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PubMed1987

The CaATPase activity of rat-incisor odontoblast vesicles.

An homogenate of rat incisor odontoblasts had Ca2+ and Mg2+ ATPase activity and suitable storage conditions kept it stable for several days. Over 90 per cent of the activity was retained in a vesicle-rich microsomal fraction that removed about 85 per cent of the total material from the homogenate. This fraction was further characterized: the resolved Ca2+-activated ATPase activity, above the basal MgATPase activity, was 0.30 mumol Pi/min-mg total protein, and 50 per cent activated at free [Ca2+] equal 0.8 microM. This calcium dependency is consistent with an intracellular Ca2+-regulated enzymatic activity. The calcium ionophore, A23187, had no measurable effect on the CaATPase activity, which suggests that the odontoblast vesicles do not concentrate Ca2+ in a lipid bilayer compartment. Direct measurement of the uptake of 45Ca2+ by the filtration method and parallel measurements of CaATPase activity on the same preparations under identical conditions indicated that the odontoblast-derived vesicles have a coupling ratio of 0.024 Ca2+/ATP. This low coupling ratio and the lack of detectable compartmentalization of calcium indicate that the CaATPase activity of the odontoblast microsomes is not associated with a calcium pump. The [Ca2+] dependence of the activity suggests the CaATPase is under intracellular Ca2+ control, but its function is unknown.

Animals↗
PubMed1994

Immunohistochemical localization of L-type calcium channels in the developing first molar of the rat during odontoblast differentiation.

To study the presence of L-type calcium channels during the different steps of odontoblast differentiation, a specific monoclonal antibody against 1,4-dihydropyridine receptors was used in combination with avidin-biotin-peroxidase complex labelling. Staining was seen on the cell bodies of pre-odontoblasts, concentrated at the apical pole (distal portion) of functional odontoblasts and localized on cell bodies and processes of mature odontoblasts. Thus calcium channels were expressed at the onset of differentiation and maintained in the differentiated cells but with some changes of localization. It is suggested that these channels may facilitate the entry of calcium to act as a second messenger for cellular polarization or be involved in the transcellular transport of calcium to the mineralizing front.

Animals↗
PubMed2005

Activation of human dental pulp progenitor/stem cells in response to odontoblast injury.

In restorative dentistry, whilst moderate carious lesion treatment does not significantly compromise odontoblast cell survival, deep cavity preparation may lead to a partial death of these cells. However, newly formed odontoblast-like cells can replace the necrotic odontoblasts and secrete a reparative dentine matrix. Although several lines of evidence strongly suggest the presence of resting progenitor or stem cells in the dental pulp, little is known about the activation and migration of these cells in response to injury. Human immature third molars extracted for orthodontic reasons were used in this work to study the activation of progenitor/stem cells and their migration after deep cavity preparation involving in pulpal exposure using 5-bromo-2'-deoxyuridine labelling (BrdU). After incubation for 1 day, the BrdU was localised to the nuclei of cells in the perivascular area. The BrdU-immunolabelling exhibited a gradient. It was strong in the blood vessels surrounding the pulpal cavity and decreased in those away from the cavity. After incubation for 2 weeks, labelled cells were seen in the vicinity of the cavity. At 4 weeks, the immunolabelling was localised to the cavity area only. Control teeth without cavities or with shallow dentine cavities did not show any perivascular labelling after culture. These results clearly demonstrate that perivascular progenitor/stem cells can proliferate in response to odontoblast injury. They also show that these proliferating cells can migrate to the pulpal injury site in their tissue of origin simulating the situation in vivo.

Biomarkers↗
PubMed2005

Smad protein mediated transforming growth factor beta1 induction of apoptosis in the MDPC-23 odontoblast-like cell line.

OBJECTIVE: The function of apoptosis and its regulation in odontoblasts remain unclear. In this study, we characterize the possible role of transforming growth factor (TGF)-beta 1 in the induction of apoptosis and the molecular mechanisms that mediate TGF-beta1-induced apoptosis in odontoblasts. METHODS: Annexin V/propidium iodide staining, cell Death Detection ELISA and DNA ladder were used to examine the effect of TGF-beta1 on apoptosis in a mouse odontoblast-like cell line, MDPC-23. Stable cell clones expressing Smad2 or Smad3 dominant negative mutants, or wild-type Smad7 were constructed to investigate the role of Smad proteins in the mediation of apoptosis by TGF-beta1 in MDPC-23 cells. The TGF-beta1-induced transcriptional activity in stable cell clones expressing Smad proteins was analyzed by a transient transfected TGF-beta-responsive reporter gene, p3TP-Lux. RESULTS: TGF-beta1 can induce apoptotic cell death in MDPC-23 cells in a dose-dependent manner. Transfection of dominant negative mutant forms of Smad2 or Smad3 blocked TGF-beta1-induced apoptosis; moreover, the Smad3 mutant was more efficient than the Smad2 mutant. Transfection of Smad7, an inhibitory Smad, also significantly inhibited TGF-beta1-induced apoptosis of these cells. Over-expression of Smad3 dominant negative mutant or Smad7 significantly inhibited TGF-beta1-induced transcriptional activity. CONCLUSION: These results suggest that Smad proteins are involved in TGF-beta1-induced apoptosis of odontoblast cells.

Animals↗
PubMed2004

Odontoblasts: the cells forming and maintaining dentine.

Odontoblasts are tall columnar cells located at the periphery of the dental pulp. They derive from ectomesenchymal cells originated by migration of neural crest cells during the early craniofacial development. Odontoblasts form the dentine, a collagen-based mineralized tissue, through secretion of its collagenous and noncollagenous organic matrix components and by control the mineralization process. A conspicuous cell process arises from the cell body of odontoblasts and penetrates into the mineralized dentine. After dentinogenesis, odontoblasts deposit new layers of dentine throughout life and might also form a type of reactionary/reparative dentine in response to dental caries and other external factors may affect teeth.

Dentin↗
PubMed2004

Odontoblast primary cilia: facts and hypotheses.

Odontoblasts, the cells responsible for the dentine formation, are organized as a single layer of highly polarized and differentiated post-mitotic cells along the interface between the dental pulp and the mineralized tubules. They lay down the physiological secondary dentine throughout the life of the teeth. Odontoblasts play a central role in the transportation of calcium to the dentine and they possibly mediate early stages of sensory processing in teeth. A primary cilium, 9+0 configuration, have been regularly identified in a supra nuclear location. Calbindin D28k has been detected at the base of the cilium membrane. The cilium structure was positive with detyrosinated alpha tubulin antibodies in vivo and in cultured human odontoblasts. Transcripts of tektin, a protein involved in ciliogenesis, were expressed in vitro. The putative role of the primary cilium constituting a critical link between external teeth stimuli and odontoblast responses is extensively discussed.

Calbindin 1↗
PubMed2006

Formation of odontoblast-like cells from cultured human dental pulp cells on dentin in vitro.

Recent characterization of human dental pulp stem cells has shed new light on the understanding of the odontoblastic lineage. The purpose of the study was to characterize human adult dental pulp cells isolated and cultured in vitro and to examine the cell differentiation potential grown on dentin. We observed that some pulp cells isolated with an enzyme-digestion approach proliferated at a similar rate as the immortal cell line NIH 3T3. Population doubling time (PDt) for pulp cells at passage 3 was 22.6 +/- 0.5 hours and for NIH 3T3 was 23.1 +/- 2.3 hours. The pulp cells formed mineral nodules stimulated with dexamethasone or dexamethasone plus 1,25-dihydroxyvitamin D3. Pulp cells, after being seeded onto mechanically and chemically treated dentin surface, appeared to establish an odontoblast-like morphology with a cytoplasmic process extending into a dentinal tubule revealed by scanning electron microscopy analysis. Our data demonstrated the formation of cells with odontoblastic morphologies on existing dentin, suggesting that isolated human pulp stem cells may differentiate into odontoblasts on dentin in vitro.

3T3 Cells↗
PubMed2004

Site-specific effect of ascorbic acid deficiency on the structure and function of odontoblasts in the teeth of osteogenic disorder rat in vivo.

The influence of chronic L-ascorbic acid (AsA) deficiency on dentinogenesis was examined in Osteogenic Disorder Shionogi (ODS) rat, which bear inborn lack of L-gulonolactone oxidase. Weanling male rats were kept on AsA-free diet for 4 weeks until all suffered from scurvy. Control rats were given AsA in drinking water. The dentin of molars and incisors of the scorbutic rats was thinner than that in control, except for the crown-analogue (enamel-related) of incisors. Predentin in scorbutic molars showed irregular thickness, and was almost lacking in roots. In the root-analogue (cementum-related) region of scorbutic incisors, dentin displayed metachromatic incremental lines, and the thickened predentin contained collagen fibrils of irregular diameter. The odontoblasts facing the affected regions contained dilated rough endoplasmic reticulum cisternae. In the crown-analogue of scorbutic incisors, however, dentin, predentin, and odontoblasts were comparable to those of controls. These data indicate that AsA deficiency differentially affects the synthetic and/or secretory activity of odontoblasts in ODS rat teeth in a site-specific manner. The regional differences implicate the presence of putative local factor(s) in the crown-analogue of incisors that might have compensated for AsA deficiency. The odontoblasts in the crown-analogue of incisors may have different requirements for AsA from those in molars and the root-analogue of incisors.

Animals↗
PubMed2006

Emdogain-gel stimulates proliferation of odontoblasts and osteoblasts.

OBJECTIVE: The purpose of this study was to determine whether a premixed form of enamel matrix derivative (EMD), Emdogain-gel, has the same property as the original formula of EMD in stimulating the proliferation of osteoblasts and odontoblasts. STUDY DESIGN: Osteoblast cell line (MC3T3) and odontoblast cell line (MDPC) were cultured in the 6-well culture plates and treated in 4 different groups: (1) culture medium control, (2) 100 microg/mL Emdogain-gel directly added to the culture medium, (3) culture medium with a culture plate insert, and (4) 100 microg/mL Emdogain-gel added onto a culture plate insert. The culture plate insert prevented direct contact between Emdogain-gel and the cells. After 3-day incubation, cell morphology was examined and the total cell number per well was counted. Data were analyzed using 1-way ANOVA. RESULTS: Emdogain-gel significantly increased cell number of both osteoblasts and odontoblasts regardless the presence of the culture plate insert. CONCLUSION: Emdogain-gel stimulates cell proliferation of odontoblasts and osteoblasts. The direct contact between Emdogain-gel and cells is not required. Heat treatment of EMD and premix with propylene glycol alginate did not change its property of releasing bioactive molecules for promoting cell proliferation.

3T3 Cells↗
PubMed1987

Regulation of free Ca2+ by subcellular fractions of rat incisor odontoblasts.

A miniaturized Ca2+ electrode system was developed to monitor small and fast fluctuations of Ca2+ activity in the micromolar range in 100 microliters volumes. This was used to study Ca2+ influx/efflux cycling in suspensions of rat-odontoblast and liver-cell mitochondria and microsomes, as well as in whole odontoblasts with plasma membranes made permeable by digitonin. The steady-state free-Ca2+ activity maintained by mitochondria was pCa 6.2-6.4, and that of microsomes pCa 6.4-6.6. These levels were held upon repeated additions of Ca2+ and EGTA. The odontoblast mitochondria and microsomes had an intracellular Ca2+ buffering capacity similar to that of liver cells. The steady-state pCa level maintained in suspensions of digitonin-permeabilized whole odontoblasts was 6.4-6.6. Thus, this study gave no evidence for any specialized intracellular handling of Ca2+ in cells involved in mineralization.

Animals↗
PubMed2000

Localization of the e-NOS enzyme in endothelial cells and odontoblasts of healthy human dental pulp.

Nitric oxide synthases (NOS) are important enzymes present in different cells such as endothelial cells, macrophages, etc. Recently, it has been found that nitric oxide (NO) is responsible for vasodilation, blood pressure regulation, platelet aggregation, cardiac contractility, and the mediation of immunity during bacterial infections and inflammation. However, the production and role of NO in various structures of the oral cavity have not been investigated extensively. The aim of this study was to evaluate the presence of e-NOS in healthy human odontoblasts and endothelial cells of the dental pulp. Twenty healthy human dental pulps were collected and frozen and pulp slices were obtained using a cryostat. The e-NOS enzyme was revealed by immunohistochemical analysis and the enzyme level was detected by Western blotting and mRNA expression by RT-PCR. The immunohistochemical results demonstrated, for the first time, the presence of e-NOS in odontoblasts and in endothelial cells. The presence of e-NOS m-RNA was confirmed by RT-PCR and the expression of the protein by Western blotting. These results clearly show that the e-NOS enzyme is present in both odontoblasts and endothelial cells of healthy human pulp. The presence of e-NOS in the odontoblast and endothelial cells of the dental pulp may mediate local vasodilation and cell proliferation.

Blotting, Western↗
PubMed1993

Transient expression of type III collagen by odontoblasts: developmental changes in the distribution of pro-alpha 1(III) and pro-alpha 1(I) collagen mRNAs in dental tissues.

The expression of pro-alpha 1(III) and pro-alpha 1(I) collagen mRNAs in mouse and human dental tissues during tooth development and after its completion was analyzed by in situ hybridization, with use of [35S]-labeled RNA probes. The expression of pro-alpha 1(III) mRNA was also compared to that of the protein product, as localized by immunostaining with polyclonal antibodies to type III collagen and the N-terminal propeptide of type III procollagen. Contrary to many previous reports, our results suggest that odontoblasts express type III collagen. While pro-alpha 1(III) transcripts were less intensely expressed in odontoblasts than pro-alpha 1(I) transcripts, the amounts of both mRNAs increased in odontoblasts with progressing dentin formation, and decreased toward its completion. In contrast to pro-alpha 1(III) mRNA, pro-alpha 1(I) mRNA was still detectable in odontoblasts of fully developed teeth. Type III collagen immunoreactivity was observed in the early predentin, and again in predentin toward the completion of dentinogenesis, when mRNA was no longer detected. Also in the pulp, the protein product, unlike pro-alpha 1(III) mRNA, was relatively strongly expressed. Hence, these immunostaining patterns were inversely related to the expression of pro-alpha 1(III) mRNA, suggesting accumulation of the protein. The mesenchymal cells, when condensed in the region of the future mandibular bone, expressed pro-alpha 1(III) mRNA intensely, whereas osteoblasts expressed pro-alpha 1(I) but not pro-alpha 1(III) transcripts strongly. Cell type- and developmental stage-related differences in the expression of the two mRNAs suggest that type I/type III collagen ratio influences the structure of dental tissues.

Aging↗
PubMed1999

TGF-beta receptor expression in human odontoblasts and pulpal cells.

Transforming growth factor-beta (TGF-beta) isoform expression by odontoblasts leads to their sequestration within the dentine matrix, from where they may be released during caries and participate in the reparative processes. Two receptor types for TGF-beta have been implicated in TGF-beta induced signalling. The aim of this study was to characterise immunohistochemically the expression of these receptors in sound and carious human teeth to facilitate our understanding of the ability of these cells to respond to TGF-beta stimulation. Sound and carious human teeth were routinely processed and paraffin sections stained for TGF-beta receptors I and II, using the StrAviGen immunoperoxidase method. Strong specific staining for both receptor types was observed in the odontoblasts of healthy teeth with the greatest intensity seen with receptor I. Staining of weaker intensity was also observed for both receptors in the underlying cell rich area and pulp core. Similar patterns of staining were observed within carious tissues. We conclude that odontoblasts and other cells of the pulp of mature human molar teeth show the presence of both TGF-beta receptors I and II in health and disease with odontoblasts showing the strongest expression. Such findings may be important in the response of these cells to tissue injury.

Activin Receptors, Type I↗
PubMed1999

Effects of alternating and direct electrical current application on the odontoblastic layer in human teeth: an in vitro study.

AIM: The aim of this study was to investigate the influence of a low intensity alternating current on the odontoblasts and odontoblast layer and compare this with the effects of a direct current. METHODOLOGY: Teeth extracted for orthodontic reasons were immersed in physiological saline stabilized with thymol crystals. Within 1 h of extraction, an alternating or direct current was applied on the crown in the direction of the apex of the tooth for 120-360 s. The current doses were 12, 30, 60, 600, 1800, 3600, 7200, 24,000 and 144,000 microC. The teeth were fixed in Bouin or Baker fluids, the pulps removed, dehydrated and immersed in paraffin, then sectioned, stained with haematoxylin and eosin, and studied under a light microscope. RESULTS: Neither direct nor alternating current, similar to that applied in electrical caries diagnosis caused histological changes in the odontoblasts. CONCLUSIONS: There was no difference between direct and low intensity alternating current in the response of the odontoblast.

Adolescent↗
PubMed2003

Laminin alpha2 is essential for odontoblast differentiation regulating dentin sialoprotein expression.

Laminin alpha2 is subunit of laminin-2 (alpha2beta1gamma1), which is a major component of the muscle basement membrane. Although the laminin alpha2 chain is expressed in the early stage of dental mesenchyme development and localized in the tooth germ basement membrane, its expression pattern in the late stage of tooth germ development and molecular roles are not clearly understood. We analyzed the role of laminin alpha2 in tooth development by using targeted mice with a disrupted lama2 gene. Laminin alpha2 is expressed in dental mesenchymal cells, especially in odontoblasts and during the maturation stage of ameloblasts, but not in the pre-secretory or secretory stages of ameloblasts. Lama2 mutant mice have thin dentin and a widely opened dentinal tube, as compared with wild-type and heterozygote mice, which is similar to the phenotype of dentinogenesis imperfecta. During dentin formation, the expression of dentin sialoprotein, a marker of odontoblast differentiation, was found to be decreased in odontoblasts from mutant mice. Furthermore, in primary cultures of dental mesenchymal cells, dentin matrix protein, and dentin sialophosphoprotein, mRNA expression was increased in laminin-2 coated dishes but not in those coated with other matrices, fibronectin, or type I collagen. Our results suggest that laminin alpha2 is essential for odontoblast differentiation and regulates the expression of dentin matrix proteins.

Animals↗
PubMed1974

Synthesis, migration, and release of precursor collagen by odontoblasts as visualized by radioautography after (3H)proline administration.

The elaboration of dentin collagen precursors by the odontoblasts in the incisor teeth of 30-40-g rats was investigated by electron microscopy, histochemistry, and radioautography after intravenous injection of tritium-labeled proline. At 2 min after injection, when the labeling of blood proline was high, radioactivity was restricted to the rough endoplasmic reticulum, indicating that it is the site of synthesis of the polypeptide precursors of collagen, the pro-alpha chains. At 10 min, when the labeling of blood proline had already declined, radioactivity was observed in spherical portions of Golgi saccules containing entangled threads, and, at 20 min, radioactivity appeared in cylindrical portions containing aggregates of parallel threads. The parallel threads measured 280-350 nm in length and stained with the low pH-phosphotungstic acid technique for carbohydrate and with the silver methenamine technique for aldehydes (as did extracellular collagen fibrils). The passage of label from spherical to cylindrical Golgi portions is associated with the reorganization of entangled into parallel threads, which is interpreted as the packing of procollagen molecules. Between 20 and 30 min, prosecretory and secretory granules respectively became labeled. These results indicate that the cylindrical portions of Golgi saccules transform into prosecretory and subsequently into secretory granules. Within these granules, the parallel threads, believed to be procollagen molecules, are transported to the odontoblast process. At 90 min and 4 h after injection, label was present in predentin, indicating that the labeled content of secretory granules had been released into predentin. This occurred by exocytosis as evidenced by the presence of secretory granules in fusion with the plasmalemma of the odontoblast process. It is proposed that pro-alpha chains give rise to procollagen molecules which assemble into parallel aggregates in the Golgi apparatus. Procollagen molecules are then transported within secretory granules to the odontoblast process and released by exocytosis. In predentin procollagen molecules would give rise to tropocollagen molecules, which would then polymerize into collagen fibrils.

Animals↗
PubMed2006

Ultrastructural and immunocytochemical characterization of immortalized odontoblast MO6-G3.

AIM: To investigate an immortalized murine odontoblast cell line as a potential alternative for experimental studies on dentinogenesis. METHODOLOGY: The MO6-G3 cell line was investigated morphologically over 3, 7, 11 and 42 days of culture, using histochemical localization of dentine sialoprotein (DSP), alkaline phosphatase (AP), type I collagen and actin filaments, histoenzymatic staining and biochemical investigation of AP and finally, transmission and scanning electron microscopy. RESULTS: Scanning electron micrographs showed elongated cells. Accordingly, a polarized organization of odontoblasts was observed by transmission electron microscopy, identifying distinct subcellular compartments as described in vivo. The secretion apparatus, which includes cisternae of rough endoplasmic reticulum, Golgi apparatus saccules and secretion vesicles and granules, was longitudinally organized in the supranuclear compartment ending distally in the secretory pole. A cellular process was observed. The investigation of the cytoskeleton network revealed that actin microfilaments were organized in parallel stress fibre oriented depending on the longitudinal axis of the cytoplasm. Immunofluorescent labelling showed a continuous expression of type I collagen, DSP and AP. A unipolar distribution characterized intracellular DSP immunoreactivity. Histoenzymology revealed AP active sites increasing from 3 to 11 days albeit with a moderate level of activity comparatively to the in vivo situation in dental cells. CONCLUSION: This cell line MO6-G3 not only showed the criteria of odontoblast phenotype as previously reported but also the characteristic morphodifferentiation pattern of polarized odontoblasts at the cellular level but with an apparent random distribution.

Actin Cytoskeleton↗
PubMed1988

Na+/Ca2+ antiports in membranes of rat incisor odontoblasts.

When incubated in the presence of Na+ ions, dissected rat incisor odontoblasts extruded Ca2+ ions, as detected by tetracycline fluorimetry. Also odontoblast mitochondria, obtained by subcellular fractionation and monitored by ion-specific mini-electrode technique, were found to extrude Ca2+ upon addition of Na+ ions. These findings demonstrate the presence of Na+/Ca2+ antiports in the odontoblast plasma membrane as well as in the odontoblast mitochondrial inner membrane. In addition, evidence was found for an ATP-dependent Ca2+ ion extrusion across the plasma membrane in these cells.

Adenosine Triphosphate↗