Rat Incisor Enamel Research Articles

Fine Structure and Elemental Analysis of the Enamel in Andrias davidianus (Cryptobranchidae) and Onychodactylus japonicus (Hynobiidae)

Functional teeth from Andrias davidianus and Onychodactylus japonicus were prepared for transmission electron microscopy (TEM) and electron microprobe analyzer (EMPA) of the elements of the enamel layer. The enamel layer is thin and the arrangement of the enamel crystals is regular in the outer enamel layer of Onychodactylus japonicus; on the other hand, the crystals of Andrias davidianus show irregularities in different tooth regions. Based on calcium and magnesium distribution levels using electron microprobe analyzer (EMPA), the enamel layer lacks enameloid in these species. Concentrations of iron and fluoride are located in the outer enamel layer. The presence of trace elements is related to the formation and mineralization of enamel. l of Herpetology, Vol. 25, No. 2, pp. 14 46, 1991 i t 1991 Society for the Study of Amphib ans and Reptiles cture and Elemental Analysi of the Enamel in i s avidianus (Cryptobranchidae) and actylus japonicus (Hynobiidae) O,1 KAZUYUKI SHIMADA,2 ATSUYUKI YOKOI,2 The enamel layers of the amphibian tooth form prismless structures, in contrast to mammalian teeth which have rods in the enamel layer. This material is known as aprismatic enamel, and though it is very thin, it is hypermineralized, as in mammals. In mammals, the enamel crystals are chiefly composed of hydroxyapatite, with iron, fluoride and other trace elements complexed in the surface enamel layer of the tooth (Halse and Selvig, 1974; Selvig and Halse, 1975; Weatherell et al., 1975; Heap et al., 1983; Nelson et al., 1989). A number of different trace elements may be involved in the formation of the apatite crystals. The pigmented layer of rat incisor enamel contains 1-10% iron (Halse and Selvig, 1974) bound to the surface of hydroxyapatite crystals (Selvig and Halse, 1975). Trace elements may also be associated with the mineral component of amphibian enamel. Based on observations using polarizing microscopy, Schmidt (1971) reported that the enamel of Andrias japonicus contains pigments that impart a ena el layers of the amphibian too h prismles structures, in contrast to ma brown color at the tip of the tooth, which he suggested to be an iron oxide. Using transmission electron microscopy (TEM), Kawasaki and Fearnhead (1983) reported the presence of enam loid, a substance previously reported to occur only in fish, beneath the enamel layer in the teeth of the salamanders Hynobius nigrescens and Cynops pyrrhogaster. Enameloid differs from enamel in having a different embryological origin (mesoderm vs. ectoderm), and in having a much lower mineral content (Poole, 1967). The purpose of this study was to look for an interrelationship between the fine structure, concentration, and distribution of trace elements of the enamel layer in Andrias davidianus and Onychodactylus japonicus, and to ascertain the presence or absence of enameloid in these species. MATERIALS AND METHODS Thirteen adult (total length ca. 80 cm) Andrias davidianus (Cryptobranchidae) and ten adult (tocolor at the tip of the tooth, w ich he ested to be an iron oxide. Using transmiselectron microscopy (TEM), K wasaki and head (1983) reported the presence of eloid, a substance previously reported to only in fish, beneath the enamel layer in teeth of the s lamanders Hynobius nigrescens nops py rhogaster. Enameloid differs from el in having a diff rent embryological or( esoderm vs. ectoderm), and in having a lower mineral content (Poole, 1967). urpose of thi study was t look for an relationship between the fine str cture, 141 This content downloaded from 157.55.39.59 on Mon, 17 Oct 2016 04:59:01 UTC All use subject to http://about.jstor.org/terms

Differential expression of calbindin‐D 28 kDa in rat incisor ameloblasts throughout enamel development

Calbindin-D 28 kDa (CaBP 28 kDa), a vitamin D-dependent calcium-binding protein, has been associated with calcium handling by cells. We have investigated the expression of this protein in the rat incisor enamel organ, an epithelium interposed between a mineralizing matrix and connective tissue rich in blood vessels, by radioimmunoassay (RIA), Western blotting, and quantitative protein A-gold immunocytochemistry with antibodies to rat kidney CaBP 28 kDa. RIA of cytosolic extracts showed that enamel organs contained relatively high concentrations of CaBP 28 kDa (compared to kidney; see review by Christakos S., C. Gabrielides, and W.B. Rhoten 1989 Endocr. Rev., 10:3-25). Immunoblotting of proteins extracted from enamel organ strips revealed an intensely-stained band near 28 kDa throughout amelogenesis following ameloblast differentiation. Immunocytochemically, CaBP 28 kDa was localized exclusively within ameloblasts. The density of labelling increased from the presecretory stage to the secretory stage and fluctuated across the maturation stage in relation to ameloblast modulation. Ruffle-ended ameloblasts consistently showed the most intense immunoreaction. Gold particles were present throughout the cytoplasm and nuclei of ameloblasts but regions rich in rough endoplasmic reticulum or cell webs showed a higher immunolabelling. Some gold particles were also associated with the external face of the rough endoplasmic reticulum. Multivesicular bodies in maturation stage ameloblasts were occasionally immunoreactive. These data suggest that the intracellular concentration of CaBP 28 kDa is regulated throughout amelogenesis reflecting a stage-specific control of calcium homeostasis in ameloblasts.

Evidence that apatite crystals of rat incisor enamel have hexagonal cross sections

Images of nearly cross sectioned enamel crystals obtained with the transmission electron microscope were examined in detail and correlated with various possible cross-sectional shapes of the crystals. The following images were seen: 1) elongated hexagons with sharp outlines, maximal density, and minimal size (type A profile); 2) elongated hexagons with one pair of long sides of high contrast, two pairs of short sides of low contrast, high density in the center of the image, and low density towards the low contrast sides (type B profile); 3) slanted hexagons with one pair of high contrast sides, two pairs of low contrast sides (one pair of long, one pair of short sides), and the density decreasing from the center towards the low contrast sides (type C profile); this profile was seen more frequently than the type B profile; and 4) octagons with low contrast edges all around, the density decreasing from the center towards the edges (type D profile). Types B, C, and D profiles were larger than type A profiles. Similar crystal images have been described in the literature. When tilting a specimen in increments of 6 degrees, the transformation of type A to type C and type C to type D profiles was observed. Many crystal images did not change their shape greatly when tilting the section through 6 degrees. No rectangular or rhomboidal profiles were seen. It is argued that types A, B, C, and D profiles cannot be generated by apatite crystals with a parallelepiped cross section. They are easily explained, however, on the basis of crystals with hexagonal cross section.

Evidence for Uptake of Basement Membrane by Differentiating Ameloblasts in the Rat Incisor Enamel Organ

Demonstration of type-IV collagen and acid phosphatase (ACPase) was carried out in the rat incisor enamel organ after the animals were fixed by perfusion with periodate-lysine-paraformaldehyde. Their incisors were dissected out, demineralized with EDTA, and prepared into 6-microns-thick frozen sections. The sections, which had been treated by means of antibody incubation for type-IV collagen, were washed with a Trismaleate buffer, incubated in Novikoff's medium for acid phosphatase (ACPase), and then incubated in a 3, 3'-diaminobenzidine solution. After osmification, the sections were embedded in epoxy resin for electron microscopy. The plasma membranes of the distal ends of the inner-enamel-epithelial cells were relatively even and were lined with a basement membrane. Type-IV collagen was localized both in the lamina densa and in the filaments attached to the lamina densa. In differentiating ameloblasts, the remarkably undulating distal plasma membranes formed irregular shallow and deep invaginations, and small cytoplasmic processes that penetrated the basement membrane. Coated pits occurred in various parts of these undulating plasma membranes. Positive reaction to type-IV collagen was observed in the invaginations and coated pits. ACPase-positive granules, present in inner-enamel-epithelial cells, increased in number and sometimes appeared close to both shallow and deep invaginations of differentiating ameloblasts. These results indicate that type-IV collagen in the basement membrane of the enamel organ is removed and degraded by differentiating ameloblasts by means of their engulfing system.

Gradients in the Composition of Enamel and Dentin Mineral in Rat Incisors

AbstractIn order to investigate possible differences in the mineral composition of rat incisors, Wistar rats received either a zinc‐deficient or a normal diet (the latter were pair fed or ad libitum). Three incisors from each group were subjected to microanalysis by the electron microprobe. No differences were found in mineral composition between the three groups. The average molar Ca/P ratio in the enamel and the dentin were (1.62 ± 0.05) and (1.50 ± 0.05), respectively. The distribution of sodium and magnesium over the dentin was homogeneous and inhomogeneous, respectively. Dentin did not contain detectable amounts of chlorine. The gradients for sodium, magnesium and chlorine as a function of depth in enamel were similar to those reported for these elements in human and rhesus monkey enamel: sodium and magnesium had a low concentration at the enamel surface, whereas chlorine had a high concentration there.

Effects of single doses of 1-hydroxyethylidene-1,1-bisphosphonate on the mineralizing front of rat incisor enamel: A microradiographic and scanning electron microscopic study

The effects of a single dose of HEBP were examined by injecting rats subcutaneously with various doses (0.5, 1, 2.5, 5 and 10 mg P/kg body wt) and killing them either 2 or 9 days after injection. The maxillary incisors were processed for microradiography and the mandibular incisors for scanning electron microscopy after enzymatic digestion of the enamel organ remains. All doses resulted in a hypomineralized incremental band corresponding to the position of the mineralizing front at the time of injection. At 5 and 10 mg P/kg body wt, a hypomineralized lesion was found throughout the whole thickness of the enamel in an area which corresponded to the stage of late enamel secretion at the time of injection. The surface layer covering this lesion was elevated or disrupted. By scanning electron microscopy, there were three different types of lesions along the enamel surface: a “demarcation groove” corresponding to the initial enamel formation at the time of injection; an “area containing mineral globules” and a “bright band” corresponding to the stages of late secretory and final enamel formation, respectively, at the time of injection. A single injection of HEBP thus interferes with different stages of enamel formation. The findings may be explained as of the physico-chemical effects of HEBP on the mineral phase alone, but a direct effect of the drug on ameloblast function cannot be excluded.

Lectin cytochemistry on the stratum intermedium and the papillary layer in the rat incisor enamel organ.

Lectin cytochemistry was carried out to elucidate the role of stratum intermedium cells and papillary layer cells in amelogenesis, especially in the process of sugar incorporation and on membrane characteristics according to their cytodifferentiation. Regarding the lectin-reaction on the plasma membrane, little or at best a weak reaction of Con A, UEA-I, PNA, MPA and WGA was seen in stratum intermedium cells from the late differentiation stage to the early secretory stage of ameloblasts. Lectin-stainability in the stratum intermedium increased in accordance with the cytodifferentiation of ameloblasts. At the active secretory stage of ameloblasts, lectins intensely stained the plasma membranes of stratum intermedium cells. The plasma membranes of papillary layer cells at both stages of ruffle-ended and smooth-ended ameloblasts were stained by same lectins as well. The results therefore suggest that: 1) stratum intermedium cells bring about changes in the glycolipids and glycoproteins of their plasma membranes in accordance with the cytodifferentiation of ameloblasts; 2) they regulate the transport of mineral and/or organic materials between ameloblasts and extracellular fluid via highly charged plasma membranes generated by glycocalyx; 3) the cell-cell interaction of stratum intermedium cells with ameloblasts, in which carbohydrate-protein (endogenous lectin) interaction plays a significant role, is important for the cytodifferentiation of these cells. Regarding the papillary layer cells, the results suggest that they also regulate the transport of minerals by their charged plasma membranes and participate in the removal of the enamel matrix.

Crystal Morphology and Decalcification Patterns Compared in Rat and Human Enamel and Synthetic Hydroxyapatite

The purpose of this investigation was to compare morphology and dissolution patterns by ultrastructural examination of rat and human enamel crystals as well as synthetic apatite crystals. Mature enamel crystals were of particular interest, since crystal maturation appears to be inhibited in amelogenesis imperfecta. Specimens were isolated from developing and mature rat incisor enamel. Rat enamel, mature human enamel, and synthetic apatite were thin-sectioned without decalcification and examined by transmission electron microscopy. Some sections were exposed to acid, and selected synthetic apatite sections were further treated for removal of embedding plastic, followed by vacuum-shadow-coating with carbon. Results showed that cross-sections of rat, human, and synthetic crystals had a distortion in the flattened hexagonal outline in regions where the growth of one crystal impinged on another. Crystal dissolution occurred preferentially along the c-axis, producing a central defect or hole in the crystals. Preliminary studies with weak acid on mature human enamel indicate that the relatively soluble crystal core is quickly dissolved, while the outer shell remains intact over a much longer period of time. In the mature rat and human enamel, this crystal hole formation had a consistent dimension of approximately 10-nm thickness. The crystal hole dimension was the same size as crystals that are formed during the early secretory phase in rat amelogenesis. Acid-treated synthetic apatite also showed dissolution of the crystal core along the c-axis, but dimensions of the hole were not consistent.(ABSTRACT TRUNCATED AT 250 WORDS)

Tubular lysosomes in ruffle-ended ameloblasts associated with enamel maturation in rat incisor.

Trimetaphosphatase (TMPase) and cytidine-5'-monophosphatase (CMPase) were localized to investigate the lysosomal system, particularly tubular lysosomes, in ruffle-ended ameloblasts associated with maturation of enamel in rat incisor. Demineralized specimens were incubated for TMPase and for CMPase in a modified medium where cerium was used as the capture ion. Ruffle-ended ameloblasts showed distal invaginations and membrane-bound bodies filled with fine granular material, some of which displayed CMPase reaction product. Elongated tubular configurations 80-140 nm wide were distributed throughout the cytoplasm and were reactive with both TMPase and CMPase, thus characterizing these structures as lysosomes. They often contained fine granular material morphologically similar to that present in multivesicular bodies. During late enamel maturation, fewer tubular lysosomes were observed when compared to early maturation. These cytochemical results demonstrate the presence of tubular lysosomes in ruffle-ended ameloblasts, and it is suggested that they are elements of the endosomal system in these cells. These findings are also consistent with a resorptive function for ruffle-ended ameloblasts during enamel maturation.

Degradation and loss of matrix proteins from developing enamel.

The pattern and timing of the breakdown and loss of matrix proteins were studied in developing rat incisor enamel using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), fluorography, radioautography, and in vitro incubations of proteins isolated from freshly dissected, crushed pieces of enamel. For biochemical studies, the technique of Robinson et al. (1974, 1977, 1983) was used to transect the enamel organ and enamel into a series of strips at 1 mm intervals along the length of the tooth. The proteins in each strip were extracted and either quantified by Lowry analysis or applied to 12% slab (enamel) or 5-15% continuous gradient (enamel organ) SDS-polyacrylamide gels and separated by electrophoresis. The biochemical studies indicated that the amount of protein contained within an enamel strip increased gradually by volume across the secretory stage, reached a peak early during the maturation stage, and then declined rapidly thereafter. The distribution of enamel proteins on SDS-polyacrylamide gels changed markedly throughout this period. These changes included increases and decreases in the intensity of staining of proteins at certain molecular weights (e.g., 18 kDa) and the appearance and disappearance of some proteins not seen clearly near the start of the secretory stage of amelogenesis (e.g., 32 and 10 kDa). Labeling studies with 35S-methionine suggested that the "stacked" arrangement of proteins typical of forming enamel (secretory stage) actually represented a very dynamic association of proteins, with new ones being added at the top of the stack and then breaking down with time to become those seen at lower molecular weights. Across the secretory stage, new proteins were always added to the top of the stack, but during early maturation this activity slowed dramatically, allowing the breakdown of aging proteins to be visualized more clearly. Radioautographic studies with 3H-methionine indicated that the breakdown of newly secreted proteins also was correlated with a movement of label from the site of secretion into deeper, previously unlabeled, areas of forming enamel. In vitro studies revealed that the rate and degree of breakdown of enamel proteins varied markedly, depending on the stage of amelogenesis from which the proteins were extracted. Secretory stage enamel proteins showed slow in vitro degradation with accumulation of proteins near 18 kDa. Early maturation stage enamel proteins showed more rapid breakdown with little accumulation of proteins near 18 kDa, whereas late maturation stage enamel proteins showed complete degradation by 2 days of incubation in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

Banding Patterns in rat incisor enamel stained by histochemical complexing methods for calcium

A characteristic banding pattern can be visualized at the surface of the rat incisor in the maturation zone of amelogenesis by staining with glyoxal bis(2-hydroxyanil) (GBHA). Other banding patterns can be obtained with certain histological and fluorochrome stains and by radioautography following 45Ca injection. In this study, several histochemical reagents known to complex with different states of calcium were used to stain the surface of enamel. Rat incisors were quickly dissected and immediately immersed in solutions containing the following calcium-binding reagents: arsenazo III, calmagite, murexide, N,N-naphthaloylhydroxylamine, and calcein. Routinely, one contralateral lower incisor from each pair was counterstained with GBHA in order to relate each of the staining patterns to the banded distribution of maturation ameloblasts that is reflected by the characteristic GBHA staining pattern in the enamel. Each of the reagents used in this study demonstrated a staining pattern consisting of a series of broad bands running transversely and obliquely across the enamel. In all cases, the dyes stained predominantly that enamel associated with ruffle-ended ameloblasts, i.e. enamel left unstained by GBHA. Some of the reagents also stained enamel in the secretion zone. The appearance and distribution of the staining patterns reflect the banded distribution of maturation ameloblasts and appear to be controlled on a time scale related to the rapid modulation of these cells.

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 orthophosphoric 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.

Effects of a low calcium maternal and weaning diet on the thickness and microhardness of rat incisor enamel and dentine

The deposition and mineralization of incisor hard tissues have been studied in rat pups nursed by mothers on a low calcium diet or weaned with the maternal diet. Animals were killed at 30 days (control and low calcium diets; maternally fed) or 60 days (after 30 days weaning on maternal diet). The degree of mineralization of enamel and dentine was evaluated by a microhardness method on thick transverse sections. The enamel and dentine thickness, and the diameters of the incisor sections and pulp cavity were measured on microradiographs from the sections. Microhardness values of enamel were similar in groups killed after 30 days maternal feeding, but the microhardness of root enamel was 73–74% less in the low calcium-diet weaned group. Peripulpar dentine had mean microhardness values lower than controls in the group fed maternally for 30 days, whereas the whole root dentine appeared significantly less hard in the low calcium-diet weaned group than in the controls. A significant reduction of the incisor bucco-lingual diameter was observed only in this last experimental group. Enamel thickness was significantly lower in the roots of both experimental groups and in the necks of the low calcium weaned group. The reduction in dentine thickness was greater (from −30 to −56%); in the root it was more evident on the lingual aspect. Thus calcium deficiency in the mother's diet did not influence either the deposition or the mineralization of the pup's incisor enamel and dentine. However, when the offspring were weaned with the maternal calcium-deficient diet, mineralization of the tooth hard tissue was retarded.

Studies on the influx of [ 3H]-histidine and 45Ca through a surgical opening to rat incisor ameloblasts and adjacent enamel

Considerable controversy exists about the role of ameloblasts in transport of calcium to mineralizing enamel. The rate and pattern of incorporation of calcium and an enamel precursor (histidine) were studied autoradiographically by introducing the isotopes through a surgically created defect in the lower border of the rat mandible. Influx of the isotopes to both secretory and maturation ameloblasts and adjacent enamel was examined in large survey sections of the entire incisor at intervals of 3 min to 2 h. Substantial concentrations of silver grains were observed over both secretory and maturation ameloblasts within 5 min of placement of either isotope. 45Ca was also present in secretory and maturation enamel within this short time. The overall patterns of influx and uptake of both precursors were similar to those found when such isotopes have been administered parenterally. The amount of influx of 45Ca across secretory, smooth-ended, and ruffle-ended maturation ameloblasts was compared qualitatively and found to be similar. The reproducibility of this surgical technique was demonstrated as well as its usefulness in combination with survey sections for multi-method investigations of rat incisor enamel formation and mineralization.

Calmodulin immunocytochemistry in rat incisor enamel organ through its life cycle.

The enamel organ of the growing rat incisor was perfusion-fixed with a mixture of formaldehyde and glutaraldehyde and processed for indirect immunogold labeling of calmodulin on post-embedded ultrathin sections. Throughout the zones of presecretion, secretion, and maturation of enamel, specific protein A-immunogold labeling was localized on polyribosomes and those attached to endoplasmic reticulum, mitochondria, nuclear chromatin, phagolysosomes, and cytoplasm adjacent to the plasma membrane, and tonofilaments associated with desmosomes of ameloblasts and cells of outer layer of enamel organ. Golgi membranes, condensing vacuoles, secretion granules, primary lysosomes, and micropinocytotic coated vesicles were hardly labeled. In the presecretion zone, the basal lamina of the preameloblasts and the matrix vesicles and collagen fibrils of the predentin matrix were not immunoreactive. Tomes' process of secretory ameloblast and adjacent enamel crystals were labeled. In addition to the above immunoreactive structures, some phagolysosomes, ferritin granules, and the cytoplasm of the ruffled border zone of maturation ameloblast contained immunogold particles. In control sections incubated with either protein A-gold complex alone, or antiserum preabsorbed with an excess of calmodulin and protein A-gold complex, only a few gold particles were observed to be randomly associated with the tissues. These results indicate that calmodulin is present in the cells of the enamel organ through all stages of amelogenesis. Its wide distribution is consistent with its involvement in various cytoplasmic functions.

H+-K+-ATPase activity in the rat incisor enamel organ during enamel formation.

The enamel organ of growing rat incisors was perfusion-fixed with a mixture of formaldehyde and glutaraldehyde and processed for ultracytochemical demonstration of ouabain-resistant, K+-stimulated p-nitrophenylphosphatase representing the second dephosphorylative step of H-K-ATPase by use of the one-step lead method. Throughout the stages of amelogenesis, the enzymatic activity was found in the plasma membranes, mitochondrial membranes, and lysosomal structures of the cells of stratum intermedium, papillary layer, and ameloblast layer. Gap junctions and desmosomes between these cells were, however, free of reaction product or showed slight precipitates of reaction. The stellate reticulum and the outer enamel epithelium at the stage of enamel secretion were usually negative for reaction. Although secretory, transition, and ruffle-ended maturation ameloblasts showed enzymatic activity at their basolateral cell surfaces, their distal cell surfaces facing the enamel were always free of reaction product. On the other hand, the smooth-ended maturation ameloblasts seldom showed a positive reaction, except in lysosomes and along their basal cell surfaces. An energy-dispersive X-ray microanalysis of reaction products of H-K-ATPase in unosmicated tissue sections demonstrated that they were composed of lead and phosphorus, which had been released during the dephosphorylation of substrate. In cytochemical controls, the enzymatic activity was completely dependent on substrate and potassium ion, resistant to ouabain and levamisole, and inhibited by nolinium bromide, a specific inhibitor of H-K-ATPase. In addition, inorganic trimetaphosphatase as enzymatic marker of lysosome was localized in dark and pale lysosomes, phagosomes, multivesicular bodies, and ferritin-containing vesicles of the ameloblasts and the cells of stratum intermedium and papillary layer. These membrane-bound structures were also positive for H-K-ATPase reaction. These results suggest that: 1) H-K-ATPase functions to maintain an acidic internal pH of lysosomes in the enamel organ cells; and 2) H-K-ATPase localization in the plasma membranes of enamel organ cells is concerned with efflux of protons derived from cytoplasmic water.

A histochemical study on lectin binding in the immature enamel and secretory ameloblasts of rat incisors.

Four lectin-fluorescent marker conjugates (Con A-F, MPA-F, PNA-F, WGA-R) were used for visualizing their binding sites on tissue sections by fluorescent microscopy. The immature enamel, Tomes' processes and distal cytoplasm of ameloblasts were stained with MPA-F and WGA-R on paraformaldehyde-fixed and paraffin-embedded tissue sections. The MPA- and WGA-binding glycoconjugates seemed to be main components of the organic enamel matrix because of the intense fluorescence from the tissue sections. The central portion of distal cytoplasm of ameloblasts was stained with PNA-F but the immature enamel and Tomes' processes were not. This suggests the PNA-binding glycoconjugates to be intermediates during the process of modifying their oligosaccharide chains or to be elements of Golgi area. Tomes' processes, the distal cytoplasm and periphery of nuclei of ameloblasts were stained with Con A-F. The Con A-binding glycoconjugates except those localizing on Tomes' processes may also be intermediates and/or intracellular elements like the PNA-binding glycoconjugates. Cells of stratum intermedium were stained with Con A-F, MPA-F and WGA-R. Cells of stellate reticulum and outer enamel epithelium were stained with Con A-F and WGA-R. It was indicated that enamel proteins, major organic components of immature enamel, are highly possible candidates for the MPA- and WGA-binding glycoconjugates.

Distribution of fluoride in the enamel of rat incisors examined by an abrasive microsampling technique

This study was undertaken to reveal detailed changes in fluoride distribution at different developmental stages of upper incisor enamel under various fluoride administration regimes. Four groups of Wistar rats received water containing 0, 25, 50 and 100 parts/10 6 fluoride respectively for 10 weeks. Five different enamel specimens were removed from the developing enamel, excluding the matrix-formation stage. Fluoride distribution in each specimen was analysed from the surface to the enamel-dentine junction using an abrasive microsampling technique. Fluoride concentration was invariably highest at the surface and decreased sharply towards the interior at every site in both control and experimental groups. The concentration throughout the tissue increased with fluoride intake at each stage of development. The fluoride-gradient curves were similar at each of the different sites of tooth development. However, the fluoride concentration of the enamel interior was significantly higher at early maturation than at the other four sites.

Enamel crystal shape: history of an idea.

The purpose of this paper is to review evidence which casts doubt on the interpretation universally applied to hexagonal images seen in sectioned enamel. The evidence is based on two possible models to explain the hexagonal profiles seen in mammalian enamel with transmission electron microscopy. The "hexagonal ribbon" model proposes that hexagonal profiles are true cross-sections of elongated hexagonal ribbons. The "rectangular ribbon" model proposes that hexagonal profiles are caused by three-dimensional segments that are parallelepipeds contained in the Epon section. Since shadow projections of such rectangular segments give angles that are inconsistent with the hexagonal unit cell, a model based on ribbons with rhomboidal cut ends and angles of 60 and 120° is proposed. The "rhomboidal ribbon" model projects shadows with angles that are predicted by the unit cell. It is suggested that segments of such crystallites in section project as opaque hexagons on the imaging plane in routine transmission electron microscopy. Morphological observations on crystallites in sections - together with predictions from the hexagonal, rectangular, and rhomboidal ribbon models - indicate that crystallites in rat incisor enamel are flat ribbons with rhomboidal cross-sectional shape. Hexagonal images in electron micrographs of thin-sectioned enamel can result from rhomboidal-ended, parallelepiped-shaped segments of these crystallites projected and viewed as two-dimensional shadows.

Na-K-ATPase in the enamel organ: localization and possible roles in enamel formation.

Ouabain-sensitive, K-dependent p-nitrophenyl phosphatase (p-NPPase) activity was localized ultra-Ocytochemically in the lateral plasma membranes of secretory ameloblasts and the stratum intermedium and principally in the papillary layer cells of aldehyde-fixed rat incisor enamel organs by the one-step lead method. Daily intraperitoneal injection of ouabain (250 μg, 500 μg, and 1 mg/100 g body weight) for two weeks reduced p-NPPase activity in the enamel organ cells. However, the degree to which this activity was reduced appeared to vary among the experimental animals. Addition of ouabain to the cytochemical incubation medium completely inhibited p-NPPase activity in the tissues. Although long-term ouabain injection did not result in any morphological alterations of the enamel organ cells, it caused, in part, an appearance of electron-dense, homogeneous matrix-like substances (MS) in the extracellular spaces of the ameloblast layers at both the secretion and maturation stages. In addition, long-term ouabain injection appeared to have resulted in delayed maturation of enamel as measured by energy-dispersive x-ray analysis of Ca and P in surface enamel. These results suggest that Na-K-ATPase of enamel organ cells may participate in the net flow (removal) of organic matrix components and water from the enamel during the maturation stage of enamel formation. It is suggested that this flow is maintained by local osmotic gradients generated by Na-K-ATPase within the papillary layer.