Amyloid-like amelogenin nanoribbons template mineralization via a low-energy interface of ion binding sites.

Protein scaffolds direct the organization of amorphous precursors that transform into mineralized tissues, but the templating mechanism remains elusive. Motivated by models for the biomineralization of tooth enamel, wherein amyloid-like amelogenin nanoribbons guide the mineralization of apatite filaments, we investigated the impact of nanoribbon structure, sequence, and chemistry on amorphous calcium phosphate (ACP) nucleation. Using full-length human amelogenin and peptide analogs with an amyloid-like domain, films of ß-sheet nanoribbons were self-assembled on graphite and characterized by in situ atomic force microscopy and molecular dynamics simulations. All sequences substantially reduce nucleation barriers for ACP by creating low-energy interfaces, while phosphoserines along the length of the nanoribbons dramatically enhance kinetic factors associated with ion binding. Furthermore, the distribution of negatively charged residues along the nanoribbons presents a potential match to the Ca­Ca distances of the multi-ion complexes that constitute ACP. These findings show that amyloid-like amelogenin nanoribbons provide potent scaffolds for ACP mineralization by presenting energetically and stereochemically favorable templates of calcium phosphate ion binding and suggest enhanced surface wetting toward calcium phosphates in general.

SAXS of murine amelogenin identifies a persistent dimeric species from pH 5.0 to 8.0.

Amelogenin is an intrinsically disordered protein essential to tooth enamel formation in mammals. Using the latest, advanced small angle X-ray scattering (SAXS) capabilities at synchrotrons and computational models, we revisited measuring the quaternary structure of murine amelogenin as a function of pH and phosphorylation at Ser-16. The SAXS data shows that at the pH extremes, amelogenin exists as an extended monomer at pH 3.0 (Rg = 38.4 Å) and nanospheres at pH 8.0 (Rg = 84.0 Å), consistent with multiple previous observations. At pH 5.0 and above there was no evidence for a significant population of monomeric species. Instead, at pH 5.0 ∼ 80% of the population is a heterogenous dimeric species that increases to ∼ 100% at pH 5.5. The dimer population was observed at all pH > 5 conditions in dynamic equilibrium with a species in the pentamer range at pH < 6.5 and nanospheres at pH 8.0. At pH 8.0 ∼ 40% of the amelogenin remained in the dimeric state. In general, serine-16 phosphorylation of amelogenin appears to modestly stabilize the population of the dimeric species.

Recombinant human amelogenin promotes wound healing by enhancing angiogenesis.

The first barrier of the human body is the skin, and more serious harm may occur when skin wound healing is delayed. One of the components of enamel matrix proteins is amelogenin, which inhibits inflammation and promotes periodontal tissue regeneration. However, its role in skin wound healing and angiogenesis is inconclusive. Thus, this study aimed to assess the therapeutic effect of recombinant human amelogenin (rhAM) on mouse skin wounds and to determine its effect on angiogenesis and its underlying mechanism. rhAM was expressed in Escherichia coli and purified using the optimized acetic acid method. A skin injury mouse model was established to explore the effects of rhAM on skin wound healing. After treatment with rhAM for 7 days, the wound healing rate was calculated, and the therapeutic effect of rhAM on skin wounds was assessed using hematoxylin & eosin (HE), Masson, and CD31 immunofluorescence staining. The expression of growth and inflammatory factors in wound tissues were detected using Western Blot. In addition, the rhAM effects on the proliferation and migration of human umbilical vein endothelial cells (HUVEC) and mouse fibroblasts (NIH 3T3) were studied in vitro using the Cell Counting Kit-8, cell scratch, cytoskeleton staining, and qPCR. The rhAM effect on HUVEC angiogenesis and its potential mechanism was studied using tube formation and Western Blot. The results showed that the purity of the obtained rhAM was more than 90 % using the optimized acetic acid method, and high-dose rhAM treatment could improve wound healing rate in mice. Additionally, more blood vessels and collagen were produced in the skin wound, and the expression of angiopoietin-related protein 2 (ANGPTL2) and transforming growth factor (TGF)-ß1 was upregulated; however, that of interleukin-6 was down-regulated. We also found that rhAM promoted the proliferation and migration of HUVEC and NIH 3T3, the mRNA levels of vascular endothelial growth factor (VEGF), fibroblast growth factor, TGF-ß1 and ANGPTL2 in HUVEC cells were upregulated, and expression of VEGF and phosphorylation of the p38 mitogen-activated protein kinase were activated. Therefore, rhAM could promote skin wound healing by upregulating angiogenesis and inhibiting inflammation.

A photoresponsive recombinant human amelogenin-loaded hyaluronic acid hydrogel promotes bone regeneration.

OBJECTIVES: In order to evaluate the effect of methacrylated hyaluronic acid (HAMA) hydrogels containing the recombinant human amelogenin (rhAm) in vitro and in vivo. BACKGROUND: The ultimate goal in treating periodontal disease is to control inflammation and achieve regeneration of periodontal tissues. In recent years, methacrylated hyaluronic acid (HAMA) containing recombinant human amyloid protein (rhAm) has been widely used as a new type of biomaterial in tissue engineering and regenerative medicine. However, there is a lack of comprehensive research on the periodontal regeneration effects of this hydrogel. This experiment aims to explore the application of photoresponsive recombinant human amelogenin-loaded hyaluronic acid hydrogel for periodontal tissue regeneration and provide valuable insights into its potential use in this field. MATERIALS AND METHODS: The effects of rhAm-HAMA hydrogel on the proliferation of human periodontal ligament cells (hPDLCs) were assessed using the CCK-8 kit. The osteogenic differentiation of hPDLCs was evaluated through ALP staining and real-time PCR. Calvarial parietal defects were created in 4-week-old Sprague Dawley rats and implanted with deproteinized bovine bone matrix in different treatment groups. The animals were euthanized after 4 and 8 weeks of healing. The bone volume of the defect was observed by micro-CT and histological analysis. RESULTS: Stimulating hPDLCs with rhAm-HAMA hydrogel did not significantly affect their proliferation (p > .05). ALP staining and real-time PCR results demonstrated that the rhAm-HAMA group exhibited a significant upregulation of osteoclastic gene expression (p < .05). Micro-CT results revealed a significant increase in mineralized tissue volume fraction (MTV/TV%), trabecular bone number (Tb.N), and mineralized tissue density (MTD) of the bone defect area in the rhAm-HAMA group compared to the other groups (p < .05). The results of hematoxylin and eosin staining and Masson staining at 8 weeks post-surgery further supported the results of the micro-CT. CONCLUSIONS: The results of this study indicate that rhAm-HAMA hydrogel could effectively promote the osteogenic differentiation of hPDLCs and stabilize bone substitutes in the defects that enhance the bone regeneration in vivo.

Biomimetic Mineral Synthesis by Nanopatterned Supramolecular-Block Copolymer Templates.

Supramolecular structures of matrix proteins in mineralizing tissues are known to direct the crystallization of inorganic materials. Here we demonstrate how such structures can be synthetically directed into predetermined patterns for which functionality is maintained. The study employs block copolymer lamellar patterns with alternating hydrophilic and hydrophobic regions to direct the assembly of amelogenin-derived peptide nanoribbons that template calcium phosphate nucleation by creating a low-energy interface. Results show that the patterned nanoribbons retain their ß-sheet structure and function and direct the formation of filamentous and plate-shaped calcium phosphate with high fidelity, where the phase, amorphous or crystalline, depends on the choice of mineral precursor and the fidelity depends on peptide sequence. The common ability of supramolecular systems to assemble on surfaces with appropriate chemistry combined with the tendency of many templates to mineralize multiple inorganic materials implies this approach defines a general platform for bottom-up-patterning of hybrid organic-inorganic materials.

AMELX Mutations and Genotype-Phenotype Correlation in X-Linked Amelogenesis Imperfecta.

AMELX mutations cause X-linked amelogenesis imperfecta (AI), known as AI types IE, IIB, and IIC in Witkop's classification, characterized by hypoplastic (reduced thickness) and/or hypomaturation (reduced hardness) enamel defects. In this study, we conducted whole exome analyses to unravel the disease-causing mutations for six AI families. Splicing assays, immunoblotting, and quantitative RT-PCR were conducted to investigate the molecular and cellular effects of the mutations. Four AMELX pathogenic variants (NM_182680.1:c.2T>C; c.29T>C; c.77del; c.145-1G>A) and a whole gene deletion (NG_012494.2:g.307534_403773del) were identified. The affected individuals exhibited enamel malformations, ranging from thin, poorly mineralized enamel with a "snow-capped" appearance to severe hypoplastic defects with minimal enamel. The c.145-1G>A mutation caused a -1 frameshift (NP_001133.1:p.Val35Cysfs*5). Overexpression of c.2T>C and c.29T>C AMELX demonstrated that mutant amelogenin proteins failed to be secreted, causing elevated endoplasmic reticulum stress and potential cell apoptosis. This study reveals a genotype-phenotype relationship for AMELX-associated AI: While amorphic mutations, including large deletions and 5' truncations, of AMELX cause hypoplastic-hypomaturation enamel with snow-capped teeth (AI types IIB and IIC) due to a complete loss of gene function, neomorphic variants, including signal peptide defects and 3' truncations, lead to severe hypoplastic/aplastic enamel (AI type IE) probably caused by "toxic" cellular effects of the mutant proteins.

Morphological study of remineralization of the eroded enamel lesions by tyrosine-rich amelogenin peptide.

BACKGROUND: Tyrosine-rich amelogenin peptide (TRAP) is the main amelogenin digestion product in the developmental enamel matrix. It has been shown to promote remineralization of demineralized enamel in our previous study. However, direct evidence of the effect of TRAP on the morphology and nanostructure of crystal growth on an enamel surface has not been reported. This study aimed to examine the effect of TRAP on the morphology of calcium phosphate crystals grown on early enamel erosion using a pH-cycling model. METHODS: Eroded lesions were produced in human premolars by 30-second immersion in 37% phosphoric acid. Forty-five samples of eroded human premolar enamel blocks were selected and randomly divided into 3 groups: deionized water (DDW, negative control); 100 µg/mL TRAP, and 2 ppm sodium fluoride (NaF, positive control group). For 14 days, the specimens were exposed to a pH-cycling model. Using scanning electron microscopy (SEM) and atomic force microscopy (AFM) methods, the surface morphology, calcium-phosphorus ratio, and enamel surface roughness were examined. X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR) were used to assess crystal characteristics. RESULTS: After pH-cycling, compared to the two control groups, the surface of the eroded enamel of the peptide TRAP group shows a large number of new, densely arranged rod-like crystals, parallel to each other, regularly arranged, forming an ordered structure, with crystal morphology similar to that of natural enamel. The crystals are mostly hydroxyapatite (HA). CONCLUSION: This study demonstrates that the peptide TRAP modulates the formation of hydroxyapatite in eroded enamel and that the newly formed crystals resemble natural enamel crystals and promote the remineralization of enamel, providing a promising biomaterial for remineralization treatment of enamel lesions.

An miRNA derived from amelogenin exon4 regulates expression of transcription factor Runx2 by directly targeting upstream activators Nfia and Prkch.

Amel, the gene encoding the amelogenin protein involved in enamel formation, is highly alternatively spliced. When exon4 is excised, it can form a mature miRNA (miR-exon4) that has previously been suggested to indirectly regulate expression of the Runt-related transcription factor 2 (Runx2) involved in bone development in ameloblasts and osteoblasts. However, the precise mechanism of this regulation is unclear. In this study, we aimed to identify direct targets of miR-exon4. The transcription factor family nuclear factor I/A (NFI/A) is known to negatively regulate expression of Runx2 and is among the most highly predicted direct targets of miR-exon4 that link to Runx2. Immunostaining detected NFI/A in osteoblasts and ameloblasts in vivo, and reporter assays confirmed direct interaction of the Nfia 3'-UTR and miR-exon4. In addition, silencing of Nfia in MC3T3-E1-M14 osteoblasts resulted in subsequent downregulation of Runx2. In a monoclonal subclone (mi2) of MC3T3-E1 cells wherein mature miR-exon4 was functionally inhibited, we observed significantly downregulated Runx2 expression. We showed that NFI/A was significantly upregulated in mi2 cells at both mRNA and protein levels. Furthermore, quantitative proteomics and pathway analysis of gene expression in mi2 cells suggested that miR-exon4 could directly target Prkch (protein kinase C-eta), possibly leading to RUNX2 regulation through mechanistic target of rapamycin kinase activation. Reporter assays also confirmed the direct interaction of miR-exon4 and the 3'-UTR of Prkch, and Western blot analysis confirmed significantly upregulated mechanistic target of rapamycin kinase phosphorylation in mi2 cells. Taken together, we conclude that Nfia and Prkch expression negatively correlates with miR-exon4-mediated Runx2 regulation in vivo and in vitro, suggesting miR-exon4 directly targets Nfia and Prkch to regulate Runx2.

Adenomatoid odontogenic tumor: Features of ameloblastic-like epithelial cells differentiation, secretion, and the nature of tumor cells products.

BACKGROUND: This study aimed to investigate the differentiation of ameloblastic-like cells and the nature of the secreted eosinophilic materials in adenomatoid odontogenic tumors. METHODS: We studied histological and immunohistochemical characteristics of 20 cases using: cytokeratins 14 and 19, amelogenin, collagen I, laminin, vimentin, and CD34. RESULTS: Rosette cells differentiated into ameloblastic-like cells positioned face-to-face, displaying collagen I-positive material between them. Epithelial cells of the rosettes can differentiate into ameloblastic-like cells. This phenomenon probably occurs due to an induction phenomenon between these cells. The secretion of collagen I is probably a brief event. Amelogenin-positive areas were interspersed by epithelial cells in the lace-like areas, outside the rosettes and distant from the ameloblastic-like cells. CONCLUSIONS: There are at least two types of eosinophilic material in different areas within the tumor, one in the rosette and solid areas and another in lace-like areas. The secreted eosinophilic material in the rosettes and solid areas is probably a product of well-differentiated ameloblastic-like cells. It is positive for collagen I and negative for amelogenin, whereas some eosinophilic materials in the lace-like areas are positive for amelogenin. We hypothesize that the latter eosinophilic material could be a product of odontogenic cuboidal epithelial or intermediate stratum-like epithelial cells.

Sex-typing of ingested human blood meal in Anopheles stephensi mosquito based on the amelogenin gene.

Identifying the sex of human hosts of insect disease vectors, using PCR amplification of the amelogenin gene (AMEL) from the ingested blood meal is an increasingly useful technique for epidemiological studies of vector-borne diseases, as well as within the criminal justice system. Detection of DNA from ingested blood is influenced by the choice of DNA extraction method, genomic target region, type and length of PCR, and rate of degradation in the DNA samples over time. Here, we have tested two types of PCR (i.e. conventional and nested), producing differently-sized PCR products, in time-course assays targeting the human AMEL gene in Anopheles stephensi mosquitoes that were fed on human male and female blood. The fed female mosquitoes were allowed to digest at 28 °C for times ranging from 0 to 120 h. Three AMEL primer pairs were used to amplify three sequences that were 977, 539, and 106 bp for the X chromosome and 790, 355, and 112 bp for Y. We found that time since feeding had a significant negative effect on the success of PCR amplification. The shortest fragments (106 and 112 bp) were amplified for the longest time after blood feeding (up to 60 h), whereas the medium and longest loci were not amplified by conventional PCR even at 0 h. However, the nested PCR protocol, targeting the medium sequence, could detect small amounts of human DNA up to 36 h (1.5 days) after the blood meal. The shortest PCR assay standardized herein successfully detected small amounts of human DNA in female mosquitoes up to 60 h after the blood meal. This assay represents a promising tool for identifying the sex of the human host from the blood meal in field-collected female mosquitoes.

Histological evaluation following treatment of recession-type defects with coronally advanced flap and a novel human recombinant amelogenin.

OBJECTIVES: To histologically evaluate the effects of a novel human recombinant amelogenin (rAmelX) on periodontal wound healing / regeneration in recession-type defects. MATERIALS AND METHODS: A total of 17 gingival recession-type defects were surgically created in the maxilla of three minipigs. The defects were randomly treated with a coronally advanced flap (CAF) and either rAmelX (test), or a CAF and placebo (control). At three months following reconstructive surgery, the animals were euthanized, and the healing outcomes histologically evaluated. RESULTS: The test group yielded statistically significantly (p = 0.047) greater formation of cementum with inserting collagen fibers compared with the control group (i.e., 4.38 mm ± 0.36 mm vs. 3.48 mm ± 1.13 mm). Bone formation measured 2.15 mm ± 0.8 mm in the test group and 2.24 mm ± 1.23 mm in the control group, respectively, without a statistically significant difference (p = 0.94). CONCLUSIONS: The present data have provided for the first-time evidence for the potential of rAmelX to promote regeneration of periodontal ligament and root cementum in recession-type defects, thus warranting further preclinical and clinical testing. CLINICAL RELEVANCE: The present results set the basis for the potential clinical application of rAmelX in reconstructive periodontal surgery.

Polymorphisms in genes expressed during amelogenesis and their association with dental caries: a case-control study.

OBJECTIVES: Dental caries is a widespread multifactorial disease, caused by the demineralization of hard dental tissues. Susceptibility to dental caries is partially genetically conditioned; this study was aimed at finding an association of selected single nucleotide polymorphisms (SNPs) in genes encoding proteins involved in amelogenesis with this disease in children. MATERIALS AND METHODS: In this case-control study, 15 SNPs in ALOX15, AMBN, AMELX, KLK4, TFIP11, and TUFT1 genes were analyzed in 150 children with primary dentition and 611 children with permanent teeth with/without dental caries from the European Longitudinal Study of Pregnancy and Childhood (ELSPAC) cohort. RESULTS: Dental caries in primary dentition was associated with SNPs in AMELX (rs17878486) and KLK4 (rs198968, rs2242670), and dental caries in permanent dentition with SNPs in AMELX (rs17878486) and KLK4 (rs2235091, rs2242670, rs2978642), (p ≤ 0.05). No significant differences between cases and controls were observed in the allele or genotype frequencies of any of the selected SNPs in ALOX15, AMBN, TFIP11, and TUFT1 genes (p > 0.05). Some KLK4 haplotypes were associated with dental caries in permanent dentition (p ≤ 0.05). CONCLUSIONS: Based on this study, we found that although the SNPs in AMELX and KLK4 are localized in intronic regions and their functional significance has not yet been determined, they are associated with susceptibility to dental caries in children. CLINICAL RELEVANCE: AMELX and KLK4 variants could be considered in the risk assessment of dental caries, especially in permanent dentition, in the European Caucasian population.

The Dynamic Interactions of a Multitargeting Domain in Ameloblastin Protein with Amelogenin and Membrane.

The enamel matrix protein Ameloblastin (Ambn) has critical physiological functions, including regulation of mineral formation, cell differentiation, and cell-matrix adhesion. We investigated localized structural changes in Ambn during its interactions with its targets. We performed biophysical assays and used liposomes as a cell membrane model. The xAB2N and AB2 peptides were rationally designed to encompass regions of Ambn that contained self-assembly and helix-containing membrane-binding motifs. Electron paramagnetic resonance (EPR) on spin-labeled peptides showed localized structural gains in the presence of liposomes, amelogenin (Amel), and Ambn. Vesicle clearance and leakage assays indicated that peptide-membrane interactions were independent from peptide self-association. Tryptophan fluorescence and EPR showed competition between Ambn-Amel and Ambn-membrane interactions. We demonstrate localized structural changes in Ambn upon interaction with different targets via a multitargeting domain, spanning residues 57 to 90 of mouse Ambn. Structural changes of Ambn following its interaction with different targets have relevant implications for the multifunctionality of Ambn in enamel formation.

Regaining enamel color quality using enamel matrix derivative.

This study aimed to demonstrate and compare the accuracy of tooth shade selection due to the remineralized enamel crystal with enamel matrix derivative (EMD) in vitro. Etched enamel slices were immersed in four types of mineralization buffers for 16 h. Sodium fluoride (NaF) was added to final concentrations of 1-100 ppm with the mineralization buffer that demonstrated the highest mineralization efficiency. EMD was added to the mineralization buffer containing NaF to see if it has any remineralization capacities. The remineralized enamel crystal was analyzed by SEM and XRD. The tooth shade was evaluated by CIE L*a*b*. The results showed that, without NaF, plate-like nanocrystals were formed on the enamel surface, but with NaF, needle-like nanocrystals were formed. By adding EMD, a layer of well-compacted hydroxyapatite crystals was successfully precipitated onto the natural enamel surface. No significant differences were observed in the L* value of the mineralization surface pre-etching and after mineralization buffer containing NaF and EMD. A new method has been developed to recover the color quality of enamel, as well as to mineralize the tooth enamel by constructing hydroxyapatite crystals with mineralization buffers containing NaF and EMD on the etched tooth surface.

Loss of biological control of enamel mineralization in amelogenin-phosphorylation-deficient mice.

Amelogenin, the most abundant enamel matrix protein, plays several critical roles in enamel formation. Importantly, we previously found that the singular phosphorylation site at Ser16 in amelogenin plays an essential role in amelogenesis. Studies of genetically knock-in (KI) modified mice in which Ser16 in amelogenin is substituted with Ala that prevents amelogenin phosphorylation, and in vitro mineralization experiments, have shown that phosphorylated amelogenin transiently stabilizes amorphous calcium phosphate (ACP), the initial mineral phase in forming enamel. Furthermore, KI mice exhibit dramatic differences in the enamel structure compared with wild type (WT) mice, including thinner enamel lacking enamel rods and ectopic surface calcifications. Here, we now demonstrate that amelogenin phosphorylation also affects the organization and composition of mature enamel mineral. We compared WT, KI, and heterozygous (HET) enamel and found that in the WT elongated crystals are co-oriented within each rod, however, their c-axes are not aligned with the rods' axes. In contrast, in rod-less KI enamel, crystalline c-axes are less co-oriented, with misorientation progressively increasing toward the enamel surface, which contains spherulites, with a morphology consistent with abiotic formation. Furthermore, we found significant differences in enamel hardness and carbonate content between the genotypes. ACP was also observed in the interrod of WT and HET enamel, and throughout aprismatic KI enamel. In conclusion, amelogenin phosphorylation plays crucial roles in controlling structural, crystallographic, mechanical, and compositional characteristics of dental enamel. Thus, loss of amelogenin phosphorylation leads to a reduction in the biological control over the enamel mineralization process.

Joel Rosenbloom and the revolution in biomedical research.

This biography of Dr. Joel Rosenbloom is published on the occasion of the 50th anniversary of the journal. Dr. Rosenbloom presents the scientific milestones and achievements throughout his career emphasizing events that have spurred him to launch into a career in biomedical research and education. The biography spans several decades of the life and achievements of a distinguished physician scientist whose dedication to science demonstrates the development of new insights into a variety of connective tissues through technological advances and insightful approaches.

The energetic basis for hydroxyapatite mineralization by amelogenin variants provides insights into the origin of amelogenesis imperfecta.

Small variations in the primary amino acid sequence of extracellular matrix proteins can have profound effects on the biomineralization of hard tissues. For example, a change in one amino acid within the amelogenin protein can lead to drastic changes in enamel phenotype, resulting in amelogenesis imperfecta, enamel that is defective and easily damaged. Despite the importance of these undesirable phenotypes, there is very little understanding of how single amino acid variation in amelogenins can lead to malformed enamel. Here, we aim to develop a thermodynamic understanding of how protein variants can affect steps of the biomineralization process. High-resolution, in situ atomic force microscopy (AFM) showed that altering one amino acid within the murine amelogenin sequence (natural variants T21 and P41T, and experimental variant P71T) resulted in an increase in the quantity of protein adsorbed onto hydroxyapatite (HAP) and the formation of multiple protein layers. Quantitative analysis of the equilibrium adsorbate amounts revealed that the protein variants had higher oligomer-oligomer binding energies. MMP20 enzyme degradation and HAP mineralization studies showed that the amino acid variants slowed the degradation of amelogenin by MMP20 and inhibited the growth and phase transformation of HAP. We propose that the protein variants cause malformed enamel because they bind excessively to HAP and disrupt the normal HAP growth and enzymatic degradation processes. The in situ methods applied to determine the energetics of molecular level processes are powerful tools toward understanding the mechanisms of biomineralization.

Sulphur dioxide and fluoride co-exposure induce incisor hypomineralization and amelogenin upregulation via YAP/RUNX2 signaling pathway.

Sulphur dioxide (SO2) and fluoride are among the most common environmental pollutants affecting human health, and both co-exist in areas predominantly consuming coal. It is vital to analyse the combined toxicity of SO2 and fluoride, and their effects on health and the underlying mechanisms of their co-exposure have not yet been adequately assessed. In the present study, we used ICR mice and LS8 cells to investigate the toxicity of SO2 and fluoride exposure to the enamel, alone or in combination. Factorial design analysis was used to reveal the combined toxicity in vitro and in vivo. Co-exposure to SO2 and fluoride exacerbated enamel injury, resulting in more severe hypomineralization of incisor, and enamel structure disorders in mice, and could induce the accumulation of protein residue in the matrix of the enamel. Amelogenin expression was increased upon exposure to SO2 and fluoride, but enamel matrix proteases were not affected. Consistent with our in vivo results, co-exposure of SO2 and fluoride aggravated amelogenin expression in LS8 cells, and increased the YAP and RUNX2 levels. Co-exposure to SO2 and fluoride resulted in greater toxicity than individual exposure, both in vitro and in vivo, indicating that residents of areas exposed to SO2 and fluoride may have an increased risk of developing enamel damage.

The power of weak ion-exchange resins assisted by amelogenin for natural remineralization of dental enamel: an in vitro study.

This study aims to develop an innovative dental product to remineralize dental enamel by a proper combination of ion-exchange resins as controlled release of mineral ions that form dental enamel, in the presence of amelogenin to guide the appropriate crystal growth. The novel product proposed consists of a combination of ion-exchange resins (weak acid and weak base) individually loaded with the remineralizing ions: Ca2+, PO43- and F-, also including Zn2+ in a minor amount as antibacterial, together with the protein amelogenin. Such cocktail provides onsite controlled release of the ions necessary for enamel remineralization due to the weak character of the resins and at the same time, a guiding tool for related crystal growth by the indicated protein. Amelogenin protein is involved in the structural development of natural enamel and takes a key role in controlling the crystal growth morphology and alignment at the enamel surface. Bovine teeth were treated by applying the resins and protein together with artificial saliva. Treated teeth were evaluated with nanoindentation, scanning electron microscopy and energy-dispersive X-ray spectroscopy. The innovative material induces the dental remineralization creating a fluorapatite layer with a hardness equivalent to sound enamel, with the appropriate alignment of corresponding nanocrystals, being the fluorapatite more acid resistant than the original mineral. Our results suggest that the new product shows potential for promoting long-term remineralization leading to the inhibition of caries and protection of dental structures.

Amelogenesis: Transformation of a protein-mineral matrix into tooth enamel.

During enamel formation, the organic enamel protein matrix interacts with calcium phosphate minerals to form elongated, parallel, and bundled enamel apatite crystals of extraordinary hardness and biomechanical resilience. The enamel protein matrix consists of unique enamel proteins such as amelogenin, ameloblastin, and enamelin, which are secreted by highly specialized cells called ameloblasts. The ameloblasts also facilitate calcium and phosphate ion transport toward the enamel layer. Within ameloblasts, enamel proteins are transported as a polygonal matrix with 5 nm subunits in secretory vesicles. Upon expulsion from the ameloblasts, the enamel protein matrix is re-organized into 20 nm subunit compartments. Enamel matrix subunit compartment assembly and expansion coincide with C-terminal cleavage by the MMP20 enamel protease and N-terminal amelogenin self-assembly. Upon enamel crystal precipitation, the enamel protein phase is reconfigured to surround the elongating enamel crystals and facilitate their elongation in C-axis direction. At this stage of development, and upon further amelogenin cleavage, central and polyproline-rich fragments of the amelogenin molecule associate with the growing mineral crystals through a process termed "shedding", while hexagonal apatite crystals fuse in longitudinal direction. Enamel protein sheath-coated enamel "dahlite" crystals continue to elongate until a dense bundle of parallel apatite crystals is formed, while the enamel matrix is continuously degraded by proteolytic enzymes. Together, these insights portrait enamel mineral nucleation and growth as a complex and dynamic set of interactions between enamel proteins and mineral ions that facilitate regularly seeded apatite growth and parallel enamel crystal elongation.