The enzyme histochemistry of developing odontoblasts in cattle, pigs and horses

Synopsis The histochemical distribution of some hydrolytic and oxidative enzymes in developing odontoblasts and subodontoblasts in cattle, pigs and horses has been observed in cryostat sections of teeth that have been decalcified with neutral EDTA.Undifferentiated dental epithelium and immature odontoblasts of the bell stage tooth germ showed lower levels of enzymatic activity as compared with the well-developed tooth germ.When the dentine matrix began to form, the young odontoblasts appeared to have a significantly positive reaction for acid phosphatase, and gradually other enzymes developed an activity at the top of the cusp.Odontoblasts as well as subodontoblastic-rich cells showed strong enzymatic activities for hydrolytic and oxidative enzymes, that is, they were strongly reactive for alkaline and acid phosphatase and lactate and malate dehydrogenases, and moderately reactive for other oxidative enzyme systems.It is suggested that the subodontoblastic layer is concerned with the biosynthesis of dentinal matrix as well as with the odontoblasts themselves.     

Identification of a fibronectin interaction site in the extracellular matrix protein ameloblastin

Mammalian teeth are composed of hydroxyapatite crystals that are embedded in a rich extracellular matrix. This matrix is produced by only two cell types, the mesenchymal odontoblasts and the ectodermal ameloblasts. Ameloblasts secrete the enamel proteins amelogenin, ameloblastin, enamelin and amelotin. Odontoblasts secrete collagen type I and several calcium-binding phosphoproteins including dentin sialophosphoprotein, dentin matrix protein, bone sialoprotein and osteopontin. The latter four proteins have recently been grouped in the family of the SIBLINGs (small integrin-binding ligand, N-linked glycoproteins) because they display similar gene structures and because they contain an RGD tripeptide sequence that binds to integrin receptors and thus mediates cell adhesion.We have prepared all the other tooth-specific proteins in recombinant form and examined whether they might also promote cell adhesion similar to the SIBLINGs. We found that only ameloblastin consistently mediated adhesion of osteoblastic and fibroblastic cells to plastic or titanium surfaces. The activity was dependent on the intact three-dimensional structure of ameloblastin and required de novo protein synthesis of the adhering cells. By deletion analysis and in vitro mutagenesis, the active site could be narrowed down to a sequence of 13 amino acid residues (VPIMDFADPQFPT) derived from exon 7 of the rat ameloblastin gene or exons 7-9 of the human gene. Kinetic studies and RNA interference experiments further demonstrated that this sequence does not directly bind to a cell surface receptor but that it interacts with cellular fibronectin, which in turn binds to integrin receptors.The identification of a fibronectin-binding domain in ameloblastin might permit interesting applications for dental implantology. Implants could be coated with peptides containing the active sequence, which in turn would recruit fibronectin from the patient's blood. The recruited fibronectin should then promote cell adhesion on the implant surface, thereby accelerating osseointegration of the implant.     

Echinoderm Phosphorylated Matrix Proteins UTMP16 and UTMP19 Have Different Functions in Sea Urchin Tooth Mineralization

Studies of mineralization of embryonic spicules and of the sea urchin genome have identified several putative mineralization-related proteins. These predicted proteins have not been isolated or confirmed in mature mineralized tissues. Mature Lytechinus variegatus teeth were demineralized with 0.6 n HCl after prior removal of non-mineralized constituents with 4.0 m guanidinium HCl. The HCl-extracted proteins were fractionated on ceramic hydroxyapatite and separated into bound and unbound pools. Gel electrophoresis compared the protein distributions. The differentially present bands were purified and digested with trypsin, and the tryptic peptides were separated by high pressure liquid chromatography. NH₂-terminal sequences were determined by Edman degradation and compared with the genomic sequence bank data. Two of the putative mineralization-related proteins were found. Their complete amino acid sequences were cloned from our L. variegatus cDNA library. Apatite-binding UTMP16 was found to be present in two isoforms; both isoforms had a signal sequence, a Ser-Asp-rich extracellular matrix domain, and a transmembrane and cytosolic insertion sequence. UTMP19, although rich in Glu and Thr did not bind to apatite. It had neither signal peptide nor transmembrane domain but did have typical nuclear localization and nuclear exit signal sequences. Both proteins were phosphorylated and good substrates for phosphatase. Immunolocalization studies with anti-UTMP16 show it to concentrate at the syncytial membranes in contact with the mineral. On the basis of our TOF-SIMS analyses of magnesium ion and Asp mapping of the mineral phase composition, we speculate that UTMP16 may be important in establishing the high magnesium columns that fuse the calcite plates together to enhance the mechanical strength of the mineralized tooth.The largest currently extant group of sea urchins (phylum Echinodermata, class Echinoidea) is the camarodonts (1), which among other features have a unique mineralized Aristotle''s lantern structure housing the five teeth of the urchin. Most studies of mineralization processes within the echinoderms have focused on the mineralized spicules of the larval urchin (2). These give structure to the larvae but are transient and not present in the postlarval maturing animal. The main mineralized structures of the growing urchin, aside from the test and spines, are the lantern stereom and the teeth (3). The teeth are especially interesting, because they grow continuously in a vectorial fashion. The preodontoblasts originating at the aboral plumula arise from a mixed population of coelomocytes. The individual monocytes condense at the outer surface of the plumula (4, 5) just under the epithelial layer, where they fuse and become multinucleated cells, which form sheetlike syncytial layers (6). Calcium carbonate mineral forms at calcification sites between the cellular layers, with the mineral deposition related to the syncytial plasma membranes (7–9). There has been considerable discussion as to whether the chambers in which the mineral forms are within the cells or in the extracellular space between syncytia, but it is clear that the mineralization is related to the membranes enclosing the mineralization space. However, the biogenic urchin tooth calcite crystals themselves contain occluded macromolecules (10).Thus, our attention has been directed to the questions of which proteins or other macromolecules might be involved in the initiation of mineralization, and specifically where these proteins might be located. We have recently reported on protocols by which the organic components of the urchin tooth can be separated into those readily extracted from the tooth and those intimately related to and protected by the mineralized compartments (6, 11). In the present study, this fractionation scheme has been applied to the isolation of the mineral-related and mineral-bound macromolecular components from the teeth of Lytechinus variegatus, a prominent urchin found in the Gulf of Mexico.The complete genome of Strongylocentrotus purpuratus, a distantly related urchin, has been reported, and a genome-wide analysis of its putative biomineralization-related proteins has been carried out by Livingston et al. (12). Although some gene and protein sequence differences were anticipated, our approach was to use the derived S. purpuratus protein sequence data base as a guide for cloning any mineral-related and mineral-bound L. variegatus proteins found. The objective of the present work was to move from the putative proteins predicted by the genome sequencing, to examine in detail the proteins actually present in the urchin tooth that might control calcite formation and the process of mineralization. The present paper describes the isolation of two mineral-associated acidic proteins, UTMP16 and UTMP19 from L. variegatus teeth, the cloning and determination of their complete cDNA sequences, and their relation to the mineral phase.     

Production and characterization of antibodies against murine dentine phosphoprotein.

Experiments were designed to produce and characterize a polyclonal antibody directed against mouse dentine phosphoprotein, the major non-collagenous protein of the dentine extracellular matrix. Dental extracellular matrix proteins from 2-day-postnatal Swiss-Webster-mouse tooth organs were extracted with 0.5 M-acetic acid, followed by 4 M-guanidinium chloride/0.5 M-EDTA. Mouse dentine phosphoprotein yields were further increased by precipitation with 1 M-CaCl2. Final purification was achieved by excising and eluting dentine phosphoprotein polypeptide bands from preparative sodium dodecyl sulphate/urea/polyacrylamide gels. Mouse dentine phosphoprotein is a single component of approx. 72 kDa and has a characteristic amino acid composition of 33% aspartic acid and 55% serine/phosphoserine. A polyclonal antibody was raised in rabbits against purified mouse dentine phosphoprotein and was shown to be monospecific by enzyme-linked immunoabsorbent, dot-immunobinding and 'Western transfer' assays. This antibody was used to detect the expression and localization of dentine phosphoprotein in 1-day-postnatal mouse tooth organs. This antigen was localized intracellularly within the monolayer of odontoblasts, which line the perimeter of the dental papilla mesenchyme, and within the odontoblastic cell processes, which traverse the predentine matrix. Newly forming mineralized dentine matrix was also cross-reactive with the dentine phosphoprotein specific antibody. The non-mineralized predentine matrix did not contain any detectable cross-reactive antigens.     

Studies on protein synthesis during sea urchin oogenesis. I. Synthesis of histone F2b

Basic proteins were extracted from sea urchin oocytes previously incubated with 3H-lysine and then were analyzed by electrophoresis. A very radioactive band, which showed the same mobility as histone F2b, was analyzed for its amino acid composition. The results show an identity between this protein and histones F2b. In addition, an improved method of isolating large amounts of sea urchin oocytes is described.     

Binding of amelogenin to MMP-9 and their co-expression in developing mouse teeth

Amelogenin is the most abundant matrix protein in enamel. Proper amelogenin processing by proteinases is necessary for its biological functions during amelogenesis. Matrix metalloproteinase 9 (MMP-9) is responsible for the turnover of matrix components. The relationship between MMP-9 and amelogenin during tooth development remains unknown. We tested the hypothesis that MMP-9 binds to amelogenin and they are co-expressed in ameloblasts during amelogenesis. We evaluated the distribution of both proteins in the mouse teeth using immunohistochemistry and confocal microscopy. At postnatal day 2, the spatial distribution of amelogenin and MMP-9 was co-localized in preameloblasts, secretory ameloblasts, enamel matrix and odontoblasts. At the late stages of mouse tooth development, expression patterns of amelogenin and MMP-9 were similar to that seen in postnatal day 2. Their co-expression was further confirmed by RT-PCR, Western blot and enzymatic zymography analyses in enamel organ epithelial and odontoblast-like cells. Immunoprecipitation assay revealed that MMP-9 binds to amelogenin. The MMP-9 cleavage sites in amelogenin proteins across species were found using bio-informative software program. Analyses of these data suggest that MMP-9 may be involved in controlling amelogenin processing and enamel formation.     

SELECTIVE EXTRACTION OF ISOLATED MITOTIC APPARATUS : Evidence That Typical Microtubule Protein Is Extracted by Organic Mercurial

Mitotic apparatus isolated from sea urchin eggs has been treated with meralluride sodium under conditions otherwise resembling those of its isolation. The treatment causes a selective morphological disappearance of microtubules while extracting a major protein fraction, probably consisting of two closely related proteins, which constitutes about 10% of mitotic apparatus protein. Extraction of other cell particulates under similar conditions yields much less of this protein. The extracted protein closely resembles outer doublet microtubule protein from sea urchin sperm tail in properties considered typical of microtubule proteins: precipitation by calcium ion and vinblastine, electrophoretic mobility in both acid and basic polyacrylamide gels, sedimentation coefficient, molecular weight, and, according to a preliminary determination, amino acid composition. An antiserum against a preparation of sperm tail outer doublet microtubules cross-reacts with the extract from mitotic apparatus. On the basis of these findings it appears that microtubule protein is selectively extracted from isolated mitotic apparatus by treatment with meralluride, and is a typical microtubule protein.     

Expression of clock proteins in developing tooth

Morphological and functional changes during ameloblast and odontoblast differentiation suggest that enamel and dentin formation is under circadian control. Circadian rhythms are endogenous self-sustained oscillations with periods of 24h that control diverse physiological and metabolic processes. Mammalian clock genes play a key role in synchronizing circadian functions in many organs. However, close to nothing is known on clock genes expression during tooth development. In this work, we investigated the expression of four clock genes during tooth development. Our results showed that circadian clock genes Bmal1, clock, per1, and per2 mRNAs were detected in teeth by RT-PCR. Immunohistochemistry showed that clock protein expression was first detected in teeth at the bell stage (E17), being expressed in EOE and dental papilla cells. At post-natal day four (PN4), all four clock proteins continued to be expressed in teeth but with different intensities, being strongly expressed within the nucleus of ameloblasts and odontoblasts and down-regulated in dental pulp cells. Interestingly, at PN21 incisor, expression of clock proteins was down-regulated in odontoblasts of the crown-analogue side but expression was persisting in root-analogue side odontoblasts. In contrast, both crown and root odontoblasts were strongly stained for all four clock proteins in first molars at PN21. Within the periodontal ligament (PDL) space, epithelial rests of Malassez (ERM) showed the strongest expression among other PDL cells. Our data suggests that clock genes might be involved in the regulation of ameloblast and odontoblast functions, such as enamel and dentin protein secretion and matrix mineralization.     

First Insights into the Biochemistry of Tube Foot Adhesive from the Sea Urchin Paracentrotus lividus (Echinoidea, Echinodermata)

Sea urchins are common inhabitants of wave-swept shores. To withstand the action of waves, they rely on highly specialized independent adhesive organs, the adoral tube feet. The latter are extremely well-designed for temporary adhesion being composed by two functional subunits: (1) an apical disc that produces an adhesive secretion to fasten the sea urchin to the substratum, as well as a deadhesive secretion to allow the animal to move and (2) a stem that bears the tensions placed on the animal by hydrodynamism. Despite their technological potential for the development of new biomimetic underwater adhesives, very little is known about the biochemical composition of sea urchin adhesives. A characterization of sea urchin adhesives is presented using footprints. The latter contain inorganic residues (45.5%), proteins (6.4%), neutral sugars (1.2%), and lipids (2.5%). Moreover, the amino acid composition of the soluble protein fraction revealed a bias toward six amino acids: glycine, alanine, valine, serine, threonine, and asparagine/aspartic acid, which comprise 56.8% of the total residues. In addition, it also presents higher levels of proline (6.8%) and half-cystine (2.6%) than average eukaryotic proteins. Footprint insolubility was partially overcome using strong denaturing and reducing buffers, enabling the visualization of 13 proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The conjugation of mass spectrometry with homology–database search allowed the identification of six proteins: alpha and beta tubulin, actin, and histones H2B, H3, H2A, and H4, whose location and function in the adhesive are discussed but require further investigation. For the remaining unidentified proteins, five de novo-generated peptide sequences were found that were not present in the available protein databases, suggesting that they might be novel or modified proteins.     

High levels of a calcium-dependent modulator protein in spermatozoa and its similarity to brain modulator protein

Sperm from hamster, human, rooster, rabbit and sea urchin were found to contain relatively high levels of calcium-dependent modulator protein. Using rabbit sperm the modulator protein was found to be exclusively located in the sperm head fraction (nuclei + acrosomes) with no activity present in the midpiece or tail regions. The modulator protein represents approximately 12% of the total soluble protein found in the sperm head fraction and is similar to porcine and brain modulator proteins in its ability to activate brain cyclic nucleotide phosphodiesterase, its heat stability and electrophoretic migration. We have also observed modulator protein to be present in high levels in sea urchin eggs.     

Apoptosis in developmental and repair-related human tooth remodeling: a view from the inside

Apoptosis is a key phenomenon in the regulation of the life span of odontoblasts, which are responsible for dentin matrix production of the teeth. The mechanism controlling odontoblasts loss in developing, normal, and injured human teeth is largely unknown. A possible correlation between apoptosis and dental pulp volume reduction was examined. Histomorphometric analysis was performed on intact 10 to 14 year-old premolars to follow dentin deposition and evaluate the total number of odontoblasts. Apoptosis in growing healthy teeth as well as in mature irritated human teeth was determined using a modified TUNEL technique and an anti-caspase-3 antibody. In intact growing teeth, the sequential rearrangement of odontoblasts into a multi-layer structure during tooth crown formation was correlated with an apoptotic wave that leads to the massive elimination of odontoblasts. These data suggest that apoptosis, coincident with dentin deposition changes, plays a role in tooth maturation and homeostasis. Massive apoptotic events were observed after dentin irritation. In carious and injured teeth, apoptosis was detected in cells surrounding the lesion sites, as well as in mono-nucleated cells nearby the injury. These results indicate that apoptosis is a part of the mechanism that regulate human dental pulp chamber remodeling during tooth development and pathology.     

Colocalization of dentin matrix protein 1 and dentin sialoprotein at late stages of rat molar development.

Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) are acidic proteins found in the extracellular matrices of bones and teeth. Recent data from gene knockouts, along with those of gene mutations, indicate that these two phosphoproteins are critical for bone and tooth development and/or maintenance. However, the precise functions of the two proteins have not been elucidated. In order to gain insights into their functions in tooth formation, we performed systematic, comparative investigations on the immunolocalization of DMP1 and dentin sialoprotein (DSP, a cleaved fragment of DSPP), using the rat first molar at different developmental stages as a model. Immunohistochemistry (IHC) was performed with specific, monoclonal antibodies against the COOH-terminal fragments of DMP1 and against DSP. In 1-day- and 1-week-old rats, weak immunoreactions for DMP1 were observed in dentinal tubules while stronger reactions for DSP were seen in the tubules and predentin. In rats older than 2 weeks, immunoreactions for DMP1 were found in dentinal tubules, predentin and odontoblasts. In 5-week- and 8-week-old rats, strong immunoreactions for DMP1 were widely distributed in odontoblasts and predentin. The distribution pattern of DSP was strikingly similar to that of DMP1 after 2 weeks and the localization of each was distinctly different from that of bone sialoprotein (BSP). The unique colocalization of DMP1 and DSPP in tooth development suggests that the two proteins play complementary and/or synergistic roles in formation and maintenance of healthy teeth.     

Mapping of magnesium and of different protein fragments in sea urchin teeth via secondary ion mass spectroscopy

Mature portions of sea urchin are comprised of a complex array of reinforcing elements yet are single crystals of high and very high Mg calcite. How a relatively poor structural material (calcite) can produce mechanically competent structures is of great interest. In teeth of the sea urchin Lytechinus variegatus, we recorded high-resolution secondary ion mass spectrometry (SIMS) maps of Mg, Ca ,and specific amino acid fragments of mineral-related proteins including aspartic acid (Asp). SIMS revealed strong colocalization of Asp residues with very high Mg. Demineralized specimens showed serine localization on membranes between crystal elements and reduced Mg and aspartic acid signals, further emphasizing colocalization of very high Mg with ready soluble Asp-rich protein(s). The association of Asp with nonequilibrium, very high magnesium calcite provides insight to the makeup of the macromolecules involved in the growth of two different composition calcites and the fundamental process of biomineralization.     

Soluble microtubule proteins of the sea urchin embryo: partial characterization of the proteins and behavior of the pool in early development

A partial characterization of the soluble microtubule proteins of sea urchin eggs and embryos is presented. Vinblastine precipitation yielded a pellet with a high colchicine binding activity. This precipitate when electrophoresed on an alkaline SDS/urea gel system yields two protein bands which correspond to molecular weights of 57,000 ± 2000 and 52,000 ± 2000. These values are very close to our values and to the published values for axonemal microtubule proteins. Electrophoresis of the vinblastine precipitated proteins on a neutral SDS system without urea yielded only one band with an apparent molecular weight of 52,000 ± 2000. The amino acid composition of the vinblastine-precipitated microtubule protein was determined to be similar to that of axonemal protein.The pool of microtubule proteins was found to remain constant in size throughout early development in both control and actinomycin-treated embryos. Soluble microtubule proteins comprise about 0.37% of the total protein of the sea urchin (Arbacia) egg. Approximately 20% of the total microtubule protein in the egg appears to be particle bound.     

A tuftelin-interacting protein (TIP39) localizes to the apical secretory pole of mouse ameloblasts

Enamel biomineralization is a complex process that involves interactions between extracellular matrix proteins. To identify proteins interacting with tuftelin, a potential nucleator of enamel crystallites, the yeast two-hybrid system was applied to a mouse tooth expression library and a tuftelin-interacting protein (TIP) was isolated for further characterization. Polyclonal antibodies were prepared against two recombinant variants of this protein. Both antibodies identified a major protein product in tooth organs at 39 kDa, and this protein has been called TIP39. Northern analysis showed TIP39 messenger RNA in multiple organs, a pattern similar to that of tuftelin messenger RNA. In situ hybridization of mandibles of 1-day-old mice detected TIP39 RNA in secretory ameloblasts and odontoblasts. Immunolocalization of TIP39 and tuftelin in cultured ameloblast-like cells showed that these two proteins colocalize. Within the developing tooth organ, TIP39 and tuftelin immunolocalized to the apical pole of secretory ameloblasts (Tomes' processes) and to the newly secreted extracellular enamel matrix. TIP39 amino acid sequence appears to be highly conserved with similarities to proteins in species as diverse as yeast and primates. Available sequence data and the findings reported here suggest a role for TIP39 in the secretory pathway of extracellular proteins.     

THE TEETH OF HYPOGEOPHIS ROSTRATUS (AMPHIBIA, APODA) AND TOOTH STRUCTURE IN THE AMPHIBIA

The bipartite nature of the tooth has been recently used to demonstrate the close relationship between the three existing groups of Amphibia. However, Considerable doubt has remained as to the composition of the two parts of the tooth and the way in which they are linked.
It order to clearify the position the teeth in Hypogeophis were examined. It is clear that the bulk of the crown and the pedicel is composed of dentine which is produced by a continuous layer of odontoblasts. This layer of cells is also responsible for the formation of the fibrous ring which joins the two portions of the tooth. This ring is regarded as an area where the odontoblasts produce fibres and little or no matrix.     

High mobility group nonhistone chromosomal proteins of the developing sea urchin embryo

The high mobility group or HMG proteins are nonhistone chromosomal proteins that have been found in relatively high amounts in nuclei of many tissues. A number of studies have shown that some of these proteins are preferentially associated with actively transcribed regions of the genome and may play a role in maintaining these regions in an active state. In this study, we undertook an investigation of the high mobility group proteins from the sea urchin, Stronglyocentrotus purpuratus. Initially the putative sea urchin HMGs were extracted from isolated nuclei of hatching blastula-stage embryos with 5% perchloric acid (PCA). The major proteins in this extract were characterized according to their electrophoretic mobility, amino acid composition, and association with isolated deoxyribonucleoprotein particles. The results indicate there is only one "major" sea urchin HMG protein, termed P2 in this paper. An estimate of the amount of P2 in relation to the inner histones, however, was low compared to what has been found for other HMG proteins. Of the other major 5% PCA-extractable proteins, one was identified as the cleavage stage H1. Another protein apparently resulted from H3 contamination in the 5% PCA extract, and the fourth major protein did not have all the characteristics of an HMG. In particular, it was not found associated with nucleosomal particles. The HMG proteins from other developmental stages were then examined. Five percent PCA extracts of nuclei from unfertilized eggs, 2-cell, 16-cell, hatching blastula, gastrula, and pluteus stages were analyzed on SDS- and acetic acid-urea gels. This analysis indicated that P2 exists in two different forms differing slightly in charge. The less basic form was found in the egg, 2-cell and 16-cell extracts. At the hatching blastula stage, both forms were present and by pluteus stage, the more basic form predominated. It appears that P2 is undergoing a developmental change from a less to more basic form. The presence of P2 in the 5% PCA extract of egg nuclei is proof that P2 does not initially appear sometime during embryogenesis but is already in the egg nucleus prior to fertilization.     

Characterization of a cDNA encoding a protein involved in formation of the skeleton during development of the sea urchin Lytechinus pictus.

In order to investigate the role of proteins in the formation of mineralized tissues during development, we have isolated a cDNA that encodes a protein that is a component of the organic matrix of the skeletal spicule of the sea urchin, Lytechinus pictus. The expression of the RNA encoding this protein is regulated over development and is localized to the descendents of the micromere lineage. Comparison of the sequence of this cDNA to homologous cDNAs from other species of urchin reveal that the protein is basic and contains three conserved structural motifs: a signal peptide, a proline-rich region, and an unusual region composed of a series of direct repeats. Studies on the protein encoded by this cDNA confirm the predicted reading frame deduced from the nucleotide sequence and show that the protein is secreted and not glycosylated. Comparison of the amino acid sequence to databases reveal that the repeat domain is similar to proteins that form a unique beta-spiral supersecondary structure.     

Immunohistochemical localization of a tenascin-like extracellular matrix protein in sea urchin embryos

Summary We have used polyclonal antisera raised against vertebrate tenascin to identify and localize tenascin-like proteins in the developing sea urchin. These antisera recognize high-molecular weight proteins on immunoblots of sea urchin embryo homogenates that are similar in size and appearance to tenascin from vertebrates. These proteins appear as a doublet with an apparent molecular weight of 150 kDa and a larger, broad band with an apparent molecular weight of 350 kDa. Whole mounts of sea urchin embryos and larvae were stained with one of these antisera. The anti-tenascin stained the surface of primary mesenchyme cells during their phase of active migration. This staining was sensitive to detergent, suggesting that the protein recognized by the antiserum was associated with the cell surface. During later stages of development, the bulk of the antitenascin staining was found dispersed throughout the blastocoel matrix, and was no longer sensitive to detergent. We conclude that sea urchins express tenascin-like proteins during early stages of development, and that these proteins may play a role associated with primary mesenchyme cell morphogenesis.     

Comparison of Odontogenic Differentiation of Human Dental Follicle Cells and Human Dental Papilla Cells

Classical tooth development theory suggests that dental papilla cells (DPCs) are the precursor cells of odontoblasts, which are responsible for dentin development. However, our previous studies have indicated that dental follicle cells (DFCs) can differentiate into odontoblasts. To further our understanding of tooth development, and the differences in dentinogenesis between DFCs and DPCs, the odontogenic differentiation of DFCs and DPCs was characterized in vitro and in vivo. DFCs and DPCs were individually combined with treated dentin matrix (TDM) before they were subcutaneously implanted into the dorsum of mice for 8 weeks. Results showed that 12 proteins were significantly differential, and phosphoserine aminotransferase 1 (PSAT1), Isoform 2 of hypoxia-inducible factor 1-alpha (HIF1A) and Isoform 1 of annexin A2 (ANXA2), were the most significantly differential proteins. These proteins are related to regulation of bone balance, angiogenesis and cell survival in an anoxic environment. Both DFCs and DPCs express odontogenic, neurogenic and peridontogenic markers. Histological examination of the harvested grafts showed that both DFCs and DPCs form pulp-dentin/cementum-periodentium-like tissues in vivo. Hence, DFCs and DPCs have similar odontogenic differentiation potential in the presence of TDM. However, differences in glucose and amino acid metabolism signal transduction and protein synthesis were observed for the two cell types. This study expands our understanding on tooth development, and provides direct evidence for the use of alternative cell sources in tooth regeneration.