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Rabbit polyclonal antibodies against bovine amelogenins and enamelins which did not show any cross-reaction were raised, and ultrathin sections of rat incisors were examined by the protein A-gold and ABC methods. The immunoreactivity of amelogenins was found in dense granules in the intercellular spaces between preameloblasts, and later over the fine- and coarse-textured material. The immunoreactivity was present over the cell organelles associated with the secretory pathway, as well as pale and dark lysosomes of the presecretory and secretory ameloblasts. Here the enamel was immunolabeled in the intercrystal spaces. The immunoreactivity in multivesicular bodies was stronger in preameloblasts than in secretory ameloblasts. In the region of second ruffle-ended ameloblasts at the maturation stage, the immunolabeling was intense in the ruffled-border, but in the rough endoplasmic reticulum and Golgi apparatus, the immunolabeling was much weaker than at the secretory stage. The immunolabeling for enamelins showed essentially the same intracellular topographical pattern as that for amelogenins by the secretory stage, but was weaker. The immunoreactivity was found mainly attached to the enamel crystals. Double immunostaining of amelogenins and enamelins revealed that both immunoreactivities were present over the same cell organelles associated with secretion and lysosomal systems. It is suggested that the presecretory and secretory ameloblasts are actively involved in the secretion, degradation and resorption of enamel proteins, and that multivesicular bodies and lysosomes in the cells take part in these processes. Ameloblasts are considered to be related to the synthesis of enamelins.

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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.

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Scanning electron microscopy was used to distinguish the topographical characteristics of two maturation ameloblast types in freeze-dried blocks of enamel organ tissue. This distinction was based primarily upon the configuration of the distal ends of the ameloblasts and the presence or absence of wide intercellular spaces. Energy dispersive x-ray spectrometry was applied to compare calcium levels in various regions of tissue identified as constituting either ruffle-ended or smooth ended ameloblasts. Greater levels of calcium were found in the distal ends of the ruffle-ended cells than in their proximal ends. In addition, greater calcium levels were found in the distal ends of the ruffle-ended cells than the distal ends of the smooth-ended cells. The higher calcium levels in ruffle-ended cells correlates with the view that these cells are actively involved in control of movement of calcium to the enamel front.

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We investigated the ultrastructural distribution of calcium in several kinds of hard tissue forming cells (secretory and maturation ameloblasts, odontoblasts osteoblasts, chondrocytes, and osteodentine forming cells) of mammals, amphibians, and fish by use of the potassium pyroantimonate technique. The calcium distribution pattern is compared among these cells, and its biological significance is discussed. Except for mammalian odontoblasts, all types of the hard tissue forming cells exhibited fundamentally the same distribution pattern of calcium; the antimonate reaction product was mainly localized on the inner face of the plasmalemma and inside mitochondria. On the other hand, in mammalian odontoblasts, the reaction product was found within secretory granules and in the intercellular spaces. Thus, the calcium distribution pattern in odontoblasts of lower vertebrates differed from that of mammalian odontoblasts and was similar to that of the osteoblasts or chondrocytes of the vertebrates examined. The differences in calcium distribution pattern among these hard tissue forming cells were not related to their origin, ectodermal or mesodermal (ectomesenchymal). We suggest on the basis of previous studies cited in this paper and of the present data that they are closely associated with the phylogeny and physiological system of Ca-ATPase.

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We investigated the ultrastructural distribution and histochemical properties of sulfated glycoconjugates, which could be preserved by glutaraldehyde fixation, in secretory ameloblasts and developing enamel matrix, by use of the high iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining and enzymatic digestion methods. Large type HID-TCH-SP stain deposits, approximately 10 nm in diameter, were detected on the interdigitating cell membrane of Tomes' process, inside some secretory granules, on the lateral cell membrane of stratum intermedium, in the basement membranes associated with outer enamel epithelium and endothelial cells of capillary, within the so-called hole region, and in the enamel matrix near future enamel-cement junction. A few large type stain deposits were, however, randomly distributed over the whole layer of enamel matrix. Small type stain deposits smaller than 5 nm in diameter were localized within some secretory granules and Golgi vesicles of ameloblasts and on the surface layer of developing enamel matrix. While the large type HID-TCH-SP stain deposits associated with the basement membranes and on the lateral cell membrane of stratum intermedium were susceptible to heparitinase, the others resisted enzymatic digestion not only by heparitinase but also by testicular hyaluronidase and chondroitinase ABC, indicating that they represent sulfated glycoconjugates other than heparan sulfate, chondroitin sulfate A, dermatan sulfate, or chondroitin sulfate C. On the other hand, HID-TCH-SP stain deposits within the secretory granules of odontoblasts and in the predentine matrix were susceptible to testicular hyaluronidase. Thus, it was confirmed that the composition of sulfated glycoconjugates secreted into the developing enamel matrix differs essentially from that of sulfated glycoconjugates associated with dentinogenesis.

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Thin sections of aldehyde-fixed, undecalcified, embedded rat incisor enamel organ were incubated with sheep antiserum to bovine testes calmodulin to reveal the sites of antigen-antibody reaction at the ultrastructural level in secretory ameloblasts using the protein A-gold immunocytochemical technique. Specific immunolabelling was localized intensely on free polyribosomes and those attached to rough-surfaced endoplasmic reticulum but only rarely observed in the cisternal space. The nuclei, mitochondria, cytosol, and plasma membranes were also immunoreactive. The Golgi membranes and related vesicles, secretion granules, and lysosomes were unlabelled. The proximal and distal cell web junctional complex systems were not immunoreactive. These findings suggest that calmodulin location reflects its synthetic site and multifunctional roles in the immunolabelled cytoplasmic components of secretory ameloblasts.

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The amelogenins are a complex mixture of hydrophobic proteins that are the major organic component of developing enamel. To study the molecular mechanisms underlying the heterogeneity of the amelogenins we isolated cDNA clones encoding these proteins. The clones were definitively identified by hybrid-selected translation experiments and by comparison of the DNA sequence with the protein-derived amino acid sequence. Southern hybridization of bovine genomic DNA indicated that amelogenin is a single copy gene. However, Northern hybridization experiments distinctly showed two major species of mRNA, each of which were sufficiently large enough to encode the highest known molecular weight species of amelogenin proteins. Furthermore, immunoprecipitation of hybrid-selected translation products using isolated amelogenin cDNA showed multiple, translated protein products. These data are supportive of a differential mRNA processing mechanism involved in generating a heterogeneous family of amelogenin matrix proteins from a single gene.

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Freeze-dried rat incisors were examined by high-resolution scanning electron microscopy (SEM) combined with energy-dispersive x-ray microanalysis (EDX) for determination of the correlation between the morphology of the enamel organ and the concentrations in the adjacent developing enamel matrix of calcium (Ca), phosphorus (P), and sulfur (S), as well as the Ca/P ratio. In SEM examination of the freeze-dried enamel organ, it was possible to identify the stages of enamel secretion, transition, and maturation, and furthermore to identify ruffle-ended and smooth-ended maturation ameloblasts. EDX analysis of the outer layer of forming and maturing enamel was carried out from the apical to the incisal end at interval points of approximately 50 micron. Ca and P concentrations increased gradually and continuously from the secretion zone to the end of the maturation zone, but never showed a steep rise in any of the zones examined. Maturing enamel overlaid by either ruffle-ended or smooth-ended maturation ameloblasts showed similar Ca and P concentrations. Throughout the outer enamel layer, the Ca/P molar ratio was fairly constant. Sulfur concentration began to decrease in the zone of enamel secretion, and was no longer detected in the middle of the maturation zone.

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Actin has been localized in Tomes' processes of the ameloblasts by the immunoperoxidase method using antiserum against purified rat actin. The zone of initial mineralization in dentin also gave a positive reaction. The possibility exists that actin may have a role in local accumulation of calcium during mineralization of various calcified tissues.

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A combined HCl-collagenase digestion technique and scanning electron microscopy were used to isolate the enamel organ and to confirm the presence of maturation ameloblasts of both ruffle-ended (RA) and smooth-ended (SA) types on maturing enamel in kitten permanent tooth germs. EDTA perfusion of animals fixed with aldehyde produced two or three belt-like shallow grooves (from 30 to 100 micron wide) running horizontally through the maturing enamel surface, coinciding closely with the SA distribution pattern. In animals that had been perfusion-fixed with unbuffered osmium tetroxide containing 2.5% potassium pyroantimonate, SEM-EDX analysis detected K in a superficial enamel layer overlaid by the SA layer. Potassium concentration decreased gradually toward the deeper layers. Very little K penetrated the enamel under the RA layer. Energy-dispersive x-ray analysis of Ca and P concentrations in the enamel revealed an even distribution of these elements throughout the superficial layer of maturing enamel. These results suggest that the SA layer forms an access route for K and EDTA and that, in spite of the obvious morphological and functional differences between RA and SA, the maturing enamel surfaces overlaid by these two cell types show similar degrees of mineralization.

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Potassium pyroantimonate-osmium tetroxide cytochemistry has been used to study the distribution of ionic calcium in hamster tooth germs during cell differentiation and during early dentinogenesis and amelogenesis. Before the onset of mineralization, pyroantimonate (PA) reaction product was found in the nucleus of differentiating preameloblasts and preodontoblasts. In the predentin, it was preferentially located along striated collagen fibrils, lying perpendicular to the basal lamina. At the onset of mineralization, a pronounced increase of PA reaction product was evident in the predentin and on the plasma membrane and in mitochondria of both preodontoblasts and preameloblasts opposite the mineralizing mantle dentin. During early enamel mineralization, PA reaction product was present in the "growing" crystal ends, while in the secretory ameloblasts, most of the PA reaction product was localized on the cytoplasmic side of the apical plasma membranes and in mitochondria. When Tomes' processes developed, PA reaction product, both cytoplasmic and membrane bound, was low or absent deep in the processes, but gradually increased toward the apical terminal web. A corresponding gradient of PA reaction product was observed on the opposing enamel crystallites. From this study we conclude that both preodontoblasts and preameloblasts seem to be involved in calcium acquisition necessary for the early stages of mantle dentin mineralization. Tomes' processes seem to regulate the entry of calcium into the enamel mineralization front.

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Actin filaments decorated with heavy meromyosin in enamel-secreting ameloblasts of rat incisors were examined. Proximal and distal terminal webs contained actin filaments; some were arrayed in diverse directions to each other. Some were associated with coated and uncoated vesicles. These actin filaments may relate to the sideways movement of secreting ameloblasts, suggesting that the distal terminal web is an apparatus generating a force by which ameloblasts can slide over each other.

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The lectin-binding patterns of the cells involved in amelogenesis and dentinogenesis in developing teeth of rats were studied. Undifferentiated odontogenic epithelia exhibited very slight staining with almost all of the lectins examined. The lectin-staining affinities of secretory ameloblasts could be divided into two categories: Concanavalin-A (Con-A), Wheat germ agglutinin (WGA) and Soybean agglutinin (SBA) binding occurred from the middle to apical cytoplasm, whereas Ricinus communis agglutinin-I (RCA-I) and Ulex europeus I (UEA-I) binding predominated in the basal regions. The cells of the stratum intermedium exhibited relatively strange lectin staining, which appeared to be dependent on ameloblastic maturation. The basement membranes in undifferentiated epithelia were markedly positive for lectin binding. Odontoblasts showed moderate Con-A staining on the apical side of the cells, as well as slight-to-moderate reactions with WGA and SBA. Pulp cells and dental papillae showed slight-to-moderate lectin staining, and predentin and dentin were also moderately positive for Con-A and RCA-I binding and slightly so for WGA and SBA. The lectin-binding affinities were enhanced during the formation of enamel and dentin, and appeared to be dependent on the degree of cellular differentiation in ameloblasts and odontoblasts.

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Antiserum to the 28-kilodalton vitamin D-dependent calcium-binding protein (CaBP) was used to localize CaBP in histologic sections of the continuously erupting incisor in mandibles obtained from normal rats. With the peroxidase--anti-peroxidase technique, no CaBP was detected in undifferentiated ameloblasts or in those which had become columnar and were facing pulp. Calcium-binding protein was first noted in the cytoplasm of random ameloblasts facing dentin in the presecretion zone. As the ameloblasts became more mature in the zone of enamel secretion, CaBP was uniformly present in their cytoplasm. Ameloblasts with Tome's processes clearly contained CaBP in these processes as well as in the cell-body cytoplasm. Near the later developmental stages of the zone of enamel secretion, some of the adjacent underlying cells of the stratum intermedium also contained CaBP in their cytoplasm. In some stratum intermedium cells and papillary cells, CaBP extended into the zone of enamel maturation, but not to the end of that zone. Cytoplasmic CaBP continued to be present in ameloblasts as they progressed through the zone of enamel maturation to the final, shortened cells at the gingival margin of the erupting incisor. No CaBP was detected in odontoblasts, pulpal cells, the stellate reticulum, or the outer dental epithelium.

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X-ray micro-analysis and electron energy loss analysis were used to confirm cytochemical localization by potassium pyro-antimonate of calcium in secretory ameloblasts. Neither intercellular calcium concentration between ameloblasts nor over-all calcium levels of the enamel organ were lowered during a period of inhibition of enamel mineralization by injected fluoride. Calcium concentrations in mitochondria and secretory granules were reduced.

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It may be inferred from this observation, that all of the phosphorus necessary to the formation of the enamel, reaches it, during its formation process, by way of compounds, formed in the ameloblastic layer, and containing only part of the calcium necessary to the crystallisation of the hydroxyapatite. The remainder of the calcium may attain, by another route, the enamel under formation where the hydroxyapatite crystallises.

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The distribution of Calcium and Phosphorus and of Na, K, S and Cl was studied in the mineralizing matrices and strata of ameloblasts and odontoblasts in developing mouse molars (5-14 days). Sections cut in a cryostat were prepared by freeze-drying and examined in an SEM by the method of energy dispersive x-ray analysis. In enamel a gradient of mineralization was observed with respect to age and topography. Progesssive loss of sulfur was also demonstrated. Less striking mineralization gradients were found in dentin. Predentin accumulated Ca at a concentration about 2% that of dentin and the Ca/P ratio was lower than that for apatite. Significant concentrations of calcium were localized in ameloblast and odontoblast strata. The concentration increased five-fold in ameloblasts as the cells matured and enamel mineralization entered the final phases, levels in odontoblasts remained stable. With age in both cellular strata, potassium counts decreased. In maturing ameloblasts the concentrations of sodium and chloride rose.

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In order to elucidate the cytochemical properties of the membranous structure between enamel and ameloblasts of the rat incisor at the maturation stage, chromic phosphotungstic acid (Cr-PTA) and periodic acid-silver methenamine (PA-silver) techniques for electron microscopy were employed in combination with a digestion test with hyaluronidase, neuraminidase, collagenase or trypsin. Also, acid phosphatase activity of ameloblasts at the maturation stage was examined with a modified GOMORI's metal salt method. An intensely Cr-PTA reactive band approximately 0.1 micron thick appeared along the surface layer of enamel at the transitional stage, and at the very beginning of the maturation stage another intensely Cr-PTA reactive band which was seen by uran-lead stain to be a delicate electron-dense membranous structure appeared as well between enamel and ameloblasts. A lot of cytoplasmic small vesicles or tubular structures, both intensely reactive to Cr-PTA, were observed near the apical membranes of the overlying ameloblasts indicating that those organelles must have been responsible for the secretion of the latter band. Acid phosphatase activity was clearly demonstrated at Cr-PTA reactive large vesicles in the cytoplasm of those cells. The PA-silver staining technique manifested a band heavily deposited with silver grains along the surface layer of enamel, i.e., where the former band existed, but showed no particular reaction at the latter, the band-like layer between enamel and ameloblasts. Hyaluronidase or neuraminidase treatment remarkably decreased the Cr-PTA reaction of the latter band. Trypsin or collagenase treatment, on the other hand, not only eliminated the Cr-PTA reaction but digested the band itself. These results suggest that the membranous structure between enamel and ameloblasts of a rat incisor is not so-called enamel cuticle but a basal lamina produced by overlying ameloblasts and that the basal lamina contains collagenous components even though it lies on enamel.

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