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BACKGROUND: Previous studies have shown a correlation between fluoride concentrations in urine and community water fluoride concentrations. However, there are no studies of the relationship between community water fluoridation, urine, serum, and amniotic fluid fluoride concentrations in pregnant women in the US. The aim of this study was to determine the relationship between maternal urine fluoride (MUF), maternal urine fluoride adjusted for specific gravity (MUFSG), maternal serum fluoride (MSF), amniotic fluid fluoride (AFF) concentrations during pregnancy, and community water fluoridation in Northern California. METHODS: Archived samples of urine, serum and amniotic fluid collected from second trimester pregnant women in Northern California from 47 different communities in Northern California and one from Montana (n = 48), were analyzed for fluoride using an ion specific electrode following acid microdiffusion. Womens addresses were matched to publicly reported water fluoride concentrations. We examined whether fluoride concentrations in biospecimens differed by fluoridation status of the community water, and determined the association between water fluoride concentrations and biospecimen fluoride concentrations using linear regression models adjusted for maternal age, smoking, Body Mass Index (BMI), race/ethnicity, and gestational age at sample collection. RESULTS: Fluoride concentrations in the community water supplies ranged from 0.02 to 1.00 mg/L. MUF, MSF , and AFF concentrations were significantly higher in pregnant women living in communities adhering to the U.S. recommended water fluoride concentration (0.7 mg/L), as compared with communities with less than 0.7 mg/L fluoride in drinking water. When adjusted for maternal age, smoking status, BMI, race/ethnicity, and gestational age at sample collection, a 0.1 mg/L increase in community water fluoride concentration was positively associated with higher concentrations of MUF (B = 0.052, 95% CI:0.019,0.085), MUFSG (B = 0.028, 95% CI: -0.006, 0.062), MSF (B = 0.001, 95% CI: 0.000, 0.003) and AFF (B = 0.001, 95% CI: 0.000, 0.002). CONCLUSIONS: We found universal exposure to fluoride in pregnant women and to the fetus via the amniotic fluid. Fluoride concentrations in urine, serum, and amniotic fluid from women were positively correlated to public records of community water fluoridation. Community water fluoridation remains a major source of fluoride exposure for pregnant women living in Northern California.

The process of amelogenesis is well understood at the histomorphological level, but the regulation of crystal formation and the underlying molecular mechanisms are still largely unknown. Ameloblast cells derived from the oral epithelium regulate enamel maturation. In addition to the secretion of matrix proteins and the transport of minerals, pH regulation is also essential as the formation of hydroxyapatite crystals generates large quantities of H+ ions, which must be neutralized by the secretion of HCO3−. During the maturation phase, the phenomenon of pH cycling is of paramount importance, during which a rearrangement of tight-junctions can also be observed. Genetic or environmental impairment of transport and regulatory processes (e.g. dental fluorosis) leads to the development of enamel defects. To explore these processes, we have developed an in vitro model suitable for functional studies. Central elements of this model are that differentiated ameloblast-derived cells cultured on a permeable membrane express ameloblast-specific protein markers, form tight junctions, become functionally polarized, and are capable of vectorial HCO3− secretion. The results of our own experimental work contribute to a better understanding of the molecular mechanism of pH cycling of ameloblast cells.

The Encouraging Novel Amelogenesis Models and Ex vivo cell Lines (ENAMEL) Development workshop was held on 23 June 2017 at the Bethesda headquarters of the National Institute of Dental and Craniofacial Research (NIDCR). Discussion topics included model organisms, stem cells/cell lines, and tissues/3D cell culture/organoids. Scientists from a number of disciplines, representing institutions from across the United States, gathered to discuss advances in our understanding of enamel, as well as future directions for the field.

Tooth enamel is mineralized through the differentiation of multiple dental epithelia including ameloblasts and the stratum intermedium (SI), and this differentiation is controlled by several signaling pathways. Previously, we demonstrated that the transcriptional coactivator Mediator 1 (MED1) plays a critical role in enamel formation. For instance, conditional ablation of Med1 in dental epithelia causes functional changes in incisor-specific dental epithelial stem cells, resulting in mineralization defects in the adult incisors. However, the molecular mechanism by which Med1 deficiency causes these abnormalities is not clear. Here, we demonstrated that Med1 ablation causes early SI differentiation defects resulting in enamel hypoplasia of the Med1-deficient molars. Med1 deletion prevented Notch1-mediated differentiation of the SI cells resulting in decreased alkaline phosphatase (ALPL), which is essential for mineralization. However, it does not affect the ability of ameloblasts to produce enamel matrix proteins. Using the dental epithelial SF2 cell line, we demonstrated that MED1 directly activates transcription of the Alpl gene through the stimulation of Notch1 signaling by forming a complex with cleaved Notch1-RBP-Jk on the Alpl promoter. These results suggest that MED1 may be essential for enamel matrix mineralization by serving as a coactivator for Notch1 signaling regulating transcription of the Alpl gene.

Abstract Background Previous studies have shown a correlation between fluoride concentrations in urine and community water fluoride concentrations. However, there are no studies of the relationship between community water fluoridation, urine, serum, and amniotic fluid fluoride concentrations in pregnant women in the US. The aim of this study was to determine the relationship between maternal urine fluoride (MUF), maternal urine fluoride adjusted for specific gravity (MUFSG), maternal serum fluoride (MSF), amniotic fluid fluoride (AFF) concentrations during pregnancy, and community water fluoridation in Northern California. Methods Archived samples of urine, serum and amniotic fluid collected from second trimester pregnant women in Northern California from 47 different communities in Northern California and one from Montana (n = 48), were analyzed for fluoride using an ion specific electrode following acid microdiffusion. Women’s addresses were matched to publicly reported water fluoride concentrations. We examined whether fluoride concentrations in biospecimens differed by fluoridation status of the community water, and determined the association between water fluoride concentrations and biospecimen fluoride concentrations using linear regression models adjusted for maternal age, smoking, Body Mass Index (BMI), race/ethnicity, and gestational age at sample collection. Results Fluoride concentrations in the community water supplies ranged from 0.02 to 1.00 mg/L. MUF, MSF , and AFF concentrations were significantly higher in pregnant women living in communities adhering to the U.S. recommended water fluoride concentration (0.7 mg/L), as compared with communities with less than 0.7 mg/L fluoride in drinking water. When adjusted for maternal age, smoking status, BMI, race/ethnicity, and gestational age at sample collection, a 0.1 mg/L increase in community water fluoride concentration was positively associated with higher concentrations of MUF (B = 0.052, 95% CI:0.019,0.085), MUFSG (B = 0.028, 95% CI: -0.006, 0.062), MSF (B = 0.001, 95% CI: 0.000, 0.003) and AFF (B = 0.001, 95% CI: 0.000, 0.002). Conclusions We found universal exposure to fluoride in pregnant women and to the fetus via the amniotic fluid. Fluoride concentrations in urine, serum, and amniotic fluid from women were positively correlated to public records of community water fluoridation. Community water fluoridation remains a major source of fluoride exposure for pregnant women living in Northern California.

Background: Amelogenesis, the formation of dental enamel, is well understood at the histomorphological level but the underlying molecular mechanisms are poorly characterized. Ameloblasts secrete enamel matrix proteins and Ca2+, and also regulate extracellular pH as the formation of hydroxyapatite crystals generates large quantities of protons. Genetic or environmental impairment of transport and regulatory processes (e.g. dental fluorosis) leads to the development of enamel defects such as hypomineralization.Aims: Our aims were to optimize the culture conditions for the three-dimensional growth of ameloblast-derived HAT-7 cells and to test the effects of fluoride exposure on HAT-7 spheroid formation.Methods: To generate 3D HAT-7 structures, cells were dispersed and plated within a Matrigel extracellular matrix scaffold and incubated in three different culture media. Spheroid formation was then monitored over a two-week period. Ion transporter and tight-junction protein expression was investigated by RT-qPCR. Intracellular Ca2+ and pH changes were measured by microfluorometry using the fluorescent dyes fura-2 and BCECF.Results: A combination of Hepato-STIM epithelial cell differentiation medium and Matrigel induced the expansion and formation of 3D HAT-7 spheroids. The cells retained their epithelial cell morphology and continued to express both ameloblast-specific and ion transport-specific marker genes. Furthermore, like two-dimensional HAT-7 monolayers, the HAT-7 spheroids were able to regulate their intracellular pH and to show intracellular calcium responses to extracellular stimulation. Finally, we demonstrated that HAT-7 spheroids may serve as a disease model for studying the effects of fluoride exposure during amelogenesis.Conclusion: In conclusion, HAT-7 cells cultivated within a Matrigel extracellular matrix form three-dimensional, multi-cellular, spheroidal structures that retain their functional capacity for pH regulation and intracellular Ca2+ signaling. This new 3D model will allow us to gain a better understanding of the molecular mechanisms involved in amelogenesis, not only in health but also in disorders of enamel formation, such as those resulting from fluoride exposure.

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

The aim of the study was to investigate the efficacy of the use of xylitol-containing tooth-wipes in preventing dental caries in young children. In a double-blinded randomized controlled clinical trial, 44 mothers with active caries and their 6- to 35-month-old children were randomized to xylitol-wipe or placebo-wipe groups. The children's caries scores were recorded at baseline and 1 year. Salivary levels of mutans streptococci and lactobacilli were enumerated at baseline, 3, 6, and 12 months. Data were analyzed by intent-to-treat modeling with imputation for caries lesions and a linear mixed-effect model for bacterial levels. Significantly fewer children in the xylitol-wipe group had new caries lesions at 1 year compared with those in the placebo-wipe group (P < 0.05). No significant differences between the two groups were observed in levels of mutans streptococci and lactobacilli at all time-points. Daily xylitol-wipe application significantly reduced the caries incidence in young children as compared with wipes without xylitol, suggesting that the use of xylitol wipes may be a useful adjunct for caries control in infants (Clinicaltrials.gov registration number CT01468727).

Amelogenin gene organization varies from 6 exons (1,2,3,5,6,7) in amphibians and sauropsids to 10 in rodents. The additional exons are exons 4, 8, 9, and "4b", the latter being as yet unidentified in AMELX transcripts. To learn more about the evolutionary origin of these exons, we used an in silico approach to find them in 39 tetrapod genomes. AMEL organization with 6 exons was the ancestral condition. Exon 4 was created in an ancestral therian (marsupials + placentals), then exon 9 in an ancestral placental, and finally exons "4b" and 8 in rodents, after divergence of the squirrel lineage. These exons were either inactivated in some lineages or remained functional: Exon 4 is functional from artiodactyls onward; exon 9 is known, to date, only in rodents, but could be coding in various mammals; and exon "4b" was probably coding in some rodents. We performed PCR of cDNA isolated from mouse and human tooth buds to identify the presence of these transcripts. A sequence analogous to exon "4b", and to exon 9, could not be amplified from the respective tooth cDNA, indicating that even though sequences similar to these exons are present, they are not transcribed in these species.

Amelogenins containing exons 8 and 9 are alternatively spliced variants of amelogenin. Some amelogenin spliced variants have been found to promote pulp regeneration following pulp exposure. The function of the amelogenin spliced variants with the exons 8 and 9 remains unknown. In this study, we synthesized recombinant leucine rich amelogenin peptide (LRAP, A-4), LRAP plus exons 8 and 9 peptide (LRAP 8, 9) or exons 8 and 9 peptide (P89), to determine their effects on odontoblasts. In vivo analyses were completed following the insertion of agarose beads containing LRAP or LRAP 8, 9 into exposed cavity preparations of rat molars. After 8, 15 or 30 days' exposure, the pulp tissues were analyzed for changes in histomorphometry and cell proliferation by PCNA stainings. In vitro analyses included the effects of the addition of the recombinant proteins or peptide on cell proliferation, differentiation and adhesion of postnatal human dental pulp cells (DPCs). These studies showed that in vivo LRAP 8, 9 enhanced the reparative dentin formation as compared to LRAP. In vitro LRAP 8, 9 promoted DPC proliferation and differentiation to a greater extent than LRAP. These data suggest that amelogenin exons 8 and 9 may be useful in amelogenin-mediated pulp repair.

Enamel fluorosis has been related to an increase in the amount of amelogenin in fluorosed enamel compared with normal enamel in the maturation stage. In this study we tested the hypothesis that fluoride incorporated into carbonated apatite alters amelogenin hydrolysis. Recombinant human amelogenin (rh174) was allowed to bind to 0.15 mg of carbonated hydroxyapatite (CAP) or to fluoride-containing carbonated hydroxyapatite (F-CAP) synthesized to contain 100, 1,000, or 4,000 ppm F(-). After 3 h of digestion with recombinant human matrix metalloproteinase 20 (MMP20) or kallikrein-related peptidase 4 (KLK4), bound protein was characterized by reverse-phase high-performance liquid chromatography (HPLC). Proteolytic fragments of amelogenin formed after 24h of digestion with MMP20 of KLK 4 were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The hydrolysis, by both MMP20 and KLK4, of amelogenin bound to F100-CAP was significantly reduced in a dose-dependent manner compared with the hydrolysis of amelogenin bound to CAP. After 24 h of hydrolysis, a similar number of MMP20 cleavage sites was found for amelogenin bound to CAP and amelogenin bound to F100-CAP; however, 24 fewer KLK4 cleavage sites were identified for amelogenin bound to F100-CAP than for amelogenin bound to CAP. These results suggest that the reduced hydrolysis of amelogenins in fluorosed enamel may be partially caused by the increased fluoride content in fluoride-containing apatite, contributing to the hypomineralized enamel matrix phenotype observed in fluorosed enamel.

Abstract Background Loss of functional UBE3A, an E3 protein ubiquitin ligase, causes Angelman syndrome (AS), a neurodevelopmental disorder characterized by severe developmental delay, speech impairment, epilepsy, movement or balance disorder, and a characteristic behavioral pattern. We identified a novel UBE3A sequence variant in a large family with eight affected individuals, who did not meet the clinical AS criteria. Methods Detailed clinical examination and genetic analysis was performed to establish the phenotypic diversity and the genetic cause. The function of the mutant UBE3A protein was assessed with respect to its subcellular localization, stability, and E3 ubiquitin ligase activity. Results All eight affected individuals showed the presence of a novel maternally inherited UBE3A sequence variant (NM_130838.4(UBE3A):c.1018‐1020del, p.(Asn340del), which is in line with a genetic AS diagnosis. Although they presented with moderate to severe intellectual disability, the phenotype did not match the clinical criteria for AS. In line with this, functional analysis of the UBE3A p.Asn340del mutant protein revealed no major deficits in UBE3A protein localization, stability, or E3 ubiquitin ligase activity. Conclusion The p.(Asn340del) mutant protein behaves distinctly different from previously described AS‐linked missense mutations in UBE3A, and causes a phenotype that is markedly different from AS. This study further extends the range of phenotypes that are associated with UBE3A loss, duplication, or mutation.

ObjectiveA single Pro-70 to Thr (p.P70T) mutation of amelogenin is known to result in hypomineralised amelogenesis imperfecta (AI). This study aims to test the hypothesis that the given mutation affects the self-assembly of amelogenin molecules and impairs their ability to conduct the growth of apatite crystals.DesignRecombinant human full-length wild-type (rh174) and p.P70T mutated amelogenins were analysed using dynamic light scattering (DLS), protein quantification assay and atomic force microscopy (AFM) before and after the binding of amelogenins to hydroxyapatite crystals. The crystal growth modulated by both amelogenins in a dynamic titration system was observed using AFM.ResultsAs compared to rh174 amelogenin, p.P70T mutant displayed significantly increased sizes of the assemblies, higher binding affinity to apatite, and decreased crystal height.ConclusionPro-70 plays an important structural role in the biologically relevant amelogenin self-assembly. The disturbed regularity of amelogenin nanospheres by this single mutation resulted in an increased binding to apatite and inhibited crystal growth.

Amelogenins, the chief proteins in enamel matrix, undergo progressive degradation by matrix metalloproteinase-20 (MMP-20) to facilitate crystal growth. Proline is the most abundant residue in amelogenin and is located upstream to all MMP-20 cleavage sites in the amelogenin sequence. Pro(41) is critical for amelogenin N-terminal processing, while the role of prolines at the amelogenin C terminus have not been determined. This study sought to elucidate the effect of the C-terminal prolines on apatite binding and MMP-20 hydrolysis. To compare apatite affinity, recombinant full-length human amelogenin (rh174) and mutated variants (P156T and P164T) were incubated with hydroxyapatite (HAP), and the unbound proteins were quantified by the Bradford assay. rh174 and mutants, as well as 3 oligopeptides, including wild-type peptide and peptides containing 2 mutations, were incubated with MMP-20 both in solution and on HAP. The digested products were analyzed by SDS-PAGE, reverse-phase high-performance liquid chromatography and mass spectrometry. Mutated amelogenins displayed a significantly lower affinity to HAP than the wild type (P156T < P164T < rH174). The proline mutation at amino acid location 164 significantly reduced the initial hydrolysis of either the amelogenins in solution or the proteins bound on HAP, which was confirmed by amelogenin oligopeptide assays. It was concluded that prolines at the amelogenin C terminus are essential for the initial processing of amelogenin and amelogenin-mineral interactions.

Amelogenin with a proline 41 to threonine mutation (P41T) is hydrolyzed at a lower rate by matrix metalloproteinase 20 (MMP20), resulting in an inherited tooth enamel defect, amelogenesis imperfecta (AI). The aim of this study was to elucidate the effect of P41T on the interactions between amelogenin and MMP20, which may contribute to the formation of this type of AI. The interactions of a recombinant wild-type human amelogenin and its P41T mutant with recombinant human MMP20 were compared by substrate competition assay, pull-down assay, and surface plasmon resonance (SPR). The results showed that the binding of the P41T mutant amelogenin for MMP20 was significantly lower than that of wild-type amelogenin. Our study supports a model in which the P41T mutation reduces the interactions between amelogenin and MMP20, leading to decreased degradation of amelogenin by MMP20, and resulting in AI.

Fluorosed enamel is more porous and less mineralized, possibly related to altered amelogenin-modulated crystal growth. The purpose of this study was to examine the role of fluoride in interactions between amelogenin and apatite crystals. Recombinant human amelogenin (rh174) was bound to carbonated hydroxyapatite containing various amounts of fluoride, and analyzed by protein assay, SDS PAGE, and AFM. Interactions between rh174 and fluoride were assayed by isothermal titration calorimetry (ITC). The initial binding rate of rh174, as well as total amount of rh174 bound to fluoride-containing carbonated hydroxyapatite, was greater than that in the control carbonated hydroxyapatite. Fluoride in solution at physiologic (5.3 micromolar, or 0.1 ppm) concentrations showed no significant effect on binding, but higher fluoride levels significantly decreased protein binding. ITC showed no interactions between fluoride and rh174. These results suggest that fluoride incorporation into the crystal lattice alters the crystal surface to enhance amelogenin binding, with no direct interactions between fluoride and amelogenin.

Ectomesenchymal stem cells derived from the dental pulp are of neural crest origin, and as such are promising sources for cell therapy and tissue engineering. For safe upscaling of these cells, microcarrier-based culturing under dynamic conditions is a promising technology. We tested the suitability of two microcarriers, non-porous Cytodex 1 and porous Cytopore 2, for culturing well characterized dental pulp stem cells (DPSCs) using a shake flask system. Human DPSCs were cultured on these microcarriers in 96-well plates, and further expanded in shake flasks for upscaling experiments. Cell viability was measured using the alamarBlue assay, while cell morphology was observed by conventional and two-photon microscopies. Glucose consumption of cells was detected by the glucose oxidase/Clark-electrode method. DPSCs adhered to and grew well on both microcarrier surfaces and were also found in the pores of the Cytopore 2. Cells grown in tissue culture plates (static, non-shaking conditions) yielded 7 × 105 cells/well. In shake flasks, static preincubation promoted cell adhesion to the microcarriers. Under dynamic culture conditions (shaking) 3 × 107 cells were obtained in shake flasks. The DPSCs exhausted their glucose supply from the medium by day seven even with partial batch-feeding. In conclusion, both non-porous and porous microcarriers are suitable for upscaling ectomesenchymal DPSCs under dynamic culture conditions.

Leucine-rich amelogenin peptide (LRAP) is an alternately spliced amelogenin. LRAP is known to bind to hydroxyapatite, and has been shown to signal mesenchymal cells to proliferate, but its function in enamel formation is unclear. The purpose of this study was to determine the calcium-binding properties and structure of recombinant human LRAP (rLRAP) compared with full-length amelogenin (rH174). rLRAP and rH174 were synthesized in Escherichia coli and purified by affinity chromatography and reverse-phase high-performance liquid chromatography. Calcium binding was measured by isothermal titration calorimetry (ITC) at pH 7.5 and 25 degrees C, and raw data were analyzed by origin 7.0 software. The structure of rLRAP was analyzed by nuclear magnetic resonance (NMR) and circular dichroism (CD) in the absence or presence of Ca2+, pH 7.5 and 4.0, at 25 degrees C. Thermodynamic values showed that rLRAP had a Ca2+-binding affinity approximately 6.4-times greater than rH174. NMR and CD data revealed that rLRAP was randomly coiled, and that this structure was not altered by Ca2+, which bound to rLRAP and rH174 via ionic interactions. Unlike r174 (beta-spiral), rLRAP had a random-coiled structure. The calcium binding and structural differences between rLRAP and rH174 suggest that these proteins have different functions in enamel biomineralization.

Amelogenins are a unique group of alternatively spliced proteins. While the full-length amelogenin is known to assemble into nanospheres and alter apatite crystal growth and alignment, the function of the leucine-rich amelogenin peptide (LRAP) in biomineralization is not understood. This study tested the hypothesis that LRAP self-assembles into a supramolecular structure and guides crystal growth similarly to the full-length protein. Synthetic LRAP and recombinant full-length amelogenin (rH175) were used at different concentrations and either immobilized onto fluoroapatite substrates (FAP) or immersed into saturated calcium-phosphate solutions. The structure of the assembled protein and the height of apatite crystals formed on the FAP template were determined using atomic force microscopy. Both LRAP and rH175 assembled into nanospheres. LRAP self-assembly, however, was only observed at concentrations of >0.5 mg ml-1 and limited to sizes between 5 and 30 nm. Apatite crystal growth was not significantly affected by LRAP, while rH175 accelerated crystal growth by up to 50-fold. The increased growth rate was only observed when rH175 precipitated at concentrations of >0.8 mg ml-1. It was concluded that the ability of amelogenins to self-assemble into nanospheres and to bind to apatite in vitro is not inevitably an indication for the ability to control apatite crystal growth.

ObjectiveTo study the mechanisms which promote the interactions of amelogenin proteins with the forming mineral to establish suitable conditions for the biomimetic synthesis of enamel in vitro.DesignSaturated calcium phosphate solutions were used in conjunction with recombinant amelogenin proteins to induce mineral formation on glass-ceramics substrates containing oriented fluoroapatite crystals (FAP). The height of mineral layers formed on these substrates within 24 h was measured by atomic force microscopy (AFM).Experimental variablesThe effect of protein concentration, pH and degree of saturation (DS) on the growth of apatite mineral was evaluated. Mineralization experiments were performed at 0, 0.4 and 1.6 mg/ml amelogenin concentrations. Mineralization solutions were used at pH values of 6.5, 7.4, 8.0 and 8.8 and DS of calcium and phosphate between 9 and 13.Outcome measureHeight and morphology of mineralized layer formed on glass-ceramic substrates as determined from AFM measurements.ResultsHomogeneous nucleation and crystal growth of thin layers on the FAP were observed, when calcium and phosphate ions were added. The height of these layers grown on (001) planes of FAP was strongly dependent on the protein concentration and pH. At concentrations of 0 and 0.4 mg/ml crystal grew 5-15 nm on the FAP, while they grew approximately to 200 nm at 1.6 mg/ml. The enhanced crystal growth was observed only at pH 6.5, 7.4 and 8.0, while layers only 20 nm thick were obtained at pH 8.8. An increase in DS resulted in uncontrolled growth of calcium phosphate mineral covering large areas of the substrate.ConclusionsProtein concentration, pH and the saturation of the mineralizing solution need to be considered carefully to provide suitable conditions for amelogenin-guided growth of apatite crystals.

The formation of aligned fibrous apatite crystals in enamel is predominantly attributed to the involvement of amelogenin proteins. We developed a model to study interactions of matrix proteins with apatite mineral in vitro and tested the hypothesis that amelogenin solubility affects the ability to induce protein-guided mineralization. Crystal growth experiments were performed on fluoroapatite (FAP) glass-ceramics in mineralizing solutions containing recombinant full-length amelogenin (rH174) at different concentrations. Using atomic force microscopy, we observed that mineral precipitated randomly on the substrate, but also formed thin layers (height, 10 nm) on FAP within 24 hrs. This growth pattern was unaffected when 0.4 mg/mL of rH174 was added. In contrast, crystals grew on FAP at a rate up to 20 times higher, at 1.6 mg/mL protein. Furthermore, nanospheres and mineral bound specifically to FAP and aligned in strings approximately parallel to the c-axis of FAP, leading us to the conclusion that amelogenin proteins indeed control direction and rate of growth of apatite in enamel.

Amelogenesis imperfecta (AI) is a group of inherited disorders with defective tooth enamel formation caused by various gene mutations. One of the mutations substitutes a cytidine for an adenine in exon 6 of the X-chromosomal amelogenin gene, which results in a proline to threonine change in the expressed amelogenin. This transformation is four amino acids N-terminal to the cleavage site for enamel matrix metalloproteinase-20 (MMP-20) in amelogenin. MMP-20 releases the tyrosine rich amelogenin peptide (TRAP) from amelogenin. This study evaluated the rate at which MMP-20 hydrolyses mutated amelogenin relative to unmutated amelogenin. A full-length recombinant human amelogenin and a mutated amelogenin with a substitution of proline by threonine were expressed and purified by ammonium sulphate precipitation and reverse phase HPLC. Recombinant metalloproteinase-20 (rMMP-20) was used to digest the recombinant proteins, which resulted in fragments with a mass predicted for TRAP. The proteolytic site was also modelled as substrates by two synthetic peptides, SYGYEPMGGWLHHQ and SYGYETMGGWLHHQ, selected from residues 36 to 49 of the amino acid sequence for amelogenin and the respective X-linked amelogenin mutant. These two peptides were labelled at their N- and C-termini respectively by using rhodamine and biotin. After digestion with MMP-20, the truncated peptides were separated by avidin-labelled magnetic Dynal beads and were identified by mass spectrometry. These results demonstrated that both oligopeptides were cleaved between tryptophan and leucine, matching the TRAP cutting site found in tooth enamel. Enzyme kinetics showed that the k(cat)/K(m) of rMMP-20 against the unmutated amelogenin peptide was 21 times greater than that against the mutated peptide. This study suggests that the reduced rate of TRAP formation by a single amino acid substitution alters enamel matrix hydrolysis by MMP-20, which may result in amelogenesis imperfecta.

In addition to seven known exons of the amelogenin gene, recent studies have identified two exons downstream of amelogenin exon 7 in genomic DNA of mouse and rat. Here the spatial and temporal expression of mRNAs and of the translated proteins derived from alternative splicing of the amelogenin gene ending with exon 8 and exon 9 were examined by in situ hybridization (ISH) and immunohistochemistry (IHC). RNA signals for exons 8 and 9 were expressed in the ameloblast layer extending from early presecretory to postsecretory transitional stages of amelogenesis. IHC of amelogenin proteins that include sequences encoded by these exons demonstrated identical localization of these proteins in the ameloblast layer corresponding to RNA signals identified by ISH. There was intense immunostaining of the enamel matrix secreted by these cells. Western blotting analysis of rat enamel proteins revealed three distinct protein bands with sequences encoded by the new exons. These data confirmed the existence of the transcripts of alternatively spliced mRNAs coding for exons 8 and 9 of the amelogenin gene in rat tooth germs and suggest that the translated proteins contribute to the heterogeneity of amelogenins and have some significant roles in enamel formation and mineralization.

Amelogenesis imperfecta (AI) is a group of inherited disorders with defective tooth enamel formation caused by various gene mutations. One of the mutations substitutes a cytidine to adenine in exon 6 of the X-chromosomal amelogenin gene, which results in a proline to threonine change in the expressed amelogenin. This transformation is four amino acids N terminal to the proteinase cleavage site in amelogenin for enamel matrix metalloproteinase-20 (MMP-20), also known as enamelysin. MMP-20 effects the release of tyrosine rich amelogenin peptide (TRAP) from amelogenin. This study evaluated the rate MMP-20 hydrolyzes the putative mutated amelogenin cleavage site. The proteolytic site was modeled as a substrate by two synthetic peptides, P1 (SYGYEPMGGWLHHQ) and M1 (SYGYETMGGWLHHQ), selected from residue 36-49 of the amino acid sequence for amelogenin and the respective X-linked amelogenin mutant. Recombinant metalloproteinase-20 (rMMP-20) was used to digest the oligopeptides and the truncated peptides were separated by reversed phase HPLC and identified by mass spectrometry. The results demonstrate that both peptides are cleaved between tryptophan and leucine, matching the TRAP cutting site found in tooth enamel. However, the apparent first order rate of digestion of the mutation containing peptide by rMMP-20 was approximately 25 times slower than that of the non-mutated peptide. This study suggests that the reduced rate of TRAP formation due to a single amino acid substitution may alter enamel formation and consequently result in amelogenesis imperfecta.

TRPM7 plays an important role in cellular Ca2+, Zn2+ and Mg2+ homeostasis. TRPM7 channels are abundantly expressed in ameloblasts and, in the absence of TRPM7, dental enamel is hypomineralized. The potential role of TRPM7 channels in Ca2+ transport during amelogenesis was investigated in the HAT-7 rat ameloblast cell line. The cells showed strong TRPM7 mRNA and protein expression. Characteristic TRPM7 transmembrane currents were observed, which increased in the absence of intracellular Mg2+ ([Mg2+]i), were reduced by elevated [Mg2+]i, and were inhibited by the TRPM7 inhibitors NS8593 and FTY720. Mibefradil evoked similar currents, which were suppressed by elevated [Mg2+]i, reducing extracellular pH stimulated transmembrane currents, which were inhibited by FTY720. Naltriben and mibefradil both evoked Ca2+ influx, which was further enhanced by the acidic intracellular conditions. The SOCE inhibitor BTP2 blocked Ca2+ entry induced by naltriben but not by mibefradil. Thus, in HAT-7 cells, TRPM7 may serves both as a potential modulator of Orai-dependent Ca2+ uptake and as an independent Ca2+ entry pathway sensitive to pH. Therefore, TRPM7 may contribute directly to transepithelial Ca2+ transport in amelogenesis.

Enamelysin is a matrix metalloproteinase (MMP-20) secreted by ameloblasts, previously shown to hydrolyze recombinant amelogenin. The purpose of this study was to use recombinant MMP-20 to further investigate the specific hydrolysis of peptide fragments containing cleavage sites for tyrosine-rich amelogenin peptide (TRAP) and leucine-rich amelogenin peptide (LRAP). MMP-20 cDNA was isolated from a subtracted bovine cDNA library, reconstructed into pRSET A vector, and overexpressed in BL21 Escherichia coli. The recombinant MMP-20 was purified using Mono-S ion exchange and nickel affinity chromatography. The proteinase was renatured by dialysis against buffer containing 50 microM zinc and 5 mM calcium and autolysed to form several active fragments. The varying sizes and activities of the activated enzyme fragments appeared to be due to sequential autolysis at different location of the carboxyl terminus of the intact enzyme. Two synthetic peptides corresponding to amelogenin amino acid sequences 36-49 and 181-188 were hydrolyzed by the activated rMMP-20. Mass spectrometry and amino acid composition analysis showed that the cleavage sites were between the tryptophan and leucine (45 and 46) for TRAP and between proline and alanine (186-187) for LRAP. These results indicate that MMP-20 can be autoactivated, and activated MMP-20 has a functional role in the initial cleavage of amelogenin.

Alternative splicing of the amelogenin gene generates a family of proteins secreted by ameloblasts that are primarily responsible for tooth enamel formation. Here the presence of two additional exons, downstream of exon 7, which are followed by an alternate polyadenylation site, is reported. Long polymerase chain reaction was used with a rat genomic amelogenin template to amplify sequences downstream from the primary polyadenylation site. Within the amplified sequences, two exons of 45 bp (exon 8) and 110 bp (exon 9) were identified. The presence of these additional exons in the human and mouse genome was indicated by Southern blot analysis. Antibodies raised against a synthetic peptide corresponding to a sequence encoded by exon 9 positively stained ameloblasts in sections of developing murine and porcine unerupted teeth. These results suggest that an amelogenin protein(s) that includes sequences derived from exons 8 and 9 is synthesized by the ameloblasts.

Amelogenin proteins are a major component of the developing enamel matrix, and are likely to have a key role in the control of enamel biomineralization. The heterogeneity of amelogenin is in part due to alternative splicing of the amelogenin RNA transcripts. Several patterns of alternative splicing have been described in mouse, bovine, porcine, and human enamel, with all alternatively spliced products having homologous 5' and 3' sequences within the coding regions. In these studies, we have used anchored PCR to identify alternatively spliced amelogenin cDNA sequences in the rat. We found amelogenin cDNAs that could be divided into two groups based on their 3' sequence. Group 1 cDNAs had a novel terminal sequence that has not been previously identified, while group 2 cDNAs were similar to those previously identified in other animal species. We identified a sequence, identical to the novel 3' amelogenin cDNA sequence, in rat genomic DNA downstream from the previously identified exon 7. The putative amelogenin proteins predicted for the two groups differed in their predicted isoelectric points, with the novel group 1 proteins having a more basic isoelectric point.

During enamel development, formation of hydroxyapatite crystals and regulation of pH in the enamel matrix require massive transport of ions. Both ameloblasts and adjacent dental epithelial cells in the stellate reticulum co-express several transmembrane cotransporters/ion-exchangers for transport of ions across plasma membranes. Gap junctions (GJs) enable intercellular exchanges of ions between neighboring cells. This suggests that the ameloblasts and other cell layers of the enamel organ, form a functional unit. During the bell stage of tooth formation, the non-ameloblast dental epithelium highly expresses the Na-K-Cl cotransporter (Nkcc1). Nkcc1-null mice are associated with enamel hypomineralization and increased expression of GJ protein connexin 43 (Cx43), suggesting that reduced ion transport in the Nkcc1-null mouse is in part compensated by increased intercellular ion transport through GJs. To understand the role of GJs in ion transport and its effect on pH regulation, we examined in a mouse strain in which Cx43 was ablated selectively in DMP1 expressing cells (Cx43flox/flox mice crossed with DMP1-8kb-Cre mice), including ameloblasts. Micro-CT analysis showed that the mineral density at late maturation stage incisal enamel of the Cx43-null mice was 10% less than in controls, whereas that in dentin was unchanged. Maturation stage ameloblasts of mice lacking the pH regulating sodium/bicarbonate transporter NBCe1 (Nbce1-null), or chloride channel Cftr (Cftr-null) were found to have increased Cx43-immunostaining. These results support the possibility that GJs in the ameloblast–papillary complex at the maturation stage contribute to ion transport by enabling passage of ions directly from cells of the papillary layer into ameloblast layer. Increasing the number of GJs may partly compensate the reduction of ion-cotransporters and ion exchangers in dental epithelium.

We have recently developed a novel in vitro model using HAT-7 rat ameloblast cells to functionally study epithelial ion transport during amelogenesis. Our present aims were to identify key transporters of bicarbonate in HAT-7 cells and also to examine the effects of fluoride exposure on vectorial bicarbonate transport, cell viability, and the development of transepithelial resistance. To obtain monolayers, the HAT-7 cells were cultured on Transwell permeable filters. We monitored transepithelial resistance (TER) as an indicator of tight junction formation and polarization. We evaluated intracellular pH changes by microfluorometry using the fluorescent indicator BCECF. Activities of ion transporters were tested by withdrawal of various ions from the bathing medium, by using transporter specific inhibitors, and by activation of transporters with forskolin and ATP. Cell survival was estimated by alamarBlue assay. Changes in gene expression were monitored by qPCR. We identified the activity of several ion transporters, NBCe1, NHE1, NKCC1, and AE2, which are involved in intracellular pH regulation and vectorial bicarbonate and chloride transport. Bicarbonate secretion by HAT-7 cells was not affected by acute fluoride exposure over a wide range of concentrations. However, tight-junction formation was inhibited by 1 mM fluoride, a concentration which did not substantially reduce cell viability, suggesting an effect of fluoride on paracellular permeability and tight-junction formation. Cell viability was only reduced by prolonged exposure to fluoride concentrations greater than 1 mM. In conclusion, cultured HAT-7 cells are functionally polarized and are able to transport bicarbonate ions from the basolateral to the apical fluid spaces. Exposure to 1 mM fluoride has little effect on bicarbonate secretion or cell viability but delays tight-junction formation, suggesting a novel mechanism that may contribute to dental fluorosis.

Na+:K+:2Cl− cotransporters (NKCCs) belong to the SLC12A family of cation-coupled Cl− transporters. We investigated whether enamel-producing mouse ameloblasts express NKCCs. Transcripts for Nkcc1 were identified in the mouse dental epithelium by RT-qPCR and NKCC1 protein was immunolocalized in outer enamel epithelium and in the papillary layer but not the ameloblast layer. In incisors of Nkcc1-null mice late maturation ameloblasts were disorganized, shorter and the mineral density of the enamel was reduced by 10% compared to wild-type controls. Protein levels of gap junction protein connexin 43, Na+-dependent bicarbonate cotransporter e1 (NBCe1), and the Cl−-dependent bicarbonate exchangers SLC26A3 and SLC26A6 were upregulated in Nkcc1-null enamel organs while the level of NCKX4/SLC24A4, the major K+, Na+ dependent Ca2+ transporter in maturation ameloblasts, was slightly downregulated. Whole-cell voltage clamp studies on rat ameloblast-like HAT-7 cells indicated that bumetanide increased ion-channel activity conducting outward currents. Bumetanide also reduced cell volume of HAT-7 cells. We concluded that non-ameloblast dental epithelium expresses NKCC1 to regulate cell volume in enamel organ and provide ameloblasts with Na+, K+ and Cl− ions required for the transport of mineral- and bicarbonate-ions into enamel. Absence of functional Nkcc1 likely is compensated by other types of ion channels and ion transporters. The increased amount of Cx43 in enamel organ cells in Nkcc1-null mice suggests that these cells display a higher number of gap junctions to increase intercellular communication.

Human deciduous and permanent teeth exhibit different developmental processes, morphologies, histological characteristics and life cycles. In addition, their pulp tissues react differently to external stimuli, such as the pulp sensitivity test, dental trauma and pulp therapy materials. These suggest differences in gene expression and regulation, and in this study we compared gene-expression profiles of the human dental pulp from deciduous and permanent teeth. Pulp tissues from permanent premolars and deciduous molars aged 11-14 years were extirpated and mRNA was isolated for cDNA microarray analysis, and quantitative real-time PCR (qPCR). Other teeth were used for immunohistochemical analysis (IHC). Microarray analysis identified 263 genes with a twofold or greater difference in expression level between the two types of pulp tissue, 43 and 220 of which were more abundant in deciduous and permanent pulp tissues, respectively. qPCR analysis was conducted for eight randomly selected genes, and the findings were consistent with the cDNA microarray results. IHC confirmed that insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) was broadly expressed in deciduous dental pulp tissue, but minimally expressed in permanent dental pulp tissue. Immunohistochemical analysis showed that calbindin 1 (CALB1), leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), and gamma-aminobutyric acid A receptor beta 1 (GABRB1) were abundantly expressed in permanent predentin/odontoblasts, but only minimally expressed in deciduous dental pulp tissue. These results show that deciduous and permanent pulp tissues have different characteristics and gene expression, suggesting that they may have different functions and responses to therapies focused on pulp or dentin regeneration.

In one normal subject, J.S., fed several formula diets in a sterol balance study, only 25-58% of the ingested plant sterols were recovered from the stool. The dietary plant sterols were completely recovered from the stools of five other men. Plant sterol recovery was complete in all men when a diet of mixed general foods was consumed.Since the chief differences in composition of the formula and the diet of mixed general foods were related to the different contents of cellulose and lactose, these components were added to the formula diet of J.S., and plant sterol balance studies were then carried out. The addition of fresh celery or pulverized cellulose to the formula diet partially corrected the usual fecal loss of plant sterols (80% being recovered). Lactose in the formula was only slightly corrective. However, the addition of both cellulose and lactose led to complete recovery of the ingested plant sterols in the feces.Bacterial cultures of stools were incubated with added cholesterol-4-14C, and a linear relationship between losses of sterol during balance studies and in vitro incubations was observed; that is, a considerable loss of the labeled cholesterol from cultures after the formula diet, but not after the diet of mixed general foods. This in vitro loss was also corrected by the addition of cellulose and lactose to the formula diet.The loss of the sterol nucleus in the intestinal tract may occur at times because of the lack of certain dietary constituents. It is hypothesized that the metabolism of intestinal tract bacteria is altered when certain constituents are not present in the diet, and that these bacteria may then degrade the sterol nucleus.