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Odontoblast response under carious lesions.

The local regulation of odontoblast response to caries is viewed through initiation and elaboration of sclerotic as well as reparative dentin. Dentin tissue represents a multiple source of potent environment factors when teeth are affected by the demineralization phases of carious process. Some of them have already been identified in sound tissue (matrix glycoproteins, proteoglycans, growth factors, Bone Morphogenetic Protein) and may act on the cell through membrane receptors. Thus, the amplification in collagen synthesis and alkaline phosphatase activity previously observed during sclerotic dentin deposition can be related to the interaction between matrix signals and cell receptors such as the 165 kDa protein shown only by odontoblasts under the affected zone. Similarly, under established lesions generating cell death, the specific matrix made of odontoblasts debris and damage tissues, probably rich in active molecules, may trigger pulp cells to elaborate a cartilage-like layer (identified by type II and XI collagen) followed by odontoblast-like cells to give rise to abnormal tubular dentin. Here, odontoblast response is identical to bone-cells response to injury. What remains to be elucidated concern: The nature of signals found in carious dentin (matrix components, growth factors, bacterial products). The nature and regulation of expression of cell membrane receptors during tooth repair. How the odontoblast produces specific responses to each of these signaling molecules will be the focus of important new investigations.

Cell Differentiation↗

Odontoblast alkaline phosphatases and Ca2+ transport.

The same isoenzyme of nonspecific alkaline phosphatase (APase), assayed with p-nitrophenylphosphate (p-NPP), was shown be present in different calcifying tissues, bone, calcifying cartilage, odontoblasts and enamel organ. Indications were also found that the enzymatic degradation of inorganic pyrophosphate (PPi) in calcifying tissues is mediated by APase. By using specific APase inhibitors, it was shown that two enzymes capable of degrading ATP exist. These were characterized in dentinogenically active odontoblasts, and it was concluded that one is the classical APase, the other is a Ca2+ and Mg2+ activated ATPase, named Ca2+-ATPase. The two phosphatases were solubilized from odontoblasts and separated. The localization of APase and Ca2+-ATPase in odontoblasts was investigated by subcellular fractionation and EM histochemistry. Routine methods for fixation were found to almost completely inactivate the enzymes. By using a mild fixation technique that preserved 80% of the enzyme activity, the main localization for both APase and Ca2+-ATPase was found to be in the membranes of intercellular vesicles located in the cell body and odontoblasts process. No activity was found in the cell membranes. It is concluded that there are at least two enzymes able to degrade phosphate compounds at alkaline pH in hard tissue forming cells. One is the nonspecific alkaline phosphatase (APase; EC 3. 1. 3. 1), which is active against p-NPP, PPi, glycerophosphates and ATP among other substrates. The other is a more specific Ca2+-ATPase (EC 3. 6. 1. 3). There seems to be an intimate relation between these two enzymes in the tissue. The function of APase in biological calcification is still obscure. In contrast, the finding of an ATP dependent, intravesicularly directed, transmembranous Ca2+-transport in vesicles derived from the microsomal fraction of odontoblasts may explain the role of Ca2+-ATPase.

Alkaline Phosphatase↗

[Histoautoradiographic studies on protein synthesis by odontoblasts].

The protein-producing function of the odontoblasts was studied in 120 Wistar rats with an initial weight ranging from 30 to 40 g determining the rate of the migration of radio-active isotopes (glycine-2-14C, lysine-1-14C) from the odontoblasts to the predentine. The odontoblasts exert a protein-synthetizing function and play an active part in predentine formation. Under the influence of a cariogenic saccharose-casein diet and the simultaneous induction of experimental caries, the intensity of the migration of the labelled amino acids from the odontoblasts to the predentine increases during the first and second stages of caries, which must be interpreted as an increase of the protein-synthetizing function of the odontoblasts associated with an intensification of dentinogenesis. During the third stage of caries, the protein-producing function of the odontoblasts decreases.

Animals↗

Effects of pulp removal and organ culture on adult rat incisor odontoblasts.

The morphological integrity of adult rat incisor odontoblasts was studied to determine the effects of pulp removal and subsequent incubation. From each pair of maxillary incisors one was left intact while the pulp from the other one was removed. Both series were incubated in BGJb medium according to the Trowell method. Morphometrical measurements of the thickness of the odontoblast layer and the size and shape of the odontoblast nuclei were performed on photomicrographs of transverse sections of the incisors. After pulp removal the odontoblasts seemed disorganized and their nuclei were darker and more rounded. During incubation the odontoblasts did not recover from the effect of this pulp removal and degenerative changes were frequently seen. In the culture of the complete tooth organ the morphological integrity of the odontoblasts was much better preserved. Therefore the organ culture of intact teeth seems more favourable.

Animals↗

The association of amorphous mineral deposits with the plasma membrane of pre- and young odontoblasts and their relationship to the origin of dentinal matrix vesicles in rat incisor teeth.

Young and preodontoblasts and matrix vesicles which occur in the presecretory region of incisor teeth of growing rats were examined in stained and unstained ultrathin sections in order to characterize sites involved in the initial mineralization of dentin. Common to pre- and young odontoblasts in the presecretory region were hemispherical membrane-associated amorphous densities, measuring 5-35 nm in diameter after fixation in glutaraldehyde-osmium tetroxide or glutaraldehyde only. Amorphous densities were associated also with the limiting membranes of some vesicles in the extracellular matrix. Other vesicles in the extracellular matrix contained needle-like crystalline deposits typical of dentinal matrix vesicles. Fully differentiated odontoblasts in more incisal regions of the tooth lacked plasma membrane-associated amorphous densities. Neither amorphous nor crystalline densities were associated with any other cellular or subcellular structures in cells of the presecretory region. Flotation of ultrathin sections on solutions of EDTA or EGTA removed the amorphous densities from the plasma membranes, suggesting that the amorphous densities are calcium-containing mineral deposits. Amorphous deposits were associated with the membrane of vesicular structures protruding from the surfaces of pre- and young odontoblasts, suggesting that vesicles found in the extracellular matrix arise by budding from the plasma membranes of pre- and young odontoblasts. The occurrence of amorphous mineral deposits in association with the limiting membrane of some vesicles in the extracellular matrix, and the occurrence of needle-like mineral crystals within other matrix vesicles, suggest that an amorphous-to-crystalline phase transformation of mineral takes place within the matrix vesicle. The results of this study suggest that calcium-binding sites associated with plasma membranes of pre- and young odontoblasts act as nucleating centers for primary mineral deposition in tooth dentin.

Animals↗

A combined scanning electron microscopy and immunofluorescence study demonstrating that the odontoblast process extends to the dentinoenamel junction in human teeth.

The extent of the odontoblast cell process has been the subject of controversy for many years. Using SEM we have examined the extent and morphology of the process on dentine surfaces of human teeth which were partially demineralized and collagenase digested. Third molars were extracted and split; the dentine surface was demineralized, digested by bacterial collagenase, fixed with glutaraldehyde, postfixed in osmium tetroxide, and prepared for SEM investigation. The SEM study revealed the presence of many processlike structures which extended from the odontoblast cell bodies up to the dentinoenamel junction (DEJ). These processes demonstrated lateral and terminal branching and some of them terminated in distended spheres. We have also applied an immunofluorescence technique at the light microscope level to these exposed dentinal surfaces to localize the intracellular microtubules. For this, a second series of third molars was processed in the same manner as for the SEM up to the fixation stage. Teeth were then fixed in periodate-lysine-paraformaldehyde, postfixed in -20 degrees C acetone, and then incubated with affinity-purified rabbit antitubulin antibodies, followed by fluorescein-conjugated goat antirabbit IgGs. Intratubular immunofluorescence labelling for tubulin was evident from the odontoblast cell bodies up to the DEJ. The presence of the tubulin-containing structures extending to the DEJ supports the hypothesis that the structures observed with the SEM are odontoblast processes and that the odontoblast processes do extend to the DEJ.

Adult↗

The effect of nerve section on the incidence and distribution of gap junctions in the odontoblast layer of the cat.

Gap junctions are numerous in the odontoblast layer of the dental pulp and may link sensory axons to odontoblasts. If these junctions do link axons and odontoblasts, they, together with the axons, should disappear after cutting the pulpal nerves centrally. Under general anesthesia the inferior alveolar nerve on one side of two young adult cats was sectioned. Under general anesthesia the animals were perfused with fixative 56 hours later and the coronal dental pulp prepared for electron microscopy. Ultrathin sections were examined from the level of the pulpal cornu and levels approximately one, two, and three mm below this. The incidence of cell processes and gap junctions was measured at different distances from the pulp predentin junction, and operated and control sides compared. The odontoblast layer at the level of the cornu differed from elsewhere in having, on the control side, a greater density of cell processes and gap junctions and in having clearly recognizable axons approaching to within 5 to 10 micron of the predentin. The only statistically significant changes after nerve section occurred in this layer and consisted of a decline in the incidence of cell processes and of gap junctions that link one cell process to another. There was no significant difference between the operated and control sides in the number of gap junctions linking cell processes to recognizable cell bodies. The odontoblast layer in the pulpal cornu contained substantial numbers of unsheathed axons, many presumably en route to the dentin. These axons may participate in gap junctions that link them to other cell processes, possibly even other axons.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Translocation of enamel proteins from inner enamel epithelia to odontoblasts during mouse tooth development.

The developmental problem of how dental epithelia and/or dental papilla ectomesenchyme induce and/or up- or down-regulate tooth formation are as yet unresolved issues. We have designed studies to map the synthesis and fate pathways of secreted amelogenin proteins from Kallenbach differentiation zones II-IV during in vivo and in vitro mouse mandibular first molar tooth development (M1). Tooth organs from cap, bell, and crown stages were processed for reverse transcriptase/polymerase chain reaction (RT-PCR) and high resolution Protein A immunocytochemistry using anti-amelogenin and anti-peptide antibodies. Cap stage M1 were cultured for periods ranging from 10-21 days in vitro using either serum-less, or 15% fetal calf sera-supplemented, chemically-defined medium. Amelogenin transcripts are expressed in the mouse embryonic molar from E15 through early postnatal development. Amelogenin antigens were first detected in Kallenbach's differentiation zone II. Amelogenin proteins secreted from preameloblasts were identified along cell processes and cell surfaces of odontoblasts adjacent to forming mantle dentine extracellular matrix (ECM) prior to biomineralization. Amelogenin proteins were restricted to forming endocytotic vesicles, clathrin-coated vesicles, and lysosomes within odontoblasts. At later stages (e.g. 2 days postnatal development), enamel proteins were not identified in odontoblasts or predentine matrix following mineralization. Comparable observations for stages of development were noted for in vitro cultured tooth explants. Preameloblasts synthesize and secrete amelogenin proteins which bind to odontoblast cell surfaces possibly through the process of receptor-mediated endocytosis. We conclude that amelogenin proteins secreted from preameloblasts, prior to the initiation of biomineralization, were translocated to odontoblasts to serve as yet unknown biological functions.

Amelogenin↗

Odontoblast-specific expression of cre recombinase successfully deletes gene segments flanked by loxP sites in mouse teeth.

Embryonic or neonatal lethality of mice with targeted disruption of critical genes preclude them from further characterization of specific roles of these genes during postnatal development and aging. In order to study the molecular roles of such genes in teeth, we generated transgenic mouse lines expressing bacteriophage Cre recombinase under the control of the mouse dentin sialophosphoprotein (dspp) gene promoter. The expression of Cre recombinase protein was mainly detected in the nucleus of the odontoblasts. The efficiency of Cre activity was analyzed by crossing the Dspp-Cre mice with ROSA26 reporter (R26R) mice. The offspring with both genotypes have shown specific deletion of intervening sequences flanked by loxP sites upstream of the reporter gene, thereby facilitating the expression of the beta-galactosidase (beta-gal) gene in the teeth. The activity of beta-gal was initially observed in the odontoblasts of 1-day-old mice and increased with tooth development. Almost all of the odontoblasts have shown lacZ activity by 3 weeks of age. We could not detect Cre recombinase activity in any other cells, including ameloblasts. These studies indicate that the Dspp-Cre transgenic mice will be valuable to generate odontoblast-specific gene knockout mice so as to gain insight into the molecular roles of critical genes in the odontoblasts during dentinogenesis.

Animals↗

Voltage-gated calcium channels and nonvoltage-gated calcium uptake pathways in the rat incisor odontoblast plasma membrane.

Odontoblasts participate actively in the transport and accumulation of Ca2+ ions to the mineralization front during dentinogenesis. These cells are known to carry membrane-bound ATP-driven pumps and Na+/Ca2+ antiports for Ca2+ extrusion, but little is known about Ca2+ influx mechanisms into these cells. It has been shown that the administration of Ca2+ channel blockers in vivo strongly impairs Ca2+ uptake in the mineral phase during dentinogenesis in the rat; the present in vitro study is aimed at further elucidating odontoblast Ca2+ uptake mechanisms. Dissected rat incisor odontoblasts exhibited a pronounced fluorescence when incubated with a fluorescently-labeled (STBodipy) dihydropyridine, which is specific for voltage-gated Ca2+ channels of the L-type, and this binding was competitively abolished by nifedipine. As assayed by fluorescence spectrometry, odontoblast Ca2+ uptake was enhanced by the agonistic dihydropyridine BAYK-8644 (5 micro;M) as well as by plasma membrane depolarization in a high K+ (120 mM) medium. The Ca2+ uptake after depolarization was impaired by nifedipine (5 micro;M). When treated with the Ca2+-ATPase inhibitor cyclopiazonic acid (CPA; 10 micro;M), a nonvoltage-gated uptake of 45Ca2+ was identified. This uptake was not influenced by nifedipine (20 micro;M) but was impaired by lanthanum ions (200 micro;M). A nonvoltage-gated uptake of Mn2+ into CPA-treated cells could be traced using the fura-2 quenching technique. This CPA-induced Ca2+ flux was not caused by an alteration of the plasma membrane potential, as assayed with di-8-ANEPPS. The results demonstrate that Ca2+ flux into dentinogenically active odontoblasts occurs through voltage-gated Ca2+ channels of the L-type and by nonvoltage-gated, agonist-sensitive Ca2+ uptake pathways.

Animals↗

Parathyroid hormone (1-34) receptor-binding and second-messenger response in rat incisor odontoblasts.

Even though indirect evidence indicates that PTH exerts an anabolic effect on dentinogenesis, the existence of PTH receptors and any second-messenger response in odontoblasts have not been demonstrated. The aim of this study was to investigate whether rat incisor odontoblasts express PTH receptors, and to identify which second messenger pathway the hormone may activate. Odontoblasts were dissected from rat incisors. Amino-terminal (1-34) fragment rat PTH [rPTH(1-34)] conjugated to fluorescein isothiocyanate visualized receptor sites on the cell surface. Upon incubation of odontoblasts with rPTH(1-34), cAMP formation was increased. However, no fluctuations in intracellular calcium activity were observed upon rPTH(1-34) stimulation when using Fura-2 as a Ca2+ probe. In long-time incubations, stimulation with PTH(1-34) upregulated APase activity. The results demonstrate that rPTH(1-34) evokes an anabolic response in dentinogenically active odontoblasts, and that this may be mediated through the protein kinase A/cAMP pathway, whereas no indications for Ca2+ as a second messenger were evident.

Alkaline Phosphatase↗

Expression of connexin 43 and ZO-1 in differentiating ameloblasts and odontoblasts from rat molar tooth germs.

We studied the distribution of connexin (Cx) 43 and ZO-1 by confocal laser scanning microscopy at early stages of dentinogenesis and amelogenesis. Labeling for Cx43 was observed at early stages of differentiation in both the epithelial cells and differentiating odontoblasts. Immunolabeling was detected at the distal and medial regions of undifferentiated ameloblasts and between cells from stratum intermedium and stellate reticulum. Differentiating odontoblasts exhibited immunoreaction for this antibody at their distal end. Immunoreactivity for ZO-1 was observed at regions that correspond to the proximal and distal junctional complexes of differentiating ameloblasts. Staining for ZO-1 was observed at apical regions of odontoblasts with a punctate appearance. In more advanced stages, expression of Cx43 was more evident on ameloblasts, especially at the junctional complexes. Punctate immunolabeling for Cx43 was observed at the lateral sides of differentiating ameloblasts and between the other cells of the enamel organ. Immunoreaction for ZO-1 in ameloblasts was more evident than at the previous stage. It was also observed at the distal end of differentiated odontoblasts. The present study showed that differentiating ameloblasts and odontoblasts express Cx43 and ZO-1 as early as the start of the differentiation process. In addition, the expression of these junctional proteins increases as differentiation of cells continues.

Ameloblasts↗

Platelet-derived growth factor exerts disparate effects on odontoblast differentiation depending on the dimers in rat dental pulp cells.

Platelet-derived growth factor (PDGF) has recently been demonstrated to control the expression of alkaline phosphatase and proteoglycan synthesis of odontoblastic cells in dental pulp tissues. Although PDGF appears to be closely related to dentinogenesis, much about the mode of action of PDGF on odontoblast differentiation remains unclear. In this study, we examined the effects of three PDGF dimers (PDGF AA, AB, and BB) on odontoblastic differentiation of dental pulp cells in long-term mineralized cultures. Dental pulp cells isolated from rat lower incisors were continuously treated with each of PDGF AA, AB, and BB in separate cultures for 20 days. The three PDGF dimers suppressed alkaline phosphatase activity, osteocalcin and calcium content, and the formation of dentin-like nodules. The expression of mRNA for dentin sialoprotein (DSP) in the cells was inhibited by PDGF AA treatment, whereas PDGF AB and BB treatment stimulated the expression of DSP, even though the dentin-like nodule formation was inhibited. Although the effects of PDGF on odontoblastic differentiation varied among the dimers, the cells expressed both PDGF alpha and beta receptors, whose quantities were similar. These results suggest that PDGF exerts diverse effects on odontoblastic differentiation depending on its dimeric form. These in vitro findings explain, at least in part, the in vivo action of PDGF in dentinogenesis during the repair process of damaged dental pulp.

Alkaline Phosphatase↗

Ultrastructural changes during the life cycle of human odontoblasts.

In developing premolars there are four successive stages of odontoblasts related to their location within the tooth, namely pre-odontoblasts, secretory, transitional and aged odontoblasts. Pre-odontoblasts are at the growing tip of the root; they are immature and slightly polarized. They become fully differentiated to give rise to the most active, secretory stage when primary dentine is produced. After completion of primary dentine formation in the coronal region, the aged odontoblasts have a reduced number of organelles which are relocated within the infranuclear region. This final stage is regressive and results from an autophagic process that is prominent in the transitional stage.

Bicuspid↗

Association between the expression of murine 72 kDa type IV collagenase by odontoblasts and basement membrane degradation during mouse tooth development.

In situ hybridization was used to study the expression of the 72 kDa type IV collagenase gene and its association with morphogenesis and cell differentiation during advancing mouse tooth development. The epithelia were completely negative during all developmental stages. The dental mesenchyme was uniformly positive during the early stages of tooth morphogenesis, and no association of type IV collagenase with morphogenetic events was observed. However, at the bell stage the expression increased in differentiating preodontoblasts. Expression was intense in the odontoblasts during secretion of the first predentine matrix. The expression was, however, transient; it decreased around the time when mineralization of dentine started until it completely ceased. Transcripts for 72 kDa type IV collagenase also gradually disappeared from the dental pulp. The expression of 72 kDa type IV collagenase was also strong in the osteoblastic cell lineage. The preosteoblasts at the beginning of the formation of mandibular bone as well as the osteoblasts of the alveolar bone expressed more 72 kDa type IV collagenase than did other mesenchymal cells. The increased gene expression in the odontoblasts correlates with the disappearance of the dental basement membrane as shown by immunolabelling with antibodies against type IV collagen. The onset of increased expression in the odontoblasts preceded the disappearance of the basement membrane and at the time when type IV collagenase transcripts were lost from all odontoblasts the basement membrane was completely removed. It can be speculated that during early stages of tooth development the 72 kDa type IV collagenase acts as a gelatinase whereas during later stages, when odontoblasts and ameloblasts differentiate and the deposition of predentine and enamel matrix is initiated, the enzyme may act as a type IV collagenase and contribute to the degradation of the dental basement membrane.

Animals↗

Cholesterol in the distal portions of differentiating and fully differentiated rat odontoblasts observed by freeze-fracture.

Freeze-fracture replicas of rat molar tooth germs in conjunction with the 3-beta-hydroxysterol marker filipin were used to study the distribution of cholesterol in the distal portions of odontoblast plasma membrane. Filipin-sterol deformations (interactions) appeared as clusters interspersed with deformation-free regions on the distal plasma membrane of early differentiating odontoblasts. In fully differentiated odontoblasts, the whole distal membrane, including the process, was occupied by packed deformations with no free regions. It seems that early developing odontoblasts are poorer in cholesterol than the fully differentiated cells. As the content and/or distribution of 3-beta-hydroxysterols (cholesterol) is known to influence membrane fluidity, the low cholesterol content of early differentiating odontoblasts might be related to the fluidity required for the budding off of matrix vesicles.

Animals↗

Immunocytochemical localization of fibronectin and a 165-kDa membrane protein in the odontoblast layer under initial carious lesions in man.

The possible role of fibronectin in dental tissue repair was investigated by comparing its distribution and that of the 165-kDa fibronectin-binding membrane protein (165 kDa-FnBP) in odontoblasts underlying carious and sound dentine. By immunoperoxidase and light microscopy, fibronectin was localized in the dentine underlying the carious lesion, mainly on the surface of the tubule walls, whereas it could not be detected in neighbouring sound zones. The antibody to the 165 kDa-FnBP strongly reacted with the membrane of odontoblasts underlying the lesion, although those facing sound dentine did not express this antigen. Ultrastructurally the 165 kDa-FnBP was localized in the cell membrane at the apical portion of odontoblasts, including the process membrane, beneath the initial lesion; fibronectin was detected in the dentinal area close to the process, and also in contact with its external surface. By a high-resolution immunogold procedure, the proteins were colocalized at the external surface of odontoblast processes. These data suggest that fibronectin present in human carious dentine could modulate the behaviour of underlying odontoblasts by means of newly expressed 165 kDa-FnBP.

Adhesins, Bacterial↗

Phenotype properties of a novel spontaneously immortalized odontoblast-lineage cell line.

Here we report on the spontaneous immortalization upon serial passages of mouse fetal dental papilla cells, which present odontoblast phenotype features. The cells named odontoblast-lineage cell (OLC) produced dentin extracellular matrix proteins, such as DSP and DMP1, and maintained transcripts of various matrix components as osteopontin, BMP-4, procollagen-1, and MEPE. The addition of osteogenic differentiation medium with beta-glycerophosphate and ascorbic acid was effective for inducing calcification and mineralization in vitro in cell cultures for up to 28 days. For the first time, we investigated the expression of Lhx6 and Lhx7 genes during induced biomineralization, since these new members of LIM homeodomain proteins have been recently proposed tracking odontoblastic phenotypes. Our results indicate that beta-glycerophosphate treatment of OLC cultures decreases Lhx6 transcript levels in vitro. Our findings proved odontoblast phenotype-specificity, which demonstrates that this novel odontoblast-lineage cell line is a valuable tool for future experiments in odontology.

Animals↗