Secretory Enamel Research Articles

Role of amelogenin phosphorylation in regulating dental enamel formation

Amelogenin (AMELX), the predominant matrix protein in enamel formation, contains a singular phosphorylation site at Serine 16 (S16) that greatly enhances AMELX's capacity to stabilize amorphous calcium phosphate (ACP) and inhibit its transformation to apatitic enamel crystals. To explore the potential role of AMELX phosphorylation in vivo, we developed a knock-in (KI) mouse model in which AMELX phosphorylation is prevented by substituting S16 with Ala (A). As anticipated, AMELXS16A KI mice displayed a severe phenotype characterized by weak hypoplastic enamel, absence of enamel rods, extensive ectopic calcifications, a greater rate of ACP transformation to apatitic crystals, and progressive cell pathology in enamel-forming cells (ameloblasts). In the present investigation, our focus was on understanding the mechanisms of action of phosphorylated AMELX in amelogenesis. We have hypothesized that the absence of AMELX phosphorylation would result in a loss of controlled mineralization during the secretory stage of amelogenesis, leading to an enhanced rate of enamel mineralization that causes enamel acidification due to excessive proton release. To test these hypotheses, we employed microcomputed tomography (µCT), colorimetric pH assessment, and Fourier Transform Infrared (FTIR) microspectroscopy of apical portions of mandibular incisors from 8-week old wildtype (WT) and KI mice. As hypothesized, µCT analyses demonstrated significantly higher rates of enamel mineral densification in KI mice during the secretory stage compared to the WT. Despite a greater rate of enamel densification, maximal KI enamel thickness increased at a significantly lower rate than that of the WT during the secretory stage of amelogenesis, reaching a thickness in mid-maturation that is approximately half that of the WT. pH assessments revealed a lower pH in secretory enamel in KI compared to WT mice, as hypothesized. FTIR findings further demonstrated that KI enamel is comprised of significantly greater amounts of acid phosphate compared to the WT, consistent with our pH assessments. Furthermore, FTIR microspectroscopy indicated a significantly higher mineral-to-organic ratio in KI enamel, as supported by µCT findings. Collectively, our current findings demonstrate that phosphorylated AMELX plays crucial mechanistic roles in regulating the rate of enamel mineral formation, and in maintaining physico-chemical homeostasis and the enamel growth pattern during early stages of amelogenesis.

MMP20-ablated Induced Aberrant Mineralization in Early Secretory Enamel

MMP20-ablated Induced Aberrant Mineralization in Early Secretory Enamel

Mapping the Tooth Enamel Proteome and Amelogenin Phosphorylation Onto Mineralizing Porcine Tooth Crowns.

Tooth enamel forms in an ephemeral protein matrix where changes in protein abundance, composition and posttranslational modifications are critical to achieve healthy enamel properties. Amelogenin (AMELX) with its splice variants is the most abundant enamel matrix protein, with only one known phosphorylation site at serine 16 shown in vitro to be critical for regulating mineralization. The phosphorylated form of AMELX stabilizes amorphous calcium phosphate, while crystalline hydroxyapatite forms in the presence of the unphosphorylated protein. While AMELX regulates mineral transitions over space and time, it is unknown whether and when un-phosphorylated amelogenin occurs during enamel mineralization. This study aims to reveal the spatiotemporal distribution of the cleavage products of the most abundant AMLEX splice variants including the full length P173, the shorter leucine-rich amelogenin protein (LRAP), and the exon 4-containing P190 in forming enamel, all within the context of the changing enamel matrix proteome during mineralization. We microsampled permanent pig molars, capturing known stages of enamel formation from both crown surface and inner enamel. Nano-LC-MS/MS proteomic analyses after tryptic digestion rendered more than 500 unique protein identifications in enamel, dentin, and bone. We mapped collagens, keratins, and proteolytic enzymes (CTSL, MMP2, MMP10) and determined distributions of P173, LRAP, and P190 products, the enamel proteins enamelin (ENAM) and ameloblastin (AMBN), and matrix-metalloprotease-20 (MMP20) and kallikrein-4 (KLK4). All enamel proteins and KLK4 were near-exclusive to enamel and in excellent agreement with published abundance levels. Phosphorylated P173 and LRAP products decreased in abundance from recently deposited matrix toward older enamel, mirrored by increasing abundances of testicular acid phosphatase (ACPT). Our results showed that hierarchical clustering analysis of secretory enamel links closely matching distributions of unphosphorylated P173 and LRAP products with ACPT and non-traditional amelogenesis proteins, many associated with enamel defects. We report higher protein diversity than previously published and Gene Ontology (GO)-defined protein functions related to the regulation of mineral formation in secretory enamel (e.g., casein α-S1, CSN1S1), immune response in erupted enamel (e.g., peptidoglycan recognition protein, PGRP), and phosphorylation. This study presents a novel approach to characterize and study functional relationships through spatiotemporal mapping of the ephemeral extracellular matrix proteome.

Proteolysis by MMP20 Prevents Aberrant Mineralization in Secretory Enamel

The present study was conducted to investigate the role of proteolysis by matrix metalloproteinase 20 (MMP20) in regulating the initial formation of the enamel mineral structure during the secretory stage of amelogenesis, utilizing Mmp20-null mice that lack this essential protease. Ultrathin sagittal sections of maxillary incisors from 8-wk-old wild-type (WT), Mmp20-null (KO), and heterozygous (HET) littermates were prepared. Secretory-stage enamel ultrastructures from each genotype as a function of development were compared using transmission electron microscopy, selected area electron diffraction, and Raman microspectroscopy. Characteristic rod structures observed in WT enamel exhibited amorphous features in newly deposited enamel, which subsequently transformed into apatite-like crystals in older enamel. Surprisingly, initial mineral formation in KO enamel was found to proceed in the same manner as in the WT. However, soon after a rod structure began to form, large plate-like crystals appeared randomly within the developing KO enamel layer. As development continued, observed plate-like crystals became dominant and obscured the appearance of the enamel rod structure. Upon formation of these plate-like crystals, the KO enamel layer stopped growing in thickness, unlike WT and HET enamel layers that continued to grow at the same rate. Raman results indicated that Mmp20-KO enamel contains a significant portion of octacalcium phosphate, unlike WT enamel. Although normal in all other respects, large, randomly dispersed mineral crystals were observed in secretory HET enamel, although to a lesser extent than that seen in KO enamel, indicating that the level of MMP20 expression has a proportional effect on suppressing aberrant mineral formation. In conclusion, we found that proteolysis of extracellular enamel matrix proteins by MMP20 is not required for the initial development of the enamel rod structure during the early secretory stage of amelogenesis. Proteolysis by MMP20, however, is essential for the prevention of abnormal crystal formation during amelogenesis.

Amelogenin Exon4 Forms a Novel miRNA That Directs Ameloblast and Osteoblast Differentiation

Amelogenins constitute the major portion of secretory enamel matrix proteins and are known to be highly alternative spliced. Of all the alternatively spliced forms of amelogenins, exon4 is most commonly spliced out. Our analyses of the exon4 sequence led us to hypothesize that when spliced out, exon4 may generate a novel mature miRNA. To explore this possibility, we used in vivo mouse models (wild-type and Amel knockout mice) and in vitro cell culture to investigate the presence and function of a mature miRNA derived from exon4 (miR-exon4). When ameloblast-like cells (LS8) were transfected with an amelogenin minigene to increase amelogenin synthesis, the transfected cells synthesized miR-exon4. Introduction of a mutation in the conserved CNNC sequence required for primary miRNA recognition, downstream of the mature miR-exon4 sequence, resulted in a significantly reduced production of miR-exon4 in the transfected cells. In vivo, miR-exon4 was most highly amplified from wild-type mouse enamel organs at the secretory stage. In Amel knockout mice, an in vivo model for reduced amelogenin synthesis, we found reduced miR-exon4, with no changes in expression of enamel matrix–related genes. However, expression of Runx2 and its downstream genes Odam and Amtn were significantly downregulated. Transfection of miR-exon4 mimic to the LS8 cells also significantly upregulated Runx2. The mature miR-exon4 as well as Runx2 was also present in mouse osteoblasts with no apparent change in expression level between wild-type and Amel knockout mice. However, transfecting miR-exon4 inhibitor to the MC3T3-E1 osteoblastic cells resulted in a significant downregulation of Runx2 expression. These data indicate that when exon4 is spliced out, as occurs most of the time during alternative splicing of amelogenin pre-mRNA, a novel mature miRNA is generated from exon4. This miR-exon4 may contribute to the differentiation of ameloblasts and osteoblasts through regulation of Runx2 expression.

Expression of Tissue Inhibitors of Metalloproteinases in Developing Rat Tooth Germs

Tissue inhibitors of metalloproteinases (TIMPs) are a family of secreted molecules that were identified as natural inhibitors of matrix metalloproteinases (MMPs). Tooth histomorphogenesis and cytodifferentiation are accompanied by rapid changes in cellular organization and remodeling of the extracellular matrix, in which MMPs and TIMPs might be expected to play significant roles. This study examined the expression and localization of TIMP-1 and TIMP-2 during the molar development of rats. The expression patterns of TIMPs were determined from Sprague-Dawley rat pups including the developing molars using RT-PCR, western blot and immunofluorescent staining. Gene and protein quantification analyses showed that both TIMPs increased from the cap stage to the root stage tooth germs. In contrast, the immunofluorescent data showed that they were expressed slight differentially. TIMP-1 was strongly expressed in secretory ameloblasts and moderate immunoreactivity was observed along the basement membrane. TIMP-2 expression was also detected in the basement membrane. Although strong immunoreactivity was observed in the secretory ameloblasts and enamel matrix itself, differentiated odontoblasts showed weak reactivity. However, little reactivity for both TIMPs were detected in the cap stage tooth germs and surrounding tissues. These distinct temporospatial expression patterns of TIMPs suggest that the TIMPs may play a variety of roles including dental hard tissue formation during molar tooth development.

Fluoride effect on the secretory-stage enamel organic extracellular matrix of mice

The formation of an ordered enamel organic extracellular matrix (EOECM) seems to be a crucial step for the proper formation of the enamel mineral phase. The ordered supramolecular structure of the EOECM in the secretory stage can be analyzed using polarizing microscopy, as it is strongly birefringent. Excessive fluoride (F) ingestion during tooth development can cause enamel fluorosis, leading to increased porosity in mature enamel. We analyzed the effects of F on the birefringence of the EOECM in the A/J, CBA, and DBA/2 strains of mice given 0, 11.25, and 45 ppm of fluoride in drinking water. In the CBA and DBA/2 strains, the 11.25 and 45 ppmF groups presented a significant decrease in optical retardation (OR) when compared with the respective 0 (CBA 11.25 ppmF p = 0.0056 and 45 ppmF p < 0.0001; DBA/2 11.25 and 45 ppmF p < 0.05). ORs in A/J 0 ppmF were significantly higher than in 45 (p < 0.0001). The enamel of the A/J strain was more severely affected by fluoride than it was in the other strains of mice and exhibited the lowest levels of fluoride in plasma, whereas its normal secretory enamel presented a significantly higher protein absorbance than it did in CBA and DBA mice (p = 0.0099 and p = 0.0025, respectively). The results showed that experimental fluorosis can alter the supramolecular organization of EOECM in the secretory stage of amelogenesis and that the susceptibility to dental fluorosis seems to be influenced by the inherent characteristics of the developing enamel.

A study in situ of the effect of metallo- and serine proteinase inhibitors on the birefringence of the secretory stage enamel organic extracellular matrix

Dental enamel formation occurs extracellularly and establishment of an ordered enamel organic extracellular matrix (ECM) seems to be crucial for proper construction of the enamel mineral phase. Polarizing microscopy shows that the ordered supramolecular structure of the secretory stage enamel organic ECM exhibits strong birefringence. We reported earlier that this birefringence is lost in unfixed specimens, probably due to extensive proteolytic cleavage of enamel proteins. Therefore, we investigated the association between enamel proteinase activities by analyzing the effects of metallo- and serine proteinase inhibitors in situ on the birefringence of the secretory stage enamel organic ECM. Male rats were used in the present study. After sacrifice, distal 10 mm fragments of upper incisors were removed and immersed for 15 h under continuous shaking at 37°C in one of the following solutions: 1) 10 mM Tris, pH 8.0; 150 mM NaCl (negative control, n = 8); 2) 2% paraformaldehyde and 0.5% glutaraldehyde in 0.2 M phosphate-buffered saline (PBS), pH 7.2 (positive control, n = 5); 3) 10 mM Tris, pH 8.0; 150 mM NaCl; 2 mM 1,10-phenanthroline (n = 9); 4) 10 mM Tris, pH 8.0; 150 mM NaCl; 2 mM phenylmethyl-sulfonyl fluoride (PMSF) (n = 8); 5) 10 mM Tris, pH 8.0; 150 mM NaCl; 2 mM 1,10-phenanthroline; 2 mM PMSF (n = 9). Samples then were immersed in fixative solution for 24 h and processed to obtain 5 μm thick longitudinal sections of the secretory stage enamel organic ECM. The sections were immersed in 80% glycerin for 30 min and analyzed by transmitted polarizing light microscopy. 1,10-Phenanthroline (inhibitor of metalloproteinases) and 1,10-phenanthroline + PMSF (inhibitor of serine proteinases) clearly prevented a decrease in the optical retardation of birefringence brightness from the tissue. PMSF alone promoted a slight preservation of the birefringence exhibited by the secretory stage enamel organic ECM. Rapid loss of birefringence in secretory stage enamel organic ECM that is not fixed immediately is caused by enamel proteinases and the activity of metalloproteinases seems to lead to preliminary degradation of the enamel organic ECM, which in turn facilitates subsequent serine proteinase activity.

Birefringence of the Secretory-Stage Enamel Organic Extracellular Matrix from Rats Submitted to Successive Injections of Bisphosphonates

The aim of the present study was to assess birefringence of the secretory-stage enamel organic extracellular matrix (ECM) and mechanical properties of mature enamel from rats treated with bisphosphonates. Longitudinal sections were obtained from upper incisors of rats that had been submitted to injections of bisodic etidronate (8 mg/Kg/day), sodium alendronate (30 μg/Kg/day), or sodium chloride as control (8 mg/Kg/day) for 42 days. Sections were immersed in 80% glycerin for 30 min and optical retardation of birefringence brightness in the secretory-stage enamel organic ECM was determined in nanometers. Etidronate-treated rats exhibited extensive morphological changes in the secretory-stage enamel organic ECM inclusive nonbirefringent conspicuous incremental lines, but presented optical retardation values similar to those showed by control rats (p > 0.05). Birefringence of secretory enamel organic ECM from etidronate rats presented an irregular aspect. Alendronate-treated rats did not show morphological alterations in the secretory-stage enamel organic ECM, however, they presented significant reduction in optical retardation of birefringence brightness when compared with control and etidronate rats (p < 0.01). Alendronate and etidronate groups exhibited reductions of ≈6–10% in mature enamel cross-sectional microhardness when compared with control group (p < 0.01). Scanning electron microscopy analysis showed extensive alterations in mature enamel only from etidronate group with absence of enamel rods. The present work shows that bisphosphonates can affect the birefringence of the secretory-stage enamel organic ECM. The results presented here suggest that alterations in the supramolecular organization of the secretory-stage enamel organic ECM are a plausible mechanism by which environmental factors may cause enamel defects.

Role of 20-kDa Amelogenin (P148) Phosphorylation in Calcium Phosphate Formation in Vitro

The potential role of amelogenin phosphorylation in enamel formation is elucidated through in vitro mineralization studies. Studies focused on the native 20-kDa porcine amelogenin proteolytic cleavage product P148 that is prominent in developing enamel. Experimental conditions supported spontaneous calcium phosphate precipitation with the initial formation of amorphous calcium phosphate (ACP). In the absence of protein, ACP was found to undergo relatively rapid transformation to randomly oriented plate-like apatitic crystals. In the presence of non-phosphorylated recombinant full-length amelogenin, rP172, a longer induction period was observed during which relatively small ACP nanoparticles were transiently stabilized. In the presence of rP172, these nanoparticles were found to align to form linear needle-like particles that subsequently transformed and organized into parallel arrays of apatitic needle-like crystals. In sharp contrast to these findings, P148, with a single phosphate group on serine 16, was found to inhibit calcium phosphate precipitation and stabilize ACP formation for more than 1 day. Additional studies using non-phosphorylated recombinant (rP147) and partially dephosphorylated forms of P148 (dephoso-P148) showed that the single phosphate group in P148 was responsible for the profound effect on mineral formation in vitro. The present study has provided, for the first time, evidence suggesting that the native proteolytic cleavage product P148 may have an important functional role in regulating mineralization during enamel formation by preventing unwanted mineral formation within the enamel matrix during the secretory stage of amelogenesis. Results obtained have also provided new insights into the functional role of the highly conserved hydrophilic C terminus found in full-length amelogenin.

Enamel Formation-Biochemical Aspect-

This review shows the biochemical aspect of enamel formation focused on porcine amelogenesis.Volumetric changes in the chemical components of secretory stage enamel intimate that enamel crystals thicken as amelogenin, the major protein component, decreases. Solubility behaviors and hydrophobicity analyses of amelogenin derivatives suggest the hypothesis that cylindrical amelogenin forms micelles. This model explains how amelogenin constructs a large structure, which forms a matrix, and continuously yields space as the crystals thicken. In porcine secretory enamel, mineral volume increases with depth, gaining space for thickening the existing crystallites by shrinking amelogenin micelles through the removal of C-terminal and 13-kDa peptides from the prototype 25-kDa amelogenin, the most abundant porcine amelogenin gene product, by the action of enamelysin (MMP-20) and enamel matrix serine proteinase (EMSP1), respectively.During the secretory stage, the crystallites thicken in the deeper secretory enamel, although calcium ions are supplied by ameloblasts. Another morphological feature of the crystallites is that they are all nearly the same size within the same developmental stage. There are several enamel proteins and their cleavage products have calcium binding activity and binding affinity for crystallites. The 25-kDa amelogenin and 89-kDa enamelin, both neutral insoluble, are involved in slow crystallite growth by trapping calcium ions secreted by ameloblasts, and may control by inhibiting newly nucleated crystals between existing crystallites. The calcium binding proteins derived from the C-terminal side of sheathlin (ameloblastin/amelin) and 32-kDa enamelin, belonging to the neutral soluble fraction, act as a calcium carrier to transport calcium ions to deeper secretory stage enamel. Sheath protein derived from the N-terminal side of sheathlin is the main protein constituent of the enamel sheath.Enamelin may be involved in initial crystal formation as it is immuno-histochemically shown to be localized on or around the initial nucleated crystals in a presecretory stage matrix. This is confirmed by no crystal formation in the enamel of enamelin knock-out mice. This conflicts with the inhibition of new nucleation between the existing crystallites in advanced secretory stage enamel. Enamelin may have multi-functions which are involved in the induction of nucleation in a particular phase of the molecule and inhibition in other phases, or change its function with the products produced during each degradation step.

Amelogenin proteins of developing dental enamel.

The amelogenins of developing dental enamel are tissue-specific proteins, rich in proline, leucine, histidine and glutamyl residues, and synthesized by the ameloblast cells of the inner enamel epithelium. These proteins comprise the bulk of the extracellular matrix that becomes mineralized with a hydroxyapatite phase to become the mature enamel. Examination of the amino acid sequences of amelogenins from a range of mammals shows a high degree of evolutionary sequence conservation, suggestive of specialized function. Recently it has been shown that multiple amelogenin components, observed in the matrix, arise both by a sequence of post-secretory proteolytic processing and by the expression of alternatively spliced mRNAs generated from the amelogenin gene(s) that are located on the sex chromosomes. Although the function of these amelogenins in enamel biomineralization is unknown, physico-chemical studies of recombinant amelogenins have shown that they undergo a self-assembly process in vitro generating supra-molecular 'nanosphere' structures, and recent observations in vivo point to a functional role for the nanospheres in the ultrastructural organization of the secretory enamel matrix, conducive to the organized development of the earliest mineral crystallites.

The 17‐kDa sheath protein in enamel proteins induces cementum regeneration in experimental cavities created in a buccal dehiscence model of dogs

Commercially available enamel proteins, such as Emdogain, are clinically used for periodontal regeneration. However, the real mechanisms behind the bioactivities of enamel proteins is still unclear, as enamel proteins have multicomponents. The purpose of this in vivo study was to identify the cementum regeneration-promoting factor in enamel proteins that is clinically used for periodontal regeneration to induce cementum-promotive and osteopromotive activities. Cementum regeneration, which is an important part of periodontal regeneration, was examined in experimental cavities prepared on a buccal dehiscence model of dogs. The purification of enamel protein with cementum regeneration activity was carried out by gel filtration and ion exchange chromatographies of newly formed secretory enamel. Cementum regeneration activity was found in the aggregate comprising 13-17-kDa sheath proteins along with a small amount of amelogenins, found in the newly formed secretory enamel. In these proteins, cementum regeneration activity was detected upon application of the 17-kDa sheath protein, but not by other lower molecular-weight sheath proteins and amelogenins. However, the purified 17-kDa sheath protein induced cementum regeneration activity only in a small area, although the regenerated cementum was thick. The activity of the 17-kDa sheath protein was believed not to have been a result of contamination by growth factors such as transforming growth factor-beta1 (TGF-beta1) found in the enamel protein, as the application of TGF-beta1 induced weak cementum regeneration activity. It is concluded that the 17-kDa sheath protein itself exhibits cementum regeneration activity, although other factors may be needed to demonstrate its full ability.

Short exposure to high levels of fluoride induces stage‐dependent structural changes in ameloblasts and enamel mineralization

We tested the hypothesis that the sensitivity of forming dental enamel to fluoride (F-) is ameloblast developmental stage-dependent and that enamel mineralization disturbances at the surface of fluorotic enamel are caused by damage to late-secretory- and transitional-stage ameloblasts. Four-day-old hamsters received a single intraperitoneal dose of 2.5-20 mg NaF/kg body weight and were examined, 24 h later, by histology and histochemistry. A single dose of >or=5 mg of NaF/kg induced the formation of a hyper- followed by a hypomineralized band in the secretory enamel, without changing the ameloblast structure. At 10 mg of NaF/kg, cystic lesions became apparent under isolated populations of distorted late-secretory- and transitional-stage ameloblasts. Staining with von Kossa stain showed that the enamel under these lesions was hypermineralized. At 20 mg of NaF/kg, cystic lesions containing necrotic cells were also found in the early stages of secretory amelogenesis and were also accompanied with hypermineralization of the enamel surface. We concluded that the sensitivity to F- is ameloblast developmental stage-dependent. Groups of transitional ameloblasts are most sensitive, followed by those at early secretory stages. These data suggest that a F-induced increase in cell death in the transitional-stage ameloblasts accompanies the formation of cystic lesions, which may explain the formation of enamel pits seen clinically in erupted teeth.

Porcine sheath proteins show periodontal ligament regeneration activity

The purpose of this study was to identify the periodontal regeneration factors of enamel protein extracts that induce cementum and bone regeneration in vivo. Cementum regeneration, one aspect of periodontal ligament regeneration, was examined using a buccal dehiscence model of dogs. Enamel matrix protein fractions were prepared from developing porcine incisors. Cementum-regeneration activity was found to reside in a protein aggregate composed of amelogenins and sheath proteins extracted from newly formed secretory enamel. Cementum-regeneration activity was not observed in protein fractions containing only amelogenin or its derivatives. The sheath proteins were purified to homogeneity and tested for alkaline phosphatase (ALP)-inducing activity on human periodontal ligament (HPDL) cells. The induction of ALP was observed following application of the 17-kDa sheath protein but not of the lower-molecular-weight sheath proteins. Although transforming growth factor-beta1 also shows ALP-inducing activity, contamination with growth factors was excluded because synthetic peptides (based on the sheath protein's sequence) also showed ALP-inducing activity. The 17-kDa sheath protein showed both cytodifferentiation and cementum-regeneration activity, but it is unclear whether its cementum-regeneration activity is derived from its ALP-inducing activity on HPDL cells.

Adsorption of Amelogenin Nanopheres onto Charged Surfaces, A Model for Tooth Enamel Matrix Re-construction

AbstractAmelogenins are hydrophobic proteins that constitute more than 90% of the secretory stage enamel matrix. The assembly of amelogenin into nanospheres has been postulated to be a key factor in controlling the structural organization of the enamel extracellular matrix framework, which provides the scaffolding for the elongated and oriented growth of enamel apatite crystals. To get insight into the structure and function of amelogenin in controlling the process of crystal growth we have utilized two different approaches to investigate adsorption of amelogenin nanospheres onto charged surfaces: A) analysis of adsorption of amelogenin onto hydroxyapatite crystals by means of Langmuir Model for protein adsorption. B) analysis of amelogenin mono or multi-layer formation by sequential adsorption process onto auto-assembled polyelctrolytes films. Our data indicate that amelogenin nanospheres adsorb onto the surface of apatite crystals as binding units with defined adsorption sites. We found that amelogenin nanospheres are negatively charged and a monolayer of these nanospheres adsorbed in an irreversible way on positively ending polyelectrolyte multilayers most likely through electrostatic interactions.

Sexual dimorphism of porcine amelogenins: male-specific amelogenins have strong adsorption properties onto apatite crystals.

The present studies were undertaken to investigate the sexual dimorphism of porcine amelogenins and to gain information as to whether excesses of male amelogenins, if any, possess functional significance in protein-crystal interactions. Enamel proteins, including the intact full-length amelogenins and their degraded polypeptides, were isolated from the secretory enamel of male and female pigs. To identify the amelogenins among the separated pools of male- and female-matrix proteins, rabbit anti-C13 and C25 peptide sera were used, which reacted specifically with the conserved C-terminal domain. Immunoblotting showed that a few extra members of the amelogenins, sharing common epitopes at the C-terminus, were recognized in male products. The apparent yield of the male amelogenins was only marginal, on the basis of their stained intensities on the gel, but the secreted male amelogenins demonstrated selective (probably the strongest among the amelogenins) adsorption properties onto apatite crystals. Reflecting the general symmetric electrophoretic profiles of the male- and female-enamel proteins in toto, there were no sex-linked differences in the protein-crystal interaction and the resulting regulatory function of crystal precipitation.

Immunoblot detection and expression of enamel proteins at the apical portion of the forming root in porcine permanent incisor tooth germs.

There have been many immunohistochemical studies of enamel proteins during root formation. In the present article, the detection and expression of enamel proteins in tissue samples prepared from the apical portion of the forming root (APFR) in porcine permanent incisor tooth germs were studied. Amelogenin, enamelin, and sheathlin were detected by immunoblot analysis, but only in small amounts. The detection of their derivatives indicated their degradation. It is, at present, unclear as to which proteinases are involved in these degradations, because activity of enamel matrix serine proteinase 1 and enamelysin was not detected on gelatin and casein zymograms. The expression of enamel proteins was also proved in the APFR sample by the detection of polymerase chain reaction products of their cDNAs, and this may be related to cells of fragmentized Hertwig's epithelial root sheath. Amelogenin expression was not greater than that of enamelin and sheathlin. It was different from the expression pattern of secretory ameloblasts involved in enamel matrix formation. These results suggest that the amelogenins found in the APFR do not form a three-dimensional structure of amelogenin micelles, which has been proposed for the secretory enamel matrix structure. In this case, the enamel proteins could spread out easily following degradation into the matrix of future cementum. Some of their derivatives may play a role in the formation of the cementum.

Immunohistochemical Detection and Distribution of Enamelysin (MMP-20) in Human Odontogenic Tumors

Enamelysin is a tooth-specific protease that was initially isolated from porcine enamel organ and subsequently from human odontoblasts. Since this protease is thought to play important roles in tooth development, the evaluation of enamelysin in odontogenic tumors may aid our understanding of the histogenesis and cell differentiation of such lesions. A monoclonal antibody (203-1C7) was generated against synthesized human enamelysin oligopeptide and was used to assess the immunolocalization of enamelysin in healthy developing tooth germs and various types of odontogenic lesions. In tooth germs, enamelysin expression was detected only in the secretory enamel. Thus, 203-1C7 may serve as an enamel-specific marker in the late stage of enamel matrix development and calcification. In odontogenic lesions, strong enamelysin staining was demonstrated in the immature enamel matrix of ameloblastic fibro-odontomas and odontomas. Furthermore, enamelysin was also detected in globular amyloid masses and calcified foci in calcifying epithelial odontogenic tumors, hyaline droplets, small and large mineralized areas in adenomatoid odontogenic tumors, and a portion of ghost cells in calcifying odontogenic cysts. Positive reactivity was also observed in selected tumor cells in some of these tumors. No intracellular staining for enamelysin was detected in ameloblastomas or the ameloblastic portion of ameloblastic fibro-odontomas. Also, enamelysin was not detected in dentin, dysplastic dentinoid hyaline matrices, and cementum that were present within the tumors examined. Thus, taken together, our results suggest that the enamelysin-specific monoclonal antibody (203-1C7) may be utilized as a marker of early enamel development and that enamelysin may be involved in the pathogenesis of specific odontogenic tumors.

Time-related changes of developing enamel crystals after exposure to the tissue fluid in vivo: analysis of a subcutaneously implanted rat incisor.

To investigate the effects of tissue fluid on the growth of enamel crystals, upper and lower incisors extracted from 3-week-old Wistar rats were removed of the enamel organ, implanted subcutaneously in the dorsal portion of animals from the same litter, and harvested at 72 h or 1 week after implantation. The grafts were chemically fixed with surrounding tissues and prepared for light and electron microscopy, X-ray microanalysis, or for the immunohistochemistry of amelogenin. Mineralization of implanted enamel layers was examined by contact X-ray microradiography. The immunoreactivities for 25 kD amelogenin in immature enamel decreased sequentially, starting from the surface to the deeper layers; by 1 week after implantation, no positive reactivities remained in the entire enamel layers at the stages of matrix formation and early maturation. In accordance with the loss of enamel proteins, immature enamel gained mineral density until it attained higher radio opacity than that of the adjacent dentin by 1 week. In contrast, the radio opacity of the full thickness of the enamel at early maturation remained low except for a superficial thin layer. Electron microscopy revealed no sign of growth of original enamel crystals, but showed heavy precipitation of electron-dense fine granules of calcium phosphate in all layers of the secretory enamel and the superficial layer of enamel at early maturation, which showed high radio opacity. The Ca/P ratio and electron diffraction patterns of the granular materials precipitated between intrinsic enamel crystals indicated the property of hydroxy apatite or octacalcium phosphate though a characteristic ribbon-like profile of enamel crystals was lacking. These data indicate that the enamel organ blocks exogenous mineral precipitates in growing enamel during the stage of matrix formation and plays an essential regulatory role for fine enamel crystallites to grow into large hexagonal crystals.