Gradients of biochemical volumes in normal enamel in relation to enamel tufts

 Increasing attention has been paid to the graded mechanical properties of dental enamel, with decreasing hardness and elastic modulus and increasing toughness from outer to inner enamel (Bajaj et al., Biomaterials 2009; 30: 4037-4046; Barani et al., J Mech Behav Biomed Mater, 2012; 15:121-130)). Spatially-resolved quantitative data on the biochemical volumes from outer to inner enamel were lacking. A recent study, exploring microradiography and interpretation of enamel birefringence, reported this later data, showing decreasing mineral volume and increasing water and organic volumes from outer to inner normal enamel of human permanent teeth (Macena et al., Archives of Oral Biology, 2014, http://dx.doi.org/10.1016/j.archoralbio.2014.03.001). Mineral volume ranged from 70.6 to 98.5%, while organic volume ranged from 0.02 to 20.87%, and water volume ranged from 3.8 to 9.8%.

Figure 1. Microradiography of ground section of normal enamel showing tufts as radiolucent structures. From enamel surface to enamel-dentine junction, mineral volume decreases and water and organic volumes increase (Macena et al., Arch Oral Biol, 2014, http://dx.doi.org/10.1016/j.archoralbio.2014.03.001), creating a osmotic gradient for water transport. Enamel layer is divided into tuft area and non-tuft area, which differed markedly with regard to biochemical volumes.

     Organic and water volume are the highest ever reported for fully formed normal enamel, and indicate an osmotic gradient from outer to inner enamel. The location of the increased organica volume coincides with the organic-rich inner enamel layer shown by BSE-SEM by Dusevich et al. (Arch Oral Biol, 2012; 57:1585-1594). This is consistent with the early report that water diffuses to inner enamel following an osmotic pressure that vanishes after removal of organic matter (Atkinson, British Dental Journal, 1947, 83: 205-214). This confirms recent report that, during dehydration at room temperature, outer enamel reaches equilibrium moisture content earlier than inner enamel (Medeiros et al., J Microsc, 2013, 250: 218-227). Macena et al. also report that after heating dried enamel to 50º C and 50% of relative humidity, inner enamel rehydrated after being exposed to 25ºC and 50% RH, evidencing the osmoti pressure exerted by the graded biochemical volumes.

    The implications for transport of materials in dental enamel is that small molecules applied to the enamel surface  are expected to follow the water flow towards inner enamel. 

     Biochemical volumes were measured in relation enamel tufts. Imaginary lines were drawn from outer to inner enamel (see Fig. 1) and histological points along those lines were selected for measuring biochemicla volumes. In each ground sections, two lines were traced: one crossing a tuft (tuft line) and another (adjacent) crossing an area without tuft (control line).  Each line extended from the area where tufts could be found (tuft area; close to the EDJ, Fig 1) to the region where there were no tufts (non-tuft area). No differences were found between tuft line and control line, but the tuft areas had biochemical volumes markedly different (lower mineral volume and higher water and organic volumes) from those of the non-tuft areas. Tuft presented the same behavior of lamellae, which are crack lines lacking birefringence in ground sections. Thus, this evidence indicates that tufts are also crack lines.

Dark zone of enamel caries: conflicting evidences

      The dark zone of enamel caries is the third (from outer to inner enamel; and the second from inner to outer enaml) histological layer of enamel caries. According to Darling (British  Dental Journal, 105:119-135, 1958), this layer, which is shown under immersion in quinoline (an oil medium), is negatively birefringent under water immersion (representing less than 5% of mineral loss) and isotropic or positively birefringent in quinoline, this later feature probably because quinoline does not infiltrate and the dark zone remains filled with air or water vapour (hence a mineral loss of 2-4%). As it was interpreted that quinoline did not infiltrate into the pores, the dark zones was, and this is prevailing current thinking, interpreted as a zone with small pores. This gave enhanced interest in the remineralizaiotn of enamel caries as those reduced pores were interpreted as a sign of remineralization within the lesion. The most influential source came for an article in Nature (Poole et al., Nature, 189: 998-1000, 1961) where the particular features of the dark zone were studied with various immersion media. But there are conflicting evidences regarding the nature of the dark zone.
        In the early reports, it was referred as a demarkation zone (Applebaum E., Journal of Dental Research, 12:619-627, 1932), because it was located between the supposedly normal enamel and the bulk of the enamel caries lesion. Before Darling published his papers on the histopathology of enamel caries in polarized light microscopy, it was already published an image showing the dark zone as an opaque area under transmitted light micrsocopy illuminated with ultraviolet light (Fig. 7A of Applebaum, Dental Cosmos, 77:931-941, 1935). Opaqueness is resulted from light scattering, which, in turn, is directly proportional to the ratio of pore size by wavelength of light. Thus, this suggested that pores sizes could be large in the dark zone.
      Now with the papers by Darling, another conflicting evidence was published and it can be seen in the images provided below. In Figs. 5B and 5C of Darling (Br Dent J, 1958), a ground section is presented under immersion in quinoline (with dark and translucent zones) and air, respectively. In air, most of the enamel caries lesion is opaque, i.e., does not show any birefringence

Figure 1. Images of figures 5B (upper) and 5C (lower) of Darling (Br Dent J, 1958). In the lower image, a zone with the same color of the backgound (isotropic) is seen below the opaque (black) enamel caries lesion.

    If one combines those images, it can be possible to detect that some parts of the dark zone seen in the upper image of Fig. 1 are located in opaque enamel seen in air immersion (lower image). Figure 2 shows a montage of the two images and allows to identify the overlapping. 

Fig. 2. Combined images of ground section of enamel caries in quinoline and air (smaller rectangle) showing that parts of the dark zone (arrow) are located in the region that was opaque under air immersion.

     Darling (1958) interpreted opaque enamel under polarizing microscopy as an indication of isotropy, a mistake that has never been identified by those who cited his papers. There is a clear difference between opaque and isotropic enamel under polarizing microscopy. Opaque enamel is that one that does not present any interference colors in polarized light microscopy. In order to identify it, one has to change the backgound color from black to red (the one produced when Red I retardation filter is used) and identify those areas of enamel that remain black with a red background. Isotropic enamel is that one that presents the same color of the bakground, so that is changes from black to red following the background color (Medeiros et al., Journal of Microscopy, 246:177-189, 2012). When pore sizes are too large in relation to the wavelength of light, light scattering combined with the lack of form birefringence from the pores eliminate birefringence. In the early reports, color images were rare, and even more rare were color reprints used by those who cited the papers of others. This contributed to the misinterpretation. Another thing that contributed, as it seemed to be from the contents of the papers, was that Darling did not identify it in his papers. Had he interpreted correctly, should not have published the article in Nature (Poole et al., Nature, 189: 998-1000, 1961). One interesting fact about this is that the same ground section shown in Fig 1 above was published in a later publication (eight years later) by a different author (Silverstone LM, British Dental Journal, 461-471, 1966; figures 1 and 2 show this same ground section in quinoline, and figure 4 shows it in air immersion; the front page of the article says "original communications", but three images were previously published). In this paper, the reader can more easily identify that parts of the dark zone are located in opaque enamel. 

     Another conflicitng evidence came from the report that removal of organic matter decreased the size of the dark zone, suggesting that the pores in the dark zone are large enough to allow infiltration of quinoline  and the excess of organic matter makes the infiltration difficult (Shellis et al., Eur J Oral Sci, 110: 392-395, 2002).

     Altogether, the evidences shown above suggest that the dark zone is not a zone of remineralization.