acid-phosphatase and Tooth-Resorption

Chondroitin sulfate (CS) compounds are commonly used to manage OA symptoms. Recent literature has indicated that abnormal subchondral bone metabolism may have a role in the pathogenesis of OA. The aim of this study was to access the effects of chondroitin sulfate obtained from bovine, fish and porcine sources on human osteoclast formation and activity in vitro. Human osteoclasts were generated from blood mononuclear cells. Cells were cultured over 17 days with the addition of macrophage colony stimulating factor (M-CSF) and then stimulated with receptor activator of nuclear factor kappa B ligand from day 7. Cells were treated with the CS commencing from day 7 onwards. To assess effects on osteoclasts, tartrate resistant acid phosphatate (TRAP) expression and resorption of whale dentine assays were used. Bovine-derived CS consistently suppressed osteoclast activity at concentrations as low as 1 μg/ml. Fish and porcine CS was less consistent in their effects varying with different donor cells. All CS compounds had little effect on TRAP activity. mRNA analysis using real-time PCR of bovine CS treated cells indicated that the inhibition of activity was not due to inhibition of the late stage NFATc1 transcription factor (p > 0.05). These results are consistent with CS inhibition of mature osteoclast activity rather than the formation of mature osteoclasts. It would appear that there are differences in activity of the different CS compounds with bovine-derived CS being the most consistently effective inhibitor of osteoclast resorption, but the results need to be confirmed.

Topics: Acid Phosphatase; Animals; Bone Density Conservation Agents; Cattle; Cell Survival; Cell Transdifferentiation; Cells, Cultured; Chondroitin Sulfates; Dentin; Dietary Supplements; Down-Regulation; Fishes; Humans; In Vitro Techniques; Isoenzymes; Leukocytes, Mononuclear; Macrophage Colony-Stimulating Factor; Osteoclasts; RANK Ligand; Recombinant Proteins; Reproducibility of Results; Sus scrofa; Tartrate-Resistant Acid Phosphatase; Tooth Resorption; Whales

Resorption of deciduous teeth is not continuous, but alternates with periods of repair or rest. Dentine surfaces in periods of rest or repair resume resorption by odontoclasts during physiological root resorption of the deciduous teeth. However, no observations of such dentine surfaces have been shown. The characteristic feature of the dentine surfaces resuming resorption remains unknown. Tartrate-resistant acid phosphatase activity (TRAP) was detected on human deciduous teeth. The root resorbing surfaces on these teeth were photographed with a whole-mount light microscope, and the photographed areas were serially sectioned into 0.5 micron semithin sections. Preodontoclasts and odontoclasts were three-dimensionally reconstructed. On root resorbing surfaces, areas with small scattered TRAP-positive cells were observed among areas with many TRAP-positive resorbing odontoclasts and TRAP-negative areas. The sections showed that areas with small scattered TRAP-positive cells have features similar to those of TRAP-negative areas, but there were three kinds of characteristic TRAP-positive cells: preodontoclasts, odontoclasts forming small lacunae, and preodontoclasts, and odontoclasts with cytoplasmic processes extending to the dentine surface, which is covered with cells. These results suggest that the areas with small scattered TRAP-positive cells could be at the stage of resuming resorption, and show that the presence of preodontoclasts and odontoclasts with cytoplasmic processes extending to the covered dentine surface is a characteristic feature of the dentine surface at this stage.

Topics: Acid Phosphatase; Cell Nucleus; Dentin; Humans; Image Processing, Computer-Assisted; Models, Structural; Osteoclasts; Tooth Resorption; Tooth Root; Tooth, Deciduous

Three dental hard tissues, i.e., cementum, dentin, and enamel, are resorbed by multinucleated cells referred to as "odontoclasts." These cells have morphological and functional characteristics similar to those of bone-resorbing osteoclasts. However, concerning enamel resorption, which is a process that may occur during tooth eruption, satisfactory ultrastructural data on odontoclastic resorption are still lacking. Ultrastructural and histochemical characteristics of odontoclasts resorbing enamel of human deciduous teeth prior to shedding were examined by means of light microscopy and transmission and scanning electron microscopy. Odontoclasts that that resorbed enamel were tartrate-resistant acid phosphatase (TRAP)-positive multinucleated giant cells that were essentially the same as those that resorbed dentin and cementum. Ultrastructurally, they had numerous mitochondria, lysosomes, and free polysomes in their cytoplasm. In addition, they were characteristically rich in large cytoplasmic vacuoles containing enamel crystals in the cytoplasm opposite the ruffled border. Although they extended a well-developed, ruffled border against enamel surface, a clear zone--an area typically devoid of organelles--was rarely seen in these cells. In many cases, the cells were in very close contact with the enamel surface by the peripheral part of their cytoplasm. The enamel prisms at the resorption surface contained more loosely packed and electron-lucent enamel crystals compared with those of unresorbed, intact enamel. Furthermore, numerous thin needle- or plate-like enamel crystals that were liberated from the enamel matrix were found in the extracellular channels of the ruffled border and in various-sized cytoplasmic vacuoles in their cytoplasm. The superficial layer of the enamel matrix undergoing odontoclastic resorption stained positively with toluidine blue and for TRAP activity. The results of the present study suggest that odontoclasts resorbing enamel secrete acids as well as organic components, including hydrolytic enzymes, into the resorption zone underlying their ruffled border and that they phagocytose crystals that have been liberated from the partially demineralized enamel matrix by acids, subsequently dissolving them intracellularly.

Topics: Acid Phosphatase; Dental Enamel; Humans; Isoenzymes; Microscopy, Electron, Scanning; Molar; Organelles; Osteoclasts; Tartrate-Resistant Acid Phosphatase; Tooth Resorption; Tooth, Deciduous

Osteoclasts and odontoclasts have been considered multinucleated giant cells which resorb hard tissue by ruffled borders. Recently, the authors reported the presence of a mononuclear osteoclast and odontoclast with a ruffled border. However, the relative frequency of such cells and the distribution of the number of nuclei including mononuclear cells in them have not been elucidated. Six human deciduous teeth were used in this study. After fixation and decalcification, tartrate-resistant acid phosphatase (TRAP) activity was detected with the azo dye method, and then TRAP-positive cells were observed on resorbing areas of teeth by light microscopy. The cells for investigation were serially sectioned by semithin sections to observe the presence of resorptive lacuna and the number of nuclei. The TRAP activity was detected in both multinucleated and mononuclear odontoclasts from serial semithin sections, and 242 TRAP-positive cells which formed lacunae on dentin were investigated to determine the frequency distribution of the number of nuclei. The mean number of nuclei per cell was 5.3, and median was 4. Only 2.9% of odontoclasts were mononucleus and 93.8% had 10 or fewer nuclei. The majority of odontoclasts forming lacunae on the dentin were cells with 10 or fewer nuclei, and mononuclear odontoclasts participated in human deciduous tooth resorption together with multinucleated ones.

Topics: Acid Phosphatase; Cell Count; Cell Nucleus; Child; Giant Cells; Histocytochemistry; Humans; Isoenzymes; Male; Osteoclasts; Tartrate-Resistant Acid Phosphatase; Tooth Resorption; Tooth, Deciduous

Single-cell suspensions of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (G-PBMC) cultured in alpha minimal essential medium (alphaMEM) containing 10% fetal bovine serum formed multicellular aggregates within 24 hours. In six separate experiments, formation of aggregates appeared to be dependent on cell density per surface area, so that 5.8 +/- 1.3 aggregates formed per 1 x 10(5) cells when G-PBMC were cultured at densities greater than or equal to 1 x 10(5) cells/cm2. The frequency of aggregate formation was less than 1 per 10(5) cells when G-PBMC were cultured at densities less than 1 x 10(5) cells/cm2. Once formed, aggregates became adherent within 72 hours, and then, over the course of 21 days, released CD3/CD4/CD25-positive cells into the supernatant. This T-cell production peaked between days 7 and 14, reaching a total of 1,269 +/- 125.9 cells released per aggregate by day 21. Between days 14 and 21, the aggregates also generated macroscopic clusters of adherent mononuclear and giant multinucleated cells that stained positive for tartrate-resistant acid phosphatase (TRAP). At 4 weeks, the macroscopic foci coalesced into monolayers. Multinucleated TRAP-positive cells were distinguished from macrophage polykaryons by the absence of CD14 expression and the presence of osteoclast-specific membrane receptors for calcitonin and alphavbeta3-vitronectin. The osteoclast nature of these cells was further demonstrated by their ability to form resorption lacunae on dentine slices. Comparable osteoclast formation was not detected in cultures of normal marrow or normal nonmobilized peripheral blood.

Topics: Acid Phosphatase; Animals; Biomarkers; Bone Marrow; Bone Marrow Cells; Cattle; Cell Aggregation; Cell Differentiation; Cells, Cultured; Coloring Agents; Dentin; Granulocyte Colony-Stimulating Factor; Humans; Isoenzymes; Leukocytes, Mononuclear; Organ Specificity; Osteoclasts; Receptors, Calcitonin; Receptors, Vitronectin; Tartrate-Resistant Acid Phosphatase; Tooth Resorption

In human deciduous teeth, odontoclastic resorption takes place at the pulpal surface of the coronal dentine prior to shedding, and this resorption shows clear time-related histological changes (Sahara et al., 1992).. Using this phenomenon as an observation system, we examined the cytodifferentiation of human odontoclasts by light and electron microscopy. For a histochemical marker of odontoclast differentiation and function, tartrate-resistant acid phosphatase (TRAP) activity was determined by light and electron microscopic enzyme histochemistry.. As root resorption neared completion, TRAP-positive mononuclear cells were initially detected in the pulp chamber. They had abundant mitochondria, small lysosomes, and moderately developed rough endoplasmic reticulum throughout their cytoplasm. In these mononuclear cells, TRAP activity was localized in compartments of the biosynthetic pathway, i.e., in cisternae of the endoplasmic reticulum and Golgi lamellae, as well as small lysosomes. The TRAP-positive mononuclear cells first made contact with the predentine surface by their elongated cellular processes. After attachment, they spread out along the predentine surface and developed specialized membrane structures, clear zones, and ruffled borders. Next, they fused with each other on the predentine surface and formed typical multinucleate odontoclasts. After termination of their resorption function, the odontoclasts lost their ruffled borders and became detached from the resorbed surface. Most of the detached odontoclasts had numerous large pale vacuoles and secondary lysosomes and appeared to be in the process of degeneration.. The present study demonstrates that: (1) odontoclasts differentiated from TRAP-positive mononuclear cells, which presumably originate from circulating progenitor cells, (2) membrane specialization of odontoclasts, i.e., development of a clear zone and ruffled border, is induced following their contact with the resorption surface, (3) multinucleation of odontoclasts takes place only after their attachment to the resorption surface, (4) mature multinucleate odontoclasts can resorb predentine as well as dentine in the same way as osteoclasts resorb bone, and (5) at the end of the resorption, odontoclasts gradually lose their ruffled borders and become detached from the resorbed surface.

Topics: Acid Phosphatase; Biomarkers; Cell Differentiation; Dental Pulp; Histocytochemistry; Humans; Leukocytes, Mononuclear; Microscopy, Electron; Models, Biological; Osteoclasts; Tooth Resorption; Tooth, Deciduous

Resorption by odontoclasts of a superficial nonmineralized layer of predentine that occurs in prior to the shedding of human deciduous teeth was studied by light and electron microscopy. As resorption of the tooth roots neared completion, multinucleate cells appeared on the predentine surface of the coronal dentine between the degenerated odontoblasts, excavated characteristic resorption lacunae in the nonmineralized predentine. These multinucleate cells had the same ultrastructural characteristics as odontoclasts and histochemical demonstration of tartrate-resistant acid phosphatase activity in the multinucleate cells revealed intense staining in numerous small granules identified as lysosomes. Occasionally, the multinucleate cells simultaneously resorbed both nonmineralized and calcospherite-mineralized matrix in the predentine. The study demonstrates that multinucleate odontoclasts can resorb nonmineralized predentine matrix in vivo, probably in the same way as they resorb demineralized organic matrix in the resorption zone underlying their ruffled border.

Topics: Acid Phosphatase; Cell Nucleus; Cuspid; Dentin; Histocytochemistry; Humans; Incisor; Molar; Osteoclasts; Tooth Resorption; Tooth, Deciduous

Topics: Acid Phosphatase; Animals; Anti-Bacterial Agents; Decalcification Technique; Fluorine; Leukocytes; Male; Maxilla; Methods; Molar; Nitrobenzenes; Pulpectomy; Rats; Tooth Resorption; Transplantation, Autologous