Transmission electron microscope observations on enamel following silver methenamine staining.

Transmission electron microscope observations on porcine enamel and secretory ameloblasts showed that silver methenamine material was located inside secretory vesicles in secretory ameloblasts and along the enamel crystals inside immature enamel. It is concluded that silver methenamine is able to stain enamel proteins selectively inside these tissues.     

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.     

Autoradiographic study with [35S]-sodium sulphate of loss of sulphated glycosaminoglycans during amelogenesis in the guinea pig.

The fate of sulphated organic compounds, probably glycosaminoglycans (GAG), during amelogenesis was studied by autoradiography in molar teeth of guinea pigs that had received a single dose of 2.5 μCi/g of body weight of [³⁵S]-sodium sulphate and killed from 10 min to 168 hr after injection. The sulphation site was the Golgi region of ameloblasts, from where the sulphated compounds migrated into the young enamel matrix along the entire zone lined by secretory ameloblasts. After forming an ill-defined band along the Tomes processes and the adjacent matrix, the ³⁵S diffused through the whole thickness of the matrix, reaching the dentine-enamel junction.By correlating the silver grain concentration over matrix with the growth of the tooth. it was shown that the radioactive reaction, after attaining a maximum of intensity at 24 hr. decreased at 48 hr when the matrix moved from one region to the next, which was still related to the secretory ameloblasts. After this sharp decrease, the concentration of silver grains tended to be relatively constant and the remaining labelled compound seemed to be stable.The results were interpreted as indicating that the removal of organic material in enamel maturation is related at least in part to the secretory ameloblasts.     

An autoradiographic study of calcium movement in the enamel organ of rat molar tooth germs

The distribution and movement of calcium through the enamel organ and into the forming enamel was studied in 6-day-old rats, intravenously injected with 45Ca. To prevent dislocation of radiocalcium in the specimens, the tooth germs were rapidly frozen/freeze-substituted and processed for 45Ca autoradiography under dry conditions. At 30 s after the 45Ca injection, there was a decrease in labelling intensity progressing from the overlying connective tissue to the enamel organ and, in the secretory ameloblasts, from the proximal to distal cytoplasm. The most intense labelling was in the enamel matrix, where it was restricted to the superficial layer extending approx. 15 microns below the surface. At later times the density of silver grains over the connective tissue decreased considerably, whereas secretory ameloblasts showed an increasing intensity in the distal portions. Enamel had the heaviest labelling: the width of the labelled enamel increased gradually to only 40 microns from the surface 60 min after the injection. The use of wet emulsion over similarly prepared sections caused a severe dislocation of radiocalcium in the specimens. These findings confirm the rapid penetration of systemically administered calcium to newly formed enamel, probably due to isotopic exchange. A relatively slow diffusion through the enamel organ and into the surface layer of enamel suggests that net transport of calcium through the enamel organ is transcellular.     

Cell proliferation and apoptosis in enamelin null mice

Enamelin is a secreted glycoprotein that is critical for dental enamel formation. Ameloblasts in enamelin (Enam) null mice develop atypical features that include the absence of a Tomes' process, expanded endoplasmic reticulum, apparent loss of polarity, and pooling of extracellular matrix in all directions, including between ameloblasts and the stratum intermedium. We hypothesized that ameloblast pathological changes may be associated with increased cell apoptosis. Our objective was to assess apoptotic activity in maxillary first molars of wild-type, Enam(+/-), and Enam(-/-) mice at postnatal days 5, 7, 9, 14, and 17. Mouse maxillae were characterized by light microscopy after terminal deoxynucleotidyl transferase (TdT)-mediated biotin-dUTP nick-end labelling (TUNEL) or 5-bromo-2'-deoxyuridine (BrdU) staining. Following the initial deposition of dentin matrix, ameloblasts became highly dysplastic and no enamel crystal ribbons were deposited. Ameloblast apoptosis was observed in the Enam null mice starting in the secretory stage and with no apparent alteration in cell proliferation. We conclude that in the absence of enamelin and subsequent shutdown of enamel formation, ameloblasts undergo pathological changes early in the secretory stage that are evident as radically altered cell morphology, detachment from the tooth surface, apoptosis, and formation of ectopic calcifications both outside and inside the dystrophic enamel organ.     

Microscopy of tetracycline-induced lesions in the rat incisor enamel organ

Adult rats received each a single dose of 12.5 mg tetracycline HCl per 100 g body weight i.p. They were killed in groups of four at 8 and 16 h, 1, 2, 3, 5, 7 and 10 days after injection. The enamel organs of the lower incisors were prepared for light and electron microscopy. Lesions of the enamel organ located in the secretion zone showed the features: distorted ameloblasts bulged below the normal profile of the enamel organ; some showed fine structural characteristics of normal secretory ameloblasts; the enamel layer over the lesion remained thin; nodules of mineralized material, some with the fine structure of enamel, were present between the cells bodies and bases of the ameloblasts; at the later intervals, exudate frequently separated the ameloblasts from the enamel surface. Lesions first appeared at 16 h in the secretion zone, then enlarged and moved distally along the tooth at a rate similar to the tooth eruption rate. At 10 days, lesions were entirely within the maturation zone, and the enamel exhibited a deep hypoplastic defect. The ameloblasts in the lesion showed, rudimentarily, the characteristics of maturation ameloblasts. The nodules of ectopic enamel appeared to be hypermineralized.

It is concluded that ameloblasts in late differentiation and early secretion stages are damaged by tetracycline so that their architecture and secretory mechanism and possibly maturative activities are interfered with, but they still respond to signals to change from secretory to maturation models at the appropriate time.     

Autoradiographic investigation of the effect of 1-hydroxyethylidene-1, 1-bisphosphonate on matrix protein synthesis and secretion by secretory ameloblasts in rat incisors

Seven daily subcutaneous injections of 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) can induce enamel hypoplasia. Several enamel-free zones were observed along the crown-analogue side of rat incisors during the secretory stage of amelogenesis. Ameloblasts related to the enamel-free zones lay directly on the abnormally non-mineralized mantle dentine, whereas the adjacent ameloblasts, which were forming the enamel matrix layer, were associated with the region where mineralization of dentine was proceeding. The further purpose of this study was to investigate the synthetic and secretory activity of these two groups of ameloblasts and to trace the fate of the radioactively labelled proteins. [(3)H]-proline was administered to Wistar rats 12 h after the last injection of HEBP. Light-microscopic autoradiography was performed. Quantitative analysis indicated that the ameloblasts of the enamel-forming zones in the drug-treated group showed a distribution pattern of silver grains similar to that of the controls. The ameloblasts of the enamel-free zones also demonstrated incorporation of [(3)H]-proline at the same level. There was some labelling over the non-mineralized mantle dentine, which was supposed to indicate the penetration of ameloblast products. From these results, it is concluded that HEBP does not affect the ameloblast activity in protein synthesis. The complete failure of enamel-layer formation in some specific regions is probably due to the failure in protein secretion and protein deposition. This study provides additional evidence that the mineralization of dentine is an essential factor in successful enamel matrix secretion and deposition.     

Short exposure to high levels of fluoride induces stage-dependent structural changes in ameloblasts and enamel mineralization

We tested the hypothesis that the sensitivity of forming dental enamel to fluoride (F^–) is ameloblast developmental stage-dependent and that enamel mineralization disturbances at the surface of fluorotic enamel are caused by damage to late-secretory- and transitional-stage ameloblasts. Four-day-old hamsters received a single intraperitoneal dose of 2.5–20 mg NaF/kg body weight and were examined, 24 h later, by histology and histochemistry. A single dose of ≥ 5 mg of NaF/kg induced the formation of a hyper- followed by a hypomineralized band in the secretory enamel, without changing the ameloblast structure. At 10 mg of NaF/kg, cystic lesions became apparent under isolated populations of distorted late-secretory- and transitional-stage ameloblasts. Staining with von Kossa stain showed that the enamel under these lesions was hypermineralized. At 20 mg of NaF/kg, cystic lesions containing necrotic cells were also found in the early stages of secretory amelogenesis and were also accompanied with hypermineralization of the enamel surface. We concluded that the sensitivity to F^– is ameloblast developmental stage-dependent. Groups of transitional ameloblasts are most sensitive, followed by those at early secretory stages. These data suggest that a F-induced increase in cell death in the transitional-stage ameloblasts accompanies the formation of cystic lesions, which may explain the formation of enamel pits seen clinically in erupted teeth.     

XBP1 may determine the size of the ameloblast endoplasmic reticulum

Ameloblasts progress through defined stages of development as enamel forms on teeth. Pre-secretory ameloblasts give rise to tall columnar secretory ameloblasts that direct the enamel to achieve its full thickness. During the maturation stage, the ameloblasts shorten and direct the enamel to achieve its final hardened form. Here we ask how the volume of selected ameloblast organelles changes (percent volume per ameloblast) as ameloblasts progress through six defined developmental stages. We demonstrate that mitochondria volume peaks during late maturation, indicating that maturation-stage ameloblasts maintain a high level of metabolic activity. Also, the endoplasmic reticulum (ER) volume changes significantly as a function of developmental stage. This prompted us to ask if X-box-binding protein-1 (XBP1) plays a role in regulating ameloblast ER volume, as has been previously demonstrated for secretory acinar cells and for plasma cell differentiation. We demonstrate that Xbp1 expression correlates positively with percent volume of ameloblast ER.     

Ultrastructure of lingual enamel epithelium in rabbit permanent incisors

The recent observation that the lingual surface of the permanent rabbit incisor, in contrast to the rodent incisor, is covered with enamel near the initially formed tip, initiated a histologic study of the enamel epithelium in this region. Tooth buds from fetal New Zealand white rabbits aged 27 and 30 days in utero were processed for transmission electron microscopy. Examination of longitudinal and cross-sections revealed a transition of secretory ameloblasts to postsecretory ameloblasts on the facial aspect of the enamel organ. Incisally, the facial ameloblast layer was continuous with postsecretory ameloblasts on the lingual aspect. More apically on the lingual surface, a gradual transition between postsecretory and reduced ameloblasts occurred. This study shows that by day 27 in utero, the ameloblasts on the lingual aspect of the tooth are in the postsecretory and degenerative stages and that, by day 30, the enamel epithelium lingually has been replaced by cementum on the prismatic enamel and by connective tissue.     

Calbindin-D28 kappa localization in rat molars during odontogenesis

Specific antiserum raised against Calbindin-D28 kappa, a vitamin D-dependent calcium-binding protein (CaBP) isolated from chick intestine, was used for localization of the protein in developing rat molars. Previously, CaBP had been localized in specific cells associated with the continuously erupting rat incisor: late pre-secretory ameloblasts, secretory and maturation zone ameloblasts, stratum intermedium cells adjacent to ameloblasts in the late zone of enamel secretion, and papillary cells underlying maturation zone ameloblasts. In this study, the peroxidase anti-peroxidase technique was used for localization of the peroxidase anti-peroxidase technique was used for localization of CaBP in histological sections of rat mandibles from 18-day-old rat embryos through 20-day-old neonates. CaBP was not detected in any cells of the enamel organ, dental papilla, or dental sac during early odontogenesis from the dental lamina stage through the advanced bell stage. The protein first appeared in secretory ameloblasts which were situated opposite odontoblasts with newly secreted dentin. CaBP was present in the cytoplasm of more mature ameloblasts, but not in less mature ameloblasts. Some stratum intermedium cells subjacent to well-developed secretory and maturation zone ameloblasts also contained CaBP. The protein was not detected in odontoblasts, pulp cells, or other cells associated with the developing molars. It was also absent from the demineralized enamel and dentin matrix. In developing rat molars, the time-course of appearance of CaBP, a protein dependent for its synthesis on the vitamin D endocrine system in other organ systems, suggests a potential direct role of this hormonal system in enamel mineralization.     

Cytoskeleton,intercellular junctions,planar cell polarity,and cell movement in amelogenesis

Background

Tooth enamel is composed of highly mineralized rods surrounded by interrod crystals that are formed by ameloblasts derived from dental epithelium. Secretory ameloblasts migrate during hard tissue formation, both away from the dentin and in groups that slide past each other, resulting in rod decussation. Enamel rod decussation is commonly observed in many animal teeth including humans.

Immunocytochemical localization of enamelin proteins in developing bovine teeth

Enamelins were localized at both the light and electron microscopic level using an antienamelin monoclonal antibody and indirect immunogold methods. Bovine fetal incisors (crown-rump length 17-30 cm) were preserved in Karnovsky's fixative and embedded in Epon. For light microscopy, 2 microM thick sections were immunostained by the indirect method using the monoclonal antibody and goat anti-mouse IgG linked to 5 nM gold particles, followed by silver enhancement to increase the sensitivity of the method. For electron microscopy, thin sections were immunostained (indirect) with the antienamelin monoclonal antibody and goat anti-mouse IgG linked to 5 or 15 nM gold. Control samples were treated with an unrelated monoclonal antibody. The localization of enamelins was confined in the light microscopic sections to the extracellular enamel matrix. No gold staining was observed in the ameloblasts or other enamel organ cells even though the gold-silver technique is extremely sensitive. Ultrastructurally, enamelin was localized in the enamel extracellular matrix and associated ameloblasts. Both the crystal-containing and granular matrix were positively stained, with most gold particles being closely associated with the crystals. Counting of gold particles indicated more than 4 times as many amelogeninas enamelin-reactive antigenic sites in similar regions. Decalcification did not increase immunostaining with the anti-enamelin antibody in the extracellular matrix. Within ameloblasts, the gold particles were associated with secretory granules and Golgi complexes. Thus it appears that enamelins are synthesized in ameloblasts and secreted into the extracellular matrix in a similar manner to amelogenins and are preferentially associated with matrix hydroxyapatite crystals. Transient levels of enamelins within the ameloblasts are apparently too low to be detected by light microscopy.     

Ultrastructure of lingual enamel epithelium in rabbit permanent incisors

The recent observation thai the lingual surface of the permanent rabbit incisor, in contrast to the rodent incisor, is covered with enamel near the initially formed tip, initiated a histologic study of the enamel epithelium in this region. Tooth buds from fetal New Zealand white rabbits aged 27 and 30 days in utero were processed for transmission electron microscopy. Examination of longitudinal and cross-sections revealed a transition of secretory ameloblasts to postsecretory amelobasts on the facial aspect of the enamel organ. Incisally. the facial ameloblast layer was continuous with postsecretory ameloblasts on the lingual aspect. More apically on the lingual surface, a gradual transition between postsecretory and reduced ameloblasts occurred. This study shows that by day 27 in utero., the ameloblasts on the lingua] aspect of the tooth are in the postsecretory and degenerative stages and that, by day 30, the enamel epithelium lingually has been replaced by cementum on the prismatic enamel and by connective tissue.     

Autoradiographic evidence of cyclical entry of calcium into maturing enamel of the rat incisor tooth

Adult rats were injected intraperitoneally with radioactive calcium and allowed to survive for periods up to 60 min. Animals were then killed and the upper incisors were removed. Soft tissue was removed from the teeth by dissection and with Clorox. Teeth were then coated with photographic emulsion and, after 17 days, the emulsion on the specimens was developed. Black bands of reduced silver indicated areas of radioactivity where calcium had entered the enamel. These bands were separated by narrower bands of non-radioactive enamel. It was judged that calcium did not gain entry into the enamel in these areas of enamel at this particular time. The patterns made by the bands of calcium entry and restriction were similar to patterns which show where striated border and non-striated border maturation ameloblasts cover the maturing enamel. Maturation ameloblasts with a striated border cover a larger area of the maturing enamel than do maturation ameloblasts without a striated border. It is considered that the maturation ameloblasts with a striated border are engaged in calcium entry into the maturing enamel.     

Quantitative image analysis of hyaluronan expression in human tooth germs

The expression of hyaluronan in human tooth germs was studied by using a biotinylated hyaluronan-binding complex and quantitative digital image analysis. At the cap stage, dental papilla exhibited a moderate staining, while intense reaction was observed in the apical portion of presecretory ameloblasts, stellate reticulum, and in dental basement membrane. When the enamel and dentine matrices started to develop, a strong hyaluronan reaction was evident in the young enamel and the apical portion of secretory ameloblasts. No hyaluronan could be detected in the secretory ameloblasts and enamel matrix of the early (9-wk-old) post-natal stage. It is concluded that hyaluronan may play a transitory role in the early phase of the development of the enamel matrix organization. A very weak signal was observed in the wall of dentin tubules, whereas the rest of the dentine matrix was not stained. The odontoblasts and the pulp were also moderately stained, and these reactions gradually decreased with age, suggesting that hyaluronan may also contribute to the development of dentine matrix and pulp.     

Organic anion transport during rat enamel formation

ObjectiveThe C-terminal end of nascent amelogenin is dissociated immediately after secretion and rapidly re-absorbed by ameloblasts, presumably by endocytosis. The purpose of this study was to test whether organic anion transporters (OATs) are also involved in the re-absorption process of enamel matrix proteins via non-endocytotic pathways.Materials and methodsLocalization of OAT1, OAT2, and OAT3 in rat tooth germs was examined by immunohistochemistry using specific antibodies. Actual translocation of organic anions through the ameloblast layer was further tested by systemic tracer experiments in rats in which Lucifer Yellow (LY), a fluorescent organic anion, was used as a tracer.ResultsIn rat tooth germs, OAT2 was associated exclusively with the distal cell membranes of secretory ameloblasts where Tomes' processes were developed and disappeared when matrix formation was terminated. On the other hand, OAT1 was absent in secretory ameloblasts and was colocalized with the ruffled border of ruffle-ended ameloblasts in the maturation stage. OAT3 was undetectable in ameloblasts and located instead only in the stratum intermedium cells. Systemic administration of LY resulted in intense labeling of immature enamel and also a transient labeling of the cytosol of secretory ameloblasts immunopositive for OAT2. In the maturation stage, cytosolic labeling of LY was negligible in all cells of the enamel organs, including ameloblasts.ConclusionsThese data suggest the existence of OATs in rat tooth germs and their possible involvement in matrix re-absorption at least in the secretory stage of amelogenesis.     

Activity of γ-glutamyl transferase in developing teeth of the rat

γ-Glutarnyl transferase activity was studied with an enzyme histochemical technique in rat molar teeth at different stages of development, The odontoblasts showed an increasing activity during formation of the crown. The secretory ameloblasts did not show any activity, while activity was intense in the postsecretory ameloblasts. This activity was confined to the apical part of the cell. In the reduced ameloblasts at the enamel free cusp tips a similar activity was noted. Since γ-glutamyl transferase has been shown to be involved in endocytosis and transport of amino acids, the localization of the enzyme in developing teeth suggests an active uptake of amino acids from the enamel matrix by the ameloblasts during enamel maturation.     

Activity of gamma-glutamyl transferase in developing teeth of the rat

gamma-Glutamyl transferase activity was studied with an enzyme histochemical technique in rat molar teeth at different stages of development. The odontoblasts showed an increasing activity during formation of the crown. The secretory ameloblasts did not show any activity, while activity was intense in the postsecretory ameloblasts. This activity was confined to the apical part of the cell. In the reduced ameloblasts at the enamel free cusp tips a similar activity was noted. Since gamma-glutamyl transferase has been shown to be involved in endocytosis and transport of amino acids, the localization of the enzyme in developing teeth suggests an active uptake of amino acids from the enamel matrix of the ameloblasts during enamel maturation.