Possible Implications for Caries Dissolution of Enamel Crystalsâ. By Colin ... present between the rods as the interrod enamel and they are exactly the same but.
Review of the paper entitled, “Crystal Initiation Structures in Developing Enamel: Possible Implications for Caries Dissolution of Enamel Crystals” By Colin Robinson and Simon D. Connell. Reviewed by Hershey Warshawsky, Emeritus Professor, Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada. This paper was recommended to me by Hiroyuki Mishima and I am grateful for bringing this article to my attention. Reading this paper reaffirmed my belief that ignoring previously published literature leads to “reinvention of the wheel”. This is what I found in this paper. Below, I cited quotes from the paper with my comments below each quote. These are of regular size and shape, densely packed with their long c-axes parallel and arranged in bundles, the enamel prisms.
With reference to the crystals, the authors omitted that crystals are also present between the rods as the interrod enamel and they are exactly the same but oriented differently. Their failure to mention the interrod is a major flaw in understanding of enamel structure and formation, since the interrod enamel forms first and dictates the pattern taken by the rods. early enamel was viewed using freeze etching which examines fractured surfaces of frozen unfixed tissue (Robinson et al., 1981).
The authors assumed that TEM preparation could introduce artifacts, but freezefracture can also introduce artifacts and these were discussed fully in two papers that were completely overlooked in the literature review. These papers are available through my ResearchGate profile: Warshawsky, Bai, Nanci and Josephsen, In: Tooth Enamel IV, eds, Fearnhead and Suga, Elsevier Publisher B.V. pp. 177-182, 1984, and Bai and Warshawsky, Anat. Rec. 212: 1-16, 1985. The authors would learn much by reading and citing these papers. This revealed ∼30 nm globular structures arranged both randomly and in linear arrays.
Had the authors read these papers they would have realized that the globular structures were artifacts caused by concentrating the amorphous matrix proteins into smaller and smaller spaces as the water is converted to ice, finally resulting in globular particles. Crystals only became visible during maturation after loss of matrix protein. The globules therefore are most likely complexes of amorphousmineral stabilized by protein.
This is absolutely untrue as crystals are seen right from the beginning and my paper with Leblond (1979) was cited to prove that young crystals are seen as soon as enamel formation begins. Therefore, to speculate that the globules are minerals stabilized by protein is completely unfounded. The dimensions and arrangement of these globules suggested that they are forerunners of the crystals seen in
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maturing enamel and delineate both the size, shape, and disposition of the crystals in mature tissue (Robinson et al., 1981).
The crystals are already there from the start and therefore do not need a forerunner, the only thing that happens to the initially formed crystals is that they grow in length, thickness and width, and that delineation occurs during the secretory stage with the crystals reaching final length at the end of secretion and final thickness and width during maturation. Since these may represent imprints of original crystal initiation structures, earlier freeze etched data was reexamined at high resolution for their presence.
The authors are confusing crystal “initiation” with “crystal growth”. Initiation occurs only once for each individual crystal and that site is the DEJ. Subsequent growth does not involve initiation, but addition of mineral at the surfaces of the young crystal. Matrix proteins may be involved, but not as initiators. As speculated in the abovementioned papers, the matrix proteins may be more involved in preventing fusion of adjacent crystals. FIGURE 1 | (A) TEM freeze etched enamel showing repeated globular structures ∼30–50 nM dia in linear arrays (rat incisor) (Robinson et al., 1981) (bar = 60 nM). (B) AFM tapping mode in air of maturation stage enamel crystal showing repeated contiguous globular subunits ∼30 nM diameter (rat incisor, tapping mode in air) (Kirkham et al., 2000; Robinson et al., 2004) arrows (bar=30 nM).(C). AFM image of polished section of mature human enamel. Cross sections of enamel crystals are visible showing ∼15 nM subunits in roughly hexagonal clusters (tapping mode in air) (Robinson et al., 2004) arrows (bar =60 nM). (D) AFM image of polished section of mature human enamel. Longitudinal sections of enamel crystals are visible showing 15nm subunits, (human, tapping mode in air) longitudinal interface between subunits can be seen arrows (Robinson et al., 2004) (bar = 60 nM). (E) High resolution TEM image of freeze etched rat incisor secretory enamel showing 30–50 nM globules but comprising smaller ∼15 nM subunits, arrows
I have no idea what these pictures show, but I predict that neither do the authors. If, as we have shown in out papers in 1984 and 1985, these particles are condensation artifacts due to the conversion of water to ice and the subsequent concentrating of the amorphous proteins, then all these pictures are artifacts with no functional significance in forming the enamel crystals. Figure 2 again confuses nucleation, or initiation, with crystal growth. They are not the same; again crystals are initiated once, growth progresses through the secretory stage and into maturation. These colorful images are wild imagination, but very fanciful. Recrystallisation results in the mature enamel crystal with crystalline or chemical discontinuities at the fusion interfaces.
This is pure unsubstantiated conjecture as the crystals are completely smooth as seen in freeze-fracture replicas or fractured enamel viewed with surface SEM. Lateral fusion would lead to a discontinuity along the length of the crystal at its center, while longitudinal fusion would lead to lateral discontinuities perpendicular to the central line. These are the sites at which enamel crystals are known to dissolve preferentially during carious attack (Johnson, 1967; Yanagisawa and Miake, 2003).
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Central dissolution of crystals has been shown to be an artifact caused by electron beam damage and misinterpretation of crystal shape. See my papers on crystal structure and visualization by TEM. Lack of attention to previous literature results in perpetuation of error and impedes a true understanding of enamel. I enclose a picture of several views of “my enamel crystals”. Fig. 17 is chatter effect produced by sectioning for TEM; Fig. 18 is a freeze-fracture replica of rat incisor enamel, and Fig. 19 show isolated rat incisor enamel crystals to demonstrate their length.
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Hershey Warshawsky.
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