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PubMed1975

Leucine and fucose incorporation into odontoblast protein.

Odontoblasts isolated from rat incisors were incubated in vitro in the presence of tritiated leucine or fucose, and the amount of precursor incorporated into TCA-recipitated proteins was determined. The effect on precursor incorporation of varying the total precursor concentration was examined. The incorporation of leucine and fucose into TCA-precipitated proteins was found to be linear with time for at least 90 min. Inhibition studies with puromycin, cycloheximide and chloramphenicol demonstrated the presence of odontoblastic protein synthesis. One hour after in vivo administration of triated leucine the highest specific activity of odontoblast-predentin protein material was noted the corresponding maximum for pulp proteins was found to occur after 30 min. The in vivo incorporation of triated fucose was maximal after 4 h in both odontoblasts and pulp tissue.

Animals↗
PubMed1978

Effect of vincristine on odontoblasts in rat incisor.

Vincristine in doses of 0.1, 0.3, 0.5 and 0.7 mg/kg was administered to 60 rats in four groups. Histomorphologic investigation of the odontoblast population in the maxillary incisors revealed dose-dependent reactions consisting of (1) swelling of the odontoblasts and an accumulation of abnormal mitotic cells in the germinative parts of the pulp after 5 h, (2) a supervening necrosis and destruction of some odontoblasts and of the mitotic cells after 24 h, and (3) after 3 d, a reversal to normal structure in some parts of the odontoblast population, but a further development of the vincristine-induced changes, with severe cellular derangement and irregular predentin production, in others.

Animals↗
PubMed1997

Dentin sialoprotein (DSP) transcripts: developmentally-sustained expression in odontoblasts and transient expression in pre-ameloblasts.

Dentin sialoprotein (DSP), a 53 kDa glycoprotein, is believed to be present exclusively in dentin. Using rat and mouse digoxigenin labeled (DIG)-DSP and 35S-DSP riboprobes, and in situ hybridization techniques, we have studied the presence of DSP mRNA at specific developmental stages of dentinogenesis. In mouse and rat molars and incisors, DSP transcripts were localized in young odontoblasts associated with early stages of predentin formation, as well as in mature odontoblasts, cells with cytoplasmic extensions embedded in the forming dentin. No DSP transcripts were detected in dental pulp, enamel organ, ameloblasts, epithelial root sheath, Meckel's cartilage, alveolar bone or tibia. Furthermore, no DSP mRNA was observed in other soft tissues including heart, lung, kidney, intestine, eye, and muscle. In addition to the intense and prolonged expression by odontoblasts, DSP mRNA was transiently expressed by pre-ameloblasts in both developing molars and incisors. These observations are consistent with the results of previous immunohistochemical studies (1). The transient expression of DSP in pre-ameloblasts across from young odontoblasts suggests an involvement of DSP in epithelial-mesenchymal interactions that are crucial to later stages of tooth development.

Alveolar Process↗
PubMed1998

Effects of aFGF, bFGF, TGFbeta1 and IGF-I on odontoblast differentiation in vitro.

In this work, we investigated the effects of aFGF and bFGF alone or combined with TGFbeta1 or IGF-I on odontoblast differentiation. Trypsin-isolated dental papillae from day 17 mandibular first molar were cultured in semisolid-agar medium for 6 d. Our results demonstrated that aFGF, bFGF or combinations of these promoted cell polarization at the periphery of the dental papillae. Moreover, simultaneous addition of aFGF and TGFbeta1 to dental papillae cultures induced both polarization and functional differentiation of odontoblast-like cells, as well as extracellular matrix deposition. Combination of aFGF or bFGF with IGF-I caused cell polarization at the surface of dental papillae, but matrix secretion was restricted to a few explants. In the presence of bFGF and TGFbeta1, the explants had pronounced cell elongations but no matrix deposition. These results indicate that aFGF or bFGF is not able to induce odontoblast differentiation alone. However, both aFGF and bFGF can act synergistically with TGFbeta1 and IGF-I to strengthen their inductive effects and promote gradients of cytological and functional changes in odontoblast-like cells.

Animals↗
PubMed2006

Nestin expression in odontoblasts and odontogenic ectomesenchymal tissue of odontogenic tumours.

BACKGROUND: Nestin, one of the intermediate filaments constituting the cytoskeleton, is a marker of neural stem cells or progenitor cells. Its expression is also related to tooth development and repair of dentine. AIMS: The aim of this study was to investigate nestin expression in various odontogenic tumours and evaluate its usefulness for histopathological diagnosis. METHODS: We studied formalin fixed, paraffin embedded specimens from 129 cases of odontogenic tumours and 9 of mandibular intraosseous myxoma. After characterisation of odontogenic ectomesenchymal tissues in these tumours using antibodies to vimentin, desmin, neurofilament, and glial fibrillary acidic protein, we immunohistochemically examined nestin expression. RESULTS: No differentiation towards muscle and nervous tissues was found in the odontogenic ectomesenchymal tissues. Although almost all the ameloblastomas and malignant ameloblastomas were negative for nestin, odontogenic ectomesenchyme in the odontogenic mixed tumours demonstrated nestin immunolocalisation, particularly in the region adjacent to the odontogenic epithelium. Odontoblasts and their processes, pulp cells near the positive odontoblasts, and flat cells adhering to the dentine showed immunoreaction with nestin in the odontomas and odontoma-like component in the ameloblastic fibro-odontomas. Neoplastic cells in almost half cases of jaw myxoma and one case of odontogenic fibroma expressed nestin. CONCLUSIONS: The distribution of nestin in the odontogenic mixed tumours suggests that nestin expression in the odontogenic ectomesenchyme is upregulated by stimulation from odontogenic epithelium. In addition, nestin may also be involved in the differentiation from pulp cells to odontoblasts in odontogenic tumours. Therefore, nestin is a useful marker for the odontogenic ectomesenchyme and odontoblasts in odontogenic tumours. Nestin, one of the intermediate filaments constituting the cytoskeleton, is a marker of neural stem cells or progenitor cells. Its expression is also related to tooth development and repair of dentine.

Adolescent↗
PubMed1979

A fine-structural analysis of mouse molar odontoblast maturation.

The first mandibular molars of the Swiss albino mice, 1 through 4 days of age, were fixed in glutaraldehyde or Karnovsky's fixative. The tissues were postfixed in OSO4, dehydrated and embedded in Epon. The prepolarizing, polarizing and secretory odontoblasts were described. The prepolarizing cells, located in the vicinity of the cervical loop, were mesenchymal-like in morphology. The cells of the polarizing stage possessed organelles indicative of protein synthesis. The nucleus was located proximally. Aperiodic fibers were evident in the wide basement membrane. The secretory odontoblasts were long, slender, polarized cells closely adjoining one another. Each odontoblast possessed six morphologically discernible regions: (1) an infranuclear region, limited in size and containing few cellular organelles; (2) a nuclear region, housing the oval nucleus and a few associated lamellae of rough endoplasmic reticulum as well as a limited number of mitochondria; (3) a supranuclear rough endoplasmic reticulum region, possessing an abundance of these organelles as well as some mitochondria and secretory vesicles; (4) a Golgi region, occupying the middle third of the cell, housing the elements of an extensive Golgi apparatus which was surrounded by peripherally located profiles of rough endoplasmic reticulum; additionally, this region contained smooth endoplasmic reticulum, mitochondria, numerous secretory granules and vesicles and occasional intracellular collagen fibers; (5) an apical rough endoplasmic reticulum region, containing a rough endoplasmic reticulum component that was less extensive than its supranuclear counterpart; in addition, this region was the one richest in mitochondria and contained a plethora of secretory vesicles and granules; (6) the odontoblastic process, a region mostly void of organelles, containing various secretory products, some of which appeared to be in the process of being released extracellularly into the surrounding dentin matrix.

Animals↗
PubMed1985

Influence of fixative on the fine structure of mouse odontoblasts: a study on undemineralized tissue.

The present report describes techniques of fixation and embedding suitable for studying the fine structure of odontoblasts without demineralization. The quality of the procedures employed was verified by comparing the ultrastructural preservation of the odontoblasts prepared by simple fixation and by the double-fixation method. Simple fixation by immersion in osmium tetroxide in vacuum preserves the longitudinal arrangement of the rough endoplasmic reticulum and Golgi apparatus, showing various vesicles which often contain filamentous threads of weak electron density aligned in parallel at repeating intervals typical of odontoblastic cells. The results obtained with this method are compared to previous descriptions of the ultrastructure of odontoblasts.

Animals↗
PubMed2001

Crown morphology and pattern of odontoblast differentiation in lower molars of tabby mice.

The Tabby mutation leads to abnormal crown morphology in the developing molars. To identify cusps which were altered in number, size, and position in the first lower molars of mutant mice, we analyzed the patterning of odontoblast differentiation using morphological criteria on serial sections and 3D reconstructions. In wildtype mice, polarized and functional odontoblasts were first observed in the median L2 and B2 cusps, then in the distal cusps L3 and B3, and finally in L1, B1, and 4. In Tabby mice, terminal differentiation of odontoblasts was retarded by 24-36 hours compared with wild-type mice. Polarized odontoblasts first appeared in the most mesial part of the tooth and progressively extended distally. The mesial part of the M1 in Tabby fetuses may correspond to the L2, B2 area from wild-type mice. The ante-molar dental primordium observed in some samples would thus represent remnants of cusps L1 and B1.

Animals↗
PubMed1981

Behavior of odontoblasts and basal lamina of trypsin or EDTA-isolated mouse dental papillae in short-term culture.

Embryonic mouse first mandibular molars (day 18), containing the first overtly differentiated odontoblasts, were treated with EDTA or trypsin, to obtain isolated dental papillae. Trypsin dissociation was accompanied by disappearance of the basal lamina. EDTA-treatment removed the basal lamina from the basal surface of the epithelium, but left it associated with the dental papillae. EDTA- or trypsin-isolated dental papillae were grown for three to 24 h at the top of a plasma clot. Trypsin-isolated dental papillae rapidly lost their typical morphology, and the post-mitotic odontoblasts never became functional. EDTA-isolated dental papillae remained covered by the basal lamina for 15 to 18 h. During this period, the typical morphology was maintained, and post-mitotic odontoblasts secreted predentin. Preodontoblasts and odontoblasts went through the basal lamina and migrated at the outer surface of the basal lamina (i.e., at the side facing away from the enamel organ).

Animals↗
PubMed1989

Demonstration of physiological barrier between pulpal odontoblasts and its perturbation following routine restorative procedures: a horseradish peroxidase tracing study in the rat.

Vascular injection of the macromolecular tracer, horseradish peroxidase (HRP), was used to study the permeability of the odontoblast cell layer in developing and mature rat molar teeth, and to investigate the effect of cavity preparations on the permeability of this epithelioid cell layer in adult animals. HRP injected into the vascular system of normal animals 28 days of age and older was localized histochemically (from 5 to 90 min after injection) throughout the extracellular spaces of the maxillary dental pulps; however, the tracer did not penetrate beyond the tight junctions at the apical region of the odontoblast cell layer, and was absent from the predentin and dentin. In contrast, HRP injected into very young neonatal animals (e.g., day 3) resulted in free passage of HRP between odontoblasts and into the overlying predentin and dentin. When Class V cavities had been prepared in adult maxillary molars after HRP was injected into the blood stream, HRP reaction product penetrated the predentin and dentin immediately beneath the cavity preparation; however, adjacent, untraumatized areas of predentin and dentin in the operated teeth were devoid of reaction product. These results provide evidence that: (1) a physiological barrier develops between the distal segments of odontoblast cell bodies in normal rat molar teeth between days 15 and 28 of postnatal life, and this barrier prevents the passage of macromolecules from the pulp into the predentin and dentin; and (2) this barrier is perturbed following routine restorative procedures in adult animals.

Ameloblasts↗
PubMed2001

Utilization of MO6-G3 immortalized odontoblast cells in studies regarding dentinogenesis.

Tooth formation is the result of reciprocal instructive interactions between oral epithelium and cranial neural-crest-derived ectomesenchymal tissues. These interactions lead to the cytodifferentiation of highly specialized matrix-forming cell types, the ameloblast, odontoblast, and cementoblast, that produce the mineralized tissues enamel, dentin, and cementum, respectively. Our laboratory has been developing immortalized dental cell lines representative of these various cell types to facilitate studies on gene regulation, cell differentiation, matrix formation, and mineralization. Odontoblasts are solely responsible for the synthesis and secretion of the dentin extracellular matrix bilayer that consists of non-mineralized predentin and mineralized dentin. The mouse immortalized MO6-G3 cell line expresses the major matrix proteins associated with the odontoblast phenotype, producing a matrix that is capable of mineralization. This cell line serves as a useful tool in studies designed to explore the various processes of dentinogenesis. In this paper, we present studies using the mouse odontoblast cell line MO6-G3 as examples of the various research applications. Studies highlighted are: in vitro promoter studies investigating the tooth-specific gene regulation of the major non-collagenous dentin matrix protein, dentin sialophosphoprotein; regulation of tertiary dentin formation by cytokines, such as transforming growth factor-Beta 1; and the utilization of dentally relevant cells in dental material biocompatibility testing.

Ameloblasts↗
PubMed1984

The odontoblast process extends to the dentinoenamel junction: an immunocytochemical study of rat dentine.

The length and extent of the odontoblast cell process in dentine has been the subject of controversy for many years. Here an immunofluorescence technique has been applied at the light microscope level to rat coronal dentine to localize the intracellular components actin and tubulin. Adult rats were perfused with periodate-lysine-paraformaldehyde fixative, teeth were extracted, the molar crowns were demineralized, dehydrated, wax embedded, and 6 micron sections were prepared. The sections were postfixed in -20 degrees C acetone and then incubated with affinity-purified rabbit anti-actin or anti-tubulin antibodies, followed by fluorescein-conjugated goat anti-rabbit immunoglobulin. Intratubular immunofluorescence labeling for tubulin extended to the dentinoenamel junction, whereas labeling for actin, although extending to the dentinoenamel junction, was more prominent in the pulpal third of the rat dentine. Areas in which odontoblast processes are known not to occur, i.e., the atubular dentine, were not labeled by either antibody. The presence of actin- and tubulin-containing structures extending to the dentinoenamel junction is consistent with the hypothesis that the odontoblast process traverses the dentine for up to 3-4 mm, all the way to the dentinoenamel junction. Furthermore, the different staining patterns for actin-containing microfilaments as compared to tubulin-containing microtubules suggest that these two filamentous systems may have different roles in the function of the odontoblast process.

Actins↗
PubMed2002

Phosphate and calcium uptake by rat odontoblast-like MRPC-1 cells concomitant with mineralization.

It has been suggested that odontoblasts are instrumental in translocating Ca2+ and inorganic phosphate (Pi) ions during the mineralization of dentin. The aim of this study was to characterize cellular Pi and Ca2+ uptake in the novel rat odontoblast-like cell line mineralizing rat pulpal cell line (MRPC) 1 during mineralization to see if changes in the ion transport activity would occur as the cultures develop and begin forming a mineralized matrix. MRPC-1 cells were cultured in chemically defined medium containing ascorbate and Pi, and cultures were specifically analyzed for cellular P, and Ca2+ uptake activities and expression of type II high-capacity Na+-Pi cotransporters. The odontoblast-like phenotype of the cell line was ascertained by monitoring the expression of collagen type I and dentin phosphopoprotein (DPP). Mineralized nodule formation started at day 9 after confluency and then rapidly increased. Ca2+ uptake by the cells showed a maximum during the end of the proliferative phase (days 5-7). Pi uptake declined to a basal level during proliferation and then was up-regulated simultaneously with the onset of mineralization to a level fourfold of the basal uptake, suggesting an initiating and regulatory role for cellular Pi uptake in mineral formation. This up-regulation coincided with a conspicuously increased glycosylation of NaPi-2a, indicating an activation of this Na+-Pi cotransporter. The study showed that MRPC-1 cells express an odontoblast-like phenotype already at the onset of culture, but that to mineralize the collagenous extracellular matrix (ECM) that formed, a further differentiation involving their ion transporters is necessary.

Animals↗
PubMed1994

The relationship between odontoblasts and immunocompetent cells during dentinogenesis in rat incisors: an immunohistochemical study using OX6-monoclonal antibody.

The relationship between odontoblasts and class II major histocompatibility complex (MHC) antigen-expressing cells in the process of dentinogenesis was studied in rat lower incisors, employing immunohistochemistry using OX6-monoclonal antibody. The dental pulp contained numerous OX6-immunopositive cells that varied in morphology from dendritic to spindle under physiological conditions. Under the electron microscope, these immunopositive cells shared common cytoplasmic features, i.e., multivesicular bodies and characteristic fine tubulovesicular structures in their cytoplasm. At the early stage of dentinogenesis, OX6-immunopositive cells, presumably of the immature type, were located in the subodontoblastic layer. During active dentin formation, the OX6-immunopositive cells increased in number and appeared in the odontoblast layer, associating intimately with fenestrated capillaries situated close to the predentin. These cells showed a dendritic appearance and possessed various sizes of multivesicular bodies and characteristic fine tubulovesicular structures, but never contained typical phagosomes. On the other hand, immunopositive macrophages characterized by typical phagosomes tended to occupy the central portion of the pulp. The results suggest that most, if not all, OX6-immunopositive cells situated deep in the odontoblast layer are dendritic cells playing a role in the defense system of the dental pulp against antigenic molecules arriving from the circulation via the fenestrated capillaries. The increasing number of OX6-immunopositive or immunonegative macrophages appearing near the incisal end of the tooth is thought to be involved in the elimination of degenerated odontoblasts.

Animals↗
PubMed1995

In situ hybridization shows Dmp1 (AG1) to be a developmentally regulated dentin-specific protein produced by mature odontoblasts.

Acidic phosphorylated proteins are prominent constituents of the extracellular matrix of bone and dentin. It has been postulated that they may have important structural and regulatory roles in the process of tissue mineralization. Studies of a cDNA library, prepared from cells of the rat incisor odontoblast-pulp complex of 3 week old Sprague-Dawley rats, led to the identification of a serine-rich acidic protein, designated AG1, which appeared to be a dentin matrix component. In order to determine which cells of the odontoblast-pulp complex were responsible for the making of AG1, in situ hybridization was carried out using digoxigenin-labeled probes. The full length AG1 cDNA was subcloned into the pBluescript vector, which contains two strong promoters, T3 and T7. The sense and antisense complementary RNA (cRNA) hybridization probes were prepared by in vitro transcription using T3 and T7 polymerases in the presence of 11-dUTP. Incisor sections were obtained from rat embryos at days 16, and 20, and newborns at days 2 and 5. No AG1 mRNA was detected in the embryonic sections, but digoxigenin labeling was evident in odontoblasts secreting mineralizing dentin at postnatal days 2 and 5. Sense probes showed no hybridization. Pulp cells, Meckel's cartilage, and alveolar bone were free of hybridization with the antisense probe. Unexpectedly, a low level of digoxigenin staining was seen in the cytoplasm of secretory ameloblasts, but not in the preameloblasts, stratum intermedium or stellate reticulum of the enamel organ. These data show that AG1 expression is regulated developmentally and is restricted to secretory stage mature odontoblasts.

Alveolar Process↗
PubMed1995

Identification of regulatory elements necessary for the expression of the COL1A1 promoter in murine odontoblasts.

Recent studies have indicated that odontoblasts and osteoblasts have unique regulatory mechanisms that control COL1A1 gene expression. We are currently examining the regulation of COL1A1 gene expression in odontoblasts and have produced transgenic mice containing various collagen promoter constructs fused to the indicator gene, chloramphenicol acetyl transferase (CAT). Mandibular first molars were removed from jaws of transgenic mice. Some teeth were assayed for CAT activity (CAT diffusion assays), others were fixed and prepared for immunohistochemistry (CAT antibodies). Our results indicate the CAT activity was present in tooth germs containing promoter constructs longer than 1.719 kb. Immunoreactivity to CAT was confined to the odontoblast cell layer. No CAT activity was present in tooth germs containing a 1.670 kb construct. These data suggest that there are important regulatory elements located between -1.719 kb and -1.670 kb on the collagen promoter in odontoblasts. Examination of sequences in this region of the promoter demonstrates consensus with those known to be involved with binding of translation products of homeobox genes.

Animals↗
PubMed2003

Tooth morphogenesis and pattern of odontoblast differentiation.

The terminal differentiation of odontoblasts is controlled by the inner dental epithelium (IDE) and occurs according to a tooth-specific pattern. It requires temporospatially regulated epigenetic signaling and the expression of specific competence. The patterning of cusp formation was compared with that of odontoblast differentiation in the first lower molar in mice. Histology, immunostaining, and three dimensional reconstructions were completed by experimental approaches in vitro. The mesenchyme controls the pattern of cusp formation. During the cap-bell transition in the molar, a subpopulation of nondividing IDE cells from the enamel knot (EK) undergo a tooth-specific segregation in as many subpopulations as cusps will form. Epithelial cell-basement membrane interactions seem to be involved in the segregation of EK cells. The timing and spatial pattern of the segregation of EK cells correlate with cusps formation. However, the temporal pattern of odontoblast terminal differentiation is different. This discrepancy might result from cusp-specific differences either in the timing of the initiation of odontoblast terminal differentiation and/or in cell proliferation kinetics.

Animals↗
PubMed1992

[Cell-matrix interactions and odontoblast differentiation].

The terminal differentiation of odontoblasts requires the integrity of the cytoskeleton and is controlled by cell-matrix interactions. These interactions implicate both matrix molecules and matrix-associated growth factors. On the one hand, predentin-dentin constituents were found to initiate odontoblast differentiation and to allow the maintenance of this state; TGF-beta or related molecules are implicated. Fibronectin on the other hand can induce the differentiation of second generation odontoblasts and interacts with three high molecular weight proteins present in membrane prepared from dental mesenchymal cells. One of these proteins (165 kDa) was localized on the surface of odontoblasts and is involved in the organization of microfilaments. Two main axes of research will have to be developed in the future in order to understand how matrix molecules and growth factors interactions can be modulated in time and space by epithelial and mesenchymal cells, and how such modulations can affect the phenotype of these cells.

Cell Differentiation↗