Fig. 3.17a, b Decalcified section through carious dentin (H&E stain).
a Cross-section through carious dentin with deeply stained bacterially infected dentin tubules and liquefaction foci. A well-developed layer of tertiary dentin (TD) is also visible.
b Conjugated liquefaction foci with accumulation of multiple bacteria show the high elasticity of dentin, giving the impression that the adjacent dentinal tubules were pushed to the side.
NOTE
With breakdown of the intact enamel surface, bacteria can penetrate deeper into enamel and reach the dentin. With further progress of the caries lesion, bacteria finally penetrate and enzymatically dissolve the demineralized dentin, which replaces a part of the zone of demineralization forming the zone of bacterial penetration. The underlying zones travel deeper into the dentin.
Spread of Bacteria within Dentin
With the first bacteria crossing the EDJ, bacteria can be found within the dentinal tubules as single cells, but more often grouping together. Tubule penetration is not a uniform process where tubules fill up with bacteria at the same rate. Longitudinal sections through carious dentin reveal that the depth of infection and also the bacterial density vary markedly from tubule to tubule. However, in the zone of penetration, a high number of dentinal tubules can be found which are completely or in part densely packed with bacterial cells (~105 vital bacteria/g).15 The tubule diameter in these cases is enlarged due to demineralization and proteolytic action. Concomitantly, the intertubular side branches are also filled with bacteria. A specific histomorphological property, associated with caries only, is the local enlargement of densely filled tubules in the form of ampullae, referred to as liquefaction foci. Tubules beside a liquefaction focus are pushed to the side, bending around it (Fig. 3.17). Parallel with the incremental growth lines, clefts can be found that are also heavily infected with bacteria (Fig. 3.18). The necrotic zone contains a vast number of bacteria (~108 vital bacteria/g).15
Bacterial composition inside carious dentin is considerably different to that of dental plaque. Bacteria at the advancing front within carious dentin are acid-producing, aciduric species, predominantly lactobacilli. Within the body of the lesion bacterial composition is more complex, but dominated by strict and facultative anaerobes owing to the anaerobic conditions.47,48 Overall, lactobacilli are the dominant bacteria found in carious dentin.
Hardness of Carious Dentin
BACKGROUND
In vitro, microhardness (after Knoop or Vickers) is used extensively to evaluate the quality and extent of enamel and dentin affected by the caries process. Using micro-hardness, one should be aware that its use on dentin contains a systematic error. Microhardness of a material is measured by placing a diamond (indenter) of known geometry on a flat and polished surface for a specific time using a specific load. Following removal of the indenter, the size of the impression left behind is measured under a microscope and used for calculation of a hardness number. This method works nicely on material that is plastic rather than elastic, like most metals. It also works on more brittle material like dental enamel, because the impression leaves relatively clear edges with a clearly defined rim of built-up material and its size does not change significantly upon releasing the load and removing the indenter. The problem with dentin is that it has considerable elastic properties. The indent left behind is therefore smaller then as it was during loading, highly influenced by the load and also the delay between unloading and measurement. Also, the area around the indent recedes elastically during loading and bounces back after unloading. Therefore, microhardness numbers from dentin should not be compared as absolute values, while comparison of relative values measured under identical conditions may be a valid procedure, assuming that the plastic and elastic properties do not change within the sample. The hardness of dentin is used clinically to estimate the presence of carious dentin (Chapter 18). When considering the hardness of carious dentin it is important to bear in mind that microhardness of healthy dentin already shows great variation within a single tooth and between teeth. Typically, microhardness of sound dentin increases from the EDJ inward due to the low mineral content of the EDJ, but soon (within a few hundred micrometers) reaches its maximum. From there on, microhardness decreases continuously and significantly toward the pulp (Fig. 3.19). Knoop hardness numbers (KHNs) range approximately from up to 70 for outer dentin to 20 close to the pulp.49 This drop in KHN may partly be attributed to the larger fraction of dentinal tubules per area cross-section and larger tubular diameter close to the pulp, as well as to a lower degree of mineralization of peritubular and intertubular secondary dentin. The overall mineral content is lower close to the pulp as can be seen by microradiography (see Fig. 3.26). Carious dentin is even more variable regarding hardness, meaning that the values given here are examples and qualitative in nature and should be used with caution. In a typical situation, in which is dentin caries not fully advanced toward the pulp, it is generally the case that lesion hardness increases from the EDJ toward the sound dentin underlying carious dentin. Within the sound dentin zone underlying the caries, microhardness decreases toward the pulp as described for sound (non-carious) dentin. The sclerotic zone between the zone of demineralization and sound dentin may be slightly harder close to the zone of healthy dentin than sound dentin at the same depth.49 But more likely for a more advanced state of caries development, it has been reported that within the zone of sclerotic dentin the Knoop microhardness decreased from sound values (~40–70 KHN) at the pulpal side of the sclerotic zone to 25–30 KHN at the lesion side of the sclerotic zone.50 In this case one must assume that mineral deposition by growth of peritubular dentin or within the tubules is not as pronounced toward outer areas of this zone, because additional demineralizing effects interact in this area. Thus, demineralization does not end at the border of the demineralized zone, but may extend well into the sclerotic zone.
The hardness of sound enamel also shows some variability, decreasing toward the pulp. In a typical, artificially created enamel carious lesion, the hardness profile from the surface toward the EDJ shows some correlation with mineral content derived from microradiography. The same could be observed by nanohardness measurements using a Berkovich indenter (Fig. 3.20).51
Fig. 3.18a, b Decalcified section through carious dentin (H&E stain).
a Overview of the dentin lesion comprising all histological caries zones, including the zone of necrosis on the outside and the tertiary dentin along the pulpal wall. On the coronal side of the lesion, clefts