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PubMed1982

Effects of diazo-oxo-norleucine on cell kinetics and odontoblast differentiation in cultured embryonic mouse molars.

Diazo-oxo-norleucine (DON), an analogue of glutamine, prevented odontoblast differentiation in cultured tooth germs. Diazo-oxo-norleucine added after the onset of odontoblast differentiation, did not affect the secretion of predentine or the functional differentiation of ameloblasts. DON decreased explant volume and modified cell kinetics, decreasing mitotic index, labelling index and number of grains per nucleus; the 5 phase of the cell cycle was lengthened. These modifications of cell kinetics should be considered when interpreting the effects of DON on odontoblast differentiation.

Animals↗
PubMed1983

A scanning electron microscopic study of the odontoblast process in human coronal dentine.

It is generally accepted that the odontoblast process occupies the dentinal tubules only in the inner part of the dentine, extending approx 0.7 mm from the pulp in both animals and man. Twenty-three premolars, molars and third molars from subjects aged 11-24 yr, all caries-free or only slightly decayed, were processed immediately after extraction by one of four methods. (I) Teeth were split in liquid nitrogen and then fixed, dehydrated in ascending ethanol, and dried by critical point drying (CPD). (II) Teeth were fractured by use of a mallet and a chisel and then prepared as in Method I. (III) The root was cut off using a diamond disk or a mallet and a chisel and the crown was then fixed, fractured in liquid nitrogen, dehydrated and CPD. (IV) As Method III but following fixation the teeth were freeze-dried. All specimens were examined in the scanning electron microscope (SEM). In specimens prepared by Methods II, III or IV the odontoblast process was limited to the inner third of crown dentine. However in all the specimens prepared by Method I the odontoblast process extended to the dentine-enamel junction.

Adolescent↗
PubMed1984

The ultrastructure of the rat incisor odontoblast in organ culture.

Extracted incisors were dissected free of adherent soft tissue, split by a longitudinal incision lingually and the pulp sac carefully removed. The predentine-dentine pieces with attached odontoblasts were incubated for up to 24 h in culture medium in 5 per cent CO2, humid air at 37 degrees C. The cultures were recovered and processed for light microscopy and transmission electron microscopy. Examination of the explants showed a homogeneous layer of odontoblasts attached to the dentinal surface. The odontoblasts had a normal structure and nuclear polarity similar to that seen in vivo. There were no apparent degenerative changes.

Animals↗
PubMed1985

Inter-odontoblastic fibres in human dentine observed by scanning electron microscopy.

Crowns of healthy premolars with closed apical foramina were fixed, resin-impregnated and freeze fractured. Fragments were critical-point dried and examined by scanning electron microscopy. Radial inter-odontoblastic fibres with a diameter of 0.1-0.7 micron were observed. Most of these radial fibres originated from the odontoblast cell body, but some of them arose from the pulp. Both of them entered the predentine at the rim of dentinal tubules and their branches formed the fibrous network of predentine. The odontoblast processes had only a few short side branches at the level of predentine. Some fibrils were observed in the mineralized intertubular dentine, as well.

Adolescent↗
PubMed1991

Ultrastructure of inter-odontoblastic fibres in the rat molar.

Radially directed inter-odontoblastic collagen fibres were observed in ultra-thin sections of the radicular pulp/predentine complex of rat molars. Bundles of fibres crossed the distal junctional complexes of the odontoblasts, went through the whole thickness of the predentine and were incorporated into the mineralized dentine. Scanning electron microscopy showed that bundles of radial fibres are not found between the coronal odontoblasts of the rat molar but only in the root. The radial fibres were inserted into the predentine along vertical long-axial crests. The intercrest interval was not tightly fixed and the pattern of fibre-bundle insertion did not show a regular periodicity.

Animals↗
PubMed1993

Retardation of mouse odontoblast differentiation by heparin in vitro.

The effect of heparin was studied histologically and immunohistochemically. Tooth germs from 15-day-old mouse embryos were cultured with or without heparin. After 6 days of culture in control medium, mesenchymal cells underlying the inner enamel epithelium had differentiated into odontoblasts and secreted predentine. In medium with heparin, mesenchymal cells were undifferentiated. In medium with other glycosaminoglycans such as chondroitin sulphate, dermatan sulphate or hyaluronate, tooth germs were similar to those in control medium, as were those in medium with heparin-Sepharose absorbed serum. After 12 days of culture in the heparin medium, mesenchymal cells in some cusps had differentiated into odontoblasts and secreted predentine but in other cusps remained undifferentiated. Immunohistochemically, exogenous heparin did not prevent the deposition of type IV collagen, laminin and fibronectin in the basement membrane and extracellular matrix. These results suggest that exogenous heparin retards differentiation of odontoblasts but not by disruption of the basement membrane nor inactivation of heparin-binding growth factors present in serum.

Animals↗
PubMed1995

Neurotensin-like immunoreactivity in odontoblasts and their processes in rat maxillary molar teeth and the effect of pulpotomy.

A strong neurotensin-like immunoreactivity (NT-like IR) was detected in the odontoblast cells of the rat teeth. 4 h after a partial pulpotomy performed in two maxillary molar teeth a decreased NT-like IR was observed in the odontoblast layer located at the vicinity of the lesion together with edema and nuclear pyknosis. NT-like IR became further decreased after 24 h. After 7 days NT-like IR had almost fully disappeared with signs of necrosis of the dental pulp and infiltration of polymorphonuclear lymphocytes. It seems possible that NT like peptides in the odontoblast cell layer may play a role, e.g., in dentinogenesis and/or nociception.

Animals↗
PubMed1992

Calcium ion transport kinetics during dentinogenesis: effects of disrupting odontoblast cellular transport systems.

Due to strongly discrepant results in the literature, controversy exists about the timing of the transport of Ca2+ ions to the mineralization front during dentinogenesis and the role of the odontoblasts in this transport. The present study gives evidence, by means of autoradiography as well as by a radiochemical technique, that the transport time for Ca2+ ions into the dentin mineral phase is about 10-15 min in the rat incisor. The results also show that technical factors, such as mode of tracer injection and the use of perfusion fixation, may influence the results more or less strongly. Finally, by disturbing odontoblast microtubules, involved in intracellular transport processes, and by blocking odontoblast calcium uptake channels by nifedipine and neomycin, the Ca2+ ion transport into dentin mineral was found to be strongly impaired. This may be taken as an indication that transcellular calcium transport mechanisms have a role during dentinogenesis.

Animals↗
PubMed2006

Effect of curing regime on the cytotoxicity of resin-modified glass-ionomer lining cements applied to an odontoblast-cell line.

OBJECTIVE: The aim of this in vitro study was to evaluate the cytotoxicity of resin-modified glass-ionomer lining cements submitted to different curing regimes and applied to an immortalized odontoblast-cell line (MDPC-23). METHODS: Forty round-shaped specimens of each experimental material (Fuji Lining LC and Vitrebond) were prepared. They were light-cured for the manufacturers' recommended time (MRT = 30 s), under-cured (0.5 MRT = 15 s), over-cured (1.5 MRT = 45 s) or allowed to dark cure (0 MRT). Sterilized filter papers soaked with either 5 microL of PBS or HEMA were used as negative and positive control, respectively. After placing the specimens individually in wells of 24-well dishes, odontoblast-like cells MDPC-23 (30,000 cells/cm2) were plated in each well and incubated for 72 h in a humidified incubator at 37 degrees C with 5% CO2 and 95% air. The cytotoxicity was evaluated by the cell metabolism (MTT assay) and cell morphology (SEM). RESULTS: Fuji Lining LC was less cytotoxic than Vitrebond (p < 0.05) in all the experimental conditions. However, the cytotoxicity of Fuji Lining LC was noticeably increased in the absence of light-curing while the same was not observed for Vitrebond. The length of light-curing (15, 30 or 45 s) did not influence the toxicity of both lining materials when they were applied on the odontoblast-cell line MDPC-23. SIGNIFICANCE: The light-activation plays an important role in reducing the cytotoxicity of Fuji Lining LC. Following the manufacturer' recommendation regarding the light-curing regime may prevent toxic effect to the pulp cells.

Animals↗
PubMed2006

TLR4 mediates LPS-induced VEGF expression in odontoblasts.

Lipopolysaccharide (LPS) from gram-negative bacteria cell walls such as Prevotella intermedia and Escherichia coli induce vascular endothelial growth factor (VEGF) expression in odontoblasts, but not in undifferentiated dental pulp cells. CD14 and TLR4 are responsible for LPS signaling in macrophages, but their expression levels and function in dental pulp cells are unknown. We showed here that murine odontoblast-like cells (MDPC-23) express CD14 and TLR4 by immunohistochemistry and flow cytometry. In contrast, undifferentiated dental pulp cells (OD-21) presented low or no expression of these two receptors. MDPC-23 cells showed CD14 and TLR4 up-regulation upon exposure to LPS, as determined by real time PCR. Dominant negative murine TLR4 (DN-mTLR4) transfected MDPC-23 cells did not show upregulated VEGF expression in response to LPS stimulation. These results demonstrate that odontoblast-like cells express CD14 and TLR4, and that LPS-induced VEGF expression is mediated, at least in part, by TLR4 signaling.

Animals↗
PubMed1997

Distribution of transforming growth factor beta1-binding proteins and low-affinity receptors during odontoblast differentiation in the mouse.

Transforming growth factor-beta1 (TGF-beta1) was immunolocalized within differentiated odontoblasts and ameloblasts while LAP-beta1 was detected at the apicol pole of odonotoblasts and ameloblasts and in predentine. Anti-LAP-beta1 antibodies also stained the epithelial-mesenchymal junction (EMJ). Decorin was immunolocalized in young functional odonotoblasts and in both predentine and dentine. Biglycan was similarly distributed but absent from dentine. Immunostaining with anti-latent TGF-beta1 binding protein-1 (LTBP-1) showed fibrillar structures located at the EMJ and between predontoblasts and odontoblasts; at older states staining was restricted to the dental papilla and sac. Thus differentiated odonotoblasts express TGF-beta1 and in a more restricted manner decorin, biglycan and LAP-beta1; it can be assumed that TGF-beta1 is able to interact with the three molecules present in predentine. Earlier, LTBP-1 and LAP-beta1, both present at the EMJ, may contribute to odontoblast differentiation.

Activin Receptors, Type I↗
PubMed1998

Modulation of rat incisor odontoblast plasma membrane-associated Ca2+ with nifedipine.

In addition to the Ca2+ portion freely dissociated in the cytosol, another Ca2+ pool is associated with plasma membranes and intracellular organelle membranes. This Ca2+ portion is of importance for regulation of, among other things, the cell cycle, actin-mediated processes, and cell morphology. In the literature, dihydropyridines have been reported to influence this membrane-associated pool of Ca2+ under certain conditions. The aim of this investigation was to study possible modulations of plasma membrane-associated Ca2+ upon treatment with nifedipine in vitro in a Ca2+-transporting cell, the dentin-forming odontoblast. The membrane-associated portion of Ca2+ in dissected dentinogenically active rat incisor odontoblasts was monitored by fluorescence spectrophotometry using chlortetracycline as a probe. In addition, images of chlortetracycline-Ca2+ binding were obtained by fluorescence microscopy. It was found that membrane-associated Ca2+ decreased by the dihydropyridine nifedipine, whereas this Ca2+ pool was unaffected by the cellular polarization state, which was in contrast to cytosolic free Ca2+ as measured by fura-2. The results show that the odontoblast plasma membrane-associated Ca2+-pool can be modulated by nifedipine, thus being dependent on the conformational state of the L-type Ca2+ channels.

Animals↗
PubMed2004

Regulation of the Cell Type-specific dentin sialophosphoprotein gene expression in mouse odontoblasts by a novel transcription repressor and an activator CCAAT-binding factor.

Dentin sialophosphoprotein (DSPP) is an extracellular matrix protein that is cleaved into dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) with a highly restricted expression pattern in tooth and bone. Mutations of the DSPP gene are associated with dentin genetic diseases. Regulation of tissue-specific DSPP expression has not been described. To define the molecular basis of this cell-specific expression, we characterized the promoter responsible for the cell-specific expression of the DSPP gene in odontoblasts. Within this region, DNase I footprinting and electrophoretic mobility shift assays delineated one element that contains an inverted CCAAT-binding factor site and a protein-DNA binding site using nuclear extracts from odontoblasts. A series of competitive electrophoretic mobility shift assay analyses showed that the protein-DNA binding core sequence, ACCCCCA, is a novel site sufficient for protein binding. These two protein-DNA binding sequences are conserved at the same proximal position in the mouse, rat, and human DSPP gene promoters and are ubiquitously present in the promoters of other tooth/bone genes. Mutations of the CCAAT-binding factor binding site resulted in a 5-fold decrease in promoter activity, whereas abolishment of the novel protein-DNA binding site increased promoter activity by about 4.6-fold. In contrast to DSPP, expression levels of the novel protein were significantly reduced during odontoblastic differentiation and dentin mineralization. The novel protein was shown to have a molecular mass of 72 kDa. This study shows that expression of the cell type-specific DSPP gene is mediated by the combination of inhibitory and activating mechanisms.

Animals↗
PubMed2005

Differential regulation of dentin sialophosphoprotein expression by Runx2 during odontoblast cytodifferentiation.

Dentin sialophosphoprotein (DSPP) consists of dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). The spatial-temporal expression of DSPP is largely restricted during differentiational stages of dental cells. DSPP plays a vital role in tooth development. It is known that an osteoblast-specific transcription factor, Runx2, is essential for osteoblast differentiation. However, effects of Runx2 on DSPP transcription remain unknown. Here, we studied different roles of Runx2 in controlling DSPP expression in mouse preodontoblast (MD10-F2) and odontoblast (MO6-G3) cells. Two Runx2 isoforms were expressed in preodontoblast and odontoblast cells, and in situ hybridization assay showed that DSPP expression increased, whereas Runx2 was down-regulated during odontoblast differentiation and maturation. Three potential Runx2 sites are present in promoters of mouse and rat DSPP genes. Runx2 binds to these sites as demonstrated by electrophoretic mobility shift assay and supershift experiments. Mutations of Runx2 sites in mouse DSPP promoter resulted in a decline of promoter activity in MD10-F2 cells compared with an increase of its activity in MO6-G3 cells. Multiple Runx2 sites were more active than a single site in regulating the DSPP promoter. Furthermore, forced overexpression of Runx2 isoforms induced increases of endogenous DSPP protein levels in MD10-F2 cells but reduced its expression in MO6-G3 cells consistent with the DSPP promoter analysis. Thus, our results suggest that differential positive and negative regulation of DSPP by Runx2 is dependent on use of cytodifferentiation of dental ectomesenchymal-derived cells that may contribute to the spatial-temporal expression of DSPP during tooth development.

Animals↗
PubMed2006

Dentin matrix protein 1 regulates dentin sialophosphoprotein gene transcription during early odontoblast differentiation.

Dentin mineralization requires transcriptional mechanisms to induce a cascade of gene expression for progressive development of the odontoblast phenotype. During cytodifferentiation of odontoblasts there is a constant change of actively transcribed genes. Thus, tissue-specific matrix genes that are silenced in early differentiation are expressed during the terminal differentiation process. Dentin sialophosphoprotein (DSPP) is an extracellular matrix, prototypical dentin, and a bone-specific gene, however, the molecular mechanisms by which it is temporally and spatially regulated are not clear. In this report, we demonstrate that dentin matrix protein 1 (DMP1), which is localized in the nucleus during early differentiation of odontoblasts, is able to bind specifically with the DSPP promoter and activate its transcription. We have identified the specific promoter sequence that binds specifically to the carboxyl end of DMP1. The DNA binding domain in DMP1 resides between amino acids 420 and 489. A chromatin immunoprecipitation assay confirmed the in vivo association of DMP1 with the DSPP promoter. Interactions between DMP1 and DSPP promoter thus provide the foundation to understand how DMP1 regulates the expression of the DSPP gene.

Base Sequence↗
PubMed2002

Analysis of odontoblast gene expression using a novel approach, laser capture microdissection.

Studying the mechanisms of molecular interactions in developing tissues demands sensitive molecular biological in vivo and in vitro techniques. Laser capture microdissection (LCM) allows for the isolation of mRNA in histological sections even from single cells, thus enabling the identification of in vivo gene expression products in closely circumscribed tissue areas. The aims of this study were to assess the optimal fixation, processing, and staining conditions to retrieve RNA from microdissected odontoblasts. Fluorometric assays and RT-PCR analysis of alpha 1(I) collagen, dentin sialophosphoprotein (Dspp), and osteocalcin (OC) confirmed that the total RNA isolated from day 0 and day 3 captured odontoblasts was sufficient in quantity and quality. Our results indicate that individual odontoblasts obtained by LCM are morphologically intact and chemically unaltered, allowing accurate molecular and biochemical analyses.

Animals↗
PubMed2004

Acceleration effect of human recombinant bone morphogenetic protein-2 on differentiation of human pulp cells into odontoblasts.

Predictable pulp capping procedures remain problematic, possibly because of the lack of appropriate stimulating factors for dentin formation. The present study examines the ability of one such stimulating factor, bone morphogenetic protein-2, to accelerate the differentiation of human dental pulp cells into odontoblasts. The number and morphology of cells between groups treated with 0 and 100 ng/ml of human recombinant bone morphogenetic protein-2 (rhBMP-2) did not significantly differ. However, ALPase activity (a marker for biomineralization) in the group stimulated with rhBMP-2 was more than double that of the control group. We then measured the expression of mRNA encoding dentin sialophosphoprotein (DSPP) as a marker of odontoblasts in rhBMP-2-stimulated human pulp cells using a quantitative polymerase chain reaction. The expression of DSPP mRNA in cells stimulated for 24 h by 1000 ng/ml of rhBMP-2 was approximately 20-fold and 5-fold higher than that by stimulated by 10 and 100 ng/ml, respectively. These findings show that rhBMP-2 promoted the differentiation of human dental pulp cells into odontoblasts but did not affect cell proliferation, suggesting that rhBMP-2 may have therapeutic utility in vital pulp therapy.

Adult↗
PubMed1991

The presence of nerve fibres in the coronal odontoblast layer of teeth at various stages of root development.

Many studies have investigated the presence of nerve fibres in the coronal odontoblast layer of mature teeth. However, little information is available on the presence of these structures in immature human teeth. The pulps of 18 human maxillary and mandibular third molars at various stages of development were collected, sectioned, and stained by the immunoperoxidase technique. The results showed the presence of numerous myelinated nerve fibres in the coronal odontoblast layer of teeth with fully formed roots. Similar observations were made in teeth with half formed roots, and in those without roots. On the basis of these results, it appears that a lack of response to vitality tests in teeth with incompletely formed roots is not wholly due to the absence of nerve fibres in the coronal odontoblast layer.

Dental Pulp↗