periradicular bands and hypoplasias are visible and can be tracked across the tooth. Images are not to scale.

MLD2 and StW151: surprisingly long-forming canines
Synchrotron imaging has the considerable advantage here of yielding access to unerupted in situ teeth. Crown formation time of both MLD2 and StW151 appear to be relatively long (> 4.5 years, Table 1) compared to values published for other specimens (e.g., [48]). Our values fall at the lower end of the range reported for female great apes in [68]. The perikymata count could be comparable to that observed on the ULC of the ARA-VP-6/1 holotype of Ardipithecus ramidus that is 193 perikymata yielding a crown formation time of 4.29 or 4.82 years following the estimation of its periodicity at 7 or 8 days [78]. We would like to underline here the high variability induced by the use of periodicity ranges in the final results. This parameter has indeed been shown to be highly variable even within one single taxon [61]. The direct determination of long-period line periodicity represents a major advantage of developmental studies performed using PPC-SRμCT [6,46]. Therefore, crown formation times should be considered extremely carefully when no direct determination of periodicity is available. Moggi-Cecchi et al. [79] report a shorter crown formation time for the StW151 LLC. We suspect that their perikymata counts in the cervical area have been underestimated, as we realized that Retzius lines were much easier to identify on the 2D virtual slice than perikymata on the cervical area of the OES of this tooth (S4 Supporting Information). Nonetheless, other instances of canine crowns developing over an even longer period of time have been documented for Plio-Pleistocene South African specimens [61]. Since these crowns were not accessible or were too damaged for direct observation (both external and internal structures), these specimens could not be fully quantified from virtual 2D slices. The approach applied in the current study demonstrates how developmental information may be retrieved from unerupted teeth, even in the case of poor preservation, by combining multiple observational techniques of PPC-SRμCT. The crown formation time of the MLD2 LLC is strikingly long, and could be interpreted as resulting from errors in the perikymata counts, because of the complex surface topography of this tooth. Nevertheless, the fact that the StW151 LLC presents a crown formation time at least as long as that of MLD2, and that multiple counts of the MLD2 canine by three different observers end within a limited variability of results comparable to that of other well-preserved teeth, confirms our initial conclusion about the MLD2 LLC. Our results demonstrate that canine crown formation time in South African Australopithecines and maybe early Homo (depending on the taxonomic attribution of StW151) can sometimes be far longer and more variable than expected from previously published studies. More extensive study is necessary to assess whether such long canine crown formation times may be related to taxonomical status, sexual dimorphism, or natural variability [70,68]. In future dental developmental studies involving PPC-SRμCT, not only should individual periodicity be directly determined as in previous studies [6,46] but also a special focus should be set on determining cuspal daily secretion rates in at least one anterior and one postcanine tooth. This would constraint the reported range and take into account taxonomic and anatomical (tooth class) variability.

Stress pattern and its 3D visualization on the EDJ interface
Stress in enamel and dentine are commonly used to match teeth across a dentition as synchronous events [14,8083]. Although odontoblasts secrete dentine slightly in advance of ameloblasts secreting enamel, this difference in time can be treated as negligible for general dental development studies [84]. For the first time, we reveal the stress pattern on the EDJ resulting from subtle variations of density and topography on both sides of this interface. This is possible because phase contrast reveals this information with high sensitivity in the black and white fringes at the interface between the two materials (S13 Fig). Although matching the EDJ (Fig 7) of several teeth does not yield temporal information, as the Andresen lines are rarely visible on the EDJ interface, it creates a relative chronology of stress events that then allows one to exploit any single usable piece of developmental information (periodicity, and number of perikymata, Retzius lines in enamel, Andresen lines in dentine) within that framework (Fig 9).

Fig 9. Direct correspondence of 2D and 3D developmental information.  Matching of the incremental pattern between the standardized developmental slice of the ULC of STS2 and the 3D models of its EDJ and OES. Retzius lines or accentuated lines in the enamel on the 2D virtual slice allows the matching of a stress on the EDJ and OES. The number of long-period lines is indicated in square brackets between the major stress events highlighted on the 2D and 3D models, providing a quantitative overview of the time elapsed between stress. The long-period line count was performed on a high resolution image of the 2D slice to ensure a high definition of the growth lines. Further developmental information for this individual may be found in [61]. 


The main advantages of the 2D-3D rendering protocol presented in this study are: (i) enhanced topographic and densitometric details of the OES and stress patterns on the EDJ; (ii) enhanced visibility of developmental structures from high-quality images allowing for reasonably consistent inter- and intra-observer agreement; (iii) accurate visualization of long-period growth increments in enamel and developmental defects using a combination of virtual histological 2D slices and 3D models; (iv) novel possibilities for visualizing surfaces of well-preserved teeth still in crypt; and (v) facilitation of the matching of stress patterns across an individual's dentition.
This 2D-3D combination rendering approach for visualization of dental surfaces opens up new possibilities for detailed developmental studies on exceptional fossil hominins with well-preserved partially complete developing dentitions. We however draw the attention on the impact that resolution and partial volume effect can have on demineralized areas in the enamel subsurface, when subdivisions of perikymata are observed on the 3D renderings. On the one hand, special care has to be taken to ensure reaching robust results based on perikymata counts derived from X-ray images. In the vast majority of cases, there is no possible mistake about recognizing real perikymata on PPC-SRμCT data. Further, it allowed for the first time to determine crown formation times of two unerupted early hominin teeth that would not have been fully accessible with any other technique. These results suggest that the short formation time conventionally expected for early hominin lower canine crowns may be too restrictive; the two specimens presented here showed development times of more than 4.5 years. Dental development in general should consider using direct determination of the individual’s periodicity to avoid reporting very wide ranges. In addition, future PPC-SRμCT-based developmental studies may improve by trying to measure systematically CuDSR, which would contribute to constrain even more the reported ranges for both crown formation times and age at death. This innovative approach is being employed to generate a comprehensive and permanent digital record of developmental information in invaluable and fragile fossil hominin specimens. These developmental data will be made freely available online and will thus facilitate future comparative studies.

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