Dark and translucent zones of natural enamel caries: new article

  A new article was published on the histopathological features of the dark and translucent zones: http://onlinelibrary.wiley.com/doi/10.1002/jemt.22962/full#article-nav. New explanations for the dark and translucent zones are reported. This has implications on enamel caries remineralization and infiltration.

  THEORIES ON THE DARK AND TRANSLUCENT ZONES

1) CLASSICAL DESCRIPTION AND THEORY - DARLING A.I. (The selective attack of caries on the dental enamel. . Annals of the Royal College of Surgeons of england, 1961:354-369)
1.1    DARK ZONE:
     Location: always between the body of the lesion and the translucent zone.
     Mineral loss = 2-4%
     Pore sizes: large and small sizes, but most of them (~ > 50%) are small (innaccessible to quinoline)
     Interpretations: (a) results of further mineral loss relative to the translucent zone (Darling, 1961);
                          (b) result of remineralization of larger pores (Silverstone; Caries Res, 261-274, 1967)

1.2 TRANSLUCENT ZONE

     Location: at the fornt of the enamel caries lesions (between the dark zone and normal enamel).
     Mineral loss = 1%
     Pore sizes: large sizes (accessible to quinoline)
     Interpretations: (a) results of loss of organic material (Darling, 1961);
                          (b) result of early demineralization (Hallsworth, Robinson, Weatherell, Caries Res, 156-168, 1972).

2) NEW DESCRIPTION AND THEORY

2.1 DARK ZONE (de Mattos Brito et al., Micros Res Tech, 2017. Doi: 10.1002/jemt.22962)

     Location: within the body of the lesion and or between the body of the lesion and the translucent zone.
     Mineral loss = > 2-4%; mean mineral volume of 73% (± 8.92%; ranging from 55.15 to 91.14%)
     Pore sizes: appearance of enamel caries immersed in quinoline depends on the pore volume components:  a mixture of organic matter, firmly bound water, air, and quinoline.
     Interpretation: results of the combination of the enamel pore volumes in histological zones where transport of quinoline occurs mainly paralell to prisms' paths.

Example of component volumes at a histological site in the dark zone:
Mineral volume = 74.893%
Organic volume = 13.526%
Firmly bound water = 6.671%
Air volume = 0.982%
Quinoline volume = 3.928% (corresponds to 15.6% of the pore volume)
Total enamel volume = 100%

2.2 TRANSLUCENT ZONE

     Location: deep to the positvely birefringent (under water immersion) body of the lesion, but not necessarily at the fornt of the enamel caries lesion (lesion continues beyond the translucent zone).
     Mineral loss = > 1%; mean mineral volume of 77.83% (± 8.08%, ranging from 62.68% to 92.26%)
     Pore sizes: appearance of enamel caries immersed in quinoline depends on the pore volume components:  a mixture of organic matter, firmly bound water, air, and quinoline
     Interpretations: results of the combination of the enamel pore volumes in histological zones where transport of quinoline occurs mainly paralell to prisms' paths.

Example of component volumes at a histological site in the translucent zone:
Mineral volume = 74.684%
Organic volume = 15.094%
Firmly bound water = 6.321%
Air volume = 1.237%
Quinoline volume = 2.664% (corresponds to 10.5% of the pore volume)
Total enamel volume = 100%
 

The differences between the dark and translucent zones above are mainly due to a higher organic volume in the translucent zone. Different combinations of component volumes could result in pseudoisotropy (of the dark zone) and negative birefringence (of the translucent zone).

2.3 LATERAL CONNECTION OF PRISMS' SHEATHS

    Replacement of air by the infiltrating liquid (quinoline) is also influenced by the existence of lateral connection of prims' sheaths (the main pathways for transport of materials). Figure 1 shows two anatomical lateral relationships between prisms' sheaths: without lateral connection (A and B) and with lateral connection (C and D). Both can be found in different histological zones of the enamel caries lesion. Lateral connection facilitates the replacemt of air by quinoline, resultng in negatively birefringent zone. The lack of lateral connection results in more entrapment of air in the pores, reducing the penetration of quinoline and, thus, favoring formation of a dark zone (pseudoisotropic of positively birefrngent zone). Dark zone can be formed in areas of the body of the lesion where there are no lateral connections of prism's sheaths.
   Translucent zone lacks lateral connection, but present higher mineral and organic volumes than the dark zone, resulting in a negatively birefringent zone.

 

Fig. 1 Two types of anatomical relationships between prisms' sheaths. A and B, without lateral connection, where the main transport pathway is paralell to the axes of the prisms. C and D, with lateral connection of prisms' sheaths, resulted of increased pore sizes, where the main transport pathways run both paralell and perpendicularly to the axes of the prisms. The later configuration facilitates the replacement of air by quinoline.

    Figure 2 shows histological features of one natural enamel caries lesion under water, air, and quinoline immersion media.

Figure 2. Histological zones of natural enamel caries lesion (proximal surface) under various immersion media (water, air, and quinoline), showing dark zone involving most of the body of the lesion. 

Reference

De Mattos Brito CS, Meira KRS, De Sousa FB: Natural enamel caries in quinoline: volumetric data and pattern of infiltration. Microsc Res Tech. 2017; doi: 10.1002/jemt.22962.

Dark zones in fluorotic enamel after resin infiltration

Before application of resin infiltrant (Icon) enamel is dried. Figure 1 shows that dried fluorotic enamel appears opaque (no birefringenc) under polarizing microscopy with Red I filter (red background). After resin infiltration, the air volume is partially filled by resin infiltrant as can be evidenced by the formation of dark zones (arrows) in infiltrated fluorotic enamel.

Figure 1. Ground section of fluorotic enamel under polarizing microscopy with Red I filter (red background). Left, fluorotic enamel dried for 48 h (20º C and 50% of relative humidity), presenting opaque enamel (no birefirngence). Right, fluorotic enamle after resin infiltration for 10 min, showing elimination of opaque enamel, but formation of dark zones (arrows) in the previously opaque area. The dark zones (same red color as the background) are evidence of the partial occlusion of enamel pores by the resin.

PORE VOLUME OCCLUDED BY RESIN (INFILTRANT) IN HUMAN FLUOROTIC ENAMEL

PORE VOLUME OCCLUDED BY RESIN (INFILTRANT) IN HUMAN FLUOROTIC ENAMEL

   Polarizing microscopy helps in the quantification of the volume of hypomineralized enamel pores occluded by infiltrant (Icon). For the first time in the literature, the volume of hypomineralized enamel pores occluded by resin (infiltrant) is quantified taking into account all enamel component volumes (mineral, organic and water volumes) (Sousa et al., Arch Oral Biol, 2017; https://doi.org/10.1016/j.archoralbio.2017.06.017). Previous data on enamel occluded pore volume neglected organic and water volumes (Robinson et al., J Dent Res, 55:812-8, 1976; Robinson et al., Caries Res, 35:136-41, 2001). It is important to note the data reported by the later studies represented the proportion of the pore volume occluded by resin, NOT the actual occluded pore volume. The total pore volume measured by the procedure that neglects water and organic volumes is lower than the actual total pore volume. The proportion of the pore volume occluded by resin is the ratio of the occluded pore volume by the total pore volume. When the total pore volume is underestimated, the proportion of the pore volume occluded by resin is overestimated.

PROPORTION OF PORE VOLUME OCCLUDED BY RESIN

Figure 1 shows the total pore volume, the pore volume occluded by resin infiltrate (orange), and corresponding values of the proportions of the occluded pore volume. This a model based on the neglection of the organic and water pore volumes.

Figure 1. Proportion of the pore volume occluded by resin (infiltrant) in a model without organic and water volumes.

DIFFERENT MODELS ON THE PORE VOLUME COMPONENTS

            Figure 2 shows the different models on the pore volume components. When organic and water volumes are neglected (Fig 2A-C), the experimental (model 1) total pore volume (blue outline) is smaller than the actual total pore volume (dotted outline). The use of model 1 total pore volume increases the proportion of the pore volume occluded by resin by considering in the denominator a total pore volume lower than the actual one. Because the actual total pore volume is not measured, the actual occluded pore volume cannot be measured. When all component pore volumes are considered (model 2), both the occluded pore volume and the proportion of the pore volume occluded by resin can be measured. Figure 2B and 2E show similar occluded pore volumes, but the corresponding proportion of occluded pore volumes largely disagree.

  

Figure 2. Two models of enamel pore volume components. A-C, without organic and water pore volumes. Dotted lines and continuous lines represent the actual and measured total pore volumes, respectively. D-E, including organic matter (red), total water, firmly bound water, loosely bound water (in the pore center), the volume of resin (orange), and the air volume.

            After dehydration, loosely bound water is replaced by air, and the total pore volume is comprised by the organic, firmly bound water, and air volumes. After resin penetration, the air volume is partially replaced by the resin volume. The pore volume after infiltration comprises four component volumes: organic, firmly bound water, resin, and air.

WHAT ARE THE NEW DATA?

            Sousa et al. (Arch Oral Biol, 2017) report both the actual occluded pore volume and the proportion of the pore volume occluded by resinous infiltrant (Icon) in fluorotic enamel. Interpretation of enamel birefringence in combination with microradiography made it possible to quantify all enamel component volumes. The focus was on the proportion of the pore volume occluded by resin because it can be compared to previous data.

Fluorotic enamel component volumes after resin infiltration

   In a point at 100 mm from the enamel surface, the following values were found:

Mineral volume	74.45%
Organic volume	15.04%
Firmly bound water volume	6.92%
Air volume	1.05%
Resin volume	2.54%
Total	100%

The occluded pore volume was 2.54%, and the proportion of the pore volume occluded by resin was 9.94%. Neglecting the organic and water volumes, the proportion of the pore volume occluded by resin is 70.75%.

The mean proportion of the pore volume occluded by resin was 10% (± 4%), ranging from 1.8 to 17.7%.  The proportion of the pore volume filled by air (before infiltration) correlate strongly with the proportion of the occluded volume, being consistent with the theory of capillarity transport. The proportion of the occluded volume accounted for ~ 70% of the air volume available for infiltration. This is consistent with previous report that neglected organic and water volumes. But the total pore volume is much larger than previously reported.

            Infiltration reduces the permeability of enamel by reducing the amount of the water volume. Changes in the air volume over time are important. Reduction causes an increase in enamel permeability and enhances enamel translucency (improve esthetic outcome). Increase causes the opposite. The close contact of the resin with the water pore volume raises the question of whether the resin is degraded over time or not. The firmly bound water volume provides pathway for cariogenic acid, making it possible caries progression in infiltrated enamel.

Stereomicroscopy has low accucary for detecting dentine carious lesion depth

     New article reports new evidences on the ambiguity of dentine aspects under stereomicroscopy (so called "histology" in Cariology) with regard to degree of mineral content in dentine.

-   Stereomicroscopy has low accuracy for detecting the depth of carious lesion in dentine. Sousa et al., Eur J Oral Sci, 125(3):229-231, 2017 (DOI: 10.1111/eos.12350; http://onlinelibrary.wiley.com/doi/10.1111/eos.12350/abstract)

     Stereomicroscopic aspect is similar to the clinical visual aspect, but with microscopic resolution. The ambiguity of dentine aspects under reflected light makes it difficult to determine the severity of caries lesions both in the clinic and in the field. The link between Cariology and Social Sciences in hampered by the fact that non cavitated caries lesions can be as severe (in terms of lesion depth) as cavitated caries lesions.

Translucent dentine: microradiography X stereomicroscopy (more cases)

DENTINE ASPECT UNDER STEREOMICROSCOPY IS AMBIGUOUS REGARDING DENTINE MINERAL CONTENT

De Sousa, Frederico Barbosa

Dept. of Morphology, Health Sciences Center, Federal University of Paraiba, Joao Pessoa, Paraiba, Brazil  

 Translucent dentine (dentine aspect under stereo microscopy, SM) plays and important role in the histopathology of carious lesions. Translucent dentine is commonly regarded as an aspect of sclerotic (hypermineralized dentine). In fact, translucent dentine is ambiguous: it can be either hyper or hypomineralized dentine. Here, new cases of translucent dentine that represent demineralized dentine under microradiography (mRX) are presented. The use of SM as a gold standard technique for measuring caries lesion depth in dentine lacks sientific validity. 

Fig.1. mRX (upper) and SM images of dentine area (white arrow) that appears as translucent under SM.

Fig.2. Two dentine aspects of dentine under SM (white, translucent, and black arrows, "normal" dentine) are shown as demineralized areas under mRX.

Opaque dentin in relation to caries (Gottlieb, Dental Caries, 1946)

       Bernhard Gottlieb (one of the fathers of Oral Histology; former professor of the College of Dentistry at Baylor University, Texas, USA, and College of Dentistry at University of Vienna, Austria) studied dentin reactions to caries in his book “Dental Caries. Lea & Febiger, 1946” (out of print). Ground sections of carious teeth were analyzed by both transmission light microscopy and microradiography (known as Grenz rays at that time), and some sections presented opaque dentin in relation to caries:

Figure. Light microscopic (left) and microradiogarphic (right) images of the same ground section of the tooth crown showing that some areas of opaque dentin (area B in the figure on the left) are sclerotic dentin (A, B, and C areas in the figure on the right) and another area is demineralized dentin (area E in the figure on the right).

       This rare evidence is important for the understanding of the nature of caries lesions.

Polychromatic polarization microscopy: new technique for studying weak birefringent structures

           A new technique for analyzing structures at nanoscale with polarizing microscopy has been developed: polychromatic polarization microscopy (Shribak, Scientific Reports, 5:17340, 2015; doi:10.1038/srep17340). One of the limitations of exisitng polarizing microscopic techniques was that samples with small retardances (like biological soft tissue samples) required digital processing to be rendered visible in the field of view. The new technique allows the visualization of birefringent samples without digital processing and the structures appear colored regardless their orientation in relation to the diagonal position (position at which strutuctures present maximun brightness in conventional polarization microscopy). The resulted image of the new polychromatic polarization microscopy shows birefringent objects with different brightness (relative to their retardances) and colors (relative to the orientaiton of their slow axis). The new technique is independent of sample orientation and the image can be seen through the eyepieces without digital processing.
  The new technique opens the possiblity of studying biological samples with small retardances, with applications in the diagnosis of various diseases and imaging of low birefringent crystals (like in carious, fluortic and developing enamel).