Enamel Matrix Secretion Research Articles

Defects‐related early childhood caries as hints of possible maternal–fetal health issues: Evidence from medieval northern Italy

AbstractDevelopmental defects of enamel (DDE) are important markers of stress as they arise from the disruption of ameloblastic activity during enamel matrix secretion and mineralization. Defects on the crowns of the deciduous dentition provide insights into maternal–fetal health because they can emerge during intrauterine development as a result of gestational issues. The presence of previous defective enamel represents one of the most predisposing causes of caries development. Thus, circular caries on the deciduous dentition are considered an indicator of stress because of their ascertained relation with dental enamel defects. In bioarchaeological analysis, these lesions allow us to deepen the question of maternal–fetal health issues in the ancient period. Here, we present evidence of defects‐related early childhood caries in three infants from a medieval cemetery in northern Italy. The findings in the dentitions of the three subjects were investigated with a multi‐analytical approach, including macroscopic examination, microscopic observation, cone‐beam computed tomography (CBCT), scanning electron microscopy (SEM), and histological sections. In the specimens analyzed, the lesions observed in the dentitions appeared to have arisen during the fetal period and then protracted in the postnatal life, suggesting the implication of in utero environment and maternal health in the etiology of defects.

Smurf1 regulates ameloblast polarization by ubiquitination-mediated degradation of RhoA.

Cell polarity is essential for ameloblast differentiation and enamel formation. Smurf1 can mediate cell polarization through ubiquitination degradation of specific substrates. But it remains unclear whether Smurf1 could regulate ameloblast polarity and the underlying mechanism. Here, immuno-fluorescence staining and RT-qPCR were applied to detect the expression of Smurf1 and F-actin. A mouse lower incisor defect model was constructed. Scanning electron microscope, rat lower incisor culture, western blot, wound healing assay and trans-well migration assay were performed to detect the influence of Smurf1 knockdown on ameloblast. IF double staining, western blot and co-immunoprecipitation were conducted to detect the interaction between Smurf1 and RhoA. The in vivo experiment was also performed. We found that Smurf1 was mainly expressed in the membrane and cell cortex of ameloblast, similar to F-actin. Smurf1 expression increased along ameloblast polarization and differentiation. After knocking down Smurf1, the cytoskeleton and cell morphology changed and the cell polarity was damaged. Smurf1 regulated ameloblast polarity through ubiquitination degradation of activated RhoA in vitro. Local knockdown of Smurf1 in rat lowerincisor ameloblast resulted in ameloblast polarity loss, enamel matrix secretion disorder and chalky enamel, but RhoA inhibitor Y-27632 could reverse this effect. Collectively, Smurf1 could regulate the polarization of ameloblast through ubiquitination degradation of activated RhoA, which contributed to the knowledge of tooth development and provided new research ideas for cell polarity.

An Unusual Case of Molar Incisor Hypomineralization with a Rare Combination of Fusion, Hypodontia, and Ankyloglossia in 11-Year-Old Male

Molar-incisor hypomineralization (MIH) is a developmentally acquired dental defect that directly affects the enamel and dentin of one to four first molars, frequently associated with affected incisors. Due to alteration in matrix production, crystal arrangement and formation, enamel matrix secretion a compromised enamel structure may be seen. This condition can also be called a idiopathic enamel hypomineralization. In the article, we report a case of 11-year-old male presenting with typical features of MIH affecting all four permanent molars in combination with fusion of mandibular right permanent central and lateral incisor, hypodontia of mandibular left central incisor and ankyloglossia, and a possible management of the clinical condition.

PER2-mediated ameloblast differentiation via PPAR\u03b3/AKT1/\u03b2-catenin axis

Circadian rhythm is involved in the development and diseases of many tissues. However, as an essential environmental regulating factor, its effect on amelogenesis has not been fully elucidated. The present study aims to investigate the correlation between circadian rhythm and ameloblast differentiation and to explore the mechanism by which circadian genes regulate ameloblast differentiation. Circadian disruption models were constructed in mice for in vivo experiments. An ameloblast-lineage cell (ALC) line was used for in vitro studies. As essential molecules of the circadian system, Bmal1 and Per2 exhibited circadian expression in ALCs. Circadian disruption mice showed reduced amelogenin (AMELX) expression and enamel matrix secretion and downregulated expression of BMAL1, PER2, PPARγ, phosphorylated AKT1 and β-catenin, cytokeratin-14 and F-actin in ameloblasts. According to previous findings and our study, BMAL1 positively regulated PER2. Therefore, the present study focused on PER2-mediated ameloblast differentiation and enamel formation. Per2 knockdown decreased the expression of AMELX, PPARγ, phosphorylated AKT1 and β-catenin, promoted nuclear β-catenin accumulation, inhibited mineralization and altered the subcellular localization of E-cadherin in ALCs. Overexpression of PPARγ partially reversed the above results in Per2-knockdown ALCs. Furthermore, in in vivo experiments, the length of incisor eruption was significantly decreased in the circadian disturbance group compared to that in the control group, which was rescued by using a PPARγ agonist in circadian disturbance mice. In conclusion, through regulation of the PPARγ/AKT1/β-catenin signalling axis, PER2 played roles in amelogenin expression, cell junctions and arrangement, enamel matrix secretion and mineralization during ameloblast differentiation, which exert effects on enamel formation.

WITHDRAWN: Evaluation of expression of p63 and amelogenin in human tooth germ and ameloblastoma

BACKGROUND: The study was undertaken to evaluate the expression of p63 and amelogenin in human tooth germs and ameloblastomas by immunohistochemistry to find out whether the pattern of expression was related to differentiation of ameloblasts. MATERIALS AND METHODS: In this study fifteen human tooth germs of late bell stage, six human tooth germs of early bell stage and fifteen ameloblastoma samples were included and expression pattern of p63 and amelogenin were evaluated individually in all the samples. RESULTS: During the early bell stage, p63 expression was intense throughout the enamel organ. But, during the late bell stage, the number and intensity of p63 expression decreases in the cells of enamel organ. The peripheral cells in the ameloblastoma shows variable pattern of p63 expression. Expression of amelogenin was first evident in the presecretory ameloblasts at the cusp tip, followed by secretory ameloblasts and progresses cervically. But in ten late bell stage tooth germs, amelogenin expression was negative in the secretory ameloblasts and positive only at the secreting end i.e. tomes process. Intense staining was evident at the enamel matrix and in some dentinal tubules nearer to enamel matrix secretion. No amelogenin expression was evident in the dental papilla cells or odontoblasts throughout odontogenesis with the exception of dental follicle. Amelogenin expression was absent in eleven cases of ameloblastoma, but in four cases positive expression was evident at the peripheral cells. CONCLUSION: In the present study, the expression pattern of p63 and amelogenin in human tooth germ and ameloblastoma correlates with cytodifferentiation of ameloblasts.

THE EFFECT OF AMOXICILLIN ON THE SECRETORY STAGE OF AMELOGENESIS IN RATS

Introduction: Molar–incisor hypomineralization is a developmental enamel defect affecting first permanent molars and incisors. The intake of antibiotics during pregnancy and early childhood has been associated with molar incisor hypomineralization. Objectives: Was to determine the effect of amoxicillin on the secretory a stage of amelogenesis histologically and histomorphometrically. Materials and methods: Fourteen pregnant rats were randomly assigned to two groups that received physiological solution (control group), 250 mg/kg/day amoxicillin (study group). Directly after birth, fourteen pups representing each group received the same treatment with dose equivalent to their body weight then they were euthanized at day 7 birth. The ameloblasts of developing molars were examined histologically and histomorphometrically. Results:In day 7 after birth, the ameloblasts in control group revealed normal features of amelogenesis while in study group, numerous vacuolar like structures were seen in between secretory ameloblasts. Conclusions: Amoxicillin disrupts the normal structure of enamel and impairs the secretion of enamel matrix

Modeling enamel matrix secretion in mammalian teeth.

The most mineralized tissue of the mammalian body is tooth enamel. Especially in species with thick enamel, three-dimensional (3D) tomography data has shown that the distribution of enamel varies across the occlusal surface of the tooth crown. Differences in enamel thickness among species and within the tooth crown have been used to examine taxonomic affiliations, life history, and functional properties of teeth. Before becoming fully mineralized, enamel matrix is secreted on the top of a dentine template, and it remains to be explored how matrix thickness is spatially regulated. To provide a predictive framework to examine enamel distribution, we introduce a computational model of enamel matrix secretion that maps the dentine topography to the enamel surface topography. Starting from empirical enamel-dentine junctions, enamel matrix deposition is modeled as a diffusion-limited free boundary problem. Using laboratory microCT and synchrotron tomographic data of pig molars that have markedly different dentine and enamel surface topographies, we show how diffusion-limited matrix deposition accounts for both the process of matrix secretion and the final enamel distribution. Simulations reveal how concave and convex dentine features have distinct effects on enamel surface, thereby explaining why the enamel surface is not a straightforward extrapolation of the dentine template. Human and orangutan molar simulations show that even subtle variation in dentine topography can be mapped to the enamel surface features. Mechanistic models of extracellular matrix deposition can be used to predict occlusal morphologies of teeth.

The Enamel Phenotype in Homozygous Fam83h Truncation Mice.

BackgroundTruncation FAM83H mutations cause human autosomal dominant hypocalcified amelogenesis imperfecta (ADHCAI), an inherited disorder characterized by severe hardness defects in dental enamel. No enamel defects were observed in Fam83h null mice suggesting that Fam83h truncation mice would better replicate human mutations.MethodsWe generated and characterized a mouse model (Fam83h Tr/Tr) expressing a truncated FAM83H protein (amino acids 1–296), which recapitulated the ADHCAI‐causing human FAM83H p.Tyr297* mutation.ResultsDay 14 and 7‐week Fam83h Tr/Tr molars exhibited rough enamel surfaces and slender cusps resulting from hypoplastic enamel defects. The lateral third of the Fam83h Tr/Tr incisor enamel layer was thinner, with surface roughness and altered enamel rod orientation, suggesting disturbed enamel matrix secretion. Regular electron density in mandibular incisor enamel indicated normal enamel maturation. Only mildly increased posteruption attrition of Fam83h Tr/Tr molar enamel was observed at 7‐weeks. Histologically, the Fam83h Tr/Tr enamel organ, including ameloblasts, and enamel matrices at sequential stages of amelogenesis exhibited comparable morphology without overt abnormalities, except irregular and less evident ameloblast Tomes' processes in specific areas.ConclusionsConsidering Fam83h−/− mice showed no enamel phenotype, while Fam83h Tr/Tr (p.Tyr297*) mice displayed obvious enamel malformations, we conclude that FAM83H truncation mutations causing ADHCAI in humans disturb amelogenesis through a neomorphic mechanism, rather than haploinsufficiency.

Do Mid-Crown Enamel Formation Front Angles Reflect Factors Linked to the Pace of Primate Growth and Development?

Enamel formation front (EFF) angles represent the leading edge of enamel matrix secretion at particular points in time. These angles are influenced by rates of enamel extension (the rates at which tooth crowns grow in height), rates of enamel matrix secretion and the angles that prisms make with the enamel-dentine junction. Previous research suggests, but has not yet established, that these angles reflect aspects of primate biology related to their pace of growth and development, most notably brain and body size. The present study tested this possibility on histological sections using phylogenetically-controlled and Bonferroni-corrected analyses spanning a broad taxonomic range. Ten species were represented in the analysis of anterior teeth; 17 in the analysis of posterior (postcanine) teeth (with varying sample sizes). Also, tested was the relationship of EFF angles to striae of Retzius periodicity (long period growth rhythms in enamel) and degree of folivory, as both factors are related to primate developmental rates. Finally, several analyses were conducted to investigate whether tooth size (operationalized as EDJ length) might mediate these relationships. Central results are as follows: (1) Relationships between EFF angles and brain weight (anterior teeth) and between EFF angles and body mass (anterior and posterior teeth) are statistically significant and (2) Mid-crown EFF angles are not statistically significantly related to EDJ lengths. These results suggest that tooth size does not mediate relationships between EFF angles and brain weight/body mass and are discussed with respect to underlying enamel growth variables (especially rates of enamel extension and secretion). Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 301:125-139, 2018. © 2017 Wiley Periodicals, Inc.

Integrative Temporo-Spatial, Mineralogic, Spectroscopic, and Proteomic Analysis of Postnatal Enamel Development in Teeth with Limited Growth.

Tooth amelogenesis is a complex process beginning with enamel organ cell differentiation and enamel matrix secretion, transitioning through changes in ameloblast polarity, cytoskeletal, and matrix organization, that affects crucial biomineralization events such as mineral nucleation, enamel crystal growth, and enamel prism organization. Here we have harvested the enamel organ including the pliable enamel matrix of postnatal first mandibular mouse molars during the first 8 days of tooth enamel development to conduct a step-wise cross-sectional analysis of the changes in the mineral and protein phase. Mineral phase diffraction pattern analysis using single-crystal, powder sample X-ray diffraction analysis indicated conversion of calcium phosphate precursors to partially fluoride substituted hydroxyapatite from postnatal day 4 (4 dpn) onwards. Attenuated total reflectance spectra (ATR) revealed a substantial elevation in phosphate and carbonate incorporation as well as structural reconfiguration between postnatal days 6 and 8. Nanoscale liquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS) demonstrated highest protein counts for ECM/cell surface proteins, stress/heat shock proteins, and alkaline phosphatase on postnatal day 2, high counts for ameloblast cytoskeletal proteins such as tubulin β5, tropomyosin, β-actin, and vimentin on postnatal day 4, and elevated levels of cofilin-1, calmodulin, and peptidyl-prolyl cis-trans isomerase on day 6. Western blot analysis of hydrophobic enamel proteins illustrated continuously increasing amelogenin levels from 1 dpn until 8 dpn, while enamelin peaked on days 1 and 2 dpn, and ameloblastin on days 1–5 dpn. In summary, these data document the substantial changes in the enamel matrix protein and mineral phase that take place during postnatal mouse molar amelogenesis from a systems biological perspective, including (i) relatively high levels of matrix protein expression during the early secretory stage on postnatal day 2, (ii) conversion of calcium phosphates to apatite, peak protein folding and stress protein counts, and increased cytoskeletal protein levels such as actin and tubulin on day 4, as well as (iii) secondary structure changes, isomerase activity, highest amelogenin levels, and peak phosphate/carbonate incorporation between postnatal days 6 and 8. Together, this study provides a baseline for a comprehensive understanding of the mineralogic and proteomic events that contribute to the complexity of mammalian tooth enamel development.

The Semaphorin 4D-RhoA-Akt Signal Cascade Regulates Enamel Matrix Secretion in Coordination With Cell Polarization During Ameloblast Differentiation.

During tooth development, oral epithelial cells differentiate into ameloblasts in order to form the most mineralized tissue in the vertebrate body: enamel. During this process, ameloblasts directionally secrete enamel matrix proteins and morphologically change from low columnar cells to polarized tall columnar cells, both of which are essential for the proper formation of enamel. In this study, we elucidated the molecular mechanism that integrates ameloblast function and morphology. Immunohistochemistry revealed that the restricted expression of semaphorin 4D (Sema4D) and RhoA activation status are closely associated with ameloblast differentiation in mouse incisors. In addition, in vitro gain-of-function and loss-of-function experiments demonstrated that Sema4D acts upstream of RhoA to regulate cell polarity and amelogenin expression via the Plexin B1/Leukemia-associated RhoGEF (LARG) complex during ameloblast differentiation. Experiments in transgenic mice demonstrated that expression of a dominant-negative form of RhoA in dental epithelium hindered ameloblast differentiation and subsequent enamel formation, as well as perturbing the establishment of polarized cell morphology and vectorial amelogenin expression. Finally, we showed that spatially restricted Akt mediates between Sema4D-RhoA signaling and these downstream cellular events. Collectively, our results reveal a novel signaling network, the Sema4D-RhoA-Akt signal cascade, that coordinates cellular function and morphology and highlights the importance of specific spatiotemporally restricted components of a signaling pathway in the regulation of ameloblast differentiation. © 2016 American Society for Bone and Mineral Research.

The glycogen metabolism via Akt signaling is important for the secretion of enamel matrix in tooth development

Cells alter their energy metabolism depending on the stage of differentiation or various environments. In the ameloblast differentiation of continuous growing mouse incisors, we found temporary glycogen storage in preameloblasts before the start of enamel matrix secretion and investigated the relationship between enamel matrix secretion and glycogen metabolism. Immunohistochemistry showed that in the transitional stage from preameloblasts to secretory ameloblasts, the glycogen synthase changed from the inactive form to the active form, the expression of glycogen phosphorylase increased, and further, the levels of IGF-1, IGF-1 receptor and activated Akt increased. These results suggested that the activation of Akt signaling via IGF is linked to the onset of both glycogen metabolism and enamel matrix deposition. In the experiments using organ culture and ameloblast cell line, the activation of Akt signaling by IGF-1 stimulated glycogen metabolism through the up-regulation of Glut-1,-4 and Gsk-3β and the dephosphorylation of glycogen synthase. Subsequently, they resulted in increased enamel matrix secretion. In contrast, some inhibitors of Akt signals and glycogen synthesis/degradation down-regulated enamel matrix secretion. Taking these findings together, glycogen metabolism via Akt signaling is an essential system for the secretion of enamel matrix in ameloblast differentiation.

In vivo administration of dental epithelial stem cells at the apical end of the mouse incisor.

Cell-based tissue regeneration is an attractive approach that complements traditional surgical techniques for replacement of injured and lost tissues. The continuously growing rodent incisor provides an excellent model system for investigating cellular and molecular mechanisms that underlie tooth renewal and regeneration. An active population of dental epithelial progenitor/stem cells located at the posterior part of the incisor, commonly called cervical loop area, ensures the continuous supply of cells that are responsible for the secretion of enamel matrix. To explore the potential of these epithelial cells in therapeutic approaches dealing with enamel defects, we have developed a new method for their in vivo administration in the posterior part of the incisor. Here, we provide the step-by-step protocol for the isolation of dental epithelial stem cells and their delivery at targeted areas of the jaw. This simple and yet powerful protocol, consisting in drilling a hole in the mandibular bone, in close proximity to the cervical loop area of the incisor, followed up by injection of stem cells, is feasible, reliable, and effective. This in vivo approach opens new horizons and possibilities for cellular therapies involving pathological and injured dental tissues.

Enamel hypoplasia and its role in identification of individuals: A review of literature.

Identification of individuals is the mainstay of any forensic investigation especially in cases of mass disasters when mutilated remains are brought for examination. Dental examination helps in establishing the identity of an individual and thus, has played a vital role in forensic investigation process since long. In this regard, description on the role of enamel hypoplasia is limited in the literature. The present article reviews the literature on the enamel hypoplasia and discusses its utility in forensic identification. Enamel hypoplasia is a surface defect of the tooth crown caused by disturbance of enamel matrix secretion. Enamel defects can be congenital or acquired. In cases of mass disasters, or when the body is completely charred, putrefied and mutilated beyond recognition, the unique dental features can help in identification of the victims.

Fluctuations in surface pH of maturing rat incisor enamel are a result of cycles of H+‐secretion by ameloblasts and variations in enamel buffer characteristics (1098.1)

The large hydroxyapatite (HA) crystals in dental enamel results from a long lasting process called enamel maturation, which follows a stage of enamel matrix secretion. Thus, after the full thickness of enamel is reached, the enamel still requires about 60‐70 % mineral to be deposited by HA‐crystals growing in width and thickness. This is facilitated by the ameloblasts, which during maturation change morphology every 6‐8th hour. This study aims to test the role of the ameloblasts in maintaining enamel pH in the maturation zone in order to elucidate the role of the cyclic changes in ameloblast morphology for HA‐crystal growth.Colorimetric pH‐indicators and ratiometric fluorometry were used to measure surface pH in maturation zone enamel of rat incisors. Alternating acidic (down to pH 6.24±0.06) and alkaline zones (up to pH 7.34±0.08) corresponding to ruffled‐ended and smooth‐ended ameloblasts, respectively, were found along the tooth thus coinciding with ameloblast morphological cycles. Underlying the cyclic pattern, a gradual decrease in pH towards the incisal edge was seen. Vinblastine or FR167356 (H+‐ATPase‐inhibitor) disturbed ameloblast acid‐secretion, especially in the early parts of acidic zones.At the pH‐values observed, PO43‐ would be protonated (pKa >12) and HA dissolved. However, by molecular dynamics simulations we estimate the pKa of HPO42‐ at an ideal HA surface to be 4.3. The acidic pH measured at the enamel surface may thus only dissolve non‐perfect domains of HA crystals in which HPO42‐ is less electrostatically shielded. As a result of the repeated alkaline/acidic cycles induced by ameloblasts, near‐perfect domains will therefore gradually replace less perfect domains and large near‐perfect HA crystals will be produced.In conclusion, fluctuations in surface pH of maturing rat incisor enamel are a result of cycles of H+‐secretion by ameloblasts and variations in enamel buffer characteristics.

Expression and localization of Nell-1 during murine molar development

Nel-like molecule-1 (Nell-1) is a recently discovered secreted protein that plays an important role in osteoblast differentiation, bone formation, and bone regeneration. However, its expression and distribution during tooth development are largely unknown. The aim of this study was to investigate the expression patterns of Nell-1 during murine molar development by immunohistochemistry. Nell-1 protein was expressed during molar development in embryonic and postnatal Kunming mice, but its expression levels and patterns at various developmental stages differed. At embryonic day 13.5 (E13.5) and E14.5, Nell-1 was found in both the entire enamel organ and the underlying mesenchyme. At E16.5, it was detected in the inner and outer enamel epithelia, stratum intermedium, secondary enamel knot, and dental papilla. At E18.5, Nell-1 was expressed in the differentiating ameloblasts, differentiating odontoblasts, and stratum intermedium. Positive staining was also found in the outer enamel epithelium. At postnatal day 2.5 (P2.5), P5, and P7, Nell-1 appeared in the secretory and mature ameloblasts and odontoblasts (odontoblastic bodies and processes) as well as immature enamel. Hertwig's epithelial root sheath also stained positively at P7. At P13.5, positive staining was restricted to the reduced dental epithelium and odontoblasts, whereas Nell-1 disappeared in the mature enamel. During tooth eruption, Nell-1 was observed only in the odontoblastic bodies, odontoblastic processes, and endothelial cells of blood vessels. The spatiotemporal expression patterns of Nell-1 during murine tooth development suggest that it might play an important role in ameloblast and odontoblast differentiation, secretion and mineralization of the extracellular enamel matrix, molar crown morphogenesis, as well as root formation.

The expression pattern of FHL2 during mouse molar development

Four and a half LIM domains 2 (FHL2) functions as a transcriptional co-activator or co-repressor in a cell-type-specific manner. As a positive regulator, FHL2 plays an important role in osteoblast differentiation and bone formation. Our previous study showed that FHL2 was expressed in odontoblasts in mature human teeth under normal and pathological conditions. The purpose of this study was to investigate the spatial-temporal expression patterns of FHL2 at different stages of mouse molar development by immunohistochemistry. Our results showed that at the bud and cap stage, FHL2 was expressed both in enamel organ and the underlying mesenchyme. At the early bell stage, FHL2 appeared in the inner and outer enamel epithelium, stratum intermedium and the secondary enamel knot. Positive staining gradually converged at the cusps of dental papilla. At the late bell stage, FHL2 was expressed in the terminal differentiated ameloblasts and odontoblasts and stratum intermedium. At the postnatal day, FHL2 was detected in the secretory and mature ameloblasts and odontoblasts and mature enamel, and gradually appeared at Hertwig's epithelial root sheath and periodontal tissues. The spatial-temporal expression patterns of FHL2 from the bud stage to the postnatal day (13.5) suggested that during tooth development, FHL2 might play an important role in ameloblast and odontoblast differentiation, secretion of enamel and dentin matrix, mineralization of enamel, molar crown morphogenesis, as well as root formation.

Enamel hypoplasia in molars of sheep and goats, and its relationship to the pattern of tooth crown growth.

Enamel is the most highly mineralized and durable tissue of the mammalian body. As enamel does not undergo remodeling or repair, disturbances of enamel formation leave a permanent record in the tissue that can be used for life history reconstruction. This study reports light and scanning electron microscope findings on hypoplastic enamel defects, and on the chronology of crown growth in the molars of sheep and goats. A marked reduction of enamel extension rates in cervical compared with more cuspal crown portions of sheep and goat molars was recorded, with formation of the cervical 25% of the crown taking about the same time as that of the upper 75% of the crown. This explains the more frequent occurrence of enamel hypoplasia in cervical compared with upper and middle crown portions. Regarding the identification of hypoplastic enamel defects by external inspection, our results suggest a dependence on the type of defect and the associated presence of smaller or larger amounts of coronal cementum. Defects considered to reflect a slight to moderate impairment of secretory ameloblast function can normally be correctly diagnosed as they are not occluded by thick layers of cementum. In contrast, defects denoting a severe impairment of enamel matrix secretion can typically not be correctly identified because they are occluded by large amounts of cementum, so that neither depth nor extension of the defects can be assessed on external inspection. In these cases, microscopic analysis of tooth sections is required for a correct diagnosis of the hypoplastic enamel defects.

Combined effect of fluoride and 2,3,7,8-tetrachlorodibenzo-p-dioxin on mouse dental hard tissue formation in vitro

Fluoride interferes with enamel matrix secretion and mineralization and dentin mineralization. The most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), also impairs dental hard tissue formation and mineralization in vitro and in vivo. Our aim was to investigate in vitro whether the combined effect of sodium fluoride (NaF) and TCDD on dental hard tissue formation is potentiative. For this purpose, mandibular first and second molar tooth germs of E18 mouse embryos were cultured for 5-12days with NaF and TCDD alone at various concentrations (2.5, 5, 10, 12.5, 15, and 20μM and 5, 10, 12.5, and 15nM, respectively) to determine the highest concentrations, which alone cause no or negligible effects. Morphological changes were studied from the whole tooth photographs and histological tissue sections. The concentrations found were 15μM for NaF and 10nM for TCDD. While at these concentrations, the effects of NaF and TCDD alone were barely detectable, the effect of simultaneous exposure on dentin and enamel formation was overt; mineralization of predentin to dentin and enamel matrix secretion and mineralization were impaired. Immunohistochemical analysis revealed that the combined exposure modified amelogenin expression by odontoblasts. Morphology of ameloblasts and the expression of amelogenin indicated that ameloblasts were still secretory. The results show that NaF and TCDD have potentiative, harmful effects on the formation of dental hard tissues. Since children can be exposed to subclinical levels of fluoride and dioxins during early childhood, coincidently with mineralization of the first permanent teeth, this finding may have clinical significance.

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

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