Enamel Maturation Research Articles

Disturbed enamel formation in wild boars (Sus scrofa L.) from fluoride polluted areas in central Europe

The pathological alterations of enamel structure in the teeth of wild boars from fluoride polluted areas in N-Bohemia (Czech Republic) and S-Saxony (Germany) were studied on a macroscopic and a microscopic level. Mandibular bone fluoride concentration (mg F(-)/kg, dry wt; mean +/-SD, individuals <24 months of age) in the specimens from N-Bohemia (754.3+/-149.6) and S-Saxony (490.8+/-135.1) was significantly higher than that of controls (free of dental fluorosis), originating from the western part of Germany (304.7+/-91.0). Fluoride content in bulk enamel (mg F(-)/kg, ash wt) of fluorotic permanent teeth from N-Bohemia (382.1+/-165.2) and S-Saxony (125.0+/-38.3) was likewise significantly increased over that of non-fluorotic control teeth from W-Germany (33.6+/-26.7). Macroscopically, fluorosed wild boar enamel exhibited opacity and discoloration of varying extent, accentuated perikymata as well as hypoplastic and posteruptive surface defects. Microradiographic and scanning electron microscopic analyses revealed enamel subsurface hypomineralization, accentuated Retzius lines and occurrence of broad, hypomineralized incremental bands of abnormal structure underlying hypoplastic enamel surface defects. The presence of zones of aprismatic enamel was associated with these bands. Incremental bands with altered enamel structure and enamel surface hypoplasias, both denoting a severe disturbance during the secretory stage of amelogenesis, have previously been observed in rodents following acute parenteral fluoride dosing. It is concluded that in the chronically fluoride exposed wild boars periods of especially elevated plasma fluoride levels exerted an acute toxic effect on the secretory ameloblasts. A feature not previously reported from fluorosed enamel was the occurrence of canal-like structures that originated at the broad incremental bands and extended into the external enamel. The presence of these canals presumably results from a delay in the resumption of secretory activity by groups of ameloblasts following a fluoride insult. Based on experimental evidence in domestic pigs and in sheep, the overall subsurface hypomineralization of fluorosed wild boar enamel is attributed to a disturbance of enamel maturation. The distribution of fluorotic enamel changes within the dentition of the wild boars could be related to the developmental sequence of tooth formation in the species. Teeth whose crown formation took place prenatally (deciduous teeth) or largely pre-weaning (permanent first molars) exhibited no or only moderate fluorotic enamel alterations. Based on the extension of enamel surface hypoplasias along the coronoapical axes of the tooth crowns, the timing of excess fluoride exposure that caused a marked disruption of enamel matrix secretion was estimated in specimens with a known date of death. The results indicate that the wild boars had been exposed to a particularly severe fluoride impact during autumn and winter of their first year of life.

Fluoride effect on the activity of enamel matrix proteinases in vitro

Dental fluorosis is common in individuals exposed to different sources of fluoride during tooth development. The mechanism causing this enamel defect is still unknown. Enamel matrix proteinases play a central role in the maturation of dental enamel, and inhibition of these enzymes by fluoride has been one explanation for dental fluorosis. We have investigated the effect of fluoride on the activity of enamel matrix proteinases using a colorimetric assay, casein zymography, and an enamel protein degradation assay. Fluoride (625 microM to 10 mM) inhibited neither the enzymatic activity of the crude matrix extract nor the activity of individual enamel enzymes separated by SDS-PAGE. The proposition that fluoride could directly inhibit enzymes was not confirmed in this study.

A histological study of the effect of growth hormone on odontogenesis in the Lewis dwarf rat

The effect of growth hormone (GH) on the dentition has been described in children with pituitary dwarfism where teeth fail to form; those that do form tend to be reduced in size and the eruption potential is diminished. The aim here was to examine the effect of GH on odontogenesis via molar development in Lewis (control), dwarf (Dw) and Dw GH-treated (Dw+GH) rats aged 3, 6, 9, 12 and 15 days. Dw+GH animals received a twice-daily dose (65 μg/kg) of GH which commenced at 2 days of age. Animals were killed, mandibles removed, processed to embedding in paraffin, sectioned and stained for histological examination of molar morphology during development. Variations in enamel mineralization and root development were observed. In 6-day-old animals, enamel mineralization was delayed in Dw and Dw+GH animals. Root initiation was evident at 6 days of age in controls but was not observed until 9 days of age in Dw and Dw+GH animals. At 12 days of age, maturation of enamel in Dw and Dw+GH animals remained delayed. By 15 days of age no variation in tooth development was evident. These data indicate that enamel mineralization is affected by the level of circulating GH in the rat. A specific deficiency of GH did not appear to delay bone resorption prior to tooth emergence.

Type X collagen in human enamel development: a possible role in mineralization

Although type X collagen is one of the key molecules in endochondral ossification, no data are available on whether it is present in dental structures when mineralization is proceeding. We therefore monitored the appearance of type X collagen in tooth germs of human samples ranging in gestational age from 17-week-old fetuses to 9-week-old newborn. Using immunohistochemistry, ELISA techniques, and Western blotting, we show that type X collagen is present in human tooth germ during enamel maturation. Intense immunohistochemical staining for collagen type X was observed in the enamel and in the apical parts of secretory ameloblast at the bell stage when the dentine and enamel matrix were already under formation. The odontoblasts, the dentine, and the pulp were not stained. In the early (9-week) postnatal stage, the staining for collagen type X in the enamel matrix was diminished, and only a very weak signal could be detected in the secretory ameloblasts. A positive reaction for collagen type X was also observed in ELISA assay of extracts obtained from human embryonic enamel and hypertrophic cartilage samples. The Western blot analysis of the enamel demonstrated that size of the molecule detected by MoAb X53 is characteristic of the type X collagen. This correlates well with our immunohistochemical findings. Based on these data, we propose that type X collagen is one of the candidate molecules present in the enamel matrix that might be involved in mineralization of the enamel.

The Evolution of Enamel Microstructure: How Important Is Amelogenin?

The shape, size, and orientation of enamel prisms have heretofore been thought to be controlled solely by the shape of the Tomes' process. It is known, however, that amelogenin proteins play an important role in enamel deposition and maturation and it is possible that they contribute independently to enamel structure. Using a phylogenetic framework, we clarify the role of amelogenin proteins in the formation of enamel microstructure. We found a negative association between evolutionary changes in amelogenin protein sequences and enamel complexity: amelogenin evolution slows as enamel complexity increases. This is probably because selective constraints on amelogenin increase as enamel complexity increases. Monotremes, which have lost their adult dentition, have particularly high rates of amelogenin evolution while rodents, which have very complex enamel, have very low rates. There is a positive correlation between the number of different amelogenin proteins in a given species and the complexity of its enamel microstructure. An increased number of amelogenins may be necessary for the formation of multiple enamel types in the same tooth. Alternative splicing of amelogenin exons, which allows multiple protein products to be produced from the same gene, may be a key innovation in the diversification of enamel microstructure.

Immunohistochemical localisation of amelogenin-like proteins and type I collagen and histochemical demonstration of sulphated glycoconjugates in developing enameloid and enamel matrices of the larval urodele (Triturus pyrrhogaster) teeth.

The presence of collagen in enameloid distinguishes it clearly from true enamel, but little is known about the phylogenetic relationship between these 2 tissues. It has previously been reported that amelogenins are the principal proteins of the enamel matrix, that type I collagen and chondroitin sulphates are the predominant matrices in dentine, and that amphibian and reptilian aprismatic enamels, contain no sulphated glycoconjugates, although certain sulphated substances are secreted into mammalian prismatic enamel during matrix formation. The larval urodele (Triturus pyrrhogaster) teeth are known to be composed of enameloid, dentine, and enamel-like tissue. To characterise the tooth matrices, the localisation of amelogenin-like proteins, type I collagen, and sulphated glycoconjugates was investigated. Chondroitin sulphates and fine fibrils immunoreactive for type I collagen were elaborated as the enameloid matrix inside the dental basement membrane. After the matrix had been deposited in full thickness, coarse collagen fibrils also immunoreactive for type I collagen and chondroitin sulphates were deposited below as the first dentine matrix. Further, enamel-like matrix with no collagen fibrils or sulphated glycoconjugates but strongly immunoreactive for amelogenins was deposited on the dentine. Although no immunolabelling for amelogenins was found over the enameloid matrix, at least at the formation stage, the zone of coarse collagen fibrils of dentine was partially immunoreactive as observed in mammalian mantle dentine. From the ontogeny and matrix constituents of larval urodele teeth, it is suggested that enameloid is originally a dentine-like tissue.

Development and characterization of an SV40 immortalized porcine ameloblast-like cell line.

Enamel is secreted as a protein matrix by the ameloblasts. These same cells then control the maturation of the enamel matrix, secreting proteinases that hydrolyze proteins as mineralization progresses, until mature enamel containing less than 1% protein by weight remains. Further understanding of the factors that control ameloblast function and differentiation requires an in vitro cell culture system. In this study, we report immortalization of enamel organ epithelial cells and the selection of a cell line with characteristics of ameloblasts. Porcine enamel organ cells were dissected from unerupted porcine molars, cultured in serum-free medium, and passaged twice. These cells were transfected with an origin-of-replication defective SV40 plasmid by calcium phosphate precipitation, and a cell line with mRNA expression characteristic of ameloblasts was cloned. This cell line (PABSo-E) expressed mRNA for amelogenin, matrix metalloproteinase-20 (enamelysin), and enamel matrix serine proteinase 1 (EMSP1), but not ameloblastin. PABSo-E cells have been passaged more than 55 times, while continuing to maintain characteristics of ameloblasts. These cells will be useful for future studies of ameloblast function.

Cbfa1 is required for epithelial-mesenchymal interactions regulating tooth development in mice.

Osteoblasts and odontoblasts, cells that are responsible for the formation of bone and dentin matrices respectively, share several molecular characteristics. Recently, Cbfa1 was shown to be a critical transcriptional regulator of osteoblast differentiation. Mutations in this gene cause cleidocranial dysplasia (CCD), an autosomal dominant disorder in humans and mice characterized by defective bone formation. CCD also results in dental defects that include supernumerary teeth and delayed eruption of permanent dentition. The dental abnormalities in CCD suggest an important role for this molecule in the formation of dentition. Here we describe results of studies aimed at understanding the functions of Cbfa1 in tooth formation. RT-PCR and in situ hybridization analyses show that Cbfa1 has a unique expression pattern in dental mesenchyme from the bud to early bell stages during active epithelial morphogenesis. Unlike that observed in osteoblast differentiation, Cbfa1 is downregulated in fully differentiated odontoblasts and is surprisingly expressed in ectodermally derived ameloblasts during the maturation phase of enamel formation. The role of Cbfa1 in tooth morphogenesis is further illustrated by the misshapen and severely hypoplastic tooth organs in Cbfa1-/- mice. These tooth organs lacked overt odontoblast and ameloblast differentiation and normal dentin and enamel matrices. Epithelial-mesenchymal recombinants demonstrate that dental epithelium regulates mesenchymal Cbfa1 expression during the bud and cap stages and that these effects are mimicked by the FGFs but not by the BMPs as shown by our bead implantation assays. We propose that Cbfa1 regulates the expression of molecules in mesenchyme that act reciprocally on dental epithelium to control its growth and differentiation. Taken together, our data indicate a non-redundant role for Cbfa1 in tooth development that may be distinct from that in bone formation. In odontogenesis, Cbfa1 is not involved in the early signaling networks regulating tooth initiation and early morphogenesis but regulates key epithelial-mesenchymal interactions that control advancing morphogenesis and histodifferentiation of the epithelial enamel organ.

Proteinases in developing dental enamel.

For almost three decades, proteinases have been known to reside within developing dental enamel. However, identification and characterization of these proteinases have been slow and difficult, because they are present in very small quantities and they are difficult to purify directly from the mineralizing enamel. Enamel matrix proteins such as amelogenin, ameloblastin, and enamelin are cleaved by proteinases soon after they are secreted, and their cleavage products accumulate in the deeper, more mature enamel layers, while the full-length proteins are observed only at the surface. These results suggest that proteinases are necessary for "activating" enamel proteins so the parent proteins and their cleavage products may perform different functions. A novel matrix metalloproteinase named enamelysin (MMP-20) was recently cloned from tooth tissues and was later shown to localize primarily within the most recently formed enamel. Furthermore, recombinant porcine enamelysin was demonstrated to cleave recombinant porcine amelogenin at virtually all of the sites that have previously been described in vivo. Therefore, enamelysin is at least one enzyme that may be important during early enamel development. As enamel development progresses to the later stages, a profound decrease in the enamel protein content is observed. Proteinases have traditionally been assumed to degrade the organic matrix prior to its removal from the enamel. Recently, a novel serine proteinase named enamel matrix serine proteinase-1 (EMSP1) was cloned from enamel organ epithelia. EMSP1 localizes primarily to the early maturation stage enamel and may, therefore, be involved in the degradation of proteins prior to their removal from the maturing enamel. Other, as yet unidentified, proteinases and proteinase inhibitors are almost certainly present within the forming enamel and await discovery.

Development of dermal denticles in skates (Chondrichthyes, Batoidea): patterning and cellular differentiation.

Patterning, cellular differentiation, and developmental sequences of dermal denticles (denticles) are described for the skate Leucoraja erinacea. Development of denticles proceeds caudo-rostrally in the tail and trunk. Once three rows of denticles form in the tail and trunk, denticles begin to appear in the region of the pelvic girdle, medio-caudal to the eyes and on the pectoral fins. Although timing of cellular differentiation of denticles differs among different locations of the body, cellular development of a denticle is identical in all locations. Thickening of the epidermis as a denticle lamina marks initiation of development. A single lamina for each denticle forms, and a small group of mesenchymal cells aggregates underneath it. The lamina then invaginates caudo-rostrally to form the inner- and outer-denticle epithelia (IDE and ODE, respectively). Before nuclei of IDE cells are polarized, enameloid matrix appears between the basement membrane of the IDE and the apical surface of the pre-odontoblasts. Pre-dentin is then laid down along with collagenous materials. Von Kossa stain visualizes initial mineralization of dentin, but not enameloid. During the growth of a denticle, dense fibrous connective tissue of the dermis forms the deep dermal tissue over the dorsal musculature. Attachment fibers and tendons anchor denticles and dorsal musculature, respectively, on deep dermal tissue. Basal tissue of the denticles develops as the denticle crown grows. If the basal tissue is bone of attachment, then the cells along the basal tissue would be osteoblasts. However, these cells could not be distinguished from odontoblasts using immunolocalization of type I pro-collagen (Col I), alkaline phosphatase (APase), and neural cell adhesion molecule (N-CAM). Well-developed dentin, (not pre-dentin), the enameloid matrix (probably when it begins to mineralize), and deep dermal tissue are Verhoeff stain-positive, suggesting that these tissues contain elastin and/or elastin-like molecules. Our study demonstrates that the cellular development of denticles resembles tooth development in elasmobranchs, but that dermal denticles differ from teeth in forming from a single denticle lamina. Whether the basal tissue of denticles is bone of attachment remains undetermined. Confirmation and function of Verhoeff-positive proteins in enameloid, dentin, and deep dermal tissue remain to be determined. We discuss these issues along with an analysis of recent findings of enamel and enameloid matrices.

Posteruptive Changes of Electrical Resistance Values in Fissure Enamel of Premolars

In studies about 15–25% of the caries–free teeth proved to have as low electrical resistance values as carious teeth, due probably to incomplete enamel maturation. It was the aim of the present prospective clinical study to investigate, if an increase in electrical resistance values could be observed after the eruption of premolars. The electrical resistance measurements were performed in children who were patients of the Dental School of the University of Marburg, Germany. The first measurement was made when a premolar was erupting or when it had erupted within 12 months before. The electrical resistance measurements were repeated every 3–6 months until the 24th posteruptive month. The measuring device consisted of an AC ohmmeter modified by Gente indicating the real component of the impedance and employing a frequency of 500 Hz and an amplitude of about 1 μA. For erupting teeth a mean resistance value of 17±15 kohm was computed when the contact fluid had been NaCl (A). The corresponding values were 72±51 kohm when distilled water (B) had been applied and 392±374 kohm when the initially saliva–coated teeth had briefly been dried (C). The mean resistance values of the premolars increased continuously from the eruption up to the 15th posteruptive month. This observation was independent of the three modes of measuring that had been used. It is concluded that enamel maturation leads to an increase in electrical resistance. To improve accuracy of caries diagnosis with electrical resistance measurements, the posteruptive age of teeth should be taken into account. In addition, electrical resistance measurements offer the opportunity to observe enamel maturation in vivo noninvasively.

Biological mechanisms of dental fluorosis relevant to the use of fluoride supplements.

Fluorosis occurs when fluoride interacts with mineralizing tissues, causing alterations in the mineralization process. In dental enamel, fluorosis causes subsurface hypomineralizations or porosity, which extend toward the dentinal-enamel junction as severity increases. This subsurface porosity is most likely caused by a delay in the hydrolysis and removal of enamel proteins, particularly amelogenins, as the enamel matures. This delay could be due to the direct effect of fluoride on the ameloblasts or to an interaction of fluoride with the proteins or proteinases in the mineralizing matrix. The specific mechanisms by which fluoride causes the changes leading to enamel fluorosis are not well defined; though the early-maturation stage of enamel formation appears to be particularly sensitive to fluoride exposure. The development of fluorosis is highly dependent on the dose, duration, and timing of fluoride exposure. The risk of enamel fluorosis is lowest when exposure takes place only during the secretory stage, but highest when exposure occurs in both secretory and maturation stages. The incidence of dental fluorosis is best correlated with the total cumulative fluoride exposure to the developing dentition. Fluoride supplements can contribute to the total fluoride exposure of children, and if the total fluoride exposure to the developing teeth is excessive, fluorosis will result.

Structural and Chemical Analysis of Crystal Enamel Nucleation

AbstractThe aim of the present study is to obtain structural and chemical information about nucleation and growth of dental enamel as a function of location in secretory mouse foetus enamel. Then enamel tissues were obtained from 19-days-mouse foetus. TEM images show that enamel crystallites had the well-documented thin ribbons, or needles. They were found in the outermost zone in the vicinity of ameloblasts. Chemical analysis by EDS showed that, besides the elements of hydroxyapatite, Na, Cl, Al and Mg are presents from the beginning of enamel mineralization. All of these elements will play an important role on the physics and chemical properties in mature enamel.

Cytochrome oxidase activity in the enamel organ during amelogenesis in rat incisors.

Cytochrome oxidase (CO), one of the membrane-bound mitochondrial enzymes involved in oxidative phosphorylation, reflects the functional activity of mitochondria. Mitochondria in the enamel organ show drastic changes in localization during amelogenesis (Smith. INSERM, 1984;125:273-282). In understanding the functional aspects of the enamel organ, it is essential that one knows the exact CO activity in the respective mitochondria. The present study examines the CO activity of mitochondria in the enamel organ of rat incisors throughout the various stages of amelogenesis using light and transmission electron microscopy. CO activity was examined histochemically according to Seligman et al. (J. Cell. Biol., 1968;38:1-14) in decalcified sections of the upper and lower incisors of the rat. In the secretory stage, half of the mitochondria in the ameloblasts accumulated in the infranuclear region were reactive for CO. Both the population and CO activity of the infranuclear mitochondria of ameloblasts decreased significantly in the later stage where the enamel matrix secretion was almost complete. The CO-reactive mitochondria in the cells of the stratum intermedium (SI) gradually increased in number throughout the secretory stage. In the maturation stage, the ameloblasts contained intensively CO-reactive giant mitochondria in the proximal region and regular sized ones in the distal cytoplasm that were mostly devoid of detectable CO reactivity. The proportion of CO-reactive mitochondria in the supranuclear region and the population of mitochondria in the infranuclear regions of the smooth-ended ameloblasts were significantly higher as compared with the respective values in the ruffle-ended ameloblasts. In the late stages of enamel maturation, ameloblasts containing a large number of ferritin-filled pigment vesicles possessed numerous CO-reactive mitochondria between those vesicles in the supranuclear region, implicating an active role of the ameloblasts in iron transfer into the maturing enamel. The papillary layer cells possessed numerous intensively CO-reactive mitochondria throughout the maturation stage. A stage-related variation in the localization of CO-reactive mitochondria in the enamel organ of rat incisors was quantitatively demonstrated. It is conceivable that maturation stage ameloblasts form a functional unit with the papillary layer cells, and operate in energy-requiring events such as active ion transport to, and water and matrix protein removal from the maturating enamel. A sign of such functional integrity among the types of the enamel organ cells (ameloblasts, cells of SI, cells of stellate reticulum, and outer enamel epithelial cells) cannot be seen in the secretory stage. The secretory ameloblasts may function in matrix formation and calcium regulation in a less cooperative manner with the other cells of the enamel organ as compared to the maturation stage ameloblasts.

The use of autoradiography and cycloheximide to determine the origin of enamel proteins in the maturation ameloblasts of the rat incisor

Morphological and cytochemical studies have suggested that maturation ameloblasts participate in protein loss by absorbing and degrading enamel proteins as the enamel matures. Several immunocytochemical and autoradiographic studies have suggested other possible explanations for the presence of enamel matrix proteins in maturation ameloblasts. The weakness of these autoradiographic studies is the uncontrolled distribution of systemically injected radioactive amino acids, making it impossible to trace the source of the visualized intracellular isotope. This study used a localized technique to control the targets of the applied isotope and to identify enamel matrix proteins in the maturation ameloblasts with more confidence about their origin. The amount of labelled enamel protein was higher in maturation ameloblasts than transitional ameloblasts. When cycloheximide, a protein-synthesis inhibitor, was applied, there was no effect on the amount of labelled protein in the maturation ameloblasts. These findings support the hypothesis that maturation ameloblasts actively resorb and degrade enamel matrix proteins during enamel formation in the mandibular incisor of the rat.

Utilization of periodic markings in enamel to obtain information on tooth growth.

Enamel retains two fundamental periodic markings: the prism cross-striations, marking a diurnal rhythm of prism elongation, and the striae of Retzius, a longer-term marker of formation of the enamel surface. It is argued that, while artefacts superficially resembling prism cross-striations can be observed, these should not mislead an experienced histologist and that true cross-striations are reliable markers of the daily incremental apposition of enamel. The incremental markings can be used to elucidate several aspects of tooth crown growth. (1) Rate of enamel apposition. In some teeth, e. g., hominoid permanent teeth, the enamel apposition rate increases progressively between the inner and outer enamel, while in others the apposition rate appears to be uniform. (2) Crown formation time. This can be estimated on intact teeth, using counts of perikymata (surface traces of the striae), together with information on the time interval between successive striae. Much better estimates can be obtained from sections, using counts of cross-striations. (3) Pattern of crown formation. The rate at which new enamel formation extends over the presumptive enamel-dentine junction (extension rate) can be estimated from knowledge of the cross-striation interval together with angular measurements of prisms and striae. Multiple measurements of the extension rate within a tooth can be used to provide an estimate of crown formation time. The main application of extension rate measurements is to give quantitative information on the pattern of crown formation. In large primate teeth, the extension rate is high early in crown formation and slower later in crown formation. In smaller, more rapidly-formed teeth, a gradient in extension rate appears to be absent. Enamel thickness is negatively correlated with extension rate among primates. This seems to be a mechanism ensuring that there is sufficient time for enamel maturation to be completed before eruption takes place. Among primates it appears that variation in the prism pattern (i.e., the extent to which the outline of the pattern 3 prisms is closed or open) is correlated with the extension rate. It is postulated that the extension rate controls prism shape by influencing the morphology of the Tomes process pits during their initial formation.

Growth tracks in dental enamel.

The present paper evaluates the enamel growth tracks as tools in the chronological mapping of dental development, with special reference to hominids. Dental enamel consists of tightly packed hydroxyapatite crystals organized by differential orientation into a pattern of prisms and interprisms. The crystal organization is probably under the influence of both cellular and physico-chemical factors. The structure of mature enamel testifies to events that took place during enamel formation. The prisms are the fossilized tracks traced out by ameloblasts. The tangential diameter of ameloblasts and the central distance of prisms increase from the enamel-dentine junction to the enamel surface. Available evidence suggests that prism cross-striations are light microscopic expressions of prism varicosities and/or compositional variations, that these are due to a rhythm in enamel formation, and that this rhythm is diurnal. In human enamel the mean daily rate of enamel production is about 3.5 micron, but increases from inner to outer enamel and decreases from incisal/cuspal to cervical enamel. Conclusive evidence has shown that Retzius lines are incremental lines. Evenly spaced Retzius lines probably represent a 6-11 day rhythm in enamel formation, while other Retzius lines may be due to various types of stress. The geometry of the enamel growth tracks and their chronological significance are valuable tools in chronological mapping of dental development and for understanding temporal and spatial patterns in tooth morphogenesis. The taxonomic significance of prism packing patterns, prism decussation and enamel thickness should be clarified through further systematic descriptive research.

Enamelysin (matrix metalloproteinase-20): localization in the developing tooth and effects of pH and calcium on amelogenin hydrolysis.

The formation of dental enamel is a precisely regulated and dynamic developmental process. The forming enamel starts as a soft, protein-rich tissue and ends as a hard tissue that is over 95% mineral by weight. Intact amelogenin and its proteolytic cleavage products are the most abundant proteins present within the developing enamel. Proteinases are also present within the enamel matrix and are thought to help regulate enamel development and to expedite the removal of proteins prior to enamel maturation. Recently, a novel matrix metalloproteinase named enamelysin was cloned from the porcine enamel organ. Enamelysin transcripts have previously been observed in the enamel organ and dental papillae of the developing tooth. Here, we show that the sources of the enamelysin transcripts are the ameloblasts of the enamel organ and the odontoblasts of the dental papilla. Furthermore, we show that enamelysin is present within the forming enamel and that it is transported in secretory vesicles prior to its secretion from the ameloblasts. We also characterize the ability of recombinant enamelysin (rMMP-20) to degrade amelogenin under conditions of various pHs and calcium ion concentrations. Enamelysin displayed the greatest activity at neutral pH (7.2) and high calcium ion concentration (10 mM). During the initial stages of enamel formation, the enamel matrix maintains a neutral pH of between 7.0 and 7.4. Thus, enamelysin may play a role in enamel and dentin formation by cleaving proteins that are also present during these initial developmental stages.

Immunocytochemical and immunochemical study of enamelins, using antibodies against porcine 89-kDa enamelin and its N-terminal synthetic peptide, in porcine tooth germs.

Enamelins comprise an important family of the enamel matrix proteins. Porcine tooth germs were investigated immunochemically and immunocytochemically using two antibodies: a polyclonal antibody raised against the porcine 89-kDa enamelin (89 E) and an affinity purified anti-peptide antibody against the porcine enamelin amino-terminus (EN). Immunochemical analysis of layers of immature enamel from the matrix formation stage detected immunopositive protein bands ranging from 10 kDa to 155 kDa in the outer layer enamel sample irrespective of the antibodies used. In contrast, the middle and inner enamel layer mainly contained lower molecular weight enamelins. In immunocytochemical analyses of the differentiation stage, 89 E stained enamel matrix islands around mineralized collagen fibrils of dentin, while EN stained both enamel matrix islands and stippled material. At the matrix formation stage, both antibodies intensely stained enamel prisms located in the outer layer. In the inner layer, 89 E moderately stained enamel matrix homogeneously, while EN primarily stained the prism sheath. The intense immunoreaction over the surface layer of enamel matrix at the matrix formation stage, following staining with 89 E and EN, disappeared by the end of the transition stage and the early maturation stage, respectively. The Golgi apparatus and secretory granules in the ameloblasts from the late differentiation stage to the transition stage were immunostained by both antibodies. These results suggest that expression of enamelin continues from late differentiation to the transition stage and the cleavage of N-terminal region of enamelin occurs soon after secretion. Some enamelin degradation products, which apparently have no affinity for hydroxyapatite crystals, concentrate in the prism sheaths during enamel maturation.

Cellular and chemical events during enamel maturation.

This review focuses on the process of enamel maturation, a series of events associated with slow, progressive growth in the width and thickness of apatitic crystals. This developmental step causes gradual physical hardening and transformation of soft, newly formed enamel into one of the most durable mineralized tissues produced biologically. Enamel is the secretory product of specialized epithelial cells, the ameloblasts, which make this covering on the crowns of teeth in two steps. First, they roughly "map out" the location and limits (overall thickness) of the entire extracellular layer as a protein-rich, acellular, and avascular matrix filled with thin, ribbon-like crystals of carbonated hydroxyapatite. These initial crystals are organized spatially into rod and interrod territories as they form, and rod crystals are lengthened by Tomes' processes in tandem with appositional movement of ameloblasts away from the dentin surface. Once the full thickness of enamel has been formed, ameloblasts initiate a series of repetitive morphological changes at the enamel surface in which tight junctions and deep membrane infoldings periodically appear (ruffle-ended), then disappear for short intervals (smooth-ended), from the apical ends of the cells. As this happens, the enamel covered by these cells changes rhythmically in net pH from mildly acidic (ruffle-ended) to near-physiologic (smooth-ended) as mineral crystals slowly expand into the "spaces" (volume) formerly occupied by matrix proteins and water. Matrix proteins are processed and degraded by proteinases throughout amelogenesis, but they undergo more rapid destruction once ameloblast modulation begins. Ruffle-ended ameloblasts appear to function primarily as a regulatory and transport epithelium for controlling the movement of calcium and other ions such as bicarbonate into enamel to maintain buffering capacity and driving forces optimized for surface crystal growth. The reason ruffle-ended ameloblasts become smooth-ended periodically is unknown, although this event seems to be crucial for sustaining long-term crystal growth.