Matrix metalloproteinase 20 promotes a smooth enamel surface, a strong dentino-enamel junction, and a decussating enamel rod pattern

Mutations of the matrix metalloproteinase 20 (MMP20, enamelysin) gene cause autosomal-recessive amelogenesis imperfecta, and Mmp20 ablated mice also have malformed dental enamel. Here we showed that Mmp20 null mouse secretory-stage ameloblasts maintain a columnar shape and are present as a single layer of cells. However, the maturation-stage ameloblasts from null mouse cover extraneous nodules of ectopic calcified material formed at the enamel surface. Remarkably, nodule formation occurs in null mouse enamel when MMP20 is normally no longer expressed. The malformed enamel in Mmp20 null teeth was loosely attached to the dentin and the entire enamel layer tended to separate from the dentin, indicative of a faulty dentino-enamel junction (DEJ). The enamel rod pattern was also altered in Mmp20 null mice. Each enamel rod is formed by a single ameloblast and is a mineralized record of the migration path of the ameloblast that formed it. The enamel rods in Mmp20 null mice were grossly malformed or absent, indicating that the ameloblasts do not migrate properly when backing away from the DEJ. Thus, MMP20 is required for ameloblast cell movement necessary to form the decussating enamel rod patterns, for the prevention of ectopic mineral formation, and to maintain a functional DEJ.     

Ameloblasts require active RhoA to generate normal dental enamel

RhoA plays a fundamental role in regulation of the actin cytoskeleton, intercellular attachment, and cell proliferation. During amelogenesis, ameloblasts (which produce the enamel proteins) undergo dramatic cytoskeletal changes and the RhoA protein level is up‐regulated. Transgenic mice were generated that express a dominant‐negative RhoA transgene in ameloblasts using amelogenin gene‐regulatory sequences. Transgenic and wild‐type (WT) molar tooth germs were incubated with sodium fluoride (NaF) or sodium chloride (NaCl) in organ culture. Filamentous actin (F‐actin) stained with phalloidin was elevated significantly in WT ameloblasts treated with NaF compared with WT ameloblasts treated with NaCl or with transgenic ameloblasts treated with NaF, thereby confirming a block in the RhoA/Rho‐associated protein kinase (ROCK) pathway in the transgenic mice. Little difference in quantitative fluorescence (an estimation of fluorosis) was observed between WT and transgenic incisors from mice provided with drinking water containing NaF. We subsequently found reduced transgene expression in incisors compared with molars. Transgenic molar teeth had reduced amelogenin, E‐cadherin, and Ki67 compared with WT molar teeth. Hypoplastic enamel in transgenic mice correlates with reduced expression of the enamel protein, amelogenin, and E‐cadherin and cell proliferation are regulated by RhoA in other tissues. Together these findings reveal deficits in molar ameloblast function when RhoA activity is inhibited.     

Short exposure to high levels of fluoride induces stage-dependent structural changes in ameloblasts and enamel mineralization

We tested the hypothesis that the sensitivity of forming dental enamel to fluoride (F^–) is ameloblast developmental stage-dependent and that enamel mineralization disturbances at the surface of fluorotic enamel are caused by damage to late-secretory- and transitional-stage ameloblasts. Four-day-old hamsters received a single intraperitoneal dose of 2.5–20 mg NaF/kg body weight and were examined, 24 h later, by histology and histochemistry. A single dose of ≥ 5 mg of NaF/kg induced the formation of a hyper- followed by a hypomineralized band in the secretory enamel, without changing the ameloblast structure. At 10 mg of NaF/kg, cystic lesions became apparent under isolated populations of distorted late-secretory- and transitional-stage ameloblasts. Staining with von Kossa stain showed that the enamel under these lesions was hypermineralized. At 20 mg of NaF/kg, cystic lesions containing necrotic cells were also found in the early stages of secretory amelogenesis and were also accompanied with hypermineralization of the enamel surface. We concluded that the sensitivity to F^– is ameloblast developmental stage-dependent. Groups of transitional ameloblasts are most sensitive, followed by those at early secretory stages. These data suggest that a F-induced increase in cell death in the transitional-stage ameloblasts accompanies the formation of cystic lesions, which may explain the formation of enamel pits seen clinically in erupted teeth.     

Buffering of protons released by mineral formation during amelogenesis in mice

Regulation of pH by ameloblasts during amelogenesis is critical for enamel mineralization. We examined the effects of reduced bicarbonate secretion and the presence or absence of amelogenins on ameloblast modulation and enamel mineralization. To that end, the composition of fluorotic and non‐fluorotic enamel of several different mouse mutants, including enamel of cystic fibrosis transmembrane conductance regulator‐deficient (Cftr null), anion exchanger‐2‐deficient (Ae2a,b null), and amelogenin‐deficient (Amelx null) mice, was determined by quantitative X‐ray microanalysis. Correlation analysis was carried out to compare the effects of changes in the levels of sulfated‐matrix (S) and chlorine (Cl; for bicarbonate secretion) on mineralization and modulation. The chloride (Cl^?) levels in forming enamel determined the ability of ameloblasts to modulate, remove matrix, and mineralize enamel. In general, the lower the Cl^? content, the stronger the negative effects. In Amelx‐null mice, modulation was essentially normal and the calcium content was reduced least. Retention of amelogenins in enamel of kallikrein‐4‐deficient (Klk4‐null) mice resulted in decreased mineralization and reduced the length of the first acid modulation band without changing the total length of all acidic bands. These data suggest that buffering by bicarbonates is critical for modulation, matrix removal and enamel mineralization. Amelogenins also act as a buffer but are not critical for modulation.     

Epithelial fibroblast growth factor receptor 1 regulates enamel formation

The interaction between epithelial and mesenchymal tissues plays a critical role in the development of organs such as teeth, lungs, and hair. During tooth development, fibroblast growth factor (FGF) signaling is critical for regulating reciprocal epithelial and mesenchymal interactions. FGF signaling requires FGF ligands and their receptors (FGFRs). In this study, we investigated the role of epithelial FGF signaling in tooth development, using the Cre-loxp system to create tissue-specific inactivation of Fgfr1 in mice. In K14-Cre;Fgfr1(fl/fl) mice, the apical sides of enamel-secreting ameloblasts failed to adhere properly to each other, although ameloblast differentiation was unaffected at early stages. Prior to eruption, enamel structure was compromised in the K14-Cre;Fgfr1(fl/fl) mice and displayed severe enamel defects that mimic amelogenesis imperfecta (AI), with a rough, irregular enamel surface. These results suggest that there is a cell-autonomous requirement for FGF signaling in the dental epithelium during enamel formation. Loss of Fgfr1 affects ameloblast organization at the enamel-secretory stage and, hence, the formation of enamel.     

Organic anion transport during rat enamel formation

ObjectiveThe C-terminal end of nascent amelogenin is dissociated immediately after secretion and rapidly re-absorbed by ameloblasts, presumably by endocytosis. The purpose of this study was to test whether organic anion transporters (OATs) are also involved in the re-absorption process of enamel matrix proteins via non-endocytotic pathways.Materials and methodsLocalization of OAT1, OAT2, and OAT3 in rat tooth germs was examined by immunohistochemistry using specific antibodies. Actual translocation of organic anions through the ameloblast layer was further tested by systemic tracer experiments in rats in which Lucifer Yellow (LY), a fluorescent organic anion, was used as a tracer.ResultsIn rat tooth germs, OAT2 was associated exclusively with the distal cell membranes of secretory ameloblasts where Tomes' processes were developed and disappeared when matrix formation was terminated. On the other hand, OAT1 was absent in secretory ameloblasts and was colocalized with the ruffled border of ruffle-ended ameloblasts in the maturation stage. OAT3 was undetectable in ameloblasts and located instead only in the stratum intermedium cells. Systemic administration of LY resulted in intense labeling of immature enamel and also a transient labeling of the cytosol of secretory ameloblasts immunopositive for OAT2. In the maturation stage, cytosolic labeling of LY was negligible in all cells of the enamel organs, including ameloblasts.ConclusionsThese data suggest the existence of OATs in rat tooth germs and their possible involvement in matrix re-absorption at least in the secretory stage of amelogenesis.     

Overexpression of constitutively active MAP3K7 in ameloblasts causes enamel defects of mouse teeth

ObjectiveCompelling evidence suggests that mitogen-activated protein kinases (Mapks) play an important role in amelogenesis. However, the role of transforming growth factor (TGF)-β-activating kinase 1 (Tak1, Map3k7), which is a known upstream kinase of Mapks, during amelogenesis remains to be determined. The aim of this study was to investigate the possible involvement of Map3k7 in amelogenesis.DesignWe generated transgenic mice that produced constitutively active human MAP3K7 (caMAP3K7) under the control of amelogenin (Amelx) gene promoter. Radiography and micro-computed tomography (μCT) analysis was used to detect the radio-opacity and density of the teeth. The enamel microstructure was observed with a scanning electron microscope. Histological analysis was used to observe the adhesion between ameloblasts and residual organic matrix of the enamel. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze the expression of enamel matrix protein.ResultsThe enamel of mandibular molars in caMAP3K7-overexpressing mice displayed pigmentation and a highly irregular structure compared with the wild type littermates. Teeth of transgenic animals underwent rapid attrition due to the brittleness of the enamel layer. The microstructure of enamel, normally a highly ordered arrangement of hydroxyapatite crystals, was completely disorganized. The gross histological appearances of ameloblasts and supporting cellular structures, as well as the expression of the enamel protein amelotin (Amtn) were altered by the overexpression of caMAP3K7.ConclusionsOur data demonstrated that protein expression, processing and secretion occurred abnormally in transgenic mice overexpressing caMAP3K7. The overexpression of caMAP3K7 had a profound effect on enamel structure by disrupting the orderly growth of enamel prisms.     

Aspects of the final phase of enamel formation as evidenced by observations of superficial enamel of human third molars using scanning electron microscopy

ObjectiveEnamel structure reflects ameloblast function. By studying the structure of the superficial enamel, information about ameloblast function toward the end of the secretory stage may be obtained.DesignThe superficial enamel in midcoronal areas of acid-etched facio-lingual sections from human third molars was studied in the scanning electron microscope (SEM).ResultsA great variation was observed in occurrence of prism-free enamel. Prism-free enamel dominated in 40% (mandibular) and 47% (maxillary) of observed areas and had a mean thickness of about 30 μm. Striations in the prism-free enamel had an interstriae distance of about 3.3–3.8 μm. The angle between prisms and enamel surface was about 60°, between prisms and Retzius lines about 45° and between Retzius lines and enamel surface about 15°. The distances between regularly occurring Retzius lines and between striations in the prism-free enamel both tended to decrease toward the enamel surface. Prisms could change direction as they approached the enamel surface, mostly in cervical direction. Where Retzius lines curved and converged occlusally, prisms tended to deviate in an occlusal direction.ConclusionsJudged from the incremental lines and occurrence of prism-free enamel, ameloblasts slow down and tend to lose their Tomes’ process as they approach the end of secretion. The crystals of prism-free enamel belong to the same system as the interprism crystals of prismatic enamel. A method, based on the disposition of fine incremental lines, is suggested for evaluation of ameloblast dynamics in the last stage of enamel secretion.     

Enamel and dental anomalies in latent‐transforming growth factor beta‐binding protein 3 mutant mice

Latent‐transforming growth factor beta‐binding protein 3 (LTBP‐3) is important for craniofacial morphogenesis and hard tissue mineralization, as it is essential for activation of transforming growth factor‐β (TGF‐β). To investigate the role of LTBP‐3 in tooth formation we performed micro‐computed tomography (micro‐CT), histology, and scanning electron microscopy analyses of adult Ltbp3‐/‐ mice. The Ltbp3‐/‐ mutants presented with unique craniofacial malformations and reductions in enamel formation that began at the matrix formation stage. Organization of maturation‐stage ameloblasts was severely disrupted. The lateral side of the incisor was affected most. Reduced enamel mineralization, modification of the enamel prism pattern, and enamel nodules were observed throughout the incisors, as revealed by scanning electron microscopy. Molar roots had internal irregular bulbous‐like formations. The cementum thickness was reduced, and microscopic dentinal tubules showed minor nanostructural changes. Thus, LTBP‐3 is required for ameloblast differentiation and for the formation of decussating enamel prisms, to prevent enamel nodule formation, and for proper root morphogenesis. Also, and consistent with the role of TGF‐β signaling during mineralization, almost all craniofacial bone components were affected in Ltbp3‐/‐ mice, especially those involving the upper jaw and snout. This mouse model demonstrates phenotypic overlap with Verloes Bourguignon syndrome, also caused by mutation of LTBP3, which is hallmarked by craniofacial anomalies and amelogenesis imperfecta phenotypes.     

Presence of enamel on the lingual surface of rabbit permanent incisors

Abstract – Permanent incisors from fetal New Zealand white rabbits aged 25–30 d in utero were processed for light microscopy. Examination of transverse and longitudinal sections of demineralized specimens revealed the development of a complete enamel covering of the incisal tip and an enamel extension on the linguoincisal surface of unerupted teeth, as evidenced by the presence and distribution of ameloblasts, enamel matrix and an enamel space. Formation of lingual enamel appeared to be completed within a few days before eruption of the incisor. The transition from the enamel-covered to enamel-free areas coincided with a gradual disintegration of the orderly ameloblast layer. The findings indicate that, histologically, the linguoincisal part of the rabbit permanent incisor is characterized by amelogenesis.     

Demonstration by staining and radioautography of cyclical distributions of protein at the enamel surface in rat incisors

Staining patterns in the enamel during the maturation stage of amelogenesis reflect the banded distribution of ruffle-ended and smooth-ended ameloblasts. This study investigated the possibility that proteins at the enamel surface may be distributed cyclically according to cyclical changes in ameloblast morphology. Dissected lower rat incisors were wiped free of their enamel organs and immediately immersed in fixative containing one of the following heavy metal and histological stains: uranyl acetate, lead citrate, Coomassie blue, alcian blue and ruthenium red. Other animals were injected with [35S]methionine to label newly-formed enamel proteins. Their incisors were dissected, the enamel organs were wiped from the enamel surface, and the teeth were processed as whole mounts for radioautography. Teeth stained by heavy metals were also viewed by back-scattered electron imaging. The in-situ staining revealed that proteins were distributed in bands and stripes across maturing enamel. Radioautography revealed that the proteins in the stripes were newly-synthesized and secreted into the enamel by certain maturation ameloblasts. We conclude that the enamel organ expresses cyclical activity in part through secretion of proteins.     

Composition of mineralizing incisor enamel in cystic fibrosis transmembrane conductance regulator‐deficient mice

Formation of crystals in the enamel space releases protons that need to be buffered to sustain mineral accretion. We hypothesized that apical cystic fibrosis transmembrane conductance regulator (CFTR) in maturation ameloblasts transduces chloride into forming enamel as a critical step to secrete bicarbonates. We tested this by determining the calcium, chloride, and fluoride levels in developing enamel of Cftr‐null mice by quantitative electron probe microanalysis. Maturation‐stage enamel from Cftr‐null mice contained less chloride and calcium than did wild‐type enamel, was more acidic when stained with pH dyes ex vivo, and formed no fluorescent modulation bands after in vivo injection of the mice with calcein. To acidify the enamel further we exposed Cftr‐null mice to fluoride in drinking water to stimulate proton release during formation of hypermineralized lines. In Cftr‐deficient mice, fluoride further lowered enamel calcium without further reducing chloride levels. The data support the view that apical CFTR in maturation ameloblasts tranduces chloride into developing enamel as part of the machinery to buffer protons released during mineral accretion.     

LPA6-RhoA signals regulate junctional complexes for polarity and morphology establishment of maturation stage ameloblasts

ObjectivesLysophosphatidic acid (LPA) is a potent bioactive phospholipid that exerts various functions upon binding to six known G protein-coupled receptors (LPA_1–6); however; its role in a tooth remains unclear. This study aimed to explore the impact of the LPA/LPA receptor 6 (LPA₆)/RhoA signaling axis on maturation stage ameloblasts (M-ABs), which are responsible for enamel mineralization.MethodsThe expression of LPA₆ and LPA-producing synthetic enzymes during ameloblast differentiation was explored through immunobiological analysis of mouse incisors and molars. To elucidate the role of LPA₆ in ameloblasts, incisors of LPA₆ KO mice were analyzed. In vitro experiments using ameloblast cell lines were performed to validate the function of LPA-LPA₆-RhoA signaling in ameloblasts.ResultsLPA₆ and LPA-producing enzymes were strongly expressed in M-ABs. In LPA₆ knockout mice, M-ABs exhibited abnormal morphology with the loss of cell polarity, and an abnormal enamel epithelium containing cyst-like structures was formed. Moreover, the expression of E-cadherin and zonula occludens-1 (ZO-1) significantly decreased in M-ABs. In vitro experiments demonstrated that LPA upregulated the expression of E-cadherin, ZO-1, and filamentous actin (F-actin) at the cellular membrane, whereas LPA₆ knockdown decreased their expression and changed cell morphology. Furthermore, we showed that RhoA signaling mediates LPA-LPA₆-induced junctional complexes.ConclusionsThis study demonstrated that LPA-LPA₆-RhoA signaling is essential for establishing proper cell morphology and polarity, via cell–cell junction and actin cytoskeleton expression and stability, of M-ABs. These results highlight the biological significance of bioactive lipids in a tooth, providing a novel molecular regulatory mechanism of ameloblasts.     

The ultrastructure of human enamel organs

The ultrastructural features of enamel organs from 3 human fetus were examined in this study. There were few and simple organelles in the inner enamel epithelium, this imply that these cells were not active ones. Comparing with the inner enamel epithelium, the ameloblasts showed active functional condition. They had a large amount of mitochondria, enlarged rough endoplasmic reticulum and well developed Golgi apparatus in the distal cytoplasm. On the top of the ameloblasts, Tomes' processes were formed. In the Tomes' processes, there were numerous vesicles containing "stippled material". This kind of material also could be seen in the spaces between the Tomes' processes and the enamel matrix, and in the vesicles of the Golgi apparatus. Ultrastructurally, each enamel matrix invagination received a large part of process of an ameloblast and a small part of another ameloblast. But the meaning of this structure were unclear. Tight junctions were found at the distal part of the ameloblasts. The stratum intermedium cells, beside the ameloblasts, had a lot of small processes protecting into the intercellular spaces. Gap junctions were found between all enamel organ cells, and illustrated that there were active material transmissions between these cells.     

Sheath proteins: synthesis,secretion, degradation and fate in forming enamel

We investigated expression of ameloblastin and sheathlin, recently cloned enamel matrix proteins from the rat and pig, in forming enamel immunocytochemically and immunochemically, using region-specific antibodies. The results obtained from the rat and pig were essentially the same. Antibodies which recognize the N-terminal region stained the secretory machinery of the secretory ameloblast and the entire thickness of the enamel matrix, especially the peripheral region of the enamel rod. Immunostained protein bands were observed near 65 or 70 kDa and below 20 kDa. C-terminal-specific antibodies stained the secretory machinery of the ameloblast and the immature enamel adjacent to the secretion sites. Immunostained protein bands were found ranging from 25 to 70 kDa. Antibodies which recognize a region in the protein just prior to the C-terminal region stained the cis-side of the Golgi apparatus but not the enamel matrix. Immunostained protein bands were observed of about 55 kDa. These results suggest that post-translational and post-secretory modifications of ameloblastin and sheathlin are similar to each other, and further showed that their cleaved N-terminal polypeptides concentrate in the prism sheath. We propose that sheathlin and ameloblastin share the same role in amelogenesis and should be classified as sheath proteins.     

Importance of bicarbonate transport in pH control during amelogenesis — need for functional studies

Dental enamel, the hardest mammalian tissue, is produced by ameloblasts. Ameloblasts show many similarities to other transporting epithelia although their secretory product, the enamel matrix, is quite different. Ameloblasts direct the formation of hydroxyapatite crystals, which liberate large quantities of protons that then need to be buffered to allow mineralization to proceed. Buffering requires a tight pH regulation and secretion of bicarbonate by ameloblasts. Many investigations have used immunohistochemical and knockout studies to determine the effects of these genes on enamel formation, but up till recently very little functional data were available for mineral ion transport. To address this, we developed a novel 2D in vitro model using HAT‐7 ameloblast cells. HAT‐7 cells can be polarized and develop functional tight junctions. Furthermore, they are able to accumulate bicarbonate ions from the basolateral to the apical fluid spaces. We propose that in the future, the HAT‐7 2D system along with similar cellular models will be useful to functionally model ion transport processes during amelogenesis. Additionally, we also suggest that similar approaches will allow a better understanding of the regulation of the cycling process in maturation‐stage ameloblasts, and the pH sensory mechanisms, which are required to develop sound, healthy enamel.     

Incremental lines in mouse molar enamel

ObjectiveThe purpose of the present study was to investigate the occurrence and periodicity of enamel incremental lines in mouse molars in an attempt to draw attention to some key questions about the rhythm in the activity of the secreting ameloblasts during formation of mouse molar enamel.MethodsThe mouse molars were ground, etched, and studied using scanning electron microscopy.ResultsLines interpreted as incremental lines generally appeared as grooves of variable distinctness, and were only observed cervically, in the region about 50–250 μm from the enamel–cementum junction. The lines were most readily observable in the outer enamel and in the superficial prism-free layer, and were difficult to identify in the deeper parts of enamel, i.e. in the inner enamel with prism decussation. However, in areas where the enamel tended to be hypomineralized the incremental lines were observed as clearly continuous from outer into inner enamel. The incremental lines in mouse molar enamel exhibited an average periodicity of about 4 μm, and the distance between the lines decreased towards the enamel surface.ConclusionsWe conclude that incremental lines are to some extent visible in mouse molar enamel. Together with data from the literature and theoretical considerations, we suggest that they probably represent a daily rhythm in enamel formation. This study witnesses the layered apposition of mouse molar enamel and supports the theory that circadian clock probably regulates enamel development.