-
Journal of Dental Research Jan 2018Biomimetic synthesis of artificial enamel is a promising strategy for the prevention and restoration of defective enamel. We have recently reported that a hydrogel...
Biomimetic synthesis of artificial enamel is a promising strategy for the prevention and restoration of defective enamel. We have recently reported that a hydrogel system composed of chitosan-amelogenin (CS-AMEL) and calcium phosphate is effective in forming an enamel-like layer that has a seamless interface with natural tooth surfaces. Here, to improve the mechanical system function and to facilitate the biomimetic enamel regrowth, matrix metalloproteinase-20 (MMP-20) was introduced into the CS-AMEL hydrogel. Inspired by our recent finding that MMP-20 prevents protein occlusion inside enamel crystals, we hypothesized that addition of MMP-20 to CS-AMEL hydrogel could reinforce the newly grown layer. Recombinant human MMP-20 was added to the CS-AMEL hydrogel to cleave full-length amelogenin during the growth of enamel-like crystals on an etched enamel surface. The MMP-20 proteolysis of amelogenin was studied, and the morphology, composition, and mechanical properties of the newly grown layer were characterized. We found that amelogenin was gradually degraded by MMP-20 in the presence of chitosan. The newly grown crystals in the sample treated with MMP-20-CS-AMEL hydrogel showed more uniform orientation and greater crystallinity than the samples treated with CS-AMEL hydrogel without MMP-20. Stepwise processing of amelogenin by MMP-20 in the CS-AMEL hydrogel prevented undesirable protein occlusion within the newly formed crystals. As a result, both the modulus and hardness of the repaired enamel were significantly increased (1.8- and 2.4-fold, respectively) by the MMP-20-CS-AMEL hydrogel. Although future work is needed to further incorporate other enamel matrix proteins into the system, this study brings us one step closer to biomimetic enamel regrowth.
Topics: Amelogenin; Biomimetic Materials; Calcium Phosphates; Chitosan; Dental Enamel; Humans; Hydrogels; Matrix Metalloproteinase 20; Recombinant Proteins
PubMed: 28846464
DOI: 10.1177/0022034517728504 -
Journal of Dental Research Aug 2020Ameloblastin (Ambn) has the potential to regulate cell-matrix adhesion through familiar cell-binding domains, but the proposed sequence motifs are not highly conserved...
Ameloblastin (Ambn) has the potential to regulate cell-matrix adhesion through familiar cell-binding domains, but the proposed sequence motifs are not highly conserved across species. Here, we report that Ambn binds to ameloblast-like cell membranes through a highly evolutionary conserved amphipathic helix-forming (AH) motif encoded by exon 5. We applied high-resolution confocal microscopy to show colocalization of Ambn with ameloblast membrane surfaces in developing mouse incisors. Using a series of Ambn-derived peptides and Ambn variants, we showed that Ambn binds to cell membranes through a motif within the sequence encoded by exon 5. Using peptides derived from the N- or C-termini of this sequence, as well as Ambn variants that lacked or had a disrupted AH motif, we demonstrated that the AH motif located at the N-terminus of the sequence is involved in cell-Ambn adhesion. Sequence analysis revealed that this highly conserved AH motif is absent from other enamel matrix proteins, including amelogenin, enamelin, and amelotin. Collectively, these data suggest that Ambn binds to the cell surface membrane via a helix-forming motif and provide insight into the molecular mechanism and function of Ambn in enamel cell-matrix interaction.
Topics: Ameloblasts; Amelogenin; Animals; Cell Communication; Dental Enamel; Dental Enamel Proteins; Incisor; Mice
PubMed: 32401578
DOI: 10.1177/0022034520918521 -
Scientific Reports Mar 2016Enamel, the outermost layer of teeth, is an acellular mineralized tissue that cannot regenerate; the mature tissue is composed of high aspect ratio apatite nanocrystals...
Enamel, the outermost layer of teeth, is an acellular mineralized tissue that cannot regenerate; the mature tissue is composed of high aspect ratio apatite nanocrystals organized into rods and inter-rod regions. Amelogenin constitutes 90% of the protein matrix in developing enamel and plays a central role in guiding the hierarchical organization of apatite crystals observed in mature enamel. To date, a convincing link between amelogenin supramolecular structures and mature enamel has yet to be described, in part because the protein matrix is degraded during tissue maturation. Here we show compelling evidence that amelogenin self-assembles into an amyloid-like structure in vitro and in vivo. We show that enamel matrices stain positive for amyloids and we identify a specific region within amelogenin that self-assembles into β-sheets. We propose that amelogenin nanoribbons template the growth of apatite mineral in human enamel. This is a paradigm shift from the current model of enamel development.
Topics: Amelogenin; Amyloidogenic Proteins; Animals; Dental Enamel; Humans; Kallikreins; Mice; Models, Molecular; Protein Structure, Secondary
PubMed: 27009419
DOI: 10.1038/srep23105 -
Scientific Reports Jan 2023Highly specialized enamel matrix proteins (EMPs) are predominantly expressed in odontogenic tissues and diverged from common ancestral gene. They are crucial for the...
Highly specialized enamel matrix proteins (EMPs) are predominantly expressed in odontogenic tissues and diverged from common ancestral gene. They are crucial for the maturation of enamel and its extreme complexity in multiple independent lineages. However, divergence of EMPs occured already before the true enamel evolved and their conservancy in toothless species suggests that non-canonical functions are still under natural selection. To elucidate this hypothesis, we carried out an unbiased, comprehensive phenotyping and employed data from the International Mouse Phenotyping Consortium to show functional pleiotropy of amelogenin, ameloblastin, amelotin, and enamelin, genes, i.e. in sensory function, skeletal morphology, cardiovascular function, metabolism, immune system screen, behavior, reproduction, and respiratory function. Mice in all KO mutant lines, i.e. amelogenin KO, ameloblastin KO, amelotin KO, and enamelin KO, as well as mice from the lineage with monomeric form of ameloblastin were affected in multiple physiological systems. Evolutionary conserved motifs and functional pleiotropy support the hypothesis of role of EMPs as general physiological regulators. These findings illustrate how their non-canonical function can still effect the fitness of modern species by an example of influence of amelogenin and ameloblastin on the bone physiology.
Topics: Animals; Mice; Amelogenin; Dental Enamel Proteins
PubMed: 36702824
DOI: 10.1038/s41598-023-28388-4 -
Journal of Dental Research Oct 2012Amelogenin, the major protein of forming dental enamel, plays a crucial role in the biomineralization of this tissue. Amelogenin is soluble at low pH and self-assembles...
Amelogenin, the major protein of forming dental enamel, plays a crucial role in the biomineralization of this tissue. Amelogenin is soluble at low pH and self-assembles to form higher order structures at physiological pH. To understand the mechanisms of its assembly and interactions with calcium phosphate mineral, we conducted FTIR spectroscopy (FTIRS) studies of pH-triggered assembly of recombinant porcine amelogenin rP172 and its interactions with mature hydroxyapatite and apatitic mineral formed in situ. Analysis of our data indicated that rP172 at pH 3.0 exists in an unfolded disordered state, while increases in pH led to structural ordering, manifested by increases in intra- and intermolecular β-sheet structures and a decrease in random coil and β-turns. Amelogenin assembled at pH 7.2 was also found to contain large portions of extended intramolecular β-sheet and PPII. These FTIRS findings are consistent with those previously obtained with other techniques, thus verifying the validity of our experimental approach. Interestingly, interactions with mineral led to a reduction in protein structural organization. The findings obtained show that amelogenin has intrinsic structural flexibility to accommodate interactions with both forming and mature calcium phosphate mineral phases, providing new insights into the potential importance of amelogenin-mineral interactions in enamel biomineralization.
Topics: Amelogenesis; Amelogenin; Animals; Calcium Phosphates; Durapatite; Hydrogen-Ion Concentration; Protein Structure, Secondary; Recombinant Proteins; Spectroscopy, Fourier Transform Infrared; Sus scrofa
PubMed: 22933608
DOI: 10.1177/0022034512457371 -
PloS One 2012Amelogenin is an extracellular protein first identified as a matrix component important for formation of dental enamel during tooth development. Lately, amelogenin has...
Amelogenin is an extracellular protein first identified as a matrix component important for formation of dental enamel during tooth development. Lately, amelogenin has also been found to have positive effects on clinical important areas, such as treatment of periodontal defects, wound healing, and bone regeneration. Here we present a simple method for purification of recombinant human amelogenin expressed in Escherichia coli, based on the solubility properties of amelogenin. The method combines cell lysis with recovery/purification of the protein and generates a >95% pure amelogenin in one step using intact harvested cells as starting material. By using amelogenin as a fusion partner we could further demonstrate that the same method also be can explored to purify other target proteins/peptides in an effective manner. For instance, a fusion between the clinically used protein PTH (parathyroid hormone) and amelogenin was successfully expressed and purified, and the amelogenin part could be removed from PTH by using a site-specific protease.
Topics: Amelogenin; Escherichia coli; Gene Expression; Humans; Parathyroid Hormone; Recombinant Fusion Proteins
PubMed: 22442680
DOI: 10.1371/journal.pone.0033269 -
Frontiers in Bioscience (Landmark... Sep 2022The latest development in molecular biology has offered an opportunity to construct multiplex panel with better applicability for forensic purpose, and a self-developed...
BACKGROUND
The latest development in molecular biology has offered an opportunity to construct multiplex panel with better applicability for forensic purpose, and a self-developed 64-plex panel, including 59 autosomal diallelic InDels, 2 miniSTRs, 2 Y-InDels, and an Amelogenin gene, was validated to be an effective forensic tool in the previous study.
METHODS
By applying the 64-plex panel for DNA profiling, the obtained genotypes and the corresponding frequency data were used to investigate the forensic characteristics and population genetic structures of the Chinese Manchu group from the Inner Mongolia Autonomous Region and the Chinese Zhuang group from the Yunnan province.
RESULTS
The 64-plex panel was qualified to perform human identification and paternity testing with the combined powers of discrimination of 0.99999999999999999999999999758 and 0.99999999999999999999999999691; and cumulative probabilities of exclusion of 0.99999866 and 0.99999880 in the studied Manchu and Zhuang groups, respectively. Relatively closer genetic relationships were found between the Chinese Manchu group and Han population in Beijing; and between the Chinese Zhuang group and Vietnamese Kinh population.
CONCLUSIONS
It could be indicated from the results that, with the preliminary ability to distinguish ancestral components from all the studied groups, the 64-plex panel can not only serve as a robust forensic panel in the Manchu and Zhuang groups, but also offer genetic insights into the genetic differentiations and substructures of these populations.
Topics: Amelogenin; Asian People; China; Genetic Background; Genetics, Population; Humans
PubMed: 36224004
DOI: 10.31083/j.fbl2709258 -
Journal of Oral Biology and... 2022Vitality of the dentin-pulp complex depends on cell activity and signalling processes. Amelogenin protein regulates cell signalling pathways during tooth development by...
Vitality of the dentin-pulp complex depends on cell activity and signalling processes. Amelogenin protein regulates cell signalling pathways during tooth development by activating the Wnt/β-catenin intercellular signalling pathway. This study aimed to regenerate a vascularized pulp using recombinant amelogenin protein, in necrotic root canals by cell homing. Access opening was performed for a total of 120 root canals and were left open to become contaminated with oral microbes for 14 days then cleaned. Canals were divided into 2 groups; in the First group, the canals were filled with amelogenin, while in the 2nd group the canals were left empty. Samples were evaluated histologically and with immunodetection of Sox2, Oct4, Vascular endothelial growth factor (VEGF), Wnt1a, Wnt 3a, Wnt 10b, and Glial Fibrillary Acidic Proteins (GFAP). IC50 was used to determine the cytotoxicity of amelogenin. Regenerated dense cellular tissue was seen in the apical part of amelogenin-treated root canals, and regenerated delicate vascularized tissue was observed in the radicular and pulp chamber. Cells found in the regenerated soft tissue expressed Wnt family members that regulate stem cell pluripotency. Also, Sox2 and Oct4, Pluripotency markers, could be identified in the newly formed apical papilla and dental follicle. Furthermore, VEGF in the regenerated pulps indicated neovascularization. While the GFAP immune reactivity demonstrated that the neuro-sensory organ was being replicated in the regenerated dental pulps. Finally, IC50 test showed that recombinant amelogenin protein has a safe dose at high-level concentrations. Recombinant amelogenin protein induces pulp regeneration most likely from the Sox2 identified stem cells within the apical papilla and can enhance apex formation in non-vital immature teeth.
PubMed: 36097610
DOI: 10.1016/j.jobcr.2022.08.008 -
Journal of the California Dental... Dec 2009Regulated gene expression assembles an extracellular proteinaceous matrix to control biomineralization and the resultant biomechanical function of tooth enamel. The... (Review)
Review
Regulated gene expression assembles an extracellular proteinaceous matrix to control biomineralization and the resultant biomechanical function of tooth enamel. The importance of the dominant enamel matrix protein, amelogenin (Amel); a minor transiently expressed protein, dentin sialoprotein (Dsp); an electrogenic sodium bicarbonate cotransporter (NBCe1); the timely removal of the proteinaceous matrix by a serine protease, Kallikrein-4 (Klk4); and the late-stage expression of Amelotin (Amtn) on enamel biomechanical function were demonstrated and measured using mouse models.
Topics: Amelogenin; Animals; Biomechanical Phenomena; Dental Enamel; Dental Enamel Proteins; Extracellular Matrix Proteins; Gene Expression Regulation; Kallikreins; Mice; Models, Animal; Phosphoproteins; Sialoglycoproteins; Sodium-Bicarbonate Symporters; Tooth Calcification
PubMed: 20066874
DOI: No ID Found -
Archives of Oral Biology Jun 2010Lysosome-associated membrane protein-1 (LAMP-1) has been suggested to be a cell surface receptor for a specific amelogenin isoform, leucine-rich amelogenin peptide or...
OBJECTIVES
Lysosome-associated membrane protein-1 (LAMP-1) has been suggested to be a cell surface receptor for a specific amelogenin isoform, leucine-rich amelogenin peptide or LRAP. However, it is unclear if LAMP-1 is an amelogenin receptor for dental mesenchymal cells. The goal of this study was to determine if LAMP-1 serves as a cell surface binding site for full length amelogenin on tooth root/periodontium associated mesenchymal cells.
DESIGN
Murine dental follicle cells and cementoblasts (OCCM-30) were cultured for 2 days followed by addition of full length recombinant mouse amelogenin, rp(H)M180. Dose-response (0-100 microg/ml) and time course (0-120 min) assays were performed to determine the optimal conditions for live cell surface binding using immunofluorescent microscopy. A competitive binding assay was performed to determine binding specificity by adding Emdogain (1 mg/ml) to the media. An antibody against LAMP-1 was used to detect the location of LAMP-1 on the cell surface and the pattern was compared to cell surface bound amelogenin. Both amelogenin and cell surface LAMP-1 were immuno-co-localized to compare the amount and distribution pattern.
RESULTS
Maximum surface binding was achieved with 50 microg/ml of rp(H)M180 for 120 min. This binding was specific as demonstrated by competitive inhibition (79% lower) with the addition of Emdogain. The binding pattern for rp(H)M180 was similar to the distribution of surface LAMP-1 on dental follicle cells and cementoblasts. The high co-localization coefficient (0.92) for rp(H)M180 and LAMP-1 supports rp(H)M180 binding to cell surface LAMP-1.
CONCLUSIONS
The data from this study suggest that LAMP-1 can serve as a cell surface binding site for amelogenin on dental follicle cells and cementoblasts.
Topics: Amelogenin; Animals; Binding Sites; Binding, Competitive; Cells, Cultured; Dental Cementum; Dental Enamel Proteins; Dental Sac; Lysosomal Membrane Proteins; Mesoderm; Mice; Periodontal Ligament; Protein Binding; Protein Isoforms; Recombinant Proteins; Regeneration; Tooth Root
PubMed: 20382373
DOI: 10.1016/j.archoralbio.2010.03.009