Effect of kallikrein 4 loss on enamel mineralization: comparison with mice lacking matrix metalloproteinase 20

Enamel formation depends on a triad of tissue-specific matrix proteins (amelogenin, ameloblastin, and enamelin) to help initiate and stabilize progressively elongating, thin mineral ribbons of hydroxyapatite formed during an appositional growth phase. Subsequently, these proteins are eradicated to facilitate lateral expansion of the hydroxyapatite crystallites. The purpose of this study was to investigate changes in enamel mineralization occurring in mice unable to produce kallikrein 4 (Klk4), a proteinase associated with terminal extracellular degradation of matrix proteins during the maturation stage. Mice lacking functional matrix metalloproteinase 20 (Mmp20), a proteinase associated with early cleavage of matrix proteins during the secretory stage, were also analyzed as a frame of reference. The results indicated that mice lacking Klk4 produce enamel that is normal in thickness and overall organization in terms of layers and rod/inter-rod structure, but there is a developmental defect in enamel rods where they first form near the dentinoenamel junction. Mineralization is normal up to early maturation after which the enamel both retains and gains additional proteins and is unable to mature beyond 85% mineral by weight. The outmost enamel is hard, but inner regions are soft and contain much more protein than normal. The rate of mineral acquisition overall is lower by 25%. Mice lacking functional Mmp20 produce enamel that is thin and structurally abnormal. Relatively high amounts of protein remain throughout maturation, but the enamel is able to change from 67 to 75% mineral by weight during maturation. These findings reaffirm the importance of secreted proteinases to enamel mineral acquisition.     

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     

TGF-? Regulates Enamel Mineralization and Maturation through KLK4 Expression

Transforming growth factor-ß (TGF-ß) signaling plays an important role in regulating crucial biological processes such as cell proliferation, differentiation, apoptosis, and extracellular matrix remodeling. Many of these processes are also an integral part of amelogenesis. In order to delineate a precise role of TGF-ß signaling during amelogenesis, we developed a transgenic mouse line that harbors bovine amelogenin promoter-driven Cre recombinase, and bred this line with TGF-ß receptor II floxed mice to generate ameloblast-specific TGF-ß receptor II conditional knockout (cKO) mice. Histological analysis of the teeth at postnatal day 7 (P7) showed altered enamel matrix composition in the cKO mice as compared to the floxed mice that had enamel similar to the wild-type mice. The µCT and SEM analyses revealed decreased mineral content in the cKO enamel concomitant with increased attrition and thinner enamel crystallites. Although the mRNA levels remained unaltered, immunostaining revealed increased amelogenin, ameloblastin, and enamelin localization in the cKO enamel at the maturation stage. Interestingly, KLK4 mRNA levels were significantly reduced in the cKO teeth along with a slight increase in MMP-20 levels, suggesting that normal enamel maturation is regulated by TGF-ß signaling through the expression of KLK4. Thus, our study indicates that TGF-ß signaling plays an important role in ameloblast functions and enamel maturation.     

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.     

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.     

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.     

Action of Urea Solutions on Unerupted and Erupted Teeth: An Investigation on Late Maturation of Human Enamel1

Replicas of unerupted and erupted human enamel submitted to protein-disrupting solutions showed: (1) an inequal distribution of porosities in the subsurface; (2) such porosities were restricted gradually in each enamel subunit (enamel segments limited by two perikymata). First the whole surface was covered by a network, reduced at a later stage to half of the surface and ultimately the porosities were present only in the perikymata. This paper confirms the occurrence of a late maturation at stage where cells are no longer active and details the time-dependent involution of these porosities.     

Odontogenic ameloblast-associated and amelotin are novel basal lamina components

Odontogenic ameloblast-associated (ODAM) and amelotin (AMTN) are secreted by maturation stage ameloblasts and accumulate at the interface with enamel where an atypical basal lamina (BL) is present. This study aimed at determining and quantifying the ultrastructural distribution of ODAM and AMTN at the cell–tooth interface. Ultrathin sections of enamel organs from the early to mid- and late maturation stage of amelogenesis were processed for immunogold labeling with antibodies against ODAM, AMTN or with the lectins wheat germ agglutinin, Helix pomatia agglutinin (HPA) and Ricinus communis I agglutinin. Immunolabeling showed that both ODAM and AMTN localized to the BL. Quantitative analyses indicated that at the beginning of maturation there is a concentration of ODAM on the cell side of the BL while AMTN appears more concentrated on the enamel side. In the late maturation stage, such differential distribution is no longer apparent. All three lectins are bound to the BL. Competitive incubation with native lectins did not affect the binding efficiency of ODAM; however, AMTN binding was significantly reduced after incubation with HPA. In conclusion, ODAM and AMTN are bona fide components of the BL associated with maturation stage ameloblasts and they organize into different subdomains during the early maturation stage. The data also suggest that the BL is a dynamic structure that rearranges its organization as enamel maturation advances. Finally, the abrogation of AMTN antibody labeling by HPA supports the presence of O-linked sugars in the molecule and/or its close association with other O-glycosylated molecules.     

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.     

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.     

A genetic model for the secretory stage of dental enamel formation

The revolution in genetics has rapidly increased our knowledge of human and mouse genes that are critical for the formation of dental enamel and helps us understand how enamel evolved. In this graphical review we focus on the roles of 41 genes that are essential for the secretory stage of amelogenesis when characteristic enamel mineral ribbons initiate on dentin and elongate to expand the enamel layer to the future surface of the tooth. Based upon ultrastructural analyses of genetically modified mice, we propose a molecular model explaining how a cell attachment apparatus including collagen 17, α6ß4 and αvß6 integrins, laminin 332, and secreted enamel proteins could attach to individual enamel mineral ribbons and mold their cross-sectional dimensions as they simultaneously elongate and orient them in the direction of the retrograde movement of the ameloblast membrane.     

Phase Transformations in a Human Tooth Tissue at the Initial Stage of Caries

The aim of the paper is to study phase transformations in solid tissues of the human teeth during the development of fissure caries by Raman and fluorescence microspectroscopy. The study of the areas with fissure caries confirmed the assumption of the formation of a weak interaction between phosphate apatite enamel and organic acids (products of microorganisms). The experimental results obtained with by Raman microspectroscopy showed the formation of dicalcium phosphate dihydrate - CaHPO₄-2H₂O in the area of mural demineralization of carious fissure. A comparative analysis of structural and spectroscopic data for the intact and carious enamel shows that emergence of a more soluble phase - carbonate-substituted hydroxyapatite - is typical for the initial stage of caries. It is shown that microareas of dental hard tissues in the carious fissure due to an emerging misorientation of apatite crystals have a higher fluorescence yield than the area of the intact enamel. These areas can be easily detected even prior to a deep demineralization (white spot stage) for the case of irreversibly changed organomineral complex and intensive removal of the mineral component.     

Partial rescue of the amelogenin null dental enamel phenotype

The amelogenins are the most abundant secreted proteins in developing dental enamel. Enamel from amelogenin (Amelx) null mice is hypoplastic and disorganized, similar to that observed in X-linked forms of the human enamel defect amelogenesis imperfecta resulting from amelogenin gene mutations. Both transgenic strains that express the most abundant amelogenin (TgM180) have relatively normal enamel, but strains of mice that express a mutated amelogenin (TgP70T), which leads to amelogenesis imperfecta in humans, have heterogeneous enamel structures. When Amelx null (KO) mice were mated with transgenic mice that produce M180 (TgM180), the resultant TgM180KO offspring showed evidence of rescue in enamel thickness, mineral density, and volume in molar teeth. Rescue was not observed in the molars from the TgP70TKO mice. It was concluded that a single amelogenin protein was able to significantly rescue the KO phenotype and that one amino acid change abrogated this function during development.     

Runx2条件性敲除小鼠釉质缺陷的部分挽救

该研究旨在探讨外源性Runx2过表达对小鼠成釉细胞Runx2敲除导致的釉质缺陷的挽救作用。采用免疫组化验证Runx2在Runx2条件性敲除且人源性Runx2过表达小鼠成釉细胞中的表达。HE染色观察成熟期成釉细胞形态及釉质基质蛋白残余。用体视显微镜和扫描电镜观察小鼠牙齿表面形态和釉柱结构。结果显示,RUNX2蛋白在出生后10天龄Tg;cKO小鼠成熟早期成釉细胞中成功表达。15天龄Tg;cKO小鼠与cKO小鼠相比,成熟晚期成釉细胞形态及排列未见明显改善,但釉质基质蛋白残余量明显减少。3月龄Tg;cKO小鼠与cKO小鼠相比,釉质磨耗减轻,釉柱间孔隙减少,釉柱排列更规则。该研究结果表明,人源性Runx2过表达可部分挽救小鼠成釉细胞Runx2敲除导致的釉质缺陷。     

Transmission FT-IR microspectroscopy of mineral phases in calcified tissues

Fourier-transform infrared microspectroscopy (FT-IRM) was used to study bone mineralization processes in an in vivo model and in enamel in osteogenesis imperfecta. Finally, the ability of FT-IRM to map new bone formed in implanted macroporous calcium phosphate biomaterial from sections was reported for the first time. FTIRM allowed the correlation of the microstructure of bone formation in the in vivo model with modifications in carbonate and phosphate environments of the mineral phases during maturation. FT-IRM analysis on enamel sections revealed changes in the mineral environment of carbonate and phosphate ions and probably in the size of enamel crystals. These modifications contributed to the fragility of enamel in osteogenesis imperfecta. The infrared functional group imaging of a part of implanted biomaterial and the bone ingrowth provided the visualization of chemical modifications occurring in biomaterial implants at 20 μm spatial resolution. The use of FT-IRM, in conjunction with appropriate sampling methods and data analysis should provide further insight into the molecular structure of mineral phases of calcified tissues and help to elucidate mineralization processes, skeletal disorders and properties of the biomaterials used as bone substitute.     

Amelogenin-deficient mice display an amelogenesis imperfecta phenotype.

Dental enamel is the hardest tissue in the body and cannot be replaced or repaired, because the enamel secreting cells are lost at tooth eruption. X-linked amelogenesis imperfecta (MIM 301200), a phenotypically diverse hereditary disorder affecting enamel development, is caused by deletions or point mutations in the human X-chromosomal amelogenin gene. Although the precise functions of the amelogenin proteins in enamel formation are not well defined, these proteins constitute 90% of the enamel organic matrix. We have disrupted the amelogenin locus to generate amelogenin null mice, which display distinctly abnormal teeth as early as 2 weeks of age with chalky-white discoloration. Microradiography revealed broken tips of incisors and molars and scanning electron microscopy analysis indicated disorganized hypoplastic enamel. The amelogenin null phenotype reveals that the amelogenins are apparently not required for initiation of mineral crystal formation but rather for the organization of crystal pattern and regulation of enamel thickness. These null mice will be useful for understanding the functions of amelogenin proteins during enamel formation and for developing therapeutic approaches for treating this developmental defect that affects the enamel.     

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.     

WDR72 models of structure and function: A stage-specific regulator of enamel mineralization

Amelogenesis Imperfecta (AI) is a clinical diagnosis that encompasses a group of genetic mutations, each affecting processes involved in tooth enamel formation and thus, result in various enamel defects. The hypomaturation enamel phenotype has been described for mutations involved in the later stage of enamel formation, including Klk4, Mmp20, C4orf26, and Wdr72. Using a candidate gene approach we discovered a novel Wdr72 human mutation in association with AI to be a 5-base pair deletion (c.806_810delGGCAG; p.G255VfsX294). To gain insight into the function of WDR72, we used computer modeling of the full-length human WDR72 protein structure and found that the predicted N-terminal sequence forms two beta-propeller folds with an alpha-solenoid tail at the C-terminus. This domain iteration is characteristic of vesicle coat proteins, such as beta′-COP, suggesting a role for WDR72 in the formation of membrane deformation complexes to regulate intracellular trafficking. Our Wdr72 knockout mouse model (Wdr72^−/−), containing a LacZ reporter knock-in, exhibited hypomineralized enamel similar to the AI phenotype observed in humans with Wdr72 mutations. MicroCT scans of Wdr72^−/− mandibles affirmed the hypomineralized enamel phenotype occurring at the onset of the maturation stage. H&E staining revealed a shortened height phenotype in the Wdr72^−/− ameloblasts with retained proteins in the enamel matrix during maturation stage. H⁺/Cl⁻ exchange transporter 5 (CLC5), an early endosome acidifier, was co-localized with WDR72 in maturation-stage ameloblasts and decreased in Wdr72^−/− maturation-stage ameloblasts. There were no obvious differences in RAB4A and LAMP1 immunostaining of Wdr72^−/− mice as compared to wildtype controls. Moreover, Wdr72^−/− ameloblasts had reduced amelogenin immunoreactivity, suggesting defects in amelogenin fragment resorption from the matrix. These data demonstrate that WDR72 has a major role in enamel mineralization, most notably during the maturation stage, and suggest a function involving endocytic vesicle trafficking, possibly in the removal of amelogenin proteins.