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PubMed2004

[Expression of matrixmetalloproteinase-8 on the bell-stage in human and rat tooth development].

OBJECTIVE: To study the expression of MMP-8 in human and rat tooth development. METHODS: Immunohistochemistry was used to detect the localization of MMP-8 protein while in situ hybridization was used to examine the expression of MMP-8 mRNA. RESULTS: The expression of MMP-8 protein was localized in odontoblast and dentin matrix at the later bell stage in human tooth germ. The dentin was denser close to the pulp cavity. The expression of MMP-8 mRNA was found in very few polarized odontoblast at the early bell stage and all polarized odontoblast at the later bell stage in rat tooth germ. CONCLUSION: The results suggested that MMP-8 involved in dentin matrix rebuilding in the process of dentin formation in human and rat dental development.

Animals↗
PubMed1992

Molecular control of dentinogenesis: a reaction.

This paper represents an invited reaction to three papers presented at the International Conference on Pathobiology of the Dentin/Pulp Complex, June, 1991. Repair dentinogenesis following transplantation into normal and germs free animals are correlated with results elucidating the expression of dentin phosphoproteins, collagen, and osteocalcin. The importance of transcription and translation controls of dentin matrix components are discussed and reviewed. In addition, possible implications of a molecular chaperone protein, Hsp47, in controlling dentinogenesis is introduced. Future research directions are developed and include: (a) identification of odontoblast precursors; (b) delineation of markers for odontoblasts at varying degrees of differentiation; (c) characterization of environmental conditions leading to odontoblast differentiation; (d) determination of the nature of repair and regenerated tissues; (e) elucidation of transcription and translation control factors, and (f) mapping the human genome for dentin matrix constituents.

Collagen↗
PubMed1991

The development of enamel tubules during the formation of enamel in the marsupial Monodelphis domestica.

In Monodelphis domestica, although both processes from odontoblasts and projections from ameloblasts were found in developing enamel, the majority of the contents of enamel tubules were probably processes that originated from odontoblasts. Processes from odontoblasts penetrating into enamel touched part of the ameloblasts in the stage of enamel formation. No specialised cell junctions were seen at the adherence between the two. There were no enamel tubules in the aprismatic and pseudoprismatic enamel layer. It is likely that enamel tubules exist only in prismatic enamel. The majority of enamel tubules seemed to contain no cell processes in the stage of enamel maturation, indicating that the cell processes in the tubules probably degenerate after the stage of enamel formation.

Amelogenesis↗
PubMed1983

Ultrastructural study of osteodentin formation induced by irradiation.

A single dose of 16,000 rads was delivered to a 1 X 1.5 cm area of the cheek of 2 day old Wistar rats. The lower first molar region was removed 1, 3, 5 and 8 days postirradiation and processed for light and electron microscopy observation. The most distinct structural feature at optical level was osteodentin production in the dental papilla, which began at the level of the initiation of Hertwig's epithelial sheath and continued until the pulpal area was completely filled by osteodentin. At the ultrastructural level, ameloblasts exhibited large lipidic vacuoles and irregular intracytoplasmic secretory granules. Odontoblasts underwent multiple regressive alterations with vacuolization and numerous myelin figures. Pulpal cells exhibited a capacity for odontoblastic differentiation with formation of one or more odontoblastic processes and dentin matrix secretion. Thus, irradiation resulted in stimulation of a great potential for dentin production in these cells.

Animals↗
PubMed1981

Ultrastructural location of Streptococcus mutans and Streptococcus sanguis antigens in carious human dentine.

The ultrastructural localization of bacterial antigens of Streptococcus sanguis and of the various serotypes a,b,c,d,e,f and g of Streptococcus mutans was studied in human carious dentine using the indirect peroxidase-antiperoxidase method with appropriate controls. No positive staining was seen in adjacent normal dentine. In the inner dentine underlying the cytoplasm of fibroblasts and Schwann cells of unmyelinated nerve fibrils. In sclerosed tubules, or on the plasmalemma of the odontoblast process or on both structures. Only in the odontoblast, facing the carious cone, were dense stainings noted in vacuoles of various sizes located in the Golgi apparatus in juxta-nuclear position and in their odontoblast processes. In pulpal regions, underlying the carious cone, dense vacuoles were also observed in the cytoplasm of fibroblasts and Schwann cells of unmyelinated nerve fibrils. In sclerosed tubules, electron dense deposits were noted in the lumen and the walls of the calcified tubules. In the outer carious dentine, invaded by micro-organisms, positive antigenic stainings were observed in the cell wall and capsular material of a great number of micro-organisms as well as in the interbacterial matrix.

Antigens, Bacterial↗
PubMed1999

Subtilisin-like proprotein convertase PACE4 (SPC4) is a candidate processing enzyme of bone morphogenetic proteins during tooth formation.

The temporospatial expression of PACE4, a member of the mammalian subtilisin-like proprotein convertase family, in the developing rat molar tooth was determined by in situ hybridization. At the initiation stage of tooth development, PACE4 mRNA was weakly expressed in the dental lamina, whereas the mesenchymal cells intensely expressed the PACE4 transcript. At the bud stage, high-level expression of PACE4 mRNA was found in the dental epithelium and condensed dental mesenchyme. Its expression became more localized in the differentiating ameloblasts during cap and early bell stages. In the newborn rats, PACE4 mRNA was localized in the ameloblasts and odontoblasts, but its expression became weaker with advancing development, showing apparent association with the differentiation and establishment of functional ameloblasts and odontoblasts. These expression patterns of PACE4 were very similar to those of several bone morphogenetic proteins (BMPs) reported previously. Because BMPs, which are primarily involved in the morphogenesis in tooth formation, are synthesized as inactive precursors and activated by limited proteolysis at the consensus Arg-X-X-Arg maturation site, the present observations suggest that PACE4 is possibly a candidate proBMP convertase that acts during tooth formation.

Ameloblasts↗
PubMed1996

Light microscopic detection of sugar residues in rabbit embryo teeth with lectin-horseradish peroxidase conjugates.

We investigated the binding of five HRP-conjugated lectins to rabbit tooth germs at the cap and late bell stages of development. The results revealed some changes in the glycosylation patterns of the glycoconjugates. Sugar residues, such as alpha-D-mannose, methyl-D-glucose, N-acetylglucosamine, beta-D-galactosamine, D-galactose, and sialic acid, were detectable in some components of the tooth germs. The most conspicuous developmental change was increased binding of Con A and WGA. These lectins showed, at the cap stage, moderate binding to the (pre)-ameloblasts and (pre)-odontoblasts. With further development to the late bell stage, but prior to the achievement of well-defined morphological-functional characteristics, the odontoblasts and ameloblasts displayed considerable amounts of alpha-D-mannose, alpha-D-glucose as well as beta-D-acetylglucosamine and sialic acid. Appropriate control studies confirmed the specificity of the binding of the lectins. Two lectins (DBA and PNA) with known specificity for N-acetylgalactosamine groups were bound by the basement membranes in tooth germs at the cap stage. A third lectin (RCA) with the same specificity did not produce any detectable staining in the same material. Further studies must be planned to determine the specific functions and significance of lectin-HRP-binding glycoconjugates in odontogenesis.

Animals↗
PubMed1976

Fine structure of differentiating ameloblasts in the kitten.

The fine structure of differentiating ameloblasts was studied in the lower second molar of 1-week-old kittens after perfusion fixation with and without subsequent decalcification. The differentiation zone was divided into three phases. In Differentiation 1, ameloblasts are about 27 mum long and face an uninterrupted basal lamina. The predentin adjacent to the basal lamina contains a few collagen fibrils oriented mainly at right angles to the ameloblast surface. The specialized predentin forms a well-defined layer, up to 1.5 mum thick, referred to as the junctional layer. In Differentiation 2, ameloblast processes extend through the basal lamina and the thickness of the junctional layer. The processes consist of cytoplasmic sheets forming a honeycomb-like network. Dentin starts to calcify after process-formation is underway. Two distinct types of odontoblast processes, having different shapes and contents, come in contact with the ameloblasts and push into the ameloblastic layer. In Differentiation 3, stippled material appears in the extracellular spaces between ameloblasts. Later, stippled material-like substances appear in the predentin close to the ameloblast apex and close to odontoblast processes within the dentin. Ameloblasts now are up to 40 mum high. Enamel secretion starts in small circumscribed areas which gradually enlarge, leading to the disappearance of the ameloblast processes. These findings are compared with results obtained in other species, including man, and their possible functional significance is discussed.

Ameloblasts↗
PubMed1994

Type III collagen is a major component of interodontoblastic fibers of the developing mouse molar root.

BACKGROUND: Recently, collagenous interodontoblastic fibers (IOF) were reported in some particular developmental stages and/or locations of the tooth. However, it remained unclear whether these fibers were identical to so-called von Korff fibers. METHODS: To clarify this issue, we examined the developing mouse molar by three-dimensional reconstruction of 8 confocal images within a 6 micron-thick section using laser scanning confocal microscopy, and confirmed our findings using immunoelectron microscopy. RESULTS: In the root pulp during circumpulpal dentin formation, the IOF stained weakly for type I collagen, but stained strongly for type III collagen by a double-staining technique. It could be clearly seen that many immunoreactive fibers ran spirally among the odontoblasts and entered the predentin. This distribution pattern of IOF was similar to that of the classical von Korff fibers. Furthermore, the existence of anti-type III reactive collagen fibrils between odontoblasts was confirmed, whereas IOF were not observed in the coronal pulp during circumpulpal dentin formation. CONCLUSIONS: This study presents for the first time, immunohistochemical observations which demonstrate the presence of IOF at least during root circumpulpal dentin formation and which reveal that type III collagen is a major component of IOF.

Animals↗
PubMed2005

PDGFR-alpha signaling is critical for tooth cusp and palate morphogenesis.

Platelet-derived growth factor receptor alpha (PDGFR-alpha) and PDGF ligands are key regulators for embryonic development. Although Pdgfralpha is spatially expressed in the cranial neural crest (CNC)-derived odontogenic mesenchyme, mice deficient for Pdgfralpha are embryonic lethal, making it impossible to investigate the functional significance of PDGF signaling in regulating the fate of CNC cells during tooth morphogenesis. Taking advantage of the kidney capsule assay, we investigated the biological function of PDGF signaling in regulating tooth morphogenesis. Pdgfralpha and Pdgfa are specifically and consistently expressed in the CNC-derived odontogenic mesenchyme and the dental epithelium, respectively, throughout all stages of tooth development, suggesting a paracrine function of PDGF signaling in regulating tooth morphogenesis. Highly concentrated expression patterns of Pdgfralpha and Pdgfa are associated with the developing dental cusp, suggesting possible functional importance of PDGF signaling in regulating cusp formation. Loss of the Pdgfralpha gene does not affect proper odontoblasts proliferation and differentiation in the CNC-derived odontogenic mesenchyme but perturbs the formation of extracellular matrix and the organization of odontoblast cells at the forming cusp area, resulting in dental cusp growth defect. Pdgfralpha-/- mice have complete cleft palate. We show that the cleft palate in Pdgfralpha mutant mice results from an extracellular matrix defect within the CNC-derived palatal mesenchyme. The midline epithelium of the mutant palatal shelf remains functionally competent to mediate palatal fusion once the palatal shelves are placed in close contact in vitro. Collectively, our data suggests that PDGFRalpha and PDGFA are critical regulators for the continued epithelial-mesenchymal interaction during tooth and palate morphogenesis. Disruption of PDGFRalpha signaling disturbs the growth of dental cusp and interferes with the critical extension of palatal shelf during craniofacial development.

Animals↗
PubMed1998

Delta-notch signaling in odontogenesis: correlation with cytodifferentiation and evidence for feedback regulation.

Recent data suggest that dental cells utilize the evolutonarily conserved Notch-mediated intercellular signaling pathway to regulate their fates. Here we report on the expression and regulation of Delta1, a transmembrane ligand of the Notch receptors, during mouse odontogenesis. Delta1 is weakly expressed in dental epithelium during tooth initiation and morphogenesis, but during cytodifferentiation, expression is upregulated in the epithelium-derived ameloblasts and the mesenchyme-derived odontoblasts. The expression pattern of Delta1 in ameloblasts and odontoblasts is complementary to Notch1, Notch2, and Notch3 expression in adjacent epithelial and mesenchymal cells. Notch1 and Notch2 are upregulated in explants of dental mesenchyme adjacent to implanted cells expressing Delta1, suggesting that feedback regulation by Delta-Notch signaling ensures the spatial segregation of Notch receptors and ligands. TGFbeta1 and BMPs induce Delta1 expression in dental mesenchyme explants at the stage at which Delta1 is upregulated in vivo, but not at earlier stages. In contrast to the Notch family receptors and their ligand Jagged1, expression of Delta1 in the tooth germ is not affected by epithelial-mesenchymal interactions, showing that the Notch receptors and their two ligands Jagged1 and Delta1 are subject to different regulations.

Animals↗
PubMed1991

A modification of tooth germ cultivation in vitro and in ovo.

Mandibular molar anlages excised from 17-day mouse foetuses were cultured in vitro or in ovo (on the chorioallantoic membrane). In both cases, the explants were underlain either with a Millipore filter or with a piece of fibrin foam. Tooth germs were harvested after 7 days of cultivation and processed histologically. Spatial arrangement was highly preserved in the tooth germs cultured in vitro on fibrin foam. In vitro cultures on Millipore filters revealed significant flattening of tooth germs, caused especially by the collapse of enamel organ and the pulp. The cytodifferentiation of tooth germs cultured in vitro on both substrates (Millipore filter, fibrin foam) was characterized by the presence of odontoblasts, polarizing ameloblasts and predentine. The cytodifferentiation of tooth germs cultured in ovo on Millipore filters placed on chorioallantoic membrane was characterized by the presence of odontoblasts, ameloblasts, predentine, dentine and enamel. However, the flattening of these explants was identical with the changes of the explants cultured on Millipore filters in vitro. In ovo cultivation on the fibrin foam failed to bring satisfactory results.

Ameloblasts↗
PubMed1978

Intercellular attachments between calcified collagenous tissue forming cells in the rat.

Osteoblasts of the young rat cranium, and cementoblasts and odontoblasts of young rat molars were prepared by ethanol freeze-fracture prior to critical point drying for scanning electron microscopy (SEM) as well as conventional transmission electron microscopy (TEM) techniques. Critical point drying causes shrinkage which separates the lateral intercellular contacts between neighbours in the same sheet in the case of cementoblasts and osteoblasts, but not those between odontoblasts. These differences are considered to be of functional significance and need to be taken into consideration when formulating theories of calcium influx into the mineralizable matrix of the respective tissues.

Animals↗
PubMed1985

Lectin-binding patterns in the developing tooth.

The lectin-binding patterns of the cells involved in amelogenesis and dentinogenesis in developing teeth of rats were studied. Undifferentiated odontogenic epithelia exhibited very slight staining with almost all of the lectins examined. The lectin-staining affinities of secretory ameloblasts could be divided into two categories: Concanavalin-A (Con-A), Wheat germ agglutinin (WGA) and Soybean agglutinin (SBA) binding occurred from the middle to apical cytoplasm, whereas Ricinus communis agglutinin-I (RCA-I) and Ulex europeus I (UEA-I) binding predominated in the basal regions. The cells of the stratum intermedium exhibited relatively strange lectin staining, which appeared to be dependent on ameloblastic maturation. The basement membranes in undifferentiated epithelia were markedly positive for lectin binding. Odontoblasts showed moderate Con-A staining on the apical side of the cells, as well as slight-to-moderate reactions with WGA and SBA. Pulp cells and dental papillae showed slight-to-moderate lectin staining, and predentin and dentin were also moderately positive for Con-A and RCA-I binding and slightly so for WGA and SBA. The lectin-binding affinities were enhanced during the formation of enamel and dentin, and appeared to be dependent on the degree of cellular differentiation in ameloblasts and odontoblasts.

Ameloblasts↗
PubMed2000

Establishment and characterization of a culture system for enzymatically released rat dental pulp cells.

To establish a cell culture system that reflects the dentin formation in dental pulp tissue, we used dental pulp cells enzymatically isolated from rat incisor teeth. During the 20-day culture period, the cells exhibited various phenotypes of the odontoblast differentiation process, from the immature stage to the terminal mineralization stage. The cells began to form the mineralized nodules from day 10, and the nodules became larger by day 20. Alkaline phosphatase (ALP)-positive cells surrounded the mineralized nodules. The ALP activity in the cell layers was maximal on day 5, and gradually decreased to day 20. The calcium content in the cell layers was very low by day 10, and significantly increased from day 15. Sulfated glycosamino-glycans (GAGs) contained in the cell layers increased by day 15, but the content then decreased by day 20. The dental pulp cells produced a small amount of osteocalcin that was released into the culture medium by day 10, and the amount secreted increased markedly from day 15. The expression of osteocalcin and parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) receptor mRNA was evident as early as day 5, and the expression of each gradually increased with the number of days in culture. Dentin matrix protein (Dmp1) mRNA gene transcripts were identified by use of the reverse transcription polymerase chain reaction (RT-PCR) in the cells throughout the culture period. The present results demonstrate that this culture system is useful for studying the differentiation process of the odontoblast-like cells.

Alkaline Phosphatase↗
PubMed2006

In vitro characterization of human dental pulp cells: various isolation methods and culturing environments.

Our purpose was to characterize human dental pulp cells isolated by various methods and to examine the behavior of cells grown under various conditions for the purpose of pulp/dentin tissue engineering and regeneration. We compared the growth of human pulp cells isolated by either enzyme digestion or the outgrowth method. Expression of dentin sialophosphoprotein, Cbfa1, and two types of collagen (I and III) in these cells was examined by Western blot or reverse transcription/polymerase chain reaction. Growth of pulp cells on dentin and in collagen gel was also characterized. We found that different isolation methods give rise to different populations or lineages of pulp cells during in vitro passage based on their collagen gene expression patterns. Cells isolated by enzymedigestion had a higher proliferation rate than those isolated by outgrowth. Pulp cells did not proliferate or grew minimally on chemically and mechanically treated dentin surface and appeared to establish an odontoblast-like morphology with a cytoplasmic process extending into a dentinal tubule as revealed by scanning electron microscopy. The contraction of the collagen matrix caused by pulp cells was dramatic: down to 34% on day 14. Our data indicate that (1) the choice of the pulp cell isolation method may affect the distribution of the obtained cell populations, (2) a treated dentin surface might still promote odontoblast differentiation, and (3) a collagen matrix may not be a suitable scaffold for pulp tissue regeneration because of the marked contraction caused by pulp cells in the matrix. The present study thus provides important information and a basis for further investigations pre-requisite to establishing pulp tissue engineering/regeneration protocols.

Base Sequence↗
PubMed2006

Capacity of dental pulp differentiation after tooth transplantation.

Under pathological conditions, dental pulp elaborates both bone and dentin matrix in which the contribution of periodontal tissue cannot be excluded. This study has aimed to clarify the capability of dental pulp to deposit bone matrix in an auto-graft experiment by using (1) immunohistochemistry for 5-bromo-2'-deoxyuridine (BrdU) and nestin and (2) histochemistry for tartrate-resistant acid phosphatase (TRAP). Following the extraction of the molars of 3-week-old mice, the roots and pulp floor were resected and immediately transplanted into the sublingual region. On Days 5-7, tubular dentin formation commenced next to the pre-existing dentin at the pulp horn in which nestin-positive odontoblast-like cells were arranged. Up until Day 14, bone-like tissue formation occurred in the pulp chamber in which intense TRAP-positive cells appeared. These results suggest that odontoblast- and osteoblast-lineage cells reside in the dental pulp. Overall, specific dental pulp regeneration should provide fundamental knowledge for the realization of human tooth regeneration in the near future.

Acid Phosphatase↗
PubMed2005

Expression of versican and ADAMTS during rat tooth eruption.

A disintegrin and metalloprotease with thrombospondin type 1 motifs (ADAMTS) is a family of extracellular proteases and implicated in cleaving proteoglycans, such as aggrecan, versican and brevican. No information is available about expression or localization of these ADAMTSs in teeth. Versican is a large chondroitin sulfate proteoglycan that is present in a variety of connective tissue including dental pulp, dentin, cementum and periodontal ligaments. The present study was designed to investigate expression of ADAMTSs and versican during rat tooth eruption. Rat maxillary first molars in weeks 1, 2, 3, 4 and 6 were examined. The mRNA expression of ADAMTS1, ADAMTS4, ADAMTS5 and versican was localized using in situ hybridization. ADAMTS1, ADAMTS4, ADAMTS5 and versican were expressed in dental pulp cells, odontoblasts, cementoblasts, cementocytes, periodontal ligament cells, osteoblasts and osteocytes. The temporal and spatial expression pattern in these cellular phenotypes was comparable among ADAMTSs and versican. The present study suggests that dental pulp cells, odontoblasts, cementoblasts, cementocytes, periodontal ligament cells, osteoblasts and osteocytes may be involved in both production and degradation of versican with secreting ADAMTS1, ADAMTS4 and ADAMTS5.

ADAM Proteins↗