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Notes: Cytological examination of the rat incisor enamel organ with the light and electron microscope revealed a small number of ameloblasts which contained two and sometimes three or more nuclei per cell. A multinucleate ameloblast usually contained two vertically apposed nuclei situated near the base of the cell. A narrow cytoplasmic band was interposed between adjacent nuclear envelopes. The apical nucleus was often the more elongated of the two nuclei and it fitted a convexity or a concavity within the more basally positioned nucleus. In serial sections examined with the electron microscope no connections were observed between the nuclei. In animals injected with 3H-thymidine instances of multinucleate ameloblasts were found within the advancing front of labeling where only one of the nuclei contained label. Finally, quantitative analysis by nuclear counting established that multinucleate ameloblasts were 60 times more frequent within the maturation zone as in the secretory zone of amelogenesis. As well, the numbers of multinucleate ameloblasts increased progressively in the course of the maturation stage. It was concluded that multinucleate ameloblasts increase with cell age and likely arise by the process of cell fusion.

Notes: 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.

Notes: The movement of proteins into and out of enamel was followed over time using a highly sensitive microprecipitation technique to quantify the amount of TCA-insoluble radioactivity present within small pieces of freeze-dried enamel and cells (enamel organ) dissected from the mandibular incisors of rats injected with L-[35S]-methionine. Conventional image processing techniques were also used to estimate the number of silver grains over enamel and cells in radioautographs of mandibular incisors from rats similarly injected with L-[methyl3H]-methionine. Data from both techniques indicated that the average half-life for labeled proteins secreted into enamel was about 8.9 days. Typically, radioactive proteins accumulated in increasing amounts for 8 hours after which they were lost slowly up to 4 days and more rapidly thereafter when enamel formed during the secretory stage underwent maturation. The half-life for radioactive proteins in cells was only about 20.7 hours. No significant accumulation of radioactivity could be detected in the TCA-soluble or TCA-insoluble fractions of cells as enamel development proceeded. Results from this study suggest that radioautographs provide an accurate estimate of changes occurring to proteins in enamel and cells except at early time intervals (less than 1 hour) when a high percentage of total radioactivity is present within the TCA-soluble fraction of cells.

Notes: Renewal of the cell populations of the incisor was studied in 100 gm male rats injected with a single dose of 3H-thymidine and sacrificed at various times from one hour to 32 days after injection. Radioautographs showed that a cohort of labeled cells within the enamel organ, odontoblast layer, and pulp was carried passively with the erupting incisor from the apical end toward the gingival margin where the life cycle of these cells was terminated. Labeled cells in the upper and lower incisor, although traversing different absolute lengths, were found in approximately the same functional stage of their life cycle at similar times after the injection. Thus, by one and one-half days labeled ameloblasts began inner enamel secretion. By 32 days labeled ameloblasts had traversed the entire maturation zone and were located at the gingival margin. Labeled odontoblasts followed closely the movement of labeled ameloblast. The mean rate of ameloblast migration was 567 μm/day on the upper incisor and 651 μm/day on the lower. For the odontoblasts this rate was 500 μm/day (upper) and 631 μm/day (lower). Finally, it was found that as the rat aged, the duration of the life cycle for epithelial and pulp cell populations of the incisor increased because of growth within the longitudinal axis of the tooth. It was concluded that the apical end of the incisor literally “grows backward” in the bony socket, and hence, the duration of the life cycle becomes greater simply because it takes cells longer to physically reach the gingival margin.

Notes: During renewal of the enamel organ in the rat incisor cohorts of epithelial cells are transported sequentially through presecretory, secretory and maturation zones to the gingival margin where the life cycles of these cells terminate. This process was examined kinetically by determining the absolute flux of cells within each of these zones of amelogenesis. It was found that the efflux of ameloblasts, stratum intermedium and papillary layer cells from the presecretory zone was about equal to the efflux plus expected growth within the secretory zone. However, between the secretory and maturation zones about 50% more ameloblasts entered the maturation zone than were required to account for the egress at the gingival margin and the expected growth. Since there was no similar imbalance between these zones for papillary layer cells, it was concluded that this discrepancy must represent a 50% reduction in the size of the ameloblast population during the maturation stage of amelogenesis. It was calculated that a little over 25% of the loss occurred immediately at the start of maturation within the region of postsecretory transition and the remaining 25% of the loss occurred throughout the subsequent regions of the maturation zone. In addition to the kinetic analysis graphic reconstructions, or surface maps, of ameloblast nuclei were prepared. These maps illustrated the characteristics of ameloblast nuclear packing within the three zones of amelogenesis and they provided quantitative confirmation that as ameloblasts progress through the maturation zone, there is a loss of cells in an amount predicted by the kinetic analysis.

Notes: 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. Degradation of maturation stage enamel proteins could be effectively inhibited with aprotinin. These results support the concept that amelogenins are degraded into small polypeptides through the action of extracellular proteinases, at least one of which appears to be a trypsinlike serine proteinase. These results further suggest that amelogenins begin to break down within hours of their secretion. There also appears to be an increase in the activity and/or amount or type(s) of proteinases involved in the degradation of amelogenins between the secretory and maturation stages of amelogenesis.

Notes: Amelogenesis in the cat has been suggested to closely resemble enamel formation in human teeth. In order to further characterize the sequence of events leading to enamel formation in the cat, the expression and distribution of enamel proteins throughout amelogenisis were examined by postembedding immunocytochemistry using an antibody to mouse amelogenins and the high resolution protein A-gold technique. Enamel proteins were first immunodetected in ameloblasts and in the extracellular matrix during the presecretory stage. Secretory stage ameloblasts showed the most intense cellular reactivity. In these cells, protein synthetic organelles, secretory granules, and large lysosome-like structures were all intensely labeled. Extracellulary, numerous gold particles were observed over enamel and over patches of material found at the baso-lateral surfaces of these ameloblasts. During the early maturation stage, the protein synthetic organelles and secretory granules of ameloblasts still showed some immunoreactivity, although the most conspicuous labeling at this later stage was found over enamel and over material present amoung the extensive apical membrane infoldings of ruffle-ended ameloblasts. Qualitative analysis of lysosome-like elements in ameloblasts suggested that their frequency and immunoreactivity in the maturation stage were relatively lower than in the secretory stage, where some groups of cells often showed numerous large labeled structures. The enamel matrix was intensely labeled at all stages; however, cervical-occlusal and surface-depth gradients were readily apparent by conventional staining and by quantitative analysis of immunolabeling in the late secretory and early maturation stages. These data suggest that the cellular and extracellular distribution of enamel proteins in the cat is generally similar to that reported in other species, although some particularities were observed, perhaps reflecting variation in the timing of developmental parameters. © 1992 Wiley-Liss, Inc.

Notes: A three-dimensional reconstruction of the rod profiles seen in inner and outer rat incisor enamel was made from serial 1 μ cross sections of a decalcified upper incisor. The enamel rod was found to be an elongated structure which travelled incisally relative to its origin and ran continuously from the dentino-enamel junction to the enamel surface. The rod was divisible into an inner and outer enamel portion. The inner enamel portion began at the dentinoenamel junction and travelled incisally for about 20 μ with either a mesial or lateral tilt towards the outer enamel. The outer enamel portion of the rod was straight and ran incisally for about 60 μ as it gradually approached the enamel surface.Inner enamel portions of rods were arranged in rows parallel to the cross sectional plane of the incisor. All the rods in each row were tilted either mesially or laterally such that individual rods of adjacent rows crossed each other at 90°. Outer enamel portions of rods were not arranged in rows but all passed incisally parallel to one another.

Notes: Longitudinal sections through the incisors of the rat show a continuous layer of ameloblasts on the labial surface of the tooth. This layer contains the entire sequence of developmental stages in enamel production. Using 1 μm Epon sections from the upper and lower incisors of 100 gm male rats, the ameloblast layer was divided into three main zones which were themselves subdivided into regions: (1) Presecretory zone which includes (a) region of ameloblasts facing pulp, itself comprising a posterior portion (upper 172 ± 35 μm; lower 187 ± 37 μm) and an anterior portion (upper 458 ± 28 μm; lower 503 ± 36 μm); (b) region of ameloblasts facing dentin (upper 1210 ± 81 μm; lower 1381 ± 90 μm). (2) Secretory zone, (a) region of inner enamel secretion (upper 2573 ± 141 μm; lower 4274 ± 160 μm); (b) region of outer enamel secretion (upper 1211 ± 60 μm; lower 868 ± 72 μm). (3) Maturation zone (upper 7335 μm; lower 10615 μm), (a) region of postsecretory transition; (b) region of maturation proper, consisting of portions of ameloblasts with striated border and portions of ameloblasts with unmodified apices; (c) region of pigmentation; (d) region of reduced ameloblasts.These regions are readily identified using clear cut morphological criteria. Length measurements made on a group of 40 rats established the reproducibility of this classification. Therefore, this classification will be used as a basis for future studies of cell population kinetics.