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PubMed1984

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.

Amelogenesis↗
PubMedDate not supplied

Vascular endothelial growth factor (VEGF) in human tooth germ center.

Many oncogenis and tumour suppressor genes found inside normal and pathological cells are fundamental for the processes of development, proliferation and tissue differentiation. The purpose of our study is to show the presence and a possible relationship of the VEGF protein during different phases of the development of human dental germ centers. After cephalometric investigation in 8 orthodontic patients with a mean age of 13 years, (4 females and 4 males), hyperdivergence of the third molars were extracted. The 40 surgical samples were tested with monoclonal human anti-VEGFs antibodies carrying out a semi-quantitative analysis to look for a positive reaction. Reaction for anti-VEGF antibodies was detected in normal embryological tissues and in microvessels near odontogenic cells. During different phases of embryologic development of the dental bud our search showed intracytoplasmatic positive immunoreactions both in the ameloblastic and odontoblastic cells. Additionally, a positive reaction was observed for the VEGF protein in the cells of the stellate reticulum and in those endothelial tissue surrounding the microvessels in all the samples examined.

Adolescent↗
PubMed1998

Ameloblast-lineage cells of rat tooth germs proliferate and scatter in response to hepatocyte growth factor in culture.

Hepatocyte growth factor (HGF) is considered to be one of the mediators of epithelial-mesenchymal interactions during early organogenesis and to be involved in the development of murine molars. In this study, the immunohistochemical localization of HGF and of its receptor, c-Met, revealed that HGF was distributed in the proliferating mesenchymal cells in the dental papillae and that c-Met was continuously expressed in the epithelial cells during the development of rat incisors. These observations confirmed the involvement of HGF in the development of rat incisors, as demonstrated previously in molars. We then used a primary culture of ameloblast-lineage cells, prepared from mandibular incisors of young rats, to examine the direct effects of HGF on the growth and differentiation of ameloblasts. We found that HGF at 2-20 ng/ml induced a marked increase in the number of ameloblast-lineage cells and in the scattering of such cells. Our results suggest that HGF promotes the proliferation and scattering of ameloblast-lineage cells simultaneously.

Ameloblasts↗
PubMed1989

Localization of two distinct acid phosphatases in secretory ameloblasts of rat molar tooth germs.

Acid phosphatases were examined histochemically at the light- and electron-microscopic levels using para-nitrophenyl phosphate (pNPP) and beta-glycerophosphate (beta-GP) as substrates. By light microscopy, there was intense activity with pNPP in supranuclear and distal regions of the secretory ameloblast, and moderate or slight activity respectively in those regions with beta-GP. These enzyme activities were less at the late secretory stage of amelogenesis and disappeared at the transitional stage. By electron microscopy, acid phosphatase activity was seen in the trans side cisternae of the Golgi apparatus, in lysosome-like granules, and in small vesicles in the Tomes' processes. The activity with pNPP but not beta-GP was also localized at the plasma membrane (proximal, lateral and distal surface). Activity with beta-GP was completely inhibited by 1 mM sodium tartrate and by 1 mM NaF; activity with pNPP was inhibited by 1 mM NaF and 10 mM sodium tartrate, but not by 1 mM sodium tartrate. Thus there are at least two different acid phosphatases, one tartrate-sensitive and the other 1 mM tartrate-resistant, in the secretory ameloblast; the tartrate-resistant enzyme is plasma-membrane bound.

Acid Phosphatase↗
PubMed2002

Expression of connexin 43 and ZO-1 in differentiating ameloblasts and odontoblasts from rat molar tooth germs.

We studied the distribution of connexin (Cx) 43 and ZO-1 by confocal laser scanning microscopy at early stages of dentinogenesis and amelogenesis. Labeling for Cx43 was observed at early stages of differentiation in both the epithelial cells and differentiating odontoblasts. Immunolabeling was detected at the distal and medial regions of undifferentiated ameloblasts and between cells from stratum intermedium and stellate reticulum. Differentiating odontoblasts exhibited immunoreaction for this antibody at their distal end. Immunoreactivity for ZO-1 was observed at regions that correspond to the proximal and distal junctional complexes of differentiating ameloblasts. Staining for ZO-1 was observed at apical regions of odontoblasts with a punctate appearance. In more advanced stages, expression of Cx43 was more evident on ameloblasts, especially at the junctional complexes. Punctate immunolabeling for Cx43 was observed at the lateral sides of differentiating ameloblasts and between the other cells of the enamel organ. Immunoreaction for ZO-1 in ameloblasts was more evident than at the previous stage. It was also observed at the distal end of differentiated odontoblasts. The present study showed that differentiating ameloblasts and odontoblasts express Cx43 and ZO-1 as early as the start of the differentiation process. In addition, the expression of these junctional proteins increases as differentiation of cells continues.

Ameloblasts↗
PubMed1995

Enamel free areas in rodent molars--ultrastructure of basement membrane in rat tooth germ.

At the cusp tip of rodent molar, there is a region of dentin without an enamel cap. This region is called enamel free area (EFA). The surface collagen arrangement has been reported to differ between the EFA and the dentin covered with the enamel (DCE). To clarify the cause of this difference, we observed the ultrastructure of the basement membrane and the distal ends of the inner enamel epithelium (IEE) in rats. At 20 days prenatal, distal ends of IEE were relatively flat on both the DCE and EFA. Ultrastructurally, there was no difference between the basement membranes. At newborn, no marked changes were observed in the morphology of the distal end of IEE on the DCE or the EFA, but aperiodic microfibrils perpendicular to basal lamina were denser and longer on the DCE than the EFA. At 2 days postnatal, cytoplasmic extensions from distal end of IEE penetrated through basal lamina, and these extensions were more developed on the DCE than the EFA. On the DCE, collagen fibrils ran into and between cytoplasmic extensions and were arranged perpendicular to the surface. On the EFA, collagen fibrils ran parallel to the surface, and few collagen fibrils ran into and between cytoplasmic extensions. These findings suggested that the differences in the collagen arrangement between the EFA and DCE are associated with the developmental state of aperiodic microfibrils in the basal lamina beneath IEE and the morphology of the distal end of IEE.

Animals↗
PubMed1984

Long-term (8 days) effects of exposure to low concentrations of fluoride on enamel formation in hamster tooth-germs in organ culture in vitro.

Second maxillary molars of 3-4-day-old hamsters were cultured for 7-8 days in the continuous presence of fluoride (F-) or chloride in concentrations between 2.63 microM and 1.31 mM. For biochemical study, explants were labelled during the last 24 h of culture with a triple label of [3H]-proline, 45Ca and 32PO4. The 3H-labelled presumptive amelogenins were separated from the 3H-labelled dentine collagens by a three-step extraction procedure. Histologically, chronic exposure to F- had no obvious effects below 26.3 microM; at 26.3 microM of F-, a non-mineralizing enamel matrix was observed besides that of a normal mineralizing enamel. From 52 microM of F- onwards, only a non-mineralizing enamel matrix was found in decreasing amounts extracellularly as F- concentrations increased. Except for the presence of globular dentine, dentinogenesis was not obviously affected by F-. Biochemically, total synthesis of presumptive amelogenins was hardly disturbed, but their solubility was changed by chronic F- treatment; more amelogenins became formic-acid soluble at the expense of water-soluble amelogenins. Chronic exposure to F- decreased the water-soluble amelogenin fraction according to a logarithmic function of the medium F- concentration. By extrapolation, it was calculated that concentrations higher than 1-2 microM of F- affect amelogenesis in vitro. Synthesis of dentine collagen was not affected by chronic exposure to F- in vitro. Chronic exposure to F- decreased uptake of 45Ca and to a less extent trichloroacetic acid-soluble 32PO4. Chronic F- exposure may inhibit energy production in the enamel organ resulting in an impairment of enamel matrix secretion as well as that of a trans-epithelial transport mechanism for calcium.

Amelogenesis↗
PubMed1992

Demonstration of amelogenin in the enamel-free cusps of rat molar tooth germs: immunofluorescent and immunoelectron microscopic studies.

The enamel-free cusps of 1-4 day-old rat mandibular first molars were investigated using the monoclonal antibody En3 against rat amelogenin at light and electron microscopic levels in order to clarify whether the enamel-free cusp is virtually devoid of enamel. At 1 day after birth, there were presecretory ameloblast-like cells (PALCs), which were short and were not polarized, at the cusp tips. They were close to the outer enamel epithelium. Hematoxylin positive enamel matrix was not distinctly observed in the enamel-free cusp by light microscopy, but almost continuous immunofluorescence for amelogenin was detected at the interface between PALCs and dentin. The penetration of immunopositive material toward the dental pulp was also observed in the enamel-free cusp. At 4 day after birth, both in the frontal section and in the horizontal section, almost continuous immunofluorescence was recognized at the interface between PALCs and dentin in the enamel-free cusp. The penetration of amelogenin toward the dental pulp was not seen in the enamel-free cusp. By immunoelectron microscopy, immunolabelling was recognized in the Golgi apparatus of PALCs, in a layer of amorphous material at the interface between PALCs and dentin, and in stippled material-like substance in the intercellular space between PALCs. Although no basement membrane was observed beneath PALCs, they did not have Tomes' processes. These investigations suggest that PALCs in the enamel-free cusp differentiate into the secretory cells and that they can synthesize and secrete the amorphous material containing amelogenin at the interface between PALCs and dentin. The penetration of amelogenin toward the dental pulp might play a role in the interaction between PALCs and odontoblasts in the enamel-free cusp and/or the initiation of mineralization of predentin.

Ameloblasts↗
PubMed1989

[Immunohistochemistry of the distribution of alpha-tubulin in rat-incisor tooth germs and changes in it caused by vinblastine administration].

The present work used 2 groups of Wistar rats, weighing 100g each. Rats in the first group served as the controls; those in the second group were given 2 mg/kg of vinblastine in single intravenous injections. The animals were then fixed by perfusion with a mixture of 0.1% glutaraldehyde and 4% paraformaldehyde. After having been dissected out of the jaws, upper incisors were demineralized in EDTA and prepared into longitudinal sections (3 microns thick) for immunohistochemical demonstration of alpha-tubulin by means of indirect methods using an anti-alpha-tubulin monoclonal antibody as the primary antibody and peroxidase-labeled anti-mouse sheep IgG as the secondary antibody. 1. Control group. In controls, diffuse alpha-tubulin reaction was observed in the distal cytoplasm of inner enamel epithelial cells, differentiating ameloblasts, and ameloblasts in the stage of matrix formation. In the ameloblasts, the reaction was especially strong in the distal ends and Tomes processes in the matrix-formation stage. It gradually decreased until the transitional stage, in which the ameloblasts regained intense reaction to alpha-tubulin. Reaction to alpha-tubulin was generally low in the enamel-maturation stage in both smooth-ended and ruffle-ended ameloblasts but grew somewhat intense in the stage in which ferritines were precipitated in the cells. Although dental papilla cells reacted only faintly to alpha-tubulin, when they started differentiating into odontoblasts, reaction grew stronger in their distal end regions and in processes extending from the distal ends into the dentin. But this reaction decreased and ceased at a middle dentin level. Fairly high reaction was observed in the cells of the outer enamel epithelium, the stratum intermedium, the stellate reticulum, the papillary layer, the pulp and the dental sac. 2. Experimental group. Reaction to alpha-tubulin described in the preceding sections generally decreased in from 1 to 6 hours after vinblastine administration. Reaction recovery appeared to begin at from 12 to 24 hours and almost reached control degree by 48 hours after administration. Following the decrease of alpha-tubulin reaction in ameloblasts and odontoblasts, their shapes and polarities changed dramatically. In other cells, however, in spite of decreased alpha-tubulin reactions, no noticeable morphological alteration took place.

Animals↗
PubMed1981

Developmental arrest of mouse tooth germs in vitro by 6-diazo-5-oxonorleucine (DON).

Molars and incisors were excised from the mandibles of 15-day-old mouse embryos and explanted to agar-solidified Eagle's basal medium with 10% fetal calf serum and to medium containing 6-diazo-5-oxonorleucine (DON), a glutamine analog which inhibits glycoprotein and glycosaminoglycan synthesis. The concentration of DON ranged from 5-100 microgram/ml; the lowest dose at which consistent results were obtained was 75 microgram/ml. At the end of a 4-day culture period, controls displayed characteristic incisiform or molariform morphologies and possessed odontoblasts and preameloblasts. The growth and development of DON-treated germs, however, was arrested and such germs remained morphologically and histologically similar to the developmental state attained at the time of explantation. DON-treated germs also contained less abundant Alcian blue staining material than controls. Development arrest by DON was prevented by addition of glutamine but not by glucosamine or the purine analog amino imidazol carboximine. Suppressed germs resumed normal development when removed from medium containing 75 microgram/ml of DON to control medium. These results suggest that glycoprotein or glycosaminoglycans play a role in epithelial-mesenchymal inductive interactions.

Ameloblasts↗