Enamel-like tissue regeneration by using biomimetic enamel matrix proteins.

Enamel regeneration currently -is limited by our inability to duplicate artificially its complicated and well-aligned hydroxyapatite structure. The initial formation of enamel occurs in enamel organs where the ameloblasts secret enamel extracellular matrix formed a unique gel-like microenvironment. The enamel extracellular matrix is mainly composed by amelogenin and non-amelogenin. In this study, an innovative strategy was proposed to regenerate enamel-like tissue by constructing a microenvironment using biomimetic enamel matrix proteins (biomimetic EMPs) composed of modified leucine-rich amelogenin peptide (mLRAP) and non-amelogenin analog (NAA). Impressively, the regenerated enamel in this biomimetic EMPs on etched enamel surface produced prismatic structures, and showed similar mechanical properties to natural enamel. The results of X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) showed that regenerated crystal was hydroxyapatite. Molecular dynamics simulation analysis showed the binding energy between mLRAP and NAA were electrostatic forces and Van der Walls. These results introduced a promising strategy to induce crystal growth of enamel-like hydroxyapatite for biomimetic reproduction of materials with complicated hierarchical microstructures.

Deproteinization of primary enamel with sodium hypochlorite before phosphoric acid etching.

The aim of this study was to evaluate the deproteinization of primary enamel by analyzing etching pattern types, with and without the application of 5% NaOCl before acid etching with 37% H3PO4. Fifteen extracted human primary molars were randomly selected for the present in vitro study; 1mm x 1mm blocks were prepared and divided into two groups (n = 21). These groups were treated as follows: Group A- Acid Etching with 37% H3PO4 gel for 15 s; Group B- 5% NaOCl for 60 s + Acid Etching with 37% H3POfor 15 s. The specimens were prepared for scanning electron microscopy analysis. The images were evaluated for quality types I and II etching of the enamel surface using ImageJ software. Datasets were checked for normality by Kolgomorv-Smirnov test and the nonparametric unpaired Mann-Whitney test was applied. The mean surface area of type I and II etching pattern values was 1922.314 µm2for Group A and 3840.473 µm2Group B. We conclude that deproteinization with 5% NaOCl prior to acid etching can be used to increase the area of adhesion and the quality of the etching pattern.

El objetivo del estudio fue evaluar la desproteinización del esmalte primario a través de los tipos de patrones de grabado, con y sin NaOCl 5% utilizado antes del grabado ácido con H3PO4 37%. Quince dientes primarios humanos extraídos se seleccionaron al azar para el presente estudio in vitro, se prepararon bloques de 1mm x 1 mm y se dividieron en dos grupos (n = 21). Estos grupos se trataron de la siguiente manera: Grupo A: Grabado ácido con H3PO4 37% en gel durante 15 segundos; Grupo B: NaOCl 5% durante 60 segundos + Grabado ácido con H3PO4 37% durante 15 segundos. Las muestras se prepararon para el análisis de microscopía electrónica de barrido. Las imágenes obtenidas se evaluaron principalmente por la calidad de los grabados tipo I y II de la superficie del esmalte primario, utilizando el software Image J. Los datos se analizaron en cuanto a su normalidad mediante la prueba de Kolgomorv-Smirnov, se utilizó pruebas no paramétricas: Prueba de Mann-Whitney no pareada. Como resultado, se encontró que el área de superficie media de los valores de patrón de grabado de tipo I y II para el Grupo A era 1922,314 µm2 y el Grupo B era 3840,473 µm2. Finalmente, llegamos a la conclusión de que se puede usar la desproteinización con NaOCl 5% antes del grabado ácido para aumentar el área de adhesión y la calidad del patrón de grabado.

Characterization of inter-crystallite peptides in human enamel rods reveals contribution by the Y allele of amelogenin.

Proteins of the inter-rod sheath and peptides within the narrow inter-crystallite space of the rod structure are considered largely responsible for visco-elastic and visco-plastic properties of enamel. The present study was designed to investigate putative peptides of the inter-crystallite space. Entities of 1-6 kDa extracted from enamel rods of erupted permanent teeth were analysed by mass spectrometry (MS) and shown to comprise N-terminal amelogenin (AMEL) peptides either containing or not containing exon 4 product. Other dominant entities consisted of an N-terminal peptide from ameloblastin (AMBN) and a series of the most hydrophobic peptides from serum albumin (ALBN). Amelogenin peptides encoded by the Y-chromosome allele were strongly detected in Enamel from male teeth. Location of N-terminal AMEL peptides as well as AMBN and ALBN, between apatite crystallites, was disclosed by immunogold scanning electron microscopy (SEM). Density plots confirmed the relative abundance of these products including exon 4+ AMEL peptides that have greater capacity for binding to hydroxyapatite. Hydrophilic X and Y peptides encoded in exon 4 differ only in substitution of non-polar isoleucine in Y for polar threonine in X with reduced disruption of the hydrophobic N-terminal structure in the Y form. Despite similarity of X and Y alleles of AMEL the non-coding region upstream from exon 4 shows significant variation with implications for segregation of processing of transcripts from exon 4. Detection of fragments from multiple additional proteins including keratins (KER), fetuin A (FETUA), proteinases and proteinase inhibitors, likely reflect biochemical events during enamel formation.

Effect of deproteinization before and after acid etching on the surface roughness of immature permanent enamel.

OBJECTIVE: The purpose of this in vitro investigation was to assess the effect of deproteinization before and after acid etching on the surface roughness of immature human enamel of permanent teeth compared to acid etching alone using noncontact three-dimensional (3D) optical profilometer. MATERIALS AND METHODS: Forty-eight enamel blocks were randomly distributed into 4 groups (12 each) according to the surface treatment in the form of deproteinized with 2.5% sodium hypochlorite (NaOCl) before and after acid etching with 32% phosphoric acid (H3PO4) compared to application of H3PO4 alone. The surface roughness (Sa) was measured using a 3D optical noncontact surface profiler. Two specimens from each group were selected and prepared for scanning electron microscopic (SEM) analysis. Shapiro-Wilk test, one-way analysis of variance, and Tukey's honest significance difference test were used. All statistical analyses were established with a significance level of P < 0.05. RESULTS: The highest surface roughness (Sa) was recorded for Group 3/NaOCl ± H3PO4 and the lowest Sa was recorded for Group 1 (control). All surface treatments applied showed significantly greater values of surface roughness (Sa) than the enamel surfaces with no surface treatment (control). There was significant difference between control group and Group 2/H3PO4 (P = 0.002), Group 3/NaOCl ± H3PO4 (P = 0.0001), and Group 4/H3PO4 ± NaOCl (P = 0.017). There was no significant difference between Group 2/H3PO4 and Group 4/H3PO4 ± NaOCl. SEM evaluation showed different topographical features of deproteinized enamel surface. CONCLUSIONS: Deproteinizing the enamel of immature permanent teeth with 2.5% NaOCl before and after acid etching with 32% H3PO4 increased surface roughness compared to the application of H3PO4 alone.

Comparison of the capacity of enamel matrix derivative gel and enamel matrix derivative in liquid formulation to adsorb to bone grafting materials.

BACKGROUND: The use of an enamel matrix derivative (EMD) has been shown to enhance periodontal regeneration (e.g., formation of root cementum, periodontal ligament, and alveolar bone). However, in certain clinical situations, the use of EMD alone may not be sufficient to prevent flap collapse or provide sufficient stability of the blood clot. Data from clinical and preclinical studies have demonstrated controversial results after application of EMD combined with different types of bone grafting materials in periodontal regenerative procedures. The aim of the present study is to investigate the adsorption properties of enamel matrix proteins to bone grafts after surface coating with either EMD (as a liquid formulation) or EMD (as a gel formulation). METHODS: Three different types of grafting materials, including a natural bone mineral (NBM), demineralized freeze-dried bone allograft (DFDBA), or a calcium phosphate (CaP), were coated with either EMD liquid or EMD gel. Samples were analyzed by scanning electron microscopy or transmission electron microscopy (TEM) using an immunostaining assay with gold-conjugated anti-EMD antibody. Total protein adsorption to bone grafting material was quantified using an enzyme-linked immunosorbent assay (ELISA) kit for amelogenin. RESULTS: The adsorption of amelogenin to the surface of grafting material varied substantially based on the carrier system used. EMD gel adsorbed less protein to the surface of grafting particles, which easily dissociated from the graft surface after phosphate-buffered saline rinsing. Analyses by TEM revealed that adsorption of amelogenin proteins were significantly farther from the grafting material surface, likely a result of the thick polyglycolic acid gel carrier. ELISA protein quantification assay demonstrated that the combination of EMD liquid + NBM and EMD liquid + DFDBA adsorbed higher amounts of amelogenin than all other treatment modalities. Furthermore, amelogenin proteins delivered by EMD liquid were able to penetrate the porous surface structure of NBM and DFDBA and adsorb to the interior of bone grafting particles. Grafting materials coated with EMD gel adsorbed more frequently to the exterior of grafting particles with little interior penetration. CONCLUSIONS: The present study demonstrates a large variability of adsorbed amelogenin to the surface of bone grafting materials when enamel matrix proteins were delivered in either a liquid formulation or gel carrier. Furthermore, differences in amelogenin adsorption were observed among NBM, DFDBA, and biphasic CaP particles. Thus, the potential for a liquid carrier system for EMD, used to coat EMD, may be advantageous for better surface coating.

Comparative temporospatial expression profiling of murine amelotin protein during amelogenesis.

Tooth enamel is formed in a typical biomineralization process under the guidance of specific organic components. Amelotin (AMTN) is a recently identified, secreted protein that is transcribed predominantly during the maturation stage of enamel formation, but its protein expression profile throughout amelogenesis has not been described in detail. The main objective of this study was to define the spatiotemporal expression profile of AMTN during tooth development in comparison with other known enamel proteins. A peptide antibody against AMTN was raised in rabbits, affinity purified and used for immunohistochemical analyses on sagittal and transverse paraffin sections of decalcified mouse hemimandibles. The localization of AMTN was compared to that of known enamel proteins amelogenin, ameloblastin, enamelin, odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4. Three-dimensional images of AMTN localization in molars at selected ages were reconstructed from serial stained sections, and transmission electron microscopy was used for ultrastructural localization of AMTN. AMTN was detected in ameloblasts of molars in a transient fashion, declining at the time of tooth eruption. Prominent expression in maturation stage ameloblasts of the continuously erupting incisor persisted into adulthood. In contrast, amelogenin, ameloblastin and enamelin were predominantly found during the early secretory stage, while odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4 expression in maturation stage ameloblasts paralleled that of AMTN. Secreted AMTN was detected at the interface between ameloblasts and the mineralized enamel. Recombinant AMTN protein did not mediate cell attachment in vitro. These results suggest a primary role for AMTN in the late stages of enamel mineralization.

Potential role of the amelogenin N-terminus in the regulation of calcium phosphate formation in vitro.

N-terminal and C-terminal (CT) domains of amelogenin have been shown to be essential for proper enamel formation. Recent studies have also suggested that although the C-terminus plays an apparent role in protein-mineral interactions, other amelogenin structural domains are involved. The objective was to explore the role of the amelogenin N-terminus in the regulation of calcium phosphate formation in vitro. Spontaneous mineralization studies were carried out using the phosphorylated (+P) and nonphosphorylated (-P) N-terminus of the leucine-rich amelogenin peptide (LRAP) that lacks the hydrophilic CT domain. Mineralization progress was monitored via changes in solution pH. Mineral phases formed were characterized using TEM, selected area electron diffraction, and FT-IR. In controls, amorphous calcium phosphate was initially formed and subsequently transformed to randomly oriented hydroxyapatite (HA) plate-like crystals. In contrast to the control, LRAP(+P)-CT stabilized ACP formation for >1 day, while LRAP(-P)-CT accelerated the transformation of ACP to HA but had little effect on crystal shape or orientation. In conclusion, the N-terminal domain found in LRAP, as in amelogenins, appears to have the capacity to interact with forming calcium phosphate mineral phases. Results suggest that the N-terminal domain of amelogenin may play a direct role in early stages of enamel formation.

Enamel deproteinization before acid etching--a scanning electron microscopic observation.

OBJECTIVES: This study was undertaken to evaluate the topographical features of enamel surface deproteinized with sodium hypochlorite (NaOCl) and etched with phosphoric acid (H3PO4) compared to phosphoric acid alone using Scanning Electron Microscopic (SEM) Analysis. STUDY DESIGN: 30 enamel blocks of 1 mm2 from ten human sound extracted permanent molars were obtained and treated as under: Group 1 (10 blocks): Enamel surface was etched with 37% H3PO4 gel for 15 seconds. Group 2 (10 blocks): Enamel surface was treated with 5.25% NaOCl for 60 seconds and then etched with 37% H3PO4 gel for 15 seconds. 10 enamel blocks were included in the control group where no treatment was carried out. The samples were subjected to SEM analysis and 5 microphotographs of each sample were obtained at 500X magnification and evaluated for the quality of etching pattern of the enamel in percentage (%) using Auto-CAD 2007 software. RESULTS: Mean values of etching pattern in Group 1 being 55.76% and Group 2 being 53.58%. No significant difference was observed between the two groups. CONCLUSION: The use of 37% phosphoric acid for 15 seconds still remains the best method for pretreatment of enamel.

Enamel deproteinization and its effect on acid etching: an in vitro study.

PURPOSE: The goal of this in vitro study was to identify the topographical features of the enamel surface deproteinized and etched with phosphoric acid (H3PO4) compared to phosphoric acid alone. MATERIALS AND METHOD: Ten extracted lower first and second permanent molars were polished with pumice and water, and then divided into 4 equal buccal sections having similar physical and chemical properties. The enamel surfaces of each group were subjected to the following treatments: Group A: Acid Etching with H3PO4 37% for 15 seconds. Group AH1: Sodium Hypochlorite (NaOCl) 5.25% for 30 seconds followed by Acid Etching with H3PO4 37% for 15 seconds. Group AH2; Sodium Hypochlorite (NaOCl) 5.25% for 60 seconds followed by Acid Etching with H3PO4 37% for 15 seconds. Results showed that group AH2 etching technique reached an area of 76.6 mm2 of the total surface, with a 71.8 mm2 (94.47%), type 1 and 2 etching pattern, followed by group AH1 with 55.9 mm2 out of 75.12 mm2 (74.1%), and finally group A with only 36.8 mm2 (48.83%) out of an area of 72.7 mm2. A significant statistical difference (P < 0.05) existed between all groups, leading to the conclusion that enamel deproteinization with 5.25% NaOCl for 1 minute before H3PO4, etching increases the enamel conditioning surface as well as the quality of the etching pattern.

The structure and orientation of the C-terminus of LRAP.

Amelogenin is the predominant protein found during enamel development and is thought to be the biomineralization protein controlling the unique elongated hydroxyapatite crystals that constitute enamel. The secondary structure of biomineralization proteins is thought to be important in the interaction with hydroxyapatite. Unfortunately, very little data are available on the structure or the orientation of amelogenin, either in solution or bound to hydroxyapatite. The C-terminus contains the majority of the charged residues and is predicted to interact with hydroxyapatite; thus, we used solid-state NMR dipolar recoupling techniques to investigate the structure and orientation of the C-terminus of LRAP, a naturally occurring splice variant of full-length amelogenin. Using (13)C{(15)N} Rotational Echo DOuble Resonance (REDOR), the structure of the C-terminus was found to be largely random coil, both on the surface of hydroxyapatite as well as lyophilized from solution. The orientation of the C-terminal region with respect to hydroxyapatite was investigated for two alanine residues (Ala(46) and Ala(49)) using (13)C{(31)P} REDOR and one lysine residue (Lys(52)) using (15)N{(31)P} REDOR. The residues examined were found to be 7.0, 5.7, and 5.8 A from the surface of hydroxyapatite for Ala(46), Ala(49), and Lys(52), respectively. This provides direct evidence that the charged C-terminus is interacting closely with hydroxyapatite, positioning the acidic amino acids to aid in controlling crystal growth. However, solid-state NMR dynamics measurements also revealed significant mobility in the C-terminal region of the protein, in both the side chains and the backbone, suggesting that this region alone is not responsible for binding.

[Biomimetics of crystal texture in dental enamel prism by self-assembly oligopeptide].

OBJECTIVE: To evaluate the possibility of self-assembly oligopeptide(T2) for dental enamel biomimetics, especially for the prism's crystal texture since it could prompt calcium phosphate precipitated in gel carrier. METHODS: SEM (Scanning electron microscope) and TEM (Transmission electron microscope) were used to observe the morphologic presentation and ED(Electron diffraction) to crystal texture comparing with the human molar enamel powder. RESULTS: (a) Flake-like and needle-like octacalcium phosphate precipitated in the gel carrier with self-assemble oligopeptide(T2). They transformed into rod-like hydroxyapatite crystals gradually in the following 2-4 weeks. (b) The rod-like hydroxyapatite may arrange or grow into bundles which are similar to the human enamel prisms in both appearance and size. (c) The rod-like hydroxyapatite showed polycrystal while the enamel prisms showed monocrystal under examination of ED. CONCLUSION: The self-assemble oligopeptide(T2) could regulate the speed of nucleation and crystallization of hydroxyapatite in morphology and crystalline size. Thus, the self-assembly oligopeptide and the gel carrier mineralization system could be primarily applied in biomimetic use for the crystallization of hydroxyapatite in dental prism in vitro.

Assembly and processing of an engineered amelogenin proteolytic product (rP148).

The purpose of this study was to express, characterize, and investigate the self-assembly of a recombinant porcine amelogenin lacking the hydrophilic 24 C-terminal amino acids (rP148). To gain further insight into the function of amelogenin processing during enamel mineralization, this protein was also used as a substrate to examine the action of matrix metalloproteinase-20 (MMP-20). The assembly properties of rP148 were monitored by dynamic light scattering (DLS). In general, rP148 molecules assemble into monomers, dimers, oligomers, and some nanosphere-like particles. Depending on the solution conditions, large aggregates were also observed. Matrix metalloproteinase-20 cleaved the rP148 molecule at a few sites, creating a number of different products, including the tyrosine-rich amelogenin polypeptide (TRAP). Our data suggest that although rP148 self-assembles into small particles, its assembly properties are different from those of the full-length rP172, indicating that the C-terminal 24 amino acids play a critical role in nanosphere assembly. We further demonstrate that MMP-20 digests rP148 in a manner that generates a similar proteolytic pattern, as would be expected to occur in vivo.

The effects of fluoride on the nanostructure and surface pK of enamel crystals: an atomic force microscopy study of human and rat enamel.

Atomic force microscopy (AFM) studies have revealed 30-40 nm-wide regular positively charged bands across maturation-stage rat enamel crystals. Low pH resolved these into positively charged spherical domains of approximately 30 nm diameter. Crystal surface pK values from adhesion force titrations were approximately 6.5. The effect of fluoride on this pK value and on the nanostructure of fluorosed human enamel crystals has not been reported. The nanostructure and surface chemistry (pK) of normal and fluorotic human and of fluoride-treated rat maturing enamel crystals was examined. Enamel was sectioned and polished, prior to examination, using AFM in height and friction modes. High-resolution height images revealed 30 nm-diameter spherical domains within crystals, arranged as layers of hexagons or as a shallow spiral. Fluorotic enamel showed similar, but less well ordered, nanodomains. These could represent an arrangement of original initiation sites or binding sites for modulating matrix proteins. Surface pK was derived from adhesion-force measurements between functionalized tips (OH or COOH) and crystal surfaces between pH 2 and pH 10. pK values of approximately 6.5 for normal crystals were reduced to approximately 5.5 after fluoride treatment. Reduction in surface pK by fluoride might indicate lowered protonation with possible effects on matrix protein binding.

Imaging RNA polymerase-amelogenin gene complexes with single molecule resolution using atomic force microscopy.

The AMELX gene encoding the enamel matrix protein, amelogenin, is located within (and in the opposite orientation to) the first intron of the ARHGAP6 gene, which encodes a GTPase-activating protein. The orientation of these two genes with respect to each other raises the possibility that they may undergo simultaneous convergent transcription during amelogenesis. The aim of this study was to use atomic force microscopy (AFM) to study a transcriptionally active amelogenin DNA template and to investigate the binding of RNA polymerase to convergently aligned promoters. Images of RNA polymerases stalled on DNA templates were obtained following incubation of the template with RNA polymerases and ribonucleotide triphosphates. A linear DNA template incorporating an intact rat amelogenin cDNA flanked by convergently aligned coliphage T7 and T3 promoters was constructed and shown to be transcriptionally active in vitro. Atomic force microscopy images of transcription complexes revealed globular structures, corresponding to single RNA polymerase molecules bound at specific locations on the DNA templates. These results indicate that AFM allows the visualization of individual RNA polymerases on DNA templates, offering a realistic approach to investigating the concept of convergent transcription of nested genes, which may lead to an understanding of whether the simultaneous expression of AMELX and ARHGAP6 is possible during the formation of tooth enamel.

Co-operative mineralization and protein self-assembly in amelogenesis: silica mineralization and assembly of recombinant amelogenins in vitro.

An amorphous silica mineralization technique was used to produce inorganic/protein composites to elucidate the structure and mechanism of formation of amelogenin assemblies, which may play an important role in regulating enamel structure during the initial stages of amelogenesis. Full-length recombinant amelogenins from mouse (rM179) and pig (rP172) were investigated along with key degradation products (rM166 and native P148) lacking the hydrophilic C terminus found in parent molecules. The resulting products were examined using transmission electron microscopy and/or small-angle X-ray scattering. Using protein concentrations of 0.1-3 mg ml-1, large monodisperse spheres of remarkably similar mean diameters were observed using rM179 (124+/-4 nm) and rP172 (126+/-7 nm). These spheres also exhibited 'internal structure', comprising nearly spherical monodisperse particles of approximately 20 nm in diameter. In the presence of rM166, P148, and bovine serum albumin (control), large unstructured and randomly shaped particles (250-1000 nm) were observed. Without added protein, large dense spherical particles of silica (mean approximately 500 nm) lacking internal structure were produced. These findings demonstrate that full-length amelogenins have the ability to form higher-order structures, whereas amelogenins that lack the hydrophilic C terminus do not. The results also suggest that full-length amelogenin can guide the formation of organized mineralized structures through co-operative interactions between assembling protein and forming mineral.

On the formation of amelogenin microribbons.

We recently reported the remarkable spontaneous self-assembly and hierarchical organization of amelogenin 'microribbons' and their ability to facilitate oriented growth of apatite crystals in vitro. In a letter of correction we communicated the finding that the X-ray diffraction pattern reported in our original report was that of cellulose contaminant and not amelogenin microribbon. We have re-evaluated our data and confirmed the protein nature of the microribbons using Fourier transform infrared and Raman microspectroscopy. Some microribbons were remarkably similar in their morphology to that of cellulose fibers. The size distribution of amelogenin microribbons was wider, particularly in width and length, and generally smaller than those originally reported. Here we present additional detailed information on the formation of a series of intermediate hierarchical structures of amelogenin assemblies prior to the formation of microribbon. The most significant finding was that full-length amelogenin nanospheres had a tendency to assemble into collinear arrays whose function is assumed to be critical at the initial stage of enamel mineral deposition. The present data gives an insight into the step-by-step assembly process of amelogenin from nanometer scale molecules to micrometer scale organized structures that can be used as templates for controlled and oriented growth of apatite mineralization in vitro.

Control of apatite crystal growth by the co-operative effect of a recombinant porcine amelogenin and fluoride.

Recently, we used native amelogenins extracted from developing pig enamel to examine the combined effect of fluoride and amelogenins on the growth of octacalcium phosphate (OCP) and apatite crystals. The purpose of the present study was to investigate this combined effect using a highly purified recombinant amelogenin. We applied porcine amelogenin (rP172) and fluoride in a dual-membrane system as a model for tooth enamel formation. The combination of rP172 and fluoride in this system resulted in the formation of rod-like apatite crystals. On the other hand, without fluoride, rod-like OCP crystals of a comparable size were formed, and rather large hexagonal prisms of mixed crystals of OCP and apatite grew without amelogenins. Thus, highly purified and homogeneous recombinant amelogenin, in co-operation with F, regulated the mineral phase, habit, and size of crystals in the same manner as the extracted heterogeneous porcine amelogenins. We suggest that in both cases the control over the crystal phase and morphology was a direct effect of amelogenin protein serving as a scaffold for apatite mineralization.

Anisotropic properties of the enamel organic extracellular matrix.

Enamel biosynthesis is initiated by the secretion, processing, and self-assembly of a complex mixture of proteins. This supramolecular ensemble controls the nucleation of the crystalline mineral phase. The detection of anisotropic properties by polarizing microscopy has been extensively used to detect macromolecular organizations in ordinary histological sections. The aim of this work was to study the birefringence of enamel organic matrix during the development of rat molar and incisor teeth. Incisor and molar teeth of rats were fixed in 2% paraformaldehyde/0.5% glutaraldehyde in 0.2 M phosphate-buffered saline (PBS), pH 7.2, and decalcified in 5% nitric acid/4% formaldehyde. After paraffin embedding, 5-microm-thick sections were obtained, treated with xylene, and hydrated. Form birefringence curves were obtained after measuring optical retardations in imbibing media, with different refractive indices. Our observations showed that enamel organic matrix of rat incisor and molar teeth is strongly birefringent, presenting an ordered supramolecular structure. The birefringence starts during the early secretion phase and disappears at the maturation phase. The analysis of enamel organic matrix birefringence may be used to detect the effects of genetic and environmental factors on the supramolecular orientation of enamel matrix and their effects on the structure of mature enamel.

Dynamic light scattering study of an amelogenin gel-like matrix in vitro.

Amelogenin self-assembly is critical for the structural organization of apatite crystals during enamel mineralization. The aim of the present study was to investigate the influence of temperature and protein concentration on the aggregation of amelogenin nanospheres at high protein concentrations (>4.4 mg ml-1) in order to obtain an insight into the mechanism of amelogenin self-assembly to form higher-order structures. Amelogenins were extracted from enamel scrapings of unerupted mandibular pig molars. The dynamics of protein solutions were measured using dynamic light scattering (DLS) as a function of temperature and at acidic pH. At pH 4-5.5, three kinds of particles were observed, ranging in size from 3 to 80 nm. At pH 6, heating the solution above approximately 30 degrees C resulted in a drastic change in the solution transparency, from clear to opaque. Low pH showed no aggregation effect, whilst solutions at a slightly acidic pH exhibited diffusion dynamics associated with the onset of aggregation. In addition, at the same temperature range, the hydrodynamic radii of the aggregates increased drastically, by almost one order of magnitude. These observations support the view that hydrophobic interactions are the primary driving force for the pH- and temperature-sensitive self-assembly of amelogenin particles in a 'gel-like' matrix. The trend of self-assembly in a 'gel-like matrix' is similar to that in solution.

The effect of recombinant mouse amelogenins on the formation and organization of hydroxyapatite crystals in vitro.

Amelogenin is the most abundant protein in developing dental enamel. It is believed to play an important role in the regulation of the growth and organization of enamel crystals. Amelogenin, unlike many other proteins found in biominerals, is mostly hydrophobic except for a 13 amino acid hydrophilic C-terminal domain. To clarify the role of amelogenin in enamel mineralization, we designed calcium phosphate crystal growth experiments in the presence of recombinant amelogenins with or without the charged C-terminal domain. The shape and organization of the crystals were examined by TEM in bright field and diffraction modes. It was found that both full-length and truncated amelogenin inhibit crystal growth in directions normal to the c-axis. At the same time, crystallites organized into parallel arrays only in the presence of the full-length amelogenin in monomeric form. Pre-assembled amelogenins had no effect on crystals organization. These results imply that the hydrophobic portion of amelogenin plays a role in an inhibition of crystal growth, whereas the C-terminal domain is essential for the alignment of crystals into parallel arrays. Our data also suggest that nascent enamel structure emerges as a result of cooperative interactions between forming crystals and assembling proteins.