Figure 1: Map showing the location of sites sampled. (Image credit: H. Hollund)
Figure 2: Schematic drawing of the longitudinal cross-section of a tooth. (Image credit: H. Hollund)
Figure 3: Acellular cementum and dentine. The asterisk marks what is called the granular layer of Tomes. Several interpretations exist for these structures. Recently it has been described as an enamel-like, hypermineralised zone containing little organics (Cherian 2011). Note the thinning of the dentinal tubuli close to the cementum and their fine lateral branches. (Image credit: H. Hollund)
Figure 4: Cellular cementum with cementocyte lacunae (arrow). These are the lacunae of the former cementocytes, collagen-producing cells that become entrapped in the extracellular matrix they excrete during cementogenesis. The canaliculi of cementocytes communicate but are not part of an inter-connected network that extends all the way to the surface, as is the case for bone. Nourishment is believed to occur by diffusion and the deeper cementocytes may not be vital. Most of the canaliculi point toward the tooth surface (Nanci 2003). The black asterisk marks the cementum surface and the white the granular layer of Tomes at the cemento-dentinal junction. (Image credit: H. Hollund)
Figure 5: The dentino-enamel junction appear as a well-defined scalloped border (asterisk). (Image credit: H. Hollund)
Figure 6: Tufts (arrow) crossing the dentino-enamel junction (asterisk). Tufts appear as hair-thin brushes that branch out from the junction and into the enamel. The tufts contain greater concentrations of enamel proteins than the rest of the enamel (Nanci 2003). (Image credit: H. Hollund)
Figure 7: Spindles (arrow) crossing the dentino-enamel junction (asterisk) are remains of odontoblast processes that become trapped when enamel formation begins (Nanci 2003). (Image credit: H. Hollund)
Figure 8: Scanned thin-section of sample EIN-15 showing the pattern of microbial tunnelling. The tunnels (dark areas) are mainly found within the cementum, the apex (root tip) and along the root canal. (Image credit: H. Hollund)
Figure 9: Scanned thin-section of sample EIN-22. Here, the attack is more extensive than in EIN-15 (Figure 8) and the microbes are also entering from the surface of the pulp cavity penetrating the whole way to the dentino-enamel junction. (Image credit: H. Hollund)
Figure 10: Micrograph of sample EIN-23. The cementum is completely bioeroded, mainly in the form of globular MFD. Despite the cemento-dentinal junction (black asterisk), the bacteria are penetrating into the dentine. The micro-organisms seem to enter the tooth at the cementum surface (white asterisk). Tunnelling into the dentine is in the form of enlarged dentinal tubuli (arrow) with branches. See also detail in Figure 16. (Image credit: H. Hollund)
Figure 11: Micrograph of sample EIN-05 showing globular-shaped tunnels within the dentine. These are of the same size and shape as the linear longitudinal tunnels (approximately 10 µm) and budded tunnels (up to 100s of µm) seen in bone. In the middle of the image a pristine area of dentine can be seen, with the dentinal tubuli clearly visible. (Image credit: H. Hollund)
Figure 12: Micrograph of sample EIN-16. Destructive foci are observed as several connected oblong shapes. (Image credit: H. Hollund)
Figure 13: Micrograph of sample EIN-23. Elongated destructive foci (arrows) stretch out from cementocyte lacunae and cancaliculi. (Image credit: H. Hollund)
Figure 14: Micrograph of sample EIN-23. Three destructive foci within the cementum where the micro-organisms seem to have entered from the cementum surface. The white asterisks mark the cementum surface and the cemento-dentinal junction. (Image credit: H. Hollund)
Figure 15: Micrograph of sample EIN-04. What can be described as Wedl type 2 tunnels are found within the cementum. These appear as enlarged canaliculi, first described in bone by Trueman and Martill (2002). However, these may also be caused by acid etching, as suggested by Fernández-Jalvo et al. (2010) for bone samples. This may also be the case here (black arrow). Alternatively, the canaliculi may appear enlarged whereas this is caused by staining along the canaliculi. The fine central line may be the original canaliculi and the broader darker band being caused by infiltrations. The white arrow indicates an unaffected cementocyte lacuna. (Image credit: H. Hollund)
Figure 16: Micrograph of sample EIN-23. Destructive foci in the dentine appear as enlarged dentinal tubuli and lateral branches. (Image credit: H. Hollund)
Figure 17: Micrograph of sample EIN-23. Destructive foci in the form of enlarged lateral branches of the dentinal tubuli. (Image credit: H. Hollund)
Figure 18: Micrograph of sample EIN-22. An area with large, globular MFD with a grainy interior can be seen in the middle of the image (asterisk). The pattern of the dentine microstructure is visible within the foci, in a way similar to lamellar tunnels in bone (Hackett 1981) and what was observed by Bell et al. (1991) in dentine. See also SEM-BSE images in Figures 19 and 20. (Image credit: H. Hollund)
Figure 19: SEM-BSE image of the large MFD seen in Figure 18. The SEM-BSE images show that the destructions consist of a demineralised area with a border of more highly mineralised material. See SEM-BSE image in Figure 20 for higher magnification. (Image credit: H. Hollund)
Figure 20: SEM-BSE image of the large MFD seen in Figure 18 and 19. At this level of magnification it is possible to see that the interior of the MFD contains numerous fine pores (roughly 1 micron across), as found within bone MFD by Jackes et al. (2001), believed to represent bacterial tunnels. (Image credit: H. Hollund)
Figure 21: Micrograph of sample EIN-06. As pathologies will change the physico-chemical and/or microstructural characteristics of teeth, these may also affect the pattern of microbial attack. Here, a circular deformation is visible within the dentine; a pulp stone. These may arise as an age change or accompany inflammatory or degenerative changes in the pulp. Calcification of the pulp results in the formation of discrete, approximately circular mineralised masses which may later be incorporated into the dentine (Marsland and Browne 1975). The microbial tunnelling can be seen to follow the circular lamellar microstructure of the imbedded pulp stone. (Image credit: H. Hollund)
Figure 22: Micrograph of sample EIN-15. This sample displays another commonly occurring pathology, a developmental defect leading to zones of interglobular and globular dentine. These are areas of unmineralised or hypomineralised dentine where globular zones of mineralisation have failed to fuse into a homogeneous mass within mature dentine. The condition is especially prevalent in human teeth as a result of vitamin D deficiency or exposure to high levels of fluoride at the time of dentine formation (Nanci 2003). In a few samples, microbes seem to have targeted the less mineralised inter-globular dentine, as can be seen in this micrograph. The two arrows mark a cluster of MFD located in an area with inter-globular dentine. Inter-globular areas are indicated with an asterisk. These MFD are located in an area of the crown dentine, directly beneath an enamel lesion. This tooth displays otherwise dense bioerosion around the root canal and in the cementum, while there are few MFD above the pulp cavity. This may suggest that the MFD in this area were caused by micro-organisms that entered via the lesion. (Image credit: H. Hollund)
Figure 23: Micrograph of sample CDU-15, viewed in polarized light. This is a Neolithic cattle tooth, the only sample to display clearly identifiable fungal tunnelling. Round, transparent fungal structures can be seen, as well as hyphae/tunnels penetrating into the dentine from the pulp cavity surface, appearing as the characteristic Wedl type tunnels (Hackett 1981; Marchiafava et al. 1974; Jans 2005). The hyphae/tunnels are clearly visible in polarized light as they are lined with a birefringent material, possibly calcite as this is a known precipitate on and in soil fungal structures (Burford et al. 2006). See also Figures 40 to 43. (Image credit: H. Hollund)
Figure 24: Micrograph of ZWO-01. This human tooth contained many framboidal pyrite (FeS2) grains on the surface of the pulp cavity and root canals, in places almost completely filling the root canals. The cementum and dentine of this tooth is stained yellow and orange indicating infiltration by iron compounds, possibly a result of partial oxidation of pyrite (see discussion in Hollund et al. 2012b). See also detail in Figure 25. (Image credit: H. Hollund)
Figure 25: Micrograph of ZWO-01. Detail of the same tooth as shown in Figure 24. This human tooth contained many framboidal pyrite (FeS2) grains on the surface of the pulp cavity and root canals, in places almost completely filling the root canals. The cementum and dentine of this tooth is stained yellow and orange indicating infiltration by iron compounds, possibly a result of partial oxidation of pyrite (see discussion in Hollund et al. 2012b). (Image credit: H. Hollund)
Figure 26: Micrograph showing an area of the enamel surface of ZWO-01 which is stained black and orange, possibly infiltrated by pyrite and iron oxides (see also Figures 24 and 25). (Image credit: H. Hollund)
Figure 27: Micrograph of sample EIN-10 in polarized light. Empty microbial tunnels are filled with a birefringent material. Analysis by SEM-EDX detected calcium, carbon and oxygen (Figure 29); most likely calcite (see also Figure 28). (Image credit: H. Hollund)
Figure 28: SEM-BSE image of sample EIN-10. Empty MFD are filled by dense material, probably calcite, as suggested by the birefringence in polarized light (see Figure 27) and SEM-EDX analysis (Figure 29). (Image credit: H. Hollund)
Figure 29: SEM-EDX results; chemical spot analysis of inclusions in empty MFD, sample EIN-10 (see Figures 26-28). (Image credit: H. Hollund)
Figure 30: Micrograph of sample EIN-13 in normal light. Small (approximately 14 μm) orange and translucent pyramid-shaped nodules are found, sitting on the surface of the pulp cavity and root canal. Some of the dentinal tubuli stretching out from the cavity are also filled with orange material. This material shows birefringence in polarized light (Figure 31) and was found by SEM-EDX to contain calcium, carbon, oxygen and sometimes manganese, iron and phosphorus (Figure 32). (Image credit: H. Hollund)
Figure 31: Micrograph of sample EIN-13 in polarized light, showing that some of the inclusive material seen in Figure 30 is birefringent. This material was found by SEM-EDX to contain calcium, carbon, oxygen and sometimes manganese, iron and phosphorus (Figure 32). (Image credit: H. Hollund)
Figure 32: SEM-EDX results; chemical spot analysis of inclusions in EIN-13 (see Figures 30 and 31). (Image credit: H. Hollund)
Figure 33: Micrograph of sample EIN-13. The sample contains orange perfectly round inclusions (arrows) and roughly circular stains (see Figure 34). These were found by SEM-EDX to contain iron and manganese (Figure 35). The inclusions are found within empty MFD in the cementum and dentine, whereas the stains mainly seem to follow the length of the dentinal tubuli. (Image credit: H. Hollund)
Figure 34: Micrograph of sample EIN-13. The sample contains roughly circular stains in addition to the round inclusions seen in Figure 34. Both stains and inclusions were found by SEM-EDX to contain iron and manganese (Figure 35). The inclusions are found within empty MFD in the cementum and dentine, whereas the stains mainly seem to follow the length of the dentinal tubuli. (Image credit: H. Hollund)
Figure 35: SEM-EDX results, inclusions and stains in EIN-13 (see also micrographs, Figures 33 and 34). (Image credit: H. Hollund)
Figure 36: Micrograph of sample EIN-09 in polarized light, showing starch-grains within a crack in the cementum surface. The grains display the starch-specific extinction cross in polarized light (Piperno et al. 2004). (Image credit: H. Hollund)
Figure 37: Micrograph of sample EIN-08, with remains of fungal fruiting bodies and fungal hyphae (arrows) within the pulp cavity. (Image credit: H. Hollund)
Figure 38: Fungal fruiting body on pulp cavity surface of EIN-24. The hyphae is located in a crack in the dentine. (Image credit: H. Hollund)
Figure 39: Yeast cells within the pulp cavity of cattle tooth CDU-15. This double form is a yeast cell reproducing by a division process called budding. (Image credit: H. Hollund)
Figure 40: Micrograph of sample CDU-15 showing transparent, round fungal structures within the pulp cavity. Tunnels (white arrows) can be seen penetrating the dentine. As seen in Figure 23, the hyphae/tunnels are lined with birefringent material, probably calcite and are thus visible in polarized light (Figure 41). (Image credit: H. Hollund)
Figure 41: Micrograph of sample CDU-15, same view as Figure 40 but in polarized light. The tunnels, which seem to be caused by the action of fungal hyphae, are lined with a birefringent material that is visible in polarized light (see also detail in Figure 23). (Image credit: H. Hollund)
Figure 42: Micrograph of sample CDU-15, viewed in normal light. In normal light, fungal fruiting bodies (arrows) are seen within pores/cracks in the dentine, associated with masses of orange and translucent material. This material displays a bright white birefringence in polarized light. As with the fungal hyphae seen in Figure 41, one of the fungal fruiting bodies is lined with the material. No analyses were carried out but it is likely that this is calcite, with some compounds containing iron and/or manganese, as seen for sample EIN-13 (Figures 30, 31, 32) and EIN-10 (Figures 27, 28, 29). (Image credit: H. Hollund)
Figure 43: Micrograph of sample CDU-15, viewed in polarized light, showing that some of the fungal fruiting bodies (arrows) also seen in Figure 42, are lined with a bright white birefringent material. No analyses were carried out but it is likely that this is calcite, with some compounds containing iron and/or manganese, as seen for sample EIN-13 (Figures 30, 31, 32) and EIN-10 (Figures 27, 28, 29). (Image credit: H. Hollund)
Figure 44: Micrograph of sample CDU-15 showing fungal spores within the pulp cavity. (Image credit: H. Hollund)
Figure 45: Scanned thin-section of EIN-17 illustrating the effects of an extreme corrosive environment that has caused generalised destruction and complete loss of a large part of the roots. Despite this, the dentine directly beneath the enamel crown remains well-preserved, testifying to the resilience of enamel and the efficient protection it provides. A caries lesion can also be seen in the enamel. This has caused some staining and reaction in the dentine. (Image credit: H. Hollund)
Figure 46: Micrograph of sample EIN-11 showing the effect of generalised destruction on the dentinal microstructure. The striated structure of the dentine has 'faded' in the upper half of the image. In the lower half, the tubuli and their lateral branches, appear stained and possibly enlarged. It is not possible to determine whether or not this is due to acid etching alone, or bioerosion prior to the etching. (Image credit: H. Hollund)
Figure 47: Micrograph of sample EIN-03. An area of generalised destruction, including staining and large cracks is shown. The cementum is lost in some areas and is detaching at the cemento-dentine junction (arrow). Sample preparation may also have caused part of this. The asterisk marks the root canal. (Image credit: H. Hollund)
Figure 48: Micrograph of EIN-08. Micro-fissures are present in the enamel. The enamel of the teeth studied was frequently cracked to different degrees. (Image credit: H. Hollund)
Figure 49: Micrograph of sample EIN-04. In this tooth the crown enamel has been worn down to the dentine during life and the exposed dentine is affected by orange staining and generalised destruction (white asterisk). This is at least partially pathological, seen by the tertiary, or reparative, dentine (black asterisk). This will form as reaction to stimuli such as caries or attrition and will be produced only by the cells affected by the stimulus (Nanci 2003). (Image credit: H. Hollund)
Figure 50: Micrograph of sample EIN-17. The white asterisk marks an enamel caries lesion, clearly identifiable with a reaction (orange staining) in the dentine underneath (Marsland and Browne 1975). It is not possible to determine whether or not the enamel surface etching and staining (black asterisk) is a smooth surface lesion, or diagenetic in origin. (Image credit: H. Hollund)
Figure 51: Micrograph of sample EIN-20. An extensive lesion of the dentine has undermined the enamel adjacent to the fissure. Such lesions may cause demineralisation of a large area directly underneath it (Marsland and Browne 1975). The lesion contains inorganic diagenetic inclusions. (Image credit: H. Hollund)
Figure 52: Micrograph of sample EIN-20. Three large pulp stones (asterisks) within the pulp cavity. These may arise as an age change or accompany inflammatory or degenerative changes in the pulp. Calcification of the pulp results in the formation of discrete, approximately circular mineralised masses (Marsland and Browne 1975). Finally, these may be completely incorporated into the dentine, as seen in Figure 21. (Image credit: H. Hollund)
Figure 53: Micrograph of sample SAI-04. A cluster of small pulp stones within the pulp cavity. (Image credit: H. Hollund)
Figure 54: Micrograph of sample EIN-10. A thick layer of calculus (white asterisk) overlies the enamel (black asterisk). (Image credit: H. Hollund)
Figure 55: Micrograph of sample EIN-12. Calculus overlying the cementum just below the enamel-cementum junction (asterisk). (Image credit: H. Hollund)
Figure 56: Tunnel-like features (white arrows) within the enamel of CDU-15 (cattle tooth). Could this be evidence of bioerosion in the enamel? The dentine is affected by intense bioerosion, going right up to the dentino-enamel junction whereas the enamel also exhibits extensive cracking. The tunnel-like features appear to emerge from the cracks. In Figure 56 the dark area does indicate cracks/tunnels filled with some opaque material. It is difficult to tell the features apart and further investigation would be needed to establish the nature of these. See also detail at higher magnification in Figure 57. (Image credit: H. Hollund)
Figure 57: Tunnel-like features (white arrows) within the enamel of CDU-15 (cattle tooth). Could this be evidence of bioerosion in the enamel? See also Figure 56. (Image credit: H. Hollund)
Figure 58: SEM-BSE images of globular cavities in the enamel of EIN-22. These are roughly 10-30 micron large, some empty, some containing inclusions. The cavities are located relatively close to the dentino-enamel junction and are sometimes associated with cracks. In several places the dentine is heavily bioeroded up to the dentino-enamel junction, where smaller cavities can be seen just on the border (Figure 61). See also Figures 59, 60 and 61. (Image credit: H. Hollund)
Figure 59: SEM-BSE images of globular cavities in the enamel of EIN-22. These are roughly 10-30 micron large, some empty, some containing inclusions. The cavities are located relatively close to the dentino-enamel junction and are sometimes associated with cracks. In several places the dentine is heavily bioeroded up to the dentino-enamel junction, where smaller cavities can be seen just on the border (Figure 61). See also Figures 58, 60 and 61. (Image credit: H. Hollund)
Figure 60: SEM-BSE images of globular cavities in the enamel of EIN-22. These are roughly 10-30 micron large, some empty, some containing inclusions. The cavities are located relatively close to the dentino-enamel junction and are sometimes associated with cracks. In several places the dentine is heavily bioeroded up to the dentino-enamel junction, where smaller cavities can be seen just on the border (Figure 61). See also Figures 58, 59 and 61. (Image credit: H. Hollund)
Figure 61: SEM-BSE images of globular cavities in the enamel of EIN-22. These are roughly 10-30 micron large, some empty, some containing inclusions. The cavities are located relatively close to the dentino-enamel junction and are sometimes associated with cracks. In several places the dentine is heavily bioeroded up to the dentino-enamel junction. Here, smaller cavities can be seen just on the border. See also Figures 58, 59 and 60. (Image credit: H. Hollund)
Figure 62: SEM-BSE images of globular cavities in the enamel of EIN-22. These are roughly 10-30 micron large, some empty, some containing inclusions, relatively close to the dentino-enamel junction and sometimes associated with cracks. (Image credit: H. Hollund)
Figure 63: Micrograph showing the remains of an insect within human tooth CAS-08. The insect must have been located in the pulp cavity, but as a result of how the tooth was cut it appears here as superimposed on the dentine. Our knowledge does not allow us to identify species and we also do not know how it got there; during decomposition, or perhaps post-excavation, in storage. Identifying such remains may give important information on taphonomy, season of burial etc. (Image credit: H. Hollund)
Figure 64: Micrograph showing unknown inclusions in a crack in CAS-08. Considering that this tooth also contained the remains of an insect (Figure 63), could these be insect eggs? See also Figure 65. (Image credit: H. Hollund)
Figure 65: Micrograph showing unknown inclusions in a crack in CAS-08. Considering that this tooth also contained the remains of an insect (Figure 63), could this be an insect egg? See also Figure 64. (Image credit: H. Hollund)
Figure 66: Micrograph of inclusions within the pulp cavity of EIN-23. These inclusions represent a variety of sizes and shapes. Most are probably fungal spores, spore sacks and yeast cells. The possibility that some of the circular features are air bubbles cannot be excluded. See also Figures 67 and 68. (Image credit: H. Hollund)
Figure 67: Micrograph of inclusions within the pulp cavity of EIN-23. These inclusions represent a variety of sizes and shapes. Most are probably fungal spores, spore sacks and yeast cells. The possibility that some of the circular features are air bubbles cannot be excluded. The dark circle in the middle of the image is an air bubble. See also Figures 66 and 68. (Image credit: H. Hollund)
Figure 68: Micrograph of inclusions within the pulp cavity of EIN-23. These inclusions represent a variety of sizes and shapes. Most are probably fungal spores, spore sacks and yeast cells. The possibility that some of the circular features are air bubbles cannot be excluded. The dark circle in the middle of the image is an air bubble. See also Figures 66 and 67. (Image credit: H. Hollund)
Figure 69: Micrograph of sample EIN-10 showing cuffed circular shapes located along the length of two dentinal tubuli. Some of the circles appear directly on the tubuli, others at the end of what looks like enlarged lateral branches. In addition a possible microbial tunnel, similar to what has been observed in many of the teeth, is emerging from one of the tubuli (arrow). The globular shape at the end of the tubuli where it meets the layer of Tomes and the cemento-dentinal junction, may also be an MFD (asterisk). (Image credit: H. Hollund)
Figure 70: This micrograph of an area of cementum in EIN-23 shows cementocyte lacunae that seem bloated and enlarged, with tree-like structures and fine branches emerging from them. Could these be MFD caused by microbes? (Image credit: H. Hollund)
Figure 71: Graph showing the biomodal distribution of the Oxford histological index (OHI) values for bone and teeth from the site of Eindhoven. (Image credit: H. Hollund)