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In order to compare its characteristics with those of jaw tooth collar enamel, normally developing and experimentally regenerating ganoine from ganoid scales of Lepisosteus oculatus (spotted gar), an actinopterygian fish species, was examined by Western blotting and immunohistochemistry. Amelogenin, a major enamel matrix protein (EMP), is widely found from sarcopterygian fish to mammals. Therefore, we used antimammalian amelogenin antibodies and antisera: an antibody against bovine amelogenin; antiserum against porcine amelogenin; and region-specific antibodies or antiserum against the C-terminus, middle region, or N-terminus of porcine amelogenin in this study. Positive immunoreactivity with the antibody against bovine amelogenin, antiserum against porcine amelogenin, and the middle and C-terminal region-specific antibodies was detected in both normally developing and regenerating ganoine matrix, as well as in granules found within inner ganoine epithelial cells. These immunohistochemical analyses indicated that the Lepisosteus ganoine matrix contains EMP-like proteins with epitopes similar to mammalian amelogenins. In Western blotting analyses of regenerating ganoid scales with the antibovine amelogenin antibody, two protein bands with molecular weights of approximately 78 and 65 kDa were detected, which were similar to those found in Lepisosteus tooth enamel. Our study suggests that in Lepisosteus, EMP-like proteins in the ganoine matrix corresponded to those in tooth enamel. However, it was revealed that the 78 and 65 kDa EMP-like proteins were different from 27 kDa bovine amelogenin.

Enameloid is a well-mineralized tissue covering the tooth surface in fish and it corresponds to the outer-most layer of dentin. It was reported that both dental epithelial cells and odontoblasts are involved in the formation of enameloid. Nevertheless, the localization and timing of secretion of ectodermal enamel matrix proteins in enameloid are unclear. In the present study, the enameloid matrix during the stages of enameloid formation in spotted gar, Lepisosteus oculatus, an actinopterygian, was examined mainly by transmission electron microscopy-based immunohistochemistry using an anti-mammalian amelogenin antibody and antiserum. Positive immunoreactivity with the antibody and antiserum was found in enameloid from the surface to the dentin-enameloid junction just before the formation of crystallites. This immunoreactivity disappeared rapidly before the full appearance of crystallites in the enameloid during the stage of mineralization. Immunolabelling was usually found along the collagen fibrils but was not seen on the electron-dense fibrous structures, which were probably derived from matrix vesicles in the previous stage. In inner dental epithelial cells, the granules in the distal cytoplasm often showed positive immunoreactivity, suggesting that the enamel matrix protein-like proteins originated from inner dental epithelial cells. Enamel matrix protein-like proteins in the enameloid matrix might be common to the enamel matrix protein-like proteins previously reported in the collar enamel of teeth and ganoine of ganoid scales, because they exhibited marked immunoreactivity with the same anti-mammalian amelogenin antibodies. It is likely that enamel matrix protein-like proteins are involved in the formation of crystallites along collagen fibrils in enameloid.

Although most fish have no enamel layer in their teeth, those belonging to Lepisosteus (gars), an extant actinopterygian fish genus, do and so can be used to study amelogenesis. In order to examine the collar enamel matrix in gar teeth, we subjected gar teeth to light and electron microscopic immunohistochemical examinations using an antibody against bovine amelogenin (27 kDa) and antiserum against porcine amelogenin (25 kDa), as well as region-specific antibodies and antiserum against the C-terminus and middle region, and N-terminus of porcine amelogenin, respectively. The enamel matrix exhibited intense immunoreactivity to the anti-bovine amelogenin antibody and the anti-porcine amelogenin antiserum in addition to the C-terminal and middle region-specific antibodies, but not to the N-terminal-specific antiserum. These results suggest that the collar enamel matrix of gar teeth contains amelogenin-like proteins and that these proteins possess domains that closely resemble the C-terminal and middle regions of porcine amelogenin. Western blot analyses of the tooth germs of Lepisosteus were also performed. As a result, protein bands with molecular weights of 78 kDa and 65 kDa were clearly stained by the anti-bovine amelogenin antibody as well as the antiserum against porcine amelogenin and the middle-region-specific antibody. It is likely that the amelogenin-like proteins present in Lepisosteus do not correspond to the amelogenins found in mammals, although they do possess domains that are shared with mammalian amelogenins.

Objectives Phosphoric acid (PA) etching used in etch-and-rinse adhesives is known to activate host-derived dentinal matrix-metalloproteinases (MMPs) and increase dentinal permeability. These two phenomena will result, respectively; in degradation of dentine-adhesive bond and leaching of some monomers especially 2-hydroxyethyl methacrylate (HEMA) into the pulp that would negatively affect the viability of pulpal cells. This study is the first to investigate the inhibitory effect of non-protein thiols (NPSH); namely reduced glutathione (GSH) and N -acetylcysteine (NAC) on dentinal MMPs and compare their effects on HEMA cytotoxicity. Methods Dentine powder was prepared from human teeth, demineralized with 1% PA and then treated with 2% GSH, 2% NAC or 2% chlorhexidine (CHX). Zymographic analysis of extracted proteins was performed. To evaluate the effect of GSH, NAC and CHX on HEMA cytotoxicity, solutions of these compounds were prepared with or without HEMA and rat pulpal cells were treated with the tested solutions for (6 and 24 h). Cells viability was measured by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cytotoxicity data were analysed by one-way ANOVA and Tukey post hoc tests ( p Results The inhibitory effect of GSH and NAC on dentinal MMPs was confirmed. GSH showed similar effectiveness to NAC regarding HEMA cytotoxicity inhibition. Conclusion NPSH were effective to inhibit dentinal MMPs and HEMA cytotoxicity. Clinical significance The tested properties of NPSH provide promising clinical use of these agents which would enhance dentine-bond durability and decrease post-operative sensitivity.

Previously, we have demonstrated that the extracellular matrix from dentin affects osteoclastic activity in co-culture between osteoclast and osteoblast-rich fraction from mouse marrow cells. In the present study, we aimed to investigate the mechanisms of dentin matrix extract-induced osteoclastogenesis in mouse bone marrow macrophages (BMMs). Dentin proteins were extracted from bovine incisor root dentin using 0.6 M HCl. BMMs were cultured in α-MEM containing macrophage colony-stimulating factor/receptor activator of nuclear factor kappa-B ligand in the presence or absence of dentin matrix extract. Tartrate-resistant acid phosphatase (TRAP)-positive cell number, total TRAP activity, and the mRNA levels of osteoclast-related genes, assayed by real-time RT-PCR, were determined as markers of osteoclastogenesis. A neutralizing antibody against transforming growth factor-β1 (TGF-β1), SB431542, a TGF-β receptor inhibitor, and ELISA were used to determine the role of TGF-β1. We observed increases in TRAP-positive cell number, TRAP activity, and the mRNA levels of osteoclast-related genes of BMMs cultured with dentin extract. The use of a neutralizing antibody against TGF-β1 or SB431542 inhibited the inductive effect of dentin extract, suggesting TGF-β1 involvement. The addition of exogenous TGF-β1, but not bone morphogenic protein-2, also increased osteoclastogenesis, corresponding to the ELISA determination of TGF-β1 in the dentin extract. In conclusion, our results indicate that proteins from dentin matrix have an inductive effect in osteoclastogenesis, which is mediated, in part, by TGF-β1.

Warunee Pluemsakunthai1), Shinji Kuroda2,*), Hitoyata Shimokawa3) and Shohei Kasugai1) 1)Oral Implantology and Regenerative Dental Medicine, Global Center of Excellence (G-COE) Program, Tokyo Medical and Dental University, Tokyo, Japan 2)Oral Implantology and Regenerative Dental Medicine, Tokyo Medical and Dental University, Tokyo, Japan 3)Pediatric dentistry, Tokyo Medical and Dental University, Tokyo, Japan

In the rat tibial bone marrow cavity, following colchicine injection, there is a phase of osteogenesis in which bone trabeculae replace the necrotic bone marrow tissues and fill the marrow cavity. The newly formed bone is subsequently resorbed by osteoclasts and normal bone marrow is restored. In this study, we correlated these morphologic events with the pattern of gene expression of bone sialoprotein (BSP), an extracellular matrix protein in mineralized tissues, to elucidate the possible functions of BSP in bone formation and resorption in vivo. The expressions of osteopontin (OPN) and type I collagen were also examined. Northern hybridization of the tibia demonstrated that OPN mRNA was gradually increased and expressed at a maximal level 10 days after colchicine injection (during the bone resorption process), while BSP mRNA expression already reached a maximal level at day 6 (during the initial process of bone formation). Its expression was, thus, quite temporary at the beginning of bone formation and different from that of type I collagen, which was continually elevated from days 6 to 10. In situ hybridization of the newly formed bone induced in the tibia revealed that BSP mRNA was evenly expressed in most osteoblasts and osteocytes, moreover in interconnecting colonies of spindle-shaped cells, possibly preosteoblasts, at day 6. At day 10, however, its expression became restricted to some cells on the bone surfaces, some osteoblasts, and most osteoclasts. These observations suggest that BSP may play an important role mainly in the initiation of bone formation and is also associated with the functions of osteoclast in vivo.

Physiological root resorption is a phenomenon that normally takes place in deciduous teeth; root resorption of permanent teeth occurs only under pathological conditions. The molecular mechanisms underlying these processes are still unclear. Our previous study showed that osteoclasts cultured on deciduous dentine exhibited a higher degree of resorption and higher levels of cathepsin K and MMP-9 mRNA than osteoclasts cultured on permanent dentine. These results could be because of different susceptibilities to acid and the different organic matrices between deciduous and permanent dentine. Thus, the purpose of this study was to investigate the effect of dentine extracts from bovine deciduous and permanent dentine on osteoclast activity. Osteoclasts, obtained from mouse bone marrow cells co-cultured with an osteoblast-rich fraction in the presence of 1,25-(OH)(2)-vitamin D3 and PGE2, were incubated with or without 0.6 M HCl extracts from bovine deciduous or permanent dentine for 48 h. TRAP positive cell number, TRAP activity, the areas of resorption pits, and mRNA levels of TRAP, v-ATPase, calcitonin receptor, cathepsin K, and MMP-9 were examined. The results illustrated that TRAP activity, the resorbed area, and the mRNA levels of osteoclast marker genes seemed to be suppressed by both deciduous and permanent dentine extracts. These findings indicate that some factors that suppress osteoclast activity are contained in both deciduous and permanent dentine extracts. Although there was no significant difference in osteoclast activity between deciduous and permanent dentine extracts, osteoclasts incubated with permanent dentine extracts tend to exhibit less resorption activity than those incubated with deciduous dentine extracts. However, we could not clearly explain the causes of this.

Membrane type 1-matrix metalloproteinase (MT1-MMP) is a membrane-bound matrix metalloproteinase capable of mediating pericellular proteolysis of extracellular matrix components. In osteoclasts, the localization of MT1-MMP has been reported at the tips of specialized membrane protrusions (podosomes and lamellipodia) so that osteoclasts might use MT1-MMP to perform focal proteolysis and move through the extracellular matrix to the bone surface. The objectives of this study were to investigate an association of MT1-MMP in physiological root resorption of the deciduous tooth by reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analysis, and to identify MT1-MMP-producing cell during deciduous tooth resorption by in situ hybridization and immunohistochemistry. RT-PCR and Northern blot analysis revealed the exclusively high expression of MT1-MMP mRNA in bovine root-resorbing tissue, which lies between the root of the deciduous tooth and its permanent successor. Expression of MT1-MMP mRNA was seen in odontoclasts aligning in the surface layer of the root-resorbing tissue at sites of root resorption. Furthermore, immmunohistochemistry also confirmed the localization of MT1-MMP protein to the odontoclasts. The present identification of MT1-MMP in odontoclasts during deciduous tooth resorption might be relevant to the migration activity that these cells have to gain access to the root surface.

Smad3 is an intracellular signaling molecule that mediates the signal from transforming growth factor-beta (TGF-beta) and activin receptors. In this study, we reveal hypomineralized enamel in mice with the targeted deletion of the Smad3 gene. The Smad3 (-/-) mice had chalky white incisor enamel, while the enamel of the wild-type or Smad3 (+/-) mice was yellow-brown. Histological analysis of the undecalcified sections showed that the enamel thickness of the maxillary incisors in the Smad3 (-/-) mice was similar to that of the wild-type and Smad3 (+/-) mice while that the enamel of the maxillary molars in Smad3 (-/-) mice was disrupted in places. Microcomputed tomography (microCT) analysis revealed that the mineralization of the maxillary incisors and mandibular molars in the Smad3 (-/-) mice showed significant reduction in the degree of mineralization when compared to that of the wild-type and Smad3 (+/-) mice. Scanning electron microscopic (SEM) analysis of the mandibular incisors revealed that the enamel surface of the Smad3 (-/-) mice was irregular and disrupted in places and showed images similar to decalcified mature enamel. The histological analysis of the decalcified sections showed that distinct morphological changes in the ameloblasts at the secretory and maturational stages were not observed between the Smad3 (-/-) and Smad3 (+/-) or wild-type mice, while the enamel matrix was observed in the decalcified sections of the mandibular molars in the Smad3 (-/-) mice. These results suggested that Smad3 was required for enamel biomineralization, and TGF-beta and activin signaling might be critical for its process.

The condylar cartilage, an important growth site in the mandible, shows characteristic modes of growth and differentiation, unlike the limb bud cartilage. To elucidate the mechanism of chondrogenesis at the condylar cartilage, we analyzed the effects of basic fibroblast growth factor (bFGF) on the growth and development of mouse mandibular condyle using serum-free organ culture and on the expression of genes related to the chondrogenesis. Further, we investigated the localization of bFGF in cultured condyle by immunohistochemistry. The present immunohistochemical observations showed that bFGF is localized in the extracellular matrix of the mesenchymal condylar anlage, the perichondrium and the proliferative cell zone, and that immunostaining was diminished in the metachromatically stained area. In the condyle culture with added recombinant human bFGF (rhbFGF) for 5 days, the area occupied by hypertrophic chondrocytes in the mandibular condylar cartilage was reduced. A reverse transcription-polymerase chain reaction (RT-PCR) assay also showed that the mRNA levels of aggrecan and type X collagen were reduced compared with nontreated tissues. Treatment with rhbFGF for 2 days decreased cell proliferation in the perichondrium, and bFGF downregulated the Indian hedgehog (Ihh), parathyroid hormone-related protein (PTHrP), bone morphogenetic protein 4 (BMP4), and core-binding factor alpha1 (Cbfa1) expression in the RT-PCR assay. These findings suggest that bFGF has the ability for inhibitory regulation of condylar growth, via the inhibition of proliferation and differentiation of chondrocytes, and that this inhibitory regulation is related to the downregulation of growth factors and transcription factors.

Background: Odontoma-like structures are formed in the jaw bone of osteopetrotic (op/op) mice, which have a congenital deficiency in osteoclastic differentiation due to the absence of functional macrophage colony-stimulating factor (M-CSF). Methods: To clarify the histopathogenesis of tooth malformation and odontoma-like structures, a 2-year postnatal process of development of the op/op mandibular incisor was examined radiologically and histologically. At the same time, extracellular matrix (ECM) remodeling around tooth germs was analyzed immunohistochemically. Results: Abnormal forms of op/op tooth germ were noticeable even at 3 days after birth on a radiogram. Histologically, op/op mice were clearly distinguished by the disappearance of dental follicular space at 3 days. With aging, bone trabeculae, which were not remodeled, penetrated into op/op tooth germs and divided them into several daughter germs, which were recognized as odontomas. In mandibular incisor bodies, the immature ECM components, such as heparan sulfate proteoglycan and tenascin, were preserved diffusely in the dental papilla/pulp, which indicates that maturation of the stroma does not take place in op/op mandibular incisors. Conclusion: The observation suggests that the disturbed morphogenesis of op/op tooth germs is functionally explained by the disordered immunolocalization of ECM molecules, and that the dental follicular space is essential for normal tooth development because it prevents bone penetration into the tooth germs.

In order to examine the functional differentiation of tumor cells of adenomatoid odontogenic tumor (AOT) as ameloblasts and to determine the participation of the extracellular matrix (ECM) in the formation of its characteristic histologic architecture, tissue samples from five cases of adenomatoid odontogenic tumor were examined by immunohistochemical staining for enamel proteins and ECM molecules. Amelogenin, enamelin, laminin, heparan sulfate proteoglycan, fibronectin, collagen type IV and type V were immunolocalized within the luminal space and along the inner rim of duct-like structures. Eosinophilic hyaline droplets within the whorled or rosette masses of tumor cells showed basically the same staining pattern as the luminal contents. High columnar tumor cells that formed duct-like structures were immunopositive for amelogenin, while the staining intensity decreased with flattening of the cells, which was a result of luminal growth. The findings suggest that the constituent cells of duct-like structures are differentiated once to ameloblasts but fail to mature further due instead to increased production of ECM molecules and due to their retention in the lumina. It is possible to regard these special structures in AOT as stromal pseudocysts.

Previous studies have demonstrated the morphological similarity of the enamel-like layer found in the teeth of the coelacanth, lungfish and gar-pike to the enamel of tetrapods. In order to clarify the phylogenetic continuity between both structures, tooth germs of the gar-pike were immunocytochemically studied using an anti-bovine amelogenin polyclonal antibody. Intense immunoreaction was shown over the enamel-like matrix layer. Certain cell organelles associated with the secretory pathway of the ameloblasts were recognized as immunoreactive. These results indicate that the enamel-like layer of the gar-pike is a tissue homologous with the mammalian enamel because both possess a common, amelogenin-like substance.

In the snake, Elaphe quadrivirgata, the occurrence of amelogenin was immunohistochemically demonstrated in the enamel of developing tooth germs. Teeth of the snake are covered with a thin true enamel layer about 1-2 microns in thickness. Light and electron microscopic immunohistochemistry indicated an intense amelogenin immunoreactivity occurring in the enamel layer during the secretory stage of tooth development. Cloning and cDNA sequence of snake amelogenin was performed by RT-PCR. The amino acid sequence of the snake amelogenin cDNA--in its portion corresponding to the area from exon 5 to exon 7 of human X189 amelogenin gene--showed 45% homology with humans. Regions of both the N-terminus and C-terminus were well conserved. Furthermore, the positions of prolin in the amino acid alignment of the snake amelogenin corresponded well with those of human amelogenin. It is suggested that prolin is an essential constituent of amelogenin and therefore its positions in the molecule have been conserved after the evolutionary divergence of reptiles and mammals. This study using reptiles is the first detection of specific amelogenin immunoreactivity by high resolutional immunoelectron microscopy and the first cloning of amelogenin cDNA in a non-mammalian animal.

Both the expression of amelogenin mRNA and secretion of amelogenin were investigated in rat molars by in situ hybridization and immunohistochemistry. Probes were designed by multiple-labeling of oligonucleotide probes for in situ hybridization. Amelogenin mRNA first appeared in differentiating ameloblasts of the distal region and some inner enamel epithelial cells of enamel-free area (EFA cells) of the second cusp at postnatal day 0. At the same time, amelogenin protein was detected in the extracellular matrix between dentin and differentiating ameloblasts and in some EFA cells of the second cusp. At postnatal day 1–3, amelogenin was expressed in the secretory ameloblasts, and in the matrix beneath these cells. Both amelogenin mRNA and amelogenin were detected in the EFA cells and their extracellular matrix. After postnatal day 5, amelogenin mRNA and amelogenin were detected in the secretory ameoloblasts and extracellular matrix in the enamel-forming region, respectively. At this time, amelogenin mRNA was not detected in the EFA cells, but a small amount of amelogenin was found in the matrix beneath the EFA cells. These findings suggest that EFA cells differentiate into amelogenin-secreting cells, i.e. ameloblasts, but that the secretion lasts for only a short period at the early stage of tooth development.

Tooth abnormalities occur in microphthalmic (mi/mi) mice. The elongated odontogenic epithelium is interrupted by unresorbed bone at the basal end of the mi/mi incisor, with the epithelium gathered into cell clusters. These clusters develop to odontoma-like masses. To identify the origin of the cell types of these odontoma-like masses, the localization of osteonectin (Osn), osteocalcin (Osc), osteopontin (Osp), matrix Gla protein (MGP) and amelogenin (Am) mRNA in the process of tooth development in mi/mi and +/+ mice was investigated by means of in situ hybridization. Decalcified mandibles of neonatal, 5-, 10-, 14-day-old mice were examined. Osn and Osc mRNA, which localized in osteoblasts and odontoblasts, were also detected in the cells of odontoma-like masses in mi/mi mice. The cells expressing these mRNA were short, columnar and odontoblast-like. Am mRNA was detected in ameloblasts. In mi/mi mice, Am mRNA was also detected in ameloblastic cell clusters, which were formed by the tall columnar cells in the odontoma-like masses. No apparent Osp mRNA expression was detected in the masses. These results indicated that even in odontogenic abnormal cells resulting from physical obstruction in mi/mi mice, the genes that are involved in normal tooth development were still expressed.

The concentration and distribution of alpha 2HS glycoprotein in teeth from patients with heritable dentin disorders were examined utilizing immunological techniques. ELISA for the quantitative determination showed lower concentrations of alpha 2HS glycoprotein in dentins of patients with heritable disorders than in the control; some cases contained very low and others intermediate concentrations. Immunohistochemical findings indicated that the concentration of this protein was correlated to the regularity of the dentinal tubules. This result suggests that alpha 2HS glycoprotein is incorporated into dentin in concomitance with the development of the dentinal tubule, especially with the deposition of peritubular dentin. Incorporation of this protein into dentin is thought to involve the function of odontoblasts.

This is the first detailed report about the collar enamel of the teeth of Polypterus senegalus. We have examined the fine structure of the collar enamel and enamel organ of Polypterus during amelogenesis by light and transmission electron microscopy. An immunohistochemical analysis with an antibody against bovine amelogenin, an antiserum against porcine amelogenin and region-specific antibodies or antiserum against the C-terminus, middle region and N-terminus of porcine amelogenin has also been performed to examine the collar enamel matrix present in these teeth. Their ameloblasts contain fully developed Golgi apparatus, rough endoplasmic reticulum and secretory granules. During collar enamel formation, an amorphous fine enamel matrix containing no collagen fibrils is found between the dentin and ameloblast layers. In non-demineralized sections, the collar enamel (500 nm to 1 μm thick) is distinguishable from dentin, because of its higher density and differences in the arrangement of its crystals. The fine structural features of collar enamel in Polypterus are similar to those of tooth enamel in Lepisosteus (gars), coelacanths, lungfish and amphibians. The enamel matrix shows intense immunoreactivity to the antibody and antiserum against mammalian amelogenins and to the middleregion- and C-terminal-specific anti-amelogenin antibodies. These findings suggest that the proteins in the enamel of Polypterus contain domains that closely resemble those of bovine and porcine amelogenins. The enamel matrix, which exhibits positive immunoreactivity to mammalian amelogenins, extends to the cap enameloid surface, implying that amelogenin-like proteins are secreted by ameloblasts as a thin matrix layer that covers the cap enameloid after enameloid maturation.

Immunohistochemical localization of two enamel proteins, amelogenin and enamelin, in comparison with that of keratin, was determined in odontogenic tumors and the allied lesions in order to verify functional differentiation of the tumor cells as ameloblasts. Amelogenin and enamelin were demonstrated in small mineralized foci and in the tumor cells surrounding them in adenomatoid odontogenic tumor (AOT), calcifying epithelial odontogenic tumor (CEOT), and calcifying odontogenic cyst (COC). Hyaline droplets in AOT showed positive staining for both enamel proteins. These mineralized and hyaline materials were not positive for keratin, although tumor cells were positive. On the other hand, no immunoreaction for enamel proteins was obtained in ameloblastoima and odontogenic epithelial cell nests within myxoma and epulis. The results suggest that tumor cells of AOT and CEOT and lining epithelial cells of COC show ameloblastic differentiation in part, but that ameloblastoma cells do not attain functional matauration as secretory phase ameloblasts.

The polymerase chain reaction was used to amplify bovine tooth amelogenin cDNA, resulting in several products which were separated by agarose gel electrophoresis. Sequence determination of one of the products revealed that it encoded an amino acid sequence identical to that of a small leucine-rich amelogenin polypeptide (LRAP) previously characterized by protein sequencing. Comparison of the nucleotide sequence of this cDNA with that determined for the cloned bovine amelogenine gene strongly suggested that the LRAP transcript resulted from alternative splicing of the primary transcript of this gene, thus explaining the origin of the puzzling LRAP sequence. Analysis of the structure of LRAP suggests that the polypeptide might exhibit interesting properties relative to hydroxy apatite crystal formation.

Monoclonal antibody against bovine serum α2HS glycoprotein was raised in mice and the localization of this glycoprotein in bovine and human teeth was examined by immunohistochemical techniques on undemineralized ground sections after surface etching with dilute HCl. Positive reactions were found in both bovine and human peritubular dentin, whereas virtually negative staining was detected in the intertubular dentin. When the ground section was examined without surface etching, no reaction was found in any area of mineralized dentin. These results suggest that α2HS glycoprotein is present mainly in the peritubular dentin associated with the mineral phase. The presence of this component in dentin may not be due simply to nonspecific binding to calcium phosphate crystals.

Many clinical reports suggest that deciduous teeth exhibit a greater susceptibility to resorption than permanent ones. To examine the difference between deciduous and permanent dentine in their susceptibility to osteoclastic resorption, osteoclast-like cells (OCLs) were cultured on deciduous and permanent dentine slices. The number, area, depth, and volume of resorption pits were then measured, using image-analyzing systems. We measured the level of degraded collagen (cross-linked N-telopeptide of type I collagen; NTx) in culture medium using an enzyme-linked immunosorbent assay (ELISA). The levels of cathepsin K, matrix metallo proteinase (MMP)-9, and MMP-13 mRNAs in the cells attached to dentine were also analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR). Deciduous dentine slices exhibited a significant (twofold) increase in resorbed area compared with the permanent slices. Three-dimensional analysis revealed that the volume of pits in deciduous dentine differed significantly (fourfold) compared to that in the permanent dentine. The depth of pits also followed the same trend. However, there was no significant difference in the number of pits or osteoclasts on the dentine slices. The NTx level in deciduous media was significantly more than that in permanent media. The mRNA levels also followed the same trend. These results suggest that deciduous dentine is more susceptible to resorption than permanent dentine and signals from the substrate play an important role in physiological resorption.

Dentin matrix protein 1 (DMP1) is an Arg-Gly-Asp-containing acidic phosphoprotein that was originally identified from a rat incisor cDNA library and was thought to be a dentin-specific protein. DMP1 was later shown to express in a number of hard tissue-forming cells, including osteoblasts, osteocytes, ameloblasts, and cementoblasts, and was considered to play important roles in mineralization. Further, DMP1 gene expression was also detected in fetal bovine brain and in newborn mouse brain. These findings indicate the possibility of DMP1 expression in other soft tissues. In the present study, to clarify the significance of DMP1 expression in nonmineralized tissues, we made a specific antibody to mouse DMP1 peptides and demonstrated that DMP1 protein was localized in mouse brain, pancreas, and kidney by immunohistochemistry. Further DMP1 mRNA was detected in nonmineralized mouse tissues including liver, muscle, brain, pancreas, and kidney by RT-PCR. Based on the evidence that the localization and the expression of DMP1 are not restricted to mineralized tissues, we assume that DMP1 may have functions other than the regulation of mineralization.

The tooth in the gar-pike, Lepisosteus oculatus, an actinopterygian fish, is characterized by the occurrence of both enamel and enameloid, the former covering the tooth shaft and the latter, the tooth cap. Our previous research demonstrated that the enamel in this species was, as in the lungfish, immunoreactive for amelogenin, indicating its homologous nature with the mammalian tooth enamel, whereas the enameloid was completely immunonegative. The present study demonstrates that, during the early maturation stage of the enameloid formation, the inner enamel epithelial cells (IEECs) synthesize through a well-developed Golgi apparatus a fine-granular substance which is intensely immunoreactive for amelogenin. This substance was accumulated in a large saccule extended in a suprabasal zone of the cell; we were unable to find any morphological sign indicating a connection of the substance with the enameloid matrix. The abortive secretion of the enamel matrix-like substance in the IEEC during an enameloid formation was considered to be an instance of rudimental enamel formation. In the gar-pike, the synthesis of amelogenin in the IEEC has been demonstrated to occur independently from that of the enameloid matrix. The present findings demonstrate a prominent difference between the tooth enamel and enameloid.

Amelogenesis imperfecta is a hereditary disease of the enamel that is unassociated with generalized defects. Cases of the condition are clinically classified into three groups: hypoplastic, hypomaturation, and hypocalcified. In this study, soluble protein fractions of the enamel from three patients with hypocalcified amelogenesis imperfecta were examined through the use of immunochemical and biochemical techniques. In immunochemical analyses done with a polyclonal anti-amelogenin antibody, all samples from enamel in which there was amelogenesis imperfecta were found to contain considerable amounts of amelogenin peptides. When an enamel sample from one patient was examined by Western-blot transfer and immunobinding analysis, the amelogenin fraction was found to consist of a 26-kDa molecule thought to be normally present in the outer layer of secretory-stage enamel. This enamel was also found to contain albumin as one of the major constituents of the protein fraction. These results suggest that hypocalcified amelogenesis imperfecta may in part be caused by a disturbance in matrix protein degradation during the maturation phase.

Gene expression and localization of amelogenin were studied in the developing rat incisor by the methods of in situ hybridization and immunohistochemistry. ISH revealed the first expression of amelogenin mRNA in the inner enamel epithelium of the cervical loop. The signals were clearly observed in pre-ameloblasts in the region bordering on predentin formation and became more intense toward the cells on the initial enamel matrix secretion. The maximal signals were found in the cytoplasm of secretory ameloblasts. From the terminal secretion zone, the signals then became gradually weaker toward the incisal edge but were still evident in the cytoplasm of shortening, transitional ameloblasts and those at the early maturation stage. No signals were found in the cells of the stratum intermedium and stellate reticulum throughout amelogenesis. Immunohistochemistry by means of an antibody against amelogenin C-telopeptide consisting of 12 amino acids revealed immunoreaction in the secretory ameloblasts reacting to the ISH. When a polyclonal antibody against amelogenin was used, immunoreaction was found in the distal ends of ruffle-ended ameloblasts (RA) in the maturation zone. Those results indicated that amelogenin is synthesized by ameloblastic cells from the inner enamel epithelium to the early maturation stage and is then resorbed by the RA.

A genomic DNA clone (14.5 kbp) for amelogenin, a tooth enamel specific protein, has been isolated from a mouse genomic library. The nucleotide structure of this DNA was determined by the polymerase chain reaction and sequence analysis. The result indicated that the cloned DNA contained all the exons in addition to approximately 5.5 kbp of upstream sequence.

The present studies were undertaken to investigate the presence of common epitopes of mammalian amelogenins at the C-terminus and the possible functional importance of the conserved C-terminal domain in enamel mineralization during mammalian amelogenesis. Enamel proteins, including the intact amelogenins and their degraded polypeptides, were isolated from the secretory enamel of pig, cow, rat, and rabbit incisors. Rabbit and rat antipeptide sera, as well as rat anti-25 kD and 20 kD pig amelogenin sera, were used to identify the amelogenins among the isolated matrix proteins of each of the animal species. The antipeptide sera were developed previously (Aoba et al. [19]) using as immunogens the two synthetic peptides, C13 and C25, which correspond to the last 12 (plus Cys for KLH-conjugation) and 25 amino acid residues of pig intact amelogenin, respectively. Reactivity of the enamel proteins with each antiserum was examined by Western blot analysis. The results of immunoblotting showed that a few enamel matrix proteins in each of the mammalian species were recognized by the anti-C13 serum, specifically, pig amelogenin at 25 kD (and trace components at 27, 22, and 18 kD), cow amelogenin at 28 kD (trace components at 26, 22, 19, and 14 kD), rat amelogenins at 28 and 26 kD (and a trace component at 20 kD), and rabbit amelogenins at 24 and 21 kD (and a trace at 13 kD). The anti-C25 serum reacted additionally with pig amelogenin at 23 kD, cow amelogenin at 27 kD (a major matrix constituent), and rabbit protein at 19 kD. The anti-pig 20 kD amelogenin (lacking the last 25 amino acid residues at the C-terminus) serum reacted with a large number of pig, cow, and rat amelogenins but, interestingly, with none of the rabbit enamel proteins. Probing of rat enamel proteins with Maclura pomifera lectin showed the heterogeneity of glycosylation of rat amelogenins, particularly between the 28 and 26 kD intact amelogenins. In parallel adsorption studies, part of the enamel protein samples isolated from each of the species was used as adsorbates to investigate the selective adsorption of amelogenins onto hydroxyapatite. Immunoblot analysis of the proteins adsorbed onto the crystals revealed that the mammalian amelogenins having the common epitopes at the C-terminus, in general, adsorb preferentially onto hydroxyapatite. The adsorption affinity of the degraded amelogenins decreased significantly with the loss of reactivity toward the anti-C13 serum. The overall results support the contention that the intact mammalian amelogenins, including rat and rabbit amelogenins, share common epitopes at the C-terminus and that the conserved C-terminal domain plays an important role in setting the molecular structures of the intact amelogenins so as to facilitate the protein-enamel mineral interaction.

Dentin phosphoprotein (DPP) and dephosphorylated DPP (deP-DPP) were isolated from developing bovine incisor and purified by DEAE-Sephacel and Sepharose CL-6B chromatography. Intact DPP showed partial resistance to proteolytic cleavage; while deP-DPP was completely susceptible to protease digestion. This result suggested that phosphate residues within DPP molecule may protect its structural integrity. DPP was also very sensitive to heat. When DPP was heated at 100°C in the presence of sodium dodecyl sulfate, increasing concentrations of SDS resulted in a protective effect from degradation. An analysis of DPP biosynthesis was carried out by isolation of the odontoblast tissue/cells attached to forming dentin or scraped from dentin surface followed by incubation with radioactive amino acids and phosphate. DPPs produced in vitro were larger in size than those DPPs extracted from mature dentin matrix. To determine the size of the unprocessed form of DPP, Xenopus leavis oocytes were incubated in [ 32 p] -phosphate-containing medium after microinjection of poly(A + mRNA extracted from the bovine odontoblasts. Radioactive DPP analyzed by two-dimensional polyacrylamide gel electrophoresis and immunoprecipitation with an anti-DPP monoclonal antibody was shown to have an apparent M r of 150-160 k. These data imply that changes in molecular size of DPP may occur by post-translational cleavage or other modification such as degradation within dentin matrix in vivo .

The effect of enamel matrix proteins on the seeded growth of enamel apatite crystals was studied in stable supersaturated solutions at pH 7.4 and 37°C. Of the two major protein classes in the enamel matrix, the enamelins were considerably more effective than the amelogenins in retarding seeded growth. However, the amelogenin species that did show significant inhibitory activity are those known to be lost first from the enamel matrix during the rapid mineralization stage of enamel maturation.

Rabbit polyclonal antibodies against bovine amelogenins and enamelins which did not show any cross-reaction were raised, and ultrathin sections of rat incisors were examined by the protein A-gold and ABC methods. The immunoreactivity of amelogenins was found in dense granules in the intercellular spaces between preameloblasts, and later over the fine- and coarse-textured material. The immunoreactivity was present over the cell organelles associated with the secretory pathway, as well as pale and dark lysosomes of the presecretory and secretory ameloblasts. Here the enamel was immunolabeled in the intercrystal spaces. The immunoreactivity in multivesicular bodies was stronger in preameloblasts than in secretory ameloblasts. In the region of second ruffle-ended ameloblasts at the maturation stage, the immunolabeling was intense in the ruffled-border, but in the rough endoplasmic reticulum and Golgi apparatus, the immunolabeling was much weaker than at the secretory stage. The immunolabeling for enamelins showed essentially the same intracellular topographical pattern as that for amelogenins by the secretory stage, but was weaker. The immunoreactivity was found mainly attached to the enamel crystals. Double immunostaining of amelogenins and enamelins revealed that both immunoreactivities were present over the same cell organelles associated with secretion and lysosomal systems. It is suggested that the presecretory and secretory ameloblasts are actively involved in the secretion, degradation and resorption of enamel proteins, and that multivesicular bodies and lysosomes in the cells take part in these processes. Ameloblasts are considered to be related to the synthesis of enamelins.

Molecular cloning of a bovine amelogenin cDNA was accomplished by construction of a cDNA expression library (λgt11 cDNA library) from the bovine ameloblast mRNA and then screening of the library with antibodies to bovine amelogenins. The complete primary structure of an amelogenin was deduced from cloned cDNA. One of the cDNA clones isolated from a bovine ameloblast phage λgt11 library had an 864-base-pair-long insert that encoded a protein with 216 amino acid residues. This cDNA clone appears to represent the complete coding region of amelogenin mRNA, including a putative AUG initiation codon and a signal peptide sequence. The predicted bovine amelogenin sequence has 87% amino acid homology with murine amelogenin.

Enamelins were extracted from developing bovine enamel with 0.5 M EDTA, 4 M guanidine HCl, and purified by DEAE-Sephacel, Sephacryl S-200, and high-performance gel filtration chromatography. Four distinct enamelins having molecular weights of 70, 45, 30, and 28 K daltons were isolated. Their amino acid compositions were found to be rich in Pro, Glu, Gly, and Asp. Low molecular weight enamelins (45, 30, and 28 K) were more abundant in Pro, Gly, and Phen. Two-dimensional electrophoretic pattern of enamelins revealed several spots that immunoreacted to monoclonal anti-enamelin antibody raised in mice. Enamelins were found to be comparised of heterogeneous proteins as well as amelogenins. Biosynthesis of enamelins was investigated by incubating the bovine ameloblast cell layer, and several radioactive enamelins were identified by the use of two-dimensional electrophoresis. The data in this study suggest that enamelins were synthesized by the ameloblasts.

A specific enzyme-linked immuno-adsorbent assay for enamel protein of the developing tooth was devised, using antibody against antigen prepared from immature bovine enamel. Samples containing 5–500 ng of amelogenin protein in 50 μl gave consistent results. Bovine enamelin cross-reacted with the antibody but was less reactive than amelogenin. Proteins in other tissues or fluids did not react to the antibody.