Crown formation times of deciduous teeth and age at death in neolithic newborns / Tiempos de formación de la corona de los dientes deciduos y edad al morir en neonatos neolíticos

SUMMARY: The aim of the present study was to investigate the possibility of estimating crown formation times of immature deciduous teeth and age at death in Neolithic newborns. In the Neolithic-Mesolithic transition, the health of the population deteriorated. Leaving the intrauterine environment for the newborn is the first obstacle in the process of adaptation and survival in the outside world. The fetus is protected by the mother's immune system and receives the necessary nutrients through the umbilical cord, but external factors indirectly affect its development. At birth deciduous teeth are not fully formed and are only partially mineralized. Variations in the rhythmic activity of ameloblasts and the secretion of the enamel matrix lead to the formation of incremental lines in the enamel. The sample consisted of unerupted deciduous teeth removed from the baby jaws from Neolithic archaeological graves, LepenskiVir Serbia. The skeletal age of the babies was from 38 to 40 gestational weeks. The daily enamel apposition rate was obtained for each tooth. The age of individuals was estimated using crown formation time. The average value of daily secretion rates for the primary teeth from the Neolithic age was 3.78 µm. There was no statistically significant difference in age at death determined by skeletal age assessment and crown formation time. Three babies were born preterm. The results of the present study show that the calculation of the time required for the formation of deciduous tooth enamel is applicable to archaeological samples of newborns.The age estimation using crown formation time together with the analysis of other anthropological parameters, can contribute to a more accurate determination of neonatal death in anthropological, archaeological and forensic contexts.

RESUMEN: El objetivo del estudio fue investigar la posibilidad de estimar el tiempo de formación de la corona de los dientes deciduos inmaduros y la edad, al momento de la muerte en neonatos neolíticos. Durante la transición Neolítico-Mesolítico, la salud de la población deterioró significativamente. Para el recién nacido dejar el medio intrauterino es el primer obstáculo en el proceso de adaptación y supervivencia en el mundo exterior. El feto está protegido por el sistema inmunológico de la madre y recibe los nutrientes necesarios a través del cordón umbilical, pero factores externos afectan indirectamente su desarrollo. Al nacer, los dientes deciduos no están completamente formados y solo están parcialmente mineralizados. Las variaciones en la actividad rítmica de los ameloblastos y la secreción de la matriz del esmalte conducen a la formación de líneas incrementales en el esmalte. La muestra consistió en dientes sin erupción extraídos de las mandíbulas de neonatos de tumbas arqueológicas neolíticas, LepenskiVir Serbia. La edad esquelética de los bebés fue de 38 a 40 semanas de gestación. Se obtuvo la tasa diaria de aposición de esmalte para cada diente. La edad de los individuos se estimó utilizando el tiempo de formación de la copa. El valor promedio de las tasas de secreción diaria para los dientes temporales del Neolítico fue de 3,78µm. No hubo diferencia estadísticamente significativa en la edad al momento de la muerte determinada por la evaluación de la edad esquelética y el tiempo de formación de la corona. Tres bebés nacieron prematuros. Los resultados del presente estudio muestran que el cálculo del tiempo requerido para la formación del esmalte dental deciduo es aplicable a muestras arqueológicas de recién nacidos. La estimación de la edad utilizando el tiempo de formación de la corona junto con el análisis de otros parámetros antropológicos, puede contribuir a una mayor determinación precisa de la muerte neonatal en contextos antropológicos, arqueológicos y forenses.

Odontogenesis-associated phosphoprotein truncation blocks ameloblast transition into maturation in OdaphC41*/C41* mice.

Mutations of Odontogenesis-Associated Phosphoprotein (ODAPH, OMIM *614829) cause autosomal recessive amelogenesis imperfecta, however, the function of ODAPH during amelogenesis is unknown. Here we characterized normal Odaph expression by in situ hybridization, generated Odaph truncation mice using CRISPR/Cas9 to replace the TGC codon encoding Cys41 into a TGA translation termination codon, and characterized and compared molar and incisor tooth formation in Odaph+/+, Odaph+/C41*, and OdaphC41*/C41* mice. We also searched genomes to determine when Odaph first appeared phylogenetically. We determined that tooth development in Odaph+/+ and Odaph+/C41* mice was indistinguishable in all respects, so the condition in mice is inherited in a recessive pattern, as it is in humans. Odaph is specifically expressed by ameloblasts starting with the onset of post-secretory transition and continues until mid-maturation. Based upon histological and ultrastructural analyses, we determined that the secretory stage of amelogenesis is not affected in OdaphC41*/C41* mice. The enamel layer achieves a normal shape and contour, normal thickness, and normal rod decussation. The fundamental problem in OdaphC41*/C41* mice starts during post-secretory transition, which fails to generate maturation stage ameloblasts. At the onset of what should be enamel maturation, a cyst forms that separates flattened ameloblasts from the enamel surface. The maturation stage fails completely.

Sonic Hedgehog Signaling and Tooth Development.

Sonic hedgehog (Shh) is a secreted protein with important roles in mammalian embryogenesis. During tooth development, Shh is primarily expressed in the dental epithelium, from initiation to the root formation stages. A number of studies have analyzed the function of Shh signaling at different stages of tooth development and have revealed that Shh signaling regulates the formation of various tooth components, including enamel, dentin, cementum, and other soft tissues. In addition, dental mesenchymal cells positive for Gli1, a downstream transcription factor of Shh signaling, have been found to have stem cell properties, including multipotency and the ability to self-renew. Indeed, Gli1-positive cells in mature teeth appear to contribute to the regeneration of dental pulp and periodontal tissues. In this review, we provide an overview of recent advances related to the role of Shh signaling in tooth development, as well as the contribution of this pathway to tooth homeostasis and regeneration.

ADAM10 is Expressed by Ameloblasts, Cleaves the RELT TNF Receptor Extracellular Domain and Facilitates Enamel Development.

MMP20 cleaves cadherins and may facilitate cell movement, however MMP20 is not known to cleave tight junction or desmosome proteins. Ameloblasts had not previously been screened for membrane anchored proteases that could contribute to cell movement. Here we performed a PCR screen for proteolyticlly active A Disintegrin And Metalloproteinase (ADAM) family members. These proteinases are termed sheddases because they have a transmembrane domain and their catalytic domain on the cell surface can function to release anchored proteins. Significantly, ADAMs can be targeted to specific substrates on the cell membrane through their interaction with tetraspanins. Six ADAMs (ADAM8, 9, 10, 15, 17, 19) were expressed in mouse enamel organs. We show that Adam10 expression begins in the apical loop, continues through the secretory stage and abruptly ends at the transition stage when ameloblast migration ceases. ADAM10 cleaves cadherins and tight junction plus desmosome proteins and is well characterized for its role in cell movement. ADAM10 facilitated LS8 cell migration/invasion through a Matrigel coated membrane and we demonstrate that ADAM10, but not ADAM17 cleaves the RELT extracellular domain. This striking result is significant because RELT mutations cause amelogenesis imperfecta (AI) and this directly links ADAM10 to an important role in enamel development.

Peptide-Mediated Biomimetic Regrowth of Human Enamel In Situ.

Mimicking the dynamics of mineral loss and gain involved in dental caries formation can help us evaluate and compare the mineralization efficacy of different treatment agents used in enamel remineralization. Here, we offer an abridged study design outlining the preparation of tooth samples, creation of artificial dental lesions, application of a peptide, and characterization of the regrown enamel-like mineral layer.

Numerical investigations of bone remodelling around the mouse mandibular molar primordia.

The formation of the alveolar bone, which houses the dental primordia, and later the roots of tooth, may serve as a model to approach general questions of alveolar bone formation. In this respect, this study aimed to investigate the potential interactions between the alveolar bone formation and tooth eruption by using finite element (FE) methods, and to figure out whether the expanding tooth systems induce shear stresses that lead to alveolar bone formation. 3D geometric surface models were generated from the 3D histological data of the heads of mice (C57 Bl/6J) ranging from stages embryonic (E) to postnatal (P) stages E15 to P20 using the reconstruction software 3-Matic. Bone, dentin, enamel and dental follicle around the primordia were generated and converted into 3D FE models. Models were imported into the FE software package MSC.Marc/Mentat. As material parameters of embryonic dentine, pulp, enamel, dental follicle, and bony structures basically are unknown, these were varied from 1% to 100% of the corresponding known material parameters for humans and a sensitivity analysis was performed. Surface loads were applied to the outside surface of dental follicle ranging from 0.1 to 5.0N/mm2. The validity of the model was analysed by comparing the activity pattern of the alveolar bone as determined in the histological study with the loading pattern from the numerical analysis. The results show that when varying the surface loads, the distribution of shear stresses remained same, and while varying the material properties of the hard tissues, the location of highest shear stresses remained stable. Comparison of the histologically determined growth regions with the distribution of shear stresses computed in the numerical model showed a very close agreement. The results provide a strong proof to support Blechschmidt's hypothesis that the bone in general is created under the influence of shear forces.

Ablation of Runx2 in Ameloblasts Suppresses Enamel Maturation in Tooth Development.

Runt-related transcription factor 2 (Runx2) is involved in the early stage of tooth development. However, only few studies have reported the role of Runx2 in enamel development, which may be attributed to that Runx2 full knockout mice cannot survive after birth. In the present study, we successfully established a Runx2-deficient mouse model using a conditional knockout (cKO) method. We observed a significant reduction in the degree of mineralization and the decreased size of enamel rods in cKO mice. Histological analysis showed the retained enamel proteins in enamel layer at maturation stage in cKO molars. Further analysis by qRT-PCR revealed that the expressions of genes encoding enamel structure proteins, such as amelogenin (AMELX), ameloblastin (AMBN) and enamelin (ENAM), were increased in cKO enamel organs. On the other hand, the expression of kallikrein-related peptidase-4 (KLK4) at the mRNA and protein levels was dramatically decreased from late secretory stage to maturation stage in cKO enamel organs, while the expression of matrix metalloproteinase-20 (MMP-20) was not significantly altered. Finally, immunohistochemistry indicated that the uptake of amelogenins by ameloblasts was significantly decreased in cKO mice. Taken together, Runx2 played critical roles in controlling enamel maturation by increasing synthesis of KLK4 and decreasing synthesis of AMELX, AMBN and ENAM.

The effect of a single injection of irinotecan on the development of enamel in the Wistar rats.

Cancer is the second most frequent cause of death in children. Because the prognosis for childhood malignancies has improved, attention has now focused on long-term consequences of cancer treatment. The immediate effects of chemotherapy on soft tissues have been well described; however, there is less information about long-term effects of chemotherapy on the development of dental tissues. To test the association between the effect of chemotherapy on enamel development, we examined two groups of rats: one that had received an intraperitoneal dose of 200 mg/kg of irinotecan, whereas the other (control) group had received vehicle only. Rats were killed at 6, 48 and 96 hr post-injection; the mandibles dissected out, fixed for histological evaluation and scanned for mineralization defects by Micro-CT. Our results showed structural changes in the ameloblast layer along with a significant reduction in mineralization and thickness of enamel at 96 hr after chemotherapy. These data demonstrate that irinotecan induces structural changes in forming enamel that become apparent after anticancer chemotherapy treatment.

Bicarbonate Transport During Enamel Maturation.

Amelogenesis (tooth enamel formation) is a biomineralization process consisting primarily of two stages (secretory stage and maturation stage) with unique features. During the secretory stage, the inner epithelium of the enamel organ (i.e., the ameloblast cells) synthesizes and secretes enamel matrix proteins (EMPs) into the enamel space. The protein-rich enamel matrix forms a highly organized architecture in a pH-neutral microenvironment. As amelogenesis transitions to maturation stage, EMPs are degraded and internalized by ameloblasts through endosomal-lysosomal pathways. Enamel crystallite formation is initiated early in the secretory stage, however, during maturation stage the more rapid deposition of calcium and phosphate into the enamel space results in a rapid expansion of crystallite length and mineral volume. During maturation-stage amelogenesis, the pH value of enamel varies considerably from slightly above neutral to acidic. Extracellular acid-base balance during enamel maturation is tightly controlled by ameloblast-mediated regulatory networks, which include significant synthesis and movement of bicarbonate ions from both the enamel papillary layer cells and ameloblasts. In this review we summarize the carbonic anhydrases and the carbonate transporters/exchangers involved in pH regulation in maturation-stage amelogenesis. Proteins that have been shown to be instrumental in this process include CA2, CA6, CFTR, AE2, NBCe1, SLC26A1/SAT1, SLC26A3/DRA, SLC26A4/PDS, SLC26A6/PAT1, and SLC26A7/SUT2. In addition, we discuss the association of miRNA regulation with bicarbonate transport in tooth enamel formation.

Ion Transport by Ameloblasts during Amelogenesis.

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 Ca2+. By cyclically interrupting Ca2+ 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 Ca2+-poor crystals, removing impurities as Mg2+ and carbonates, and recrystallizing into more acid-resistant crystals.

Circadian Rhythm Regulates Development of Enamel in Mouse Mandibular First Molar.

Rhythmic incremental growth lines and the presence of melatonin receptors were discovered in tooth enamel, suggesting possible role of circadian rhythm. We therefore hypothesized that circadian rhythm may regulate enamel formation through melatonin receptors. To test this hypothesis, we examined expression of melatonin receptors (MTs) and amelogenin (AMELX), a maker of enamel formation, during tooth germ development in mouse. Using qRT-PCR and immunocytochemistry, we found that mRNA and protein levels of both MTs and AMELX in normal mandibular first molar tooth germs increased gradually after birth, peaked at 3 or 4 day postnatal, and then decreased. Expression of MTs and AMELX by immunocytochemistry was significantly delayed in neonatal mice raised in all-dark or all-light environment as well as the enamel development. Furthermore, development of tooth enamel was also delayed showing significant immature histology in those animals, especially for newborn mice raised in all daylight condition. Interestingly, disruption in circadian rhythm in pregnant mice also resulted in delayed enamel development in their babies. Treatment with melatonin receptor antagonist 4P-PDOT in pregnant mice caused underexpression of MTs and AMELX associated with long-lasting deficiency in baby enamel tissue. Electromicroscopic evidence demonstrated increased necrosis and poor enamel mineralization in ameloblasts. The above results suggest that circadian rhythm is important for normal enamel development at both pre- and postnatal stages. Melatonin receptors were partly responsible for the regulation.

Full Spectrum of Postnatal Tooth Phenotypes in a Novel Irf6 Cleft Lip Model.

Clefting of the lip, with or without palatal involvement (CLP), is associated with a higher incidence of developmental tooth abnormalities, including hypodontia and supernumerary teeth, aberrant crown and root morphologies, and enamel defects, although the underlying mechanistic link is poorly understood. As most CLP genes are expressed throughout the oral epithelium, the authors hypothesized that the expression of CLP genes may persist in the dental epithelium and thus, in addition to their earlier role in labiopalatine development, may play an important functional role in subsequent tooth patterning and amelogenesis. To address this, the authors generated a unique conditional knockout model involving the major CLP gene, Irf6, that overcomes the previously reported perinatal lethality to enable assessment of any posteruption dental phenotypes. A dental epithelium-specific Irf6 conditional knockout (Irf6-cKO) mouse was generated via a Pitx2-Cre driver line. Dental development was analyzed by microcomputed tomography, scanning electron microscopy, histology, immunohistochemistry, and quantitative polymerase chain reaction. Irf6-cKO mice displayed variable hypodontia, occasional supernumerary incisors and molars, as well as crown and root patterning anomalies, including peg-shaped first molars and taurodontic and C-shaped mandibular second molars. Enamel density was reduced in preeruption Irf6-cKO mice, and some shearing of enamel rods was noted in posteruption incisors. There was also rapid attrition of Irf6-cKO molars following eruption. Histologically, Irf6-cKO ameloblasts exhibited disturbances in adhesion and polarity, and delayed enamel formation was confirmed immunohistochemically. Altered structure of Hertwig's epithelial root sheath was also observed. These data support a role for IRF6 in tooth number, crown and root morphology and amelogenesis that is likely due to a functional role of Irf6 in organization and polarity of epithelial cell types. This data reinforce the notion that various isolated tooth defects could be considered part of the CLP spectrum in relatives of an affected individual.

Infiltrante resinoso vs Microabrasão no manejo de lesões de mancha branca: relato de caso / Resin infiltration vs microabrasion in white-spot lesions treatment: case report

Objetivo: Apresentar caso clínico de tratamento estético das lesões de mancha branca após tratamento ortodôntico nos dentes superiores anteriores através de duas técnicas minimamente invasivas usando o sistema de infiltração de resina e microabrasão. Relato de caso: Paciente com 18 anos de idade apresentava lesões de manchas brancas inativas nos dentes 11, 12, 13, 21, 22, 23. O sistema de infiltrante de resina Icon (DMG, Hamburgo, Alemanha) foi utilizado nos dentes 11, 12, 13, enquanto os dentes 21, 22, 23 foram submetidos à microabrasão com Whiteness RM (FGM, Joinville, Santa Catarina, Brasil).Ambos os protocolos foram utilizados de acordo com as recomendações do fabricante.Nos dentes 21 e 22, o produto para microabrasão foi aplicado com espátula que acompanha o kit, enquanto que no dente 23 utilizou-se o mesmo produto aplicado com taça de borracha em baixa rotação, todos os procedimentos executados por um único operador. A microabrasão com taça de borracha proporcionou uma superfície mais lisa e homogênea.Ambos os produtos tiveram resultados satisfatórios na resolução estética das lesões de mancha branca após um ano de acompanhamento. Conclusão: Os dois produtos apresentam bom desempenho e resolutividade para os problemas estéticos de lesões de manchas brancas inativas, entretanto deve-se levar em conta o tempo clínico, toxicidade, a atividade da lesão e a possibilidade de desgaste da estrutura dentária.

Objective: To present a case report of aesthetic treatment of post orthodontic white--spot lesions in the anterior superior teeth through two minimally invasive technique susing resin infiltration system or microabrasion. Case report: Patient 17 years of age had inactive white-spots lesions on teeth 11, 12, 13, 21, 22, 23. The teeth 11, 12, 13 received the resin infiltrating system Icon (DMG, Hamburg, Germany) while the teeth 21, 22, 23were submitted to microabrasion with Whiteness RM (FGM, Joinville, Santa Catarina, Brazil). Both protocols were used according to manufacturer’s recommendations. In the teeth 21 and 22, the product of the microabrasion was applied with a spatula that accompanied the kit, while the tooth 23 received the same product applied with a rubbercup at low speed turbine, all by a single operator. The microabrasion with rubber cupoccasioned a more smooth and homogeneous surface. Both products had satisfactory results in aesthetic resolution of the white-spots lesions, for a 1-year follow-up. Conclusion:Although both products are able to resolve aesthetic problems of inactive white-spots lesions, it should be taken into account the clinical time, toxicity, the activity ofthe lesion and the possibility of wear of the tooth structure.

Beta-catenin is essential for ameloblast movement during enamel development.

Beta-catenin is a multifunctional protein that plays key roles in cadherin-based cell adherens junctions and in the Wnt signaling pathway. The canonical Wnt/ß-catenin pathway can regulate transcription factors that control cell movement/invasion. We investigated whether ß-catenin regulates ameloblast movement through canonical Wnt signaling. The morphological and physical properties of enamel were assessed in enamel from control and ß-catenin conditional knockout (cKO) mice. Ameloblast-lineage cells (ALC) were used to investigate the potential roles of ß-catenin in cell migration and in E-cadherin expression. Compared with controls, incisors from ß-catenin cKO mice were short, blunt, and where enamel was present, it was soft and malformed. Scanning electron microscopy revealed a dysplastic rod pattern within the enamel of incisors from ß-catenin cKO mice, and Vickers microhardness measurements confirmed that mice with ß-catenin ablated from their enamel organ had enamel that was significantly softer than normal. Amelogenesis was disrupted in the absence of ß-catenin and the ameloblasts did not differentiate properly. We further demonstrated that migration of ALCs was inhibited in vitro and that E-cadherin expression was significantly up-regulated when ALCs were treated with the ß-catenin inhibitor, ICG-001. Beta-catenin ablation causes enamel malformation in mice and this phenotype may occur, in part, by a lack of ameloblast differentiation and/or movement necessary to form the decussating enamel rod structure.

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-3ß 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.

The Role of Chronic Exposure to Amoxicillin/Clavulanic Acid on the Developmental Enamel Defects in Mice.

Amoxicillin used in early childhood may be associated with enamel hypomineralization. Our aim was to assess disturbances of amelogenesis in mice lower incisors induced by chronic administration of amoxicillin/clavulanic acid (AMC). Twenty-eight C57BL/6 male mice, of similar age, randomly divided into a control and 3 treatment groups (n = 7) received subcutaneous injection, once per day, for 60 days: 50, 100, and 150 mg/kg BW of AMC. Scanning electron microscopy/energy dispersive X-ray spectroscopy analysis in AMC treatment groups showed higher content in F and a decrease in P and Ca. Morphology changes ranged from scratched patterns, and small isolated pits-like enamel loss, to generalized demineralized enamel surface, giving a rough, foamy, scaly, or even cracked eggshell appearance to the affected areas. Histological analysis showed disturbances of maturation ameloblasts, which were less organized, with increased amounts of clear vacuoles in the cytoplasm and slightly more elongated and less condensed nucleus. Additionally, they were often detached from the enamel matrix. Transitional ameloblasts formed underlying the cysts of varied sizes. In conclusion, AMC dose-dependently affect ameloblast functions especially in the maturation phase, causing hypomineralized enamel formation with quantitative and/or qualitative defects.

SLC26A Gene Family Participate in pH Regulation during Enamel Maturation.

The bicarbonate transport activities of Slc26a1, Slc26a6 and Slc26a7 are essential to physiological processes in multiple organs. Although mutations of Slc26a1, Slc26a6 and Slc26a7 have not been linked to any human diseases, disruption of Slc26a1, Slc26a6 or Slc26a7 expression in animals causes severe dysregulation of acid-base balance and disorder of anion homeostasis. Amelogenesis, especially the enamel formation during maturation stage, requires complex pH regulation mechanisms based on ion transport. The disruption of stage-specific ion transporters frequently results in enamel pathosis in animals. Here we present evidence that Slc26a1, Slc26a6 and Slc26a7 are highly expressed in rodent incisor ameloblasts during maturation-stage tooth development. In maturation-stage ameloblasts, Slc26a1, Slc26a6 and Slc26a7 show a similar cellular distribution as the cystic fibrosis transmembrane conductance regulator (Cftr) to the apical region of cytoplasmic membrane, and the distribution of Slc26a7 is also seen in the cytoplasmic/subapical region, presumably on the lysosomal membrane. We have also examined Slc26a1 and Slc26a7 null mice, and although no overt abnormal enamel phenotypes were observed in Slc26a1-/- or Slc26a7-/- animals, absence of Slc26a1 or Slc26a7 results in up-regulation of Cftr, Ca2, Slc4a4, Slc4a9 and Slc26a9, all of which are involved in pH homeostasis, indicating that this might be a compensatory mechanism used by ameloblasts cells in the absence of Slc26 genes. Together, our data show that Slc26a1, Slc26a6 and Slc26a7 are novel participants in the extracellular transport of bicarbonate during enamel maturation, and that their functional roles may be achieved by forming interaction units with Cftr.

Ameloblast Modulation and Transport of Cl⁻, Na⁺, and K⁺ during Amelogenesis.

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.

p38α MAPK is required for tooth morphogenesis and enamel secretion.

An improved understanding of the molecular pathways that drive tooth morphogenesis and enamel secretion is needed to generate teeth from organ cultures for therapeutic implantation or to determine the pathogenesis of primary disorders of dentition (Abdollah, S., Macias-Silva, M., Tsukazaki, T., Hayashi, H., Attisano, L., and Wrana, J. L. (1997) J. Biol. Chem. 272, 27678-27685). Here we present a novel ectodermal dysplasia phenotype associated with conditional deletion of p38α MAPK in ectodermal appendages using K14-cre mice (p38α(K14) mice). These mice display impaired patterning of dental cusps and a profound defect in the production and biomechanical strength of dental enamel because of defects in ameloblast differentiation and activity. In the absence of p38α, expression of amelogenin and ß4-integrin in ameloblasts and p21 in the enamel knot was significantly reduced. Mice lacking the MAP2K MKK6, but not mice lacking MAP2K MKK3, also show the enamel defects, implying that MKK6 functions as an upstream kinase of p38α in ectodermal appendages. Lastly, stimulation with BMP2/7 in both explant culture and an ameloblast cell line confirm that p38α functions downstream of BMPs in this context. Thus, BMP-induced activation of the p38α MAPK pathway is critical for the morphogenesis of tooth cusps and the secretion of dental enamel.

Associação dos indicadores socioeconômicos, fatores pre e perinatais na ocorrência de defeitos de desenvolvimento de esmalte na dentição decídua: estudo de base populacional / Socioeconomic indicators, pre and perinatal factors associated with developmental defects of enamel in primary teeth: a population-based study

Os defeitos de desenvolvimento de esmalte (DDE) são alterações comuns na dentição decídua e podem estar associados a intercorrências nos períodos pré, peri e pós natais bem como a indicadores socioeconômicos. A literatura é escassa de evidências científicas de base populacional, sendo que a maioria das pesquisas é realizada principalmente com amostras específicas. O objetivo deste estudo foi estimar a prevalência de DDE em crianças de cinco anos de idade e verificar a associação com prematuridade, peso ao nascer, intercorrências na gravidez (síndrome hipertensivo, infeções urinarias e diabetes gestacional) e indicadores socioeconômicos (renda mensal per capita, escolaridade materna, tipo de escola frequentada pela criança). Realizou-se um estudo transversal representativo com uma amostra de 1350 crianças de cinco anos de idade em Belo Horizonte - MG. Brasil. Após o consentimento dos pais, as crianças foram examinadas para o diagnóstico do DDE utilizando os critérios do índice DDE modificado (FDI,1992), e, através de um questionário, as mães forneceram os dados socioeconômicos bem como peso ao nascer, tempo de gestação e às intercorrências durante a gravidez relacionadas a parto pre-termo e baixo peso. A análise dos dados foi realizada utilizando-se o programa SPSS para Windows versão 19.0 e incluiu a distribuição de frequência, qui-quadrado de Pearson e Teste Exato de Fisher e regressão de Poisson com variância robusta adotando um valor de p <0,05. A prevalência de DDE foi de 40,6%. A opacidade demarcada foi o tipo de defeito mais frequente (22,7%) Através da análise bivariada, verificou-se associação estatisticamente significativa entre o DDE e gênero, peso ao nascer, idade da mãe durante a gestação, escolaridade materna, intercorrência na gravidez (síndrome hipertensivo) e tipo de escola da criança. A partir do modelo de regressão de Poisson ajustado, observou-se maior prevalência de DDE entre as crianças do gênero masculino (RP: 1,177, 95% IC 1,033 ­ 1,342), com baixo peso ao nascer (RP: 1,387; 95% IC 1,161 ­ 1,656) e muito baixo peso ao nascer (RP: 1,667; 95% IC: 1,150 ­ 2,416). A partir destes resultados conclui-se que os defeitos de desenvolvimento de esmalte foram mais comuns entre as crianças do gênero masculino, com peso ao nascer baixo ou muito baixo

The developmental defects of enamel (DDE) are common changes in the primary dentition and may be associated with complications in the pre, peri and post-natal care as well as with socioeconomic indicators. Literature is scarce in scientific evidence in population base, once the majority of studies are mainly performed with specific samples. The aim of this cross-sectional study was to estimate the prevalence of DDE in five years old children and the association with premature, complications during pregnancy birth weight and socioeconomic indicators (family income, maternal education, type of preschool attended by children). A cross-sectional study was conducted with a representative sample of 1350 children of five years old of Belo Horizonte - MG. Brazil. After parental consent, children were examined for the diagnosis of DDE using DDE index modificated (FDI, 1992), and through a questionnaire mothers provided socioeconomic data, birth weight and premature and complications during pregnancy. Data analysis was performed using SPSS for Windows 19.0, and included frequency distribution, chi- square test and Fisher's exact test and Poisson regression with robust variance (p < 0.05). The prevalence of DDE was 40.6 %. The demarcated opacity was the most common type of defect (22.7%) by bi-variate analysis; there was also a statistically significant association between developmental defects of enamel and gender, weight birth, complications during pregnancy (Arterial hypertension), mother´s age in the pregnancy, maternal education, type of school of children. The Poisson regression model adjusted showed that there was a higher prevalence of enamel defects among male children (PR: 1.177, 95% CI 1.033 to 1.342), low weight (PR: 1.387, 95% CI 1.61 to 1.656) and very low (PR: 1.667, 95% CI: 1.150 to 2.416). Out of these results it is concluded that the development of enamel defects were more common among male children with low or very low birth weight