1.

Introduction

Dental calculus, also known as tartar, is increasingly targeted in archeological studies for its ability to preserve objects and residues that can provide information about past human diets and behaviors.^1–4 Calculus is formed when oral plaque, a bacterial pellicle, is mineralized through the precipitation of salivary calcium phosphates.^5–7 The mineralized surface of the forming calculus is re-colonized by an oral bacterial biofilm, which is then mineralized at a later point. This process creates a layered structure, and objects that were in the oral cavity, such as microscopic botanical remains, mineral particles, and a variety of biomolecules, such as deoxyribonucleic acid (DNA), proteins, and lipids, become encased and protected within the mineral structure of the dental calculus.^2,8–12 A variety of archeological studies have isolated and identified these inclusions, to record the consumption of particular food items,^1,4 to describe oral bacterial communities,^1,11 and to reconstruct human non-dietary behavior such as paint preparation² and use of burial goods.^{1,2,5,13–15}

Despite the success of these methods, several questions about dental calculus as a record of past diets and behaviors remain unanswered. First, though it is widely understood how dental calculus formation occurs and how salivary calcium phosphates inhibit demineralization of enamel and dental decay,⁶ the mechanisms by which objects become entrapped and preserved are unclear. Second, most methods to extract and analyze the internal structure or the inclusions are destructive. In most analyses, the calculus is either entirely broken down, chemically or mechanically, to isolate the inclusions, or it is cut following histological or thin-sectioning methods.^1,5 The former methods may damage fragile inclusions, particularly calcium-based inclusions due to the use of acidic or chelating chemicals, while the latter results in the loss of most of the sample during the preparation of the section. Therefore, valuable data about the formation process of the calculus and the relative position of certain inclusions in three-dimensional (3D) space is lost. Furthermore, destructive sampling methods are not always permissible on archeological material (see Ref. 16 for a discussion).

Several researchers have explored non-destructive methods to visualize the microscopic structures and internal features of archeological and fossil materials. Scanning electron microscopy has been used to examine the internal structures and inclusions of dental calculus, but only on already-broken surfaces or histological thin sections.^1,13,17–19 Other methods, such as conventional micro-computed tomography ($\mu \mathrm{CT}$), are better suited for visualizing internal structures in undamaged specimens, and have been successfully used on other kinds of archeological specimens such as pottery and charred plant seeds.²⁰ However, using conventional $\mu \mathrm{CT}$, the intensity contrast of inclusions and structures of interest in calculus is typically too small to visualize them. High-resolution analyses of internal features are possible with propagation phase-contrast synchrotron radiation $\mu \mathrm{CT}$ (PPC-SR-$\mu \mathrm{CT}$). Although researchers have used this method to explore the microstructure in fossil teeth to understand the dental development of extinct taxa,^21–28 to date, no studies have used PPC-SR-$\mu \mathrm{CT}$ to visualize the 3D structure of dental calculus.

In this study, we report preliminary results of the analyses of two dental calculus samples scanned using PPC-SR-$\mu \mathrm{CT}$ at the beamline P07 operated by Hereon at the PETRA III storage ring (DESY, Hamburg, Germany). This pilot study aims to: (1) test whether PPC-SR-$\mu \mathrm{CT}$ enables the non-destructive visualization the inner microstructure of dental calculus in both isolated chunks and in situ samples (on teeth); (2) compare the visibility of the fine calculus inner structure between PPC-SR-$\mu \mathrm{CT}$ and conventional $\mu \mathrm{CT}$; (3) see whether inclusions can be visualized and identified; and (4) explore whether the fine microstructure of the calculus deposits can be observed and the formation process be discussed. Because this pilot study also aims at introducing this approach using x-ray imaging to archeologists, we provide detailed explanations and technical parameters.

2.

Materials

We examined two samples of dental calculus: one contemporary (CM03) and one archeological (FK01) (Fig. 1). CM03 was removed from a 36-year-old male smoker as part of their clinical management. It was collected from the supra-gingival buccal surface of the mandibular left second premolar and mandibular left first molar. This calculus deposit had formed over the course of 1 year and was removed atraumatically from the patient, avoiding any blood contamination of the sample. Tobacco smoking is known to have a strong impact on dental calculus deposition in the clinical literature,^29,30 which could account for the non-negligible amount of calculus formed on these teeth within 1 year. The ethical approval to collect and study this material was granted by the Research Ethics Committee of the Cork Teaching Hospitals, Ireland (2018 meeting).

Fig. 1

(a) Macrophotograph of the archeological dental calculus sample on the tooth (FK01). The tooth is from Cist 2, Fourknocks II (Ireland); (b) macrophotograph of the modern isolated dental calculus sample from a clinical setting (CM03); and (c) CM03 positioned and fixed on the sample holder with beeswax. The samples were mounted above the sample holder to minimize attenuation during scanning. The scale bar applies to all subfigures and is 1 cm.

FK01 formed on the upper right second molar of a roughly 25-year-old female individual recovered from a Chalcolithic burial from Fourknocks II in Ireland.³¹ This individual was buried in a secondary cist (Cist 2) inserted into the covering mound of a Neolithic Passage Tomb. The burial has been directly dated to 2450–2030 cal BC (UBA-29672 $3780\pm 53$).³² The calculus was not removed from the tooth before scanning; instead, the whole tooth was mounted on the sample holder. It is not possible to determine the length of time over which this calculus deposit was formed but given the absence of modern dental hygiene practices in prehistory, it is likely that this deposit represents years to decades of the individual’s life.

3.

Methods

4.

Results

4.2.

Calculus Microstructures

Based on the exploration of the high precision 3D models of the surface of the calculus, we could identify interesting and relevant microfeatures. First, in the isolated calculus chunk (CM03), it was straightforward to identify the surface of the dental calculus that once adhered to the tooth surface by its smooth profile. This interior surface also retained negative impressions of regular and periodic incremental markers (Fig. 2), much like a high-quality dental cast. In the middle, at the junction with the free rim of the specimen, a wavy line of substantial thickness marks the gingival line (Fig. 2). Above this, on the surface of the calculus in direct contact with the enamel crown, periodic grooves show an average spacing ranging from 38 to $65.4\mu \mathrm{m}$ in the most cervical part, and from 92 to $104\mu \mathrm{m}$ in what corresponds to the middle third of the crown enamel (Fig. 2). These were identified as negative impressions of perikymata (i.e., enamel growth markers), in agreement with the wide ranges of perikymata spacing values provided for ancient human teeth.⁴¹ Below the impression of the gingival line, fainter curved lines represent the impression of periradicular bands, which are root dentine growth markers. Since these bands are often covered by a thin layer of cementum, it is not reliable to measure their spacing, due to the uncertainty of clearly identifying two consecutive periradicular lines, and to the higher probability of seeing only accentuated lines.^26,42,43 The surface in contact with the oral environment could be readily distinguished by its complex texture.

Fig. 2

Lingual view of the 3D rendering (Phong algorithm, colored lights; see Ref. 26 for details) of the CM03 modern calculus specimen. Part of the calculus was in direct contact with either the mandibular second premolar or first permanent molar. This left negative imprints of the growth increments on the surface of the enamel crown (termed “perikymata”; green lines), of the gum line (blue arrows), and of dentine accentuated incremental features on the tooth root (called periradicular bands; red arrows). Average spacing between structures identified as perikymata are consistent with published values⁴¹: (a) 93, (b) 103, (c) 63, (d) 53, (e) 42, and (f) $44\mu \mathrm{m}$.

Using virtual histology techniques, and especially 2D thick-slabs (i.e., the averaging of multiple contiguous 2D slices to reach a desired slab thickness), the scans also allow us to visualize the complex interior structures of the calculus that are related to how it formed on the tooth. Both samples show multiple levels of layering, which exhibit as alternating heterogeneous thick layers with increments of varying density. Unexpectedly, many of these layers are not parallel to the calculus surface, the tooth surface, or other layers. Instead, they occur as highly convoluted, marbled forms (Figs. 3Fig. 4–5). Figure 3 shows the two major types of layers that can be distinguished in dental calculus: the very thin and contrasting layers of a few microns thickness, and broader, more homogeneous, light gray layers reaching several tens of microns in thickness. Some of the layers curve around inclusions. Notably, the complexity of this layering is only apparent at the highest resolutions, which is only possible with PPC-SR-$\mu \mathrm{CT}$. Figure 6 shows this point by comparing the virtual sections of both the CM03 modern and FK01 archeological specimens scanned using a conventional x-ray lab source and a synchrotron x-ray source with phase-contrast imaging. Although the presence of the layers is indicated in the conventional $\mu \mathrm{CT}$ scans, only with phase contrast imaging is it possible to visualize their fine microstructure and borders. Figure 4 shows how these layers become optimally observable when individual PPC-SR-$\mu \mathrm{CT}$ slices are averaged, although within limits. Indeed, when the virtual thickness is comparable with those of histological sections of calculus, the layers and other microfeatures appear blurred (Fig. 4).

Fig. 3

3D model of the CM03 specimen [(a) Phong algorithm, colored lights] showing the location of the $24\mu \mathrm{m}$-thick virtual 2D sections in the three orthogonal planes of space (b) blue; (c) green; (d) red. This reveals the extensive incremental structure of this modern calculus involving fine and scalloped growth layers of various thickness, likely resulting from differences in deposition rates, with convoluted patterning, not parallel to the tooth surface, nor to the calculus surface. Some broader layers show a slightly higher density and a more homogeneous structure (light gray). R: vegetal rod inclusion, shown in Fig. 8. S: poppy seed inclusion, shown in Fig. 7.

Fig. 4

Variation in virtual slice thickness from a single reconstructed PPC-SR-$\mu \mathrm{CT}$ slice (with a binning of 2: $2.4\text{-}\mu \mathrm{m}$ thick) to the averaging of multiple slices to reach from 4.8 to $100\mu \mathrm{m}$ in thickness (a)–(h), in the modern calculus sample CM03. It is noteworthy that there is a considerable loss of details for slice thicknesses comparable with values used in classical histology (from 24 to $100\mu \mathrm{m}$). Slices that are 12- to $18\text{-}\mu \mathrm{m}$ thick seem to best preserve details with an enhanced visibility due to the noise reduction resulting from the averaging of multiple slices (see Ref. 40 for details).

Fig. 5

Archeological specimen FK01 shows a layered structure similar to that observed in CM03 (Fig. 3). On the $12\text{-}\mu \mathrm{m}$-thick virtual slices (a)–(c), taphonomic processes may have induced local and partial demineralization of the layers. They nevertheless preserve their convoluted shapes that diverge from both the tooth and calculus surface. E: enamel.

Fig. 6

Comparisons of conventional $\mu \mathrm{CT}$ and PPC-SR-$\mu \mathrm{CT}$ results. (a)–(c) are the archeological specimen FK01, and (d)–(i) are the modern clinical sample CM03. (a), (d), and (g) were captured using conventional $\mu \mathrm{CT}$ with a $5.86\mu \mathrm{m}$ slice thickness, while (b), (e), and (h) show PPC-SR-$\mu \mathrm{CT}$ scans at $2.5\mu \mathrm{m}$, and (c), (f), and (i) are virtual sections averaging several slices to reach a thickness of $5.86\mu \mathrm{m}$. The difference between the two types of PPC-SR-$\mu \mathrm{CT}$ is negligible, while the fine microstructure of the dental calculus, especially the layers, are much easier to identify than on the conventional $\mu \mathrm{CT}$ data.

The modern calculus sample (CM03) also retains voids that seemingly follow intra-layer spaces and are of highly variable sizes, from a few tens to several hundreds of microns in diameter. The smaller voids often have a very elongated shape and scalloped outline, and lie roughly parallel to the layers of calculus, while the larger ones have irregular outlines, and possibly are the result of the decay or dissolution of organic inclusions. Voids also occur in the archeological sample FK01, but these are much larger (up to $>1\mathrm{mm}$), much more irregular, and often communicate with each other and open to the exterior.

In both the clinical and archeological samples, cracks are often seen running parallel to the layers of calculus, likely because these are areas of mechanical weakness. Cracks that run perpendicular to the calculus layers and connect the tooth surface (crown enamel or root dentine) to the exterior also occur. Although FK01 appears as an extremely well-preserved sample of calculus on its exterior (Fig. 1), it displays far more cracks and discontinuities inside than does CM03 (Fig. 5).

4.3.

Inclusions

The better quality of the CM03 scan allows for a finer observation of inclusions. Some inclusions were remarkably well-preserved and easy to identify. These included a reniform object, roughly $1225\times 1005\times 770\mu \mathrm{m}$, with a thick outer shell, amorphous interior, and a scalloped surface, found in CM03 (Fig. 7). Based on the 3D structure and by comparison with published images (Ref. 44; and see Figs. 3 and 7 in Ref. 45), this was identified as a poppy seed (Papaver cf. somniferum). The seed coat (SC) is well preserved, and easily identified in 2D and 3D due to its characteristic textured surface. The endosperm (E) is preserved and visible as well (Fig. 7). The seed is entrapped at the very surface of the calculus (toward the oral cavity), and is almost exposed along its entire length.

Fig. 7

(a) 3D model of the modern specimen CM03 with red arrow showing the location of a poppy seed (Papaver cf. somniferum); dorsal view in (b), and lateral view in (c) preserved in the calculus. Virtual 2D sections [thickness: $24\mu \mathrm{m}$; (d)–(f)] enable visualization of the detailed inner structure of the seed, preserving its seed coat (SC), and its endosperm (E). All 3D models are computed with Phong renderer and colored lights.

Other objects that are clearly inclusions to the calculus were also visible, but we are not yet able to identify their origin. These include a large, 3-mm long tube feature with multi-layer walls, which was partially exposed on the surface of CM03 (Fig. 8), and visible with the naked eye as brown in color. We also noted a smaller rod in FK01, as well as rhomboid cell-like structures, which might be plant cells (Fig. 9).

Fig. 8

(a) Location of an inclusion likely representing a (b) possible vegetal rod on CM03. The $24\text{-}\mu \mathrm{m}$-thick virtual 2D sections (d) and (e) reveal a hollow structure with multi-layered walls. Further calculus mineralization events occurred inside this inclusion after it was entrapped in the calculus.

Fig. 9

Three inclusions of an uncertain nature from FK01 shown with $12\text{-}\mu \mathrm{m}$-thick virtual sections in the three planes of space (a)–(c), (d)–(f), and (g)–(i). The first two (a)–(c) and (d)–(f) may represent aligned vegetal cells or fungi, while the third inclusion (g)–(i) looks similar to the rod identified in CM03 (Fig. 8).

In some cases, discerning the complex structures created by the mineralization of oral microbiome communities from inclusions was challenging. We observed several structures that fall into this category: honeycomb structures that occurred on the edges of voids, and rod-like features that may be pseudomorphs of exudate/transudate from bacteria, or the remnants of the bacteria themselves (Figs. 8Fig. 9Fig. 10–11; see Figs. 3G, 5 and 6 in Ref. 46).¹⁹

Fig. 10

Virtual 2D sections through CM03 showing two instances of inclusions with a honeycomb-like structure. (a)–(c) First inclusion: $18\text{-}\mu \mathrm{m}$-thick slices; (d)–(f) second inclusion: $24\text{-}\mu \mathrm{m}$-thick slices.

Fig. 11

Honeycomb-like structures observed in archeological specimen FK01. The first honeycomb structure is shown in red and indicated by a white arrow on the 3D model of FK01 (a). Virtual 2D sections [$8\text{-}\mu \mathrm{m}$-thick; (c)–(e)] show a regular pattern of empty spaces running parallel to the calculus surface. (b) 3D model depicting the negative imprint of these canals. (f)–(h) The second and (i)–(k) third honeycomb-like structures look slightly different, with less accentuated empty spaces. The third structure (l)–(n) is more similar to (b)–(e). Virtual sections (f)–(n) are $12\text{-}\mu \mathrm{m}$-thick.

5.

Discussion and Conclusion

Synchrotron radiation micro-tomography allows unparalleled insights into the inner structure of human dental calculus. This non-destructive imaging technique enables researchers to visualize inclusions in situ, without losing their complex preservational contexts. Fragile calcium-based inclusions, which are lost by dissolution in typical extraction methods, can be identified. Many of the potentially informative inclusions found in calculus (e.g., starches, phytoliths, diatoms, pollen, etc.) are $\sim 5$- to $100\text{-}\mu \mathrm{m}$ large, and therefore should be visible with the effective pixel size of $2.55\mu \mathrm{m}$. We did not identify these microremains in this preliminary study but expect that a more thorough examination of our scans would reveal such inclusions.

We show that conventional $\mu \mathrm{CT}$ scans, which are more widely available than PPC-SR-$\mu \mathrm{CT}$, may reveal some of the inner structure of dental calculus. However, phase contrast (used in PPC-SR-$\mu \mathrm{CT}$) appears necessary to reach the level of details and spatial resolution that allow the unambiguous identification of the calculus layers and clear visualization of the borders of the inclusions. Conventional $\mu \mathrm{CT}$ may however be easily used as a pre-screening step to select valuable specimens for further investigation at a synchrotron facility.

In contrast to histological and hard tissue sections, which provide layers of 3 to $50\mu \mathrm{m}$ with adjoining volume being lost,⁴⁷ PPC-SR-$\mu \mathrm{CT}$ enables us to explore the entire scanned area, or even the entire specimen for an isolated chunk of calculus. Synchrotron radiation $\mu \mathrm{CT}$ provides an infinite degree of freedom regarding slice position, orientation (i.e., rotation and translation), and thickness (Fig. 4). Furthermore, unlike thin sectioning, we are able to create sections in targeted areas of any thickness without the risk of breaking the sample, therefore enhancing reproducibility.

Synchrotron radiation micro-tomography also enables us to easily distinguish, in an isolated calculus fragment, the surface of the specimen that was in contact with the oral environment from that which was in contact with the crown enamel or root surface. The clear imprint of perikymata, periradicular bands and the gingival line on the interior surface suggests that it may be possible to use well-preserved calculus as an insight into the record of dental development (e.g., perikymata counts in preserved areas, perikymata spacing, and enamel hypoplasias) in the absence of the tooth itself.

Shifts in diet, health, or oral pollution could potentially be captured in dental calculus and identified by a change in the nature of the inclusions, in the layers’ thickness or structure (or even in chemical composition, although this is not possible to assess using PPC-SR-$\mu \mathrm{CT}$). Our results support the possibility of providing a relative chronology of inclusions based on their position within the calculus. Given that calculus develops first on the enamel surface and then builds on itself, inclusions found closer to the enamel will be older than those found near the exterior of the calculus (i.e., toward the oral environment). However, our results clearly demonstrate that the development of calculus does not follow a regular, onion-like pattern. Instead, the layering is irregular, and visually similar to that of stromatolites, with layers appearing in some areas but not in others. The periodicity of these layers is unknown, and likely varies among individuals. Given the highly irregular nature of these layers, as well as frequent presence of voids, methods such as repeated acid etching cannot accurately discern the true depositional order of the inclusions, nor capture the formation process on a fine time scale. It might be possible to use synchrotron images of calculus to follow each layer surface and establish the order of formation, to more closely constrain the relative ages of the inclusions.

Finally, the comparison of the modern and archeological samples provides some insight into the potential taphonomic processes affecting ancient calculus. While some of the differences between these samples are due to changes in the scanning parameters (see Table 1), others such as the increase in voids and cracks in FK01 are likely due to a variety of taphonomic processes, including exposure to sediments, moisture and temperature changes, and weathering. Cracks and voids occur even in modern calculus, and can be much larger even in well-preserved and solid-looking archeological specimens. These open spaces may allow decay to occur inside calculus deposits. We do note, however, that there did not appear to be any in-filling of these cracks, suggesting that larger inclusions were not introduced to the interior of the calculus. However, biomolecules that might be mobilized by water, such as proteins and DNA, may be washed out of or washed into the calculus. Further study of more modern and ancient calculus is needed to determine if taphonomic alteration to calculus is widespread.