Amelogenesis: Transformation of a protein-mineral matrix into tooth enamel

During enamel formation, the organic enamel protein matrix interacts with calcium phosphate minerals to form elongated, parallel, and bundled enamel apatite crystals of extraordinary hardness and biomechanical resilience. The enamel protein matrix consists of unique enamel proteins such as amelogenin, ameloblastin, and enamelin, which are secreted by highly specialized cells called ameloblasts. The ameloblasts also facilitate calcium and phosphate ion transport toward the enamel layer. Within ameloblasts, enamel proteins are transported as a polygonal matrix with 5 nm subunits in secretory vesicles. Upon expulsion from the ameloblasts, the enamel protein matrix is re-organized into 20 nm subunit compartments. Enamel matrix subunit compartment assembly and expansion coincide with C-terminal cleavage by the MMP20 enamel protease and N-terminal amelogenin self-assembly. Upon enamel crystal precipitation, the enamel protein phase is reconfigured to surround the elongating enamel crystals and facilitate their elongation in C-axis direction. At this stage of development, and upon further amelogenin cleavage, central and polyproline-rich fragments of the amelogenin molecule associate with the growing mineral crystals through a process termed “shedding”, while hexagonal apatite crystals fuse in longitudinal direction. Enamel protein sheath-coated enamel “dahlite” crystals continue to elongate until a dense bundle of parallel apatite crystals is formed, while the enamel matrix is continuously degraded by proteolytic enzymes. Together, these insights portrait enamel mineral nucleation and growth as a complex and dynamic set of interactions between enamel proteins and mineral ions that facilitate regularly seeded apatite growth and parallel enamel crystal elongation.     

Spatiotemporal expression of ameloblastin isoforms during murine tooth development

Ameloblasts synthesize and secrete the enamel matrix proteins (amelogenin, ameloblastin, and enamelin). This investigation examined the profiles of ameloblastin in the ameloblasts and in the enamel matrix during different postnatal (PN) days (days 0-9) of development of mouse molar, using an antibody specific for C-terminal sequence of ameloblastin (Ct; GNKVHQPQVHNAWRF). Ameloblastin is found in three different molecular sizes (37, 55, and 66 kDa) in both ameloblasts and enamel matrix during PN development. In the ameloblasts, the sequence of expression of these fractions varied. The 37-kDa fraction was observed (even before the appearances of mRNA of the proteases, enamelysin and kallikrein-4) on days 0 and 1, persisted until day 3, and was not found thereafter. Other isoforms (55 and 66 kDa) distinctly appeared in ameloblasts after day 1, reached a peak on day 5, and remained thereafter. The Ct-positive granules appeared beaded in the ameloblasts on day 3. In the extracellular matrix, a 37-kDa (but not 66- or 55-kDa) fraction was detected on days 0 and 1 and remained in the matrix throughout the PN days. The larger isoforms (55 and 66 kDa) appeared in the enamel matrix from day 3 onward. On days 0-3, but not later, the 37-kDa isoform co-localizes with amelogenin in Tomes' process and formative enamel, as revealed by laser scan confocal microscopy. Autoradiography confirmed accumulation of 3H-labeled amelogenin trityrosyl motif peptide in the region of Tomes' process and formative enamel from day 0 to 3. These observations suggest that the 37-kDa isoform interacts with amelogenin during early tooth development.     

Binding of amelogenin to MMP-9 and their co-expression in developing mouse teeth

Amelogenin is the most abundant matrix protein in enamel. Proper amelogenin processing by proteinases is necessary for its biological functions during amelogenesis. Matrix metalloproteinase 9 (MMP-9) is responsible for the turnover of matrix components. The relationship between MMP-9 and amelogenin during tooth development remains unknown. We tested the hypothesis that MMP-9 binds to amelogenin and they are co-expressed in ameloblasts during amelogenesis. We evaluated the distribution of both proteins in the mouse teeth using immunohistochemistry and confocal microscopy. At postnatal day 2, the spatial distribution of amelogenin and MMP-9 was co-localized in preameloblasts, secretory ameloblasts, enamel matrix and odontoblasts. At the late stages of mouse tooth development, expression patterns of amelogenin and MMP-9 were similar to that seen in postnatal day 2. Their co-expression was further confirmed by RT-PCR, Western blot and enzymatic zymography analyses in enamel organ epithelial and odontoblast-like cells. Immunoprecipitation assay revealed that MMP-9 binds to amelogenin. The MMP-9 cleavage sites in amelogenin proteins across species were found using bio-informative software program. Analyses of these data suggest that MMP-9 may be involved in controlling amelogenin processing and enamel formation.     

Morphological and immunocytochemical analyses on the effects of diet-induced hypocalcemia on enamel maturation in the rat incisor.

During the maturation stage of amelogenesis, the loss of matrix proteins combined with an accentuated but regulated influx of calcium and phosphate ions into the enamel layer results in the "hardest" tissue of the body. The aim of the present investigation was to examine the effects of chronic hypocalcemia on the maturation of enamel. Twenty-one-day old male Wistar rats were given a calcium-free diet and deionized water for 28 days, while control animals received a normal chow. The rats were perfused with aldehyde and the mandibular incisors were processed for histochemical and ultrastructural analyses and for postembedding colloidal gold immunolabeling with antibodies to amelogenin, ameloblastin, and albumin. The maturation stage enamel organ in hypocalcemic rats exhibited areas with an apparent increase in cell number and the presence of cyst-like structures. In both cases the cells expressed signals for ameloblastin and amelogenin. The content of the cysts was periodic acid-Schiff- and periodic acid-silver nitrate-methanamine-positive and immunolabeled for amelogenin, ameloblastin, and albumin. Masses of a similar material were also found at the enamel surface in depressions of the ameloblast layer. In addition, there were accumulations of glycoproteinaceous matrix at the interface between ameloblasts and enamel. In decalcified specimens, the superficial portion of the enamel matrix sometimes exhibited the presence of tubular crystal "ghosts." The basal lamina, normally separating ameloblasts and enamel during the maturation stage, was missing in some areas. Enamel crystals extended within membrane invaginations at the apical surface of ameloblasts in these areas. Immunolabeling for amelogenin, ameloblastin, and albumin over enamel was variable and showed a heterogeneous distribution. In contrast, enamel in control rats exhibited a homogeneous labeling for amelogenin, a concentration of ameloblastin at the surface, and weak reactivity for albumin. These results suggest that diet-induced chronic hypocalcemia interferes with both cellular and extracellular events during enamel maturation.     

Reuptake of extracellular amelogenin by dental epithelial cells results in increased levels of amelogenin mRNA through enhanced mRNA stabilization

Amelogenin is an extracellular matrix protein secreted by ameloblasts and is a major component of enamel matrix. Recently, in addition to their role in enamel formation, the biological activity of enamel proteins in the process of cell differentiation has recently become widely appreciated. In this study, we examined the biological activity of amelogenin on ameloblast differentiation. Recombinant mouse amelogenin (rm-amelogenin) enhanced the expression of endogenous amelogenin mRNA in a cultured dental epithelial cell line (HAT-7), despite a lack of increased amelogenin promoter activity. To solve this discrepancy, we analyzed the effects of rm-amelogenin on the stability of amelogenin mRNA. The half-life of amelogenin mRNA is extremely short, but in the presence of rm-amelogenin its half-life was extended three times longer than the control. Furthermore, we showed the entry of exogenous fluorescein isothiocyanate-conjugated rm-amelogenin into the cytoplasm of HAT-7 cells. It follows from our results that exogenous amelogenin increases amelogenin mRNA levels through stabilization of mRNA in the cytoplasm of HAT-7 cells. Here we speculated that during differentiation, dental epithelial cells utilize a unique mechanism for increasing the production of amelogenin, the reuptake of secreted amelogenin.     

Initial enamel crystals are not spatially associated with mineralized dentine

During epithelial-mesenchymal interactions associated with mammalian tooth development, epithelially-derived and mesenchymally-derived extracellular matrix molecules form a discrete dentine-enamel junction. The developmental and molecular processes required to form this junction are not known. To address this problem we designed studies to test the hypothesis that ectodermally-derived epithelial cells synthesize and secrete enamel proteins which function to nucleate and regulate the growth of enamel calcium phosphate crystals. Initial enamel crystals were detected separate from the adiacent dentine. Electron-microprobe analyses revealed that early enamel crystals were octacalciumphosphate or tricalciumphosphate rather than hydroxyapatite. Thereafter, enamel crystals became confluent with the adjacent, albeit significantly smaller hydroxyapatite crystals associated with mineralized dentine. Therefore, we interpret our data to indicate that de novo enamel crystal nucleation and growth are independent from the mineralization processes characterized for dentine. We further argue that gene expression of enamel protein appears to have a constitutive function during early enamel formation and that supramolecular aggregates of amelogenin and enamelin provide the microenvironment for the nucleation and crystal growth of the initial enamel matrix.     

Ameloblast differentiation in the human developing tooth: Effects of extracellular matrices

Tooth enamel is formed by epithelially-derived cells called ameloblasts, while the pulp dentin complex is formed by the dental mesenchyme. These tissues differentiate with reciprocal signaling interactions to form a mature tooth. In this study we have characterized ameloblast differentiation in human developing incisors, and have further investigated the role of extracellular matrix proteins on ameloblast differentiation. Histological and immunohistochemical analyses showed that in the human tooth, the basement membrane separating the early developing dental epithelium and mesenchyme was lost shortly before dentin deposition was initiated, prior to enamel matrix secretion. Presecretary ameloblasts elongated as they came into contact with the dentin matrix, and then shortened to become secretory ameloblasts. In situ hybridization showed that the presecretory stage of odontoblasts started to express type I collagen mRNA, and also briefly expressed amelogenin mRNA. This was followed by upregulation of amelogenin mRNA expression in secretory ameloblasts. In vitro, amelogenin expression was upregulated in ameloblast lineage cells cultured in Matrigel, and was further up-regulated when these cells/Matrigel were co-cultured with dental pulp cells. Co-culture also up-regulated type I collagen expression by the dental pulp cells. Type I collagen coated culture dishes promoted a more elongated ameloblast lineage cell morphology and enhanced cell adhesion via integrin α2β1. Taken together, these results suggest that the basement membrane proteins and signals from underlying mesenchymal cells coordinate to initiate differentiation of preameloblasts and regulate type I collagen expression by odontoblasts. Type I collagen in the dentin matrix then anchors the presecretary ameloblasts as they further differentiate to secretory cells. These studies show the critical roles of the extracellular matrix proteins in ameloblast differentiation.     

Biomimetic Precipitation of Uniaxially Grown Calcium Phosphate Crystals from Full-Length Human Amelogenin Sols

Human dental enamel forms over a period of 2 - 4 years by substituting the enamel matrix, a protein gel mostly composed of a single protein, amelogenin with fibrous apatite nanocrystals. Self-assembly of a dense amelogenin matrix is presumed to direct the growth of apatite fibers and their organization into bundles that eventually comprise the mature enamel, the hardest tissue in the mammalian body. This work aims to establish the physicochemical and biochemical conditions for the synthesis of fibrous apatite crystals under the control of a recombinant full-length human amelogenin matrix in combination with a programmable titration system. The growth of apatite substrates was initiated from supersaturated calcium phosphate solutions in the presence of dispersed amelogenin assemblies. It was shown earlier and confirmed in this study that binding of amelogenin onto apatite surfaces presents the first step that leads to substrate-specific crystal growth. In this work, we report enhanced nucleation and growth under conditions at which amelogenin and apatite carry opposite charges and adsorption of the protein onto the apatite seeds is even more favored. Experiments at pH below the isoelectric point of amelogenin showed increased protein binding to apatite and at low Ca/P molar ratios resulted in a change in crystal morphology from plate-like to fibrous and rod-shaped. Concentrations of calcium and phosphate ions in the supernatant did not show drastic decreases throughout the titration period, indicating controlled precipitation from the protein suspension metastable with respect to calcium phosphate. It is argued that ameloblasts in the developing enamel may vary the density of the protein matrix at the nano scale by varying local pH, and thus control the interaction between the mineral and protein phases. The biomimetic experimental setting applied in this study has thus proven as convenient for gaining insight into the fundamental nature of the process of amelogenesis.     

Analysis of self-assembly and apatite binding properties of amelogenin proteins lacking the hydrophilic C-terminal.

Amelogenins, the major protein component of the mineralizing enamel extracellular matrix, are critical for normal enamel formation as documented in the linkage studies of a group of inherited disorders, with defective enamel formation, called Amelogenesis imperfecta. Recent cases of Amelogenesis imperfecta include mutations that resulted in truncated amelogenin protein lacking the hydrophilic C-terminal amino acids. Current advances in knowledge on amelogenin structure, nanospheres assembly and their effects on crystal growth have supported the hypothesis that amelogenin nanospheres provide the organized microstructure for the initiation and modulated growth of enamel apatite crystals. In order to evaluate the function of the conserved hydrophilic C-terminal telopeptide during enamel biomineralization, the present study was designed to analyze the self-assembly and apatite binding behavior of amelogenin proteins and their isoforms lacking the hydrophilic C-terminal. We applied dynamic light scattering to investigate the size distribution of amelogenin nanospheres formed by a series of native and recombinant proteins. In addition, the apatite binding properties of these amelogenins were examined using commercially available hydroxyapatite crystals. Amelogenins lacking the carboxy-terminal (native P161 and recombinant rM166) formed larger nanospheres than those formed by their full-length precursors: native P173 and recombinant rM179. These data suggest that after removal of the hydrophilic carboxy-terminal segment further association of the nanospheres takes place through hydrophobic interactions. The affinity of amelogenins lacking the carboxy-terminal regions to apatite crystals was significantly lower than their parent amelogenins. These structure-functional analyses suggest that the hydrophilic carboxy-terminal plays critical functional roles in mineralization of enamel and that the lack of this segment causes abnormal mineralization.     

Enamel biomineralization defects result from alterations to amelogenin self-assembly

Enamel formation is a powerful model for the study of biomineralization. A key feature common to all biomineralizing systems is their dependency upon the biosynthesis of an extracellular organic matrix that is competent to direct the formation of the subsequent mineral phase. The major organic component of forming mouse enamel is the 180-amino-acid amelogenin protein (M180), whose ability to undergo self-assembly is believed to contribute to biomineralization of vertebrate enamel. Two recently defined domains (A and B) within amelogenin appear essential for this self-assembly. The significance of these two domains has been demonstrated previously by the yeast two-hybrid system, atomic force microscopy, and dynamic light scattering. Transgenic animals were used to test the hypothesis that the self-assembly domains identified with in vitro model systems also operate in vivo. Transgenic animals bearing either a domain-A-deleted or domain-B-deleted amelogenin transgene expressed the altered amelogenin exclusively in ameloblasts. This altered amelogenin participates in the formation an organic enamel extracellular matrix and, in turn, this matrix is defective in its ability to direct enamel mineralization. At the nanoscale level, the forming matrix adjacent to the secretory face of the ameloblast shows alteration in the size of the amelogenin nanospheres for either transgenic animal line. At the mesoscale level of enamel structural hierarchy, 6-week-old enamel exhibits defects in enamel rod organization due to perturbed organization of the precursor organic matrix. These studies reflect the critical dependency of amelogenin self-assembly in forming a competent enamel organic matrix and that alterations to the matrix are reflected as defects in the structural organization of enamel.     

Maturation ameloblasts of the porcine tooth germ do not express amelogenin

 Amelogenins are the most abundant constituent in the enamel matrix of developing teeth. Recent investigations of rodent incisors and molar tooth germs revealed that amelogenins are expressed not only in secretory ameloblasts but also in maturation ameloblasts, although in relatively low levels. In this study, we investigated expression of amelogenin in the maturation stage of porcine tooth germs by in situ hybridization and immunocytochemistry. Amelogenin mRNA was intensely expressed in ameloblasts from the differentiation to the transition stages, but was not detected in maturation stage ameloblasts. C-terminal specific anti-amelogenin antiserum, which only reacts with nascent amelogenin molecules, stained ameloblasts from the differentiation to the transition stages. This antiserum also stained the surface layer of immature enamel at the same stages. At the maturation stage, no immunoreactivity was found within the ameloblasts or the immature enamel. These results indicate that, in porcine tooth germs, maturation ameloblasts do not express amelogenins, suggesting that newly secreted enamel matrix proteins from the maturation ameloblast are not essential to enamel maturation occurring at the maturation stage. Accepted: 14 January 1999     

Rat <Emphasis Type="Italic">wct</Emphasis> mutation prevents differentiation of maturation-stage ameloblasts resulting in hypo-mineralization in incisor teeth

A recent study provided genetic and morphological evidence that rat autosomal-recessive mutation, whitish chalk-like teeth (wct), induced tooth enamel defects resembling those of human amelogenesis imperfecta (AI). The wct locus maps to a specific interval of rat chromosome 14 corresponding to human chromosome 4q21 where the ameloblastin and enamelin genes exist, although these genes are not included in the wct locus. The effect of the wct gene mutation on the enamel matrix synthesis and calcification remains to be elucidated. This study clarifies how the wct gene mutation influences the synthesis of enamel matrix and its calcification by immunocytochemistry for amelogenin, ameloblastin and enamelin, and by electron probe micro-analysis (EPMA). The immunoreactivity for enamel proteins such as amelogenin, ameloblastin, and enamelin in the ameloblasts in the homozygous teeth was the same as that in the heterozygous teeth from secretory to transitional stages, although the homozygous ameloblasts became detached from the enamel matrix in the transitional stage. The flattened ameloblasts in the maturation stage of the homozygous samples contained enamel proteins in their cytoplasm. Thus, the wct mutation was found to prevent the morphological transition of ameloblasts from secretory to maturation stages without disturbing the synthesis of enamel matrix proteins, resulting in the hypo-mineralization of incisor enamel and cyst formation between the enamel organ and matrix. This mutation also prevents the transfer of iron into the enamel.     

Interaction between the enamel matrix proteins amelogenin and ameloblastin

Enamel matrix consists of amelogenin and non-amelogenins. Though amelogenin is not involved in nucleation of minerals, the enamel mineralization is impaired when amelogenin or other matrix protein (ameloblastin/enamelin) genes are mutated. We hypothesize that amelogenin may promote enamel mineralization by interacting with the calcium-binding matrix proteins. Specific binding of amelogenin to N-acetylglucosamine (GlcNAc), GlcNAc-mimicking peptides (GMps), and their carrier proteins and the identification of amelogenin-trityrosyl-motif-peptide (ATMP) as a GlcNAc/GMp-binding domain in amelogenin favor the hypothesis. This study tested the interaction of amelogenin with ameloblastin, a carrier of GMp sequence at intermittent sites. Neither GlcNAc nor sialic acids were identified in the recombinant-ameloblastin. Amelogenin bound to recombinant-ameloblastin in both Western blots and in ELISA. More specifically, [(3)H]ATMP bound to both recombinant and native ameloblastins. Dosimetry and Scatchard analyses showed the specific interaction between ATMP and ameloblastin, suggesting that amelogenin may interact with ameloblastin to form a heteromolecular assembly.     

Cytochemical and biochemical characterization of glycoproteins in forming and maturing enamel of the rat incisor

Biochemical and histochemical studies have shown the presence of various carbohydrates in enamel. Using lectin-gold cytochemistry, we have examined the distribution of glycoconjugates containing N-acetyl-D-galactosamine (GalNAc) and/or N-acetyl-glucosamine (GlcNAc)/N-acetyl-neuraminic acid (NeuNAc) residues in rat incisor ameloblasts and in forming and maturing enamel embedded in Lowicryl K4M, LR Gold, and LR White resins. The enamel proteins that contain these carbohydrate moieties were further characterized by lectin blotting. All three resins allowed, albeit to a variable degree, detection of the binding sites for Helix pomatia agglutinin (HPA) and wheat germ agglutinin (WGA) GalNAc, and GlcNAc/NeuNAc, respectively. In general, Lowicryl K4M permitted more intense reactions with both lectins. Lectin binding was observed over the rough endoplasmic reticulum (weak labeling with WGA), the Golgi apparatus, lysosomes, secretory granules, and the enamel matrix. These compartments were shown by double labeling with WGA and anti-amelogenin antibody, and by previous immunocytochemical studies, to contain enamel proteins. Furthermore, WGA binding was more concentrated at the growth sites of enamel. Lectin blotting showed that several proteins in the amelogenin group were glycosylated and contained the sugars GalNAc and GlcNAc/NeuNAc. Fewer proteins were stained by HPA than by WGA, and the staining pattern suggested that the extracellular proteins recognized by these two lectins are processed differently. The HPA-reactive proteins were lost by or during the early maturation stage, whereas many of the WGA-reactive proteins persisted into the mid maturation stage. The heterogeneous staining of certain protein bands observed with WGA suggests that they contain more than one component. Two distinct glycoproteins containing GlcNAc/NeuNAc also appeared during the maturation stage. These results are consistent with the notion that ameloblasts produce an extracellular matrix composed mainly of glycosylated amelogenins which are differently processed throughout amelogenesis.     

Immunochemical characterization of a chicken egg yolk antibody to secretory forms of rat incisor amelogenin.

Amelogenins represent the major component of the organic matrix of enamel, and consist of several intact and degraded forms. A precise knowledge of their respective distributions throughout the enamel layer could provide some insight into their functions. To date, no antibody exists that can selectively detect the secretory forms of amelogenin. In this study we used the chicken egg yolk system to generate an antibody to recombinant mouse amelogenin. Immunoblots of whole homogenates from rat incisor enamel organs and enamel showed that the resulting antibody (M179y) recognized proteins corresponding to the five known secretory forms of rat amelogenin. Immunogold cytochemistry demonstrated that reactivity was restricted to ameloblasts and enamel. Secretory forms of amelogenin persisted in significant amounts throughout the enamel layer. The density of labeling was highest over the surface portion of the enamel layer, but enamel growth sites in this region showed a localized paucity of gold particles. Immunoreactivity was lowest over the mid-portion of the layer and increased moderately near the dentino-enamel junction. These results indicate that intact forms of amelogenin probably have a more complex distribution in the enamel layer than was heretofore suspected.     

Enamel protein biosynthesis and secretion in mouse incisor secretory ameloblasts as revealed by high-resolution immunocytochemistry

Mouse secretory ameloblasts express a number of enamel proteins, which have been divided into amelogenin and enamelin subfamilies. We have used polyclonal antibodies to murine amelogenins to reveal enamel proteins in mouse ameloblasts using the protein A-gold immunocytochemical technique. Specific immunolabeling was detected over the extracellular enamel matrix and over the rough endoplasmic reticulum, the saccules of the Golgi apparatus, and the secretory granules of the ameloblasts. In addition, some lysosome-like granules were also labeled. Only background labeling was obtained over mitochondria, nuclei, cytosol, adjacent odontoblasts, and dentin. Quantitation of the intensity of labeling showed the presence of an increasing gradient along the secretory pathway, which may correspond to the concentration or the maturation of these proteins as they are processed by the cell. These findings indicate that the ameloblast displays an intracellular distribution of its secretory products similar to that of other merocrine secreting cells. The presence of enamel proteins in lysosomes suggests that crinophagy and/or resorption occurs in these cells.