Response to the critical comments on the article entitled "Organization of Crystals in Enamel".
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Animals↗
Biomedical subjects
H Warshawsky
Publications and source records attributed to H Warshawsky.
Cyclical incorporation of 33P into rat incisor enamel in vivo as visualized by whole-mount radioautography.
Phosphorus uptake during amelogenesis was investigated in the continuously erupting rat incisor. Five minutes after intravenous injection of 33P-labelled ortho phosphoric acid, whole-mount radioautography of entire incisors revealed heavy labelling in the form of bands and narrow parallel stripes at the surface of the enamel in the maturation zone. There was relatively little labelling over enamel in the secretion zone and over pigmented enamel. Thus 33P is incorporated cyclically into maturing enamel and is visualized as (1) a banded pattern that reflects the modulation of ruffle-ended and smooth-ended maturation ameloblasts and (2) a striped pattern that reflects the distribution of newly-formed protein secreted by maturation ameloblasts. Presumably these P incorporation patterns are closely related to other cyclical events known to occur during enamel maturation.
Ameloblasts↗
Effects of acute doses of sodium fluoride on the morphology and the detectable calcium associated with secretory ameloblasts in rat incisors.
Fluoride in high concentrations is known to have an adverse effect on the formation of enamel. The effect of a single injection of two concentrations of sodium fluoride on inner enamel secretory ameloblasts was investigated morphologically by electron microscopy and functionally by assessing the location and relative amount of available calcium, using the potassium pyroantimonate method. The results showed that acute doses of fluoride interfere with the normal function of secretory ameloblasts. The increase in the population of lysosome-like structures observed after fluoride administration is suggestive of defects in the synthetic pathway. Concomitant with the effect of fluoride on secretory ameloblasts is an inhibition of enamel formation, resulting in incomplete enamel rods and leaving large remnants of Tomes' processes buried in the enamel. The distribution of the calcium pyroantimonate deposits found tends to support the concept of calcium traveling between the cells to the enamel. Acute doses of fluoride also reduce the amount of calcium available for complexing with pyroantimonate in the intercellular region.
Ameloblasts↗
Earliest enamel deposits of the rat incisor examined by electron microscopy, electron diffraction, and electron probe microanalysis.
In order to describe initial events in enamel mineralization and to help characterize inorganic-organic interactions in this tissue, the earliest rod and interrod enamel in mandibular incisors from normal young adult (100 gm) rats, perfused with 100% ethylene glycol, has been studied by transmission electron microscopy, selected area electron diffraction, and high-spatial-resolution electron probe microanalysis. Diffraction and probe data were correlated precisely from the same extracellular regions of the tissue. Sites were examined progressively as a function of location a) from the most recently deposited enamel adjacent to ameloblasts toward the dentin-enamel junction and b) from the apical portion of the tooth longitudinally toward its incisal end. Electron diffraction patterns consistent with that of a poorly crystalline hydroxyapatite were generated at all locations. Diffraction characteristics changed only slightly toward that of more crystalline hydroxyapatite at different locations. Earliest apical enamel generated molar Ca/P ratios in a range of 0.99-1.46 (average 1.24 +/- 0.15). Molar Ca/P ratios of the first enamel interrod elements increased from approximately 1.24 at ameloblast-enamel boundaries to approximately 1.40 at the dentin-enamel junction, small changes corresponding to those observed in electron diffraction characteristics.
Animals↗
A simple method for the preparation of isolated enamel crystallites for transmission electron microscopy.
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Animals↗
Demonstration by staining and radioautography of cyclical distributions of protein at the enamel surface in rat incisors.
Staining patterns in the enamel during the maturation stage of amelogenesis reflect the banded distribution of ruffle-ended and smooth-ended ameloblasts. This study investigated the possibility that proteins at the enamel surface may be distributed cyclically according to cyclical changes in ameloblast morphology. Dissected lower rat incisors were wiped free of their enamel organs and immediately immersed in fixative containing one of the following heavy metal and histological stains: uranyl acetate, lead citrate, Coomassie blue, alcian blue and ruthenium red. Other animals were injected with [35S]methionine to label newly-formed enamel proteins. Their incisors were dissected, the enamel organs were wiped from the enamel surface, and the teeth were processed as whole mounts for radioautography. Teeth stained by heavy metals were also viewed by back-scattered electron imaging. The in-situ staining revealed that proteins were distributed in bands and stripes across maturing enamel. Radioautography revealed that the proteins in the stripes were newly-synthesized and secreted into the enamel by certain maturation ameloblasts. We conclude that the enamel organ expresses cyclical activity in part through secretion of proteins.
Ameloblasts↗
In vivo demonstration of cell types in bone that harbor epidermal growth factor receptors.
The binding and internalization of [125I]iodoepidermal growth factor (EGF) by bone cells of the rat was demonstrated in situ by quantitative radioautography. Specific binding sites were observed on a cell profile enriched in endocytic components, including lysosome-like structures, a rough endoplasmic reticulum-rich cell profile, and a cell profile that histologically resembles an undifferentiated precursor cell. By the criteria of gel filtration and precipitability by trichloroacetic acid, most of the bound [125I]iodo-EGF was considered intact. By morphological criteria none of the cell profiles that bound [125I]iodo-EGF corresponded to fully formed osteoclasts or osteoblasts. The endocytic cell was found in the epiphyseal plate between the invading capillary and the transverse and longitudinal cartilage septa as well as near osteoclasts in the zone of mixed spicules. The rough endoplasmic reticulum-rich cell was present in vacated chondrocyte lacunae of the epiphyseal plate close to the metaphysis, and the poorly differentiated cell was observed between the mixed spicules of the metaphysis. Similar cell types were also found in the alveolar bone surrounding the incisors. These cells may be the origin of established bone cell lines that harbor high concentrations of EGF receptors and may also be responsible for the humoral hypercalcemia in response to the reported actions of injected EGF or transforming growth factor-alpha as well as that of malignancy.
Alveolar Process↗
Specific binding sites for transferrin on ameloblasts of the enamel maturation zone in the rat incisor.
During enamel maturation in rodents, an iron-containing pigment is deposited into the surface layer of the enamel. Maturation zone ameloblasts presumably are responsible for this deposition. The presence of large amounts of ferritin in the cytoplasm of these cells suggests that they receive iron, presumably from circulating transferrin. An in vivo radioautographic binding assay using iodinated transferrin was used to determine if indeed maturation ameloblasts possess transferrin receptors at their cell surfaces. Experimental rats received systemic injections of labeled transferrin while control rats received injections of labeled transferrin plus a large excess of unlabeled transferrin in order to compete with the labeled transferrin for available specific receptors. Light microscope radioautography showed that ruffle-ended ameloblasts (RAs) of the enamel maturation zone had a high density of specific receptors for transferrin relative to smooth-ended ameloblasts (SAs). Electron microscopy and energy-dispersive X-ray spectroscopy confirmed the presence of ferritin and iron, respectively, within these cells. It is postulated that the iron responsible for enamel pigmentation is transported by transferrin to maturation ameloblasts and is bound to specific transferrin receptors found mostly on RAs and that the modulation of these cells into SAs results in a loss of most of these receptors.
Ameloblasts↗
Analysis of crystallite shape in rat incisor enamel.
The hydroxyapatite crystallites of mammalian enamel appear as hexagons when seen in cross-sections examined with the transmission electron microscope. Using goniometric transmission electron microscopy, stereo-pair electron micrographs and freeze-fracture replicas, two models have been proposed to explain the hexagonal crystallite profile. The "hexagonal ribbon" model proposes that hexagonal profiles are true cross-sections of elongated hexagonal ribbons. The "rectangular ribbon"model proposes that crystallite profiles are three-dimensional rectangular segments (parallelepipeds), which are contained in the Epon sections and project as opaque hexagons in routine transmission electron micrographs. Morphological observations together with predictions from models indicate that the crystallites in rat incisor enamel are flat ribbons with rectangular cross-sectional profiles. The hexagonal images seen in electron micrographs of thin sections of enamel result from viewing parallelepiped-shaped segments of these crystallites as two-dimensional shadows.
Animals↗
The effects of vinblastine on the secretory ameloblasts: an ultrastructural, cytochemical, and immunocytochemical study in the rat incisor.
Secretory ameloblasts synthesize the organic matrix of enamel and secrete it at two distinct "putative secretory sites" characterized by membrane infoldings (Nanci and Warshawsky, 1984a). The antimicrotubular agent vinblastine sulphate interferes with secretion. We have examined the effect of this drug on the ameloblast secretory sites and re-evaluated the effect on the intracellular organization of the cell by using conditions that optimize fixation, cytochemistry (ZI0), and immunocytochemistry. Associated with the disappearance of secretory granules and Golgi-related structures from Tomes' process was the loss of membrane infoldings at secretory sites. The Golgi apparatus appeared fragmented and numerous granule clusters were found throughout the cell body. These clusters were often seen in relation to extracellular patches of material in which no crystallites were seen. Immunocytochemistry revealed the presence of enamel proteins in the protein synthetic organelles, including various granule types, in lysosomes and in the extracellular patches. These data suggest that ameloblasts under the effect of vinblastine carry on secretory activities, but the product is not routed to the usual sites. It was confirmed that membrane infoldings characterize the sites where enamel proteins are normally secreted.
Ameloblasts↗
A radioautographic study of the effects of vinblastine on the fate of injected 45calcium and [125I]-insulin in the rat incisor.
To examine the effect of vinblastine on the movement of calcium and macromolecules through the enamel organ in the secretion zone and through the odontoblast layer, 45CaCl2 and [125I]-insulin were used as radioautographic tracers. Vinblastine did not alter the localization of either labelled Ca or insulin in the enamel organ and underlying enamel, but eliminated both labels in the pulp, odontoblasts and dentine. It is concluded that vinblastine has no effect on the passage of Ca and macromolecules in the enamel organ and secretory ameloblast layers, whereas its effect on the pulp and odontoblasts prevents passage of these tracers into the predentine and dentine.
Animals↗
Enamel crystal shape: history of an idea.
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Animals↗
External shape of enamel crystals.
Biological hydroxyapatite crystals are either small, as in bone and dentin, or large as in enamel. Enamel crystallites are unique since each is initiated and grows in length, thickness and width until the entire layer of enamel is secreted. In maturation, these extremely long crystallites grow only in thickness and width. Crystal growth in vitro follows physico-chemical principles, but lacks biological intelligence; in vivo this intelligence is contributed by protein templates. The location of the organic template in enamel is congruent with the crystallite, constituting the crystal ghost. Since crystals cannot accommodate proteins, the explanation is logically inconsistent. In sections, enamel crystallites, appear hexagonal, and this is interpreted as their cross-sectional shape. Since this hexagonal image also contains the crystal ghost, the notion that hexagons do not represent true cross-sections was explored with models of crystallite shape. Hexagonal rods were compared to rectangular or rhombohedral rods. Whereas segments of hexagonal rods in the section should project as octagons at the electron microscope imaging plane, and octagonal profiles are never found, rectangular or rhombohedral rod segments project as hexagons. Assuming the organic template covers the crystal exterior, the projected rhombohedral segment, appearing hexagonal, would seem to contain the protein, hence explaining the apparent presence of the crystal ghost.
Animals↗
In vivo demonstration by radioautography of binding sites for insulin in liver, kidney, and calcified tissues of the rat.
An in vivo binding assay using radioautography was employed to visualize insulin receptors in rat tissues. Two and one-half minutes after the intravenous injection of 125I-insulin, free hormone was separated from bound hormone by whole body perfusion with lactated Ringer's solution followed by perfusion with glutaraldehyde. The localization of bound hormone, fixed in situ by perfusion with glutaraldehyde, was determined. Nonspecific binding of labeled insulin was noted in the proximal convoluted tubules of the kidney cortex, prebone and adjacent bone, predentin and adjacent dentin, and enamel. Specific binding sites were observed at the periphery of hepatocytes, over osteoblasts, and in relation to the endothelial cells of fenestrated capillaries within the papillary layer of the maturation zone of the incisors.
Animals↗
Calcitonin receptor binding as a marker of osteoclast heterogeneity in osteopetrotic rodents.
We have employed a radioautographic technique to examine in vivo receptor binding of calcitonin to osteoclasts in four rodent mutants with osteopetrosis. 125I-Labeled calcitonin was injected intravenously alone or with excess unlabeled calcitonin to osteopetrotic (op/op), osteosclerotic (oc/oc), and microphthalmic (mi/mi) mice and to incisor absent (ia/ia) rats. Similar experiments were performed simultaneously in phenotypically normal littermates. Specific binding of calcitonin to receptors on osteoclasts and osteoclast morphology were then examined by light and electron microscope radioautography. Calcitonin binding was increased in mi/mi mice, where osteoclasts were abundant but reduced in size, and was also increased in op/op mice in association with an undulated and redundant osteoclast cell membrane. Binding of the hormone was markedly diminished on osteoclasts of oc/oc mice and ia/ia rats. Thus, in these rodent models of osteopetrosis all of which manifest reduced skeletal remodeling and share a recessive pattern of inheritance, considerable heterogeneity of osteoclast characteristics was demonstrable. Although calcitonin may play no primary pathogenetic role in most forms of this disease, calcitonin receptor binding is a morphological and functional marker of osteoclasts that can be used in assessing the pathophysiology of disorders of bone remodeling.
Animals↗
Modification of the enamel maturation pattern by vinblastine as revealed by glyoxal bis(2-hydroxyanil) staining and 45calcium radioautography.
Patterns characteristic of enamel maturation can be visualized at the surface of the rat incisor by staining with glyoxal bis(2-hydroxyanil) (GBHA) and radioautography following 45calcium injection. In this study, the effects of vinblastine on enamel maturation were monitored by these two methods. At 4 h after injection of vinblastine, the darkly-stained GBHA bands had widened incisally into the interband regions when compared to normal, control teeth. Radioautography at 5 min after calcium injection in vinblastine-treated animals (4 h) showed a modified maturation pattern of weaker labeling and less distinct banding. At 8 h after vinblastine injection, most of the enamel stained uniformly with GBHA, and bands and interband regions could not be resolved. Radioautography at 5 min after calcium injection showed that the 8 h vinblastine treatment removed the banding pattern, leaving only a weakly-labeled area. Vinblastine is known to destroy and prevent the formation and turnover of microtubules, and hence the formation of ruffled borders of ruffle-ended ameloblasts (Akita et al. 1983). The concomitant decrease in calcium incorporation implies that events taking place in relation to the ruffled border may affect calcium exchange or accretion within the enamel.
Aging↗
Penetration of various molecular-weight proteins into the enamel organ and enamel of the rat incisor.
During enamel maturation, most of the organic matrix is removed as the mineral content increases; it is postulated that proteolytic enzymes within enamel break down large proteins into more mobile fragments. To predict how such fragments might leave the enamel, the entry and penetration of various proteins into it was examined. Rats (100 g) were injected via the external jugular vein with 125I-iodinated calcitonin (3600), insulin (5700), epidermal growth factor (EGF; 6100) and albumin (68,000). They were killed after 10 min and radioautographs made to visualize these molecules in the incisor enamel organ and enamel. In addition, dissected incisors were wiped free of their enamel organs, dipped in the iodinated protein solutions for 10 min, and processed for radioautography. In all dipped teeth, except those exposed to albumin, there was a gradient of silver-grain density over the entire thickness of enamel in both the secretion and maturation zones. In all injected animals, enamel labelling in the secretion zone was only slightly above background. In the maturation zone of animals injected with calcitonin and insulin, many grains were over enamel adjacent to smooth-ended ameloblasts but not ruffle-ended ones. Animals injected with EGF and albumin had no labelled enamel in the maturation zone. Thus dipped rat incisor enamel was permeable to proteins with molecular weights as high as 6100. Localization of injected proteins indicates that the enamel organ restricts their passage into enamel, but proteins with molecular weights as high as 5700 may pass into enamel through or between smooth-ended ameloblasts. As exogenous proteins readily diffused into the enamel, it seems likely that enamel proteins of similar size can leave enamel by a similar route.
Animals↗
Effects of various agents on staining of the maturation pattern at the surface of rat incisor enamel.
In the maturation zone, two types of ameloblasts are arranged as bands across the rat incisor; these corresponded with a staining pattern at the surface which reflected the alternating pattern of ruffle-ended and smooth-ended ameloblasts. PAS stain showed bands and stripes similar to those following glyoxal bis(2-hydroxyanil) (GBHA) staining; these stains visualize the organic (PAS) and inorganic (GBHA) components of the maturation pattern. To further elucidate the nature of these bands, dissected rat incisors were treated with various agents prior to staining with GBHA or PAS. Guanidine for 2 h at room temperature showed no maturation pattern when stained with GBHA, as did teeth treated with EDTA and a bisophosphonate (HEBP). Hydrochloric acid and nitric acid removed the layer of outer enamel and incisors did not stain with GBHA, suggesting that the staining is a surface-related phenomenon. As staining was abolished by either the removal of mineral (EDTA) or protein (guanidine), the concurrent localization of non-crystal-bound calcium by GBHA staining, and of glycoprotein by PAS staining, indicates that calcium is associated with glycoprotein at the surface of the enamel.
Ameloblasts↗