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Neurite outgrowth inhibitor A (Nogo-A) is a major player in neural development and regeneration and the target of clinical trials aiming at promoting the regeneration of the central nervous system upon traumatic and ischemic injury. In this work, we investigated the functions of Nogo-A during tooth development to determine its role in dental physiology and pathology. Using immunohistochemistry and in situ hybridization techniques, we showed that Nogo-A is highly expressed in the developing mouse teeth and, most specifically, in the ameloblasts that are responsible for the formation of enamel. Using both Nogo-A knockout and K14-Cre;Nogo-A fl/fl transgenic mice, we showed that Nogo-A deletion in the dental epithelium leads to the formation of defective enamel. This phenotype is associated with overexpression of a set of specific genes involved in ameloblast differentiation and enamel matrix production, such as amelogenin, ameloblastin and enamelin. By characterising the interactome of Nogo-A in the dental epithelium of wild-type and mutant animals, we found that Nogo-A directly interacts with molecules important for regulating gene expression, and its deletion disturbs their cellular localisation. Furthermore, we demonstrated that inhibition of the intracellular, but not cell-surface, Nogo-A is responsible for gene expression modulation in ameloblasts. Taken together, these results reveal an unexpected function for Nogo-A in tooth enamel formation by regulating gene expression and cytodifferentiation events., (© 2024. The Author(s).)

Objective: Our aim was to evaluate by enamel microstructure analysis two hypotheses that would explain the early dental eruption in the Bakaparticularity, a shorter crown formation time and/or earlier onset of crown formation., Design: Deciduous canines corresponds to the best teeth to perform the analysis of enamel microstructure. Longitudinal ground sections of 21 deciduous canines from 12 individuals were studied with transmitted light microscopy. Cross-striations, striaes of Retzius (SR) and the neonatal line (NNL) enable to establish the prenatal crown formation time (preCFT), the postnatal crown formation time (postCFT), the crown formation time (CFT) as well as the daily secretion rate (DSR) and the enamel extension rate (EER) and their variation along crown formation., Results: The DSR and the EER in the Baka are similar than in other populations with an average DSR of 3.26 µm and EER of 18.18 µm. The preCFT was 154 days, the postCFT 265 days and CFT 419 days. Comparison with other population does not show difference in CFT. However, the preCFT and the postCFT differ, the first is higher and the second lower in the Baka than in other populations. Furthermore, the number of prenatal areas of enamel was greater in the Baka., Conclusion: Our analysis suggests that the Baka does not distinguish by a different CFT but the onset of crown formation is earlier than in other groups. Therefore, the early dental eruption in the Baka results from an earlier onset of crown formation., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Circadian rhythms are self-sustaining oscillations within biological systems that play key roles in a diverse multitude of physiological processes. The circadian clock mechanisms in brain and peripheral tissues can oscillate independently or be synchronized/disrupted by external stimuli. Dental enamel is a type of mineralized tissue that forms the exterior surface of the tooth crown. Incremental Retzius lines are readily observable microstructures of mature tooth enamel that indicate the regulation of amelogenesis by circadian rhythms. Teeth enamel is formed by enamel-forming cells known as ameloblasts, which are regulated and orchestrated by the circadian clock during amelogenesis. This review will first examine the key roles of the circadian clock in regulating ameloblasts and amelogenesis. Several physiological processes are involved, including gene expression, cell morphology, metabolic changes, matrix deposition, ion transportation, and mineralization. Next, the potential detrimental effects of circadian rhythm disruption on enamel formation are discussed. Circadian rhythm disruption can directly lead to Enamel Hypoplasia, which might also be a potential causative mechanism of amelogenesis imperfecta. Finally, future research trajectory in this field is extrapolated. It is hoped that this review will inspire more intensive research efforts and provide relevant cues in formulating novel therapeutic strategies for preventing tooth enamel developmental abnormalities., (© 2024. The Author(s).)

At maturation stage of enamel development, a specialized basal lamina (sBL) was built between ameloblasts and enamel. After the teeth eruption, the ameloblasts transform into the inner cell layer of junctional epithelium. The inner cell layer forms the internal basal lamina of junctional epithelium. However, the composition of the sBL and internal basal lamina was not clarified. The objective of our study was to make a description of the localization of amelotin (AMTN), laminin γ2 (LAMC2) and Odontogenesis-associated phosphoprotein (ODAPH) on the sBL and internal basal lamina. In immunohistochemical study, AMTN, LAMC2 and ODAPH were detected on the sBL at maturation stage. AMTN was also detected in ameloblasts at maturation stage. The expression of AMTN decreased from early-to-late maturation stage. In contrast, the expression of LAMC2 and ODAPH was stable. Immunofluorescence double-staining showed the localization of AMTN was close to enamel surface. However, the localization of ODAPH was close to ameloblasts. LAMC2 and ODAPH were observed on internal basal lamina of junctional epithelium. In contrast, no expression of AMTN was detected on internal basal lamina of junctional epithelium. Our results suggested that ODAPH might participate in enamel maturation and periodontal health, which might provide a better understanding of enamel defects and periodontal disease in clinic., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The aim of the study was to see whether the length of the enamel secretion zone in unimpeded rat incisors, measured precisely, is in agreement with the observed decrease in enamel thickness. Unimpeded eruption of mandibular incisors of five experimental and two control rats was induced by cutting off the erupted part of the incisors three times per week for 5 weeks. The length of the zone of enamel secretion in unimpeded and impeded control incisors was measured on longitudinal and serial transverse histological sections of fixed, demineralised and embedded hemimandibles. Impeded contralateral incisors were also included in the study. The length of the zone of enamel secretion in unimpeded incisors showed an increase to 8,398 ± 558 µm, that is 161% of the length in control incisors (5,213 ± 95 µm). The contralateral incisor showed a reduction in eruption rate, in length of the secretion zone, and the whole tooth was shifted somewhat apically. The measured length of the secretion zone is in agreement with the observed thickness of enamel (98 µm) in unimpeded incisors. The reduced eruption rate and the apical shift of the contralateral incisor are probably due to an increased occlusal load., (© 2020 The Authors. Anatomia, Histologia, Embryologia published by Blackwell Verlag GmbH.)

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., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.)

Enamel is a highly calcified tissue. Its formation requires a progressive and dynamic system for the regulation of electrolyte concentration by enamel epithelia. A critical function of enamel epithelial cells, ameloblasts, is the secretion and movement of electrolytes via various channels and transporters to develop the enamel tissue. Enamel formation generates protons, which need to be neutralised. Thus, ameloblasts possess a buffering system to sustain mineral accretion. Normal tooth formation involves stage-dependent net fluctuations in pH during amelogenesis. To date, all of our information about ion transporters in dental enamel tissue is based solely on immunostaining-expression techniques. This review critically evaluates the current understanding and recent discoveries and physiological role of ion channels and transporters, Mg 2+ transporters, and Ca 2+ regulatory proteins during amelogenesis in enamel formation. The ways in which ameloblasts modulate ions are discussed in the context of current research for developing a novel morphologic-functional model of enamel maturation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Ameloblasts responsible for enamel formation express matrix metalloproteinase 20 (MMP20), an enzyme that cleaves enamel matrix proteins, including amelogenin (AMELX) and ameloblastin (AMBN). Previously, we showed that continuously erupting incisors from transgenic mice overexpressing active MMP20 had a massive cell infiltrate present within their enamel space, leading to enamel mineralization defects. However, effects of MMP20 overexpression on mouse molars were not analyzed, although these teeth more accurately represent human odontogenesis. Therefore, MMP20-overexpressing mice ( Mmp20 +/+ Tg + ) were assessed by multiscale analyses, combining several approaches from high-resolution micro-computed tomography to enamel organ immunoblots. During the secretory stage at postnatal day 6 (P6), Mmp20 +/+ Tg + mice had a discontinuous ameloblast layer and, unlike incisors, molar P12 maturation stage ameloblasts abnormally migrated away from the enamel layer into the stratum intermedium/stellate reticulum. TOPflash assays performed in vitro demonstrated that MMP20 expression promoted β-catenin nuclear localization and that MMP20 expression promoted invasion through Matrigel-coated filters. However, for both assays, significant differences were eliminated in the presence of the β-catenin inhibitor ICG-001. This suggests that MMP20 activity promotes cell migration via the Wnt pathway. In vivo, the unique molar migration of amelogenin-expressing ameloblasts was associated with abnormal deposition of ectopic calcified nodules surrounding the adherent enamel layer. Enamel content was assessed just prior to eruption at P15. Compared to wild-type, Mmp20 +/+ Tg + molars exhibited significant reductions in enamel thickness (70%), volume (60%), and mineral density (40%), and MMP20 overexpression resulted in premature cleavage of AMBN, which likely contributed to the severe defects in enamel mineralization. In addition, Mmp20 +/+ Tg + mouse molar enamel organs had increased levels of inactive p-cofilin, a protein that regulates cell polarity. These data demonstrate that increased MMP20 activity in molars causes premature degradation of ameloblastin and inactivation of cofilin, which may contribute to pathological Wnt-mediated cell migration away from the enamel layer.

FAM83H was identified as the major causative gene for autosomal dominant hypocalcified amelogenesis imperfect (ADHCAI). The pathogenic mechanism of FAM83H in ADHCAI remains elusive. The present study aims to investigate the effect of Fam83h mutation on the mineralization of mouse ameloblast cell line LS8 and to explore the possible pathogenesis of ADHCAI. Lentivirus package was performed for the plasmids with mouse Fam83h mutant cDNA (c.1186C > T, M3) and empty vector (Control) and transfected into LS8, which were divided into M3-FLAG and Control groups. Immunoprecipitation, western-blot and immunofluorescence were performed to detect the expression and subcellular localization of Fam83 h, CK1α and β-catenin. ALP activity, ALP staining, expression of the mineralization factors were detected in two groups during mineralization induction. Expression of the mineralization factors was also detected in M3-FLAG and LS8 exposing to pyrvinium pamoate. Compared with the Control, the Fam83h mutation altered the expression and localization of Fam83 h, CK1α and β-catenin in LS8, inhibited the mineralization and down-regulated the expression of mineralization factors in M3-FLAG. Pyrvinium pamoate, an inhibitor of the Wnt/β-catenin signaling pathway, up-regulated expression of mineralization factors in LS8 and rescued the inhibited mineralization in M3-FLAG. The results indicated that the Fam83h mutation could inhibit the mineralization in ameloblasts by activating Wnt/β-catenin signaling pathway., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Transient receptor potential melastatin-7 (TRPM7) is a bi-functional protein containing a kinase domain fused to an ion channel. TRPM7 is highly expressed in ameloblasts during tooth development. Here we show that TRPM7 kinase-inactive knock-in mutant mice (TRPM7 KR mice) exhibited small enamel volume with opaque white-colored incisors. The TRPM7 channel function of ameloblast-lineage cells from TRPM7 KR mice was normal. Interestingly, phosphorylation of intracellular molecules including Smad1/5/9, p38 and cAMP response element binding protein (CREB) was inhibited in ameloblasts from TRPM7 KR mice at the pre-secretory stage. An immunoprecipitation assay showed that CREB was bound to TRPM7, suggesting that direct phosphorylation of CREB by TRPM7 was inhibited in ameloblast-lineage cells from TRPM7 KR mice. These results indicate that the function of the TRPM7 kinase domain plays an important role in ameloblast differentiation, independent of TRPM7 channel activity, via phosphorylation of CREB.

Enamelin (ENAM) has three putative phosphoserines (pSers) phosphorylated by a Golgi-associated secretory pathway kinase (FAM20C) based on their distinctive Ser-x-Glu (S-x-E) motifs. Fam20C-knockout mice show severe enamel defects similar to those in the Enam-knockout mice, implying an important role of the pSers in ENAM. To determine the role of pSer 55 in ENAM, we characterized ENAM Rgsc514 mice, in which Ser 55 cannot be phosphorylated by FAM20C due to an E 57 >G 57 mutation in the S-x-E motif. The enamel microstructure of 4-week-old mice was examined by scanning electron microscopy. The teeth of 6-day-old mice were characterized by histology and immunohistochemistry. The protein lysates of the first lower molars of 4-day-old mice were analyzed by Western immunoblotting using antibodies against ENAM, ameloblastin and amelogenin. ENAM Rgsc514 heterozygotes showed a disorganized enamel microstructure, while the homozygotes had no enamel on the dentin surface. The N-terminal fragments of ENAM in the heterozygotes were detained in the ameloblasts and localized in the mineralization front of enamel matrix, while those in the WT mice were secreted out of ameloblasts and distributed evenly in the outer 1/2 of enamel matrix. Surprisingly, the ~15  kDa C-terminal fragments of ameloblastin were not detected in the molar lysates of the homozygotes. These results suggest that the phosphorylation of Ser 55 may be an essential posttranslational modification of ENAM and is required for the interaction with other enamel matrix molecules such as ameloblastin in mediating the structural organization of enamel matrix and protein-mineral interactions during enamel formation.International Journal of Oral Science (2017) 9;e4; doi:10.1038/ijos.2017.41; published online 29 November 2017.

Lanzhousaurus magnidens, a large non-hadrosauriform iguanodontian dinosaur from the Lower Cretaceous Hekou Group of Gansu Province, China has the largest known herbivorous dinosaur teeth. Unlike its hadrosauriform relatives possessing tooth batteries of many small teeth, Lanzhousaurus utilized a small number (14) of very large teeth (~10 cm long) to create a large, continuous surface for mastication. Here we investigate the significance of Lanzhousaurus in the evolutionary history of iguanodontian-hadrosauriform transition by using a combination of stable isotope analysis and CT imagery. We infer that Lanzhousaurus had a rapid rate of tooth enamel elongation or amelogenesis at 0.24 mm/day with dental tissues common to other Iguanodontian dinosaurs. Among ornithopods, high rates of amelogenesis have been previously observed in hadrosaurids, where they have been associated with a sophisticated masticatory apparatus. These data suggest rapid amelogenesis evolved among non-hadrosauriform iguanodontians such as Lanzhousaurus, representing a crucial step that was exapted for the evolution of the hadrosaurian feeding mechanism.

The role of Ras signaling during tooth development is poorly understood. Ras proteins-which are activated by many upstream pathways, including receptor tyrosine kinase cascades-signal through multiple effectors, such as the mitogen-activated protein kinase (MAPK) and PI3K pathways. Here, we utilized the mouse incisor as a model to study how the MAPK and PI3K pathways regulate dental epithelial stem cells and amelogenesis. The rodent incisor-which grows continuously throughout the life of the animal due to the presence of epithelial and mesenchymal stem cells-provides a model for the study of ectodermal organ renewal and regeneration. Utilizing models of Ras dysregulation as well as inhibitors of the MAPK and PI3K pathways, we found that MAPK and PI3K regulate dental epithelial stem cell activity, transit-amplifying cell proliferation, and enamel formation in the mouse incisor.

Hypomineralization of developing enamel is associated with changes in ameloblast modulation during the maturation stage. Modulation (or pH cycling) involves the cyclic transformation of ruffle-ended (RE) ameloblasts facing slightly acidic enamel into smooth-ended (SE) ameloblasts near pH-neutral enamel. The mechanism of ameloblast modulation is not clear. Failure of ameloblasts of Cftr-null and anion exchanger 2 ( Ae2)-null mice to transport Cl - into enamel acidifies enamel, prevents modulation, and reduces mineralization. It suggests that pH regulation is critical for modulation and for completion of enamel mineralization. This report presents a review of the major types of transmembrane molecules that ameloblasts express to transport calcium to form crystals and bicarbonates to regulate pH. The type of transporter depends on the developmental stage. Modulation is proposed to be driven by the pH of enamel fluid and the compositional and/or physicochemical changes that result from increased acidity, which may turn RE ameloblasts into SE mode. Amelogenins delay outgrowth of crystals and keep the intercrystalline space open for diffusion of mineral ions into complete depth of enamel. Modulation enables stepwise removal of amelogenins from the crystal surface, their degradation, and removal from the enamel. Removal of matrix allows slow expansion of crystals. Modulation also reduces the stress that ameloblasts experience when exposed to high acid levels generated by mineral formation or by increased intracellular Ca 2+ . By cyclically interrupting Ca 2+ transport by RE ameloblasts and their transformation into SE ameloblasts, proton production ceases shortly and enables the ameloblasts to recover. Modulation also improves enamel crystal quality by selectively dissolving immature Ca 2+ -poor crystals, removing impurities as Mg 2+ and carbonates, and recrystallizing into more acid-resistant crystals.

Exposure of forming enamel to fluoride results into formation of hypomineralized enamel. We tested whether enamel hypomineralization was caused by lower expression of the NCKX4/SLC24A4 Ca 2+ -transporter by ameloblasts. Three commercial antibodies against NCKX4 were tested on enamel organs of wild-type and Nckx4-null mice, one of which (a mouse monoclonal) was specific. This antibody gave a prominent staining of the apical plasma membranes of maturation ameloblasts, starting at early maturation. The layer of immuno-positive ameloblasts contained narrow gaps without immunostaining or with reduced staining. In fluorotic mouse incisors, the quantity of NCKX4 protein in ameloblasts as assessed by western blotting was not different from that in non-fluorotic ameloblasts. However, immunostaining of the apical plasma membranes of fluorotic ameloblasts was strongly reduced or absent suggesting that trafficking of NCKX4 to the apical membrane was strongly reduced. Exposure to fluoride may reduce NCKX4-mediated transport of Ca 2+ by maturation stage ameloblasts which delays ameloblast modulation and reduces enamel mineralization., Competing Interests: A. L. J. J. Bronckers, R. Jalali, J. Lytton declare that they have no conflict of interest. Human and Animal Rights and Informed Consent The authors confirm that the animal studies were approved and carried out according to national guidelines.

Recent studies have shown that native phosphorylated full-length porcine amelogenin (P173) and its predominant cleavage product (P148) can inhibit spontaneous calcium phosphate formation in vitro by stabilizing an amorphous calcium phosphate (ACP) precursor phase. Since full-length amelogenin undergoes proteolysis by matrix metalloproteinase 20 (MMP20, enamelysin) soon after secretion, the present study was conducted to assess the effect of amelogenin proteolysis on calcium phosphate formation. Calcium and phosphate were sequentially added to protein solutions without and with added MMP20 (ratio = 200:1) under physiological-like conditions of ionic strength (163 mM) in 50 mM Tris-HCl (pH 7.4) at 37 °C. Protein degradation with time was assessed by gel-electrophoresis, and mineral products formed were characterized by transmission electron microscopy (TEM). MMP20 was found to cleave P173 to primarily generate P148, along with P162, P46-148, and P63/64-148. In sharp contrast, MMP20 did not cleave P148. In addition, the formation of well-aligned bundles of enamel-like hydroxyapatite (HA) crystals was promoted in the presence of P173 with added MMP20, while only ACP particles were seen in the absence of MMP20. Although P148 was found to have a somewhat lower capacity to stabilize ACP and prevent HA formation compared with P173 in the absence of MMP20, essentially no HA formation was observed in the presence of somewhat higher concentrations of P148 regardless of MMP20 addition, due to the lack of observed protein proteolysis. Present findings suggest that ACP transformation to ordered arrays of enamel crystals may be regulated in part by the proteolysis of full-length native amelogenin, while the predominant amelogenin degradation product in developing enamel (e.g., P148) primarily serves to prevent uncontrolled mineral formation during the secretory stage of amelogenesis., Competing Interests: The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article., (© International & American Associations for Dental Research 2016.)

FAM20C is a newly identified kinase on the secretory pathway responsible for the phosphorylation of serine residues in the Ser-x-Glu/pSer motifs in several enamel matrix proteins. Fam20C-knockout mice showed severe enamel defects very similar to those in the ameloblastin ( Ambn)-knockout mice, implying that phosphoserines may have a critical role in AMBN function. To test this hypothesis, we generated amelogenin ( Amel) promoter-driven Ambn-transgenic mice, in which Ser 48 , Ser 226 , and Ser 227 were replaced by aspartic acid (designated as D-Tg) or alanines (designated as A-Tg). The negative charge of aspartic acid is believed to be able to mimic the phosphorylation state of serine, while alanine is a commonly used residue to substitute serine due to their similar structure. Using Western immunoblotting and quantitative polymerase chain reaction, the authors identified transgenic lines expressing transgenes somewhat higher (Tg+) or much higher (Tg++) than endogenous Ambn. The lower incisors collected from 7-d-old and 7-wk-old mice were analyzed by histology, scanning electron microscopy, immunohistochemistry, and Western immunoblotting to examine the morphology and microstructure changes in enamel, as well as the expression pattern of enamel matrix proteins. The A-Tg+ and A-Tg++ mice displayed severe enamel defects in spite of the expression level of transgenes, while the D-Tg+ and D-Tg++ mice showed minor to mild enamel defects, indicating that the D-Tg transgenes disturbed enamel formation less than the A-Tg transgenes did. Our results suggest that the phosphorylation state of serines is likely an essential component for the integrity of AMBN function., Competing Interests: The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

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., (© 2016 Eur J Oral Sci.)

Human dental enamel, the hardest tissue in the body, plays a vital role in protecting teeth from wear as a result of daily grinding and chewing as well as from chemical attack. It is well established that the mechanical strength and fatigue resistance of dental enamel are derived from its hierarchical structure, which consists of periodically arranged bundles of hydroxyapatite (HAP) nanowires. However, we do not yet have a full understanding of the in vivo HAP crystallization process that leads to this structure. Mg(2+) ions, which are present in many biological systems, regulate HAP crystallization by stabilizing its precursor, amorphous calcium phosphate (ACP), but their atomic-scale distribution within HAP is unknown. We use atom probe tomography to provide the first direct observations of an intergranular Mg-rich ACP phase between the HAP nanowires in mature human dental enamel. We also observe Mg-rich elongated precipitates and pockets of organic material among the HAP nanowires. These observations support the postclassical theory of amelogenesis (that is, enamel formation) and suggest that decay occurs via dissolution of the intergranular phase. This information is also useful for the development of more accurate models to describe the mechanical behavior of teeth.

Introduction: Our aim in this study was to investigate the association between dental mineralization stages and the periods of the pubertal growth spurt (PGS)., Methods: The sample included panoramic and hand-wrist radiographs from 491 subjects (222 boys, 269 girls) aged 7 to 17 years. Dental development was rated, and skeletal maturation was evaluated. The relevant associations were investigated by analysis of ordinal multinomial logistic regression., Results: The second molar (odds ratio [OR] = 4.34) and the first premolar (OR = 2.45) were the best growth predictors for girls. For boys, the second molar (OR = 6.80), second premolar (OR = 2.41), and canine (OR = 3.21) proved to be the best predictors. Stages D and E of the second molar for girls, and stages E and F for boys, corresponded to the onset of the accelerated growth spurt. Stage F of the second molar for girls and stage G for boys corresponded to the peak of the PGS. At the end of the PGS, most teeth had already attained apical closure. In girls, however, most second molars were found at stage G., Conclusions: An association exists between the dental mineralization stages and the periods of the PGS, especially for second molars. Panoramic radiographs can be used as the first diagnostic tool to estimate the pubertal growth period., (Copyright © 2016 American Association of Orthodontists. Published by Elsevier Inc. All rights reserved.)

Ameloblasts express transmembrane proteins for transport of mineral ions and regulation of pH in the enamel space. Two major transporters recently identified in ameloblasts are the Na(+)K(+)-dependent calcium transporter NCKX4 and the Na(+)-dependent HPO4 (2-) (Pi) cotransporter NaPi-2b. To regulate pH, ameloblasts express anion exchanger 2 (Ae2a,b), chloride channel Cftr, and amelogenins that can bind protons. Exposure to fluoride or null mutation of Cftr, Ae2a,b, or Amelx each results in formation of hypomineralized enamel. We hypothesized that enamel hypomineralization associated with disturbed pH regulation results from reduced ion transport by NCKX4 and NaPi-2b. This was tested by correlation analyses among the levels of Ca, Pi, Cl, Na, and K in forming enamel of mice with null mutation of Cftr, Ae2a,b, and Amelx, according to quantitative x-ray electron probe microanalysis. Immunohistochemistry, polymerase chain reaction analysis, and Western blotting confirmed the presence of apical NaPi-2b and Nckx4 in maturation-stage ameloblasts. In wild-type mice, K levels in enamel were negatively correlated with Ca and Cl but less negatively or even positively in fluorotic enamel. Na did not correlate with P or Ca in enamel of wild-type mice but showed strong positive correlation in fluorotic and nonfluorotic Ae2a,b- and Cftr-null enamel. In hypomineralizing enamel of all models tested, 1) Cl(-) was strongly reduced; 2) K(+) and Na(+) accumulated (Na(+) not in Amelx-null enamel); and 3) modulation was delayed or blocked. These results suggest that a Na(+)K(+)-dependent calcium transporter (likely NCKX4) and a Na(+)-dependent Pi transporter (potentially NaPi-2b) located in ruffle-ended ameloblasts operate in a coordinated way with the pH-regulating machinery to transport Ca(2+), Pi, and bicarbonate into maturation-stage enamel. Acidification and/or associated physicochemical/electrochemical changes in ion levels in enamel fluid near the apical ameloblast membrane may reduce the transport activity of mineral transporters, which results in hypomineralization., (© International & American Associations for Dental Research 2015.)

Objective: The permanently growing mouse incisors exhibit all stages of tooth development along their inciso-apical axis at any time. Any disturbance or injury of the ameloblasts during enamel formation or maturation may result in permanent defects in the finished enamel since the enamel does not undergo repair or remodeling after formation. In order to increase our understanding of how hypoxia affects enamel formation, we induced severe acute hypoxia in adult mice and observed its effects on the enamel in incisors., Design: Incisors from hypoxic mice were obtained 5 and 49 days after the hypoxic insult. Hypoxic and control incisors were dissected out and observed by scanning electron microscopy (SEM). Incisors were subsequently ground longitudinally or transversely, etched, and observed again by SEM. The nature and position of defects were considered in relation to the configuration and dynamics of the incisors., Results: The effect of hypoxia varied considerably, among mice, among incisors, and among ameloblasts. Affected enamel showed hypoplasia with hypomineralization or hypomineralization without hypoplasia. Vascular endothelial growth factor (VEGF) showed considerably stronger labeling in hypoxic compared to control ameloblasts., Conclusions: The present study demonstrates quantitative and qualitative defects in the enamel reflecting the vulnerability of ameloblasts toward severe acute hypoxia in mouse incisors., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The hardest mammalian tissue, dental enamel is produced by ameloblasts, which are electrolyte-transporting epithelial cells. Although the end product is very different, they show many similarities to transporting epithelia of the pancreas, salivary glands and kidney. Enamel is produced in a multi-step epithelial secretory process that features biomineralization which is an interplay of secreted ameloblast specific proteins and the time-specific transport of minerals, protons and bicarbonate. First, "secretory" ameloblasts form the entire thickness of the enamel layer, but with low mineral content. Then they differentiate into "maturation" ameloblasts, which remove organic matrix from the enamel and in turn further build up hydroxyapatite crystals. The protons generated by hydroxyapatite formation need to be buffered, otherwise enamel will not attain full mineralization. Buffering requires a tight pH regulation and secretion of bicarbonate by ameloblasts. The whole process has been the focus of many immunohistochemical and gene knock-out studies, but, perhaps surprisingly, no functional data existed for mineral ion transport by ameloblasts. However, recent studies including ours provided a better insight for molecular mechanism of mineral formation. The secretory regulation is not completely known as yet, but its significance is crucial. Impairing regulation retards or prevents completion of enamel mineralization and results in the development of hypomineralized enamel that easily erodes after dental eruption. Factors that impair this function are fluoride and disruption of pH regulators. Revealing these factors may eventually lead to the treatment of enamel hypomineralization related to genetic or environmentally induced malformation., (Copyright © 2015 IAP and EPC. Published by Elsevier B.V. All rights reserved.)

Enamel is unique. It is the only epithelial-derived mineralized tissue in mammals and has a distinct micro- and nanostructure with nanofibrous apatite crystals as building blocks. It is synthesized by a highly specialized cell, the ameloblast, which secretes matrix proteins with little homology to any other known amino acid sequence, but which is composed of a primary structure that makes it competent to self-assemble and control apatite crystal growth at the nanometer scale. The end-product of ameloblast activity is a marvel of structural engineering: a material optimized to provide the tooth with maximum biting force, withstanding millions of cycles of loads without catastrophic failure, while also protecting the dental pulp from bacterial attack. This review attempts to bring into context the mechanical behavior of enamel with the developmental process of amelogenesis and structural development, since they are linked to tissue function, and the importance of controlling calcium phosphate mineralization at the nanometer scale. The origins of apatite nanofibers, the development of a stiffness gradient, and the biological processes responsible for the synthesis of a hard and fracture-resistant dental tissue are discussed with reference to the evolution of enamel from a fibrous composite to a complex, tough, and damage-tolerant coating on dentin., (© International & American Associations for Dental Research 2015.)

Enamel is a unique tissue in vertebrates, acellular, formed on a labile scaffolding matrix and hypermineralized. The ameloblasts are epithelial cells in charge of amelogenesis. They secrete a number of matrix proteins degraded by enzymes during enamel mineralization. This ordered cellular and extracellular events imply that any genetic or environmental perturbation will produce indelible and recognizable defects. The specificity of defects will indicate the affected cellular process. Thus, depending on the specificity of alterations, the teratogenic event can be retrospectively established. Advances in the field allow to use enamel defects as diagnostic tools for molecular disorders. The multifunctionality of enamel peptides is presently identified from their chemical roles in mineralization to cell signaling, constituting a source of concrete innovations in regenerative medicine., (© 2015 médecine/sciences – Inserm.)

Amelogenins are the most abundant protein species in forming dental enamel, taken to regulate crystal shape and crystal growth. Unprotonated amelogenins can bind protons, suggesting that amelogenins could regulate the pH in enamel in situ. We hypothesized that without amelogenins the enamel would acidify unless ameloblasts were buffered by alternative ways. To investigate this, we measured the mineral and chloride content in incisor enamel of amelogenin-knockout (AmelX(-/-)) mice and determined the pH of enamel by staining with methyl-red. Ameloblasts were immunostained for anion exchanger-2 (Ae2), a transmembrane pH regulator sensitive for acid that secretes bicarbonate in exchange for chloride. The enamel of AmelX(-/-) mice was 10-fold thinner, mineralized in the secretory stage 1.8-fold more than wild-type enamel and containing less chloride (suggesting more bicarbonate secretion). Enamel of AmelX(-/-) mice stained with methyl-red contained no acidic bands in the maturation stage as seen in wild-type enamel. Secretory ameloblasts of AmelX(-/-) mice, but not wild-type mice, were immunopositive for Ae2, and stained more intensely in the maturation stage compared with wild-type mice. Exposure of AmelX(-/-) mice to fluoride enhanced the mineral content in the secretory stage, lowered chloride, and intensified Ae2 immunostaining in the enamel organ in comparison with non-fluorotic mutant teeth. The results suggest that unprotonated amelogenins may regulate the pH of forming enamel in situ. Without amelogenins, Ae2 could compensate for the pH drop associated with crystal formation., (© International & American Associations for Dental Research 2014.)

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., (© 2014 Eur J Oral Sci.)

Although a big deal of dental research is being focused to the understanding of early stages of tooth development, a huge gap exist on our knowledge on how the dental hard tissues are formed and how this process is controlled daily in order to produce very complex and diverse tooth shapes adapted for specific functions. Emerging evidence suggests that clock genes, a family of genes that controls circadian functions within our bodies, regulate also dental mineralized tissues formation. Enamel formation, for example, is subjected to rhythmical molecular signals that occur on short (24h) periods and control the secretion and maturation of the enamel matrix. Accordingly, gene expression and ameloblast functions are also tightly modulated in regular daily intervals. This review summarizes the current knowledge on the circadian controls of dental mineralized tissues development with a special emphasis on amelogenesis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Dental enamel formation depends upon the transcellular transport of Ca(2+) by ameloblasts, but little is known about the molecular mechanism, or even if the same process is operative during the secretory and maturation stages of amelogenesis. Identifying mutations in genes involved in Ca(2+) homeostasis that cause inherited enamel defects can provide insights into the molecular participants and potential mechanisms of Ca(2+) handling by ameloblasts. Stromal Interaction Molecule 1 (STIM1) is an ER transmembrane protein that activates membrane-specific Ca(2+) influx in response to the depletion of ER Ca(2+) stores. Solute carrier family 24, member 4 (SLC24A4), is a Na(+)/K(+)/Ca(2+) transporter that exchanges intracellular Ca(2+) and K(+) for extracellular Na(+). We identified a proband with syndromic hypomaturation enamel defects caused by a homozygous C to T transition (g.232598C>T c.1276C>T p.Arg426Cys) in STIM1, and a proband with isolated hypomaturation enamel defects caused by a homozygous C to T transition (g.124552C>T; c.437C>T; p.Ala146Val) in SLC24A4. Immunohistochemistry of developing mouse molars and incisors showed positive STIM1 and SLC24A4 signal specifically in maturation-stage ameloblasts. We conclude that enamel maturation is dependent upon STIM1 and SLC24A4 function, and that there are important differences in the Ca(2+) transcellular transport systems used by secretory- and maturation-stage ameloblasts., (© International & American Associations for Dental Research.)

Although most fish have no enamel layer in their teeth, those belonging to Lepisosteus (gars), an extant actinopterygian fish genus, do and so can be used to study amelogenesis. In order to examine the collar enamel matrix in gar teeth, we subjected gar teeth to light and electron microscopic immunohistochemical examinations using an antibody against bovine amelogenin (27 kDa) and antiserum against porcine amelogenin (25 kDa), as well as region-specific antibodies and antiserum against the C-terminus and middle region, and N-terminus of porcine amelogenin, respectively. The enamel matrix exhibited intense immunoreactivity to the anti-bovine amelogenin antibody and the anti-porcine amelogenin antiserum in addition to the C-terminal and middle region-specific antibodies, but not to the N-terminal-specific antiserum. These results suggest that the collar enamel matrix of gar teeth contains amelogenin-like proteins and that these proteins possess domains that closely resemble the C-terminal and middle regions of porcine amelogenin. Western blot analyses of the tooth germs of Lepisosteus were also performed. As a result, protein bands with molecular weights of 78 kDa and 65 kDa were clearly stained by the anti-bovine amelogenin antibody as well as the antiserum against porcine amelogenin and the middle-region-specific antibody. It is likely that the amelogenin-like proteins present in Lepisosteus do not correspond to the amelogenins found in mammals, although they do possess domains that are shared with mammalian amelogenins.

Enamel and enameloid, the highly mineralized tooth-covering tissues in living vertebrates, are different in their matrix composition. Enamel, a unique product of ameloblasts, principally contains enamel matrix proteins (EMPs), while enameloid possesses collagen fibrils and probably receives contributions from both odontoblasts and ameloblasts. Here we focused on type I collagen (COL1A1) and amelogenin (AMEL) gene expression during enameloid and enamel formation throughout ontogeny in the caudate amphibian, Pleurodeles waltl. In this model, pre-metamorphic teeth possess enameloid and enamel, while post-metamorphic teeth possess enamel only. In first-generation teeth, qPCR and in situ hybridization (ISH) on sections revealed that ameloblasts weakly expressed AMEL during late-stage enameloid formation, while expression strongly increased during enamel deposition. Using ISH, we identified COL1A1 transcripts in ameloblasts and odontoblasts during enameloid formation. COL1A1 expression in ameloblasts gradually decreased and was no longer detected after metamorphosis. The transition from enameloid-rich to enamel-rich teeth could be related to a switch in ameloblast activity from COL1A1 to AMEL synthesis. P. waltl therefore appears to be an appropriate animal model for the study of the processes involved during enameloid-to-enamel transition, especially because similar events probably occurred in various lineages during vertebrate evolution.

Classic tissue recombination studies have demonstrated that, in the early developing mouse tooth germ, the odontogenic potential, known as the tooth-inductive capability, resides initially in the dental epithelium and then shifts to the dental mesenchyme. However, it remains unknown if human embryonic dental tissues also acquire such odontogenic potential. Here we present evidence that human embryonic dental tissues indeed possess similar tooth-inductive capability. We found that human dental epithelium from the cap stage but not the bell stage was able to induce tooth formation when confronted with human embryonic lip mesenchyme. In contrast, human dental mesenchyme from the bell stage but not the cap stage could induce mouse embryonic second-arch epithelium as well as human keratinocyte stem cells, to become enamel-secreting ameloblasts. We showed that neither post-natal human dental pulp stem cells (DPSCs) nor stem cells from human exfoliated deciduous teeth (SHED) possess odontogenic potential or are odontogenic-competent. Our results demonstrate a conservation of odontogenic potential in mouse and human dental tissues during early tooth development, and will have an implication in the future generation of stem-cell-based bioengineered human replacement teeth.

Objective: Our aim was to study the effect of high temperature (39°C) corresponding to fever on the development of enamel in cultured mouse molars., Design: For morphological studies mandibular molar blocks from E18 mice were cultured for 11 days. After three days at 37°C the teeth were exposed to 39°C for three or five days and returned to 37°C. At the end of culture, the tooth explants were photographed. The heights of the enamel and the crown of the first molars were measured and the enamel/crown height ratio was calculated. The ratios were compared between the groups using the Mann-Whitney test. To analyze gene expression in ameloblasts and odontoblasts with RT-qPCR and in situ hybridization, part of the test explants were cultured for three days at 37°C and then five days at 39°C. Control explants were kept at 37°C for 11 or eight days., Results: The enamel/crown height ratio of the first molars cultured for five days at 39°C was smaller than that of the unexposed (P<0.001). Tooth morphology did not differ between controls and exposed teeth. In qPCR-analysis, dentine sialophosphoprotein showed clearly decreased expression. In situ hybridization showed no dentine sialophosphoprotein expression in preameloblasts. The expressions of bone morphogenic protein 4, matrix metalloproteinase 20, dentine matrix protein 1, amelogenin and osteocalcin showed a trend of downregulation., Conclusions: High culturing temperature interferes with enamel formation of mouse molars and alters the expression of some genes essential for normal enamel development., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Forensic dentists are frequently required to determine the age at death of unidentified skeletons, or to age live individuals who have no record/documentation of their chronological age. In order to be of the greatest value, the method used should have the lowest possible standard deviation and be validated for the individual's specific population group. The method most frequently used in Forensic Dentistry for the estimation of age in children, was described by Demirjian et al. The maturity standards determined were based on samples of French Canadian origin and it has been recommended by several authors that correction factors be incorporated when applying this method to different population groups. The current research was carried out on a sample of 838 black South African children. A new model for age estimation in the said population was developed, to accurately determine the chronological age from dental development. A sample of 604 black South African children was used to test the validity of the method described by Demirjian.

Enamel formation produces the most highly mineralized tissue in the human body. The growth of enamel crystallites is assisted by enamel proteins and proteinases. As enamel formation progresses from secretory to maturation stages, the composition of the matrix with its mineral and non-mineral components dynamically changes in an inverse fashion. We hypothesized that appropriately calibrated micro-computed tomography (µCT) technology is suitable to estimate the mineral content (weight and/or density) and volume comparable in accuracy with that for directly weighed and sectioned enamel. Different sets of mouse mandibular incisors of C57BL/6 mice were used for dissections and µCT reconstructions. Calibration phantoms corresponding to the range of enamel mineral densities were used. Secretory-stage enamel contained little mineral and was consequently too poor in contrast for enamel volumes to be accurately estimated by µCT. Maturation-stage enamel, however, showed remarkable correspondence for total mineral content per volume where comparisons were possible between and among the different analytical techniques used. The main advantages of the µCT approach are that it is non-destructive, time-efficient, and can monitor changes in mineral content of the most mature enamel, which is too physically hard to dissect away from the tooth.

Matrix metalloproteinase-20 (enamelysin, MMP20) is essential for dental enamel development. Seven different MMP20 mutations in humans cause non-syndromic enamel malformations, termed amelogenesis imperfecta, and ablation of Mmp20 in mice results in thin brittle enamel with a dysplastic rod pattern. Healthy enamel formation requires the sliding movement of ameloblasts in rows during the secretory stage of development. This is essential for formation of the characteristic decussating enamel rod pattern observed in rodents, and this is also when MMP20 is secreted into the enamel matrix. Therefore, we propose that MMP20 facilitates ameloblast movement by cleaving ameloblast cell-cell contacts. Here we show that MMP20 cleaves the extracellular domains of the E- and N-cadherin adherens junction proteins, that both E- and N-cadherin transcripts are expressed at significantly higher levels in Mmp20 null vs. wild-type (WT) mice, and that in Mmp20 ablated mice, high-level ameloblast N-cadherin expression persists during the maturation stage of development. Furthermore, we show that E-cadherin gene expression is down-regulated from the pre-secretory to the secretory stage, while N-cadherin levels are up-regulated. This E- to N-cadherin switch supports epithelial migration in other tissues and may be an important event necessary for the ameloblasts to start moving in rows that slide by one another.

Objective: Enamelysin (MMP20) and kallikrein 4 (KLK4) are believed to be necessary to clear proteins from the enamel matrix of developing teeth. MMP20 is expressed by secretory stage ameloblasts, while KLK4 is expressed from the transition stage throughout the maturation stage. The aim of this study is to investigate the activation of KLK4 by MMP20 and the inactivation of MMP20 by KLK4., Design: Native pig MMP20 (pMMP20) and KLK4 (pKLK4) were isolated directly from enamel scrapings from developing molars. Recombinant human proKLK4 (rh-proKLK4) was activated by incubation with pMMP20 or recombinant human MMP20 (rhMMP20), and the resulting KLK4 activity was detected by zymography. Reaction products were isolated by reverse-phase high performance liquid chromatography (RP-HPLC), and their N-termini characterized by Edman degradation. The pMMP20 was incubated with pKLK4 under mildly acidic or under physiologic conditions, and enzyme activity was analyzed by zymography. The catalytic domain of rhMMP20 was incubated with pKLK4 or recombinant human KLK4 (rhKLK4) and the digestion products were characterized by zymography and Edman degradation., Results: Both pMMP20 and rhMMP20 activated rh-proKLK4 by cleaving at the propeptide-enzyme junction used in vivo. The pMMP20 was inactivated by pKLK4 under physiologic conditions, but not under mildly acidic conditions. Both pKLK4 and rhKLK4 cleaved MMP20 principally at two sites in the catalytic domain of MMP20., Conclusions: MMP20 activates proKLK4 and KLK4 inactivates MMP20 in vitro, and these actions are likely to occur during enamel formation in vivo., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Previously, we showed that Sox2-Cre;Fam20C(fl/fl) mice in which Fam20C was ubiquitously inactivated had severe defects in dentin, enamel, and bone, along with hypophosphatemia. It remains to be determined if the enamel defects in the mice with universal inactivation of Family with sequence similarity 20-C (FAM20C) were associated with the dentin defects and whether hypophosphatemia in the knockout mice contributed to the enamel defects. In this study, we crossed Fam20C(fl/fl) mice with keratin 14-Cre (K14-Cre) transgenic mice to specifically inactivate Fam20C in the epithelial cells, including the dental epithelial cells that are responsible for forming tooth enamel. X-ray, backscattered scanning electron microscopic, and histological analyses showed that the K14-Cre;Fam20C(fl/fl) mice had severe enamel and ameloblast defects, while their dentin and alveolar bone were not significantly affected. Accordingly, serum biochemistry of the K14-Cre;Fam20C(fl/fl) mice showed normal phosphate and FGF23 levels in the circulation. Analysis of these data indicates that, while FAM20C is a molecule essential to amelogenesis, its inactivation in the dental epithelium does not significantly affect dentinogenesis. Hypophosphatemia makes no significant contribution to the enamel defects in the mice with the ubiquitous deletion of Fam20C.

Highly mineralized tooth enamel develops from an extracellular matrix chiefly comprised of amelogenins formed by splicing of 7 (human) or 9 (rodent) exons secreted from specialized epithelial cells known as ameloblasts. Here we examined the role of the 59 amino acid alternatively spliced amelogenin known as leucine rich amelogenin peptide (LRAP) on enamel formation, using transgenic murine models in which LRAP overexpression is driven by an amelogenin promoter (TgLRAP). Beginning in the secretory stage of mouse amelogenesis, we found a reduced thickness of enamel matrix and a loss of Tomes' processes, followed by upregulated amelogenin mRNA expression, inhibited amelogenin secretion and loss of cell polarity. In the presecretory stage (P0) amelogenin m180 mRNA expression was increased 58 fold along with a 203 fold increase in MMP-20 expression and 3.5 and 3.2 fold increased in respectively enamelin and ameloblastin. When LRAP was overexpressed on an amelogenin knockout mouse model, the ameloblasts were not affected. Further, expression of the global chromatin organizer and transcription factor SATB1 was reduced in secretory stage TgLRAP ameloblasts. These findings identify a cellular role for LRAP in enamel formation that is not directly related to directing enamel crystal formation as is reported to be the primary function of full length amelogenins. The effect of LRAP overexpression in upregulating amelogenins, MMP-20 and SATB1, suggests a role in protein regulation critical to ameloblast secretion and matrix processing, to form a mineralized enamel matrix., (Copyright © 2013 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Objective: The 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., Methods: The mouse molars were ground, etched, and studied using scanning electron microscopy., Results: Lines interpreted as incremental lines generally appeared as grooves of variable distinctness, and were only observed cervically, in the region about 50-250μm 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 4μm, and the distance between the lines decreased towards the enamel surface., Conclusions: We 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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Objective: Development of human tooth enamel is a part of a foetus's development; its correctness is the outcome of genetic and maternal factors shaping its prenatal environment. Many authors reported that individuals born in different seasons experience different early developmental conditions during pregnancy. In this study, we investigated the effects of season of birth and selected maternal factors on enamel thickness of deciduous incisors., Design: Dental sample comprises 60 deciduous incisors. The parents who handed over their children's teeth for research fill in questionnaires containing questions about the course of pregnancy. All teeth were sectioned in the labio-linqual plane using diamond blade (Buechler IsoMet 1000). The final specimens were observed by way of scanning electron microscopy at magnifications 80× and 320×. The thickness of total enamel (TE), prenatally (PE) and postnatally (PSE) formed enamel was measured., Results: Children born in summer and in spring (whose first and second foetal life fall on autumn and winter) have the thinnest enamel. Season of birth, number of children in family, diseases and spasmolytic medicines using by mother during pregnancy explained almost 13% of the variability of TE. Regression analysis proved a significant influence of the season of birth and selected maternal factors on the PE thickness - these factors explained over 17% of its variability. Neither of analysed variables had influenced PSE., Conclusions: Our findings suggests that the thickness of enamel of deciduous incisors depends on the season of birth and some maternal factors. The differences were observed only in the prenatally formed enamel., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Aim: To establish whether eliminating Lysyl oxidase (LOX) gene would affect dentine formation., Methodology: Newborn wild-type (wt) and homo- and heterozygous LOX knock-out (Lox(-/-) and Lox(+/-) , respectively) mice were used to study developing tooth morphology and dentine formation. Collagen aggregation in the developing dentine was examined histochemically with picrosirius red (PSR) staining followed by polarized microscopy. Because Lox(-/-) die at birth, adult wt and Lox(+/-) mouse tooth morphologies were examined with FESEM. Human odontoblasts and pulp tissue were used to study the expression of LOX and its isoenzymes with Affymetrix cDNA microarray., Results: No differences between Lox(-/-) , Lox(+/-) and wt mice developing tooth morphology were seen by light microscopy. Histochemically, however, teeth in wt mice demonstrated yellow-orange and orange-red polarization colours with PSR staining, indicating thick and more densely packed collagen fibres, whilst in Lox(-/-) and Lox(+/-) mice, most of the polarization colours were green to green-yellow, indicating thinner, less aggregated collagen fibres. Fully developed teeth did not show any differences between Lox(+/-) and wt mice with FESEM. Human odontoblasts expressed LOX and three of four of its isoenzymes., Conclusions: The data indicate that LOX is not essential in dentinogenesis, even though LOX deletion may affect dentine matrix collagen thickness and packing. The absence of functional LOX may be compensated by LOX isoenzymes., (© 2012 International Endodontic Journal.)

We hypothesize that a population of migrating cells can form patterns when changes in local strains owing to relative cell motions induce changes in cell motility. That the mechanism originates in competing rates of motion distinguishes it from mechanisms involving strain energy gradients, e.g. those generated by surface energy effects or eigenstrains among cells, and diffusion-reaction mechanisms involving chemical signalling factors. The theory is tested by its ability to reproduce the morphological characteristics of enamel in the mouse incisor. Dental enamel is formed during amelogenesis by a population of ameloblasts that move about laterally within an expanding curved sheet, subject to continuously evolving spatial and temporal gradients in strain. Discrete-cell simulations of this process compute the changing strain environment of all cells and predict cell trajectories by invoking simple rules for the motion of an individual cell in response to its strain environment. The rules balance a tendency for cells to enhance relative sliding motion against a tendency to maintain uniform cell-cell separation. The simulations account for observed waviness in the enamel microstructure, the speed and shape of the 'commencement front' that separates domains of migrating secretory-stage ameloblasts from those that are not yet migrating, the initiation and sustainment of layered, fracture-resistant decussation patterns (cross-plied microstructure) and the transition from decussating inner enamel to non-decussating outer enamel. All these characteristics can be correctly predicted with the use of a single scalar adjustable parameter.

Background: Enamel synthesis is a highly dynamic process characterized by simultaneity of matrix secretion, assembly and processing during apatite mineralization. MMP-20 is the first protease to hydrolyze amelogenin, resulting in specific cleavage products that self-assemble into nanostructures at specific mineral compositions and pH. In this investigation, enzyme kinetics of MMP-20 proteolysis of recombinant full-length human amelogenin (rH174) under different mineral compositions is elucidated., Methods: Recombinant amelogenin was cleaved by MMP-20 under various physicochemical conditions and the products were analyzed by SDS-PAGE and MALDI-TOF MS., Results: It was observed that mineral ions largely affect cleavage pattern, and enzyme kinetics of rH174 hydrolysis. Out of the five selected mineral ion compositions, MMP-20 was most efficient at high calcium concentration, whereas it was slowest at high phosphate, and at high calcium and phosphate concentrations. In most of the compositions, N- and C-termini were cleaved rapidly at several places but the central region of amelogenin was protected up to some extent in solutions with high calcium and phosphate contents., Conclusion: These in vitro studies showed that the chemistry of the protein solutions can significantly alter the processing of amelogenin by MMP-20, which may have significant effects in vivo matrix assembly and subsequent calcium phosphate mineralization., General Significance: This study elaborates the possibilities of the processing of the organic matrix into mineralized tissue during enamel development.

Fully matured dental enamel is an architecturally and mechanically complex hydroxyapatite-based bioceramic devoid of most of the organic material that was essential in its making. Enamel formation is a staged process principally involving secretory and maturation stages, each associated with major changes in gene expression and cellular function. Cellular activities that define the maturation stage of amelogenesis include ion (e.g., calcium and phosphate) transport and storage, control of intracellular and extracellular pH (e.g., bicarbonate and hydrogen ion movements), and endocytosis. Recent studies on rodent amelogenesis have identified a multitude of gene products that appear to be linked to these cellular activities. This review describes the main cellular activities of these genes during the maturation stage of amelogenesis.

The ameloblast cell layer of the enamel organ is in contact with the forming enamel as it develops into the hardest substance in the body. Ameloblasts move in groups that slide by one another as the enamel layer thickens. Each ameloblast is responsible for the formation of one enamel rod, and the rods are the mineralized trail that moving ameloblasts leave behind. Matrix metalloproteinases (MMPs) facilitate cell movement in various tissues during development, and in this review we suggest that the tooth-specific MMP, enamelysin (MMP20), facilitates ameloblast movements during enamel development. Mmp20 null mice have thin brittle enamel with disrupted rod patterns that easily abrades from the underlying dentin. Strikingly, the Mmp20 null mouse enamel organ morphology is noticeably dysplastic during late-stage development, when MMP20 is no longer expressed. We suggest that in addition to its role of cleaving enamel matrix proteins, MMP20 also cleaves junctional complexes present on ameloblasts to foster the cell movement necessary for formation of the decussating enamel rod pattern. Therefore, inactivation of MMP20 would result in tight ameloblast cell-cell attachments that may cause maturation-stage enamel organ dysplasia. The tight ameloblast attachments would also preclude the ameloblast movement necessary to form decussating enamel rod patterns.

Deletion or mutation of dentin matrix protein 1 (DMP1) leads to hypophosphatemic rickets and defects within the dentin. However, it is largely unknown if this pathological change is a direct role of DMP1 or an indirect role of phosphate (Pi) or both. It has also been previously shown that Klotho-deficient mice, which displayed a high Pi level due to a failure of Pi excretion, causes mild defects in the dentinal structure. This study was to address the distinct roles of DMP1 and Pi homeostasis in cell differentiation, apoptosis and mineralization of dentin and enamel. Our working hypothesis was that a stable Pi homeostasis is critical for postnatal tooth formation, and that DMP1 has an antiapoptotic role in both amelogenesis and dentinogenesis. To test this hypothesis, Dmp1-null (Dmp1(-/-)), Klotho-deficient (kl/kl), Dmp1/Klotho-double-deficient (Dmp1(-/-)/kl/kl) and wild-type (WT) mice were killed at the age of 6 weeks. Combinations of X-ray, microcomputed tomography (μCT), scanning electron microscopy (SEM), histology, apoptosis and immunohistochemical methods were used for characterization of dentin, enamel and pulp structures in these mutant mice. Our results showed that Dmp1(-/-) (a low Pi level) or kl/kl (a high Pi level) mice displayed mild dentin defects such as thin dentin and a reduction of dentin tubules. Neither deficient mouse line exhibited any apparent changes in enamel or pulp structure. However, the double-deficient mice (a high Pi level) displayed severe defects in dentin and enamel structures, including loss of dentinal tubules and enamel prisms, as well as unexpected ectopic ossification within the pulp root canal. TUNEL assay showed a sharp increase in apoptotic cells in ameloblasts and odontoblasts. Based on the above findings, we conclude that DMP1 has a protective role for odontoblasts and ameloblasts in a pro-apoptotic environment (a high Pi level).

Amelogenin, the major protein of forming dental enamel, plays a crucial role in the biomineralization of this tissue. Amelogenin is soluble at low pH and self-assembles to form higher order structures at physiological pH. To understand the mechanisms of its assembly and interactions with calcium phosphate mineral, we conducted FTIR spectroscopy (FTIRS) studies of pH-triggered assembly of recombinant porcine amelogenin rP172 and its interactions with mature hydroxyapatite and apatitic mineral formed in situ. Analysis of our data indicated that rP172 at pH 3.0 exists in an unfolded disordered state, while increases in pH led to structural ordering, manifested by increases in intra- and intermolecular β-sheet structures and a decrease in random coil and β-turns. Amelogenin assembled at pH 7.2 was also found to contain large portions of extended intramolecular β-sheet and PPII. These FTIRS findings are consistent with those previously obtained with other techniques, thus verifying the validity of our experimental approach. Interestingly, interactions with mineral led to a reduction in protein structural organization. The findings obtained show that amelogenin has intrinsic structural flexibility to accommodate interactions with both forming and mature calcium phosphate mineral phases, providing new insights into the potential importance of amelogenin-mineral interactions in enamel biomineralization.