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PubMed1976

Acid hydrolases in the odontoblast-predentin region of dentinogenically active teeth.

The presence of hyaluronoglucosidase (EC 3.2.1.35; hyaluronidase), beta-N-acetylglucosaminidase (EC 3.2.1.30), exo-1,4-beta-xylosidase (EC 3.2.1.37), and arylsulfatase (EC 3.1.6.1) in dentinogenically active odontoblasts isolated from the rat incisor has been demonstrated by means of biochemical methods. The possible function of these enzymes in relation to the calcification process is discussed.

Acetylglucosaminidase↗

PubMed1982

Relationship of inorganic pyrophosphatase and para-nitrophenylphosphatase activities of alkaline phosphatase in the microsomal fraction of isolated odontoblasts.

Some properties of inorganic pyrophosphatase (PPiase EC 3.6.1.1.) and para-nitrophenylphosphatase (p-NPPase EC 3.1.3.1) in the microsomal fraction of odontoblasts were investigated. The ratio of Mg2+:p-NPP and Mg2+:PPi for optimal enzyme activities was 1:1. A mutual substrate competition for PPiase and p-NPPase was described. In the presence of 0.1 mM EDTA, Mg2+ alone was not able to reactivate p-NPPase or PPiase. Instead, Zn2+ and Co2+ reactivated the PPiase, indicating they might act as cofactors for the enzyme. Mg2+ increased the PPiase activity, probably because Mg PP2-i was the true substrate for the enzyme. The diphosphonates ethane-1-hydroxy 1,1 diphosphonate (EHDP), methane diphosphonate (MDP) and dichloromethane diphosphonate (Cl2MDP) inhibited the PPiase activity.

4-Nitrophenylphosphatase↗

PubMed1998

Odontoblast differentiation: a response to environmental calcium?

The response of the dental pulp to calcium hydroxide has been well described but the process of pulpal repair leading to dentinal bridge formation appears complex and the mechanisms remain incompletely understood. Through the precise regulation of the free calcium ion in the cytosol, cells have been able to utilize anions such as phosphates for a wide range of activities such as energy production (oxidative phosphorylation). As anions are abundant in the cytosol, intracellular levels of calcium ions are kept low, several orders of magnitude less than that of the surrounding extracellular matrix. Consequently, cells are able to use calcium ions for the regulation of many cellular events. The binding of extracellular molecules such as cytokines, hormones or antibodies, with receptors on the plasma membrane may result in short- or long-term modifications to cellular metabolism, including the mechanisms of intracellular calcium homeostasis. Cell survival depends upon the ability to adapt to changes in the cell's micro-environment. Adaptation in turn results in altered cellular activity that may be interpreted as showing that the cell has become more or less specialised. In some instances this may include the resumption of mitotic activity. If the rate or magnitude of change exceeds a cell's adaptive capacity, the cell dies. Responses of cells to alterations in their environment are reviewed as they may provide an explanation for the success of calcium hydroxide in facilitating pulpal repair and the differentiation of odontoblasts.

Calcium Hydroxide↗

PubMed1983

The extent of the odontoblast process in normal and carious human dentin.

When observed by SEM, after being treated with the HCl-collagenase method, the odontoblast processes extended throughout the whole thickness of dentin in intact teeth and the whole thickness of normal and the inner carious dentin in carious teeth. Small holes and depressions were found on the processes in the transparent layer.

Adult↗

PubMed1985

An immunocytochemical study of the human odontoblast process using antibodies against tubulin, actin, and vimentin.

An immunofluorescence technique was applied at the light microscope level to human third molar coronal dentin in order to localize the intracellular components tubulin, vimentin, and actin. Third molars were split immediately upon extraction, and immersed in periodate-lysine-paraformaldehyde fixative. The crowns were demineralized, dehydrated, and wax-embedded, and 6-micron sections were prepared. The sections were post-fixed in -20 degrees C acetone, and then incubated with monoclonal mouse anti-tubulin, anti-vimentin, or anti-actin antibodies, followed by fluorescein-conjugated sheep anti-mouse immunoglobulins. Intratubular immunofluorescence labeling for tubulin and vimentin was very similar in pattern and intensity and extended to the dentino-enamel junction. In contrast, the actin labeling appeared less intense and more punctate, and was located primarily in the pulpal half of the crown, although some labeling was detectable up to the dentino-enamel junction. The presence of tubulin-, vimentin-, and actin-containing structures extending to the dentino-enamel junction supports the hypothesis that the odontoblast process does extend to the dentino-enamel junction in the human, and is in agreement with earlier studies of rat molars.

Actin Cytoskeleton↗

PubMed1998

Pex mRNA is localized in developing mouse osteoblasts and odontoblasts.

Mutations in PEX, a phosphate-regulating gene with homology to endopeptidase on the X chromosome, were recently identified in patients with X-linked hypophosphatemia (XLH), an inherited disorder of phosphate homeostasis characterized by growth retardation and rachitic and osteomalacic bone disease. To understand the mechanism by which loss of PEX function elicits the mutant phenotype, a study of its mRNA localization and ontogenesis was undertaken. Using the reverse transcriptase-nested polymerase chain reaction (RT-nested PCR) with polyA+ RNA purified from mouse testis, a 337-bp Pex cDNA fragment was generated and cloned in the pCRII plasmid. The cDNA was used to generate sense and anti-sense Pex riboprobes for in situ hybridization (ISH) and Northern analysis. To survey a large number of different tissues, sagittal sections of embryos and newborn mice were examined. ISH showed the presence of Pex mRNA in osteoblasts and odontoblasts. Pex gene expression was detectable on Day 15 of embryonic development, which coincides with the beginning of intercellular matrix deposition in bones. Finally, Northern analysis of total RNA from calvariae and teeth of 3-day-old and adult mice showed that the abundance of the 7-kb Pex transcript is decreased in adult bones and in nongrowing teeth. The present study demonstrates that Pex mRNA is expressed in bones and teeth and suggests that this putative endopeptidase plays an important role in the development of these tissues.

Aging↗

PubMed2003

TGF-beta1 induces accumulation of dendritic cells in the odontoblast layer.

TGF-beta1 released from dentin degraded by bacterial or iatrogenic agents is suspected to influence dental pulp response, including the modulation of cell migration. To determine the consequences of TGF-beta1 action on pulp immune cells, we analyzed, by immunohistochemistry, the effect of transdentinally diffusing TGF-beta1 on their localization in a human tooth slice culture model. TGF-beta1 induced an accumulation of HLA-DR-positive cells in both odontoblast and subodontoblast layers of the stimulated zone. Together with HLA-DR, these cells co-expressed Factor XIIIa and CD68, two features of immature antigen-presenting dendritic cells (DC), as well as the TGF-beta1 specific receptor TbetaRII. In contrast, no effect could be detected on the localization of either mature DC-LAMP-positive DC or of T- and B-lymphocytes. Analysis of these data suggests that TGF-beta1 released from dentin degraded by bacterial or iatrogenic agents could be involved in the immune response of the dental pulp resulting from tooth injury.

Adolescent↗

PubMed2005

Hyper-expression of osteocalcin mRNA in odontoblasts of Hyp mice.

The Hyp mouse is a murine homologue of human X-linked hypophosphatemia that displays hypo-mineralization in bone and dentin. In this study, we tested the hypothesis that the defect in Hyp mice leads to alterations in the expression of dentin matrix proteins that may be associated with the hypo-mineralization changes in the tissues. Quantitative RT-PCR analyses showed that expression of the osteocalcin gene in Hyp mice tooth germ samples was significantly higher than in wild-type mice, whereas the gene expressions of osteonectin, osteopontin, dentin matrix protein 1, and type I collagen in both types of mice were similar. Further, cultured Hyp mice tooth germ samples exhibited a higher expression of the osteocalcin gene than did those from wild-type mice, which was in accord with the results of our in vivo analysis. These findings suggest that osteocalcin mRNA is highly expressed in Hyp mice odontoblasts and may be associated with dentin hypo-mineralization.

Animals↗

PubMed1976

A comparison of ATP-degrading enzyme activities in rat incisor odontoblasts.

In active odontoblasts from the rat incisor, used as a model system for biologic calcification, two distinguishable enzyme activities capable of degrading adenosine monophosphate (ATP) exist. Once can be inhibited ny 1-tetramisole, (+/-)-2,3,5,6,-tetrahydro-6-phenylimidazo (2.1B) THIAZOLE HYDROCHLORIDE (Levamisol) and (+/-)-6(m-bromophenyl)-5.6-dehydroimidazo (2.1-b) thiazole oxalate (R823) and is probably identical with nonspecific alkaline phosphatase (EC 3.1.3.1). The activity of the other enzyme, named Ca2+-ATPase, is dependent on the presence of Ca2+ or Mg2+ and is activated by these ions. The pH optimum of Ca2+-ATPase is 9.8. The Ca2+-ATPase is unaffected by Levamisole, R 8231, ouabain, ruthenium red, Na+ and K+ ions. Maximal activity was found against ATP, whereas adenosine diphosphate, guanosine triphosphate, inosine triphosphate and adensoine monophosphate were hydrolysed at lower rate. It may be speculated that the Ca2+-ATPase is concerned with the transmembranous transport of Ca2+ ions to the mineralization front.

Adenosine Triphosphatases↗

PubMed1981

Ultrastructural localization of complex carbohydrates in odontoblasts, predentin, and dentin.

Glycosaminoglycans (GAGs) and glycoproteins (GPs) are essential components for dentinogenesis. We have examined rat odontoblasts, predentin, and dentin decalcified with EDTA and stained with: 1) Spicer's hig-iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) method for sulfated glycoconjugates, and 2) Thiéry's periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method for vicinal glycol-containing glycoconjugates. HIS-TCH-SP stained distended portions of Golgi saccules and secretory granules. The predentin contained three times the number of HID-TCH-SP stain precipitates when compared to the mineralization front of the dentin matrix. PA-TCH-SP weakly stained membranes of Golgi saccules and cisternae of rough endoplasmic reticulum (RER), whereas stronger staining was observed in secretory granules, lysosomes, and multivesicular bodies (MVBs). Collagen fibrils in predentin demonstrated moderate PA-TCH-SP staining. In contrast, strong PA-TCH-SP staining was observed on and between collagen fibrils in the mineralization front of the dentin matrix. TCH-SP controls of unosmicated specimens lacked significant staining, however, osmicated control specimens did contain some TCH-SP stain deposits in the mineralization front. These results indicate that sulfated and vicinal glycol-containing glycoconjugates are packaged in the same type of secretory granule and released into the extracellular matrix; subsequently vicinal glycol-containing glycoconjugates concentrate in the calcification front, whereas sulfated glycoconjugates accumulate in the predentin and are either removed or masked to staining in the dentin.

Animals↗

PubMed1994

Scanning electron microscopy of the organelles of rat incisor odontoblasts--in particular the tubulo-vesicular elements.

The fine structure of rat odontoblasts (OBs) showing the developmental sequence of cellular maturation along the long axis of the incisor was studied. The cytomorphological changes and relationships of the intracellular components, particularly the membranous component, were investigated mainly by scanning electron microscopy (SEM) using the AODO (A: aldehyde prefixation; O: osmium tetroxide postfixation; D: dimethyl sulfoxide (DMSO) freeze-fracture; O: osmium tetroxide maceration) method and transmission electron microscopy (TEM) using the conventional methods. The following results were obtained: 1) The elongated young OBs were highly polarized and characterized by well-developed concentric cisternal granular (rough) endoplasmic reticulum (rER) layers and Golgi complex. SEM showed the rER was continuous to the agranular (smooth) ER (sER). 2) Transformation of the concentric rER to a tubular network was closely related to the maturation and secretory activity of the OBs. 3) Many branched and elongated tubular mitochondria were observed in the supranuclear region of young OBs. On the other hand, many round-to-ovoid mitochondria were observed particularly in the distal clear area of old OBs. 4) SEM also revealed tubulo-vesicular elements, which are believed to be structures relating to secretory and absorptive functions, composed of secretory granules and small tubules, having connections with the cell membrane in both young and old OBs. Furthermore, the small tubules were continuous with the larger sER tubules in the distal clear area and cell process of the OBs. 5) Using TEM, long lysosomes, various cytosomes and multivesicular bodies (MVBs) were usually observed in the peripheral region and in the distal cytoplasm and major processes of the OBs. The present SEM and TEM studies indicated the MVBs were closely associated with sER tubules of the tubulo-vesicular elements. 6) Both TEM and SEM showed cytosegresomes containing laminated ER and other organelles particularly in the distal clear area of old OBs. 7) SEM also showed some thin lateral branchings of the OBs running along the fine matricial fibrils, as well as matrix-vesicle-like structures deeply invaginated at the basal portion of the cell processes and distributed in the predentine. The present morphological study demonstrated a three-dimensional configuration of the structures relating to secretory and absorptive functions of young and old OBs.

Animals↗

PubMed1975

Inorganic pyrophosphatase in isolated enamel organ and odontoblasts from the rat incisor.

The inorganic pyrophosphatase (PPiase) activity was determined by a colorimetric method in the odontoblasts and the parts of the enamel organ related to enamel matrix formation and enamel maturation. The effects on PPi hydrolysis by EDTA, R 8231, urea and heat treatment were found to be almost identical to those reported for nonspecific alkaline phosphatase (APase) in the same tissues. The Mg2+ activation curve for PPiase was also similar. Like those of APase, these characteristics of PPiase activity were identical in the three locations studied. It is suggested that the close similarity in the properties of PPiase and APase is due to activity of the same enzyme, a concept which is in agreement with recent biochemical and histochemical studies of calcification.

Animals↗

PubMed1977

ATP-ase activity in the odontoblastic layer of rat incisor. Determination with a radiochemical and a colorimetric method.

The ATP-splitting enzyme activity in odontoblasts isolated from rat incisors has been studied by means of a radiochemical and a colorimetric micromethod. The results with the two methods were virtually identical. The reaction was linear with time for at least 45 min. The pH optimum was found to be 9.8 independently of the ATP concentration. Maximal substrate saturation occurred at a total ATP concentration of 3 mM. Ca2+ and Mg2+ ions activated ATP degradation. F-ions did not affect the activity at low concentrations, whereas higher concentrations were inhibitory. Na+ and ions were slightly inhibitory. Urea inhibited the enzyme activity at concentrations above 1.5 M, while EDTA and EGTA were strong inhibitors at very low concentrations. When incubating in the presence of low concentrations of specific inhibitors for nonspecific alkaline phosphatase, levamisole and R8231, about 20% ATP degrading enzyme activity remained. In conclusion it is suggested that there are at least two ATP degrading phosphatases active at alkaline pH.

Adenosine Triphosphatases↗

PubMed1979

The hydrolysis rates of ATP and phosphoserine in the odontoblast-predentin region of intact and carious human teeth.

Coronal odontoblast-predentin tissue was taken from intact teeth and from teeth with carious lesions of varying depths, and the samples (20, 200 x g, saline-soluble fractions) were studied for their ability to hydrolyze adenosine triphosphate (ATP) and phosphoserine. A significantly higher rate of ATP hydrolysis at pH 9.8 was detected in enamel caries and a significantly lower rate in advanced caries than in intact teeth. The rates of the hydrolysis of phosphoserine at pH 9.8 did not differ between the various tooth groups, and the only clear trend indicating elevated enzyme activity was seen in the group of initial dentin caries. The ATP and phosphoserine hydrolysis at pH 9.8 were considered to have been due to the nonspecific alkaline phosphatase (EC 3.1.3.1) activity. In the presence of both levamisole and ouabain, maximal enzyme-dependent ATP hydrolysis was observed at pH 7.9. The remaining ATP-cleaving activity was thought to have been due to the Ca2+Mg2+-dependent ATPase (EC 3.6.1.3). The Ca2+Mg2+-dependent ATPase activities at pH 7.9 remained at constant levels in both the intact and carious groups. The parameters studied in vitro were thought to reflect changes in the mineralization rate of reparative dentin as a response to caries in vivo.

Adenosine Triphosphatases↗

PubMed1983

Effect of different inhibitors on nonspecific alkaline phosphatase in the microsomal fraction from isolated odontoblasts.

In the microsomal fraction isolated from rat incisor odontoblasts, the influence of several inhibitors on p-nitrophenyl phosphatase activity (p-NPPase) was demonstrated at pH 10.3 and compared with the influence at pH 8.8. The inhibiting effect of EDTA on the p-NPPase activity was reversed by equimolar concentrations of Zn2+. The inhibitory effect of three diphosphonates, ethane-1-hydroxydiphosphonate (EHDP), methanediphosphonate (MDP), and dichloromethanediphosphonate (Cl2MDP) was equal to that of a chelating agent. Tetramisole and levamisole exerted an inhibitory effect that was different from the complexing type. L-Cystein, but not L-ascorbic acid, inhibited p-NPPase activity with a maximum inhibitory effect at pH 10.5.

Alkaline Phosphatase↗

PubMed1984

Effects of methylmercury chloride on rat incisor odontoblasts and dentinogenesis.

Methylmercury chloride, a well-known nerve cell toxicant, was administered to 40 rats in 2 groups, in doses of 10 mg/kg and 20 mg/kg. Histomorphologic investigation of maxillary incisors after 2 and 4 days revealed granular and hydropic changes in parts of the odontoblastema. Deposition of interglobular dentin was seen after 7 and 11 days. The most vulnerable cells seemed to be the mantle dentin-producing odontoblasts, and the alterations found are probably due to interference with the rough endoplasmic reticulum.

Animals↗

PubMed1995

Molecular cloning and sequence of bovine Msx-1 homeobox-containing gene cDNA from a bovine odontoblast library.

Screening of a bovine odontoblast cDNA library from developing incisor with murine Msx-1 and Msx-2 cDNA probes led to the isolation of three positive clones. All of them encoded for a sequence of a protein containing 297 amino acids. The responsible gene was designated as bovine Msx-1 (bMsx-1) due to the high homology with the human MSX-1 and mouse Msx-1 sequences.

Amino Acid Sequence↗

PubMedDate not supplied

Effects of light-curing time on the cytotoxicity of a restorative resin composite applied to an immortalized odontoblast-cell line.

This in vitro study evaluated the cytotoxic effects of a restorative resin composite applied to an immortalized odontoblast-cell line (MDPC-23). Seventy-two round resin discs (2-mm thick and 4 mm in diameter) were light-cured for 20 or 40 seconds and rinsed, or not, with PBS and culture medium. The resin discs were divided into four experimental groups: Group 1: Z-100/20 seconds; Group 2: Z-100/20 seconds/rinsed; Group 3: Z-100/40 seconds; Group 4: Z-100/40 seconds/rinsed. Circular filter paper was used as a control material (Group 5). The round resin discs and filter papers were placed in the bottom of wells of four 24-well dishes (18 wells for each experimental and control group). MDPC-23 cells (30,000 cells/cm2) were plated in the wells and allowed to incubate for 72 hours. The zone of inhibition around the resin discs was measured under inverted light microscopy; the MTT assay was carried out for mitochondrial respiration and cell morphology was measured under SEM. The scores obtained from inhibition zone and MTT assay were analyzed with the Kruskal-Wallis followed by Dunnett tests. In Groups 1, 2, 3 and 4, the thickness of the inhibition zone was 1,593 +/- 12.82 microm, 403 +/- 15.49 microm, 1,516 +/- 9.81 microm and 313 +/- 13.56 microm, respectively. There was statistically significant difference among the experimental and control groups at the 0.05 level of significance. The MTT assay demonstrated that the resin discs of the experimental groups 1, 2, 3 and 4 reduced the cell metabolism by 83%, 40.1%, 75.5% and 24.5%. Only between the Groups 2 and 4 was there no statistically significant difference for mitochondrial respiration. Close to the resin discs, the MDPC-23 cells exhibited rounded shapes, with only a few cellular processes keeping the cells attached to the substrate or, even disruption of plasma membrane. Adjacent to the inhibition zone, the cultured cells exhibited multiple fine cellular processes on the cytoplasmic membrane organized in epithelioid nodules, similar to the morphology observed to the control group. Based on the results, the authors may conclude that the Z-100 resin composite light cured for 20 seconds was more cytopathic to MDPC-23 cells than Z-100 light cured for 40 seconds. The cytotoxic effects of the resin discs decreased after rinsing them with PBS and culture medium. This was confirmed by MTT assay and upon evaluation of the inhibition zone, which was narrower following rinsing of the resin discs.

Cell Line, Transformed↗