Secretory Amelogenesis Research Articles

PH triggered self-assembly of native and recombinant amelogenins under physiological pH and temperature in vitro

Self-assembly of the extracellular matrix protein amelogenin is believed to play an essential role in regulating the growth and organization of enamel crystals during enamel formation. This study examines the effect of temperature and pH on amelogenin self-assembly under physiological pH conditions in vitro, using dynamic light scattering, turbidity measurements, and transmission electron microscopy. Full-length recombinant amelogenins from mouse (rM179) and pig (rP172) were investigated, along with proteolytic cleavage products (rM166 and native P148) lacking the hydrophilic C-terminus of parent molecules. Results indicated that the self-assembly of full-length amelogenin is primarily triggered by pH in the temperature range from 13 to 37°C and not by temperature. Furthermore, very large assemblies of all proteins studied formed through the rearrangement of similarly sized nanospherical particles, although at different pH values: pH 7.7 (P148), pH 7.5 (rM166), pH 7.2 (rP172), and pH 7.2 (rM179). Structural differences were also observed. The full-length molecules formed apparently tightly connected elongated, high-aspect ratio assemblies comprised of small spheres, while the amelogenin cleavage products appeared as loosely associated spherical particles, suggesting that the hydrophilic C-terminus plays an essential role in higher-order amelogenin assembly. Hence, tightly controlled pH values during secretory amelogenesis may serve to regulate the functions of both full-length and cleaved amelogenins.

Effect of calcium, given before or after a fluoride insult, on hamster secretory amelogenesis in vitro

We tested the hypothesis that high-calcium medium given prior to or immediately after exposure to fluoride (F) reduces the negative effects of F on secretory amelogenesis. Hamster molar tooth germs were grown in organ culture in media with different calcium levels. Deposition of enamel matrix and matrix mineralization were monitored by incorporation of [3H]proline and uptake of 45Ca and acid-soluble 32PO4. Ameloblast structure and the occurrence of a fluorotic enamel matrix were examined by light and electron microscopy. A preculture of explants in high-calcium medium partially prevented the formation of fluorotic (non-mineralizing) enamel matrix, increased matrix secretion but could not prevent F-induced hypermineralization of the pre-exposure enamel. High-calcium medium, applied after F insult, accelerated the recovery of fluorotic matrix, improved ameloblast structure, enhanced amelogenin secretion, and increased enamel thickness. The data indicate that it might be the balance between the amount of mineral deposition and that of matrix secretion which is critical for the mineralization of newly secreted enamel. Exposure to F disturbs this balance by enhancing mineralization of the pre-exposure enamel, probably generating an excess of protons. High calcium may protect against F exposure by enhancing amelogenin secretion into the enamel space, thereby increasing the local buffering capacity at the mineralization front.

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 >or=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.

Fluoride Enhances Intracellular Degradation of Amelogenins During Secretory Phase of Amelogenesis of Hamster Teeth in Organ Culture

Amelogenins are the major protein species synthesized by secretory ameloblasts and are believed to be involved in enamel mineralization. During enamel formation, amelogenins are progressively degraded into smaller fragments by protease activity. These amelogenin fragments are removed from the enamel extracellular space, thereby enabling full mineralization of the dental enamel. Enamel from fluorotic teeth is porous and contains more proteins and less mineral than sound enamel. In this study we examined the hypothesis that fluoride (F m ) is capable of inhibiting the proteolysis of amelogenins in enamel being formed in organ culture. Hamster molar tooth germs in stages of secretory amelogenesis were pulse labeled in vitro with [ 3 H]- or [ 14 C] proline and subsequently pulse chased. The explants were exposed to F m at different days of chase (i.e., during secretory amelogenesis early after labeling, later after labeling or at stages just beyond secretory amelogenesis). Exposure of secretory stage explants to F m enhanced the release of radiolabeled fragments when F m was applied early after labeling but progressively less if applied later. In contrast, F m had no such effect in stages beyond secretion. The enhanced release of radiolabeled fragments in secretory stages was associated with a reduction of radioactivity in the soft tissue enamel organ indicating that fragmentation of enamel matrix proteins (mainly amelogenins) occurred intracellularly. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the fluorotic enamel contained less radiolabeled parent amelogenins ( M r 28 kD and 26 kD) but more low-molecular-mass fragments than enamel from control explants. Our data indicate that F m promotes intracellular degradation of the newly synthesized parent amelogenins during secretory stage. Our in vitro data do not support the concept that F m impairs extracellular proteolysis of amelogenins, either in the secretory phase or in the stage just beyond the secretory phase.

Effects of actinomycin D on developing hamster molar tooth germs in vitro.

The aim of this study was to evaluate the toxic effects of actinomycin D on the developing hamster tooth germ in organ culture. Hamster tooth germs during early secretory amelogenesis were exposed in vitro for 24 h to 10(-9) M-5 x 10(-5) M actinomycin D. Actinomycin D dose-dependently (> or = 10(-7) M) decreased the tooth germ dry weight but mineralization was affected only by doses > or = 10(-5) M. However, the uptakes of TCA-insoluble 32P and [3H]thymidine were significantly reduced dose-dependently from > or = 10(-8) M actinomycin D, indicating that the drug inhibits the synthesis of phosphate-containing macromolecules as well as DNA synthesis. Histologically, 10(-8) M actinomycin D was the lowest dose which was not toxic to any cell type in the developing tooth germ. At 10(-7) M actinomycin D, the most sensitive cells were the proliferating pre-odontoblasts followed by pre-ameloblasts; the mature secretory ameloblasts and odontoblasts appeared unaffected. Higher doses resulted in increased cytotoxicity to the secretory cells and, eventually, total degeneration of most cells. The data suggest that children treated for cancer during tooth development using anti-chemotherapy cocktails containing actinomycin D (serum levels > 10(-7) M) may develop defects later on in the mature dentition as a direct consequence of the toxicity of the drug to the tooth organ.

Degradation of hamster amelogenins during secretory stage enamel formation in organ culture

Increasing amelogenin heterogeneity during pre-eruptive enamel formation has been explained by proteolytic cleavage of a parent amelogenin, differences in posttranslational modifications, translation of multiple alternative spliced mRNA transcripts or combinations of these possibilities. We investigated the possibility of proteolytic degradation of amelogenins during secretory amelogenesis by pulse-labelling amelogenins with [3H]proline followed by a pulse chase, all under organ culture conditions. The results indicate that during pulse chase, hamster molar tooth explants rapidly released substantial amounts of the radioactivity into the culture medium, as non-trichloroacetic-acid precipitable, noncollagenous 3H-activity at the expense of radioactivity associated with the proteins in the enamel space. Simultaneously, there was a continuous mineralization of the forming enamel in vitro as shown by an increase in total calcium content of the explants. Western blotting, microdissection studies and fluorography of radiolabelled matrix proteins after SDS-PAGE indicated that after an 8-h labelling, three radioactive amelogenin species could be extracted from forming enamel, one prominent species of molecular mass 26 kDa and two less prominent ones of 28 and 22 kDa. During pulse chase more amelogenin bands with lower molecular mass became apparent, a pattern similar to that observed in vivo. Examination of amelogenin blots with the glycan assay showed that none of the hamster amelogenins stained for carbohydrate. We conclude that changes in the amelogenin profiles during enamel development of cultured hamster explants are similar to those observed in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of adriamycin on dental proteins formation and mineralization in vitro.

Second maxillary molars of 4-5 days old golden hamsters were exposed for 2 h in vitro to 1 mg/L adriamycin, rinsed and subsequently cultured up to 7 days without the drug. At days 3, 5 or 7 of culture the synthesis of extracellular tooth matrices and their mineralization were examined by measuring the incorporation of 3H-proline and the uptake of 45Ca and 32PO4 by the explants during a 24 h pulse labeling. Compared with unexposed control explants, exposure to adriamycin for the first 2 h of culture had no effect on total biosynthesis of proline-containing matrix proteins. However, at days 3 and 5 of culture it increased the quantity of water-soluble enamel matrix proteins (amelogenins). Adriamycin also strongly reduced the amount of organically-bound 32P-activity in a fraction extractable with guanidine-HC1-EDTA only, allegedly containing a mixture of mineral-associated proteins from both enamel and dentin. Since this decrease of 32P-activity coincided with the formation of osteodentin in the pulp as shown previously in histological and electron microscopical studies, it was speculated that osteodentin matrix may not contain the highly phosphorylated, dentin-specific phosphoproteins (DPP). Adriamycin also affected the uptake of 45Ca and 32PO4. At day 3 these values were slightly higher than control values but lower at days 5 and 7. It therefore appears that a 2 h exposure to adriamycin in concentrations as low as 1 mg/L causes an acceleration of secretory amelogenesis by tooth germs in vitro. It also induces pulp cells to form osteodentin.

X-ray micro-analysis of the mineralization patterns in developing enamel in hamster tooth germs exposed to fluoride in vitro during the secretory phase of amelogenesis.

The developing enamel from three-day-old hamster first maxillary (M1) molar tooth germs exposed to fluoride (F-) in vitro was analyzed for its mineral content by means of the energy-dispersive x-ray microanalysis technique. The aim of this study was to obtain semi-quantitative data on the F(-)-induced hypermineralization patterns in the enamel and to confirm that the increase in electron density observed in micrographs of F(-)-treated enamel (Lyaruu et. al., 1986, 1987b) is indeed due to an increase in mineral content in the fluorotic enamel. The tooth germs were explanted during the early stages of secretory amelogenesis and initially cultured for 24 hr in the presence of 10 ppm F- in the culture medium. The germs were then cultured for another 24 hr without F-. In order to compare the ultrastructural results directly with the microprobe data, we used the same specimens for both investigations. The net calcium counts (measurement minus background counts) in the analyses were used as a measure of the mineral content in the enamel. The aprismatic pre-exposure enamel, deposited in vivo before the onset of culture, was the most hypermineralized region in the fluorotic enamel, i.e., it contained the highest amount of calcium measured. The degree of the F(-)-induced hypermineralization gradually decreased (but was not abolished) in the more mature regions of the enamel. The unmineralized enamel matrix secreted during the initial F- treatment in vitro mineralized during the subsequent culture without F-. The calcium content in this enamel layer was in the same order of magnitude as that recorded for the newly deposited enamel in control tooth germs cultured without F-.(ABSTRACT TRUNCATED AT 250 WORDS)

Antagonism of fluoride toxicity by high levels of calcium but not of inorganic phosphate during secretory amelogenesis in the hamster tooth germ in vitro

Whether the interference by fluoride (F −) with secretory amelogenesis in vitro could be modulated by altering the levels of calcium (Ca) and inorganic phosphate (P) in the medium was investigated. Hamster first upper molar tooth germs in the secretory phase of amelogenesis were exposed to 10 μM–1.31 mM (0.2–25 parts/10 6) of F − in vitro for 2 days in the presence of either low (1.2 mM), moderate (2.1 mM) or high (4.1 mM) levels of Ca, or moderate (1.6 mM) and high (3.6 mM) levels of P. The biosynthesis and secretion of enamel matrix proteins under each of the experimental conditions were examined by labelling with [ 3H]-proline during the last 24 h of culture, and mineralization by labelling with 45Ca and [ 32P]- orthophosphate. With moderate levels of Ca and P (control medium), F − increased the uptake of 45Ca and 32P in a dose-dependent manner; F − did not inhibit the synthesis of matrix proteins but to a moderate extent impaired their secretion. In explants grown in the presence of 52 μM of F − the superficial layers of enamel matrix deposited in vitro (fluorotic matrix) failed to mineralize. Increasing P levels in the medium had no clear histological effect, whereas lowering Ca levels sometimes seemed to aggravate the F − effect. Raising Ca levels improved the histological pattern: in spite of the presence of F −, high Ca levels allowed a limited mineralization of the superficial layer of fluorotic matrix along with a strong rise in mineralization of the deeper layers of pre-exposure enamel. High Ca levels also considerably reduced the cellular changes in secretory ameloblasts induced by 52 μM of F − and slightly counteracted the inhibition of matrix secretion, as measured biochemically. Some of the effects of F − on secretory amelogenesis in vitro can thus be reversed by raising Ca levels in the medium. Therefore, the effect of F − on secretory amelogenesis in vitro seems to be primarily interference with the enamel mineralization process per se and, secondarily, an impairment of matrix secretion.

Autoradiographic, ultrastructural and biosynthetic study of the effect of colchicine on enamel matrix secretion and enamel mineralization in hamster tooth germs in vitro

First upper molar tooth germs of two to three days old hamsters were exposed in vitro to colchicine in concentrations ranging between 10 −7 and 10 −4 M in the presence of 45Ca and/or [ 3H]-proline for various times up to 18 h. Enamel mineralization was determined by chemical extraction of in vitro incorporated 45Ca and verified ultrastructurally. Quantitative autoradiography compared with water extracts from total explants radiolabelled with [ 3H]-proline showed a dose-dependent decrease of grain counts over the extracellular enamel to the similar extent as the decrease in radiolabelled amelogenins in water-extracts. It was concluded that water-extracts from total expiants represent amelogenins from the extracellular compartment. Enamel matrix secreted in vitro during exposure to high doses of colchicine failed to mineralize and the complete loss was provoked of the distal parts of the secretory ameloblasts including the distal junctional complexes. Nevertheless, the mineralizing pre-exposure enamel neither hypermineralized nor increased uptake of 45Ca. These data do not support the hypothesis that secretory ameloblasts restrict transepithelial calcium transport by directing most of the calcium ions away from the mineralization front. The biosynthetic data furthermore suggest that enamel matrix proteins, only extractable with guanidine-HCl-EDTA, change their physico-chemical nature during secretory amelogenesis in vitro either during secretion or upon their extracellular mineralization.

The effects of cadmium on secretory amelogenesis in mineralizing tooth germs in vitro: [formula omitted][formula omitted][formula omitted][formula omitted][formula omitted][formula omitted][formula omitted][formula omitted][formula omitted] Lab of Tooth Development, Dept. of Oral Cell Biology, ACTA, Vrije University, de Boelelaan 1115, 1081 HV Amsterdam, The Netherlands

The effects of cadmium on secretory amelogenesis in mineralizing tooth germs in vitro: [formula omitted][formula omitted][formula omitted][formula omitted][formula omitted][formula omitted][formula omitted][formula omitted][formula omitted] Lab of Tooth Development, Dept. of Oral Cell Biology, ACTA, Vrije University, de Boelelaan 1115, 1081 HV Amsterdam, The Netherlands

Monoclonal antibody and immunogold cytochemical localization of amelogenins in bovine secretory amelogenesis

Amelogenin enamel-protein epitopes in developing incisors were ultrastructurally localized with high specificity resolution. They formed clumps scattered over the enamel organic matrix between the hydroxyapatite crystals, and were also present over islands of stippled or granular material at the forming surface of the enamel matrix demonstrating that this material consists in part of amelogenin enamel protein. The amounts of amelogenin, as judged by labelling density, were not greater in the stippled or the surface crystal-containing matrix as compared to the enamel matrix up to 50 μm deep. Amelogenins were also localized in the rough endoplasmic reticulum, Golgi apparatus and secretory granules of the ameloblasts, which suggests they are merocrine secretions.

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 μM 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 μM 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 μM 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.

Developmental comparisons of murine secretory amelogenesis in vivo, as xenografts on the chick chorio-allantoic membrane, and in vitro.

The temporal, spatial, and cytological characteristics of secretory amelogenesis in developing mouse mandibular first molar tooth organs have been compared during in vivo odontogenesis (from the Cap Stage in Theiler stage 25 C57BL/6 embryos to 10-day-old postnatal mice), as xenografts on the chick chorioallantoic membrane (CAM) for periods up to 7 days, and as explants in chemically defined medium without serum or antibiotics for periods up to 21 days in vitro. Tooth morphogenesis and cytodifferentiation proceeded in each environmental condition in the same sequence albeit at different rates of development. In vivo and CAM xenografts were remarkably comparable in their respective expressions of dentinogenesis and amelogenesis, whereas those explants cultured in a chemically defined medium without serum or antibiotics developed at a much slower rate (e.g., 0.5 days in vivo is equivalent to 1 day in vitro). In each experimental group, secretory amelogenesis was typically first detected along the mesial-buccal cusp of the molar organ independent of which environment was evaluated. Tooth morphogenesis in vitro and as xenografts on the CAM was routinely smaller than in situ odontogenesis. In each environmental condition a “stippled” precursor ultrastructural form of enamel matrix preceded mineralization, except during in vitro cultures of tooth organs. In vitro secretory amelogenesis or dentinogenesis did not indicate morphological characteristics of mineralization; both dentine and enamel matrices did not mineralize under the permissive environmental conditions used in these experiments. Calcium hydroxyapatite crystal formations within dentine and along the dentinoenamel junction during initial enamel matrix formation were not observed during in vitro tooth organogenesis, even for periods up to 21 days in vitro. We conclude that cap stage mandibular first molar tooth organs (enamel organ epithelium and adjacent dental papilla mesenchyme) from Theiler stage 25 embryos contain all of the necessary developmental instructions to express morphogenesis and cytodifferentiation except cues for the serum-containing factors for mineralization.

An Antisera for the Fluorescent Labeling of Mouse Amelogenesis

Embryonic mammalian enamel extracellular matrix is immunogenic. Antisera has been produced in New Zealand white rabbits using 5-day-old (post-natal) C57B1/6J mandibular and maxillary incisor and molar tooth organs as immunogens. The expression of secretory amelogenesis in mouse molar tooth organs was studied from the "cap stage" (circa 17-day fetus) to the fifth day of postnatal odontogenesis using indirect immunofluorescent microscopy. The specificity of the antisera for enamel matrix secretion was unequivocal. Secretory amelogenesis was observed in molar tooth organs as early as day-2 postnatal age. These reagents and methods provide a significant strategy in studies of epithelial-mesenchymal interactions during tooth development.