Fluorosis: a new model and new insights.

Fluoride is an effective agent for the prevention of dental caries. However, the mechanism of how excessive fluoride exposure causes fluorosis remains uncertain. Zebrafish (Danio rerio) exhibit periodic tooth replacement throughout their lives, thereby providing continuous access to teeth at developmental stages susceptible to fluoride exposure. Zebrafish teeth do not contain true enamel, but consist of a hard enameloid surface. Therefore, we asked whether zebrafish could be used as a model organism for the study of dental fluorosis. Scanning electron microscopy of fluoride-treated teeth demonstrated that the enameloid was pitted and rough, and FTIR analysis demonstrated that the teeth also contained a significantly higher organic content when compared with untreated controls. Furthermore, we demonstrate for the first time that decreased expression of an important signaling molecule (Alk8) in tooth development may contribute to the observed fluorotic phenotype, and that increased cell apoptosis may also play a role in the mechanism of fluorosis.

The cadherin-catenin complex is expressed alternately with the adenomatous polyposis coli protein during rat incisor amelogenesis.

E-cadherin, a calcium-dependent cell-cell adhesion molecule, is expressed in highly specific spatiotemporal patterns throughout metazoan development, notably at sites of embryonic induction. E-cadherin also plays a critical role in regulating cell motility/adhesion, cell proliferation, and apoptosis. We have used the continuously erupting rat incisor as a system for examining the expression of E-cadherin and the associated catenins [alpha-, beta-, gamma-catenin (plakoglobin) and p120(ctn)] during amelogenesis. Using immunhistochemical techniques, we observed expression of alpha-catenin and gamma-catenin in ameloblasts throughout amelogenesis. In contrast, expression of E-cadherin, beta-catenin, and p120(ctn) was strong in presecretory, transitional, and reduced stage ameloblasts (Stages I, III, and V) but was dramatically lower in secretory and maturation stage ameloblasts (Stages II and IV). This expression alternates with the expression pattern we previously reported for the adenomatous polyposis coli protein (APC), a tumor suppressor that competes with E-cadherin for binding to beta-catenin. We suggest that alternate expression of APC and the cadherin-catenin complex is critical for the alterations in cell-cell adhesion and other differentiated cellular characteristics, such as cytoskeletal alterations, that are required for the formation of enamel by ameloblasts.

The vascular pattern in the papillary region of rat incisor and molar tooth enamel organ.

Scanning electron microscopy of the enamel organ of rat incisor and molar teeth in the maturation stage of amelogenesis revealed two vascularization patterns of the papillary layer. In one pattern, the anastomosing capillaries formed loops of varying sizes around spherical or somehwat oblong papillae. In the second pattern, the capillaries were parallel to each other embedded in furrows between long ridges of papillary cells. It is postulated that each of these two patterns may be associated with a specific stage in the process of enamel maturation.

The pathway of enamel rods at the base of cusps of human teeth.

Enamel increases in volume and surface area with distance from the dentin-enamel junction, particularly at cusps, while the number of rods remains constant. Our results indicate that the increase in volume may be due to the increased rod diameter near the tooth surface, a circumferential winding of rods in the parazone orientation of Hunter-Schreger bands, and the migration of rods from a mid-coronal origin at the dentin surface to a cuspal terminaton at the tooth surface. Increased surface area can be explained by the increased rod diameter and the oblique approach of rods to the tooth surface.

Correlation of apical and lateral membrane modulations of maturation ameloblasts.

Maturation ameloblasts of rat incisor teeth have smooth-ended and ruffle-ended apical membrane configurations. It has also been reported that maturation ameloblasts have several lateral membrane configurations. The purpose of this study was to determine the correlation between the modulations of lateral and apical cell membranes of murine incisor ameloblasts in the maturation stage of amelogenesis. Maxillary and mandibular incisors were dissected, demineralized, embedded in paraffin, sectioned and then de-paraffinized, and the enamel organs were prepared for scanning electron microscopy. Additional mouse and rat incisor enamel organs were fixed and teased apart during dehydration, then observed in the SEM. The lengths of smooth- and ruffle-ended ameloblast segments were measured, and the site, length, and frequency of each lateral membrane configuration were determined within each segment. The lateral membrane configuration with folds forming from 12 to 14 channels around the periphery of the cells was most predominant in both smooth- and ruffle-ended cells. Cells surrounded by from six to eight channels were the only other lateral membrane configuration observed in ruffle-ended ameloblasts. Smooth-ended ameloblasts had lateral membrane configurations with either dense or sparse microvillous projections in addition to both types of channel cells. The observation that channelled extracellular spaces are always associated with ruffle-ended cells suggests that channels somehow function in conjunction with the ruffled apical membrane in resorption and removal of enamel matrix proteins. The smooth-ended ameloblasts lack tight apical junctions, and their microvillous lateral membranes permit the passage of plasma fluids around cells to the maturing enamel surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum fluoride level and fluoride content of enameloid.

We investigated diverse groups of fish species to determine whether the fluorine (F) contents of the dental hard tissues were related to baseline serum F levels. Serum samples, enameloid, dentin, ganoid/enamel, and bone were analyzed for F by either electron microprobe or wet chemistry. Species were categorized into two groups based on the F content of the enameloid. One group contained greater than 2.6 wt% F in enameloid, whereas the other group had less than 0.45 wt% F in enameloid. The dentin and bone from all species (or, in skates, the cartilage), as well as the ganoid/enamel layer of a Holostean fish (alligator gar), showed consistently low F content. In those species whose teeth developed in sequential rows, the F content of enameloid increased with progressive tooth development. The serum F levels of all fish were below 0.05 microgram F/mL (2.63 mumol/L) and were not significantly related to the F content of the enameloid. The results substantiate the idea that F incorporation into enameloid is related to fish phylogeny, not food or habitat. It is suggested that specialized outer dental epithelial cell configurations may facilitate the incorporation of F into enameloid.

A scanning electron microscope study of monkey maturation-stage ameloblasts.

The maturation-stage enamel organs of Macaca arctoides and Macaca mulatta were examined in order to determine whether the cells were similar to those of the continuously erupting rat incisor. Tooth buds of the permanent dentition were fixed in formaldehyde-glutaraldehyde and post-fixed in OsO4. The enamel organs were separated from the enamel during dehydration, critical-point-dried, metal-coated, and examined in a scanning electron microscope. The results showed that there were few differences in the morphology of maturation-stage ameloblasts of these primates compared with those of other species reported in the literature. The apical plasma membranes were either smooth- or ruffle-ended, while the later membranes had maze, microvillous, or ridge configurations, also seen in rats, and an additional configuration of interdigitating bulbous extensions. The blood vessels of the papillary layer in monkeys were about 7 micron in diameter, considerably larger than those of the rat.

Scanning electron microscopy of monkey secretory- and transitional-stage enamel organ cells.

This scanning electron microscope (SEM) study of secretory- and transitional-stage enamel organ cells of the permanent dentition of Macaca mulatta and Macaca arctoides was undertaken because the topography of these cells in primates has not been described in the literature. Comparison of our results with murine enamel organ morphology reported previously revealed not only many similarities, but also some significant differences. Tooth buds of the permanent dentition were routinely prepared for SEM. Murine secretory-stage ameloblasts have been described to be 65-70 microns long, with smooth lateral membranes, but those of monkeys were only 30-35 microns tall, with four different lateral plasma membrane configurations: smooth, filamentous, longitudinally ridged, and transversely ridged. The filamentous form was most common. Cells were seen with either transverse or longitudinal ridges in the basal half, and with filamentous ridges in the apical portion; this indicates modulation between these forms. Because of the extraordinary similarity between these lateral membrane modulations and those of rat incisor maturation ameloblasts, a comparable function is proposed--namely, that monkey secretory ameloblasts function, in part, in the resorption and mineralization of enamel matrix. There were several layers of rounded stratum intermedium cells basal to monkey secretory-stage ameloblasts, but only one layer of cuboidal stratum intermedium in rodents. The stellate reticulum cells of rats and monkeys appeared attenuated, with large extracellular spaces. There was little or no reduction in cell length of monkey transitional-stage ameloblasts. The position of the nuclear bulge differentiated transitional- from secretory-stage ameloblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of tooth development by the novel type I TGFbeta family member receptor Alk8.

We have recently identified, in zebrafish, a novel type I receptor of the TGFbeta family, alk8, that participates in Bmp signaling pathways to mediate early dorsoventral patterning of neurectodermal and mesendodermal tissues. Since Bmps play significant roles in tooth specification, initiation, and differentiation, we hypothesized that alk8 may play a role in directing the Bmp-mediated epithelial mesenchymal cell interactions regulating tooth development. Immunohistochemical analysis demonstrates that Alk8 is expressed in developing zebrafish and mouse teeth. Examination of tooth development in zebrafish with disrupted alk8 signaling revealed specific defects in tooth development. Ectopic expression of constitutively active Alk8 results in the formation of elongated tooth structures, while expression of dominant-negative Alk8 results in arrested tooth development at the bud stage. These results are consistent with the established requirements for Bmp signaling in tooth development and demonstrate that Alk8 is a key regulator of tooth development.

Adenomatous polyposis coli protein is expressed in alternate stages of the ameloblast life cycle.

Mutations of the adenomatous polyposis coli gene protein (APC) are associated with familial polyposis and also sporadic colon adenomas, both preconditions to cancer formation. Some familial polyposis patients also develop Gardner's syndrome, a condition characterized by supernumerary teeth, mandibular osteomas, and other maladies. We investigated participation of APC in normal tooth development. Using a monoclonal antibody to study APC expression in the forming rat incisor, we found no APC staining in differentiating ameloblasts, then strong staining in secreting ameloblasts and stratum intermedium cells, followed by cells in the transition stage which did not stain. Intense APC staining resumes in maturation-stage ameloblasts and proximal papillary cells. APC staining disappears again in reduced ameloblasts at the conclusion of amelogenesis. APC staining was not seen in any other odontogenic cells. We report a unique system in which APC expression is upregulated and downregulated twice during the normal life cycle of ameloblasts. APC, therefore, is important in the normal maturation of both colonic epithelium and odontogenic epithelium. At this point, we cannot rule out any of the known functions of APC, which include: modulation of cell adhesion by binding to catenin, regulation of beta-catenin as a differentiative signaling molecule, and promotion of microtubule assembly. In this respect, the rat incisor enamel organ provides a unique tissue for studying the regulation and functions of APC.

Comparison of rates of enamel synthesis in impeded and unimpeded rat incisors.

Periodic intubations of rats with solutions of fluoride (F) lead to the appearance of bands of disrupted pigmentation in continuously erupting incisors. Distances between fluorotic bands reflect time intervals between intubations. In this experiment, the periodicity of fluorotic banding was used for estimation of the rate of enamel synthesis in impeded and unimpeded rat incisors. Rats kept on a low-F diet and distilled water were intubated two or four times per week with 2 mg NaF/150 g body weight. In a group of rats, one of the mandibular incisors was cut at the gingival margin after two weeks, and intubations were continued for an additional two weeks. In another group of F-intubated rats, incisors were cut or notched at the gingival margin twice, six days apart. Control rats either received the same periodic F intubations or were maintained on the low-F diet without intubation. Measurements of spacing between fluorotic bands were identical in impeded and unimpeded teeth, even though the latter erupted at a faster rate. In an unimpeded mandibular incisors, there was a significant elongation of the secretory zone and a shortening of the pigmentation zone, resulting in reduced pigmentation intensity of the erupted portions of the teeth. The results show that the rate of enamel synthesis is independent of the eruption rate.

Modification of etching patterns in bovine dental enamel.

It is presumed that the etching pattern is controlled by the residual organic content of dental enamel. Pretreatment with 1.ON NaOH sould remove the organic material and modify the etching pattern. SEM studies and other tests for physical and chemical properties show that the predicted modification of the etching pattern, when the tooth surface is pretreated with NaOH solution, occurs apparently without other changes or properties.

Role of sucrose in colonization of Streptococcus mutans in conventional Sprague-Dawley rats.

The role of sucrose in the colonization of S mutans strain 6715 in conventional Sprague-Dawley rats was studied. A diet with 56% sucrose favored the oral colonization of the test strain compared to diets with 56% glucose or fructose or to laboratory chow as determined by recoveries from extracted teeth ground in tissue grinders. S mutans strain 6715 cells became well established in all rats fed a high sucrose diet with cell inoculums ranging from 10(8) to the lowest effective dose of 10(5) CFU once orally administered; in rats on nonsucrose diets, inoculation with even the highest dose only infrequently resulted in the establishment of S mutans strain 6715. Sucrose- and glucose- grown cells appeared to behave similarly. Colonization of S mutans strain 6715 occurred in all rats fed diets with a sucrose content ranging from 56 to as low as 1%. The establishment of S mutans strain 6715 on the teeth of rats fed diets with a sucrose concentration of 0.1 or 0.01% was impaired and comparable to the diet containing 56% glucose. In rats fed a high glucose diet, uniform establishment and persistence of the test strain occurred after frequent inoculations with about 5 X 10(8) CFU. The colonization under these conditions appeared to be independent of the intestinal canal as a bacterial cell source. These data suggest the possibility that S mutans can establish itself in the human mouth in the absence of dietary sucrose. In rats fed a high glucose diet and inoculated with 10(7) CFU or less, the cells gradually disappeared from the teeth; in contrast, the test strain implanted well in rats fed the sucrose favors firmer attachment of initially weakly attached cells via in situ new glucan synthesis. S mutans strain 6715 also appeared to have some affinity for teeth in the absence of dietary sucrose that may be of ecological significance. Once firmly established in rats fed a high sucrose diet, S mutans strain 6715 maintained itself in high numbers on the teeth after a switch to a high glucose diet during a 14-week period.

Scanning electron microscope study of cat and dog enamel structure.

Scanning electron microscopy revealed several similarities as well as significant differences in the enamel structure between cat and dog teeth. Three enamel layers were present in both species; a surface rodless (aprismatic) layer, an outer layer of parallel rods (only at some sites), and an inner layer with prominent Hunter-Schreger bands. In the inner layer of both carnivores, the diameter of individual rods varied significantly and frequently their course changed abruptly with respect to neighboring rods. In dog teeth the cross-sectional shape of inner enamel rods was pleomorphic, but hexagonal in outer enamel. In contrast, cat enamel rods were rounded in both inner and outer enamel layers. Hunter-Schreger bands of cats circumscribed the teeth in relatively straight segments, but these bands showed pronounced waviness in dog teeth. In cats and dogs the surface rodless layer was structurally continuous with subjacent interrod enamel and covered all tooth surfaces with the exception of the cervical areas. The data show that the structure of inner and outer enamel layers differ between these two carnivore species and that the enamel structure of the cat was most similar to that described in humans. One principal difference between carnivore and human teeth is that the growth lines of carnivores do not terminate at perikymata on the tooth surface.

Microbiological findings in prepubertal periodontitis. A case report.

Generalized pre-pubertal periodontitis (GPP) is a rare entity that usually affects children with severe systemic diseases. We report the case of a 7-year-old male patient diagnosed with GPP, with no apparent systemic condition, who lost all his primary teeth to periodontal disease. Before extractions, while he was still in mixed dentition the subgingival plaque was collected and analyzed using DNA probes to 40 different microorganisms. Putative periodontopathogens such as Prevotella intermedia, Selenomonas noxia, Fusobacterium nucleatum, and Actinobacillus actinomycetemcomitans could be identified throughout the mouth. More intriguing was the colonization of the sulcus of some secondary teeth by potentially harmful microorganisms found in pockets of diseased adjacent primary teeth.

Ultrastructure of the dental epithelium during enameloid mineralization in a teleost fish, Cichlasoma cyanoguttatum.

Secretory-stage inner dental epithelial cells (IDE) of tooth buds deposited an unmineralized, ectodermally-derived, enameloid collagen matrix. Pharyngeal plates bearing tooth buds were fixed: some were demineralized, others treated with guanidine-EDTA, then fixed and post-fixed in osmium tetroxide with potassium ferricyanide. Thin Epon sections were viewed in a Jeol 100B TEM. Nascent enameloid crystals were orientated parallel to the collagen fibres and attained widths of 200 nm. Enameloid collagen was absent in demineralized mature enameloid. The outer dental epithelial plasma membrane was deeply invaginated forming extensive channels associated with elongated fuzzy-coated vesicles. Four configurations of IDE cells were characterized by cellular constituents, including elongated granules, Golgi complexes, multivesicular bodies, large electron-dense granules and extracellular amorphous material which was also adjacent to cells containing few organelles associated with protein synthesis, within infoldings of ruffled apical membranes and multivesicular bodies. This material was considered to be resorbed enameloid collagen, not a secretory product.

Ultrastructure of secretory ameloblasts in a monkey Macaca mulatta.

A 13-month-old animal was fixed by perfusion; the tooth buds of the permanent dentition were removed and prepared for transmission electron microscopy. Secretory ameloblasts were about 35 micrometers long and Tomes processes varied from 6 to 16 micrometers in length. The organelles included a basal nucleus, a Golgi apparatus concentrated in a spherical region about 4 micrometers in diameter in the supranuclear region, rough endoplasmic reticulum situated between the basal and apical terminal webs, and mitochondria dispersed throughout the cytoplasm, including Tomes processes. Many ameloblasts also contained a variety of granules, rosettes of small vesicles, and multivesicular bodies interpreted as parts of a lysosomal or GERL system. Stippled material was observed only at the distal end of the Tomes process of some ameloblasts. The results demonstrate that the Tomes process in the macaque was longer than reported in cats or man, otherwise the secretory ameloblast ultrastructure is similar among the three species.

Enameloid formation in two tetraodontiform fish species with high and low fluoride contents in enameloid.

Forming teeth of parrotfish and pufferfish were viewed by transmission electron microscopy to correlate cytological features of the enameloid organ with the species' fluoride (F) content in mature enameloid. Secretory-stage inner dental epithelial cells (IDE) of parrotfish (high F) and pufferfish (low F) secreted procollagen granules into the enameloid collagen matrix. The odontoblasts of both species, less numerous than IDE cells, also contained procollagen granules at the enameloid matrix formation stage. After the full thickness of enameloid matrix collagen had been deposited, enameloid crystallites formed parallel to the long axis of the enameloid collagen fibres. Concurrently, the plasma membranes of the outer dental epithelial cells (ODE) became invaginated in both species, but to a much greater extent in parrotfish. Highly undulating parrotfish ODE cells surrounded numerous fenestrated capillaries. In contrast, pufferfish ODE cells remained straight with few adjacent capillaries. Extensive tight junctions formed between ODE and IDE cells of both species, sealing the extracellular space. With increased mineralization, enameloid collagen fibres were no longer discernible. A thin layer of amorphous material, which subsequently mineralized, was secreted on to the enameloid surface by IDE cells in both species. Pufferfish odontoblasts secreted a mineralizing amorphous layer on the pulpal aspect of the enameloid. The results suggest that at the mineralization stage, a triad of cytostructural features, highly invaginated ODE cells, highly vascularized ODE cells, and extensive tight junctions are strongly correlated with high fluoride content of mature enameloid mineral. Species without any one of these features have lower fluoride in the enameloid.

Ultrastructural study of tracer permeability through the cat and ferret enamel organ.

The access of exogenous materials to the developing enamel surface has been intensively studied in rodents, but not in other mammalian species. This ultrastructural study investigates the permeability of injected horseradish peroxidase (HRP) and lanthanum tracers in cat and ferret tooth buds. In cat enamel organs fixed by immersion, lanthanum did not escape the capillaries overlying secretory stage tooth buds, but it did permeate up to the distal junctions of ruffle-ended (RA) and the proximal junctions of smooth-ended (SA) ameloblasts. Perfusion fixation with lanthanum compromised junctional integrity of cat ameloblasts at all stages of development. Similarly, HRP rarely escaped the capillaries associated with cat secretory stage enamel organs. However, unlike lanthanum, HRP was mostly confined to the vasculature of maturation stage enamel organs in immersion fixed cats at all time intervals examined. In ferrets, HRP penetrated up to, but not beyond, the distal junctional complexes of secretory ameloblasts. In maturation stage enamel organs, HRP coated the papillary and RA cells, but did not penetrate the RA distal cell junctions. HRP did permeate the extracellular spaces of SA to reach the underlying enamel surface. Ameloblasts in transitional phases of SA and RA endocytosed HRP at the distal cell surface. This data leads to several conclusions. First, HRP localization in the ferret paralleled that observed in rodents. Second, the results of cat enamel organs substantiate previous studies showing perfusion fixation can increase vascular and intercellular permeability to lanthanum. However, in cats fixed by immersion, both lanthanum and HRP were restricted to capillaries associated with the secretory stage enamel organ, and only lanthanum escaped maturation stage capillaries. It is suggested that variations in the fenestrations and distribution of capillaries associated with the cat enamel organ may differentially retain some materials and permit other materials to escape with relative ease.

The penetration of exogenous tracers through the enameloid organ of developing teleost fish teeth.

In order to determine whether exogenous materials permeate to the forming tooth enameloid matrix, teleost species were injected intramuscularly with horseradish peroxidase (HRP) or myoglobin, or; intracardially with lanthanum nitrate or HRP, then killed a predetermined intervals post-injection. Tooth bearing bones were processed for transmission electron microscopy. At the enameloid matrix formation stage, capillaries associated with the enameloid organ were few in number and rarely fenestrated. Both organic tracers reached the matrix at cervical but not coronal, regions of the teeth in all species examined. Lanthanum was rarely observed extravascularly and never extended to the enameloid matrix at the secretion stage. At the enameloid mineralization stage, fenestrated capillaries were closely associated with the outer dental epithelial cells (ODE). All tracers were observed in the plasma membrane invaginations of the ODE. Only intracardially injected HRP compromised the apical intercellular junctions of the inner dental epithelial cells (IDE) to reach the mineralizing enameloid Lanthanum did not extend past the ODE-IDE cell junctions. It is concluded that the close association of mineralization stage fenestrated capillaries with the highly invaginated ODE cells result in increased tracer penetration compared to the secretory stage. The deeper penetration of the organic tracers, compared with lanthanum, between mineralization stage IDE cells may be due to longer in vivo circulation of the former material. The apical junctions of mineralization stage IDE cells, however, remained impermeable to the organic tracers. The absence of mineral in secretory stage enameloid mineral could not be due to specialized cell junctions preventing access of molecules to the matrix. It is suggested that controlling factors other than cellular permeability initiate enameloid mineralization.