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.     

A histological study of the short-term effects of fluoride on enamel and dentine formation in hamster tooth-germs in organ culture in vitro

First maxillary molars in early stages of secretory amelogenesis were exposed in vitro to fluoride (F-) concentrations ranging between 2.63 microM and 2.63 mM for up to 3 days. In contrast to the dentine papilla, which seemed unaffected by F- in concentrations up to 1.31 mM, the enamel organ was dose-dependently extremely sensitive for F-. Young ameloblasts which became secretory in vitro were in particular sensitive and did not secrete enamel adjacent to new, in vitro-formed dentine. Hypermineralization of the dentine at the enamel-dentine junction suggested that these ameloblasts still transport and deposit minerals. 52 microM was the lowest concentration of F- that inhibited deposition of enamel in the cervical-loop region. Ameloblasts, secretory at the time of exposure to F-, in concentrations of 52 microM up to 1.31 mM secreted an abnormal, amorphous, von-Kossa-negative enamel matrix. 1.31 mM of F- was the lowest concentration which induced the formation of a hypermineralized band in the pre-exposure enamel. 2.63 mM of F- was highly toxic for the enamel organ but had only moderate effects on the dentine papilla.     

In the absence of a basal lamina,ameloblasts absorb enamel in a serumless and chemically defined organ culture system

ObjectivesTooth organ development was examined in a serumless, chemically defined organ culture system to determine whether morphological and functional development was identical to that in in vivo and serum-supplemented organ cultures.MethodsMouse mandibular first molar tooth organs at 16 days of gestation were cultured for up to 28 days in a Tronwell culture system using a serum-supplemented or serumless, chemically defined medium. After culture, specimens were processed for assessing tooth development using ultrastructural, immunohistochemical, and mRNA expression analyses.ResultsIn serum-supplemented conditions, inner enamel epithelial cells differentiated into secretory-stage ameloblasts, which formed enamel and reached the maturation stage after 14 and 21 days of culture, respectively. Ameloblasts deposited a basal lamina on immature enamel. Conversely, in serumless conditions, ameloblasts formed enamel on mineralized dentin after 21 days. Moreover, maturation-stage ameloblasts did not form basal lamina and directly absorbed mineralized enamel after 28 days of culture. RT-PCR analysis indicated that tooth organs, cultured in serumless conditions for 28 days, had significantly reduced expression levels of ODAM, amelotin, and laminin-322.ConclusionsThese results indicate that several differences were detected compared to the development in serum-supplemented conditions, such as delayed enamel and dentin formation and the failure of maturation-stage ameloblasts to form basal laminae. Therefore, our results suggest that some factors might be required for the steady formation of mineralized dentin, enamel, and a basal lamina. Additionally, our results indicate that a basal lamina is necessary for enamel maturation.     

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.     

Intercellular junctions in the cells of the human enamel organ as revealed by freeze-fracture

Examined by thin sections and freeze-fracture replication techniques, secretory ameloblasts possessed two sets of the junctional complexes at both proximal and distal ends of the cell bodies, which consisted of tight junctions and occasional gap junctions and desmosomes. The proximal tight junction was fascia occludens, whereas the distal tight junction was zonula occludens. Between adjacent ameloblasts, mature gap junctions were frequent. The stratum-intermedium cells were connected to each other and to the stellate-reticulum cells and ameloblasts by well-developed desmosomes, gap junctions and fascia or macula-type tight junctions. Stellate-reticulum cells were inter-connected by many extensive cytoplasmic processes, in which well-developed desmosomes, small gap junctions and occasional macula-type tight junctions appeared. Thus fascia or macula-type tight junctions as well as many desmosomes seem to serve in mechanical, cell-to-cell adhesion during tooth formation. Frequent and large gap junctions between adjacent stratum-intermedium cells and between the stratum intermedium and the base of the ameloblast suggest that, in relation to enamel formation, these two cell layers form a functional unit.     

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.     

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.     

Buffering of protons released by mineral formation during amelogenesis in mice

Regulation of pH by ameloblasts during amelogenesis is critical for enamel mineralization. We examined the effects of reduced bicarbonate secretion and the presence or absence of amelogenins on ameloblast modulation and enamel mineralization. To that end, the composition of fluorotic and non‐fluorotic enamel of several different mouse mutants, including enamel of cystic fibrosis transmembrane conductance regulator‐deficient (Cftr null), anion exchanger‐2‐deficient (Ae2a,b null), and amelogenin‐deficient (Amelx null) mice, was determined by quantitative X‐ray microanalysis. Correlation analysis was carried out to compare the effects of changes in the levels of sulfated‐matrix (S) and chlorine (Cl; for bicarbonate secretion) on mineralization and modulation. The chloride (Cl^?) levels in forming enamel determined the ability of ameloblasts to modulate, remove matrix, and mineralize enamel. In general, the lower the Cl^? content, the stronger the negative effects. In Amelx‐null mice, modulation was essentially normal and the calcium content was reduced least. Retention of amelogenins in enamel of kallikrein‐4‐deficient (Klk4‐null) mice resulted in decreased mineralization and reduced the length of the first acid modulation band without changing the total length of all acidic bands. These data suggest that buffering by bicarbonates is critical for modulation, matrix removal and enamel mineralization. Amelogenins also act as a buffer but are not critical for modulation.     

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.     

Effects of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) on the formation of dental hard tissue of mouse molar tooth germs in organ culture system

In the developing tooth, 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) causes hypoplasia and hypomineralization of enamel and dentine. The present study was undertaken to clarify the effects of HEBP on the formation of dental tissues of tooth germs in an organ culture system. Mandibular first molars from 17.5-day-old mouse embryos were cultured with or without 250 microM HEBP in culture medium. Cultured tooth germs were analyzed by histological examination and by immunohistochemical localization using anti-amelogenin antibody. In cultured tooth germs treated with HEBP before the commencement of calcification in dentine, calcification of dentine matrix was inhibited completely and enamel formation was not observed. Ameloblasts were directly adjacent to dentine matrix. However, immunohistochemical data indicated that these ameloblasts secreted amelogenin. In the experiments of adding HEBP to cultured tooth germs on culture day 13, calcified dentine and enamel had formed before the administration of HEBP, but the dentine matrix newly formed after the administration of HEBP had not calcified. It was confirmed by immunohistochemical observations that enamel matrix-like material had penetrated into uncalcified dentine matrix and accumulated in dental papilla of tooth germs. However, no enamel matrix-like material was observed in calcified dentine and predentine underneath the calcified dentine by immunohistochemical staining. From these results, it might be concluded that ameloblasts secreted enamel matrix in the presence of HEBP and diffused through uncalcified dentine matrix into dental papilla. These findings suggests the calcification of dentine might be essential for the physical barrier to accumulate the enamel matrix and form a distinct layer of enamel as enamel.     

Composition of mineralizing incisor enamel in cystic fibrosis transmembrane conductance regulator‐deficient mice

Formation of crystals in the enamel space releases protons that need to be buffered to sustain mineral accretion. We hypothesized that apical cystic fibrosis transmembrane conductance regulator (CFTR) in maturation ameloblasts transduces chloride into forming enamel as a critical step to secrete bicarbonates. We tested this by determining the calcium, chloride, and fluoride levels in developing enamel of Cftr‐null mice by quantitative electron probe microanalysis. Maturation‐stage enamel from Cftr‐null mice contained less chloride and calcium than did wild‐type enamel, was more acidic when stained with pH dyes ex vivo, and formed no fluorescent modulation bands after in vivo injection of the mice with calcein. To acidify the enamel further we exposed Cftr‐null mice to fluoride in drinking water to stimulate proton release during formation of hypermineralized lines. In Cftr‐deficient mice, fluoride further lowered enamel calcium without further reducing chloride levels. The data support the view that apical CFTR in maturation ameloblasts tranduces chloride into developing enamel as part of the machinery to buffer protons released during mineral accretion.     

Fine structural localization of alkaline phosphatase in relation to enamel formation in the mouse molar

Enzyme activity was localized to the stratum intermedium throughout amelogenesis but occurred in the ameloblasts only with the onset of enamel maturation. Its localization was to the striated border of the maturative ameloblast. It is suggested that the appearance of enzyme activity within the ameloblasts at this stage of enamel formation is related to calcium transport.     

Fine structure of secretory ameloblasts in kitten tooth germs, with special regard to intercellular junctions as revealed by freeze-fracture

Using both thin sectioning and freeze-fracture replication, junctional complexes at both proximal and distal ends of the cells consisted of tight junctions in close association with gap junctions and desmosomes. The tight junctions generally consisted of smooth, continuous rows of particles on the P-face and corresponding patterns of shallow grooves on the E-face of cell membranes. Though sealing of paracellular spaces around the ameloblasts in the proximal junctional complex was incomplete, there was complete sealing around the ameloblasts and well-developed meshwork structures of tight junctions in distal junctional complexes. Discontinuous and free-ending strands of tight junctions were frequent in junctional complexes, suggesting that ameloblast distal junctional complexes serve, not only as a barrier to high molecular passive substances through the ameloblast layer, but also as a channel for ions and low-molecular substances. Ameloblasts were firmly connected with stratum intermedium cells by desmosomes and gap junctions. The gap junctions on ameloblast basal and lateral surfaces probably function in intercellular transfer of ions and low-molecular substances between the stratum intermedium and ameloblasts and in control of ameloblast cytodifferentiation.