Management of the questionable carious fissure: invasive vs noninvasive techniques.
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Bacteria↗
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Results for “Tooth Calcification”
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Ca-binding domains in the odontoblast layer of rat molars and incisors under normal and pathological conditions.
We recently reported the presence of high concentrations of a Ca-binding matrix in the circumpulpal dentin of rat incisors which had been prevented from mineralization by a systemic administration of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP), a type of bisphosphonates, thus suggesting the role of the putative Ca-binding matrix in the appositional mineralization of circumpulpal dentin (TAKANO et al., 1998, 2000; OHMA et al., 2000). In this study, we examined the distribution of Ca-binding domains in the pulp tissue of normal rat teeth and its changes under the influence of HEBP, in order to identify and clarify the role of the Ca-binding matrix in the physiological process of dentin mineralization. Observation of the normal rat tooth pulp showed occasional, tiny extracellular deposits of Ca-enriched material in the odontoblast layer, associated primarily with pericapillary regions. Such deposits were immunopositive for dentin sialoprotein (DSP), displayed high levels of X-ray peaks for calcium and phosphorus, and showed a drastic increase in amount by daily injections of HEBP. A brief vascular perfusion of high Ca-containing solution in normal animals caused the extensive deposition of Ca-P complexes along the basolateral membranes of odontoblasts but not in the other regions of the pulp tissue. These data suggest the existence of DSP-enriched extracellular Ca-binding domains in the odontoblast layer and also indicate a novel Ca-binding property of the basolateral membranes of odontoblasts. Since DSP is primarily synthesized as dentin sialophosphoprotein (DSPP) and later cleaved into dentin phosphophoryn (DPP) and DSP in odontoblasts, and since DSP has no notable affinity for Ca, the sites of DSP-immunopositive Ca-P deposits in the odontoblast layer may also contain DPP, a highly phosphorylated acidic protein having a strong binding property for calcium. Characteristic Ca-binding properties seen in the odontoblast layer appear to be related to the regulation of the appositional mineralization of circumpulpal dentin.
Animals↗
Growth factors regulate expression of mineral associated genes in cementoblasts.
BACKGROUND: Knowledge of the responsiveness of cells within the periodontal region to specific bioactive agents is important for improving regenerative therapies. The aim of this study was to determine the effect of specific growth factors, insulin-like growth factor-I (IGF-I), platelet-derived growth factor-BB (PDGF-BB), and transforming growth factor-beta (TGF-beta) on cementoblasts in vitro and ex vivo. METHODS: Osteocalcin (OC) promoter driven SV40 transgenic mice were used to obtain immortalized cementoblasts. Growth factor effects on DNA synthesis were assayed by [3H]-thymidine incorporation. Northern analysis was used to determine the effects of growth factors on gene expression profile. Effects of growth factors on cementoblast induced biomineralization were determined in vitro (von Kossa stain) and ex vivo (re-implantation of cells in immunodeficient (SCID) mice). RESULTS: All growth factors stimulated DNA synthesis compared to control. Twenty-four hour exposure of cells to PDGF-BB or TGF-beta resulted in a decrease in bone sialoprotein (BSP) and osteocalcin (OCN) mRNAs while PDGF-BB also increased osteopontin (OPN) mRNA. Cells exposed to IGF-I for 24 hours exhibited decreased transcripts for OCN and OPN with an upregulation of BSP mRNA noted at 72 hours. In vitro mineralization was inhibited by continuous application of PDGF-BB or TGF-beta, while cells exposed to these factors prior to implantation into SCID mice still promoted biomineralization. CONCLUSIONS: These data indicate IGF-I, PDGF-BB, and TGF-beta influence mitogenesis, phenotypic gene expression profile, and biomineralization potential of cementoblasts suggesting that such factors alone or in combination with other agents may provide trigger factors required for regenerating periodontal tissues.
Animals↗
Leucine-rich amelogenin peptide: a candidate signaling molecule during cementogenesis.
BACKGROUND: Cementum is a critical mineralized tissue; however, control of its formation remains undefined. One hypothesis is that enamel matrix proteins/peptides secreted by ameloblasts and/or epithelial rest cells contribute to the control of cementum formation via epithelial-mesenchymal interactions. Here, we focused on determining whether or not leucine-rich amelogenin peptide (LRAP), translated from an alternatively spliced amelogenin RNA, altered cementoblast behavior. METHODS: Immortalized murine cementoblasts (OCCM-30) were exposed to LRAP and evaluated for: 1) proliferative activity; 2) gene expression using Northern blot for Cbfal (core binding factor alpha-1); OCN (osteocalcin), OPN (osteopontin), and real-time reverse transcription-polymerase chain reaction (RT-PCR) for OPG (osteoprotegerin); and RANKL (receptor activator of NF-kappaB ligand); 3) signaling pathway using inhibitors of PKA (THFA), PKC (GF109203X), and MAPK (UO126); and 4) mineralization evaluated by von Kossa and Alizarin-red. RESULTS: LRAP had no effect on cell proliferation up to 6 days, with a decrease in cell growth observed at the highest dose by 9 days versus untreated cells. LRAP down regulated OCN and up regulated OPN in a dose- and time-response fashion, and inhibited the capacity of mineral nodule formation. Transcripts for OPG were increased in LRAP-treated cells compared to control, but RANKL mRNA levels were not affected. Core binding factor alpha (Cbfa) mRNA, expressed constitutively, was not affected by LRAP. Signaling pathway assays suggested involvement of the MAPK pathway, since the addition of the MAPK inhibitor suppressed OPN expression in LRAP-treated cells. CONCLUSION: Leucine-rich amelogenin peptide appears to have a direct effect on cementoblast activity that may prove significant during development as well as in regeneration of periodontal tissues.
Animals↗
Prostanoid- and interleukin-1-induced primary genes in cementoblastic cells.
BACKGROUND: Cementum is a key component of a functional periodontal organ. However, regenerating lost cementum is difficult and often incomplete. Identifying molecular mediators of cementoblast differentiation and function should lead to better targeted treatment for periodontitis. Prostaglandins increase mineralization of murine cementoblastic OCCM cells and alveolar bone formation, whereas the cytokine interleukin-1 (IL-1) inhibits alveolar bone formation. We hypothesized that differentially induced primary genes in OCCM cells may mediate anabolic and catabolic responses. Our objective was to identify primary genes differentially induced by the synthetic prostanoid fluprostenol and IL-1 in cementoblastic cells. METHODS: Confluent OCCM cells were pretreated with the protein synthesis inhibitor cycloheximide followed by fluprostenol or IL-1 for 1.5 hours. cDNA generated from each group was used for cDNA subtraction hybridization to identify differentially induced genes. Preferential gene induction was verified by Northern blot analysis. RESULTS: Thirteen fluprostenol- and seven IL-1-regulated genes were identified. Among the fluprostenol-induced genes was mitogen-activated protein (MAP) kinase phosphatase 1 (MKP1), a negative regulator of MAP kinase signaling. To verify the cDNA subtraction hybridization results, OCCM cells were treated with fluprostenol or prostaglandin F2 (PGF2), and MKP1 mRNA levels were determined. The 0.001 to 1 microM fluprostenol and 0.01 to 1 microM PGF2 significantly induced MKP1 mRNA levels, which peaked at 1 hour of treatment and returned to baseline at 2 hours. CONCLUSIONS: Fluprostenol enhanced, whereas IL-1 inhibited, OCCM mineralization. Using cDNA subtraction hybridization, we identified primary genes that correlate with the observed anabolic and catabolic responses. These findings further our understanding of cementoblast function and suggest that differentially induced genes may mediate cementum formation and resorption.
Animals↗
Study on hypomineralized areas in hamster molars.
Hypomineralized area (HMA) scores were determined for hamster molars during tooth maturation and fluoride treatment. It was found that the molars were maturing until the animals were 45 days old, but from 45 to 74 days of age the HMA scores remained constant. Fluoride treatment decreased the HMA score. The group with the lowest HMA score was fed a cariogenic diet, inoculated with Streptococcus mutans and received topical fluoride treatment.
Animals↗
A histological study on the nature of human interglobular dentin.
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Dentin↗
Cathepsin D. Purification from rat liver and immunohistochemical demonstration in rat incisor.
Rat liver cathepsin D (EC 3.4.23.5) was purified using precipitation technique, ion exchange chromatography, molecular sieve chromatography and isoelectric focusing. Rabbit anti-rat cathepsin D IgG was prepared and rat incisor teeth were cross-sectioned in a cryostat. These sections were incubated with FITC-conjugated anti-rat cathepsin D IgG. Marked fluorescence, indicating the localization of cathepsin D, could be seen over the odontoblast and predentin area. No specific fluorescence could be dmonstrated in the pulp connective tissue proper nor in the dentin.
Animals↗
The calcium selective electrode--a possible tool in dental research.
Ion selective potentiometry is widely used for analytical purposes in biologic research. The Ca selective electrode offers an inexpensive and easily handled means for studying ionized calcium. It does not affect the process studied, and may, for example, combined with an automatic recorder, give running information on dynamic systems. The calcium electrode responds in accordance with the Nernst equation and has a theoretical range of 1--10(-6) M Ca2+. The electrode is influenced by changes in temperature, in pH and also in concentration of buffer solution. The Ca sensitive membrane is adversely affected by acetic acid but tolerates lactate/lactic acid buffers. Best linearity in calibration curves for Ca2+ in lactate buffers is found in concentrations from 10(-4) M to 10(-2) M Ca. In a 5% Na lactate/lactic acid buffer, pH 5, with 0.5 M KNO3 as an ionic strength adjuster, a straight calibration curve, Nernstian slope factor 27 mV/decade, is found from 10(-4) to 1 M Ca.
Buffers↗
The effect of early weaning on dentin formation and dentinal caries in rats.
The effect of early weaning on caries progression, dentin formation, and dentin mineralization was examined in four groups of rats. Two groups received a normal diet and were weaned on day 18 or 21, and another two received a sucrose-rich diet and were weaned on day 18 or 21. At age 35 days the lower molars were sectioned sagittally, and the areas of dentin formation and of the dentinal caries were quantified. The width of the predentin zone was measured from histologically stained sections of maxillary molars. Early weaning reduced dentin formation in the group on the high-sucrose diet only the first days; later this effect was partially caught up with. A high-sucrose diet significantly increased caries frequency and extension of caries lesions compared with a normal diet in both early weaned and normally weaned groups. The effect of early weaning on caries frequency and extension in the high-sucrose group was insignificant compared with the normally weaned group on a high-sucrose diet. The predentin zone was wider in the sucrose groups than in the control groups at the end of the experiment. These results indicate that the effect of sucrose on dentin formation was dependent on the stage of physiologic dentin formation, but early weaning as such did not affect this.
Analysis of Variance↗
An immunocytochemical study of the routes of secretion of collagen and phosphophoryn from odontoblasts into dentin.
Polyclonal antibodies to rat incisor phosphophoryns and to the amino-telopeptide of the alpha1 (I)-chain of type I collagen were used to follow the pathways of movement of collagen I (COL1) and phosphophoryns (PP) from synthesis in the odontoblast to secretion into the mineralized dentin. The antibodies were detected at the transmission electron microscopic level by their reaction with Protein A-colloidal gold conjugates. Special care was given in specimen preparation to retention of maximal antigenicity during fixation while maintaining cellular and extracellular ultrastructure at the mineralization front (MF) in nondemineralized sections. Intracellularly, COL1 and PP were detected within the endoplasmic reticulum (ER), the Golgi (G) and secretory granules (SG). However, as determined by double-immunolabeling with different size gold particles the COL1 and PP were not found together within the same ER, G or SG compartments. PP was localized within the tubular ER, round-shaped transitional vesicles, the Golgi and in narrow asymmetric SG. These asymmetric SG were found in abundance in the odontoblastic process. PP secretion from these vesicles was near the MF at the predentin-dentin boundary. COL1 was localized within rosette form ER compartments, the Golgi and in large, distinctive SG. COL1 was deposited at the cell-predentin boundary. No COL1 SG were seen within the odontoblastic process near the MF. In the region of the MF, prior to mineralization, the PP was localized along the surfaces of the COL1 fibrils of the predentin. The mineral phase etched surfaces revealed both COL1- and abundant mineral-associated PP. These data support the hypotheses that, in dentin, the interaction between COL1 and PP may initiate crystal nucleation and that additional interactions between PP and the growing crystals may modulate the crystal growth pattern and crystal size.
Animals↗
The mineral and protein content of enamel in amelogenesis imperfecta.
The purpose of this investigation was to characterize the enamel composition of teeth affected with the hereditary enamel disorders known as amelogenesis imperfecta. Teeth from 10 individuals representing all 3 major AI types (hypocalcified, n = 3; hypomaturation, n = 3; hypoplastic, n = 4) and 10 normal teeth were studied. Half of each tooth was used for histological and biochemical studies. The enamel protein content was estimated by amino acid analysis. The enamel mineral content (volume %) was determined from the calcium and/or phosphorus content. Calcium was measured using atomic absorption and phosphorus was determined colorimetrically. The mean enamel mineral content was reduced for all hypomaturation and hypocalcified AI teeth while hypoplastic AI enamel varied from normal to reduced compared with normal enamel. The enamel protein content was increased in all but one AI case (7 cases were examined for protein) compared with the normal enamel. The mineral and protein content in AI enamel showed a significant inverse relationship (R = -0.939, P = 0.001). This study shows that all three of the major AI groups can have subtypes associated with substantial decreases in the enamel mineral content, although hypomineralization appears most severe in the hypomaturation and hypocalcified AI types. The decreased mineral content was associated with an increased protein content in AI enamel. These findings provide further evidence that altered enamel mineralization in AI teeth likely involves abnormal post-secretory processing of the enamel proteins.
Amelogenesis Imperfecta↗
Effects of thyro-parathyroidectomy and parathyroidectomy upon dentinogenesis: Part I: Light microscopy.
In order to determine the differential effects of the thyroid hormones and the parathyroid hormone upon dentinogenesis in the rat incisor one control group (C) and four groups of surgically treated rats were studied: parathyroid autotransplanted (PTT), thyroidectomized (TX), parathyroidectomized (PTX), and thyro-parathyroidectomized group. One month after surgery the incisors were dissected and the tissues were prepared for light microscopy and morphometric measurements. This study revealed modifications in the TPTX rats as well as in the PTX rats: an enlargement of the predentin, alterations in the predentin appearance and the presence of mineralization defects. These results confirm that the effects observed are probably due to a PTH deficiency and/or hypocalcemia and suggest that their occurrence is associated with a determined stage of dentinogenesis in the rat.
Animals↗
Dentin matrix proteins and dentinogenesis.
The precise mechanisms involved in dentinogenesis are not understood; however, the information to date suggests that a number of highly controlled extracellular events are involved. Mature odontoblasts secrete collagen at the cell border into predentin. They synthesize and secrete other non-collagenous proteins (NCPs) at the mineralization front, possibly through odontoblastic processes. A collagen-NCP complex is formed at the predentin-dentin border and apatite crystal initiation and growth takes place. One of the research needs is to uncover the nature of this dentin collagen-NCP complex and to understand how it controls mineralization. At least three dentin specific NCPs are known: phosphophoryn(s), dentin sialoprotein (DSP) and AG1 (Dmp1). Other macromolecules are commonly made by osteoblasts and odontoblasts and participate in bone and dentin formation. Some progress in understanding dentin mineralization has been gained by focusing upon the role of phosphophoryns. These highly phosphorylated proteins are secreted at the mineralization front, where a small portion binds in the gap region of type I collagen fibrils. This portion of phosphoproteins probably initiates formation of plate-like apatite crystals. Additional phosphoryns in higher concentrations bind to the growing apatite crystals and slow their growth, possibly influencing their size and shape. Other areas which need careful investigations are those involving the mechanisms involved in odontoblast differentiation, how the synthesis of the dentin specific NCPs is controlled and the precise roles of these macromolecules in dentinogenesis. Future experimentation will focus on the gene structures for these NCPs and the mechanisms of tissue specific gene regulation.(ABSTRACT TRUNCATED AT 250 WORDS)
Apatites↗
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↗
Characterization of protein kinases involved in dentinogenesis.
Protein phosphorylation and dephosphorylation control many different cell functions as well as responses to internal and external signals. It has also been shown that highly phosphorylated acidic proteins have an important role in matrix mediated biomineralization, perhaps functioning as nucleators for crystal formation. Dentine phosphoprotein (DPP) is one of such proteins which is exclusively synthesized by the odontoblast cells and therefore a likely candidate to play a significant role in normal and abnormal dentine biomineralization. These studies are directed at characterizing the protein kinases involved in dentinogenesis and in particular the enzyme(s) responsible for DPP phosphorylation. In this report we present data which indicate that there are several different types of kinases in the odontoblast-enriched dental papilla mesenchyme (DPM), some of which can phosphorylate DPP, such as casein kinase I and II. However, a different DPP-kinase activity was identified. This enzyme(s) appears to be different from other reported kinases, and it is the only kinase that can phosphorylate both phosphorylated DPP and enzymatically dephosphorylated DPP.
Acid Phosphatase↗
Temperature sensitive simian virus 40 large T antigen immortalization of murine odontoblast cell cultures: establishment of clonal odontoblast cell line.
During tooth formation instructive epithelial-mesenchymal interactions result in the cytodifferentiation of ectomesenchymal cells into odontoblasts which produce the dentin extracellular matrix (DECM). The purpose of our study was to establish a stable murine odontoblast cell line by immortalization of odontoblasts using retrovirus transfection. In order to accomplish this goal, we utilized a previously characterized odontoblast monolayer cell culture system supportive of odontoblast cytodifferentiation from dental papilla mesenchyme (DPM), expression and secretion of a DECM and dentin biomineralization. First mandibular molars from E-18 Swiss Webster mice were dissected, the DPM isolated, and pulp cells dissociated. Pulp cells (5 x 10(5)/well) were plated as monolayers and grown in alpha-MEM supplemented with 10% FCS, 100 units/ml penicillin and streptomycin, 50 micrograms/ml ascorbic acid. Cultures were maintained for 6 days at 37 degrees C in a humidified atmosphere of 95% air and 5% CO2, with media changes every two days. Immortalization was performed using a recombinant defective retrovirus containing the temperature sensitive SV-40 large T antigen cDNA and the neomycin (G418) resistance gene recovered from CRE packaging cells. Cultures were infected for 24 h with CRE conditioned medium containing 8 micrograms/ml of polybrene, the media was replaced with selective media containing 300 micrograms/ml of G418, and the cultures incubated at 33 degrees C for one month with media changes every 3-5 days. Neomycin resistant cells were cloned by serial dilution to single cells in 96-well culture plates and grown in selection medium at 33 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Lipids in predentine and dentine.
Using two histochemical methods, malachite green-aldehyde and iodoplatinate, phospholipids were visualized in the predentine of rat incisors in the spaces located between collagen fibers and in dentine as needle-like structures located along individual or groups of mineralizing collagen fibers. The same staining pattern was seen with phospholipase A2-gold. Autoradiographic investigation using 3H choline as labelled precursor, visualized the incorporation of membrane-associated and extracellular choline-containing phosphatidyl choline and sphingomyelin. The cell and membrane-associated labelling decreased gradually between 24 and 4 days, whereas incorporation of the labelled precursor as stable extracellular matrix component was seen in dentine. In addition to these investigations, pharmacologically induced (suramine) and genetically (Krabbe's disease) lysosomal storage pathology was investigated. Defects due to lipid metabolism alterations were seen in predentine and/or in dentine. The major differences visualized here between the non-mineralized and mineralized compartments and interactions between phospholipids and proteoglycans, support the view that phospholipids as matrix components play an important role in the mechanisms of dentine formation and mineralization.
Animals↗