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Hayato Ohshima - One of the best experts on this subject based on the ideXlab platform.
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lymphoid enhancer binding factor 1 expression precedes dentin sialophosphoprotein expression during rat Odontoblast differentiation and regeneration
Journal of Endodontics, 2013Co-Authors: Mitsushiro Nakatomi, Hiroko Idayonemochi, Hayato OhshimaAbstract:Abstract Introduction The molecular mechanisms behind Odontoblast differentiation remain obscure. Lymphoid enhancer-binding factor 1 (Lef1) is a transcription factor that mediates Wnt signaling and has been suggested to regulate dentin sialophosphoprotein ( Dspp ) expression in vitro . This study aimed to clarify their precise relationship in the process of Odontoblast differentiation in vivo . Methods The detailed spatiotemporal expression patterns of Lef1 and Dspp together with other known and putative Odontoblast differentiation markers such as P21 and heat-shock protein 25 ( Hsp25 ) were examined by in situ hybridization and immunohistochemistry on paraffin sections of rat incisors and developing molars at postnatal days 1–100. To observe Odontoblast regeneration following tooth injury, a cavity was prepared on the upper first molar of 10-week-old rats and the expressions of Lef1 and Dspp were investigated. Results Following undifferentiated state expressing none of these examined markers, preOdontoblasts begun to express P21 , Lef1 and Hsp25 according to their progress of differentiation, although Dspp was undetectable. Immature Odontoblasts commenced transcribing Dspp simultaneously with dentin calcification. Lef1, Dspp and Hsp25 were co-expressed in mature Odontoblasts. In contrast to continuously growing incisors, Lef1, Dspp and P21 were down-regulated in the resting Odontoblasts in molars when primary dentin formation was completed. Remarkably, Lef1 expression also preceded Dspp expression in newly differentiated Odontoblast-like cells during the pulpal healing process after tooth injury. Conclusions Lef1 expression precedes Dspp expression without exception in both primary and reparative dentinogeneses. Our results suggest that Lef1 might play a key role in Odontoblast differentiation through regulating Dspp expression.
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expression patterns of nestin and dentin sialoprotein during dentinogenesis in mice
Biomedical Research-tokyo, 2012Co-Authors: Angela Quispesalcedo, Mitsushiro Nakatomi, Hiroko Idayonemochi, Hayato OhshimaAbstract:Differentiated Odontoblasts could not be identified by one unique phenotypic marker, but the combination of expression of dentin phosphoprotein (Dpp), dentin sialoprotein (Dsp), dentin matrix protein 1 (Dmp1), and nestin may be valuable for the assessment of these cells. However, the findings using these proteins remain controversial. This study aimed to compare two Odontoblast differentiation markers: nestin and Dsp in the process of dentinogenesis in mice. We performed immunohistochemistry and/or in situ hybridization technique for nestin and Dsp using 3-week-old incisors as well as postnatal 1-day- to 8-week-old molars. PreOdontoblasts began to express nestin and Dsp proteins and Dsp mRNA, which increased in their intensity according to the progress of Odontoblast differentiation in both incisors and developing molars. Nestin was consistently expressed in the differentiated Odontoblasts even after the completion of dentin matrix deposition. The expression of Dsp mRNA coincided with the Odontoblast secretory activity for dentin matrix deposition. In contrast, other pulpal cells, predentin matrix and dentinal tubules also showed a positive reaction for Dsp protein in addition to differentiated Odontoblasts. In conclusion, nestin is valuable as a differentiation marker for Odontoblasts, whereas Dsp mRNA is a functional marker for their secretory activity.
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Odontoblast responses to gaalas laser irradiation in rat molars an experimental study using heat shock protein 25 immunohistochemistry
European Journal of Oral Sciences, 2006Co-Authors: Yasuaki Tate, Kunihiko Yoshiba, Nagako Yoshiba, Masaaki Iwaku, Takashi Okiji, Hayato OhshimaAbstract:: Pulpal responses to gallium-aluminum-arsenide (GaAlAs) laser irradiation applied to the tooth remains to be elucidated. This study aimed to evaluate the effect of the GaAlAs laser on Odontoblasts using immunohistochemistry for heat-shock protein (HSP)-25, which labels mature and newly differentiated Odontoblasts. The mesial surface of the upper right first molar of 8-wk-old Wistar rats was lased at an output power of 0.5-1.5 W for 180 s. The animals were perfusion-fixed at intervals of 6 h to 30 d after irradiation. At 6 h to 7 d, the intensity of HSP-25-immunoreactivity was found to be disturbed in the coronal Odontoblast-layer in an energy-dependent manner. At 30 d, tertiary dentin with/without bone-like tissue was formed abundantly in the dental pulp. Statistical analysis revealed that the area occupied by the new hard tissues was significantly wider in 1.5 W-lased specimens than in 0.5 W-lased specimens. An intense HSP-25 immunoreactivity was seen in the Odontoblasts underlying the tertiary dentin, whereas immunoreactivity was weak around the bone-like tissue. It was concluded that the GaAlAs laser may induce the formation of tertiary dentin by influencing the secretory activity of Odontoblasts. However, higher energies may cause irreversible changes to the pulp, often leading to the formation of an intrapulpal bone-like tissue.
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An immunocytochemical study of pulpal responses to cavity preparation by laser ablation in rat molars by using antibodies to heat shock protein (Hsp) 25 and class II MHC antigen
Cell and Tissue Research, 2004Co-Authors: Takeshi Suzuki, Shuichi Nomura, Takeyasu Maeda, Hayato OhshimaAbstract:Initial responses of Odontoblasts and immunocompetent cells to cavity preparation by laser ablation were investigated in rat molars. In untreated control teeth, intense heat shock protein (Hsp) 25 immunoreactivity was found in the cell bodies of Odontoblasts, whereas cells immunopositive for the class II major histocompatibility complex (MHC) antigen were predominantly located beneath the Odontoblast layer in the dental pulp. Cavity preparation caused the destruction of the Odontoblast layer and the shift of most class-II-MHC-positive cells from the pulp-dentin border toward the pulp core at the affected site. Twelve hours after cavity preparation, numerous class-II-MHC-positive cells appeared along the pulp-dentin border and extended their processes deep into the exposed dentinal tubules, but subsequently disappeared from the pulp-dentin border together with Hsp-25-immunopositive cells by 24 h after the operation. By 3–5 days postoperation, distinct abscess formation consisting of polymorphonuclear leukocytes was found in the dental pulp. The penetration of masses of oral bacteria was recognizable in the dentinal tubules beneath the prepared cavity. These findings indicate that cavity preparation by laser ablation induces remarkable inflammation by continuous bacterial infections via dentinal tubules in this experimental model, thereby delaying pulpal regeneration.
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possible role of immunocompetent cells and the expression of heat shock protein 25 in the process of pulpal regeneration after tooth injury in rat molars
Journal of Electron Microscopy, 2003Co-Authors: Kuniko Nakakuraohshima, Junichi Watanabe, Shinichi Kenmotsu, Hayato OhshimaAbstract:Recent studies have established that heat shock proteins (HSPs) potentially play a role in immunosurveillance. The purpose of the present study was to clarify the relationship between the chronological changes of immunocompetent cells and the expression of HSP-25 in the process of pulpal regeneration after tooth injury in rat molars by immunocytochemistry for HSP-25 and class II major histocompatibility complex (MHC ) antigen. In untreated control teeth, intense HSP-25 immunoreactivity was found in the cell bodies of Odontoblasts. Both cavity preparation and tooth replantation caused the degeneration of the Odontoblast layer to result in the loss of HSP-25 immunoreactions in the suffered dental pulp at the early stages after tooth injury. Numerous class II MHC-positive cells appeared along the pulp-dentin border and extended their cell processes into the dentinal tubules at 12-24 h after cavity preparation and 3 days after tooth replantation. Newly differentiated Odontoblast-like cells with HSP-25 immunoreactivity were arranged at the pulp-dentin border and the class II MHC-positive cells retreated towards the subOdontoblastic layer by post-operative days 3-5 after tooth injury. Thus, the common cellular events occur during pulpal regeneration following two different experimental injuries. These findings indicate that the time course of changes in the expression of HSP-25 immunoreactivity reflects the degeneration/regeneration process of Odontoblasts and that the temporal appearance of the class II MHC-positive cells at the pulp-dentin border suggests their participation in Odontoblast differentiation as well as in initial defence reactions during the pulpal regeneration process.
Anthony J Smith - One of the best experts on this subject based on the ideXlab platform.
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lipopolysaccharide enhances decorin expression through the toll like receptor 4 myeloid differentiating factor 88 nuclear factor kappa b and mitogen activated protein kinase pathways in Odontoblast cells
Journal of Endodontics, 2012Co-Authors: Wenxi He, Qing Yu, Tiejun Qu, Zhihua Wang, Hanguo Wang, Jing Zhang, Anthony J SmithAbstract:Abstract Introduction Lipopolysaccharide (LPS) has been shown to regulate the function of Odontoblasts. However, the molecular mechanisms of the effect of LPS on Odontoblasts are poorly understood. Decorin (DCN), one of the major matrix proteoglycans, is known to affect the mineralization of teeth. In this study, we investigated whether LPS can regulate the expression of DCN in Odontoblasts and determined the intracellular signaling pathways triggered by LPS. Methods The DCN messenger RNA and protein expression changes in mouse Odontoblast-lineage cells (OLCs) were detected by real-time polymerase chain reaction (PCR) analysis and enzyme-linked immunosorbent assay (ELISA). Whether TLR4, myeloid differentiating factor 88 (MyD88), nuclear factor-kappa B (NF-κB), or mitogen-activated protein kinase (MAPK) pathways were involved in the LPS-induced DCN expression was determined by examined real-time PCR, ELISA, and luciferase activity assay. The activation of extracellular signal-regulated kinase (ERK), p38, and JNK in OLCs was measured by Western blot analysis. Results We found that the mouse OLCs expressed DCN. DCN messenger RNA was rapidly induced by LPS in a time- and dose-dependent manner. Pretreatment with a MyD88 inhibitory peptide, a TLR4 antibody, or a specific inhibitor for NF-κB or I Kappa B alpha (IκBα) significantly inhibited LPS-induced DCN expression. Moreover, the LPS-mediated increase in κB-luciferase activity in OLCs was suppressed by the overexpression of dominant negative mutants of TLR4, MyD88, and IκBα but not by a dominant negative mutant of TLR2. In addition, LPS stimulation activated the ERK, p38, and JNK MAPK pathways. The pretreatment of OLCs with specific inhibitors of the ERK, p38, and JNK MAPK pathways markedly offset the LPS-induced up-regulation of DCN expression. Conclusions Our results show that LPS stimulation can up-regulate the gene expression of DCN via the TLR4, MyD88, NF-κB, and MAPK pathways in Odontoblast cells.
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the map kinase pathway is involved in Odontoblast stimulation via p38 phosphorylation
Journal of Endodontics, 2010Co-Authors: Stephane Simon, Ariane Berdal, Anthony J Smith, P J Lumley, Paul R CooperAbstract:Abstract Introduction We have previously shown that the p38 gene is highly expressed in Odontoblasts during active primary dentinogenesis, but is drastically down-regulated as cells become quiescent in secondary dentinogenesis. Based on these observations, we hypothesized that p38 expression might be upregulated, and the protein activated by phosphorylation, when Odontoblasts are stimulated such as during tertiary reactionary dentinogenesis. Methods We stimulated immortalized, Odontoblast-like MDPC-23 cells, alone or in combination, with heat-inactivated Streptococcus mutans , EDTA-extracted dentine matrix proteins (DMPs), or growth factors, including transforming growth factor (TGF)-β1, tumor necrosis factor-α (TNF-α), and adrenomedullin (ADM). We used ELISA to measure the resulting phosphorylation of the p38 protein, as well as its degree of nuclear translocation. Results Our results suggest that the p38-MAPKinase pathway is activated during Odontoblast stimulation in tertiary dentinogenesis by both p38 phosphorylation and enhanced nuclear translocation. Conclusions Data indicate that Odontoblast behaviour therefore potentially recapitulates that during active primary dentinogenesis.
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short term in vitro effects of low frequency ultrasound on Odontoblast like cells
Ultrasound in Medicine and Biology, 2007Co-Authors: Ben A Scheven, Paul R Cooper, Jane Millard, Damien A Walmsley, Anthony J SmithAbstract:Abstract In this study, the effects of low frequency ultrasound (US) were examined on Odontoblasts, the primary cell responsible for dentinogenesis and dentine repair. An established Odontoblast-like cell line, MDPC-23, was subjected to 30 kHz ultrasound at three different power settings. US induced a marginal level of cell death (3% to 4%) at lower amplitudes rising to 25% cell death at the highest power tested. The latter was reflected in a 30% decrease in cell attachment after 4 to 24 h of culture, while the number of adherent cells was reduced by approximately 10% to 15% in the lower power groups. Cell replication after 24 h, as measured by BrdU incorporation, showed no significant changes in the US-treated groups. Gene expression analyses demonstrated a moderate dose-dependent increase in the expression of GAPDH (glyseraldehyde-3-phosphate dehydrogenase)-normalised collagen type I, osteopontin (OPN), transforming growth factor-β1 (TGFβ1) and the heat shock protein (hsp) 70. The greatest change was found in the expression of the small hsp 25/27, which showed a two- to six-fold increase following US treatment. No significant effects were observed for alkaline phosphatase (ALP) and core-binding factor A1 (CBFA1/Runx2) expression levels. This is the first report describing US effects on Odontoblasts. Further studies are warranted to elucidate US effects on Odontoblast function and to evaluate US as a therapeutic application in dentine repair. (E-mail: b.a.scheven@bham.ac.uk )
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smad protein mediated transforming growth factor β1 induction of apoptosis in the mdpc 23 Odontoblast like cell line
Archives of Oral Biology, 2005Co-Authors: Wenxi He, Shouliang Zhao, Anthony J SmithAbstract:Summary 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)-β 1 in the induction of apoptosis and the molecular mechanisms that mediate TGF-β1-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-β1 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-β1 in MDPC-23 cells. The TGF-β1-induced transcriptional activity in stable cell clones expressing Smad proteins was analyzed by a transient transfected TGF-β-responsive reporter gene, p3TP-Lux. Results: TGF-β1 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-β1-induced apoptosis; moreover, the Smad3 mutant was more efficient than the Smad2 mutant. Transfection of Smad7, an inhibitory Smad, also significantly inhibited TGF-β1-induced apoptosis of these cells. Over-expression of Smad3 dominant negative mutant or Smad7 significantly inhibited TGF-β1-induced transcriptional activity. Conclusion: These results suggest that Smad proteins are involved in TGF-β1-induced apoptosis of Odontoblast cells.
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induction and regulation of crown dentinogenesis embryonic events as a template for dental tissue repair
Critical Reviews in Oral Biology & Medicine, 2001Co-Authors: Anthony J Smith, H LesotAbstract:Close regulation of Odontoblast differentiation and subsequent secretory activity is critical for dentinogenesis during both embryogenesis and tissue repair. Some dental papilla cells achieve commitment and specific competence, allowing them to respond to epithelially derived inductive signals during the process of Odontoblast differentiation. Temporo-spatial regulation of Odontoblast differentiation is dependent on matrix-mediated interactions involving the basement membrane (BM). Experimental studies have highlighted the possible roles of growth factors in these processes. Regulation of functional activity of Odontoblasts allows for both ordered secretion of the primary dentin matrix and maintenance of vitality and down-regulation of secretory activity throughout secondary dentinogenesis. After injury to the mature tooth, the fate of the Odontoblast can vary according to the intensity of the injury. Milder injury can result in up-regulation of functional activity leading to focal secretion of a reaction...
Françoise Bleicher - One of the best experts on this subject based on the ideXlab platform.
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Mechanosensitive Ion Channels in Odontoblasts
Mechanosensitivity in Cells and Tissues, 2020Co-Authors: Henry Magloire, Bruno Allard, Marie-lise Couble, Jean-christophe Maurin, Françoise BleicherAbstract:Odontoblasts are post-mitotic cells involved in the dentine formation throughout the life of the tooth and suspected to play a role in tooth pain trans- mission. They are organized as a single layer of specialized cells along the interface between dental pulp and calcified dentinal tubules into which run a cellular exten- sion (Odontoblast process) bathed in a liquid phase. Dense sensory unmyelinated nerve fibres surrounded the Odontoblast bodies, coiled around the cell processes and give to this complex (nerve/Odontoblast) a fundamental role as a barrier regulating molecules, fluid flow, ion transferts between dentine and pulp following external stimuli (mechanical thermal, electrical, osmotic shock. ..) . Thus, this unique spatial situation of Odontoblasts closely related with nerve endings and fluid movements suggest that Odontoblasts could convert pain-evoking fluid displacement within dentinal tubules into electrical signals via at least mechanosensitive ion channels. Along this line, two kinds of mechanosensitive K + channels have been identified in human Odontoblasts: I- TREK-1 channels belonging to the two-pore-domain potassium channel family and expressed in the plasma membrane of coronal odon- toblasts; II- high-conductance Ca 2+ -activated potassium channels (KCa) activated by stretch of the membrane as well as osmotic shock. These findings strengthened by the recent evidence for excitable properties of Odontoblasts, concentration of mechanosensitive channels in the borderline between cell extension and bodies and clustering of key molecules at the site of Odontoblast-nerve contact strongly suggest that Odontoblasts may operate as sensor cells.
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Different Roles of Odontoblasts and Fibroblasts in Immunity
Journal of Dental Research, 2020Co-Authors: Marie-jeanne Staquet, Françoise Bleicher, S.h. Durand, Evelyne Colomb, Annick Romeas, Claude Vincent, Serge Lebecque, Jean-christophe FargesAbstract:Odontoblasts and fibroblasts are suspected to influence the innate immune response triggered in the dental pulp by micro-organisms that progressively invade the human tooth during the caries process. To determine whether they differ in their responses to oral pathogens, we performed a systematic comparative analysis of Odontoblast-like cell and pulp fibroblast responses to TLR2-, TLR3-, and TLR4-specific agonists (lipoteichoic acid [LTA], double-stranded RNA, and lipopolysaccharide [LPS], respectively). Cells responded to these agonists by differential up-regulation of chemokine gene expression. CXCL2 and CXCL10 were thus increased by LTA only in Odontoblast-like cells, while LPS increased CCL7, CCL26, and CXCL11 only in fibroblasts. Supernatants of stimulated cultures increased migration of immature dendritic cells compared with controls, Odontoblast-like cells being more potent attractants than fibroblasts. Analysis of these data suggests that Odontoblasts and pulp fibroblasts differ in their innate imm...
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Odontoblast primary cilia: facts and hypotheses.
Cell Biology International, 2020Co-Authors: Henry Magloire, Marie-lise Couble, Annick Romeas, Françoise BleicherAbstract: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 α 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.
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Exostosin 1 is expressed in human Odontoblasts.
Archives of Oral Biology, 2017Co-Authors: Virve Pääkkönen, Françoise Bleicher, Stina Saraniemi, Zvi Nevo, Leo TjäderhaneAbstract:Abstract Objective Dental pulp is soft connective tissue maintaining the vitality of the tooth, while Odontoblasts form the dentin. Our earlier DNA microarray analysis revealed expression of putative tumour suppressor exostosin 1 (EXT-1) in Odontoblasts. EXT-1 is essential for heparan sulphate synthesis, which may play a role in the dentin mineralization. Since the absence of the functional EXT-1 causes bone tumours, expression in Odontoblasts is interesting. Our aim was to analyse further the EXT-1 expression in human tooth. Designs DNA microarray and PCR techniques were used to study the EXT-1 expression in mature native human Odontoblasts and pulp tissue as well as in newly-differentiated cultured Odontoblast-like cells. Immunohistochemistry was performed to study EXT-1 protein in mature human teeth, teeth with incomplete root and developing teeth. Results Markedly higher EXT-1 was observed in mature Odontoblasts than in pulp at mRNA level with DNA microarray and PCR techniques. Immunohistochemistry of mature tooth revealed EXT-1 both in Odontoblasts and the predentin but not in the dentin. EXT-1 was also observed in the Odontoblasts of incomplete root, but the localization of the staining was different. In developing foetal tooth, staining was detected in ameloblasts and the basal lamina. Conclusions The detection of EXT-1 in both mature and newly-differentiated cells indicates a role in the Odontoblast function, and EXT-1 staining in the predentin indicates a function in the dentin formation. Detection of EXT-1 in developing teeth indicates a role in tooth development.
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human Odontoblast like cells produce nitric oxide with antibacterial activity upon tlr2 activation
Frontiers in Physiology, 2015Co-Authors: Jean-christophe Farges, Françoise Bleicher, Aurelie Bellanger, Maxime Ducret, Elisabeth Aubertfoucher, Beatrice Richard, Brigitte Alliotlicht, Florence CarrouelAbstract:The penetration of cariogenic oral bacteria into enamel and dentin during the caries process triggers an immune/inflammatory response in the underlying pulp tissue, the reduction of which is considered a prerequisite to dentinogenesis-based pulp regeneration. If the role of Odontoblasts in dentin formation is well known, their involvement in the antibacterial response of the dental pulp to cariogenic microorganisms has yet to be elucidated. Our aim here was to determine if Odontoblasts produce nitric oxide (NO) with antibacterial activity upon activation of Toll-like receptor-2 (TLR2), a cell membrane receptor involved in the recognition of cariogenic Gram-positive bacteria. Human Odontoblast-like cells differentiated from dental pulp explants were stimulated with the TLR2 synthetic agonist Pam2CSK4. We found that NOS1, NOS2 and NOS3 gene expression was increased in Pam2CSK4-stimulated Odontoblast-like cells compared to unstimulated ones. NOS2 was the most up-regulated gene. NOS1 and NOS3 proteins were not detected in Pam2CSK4-stimulated or control cultures. NOS2 protein synthesis, NOS activity and NO extracellular release were all augmented in stimulated samples. Pam2CSK4-stimulated cell supernatants reduced Streptococcus mutans growth, an effect counteracted by the NOS inhibitor L-NAME. In vivo, the NOS2 gene was up-regulated in the inflamed pulp of carious teeth compared with healthy ones. NOS2 protein was immunolocalized in Odontoblasts situated beneath the caries lesion but not in pulp cells from healthy teeth. These results suggest that Odontoblasts may participate to the antimicrobial pulp response to dentin-invading Gram-positive bacteria through NOS2-mediated NO production. They might in this manner pave the way for accurate dental pulp healing and regeneration.
Anne George - One of the best experts on this subject based on the ideXlab platform.
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dentin matrix protein 4 a novel secretory calcium binding protein that modulates Odontoblast differentiation
Journal of Biological Chemistry, 2007Co-Authors: Karthikeyan Narayanan, Amsaveni Ramachandran, Tanvi Muni, Anne GeorgeAbstract:Abstract Formation of calcified tissues is a well regulated process. In dentin, the Odontoblasts synthesize several biomolecules that function as nucleators or inhibitors of mineralization. To identify genes that are Odontoblast-specific, a subtractive hybridization technique was employed that resulted in the identification of a previously undescribed novel gene synthesized by the Odontoblasts. Based on the nomenclature in our laboratory, this gene has been named dentin matrix protein 4 (DMP4). The protein encoded by mouse DMP4 cDNA contained 579 amino acids, including a 26-amino acid signal peptide. Analysis of the protein sequence demonstrated the presence of a Greek key calcium-binding domain and one conserved domain of unknown function in all the species examined thus far. Calcium binding property was confirmed by 45Ca binding assays and the corresponding change in conformation by far-ultraviolet circular dichroism. Northern analysis demonstrated high expression levels of a single 3-kb mRNA transcript in tooth, whereas low expression levels were detected in other tissues. In situ hybridization analysis showed high expression levels of DMP4 in Odontoblasts and low levels in osteoblasts and ameloblasts during tooth development. Gain and loss of function experiments demonstrated that DMP4 had the potential to differentiate mesenchymal precursor cells into functional Odontoblast-like cells.
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Odontoblast cells immortalized by telomerase produce mineralized dentin like tissue both in vitro and in vivo
Journal of Biological Chemistry, 2002Co-Authors: Karthikeyan Narayanan, Amsaveni Ramachandran, Gen He, Abdullah Almushayt, Carla A Evans, Anne GeorgeAbstract:Abstract The formation of dentin provides one well accepted paradigm for studying mineralized tissue formation. For the assembly of dentin, several cellular signaling pathways cooperate to provide neural crest-derived mesenchymal cells with positional information. Further, “cross-talk” between signaling pathways from the mesenchymal derived Odontoblast cells and the epithelially derived ameloblasts during development is responsible for the formation of functional Odontoblasts. These intercellular signals are tightly regulated, both temporally and spatially. When isolated from the developing tooth germ, Odontoblasts quickly lose their potential to maintain the Odontoblast-specific phenotype. Therefore, generation of an Odontoblast cell line would be a valuable reproducible tool for studying the modulatory effects involved in Odontoblast differentiation as well as the molecular events involved in mineralized dentin formation. In this study an immortalized Odontoblast cell line, which has the required biochemical machinery to produce mineralized tissuein vitro, has been generated. These cells were implanted into animal models to determine their in vivo effects on dentin formation. After implantation, we observed a multistep, programmed cascade of gene expression in the exogenous Odontoblasts as the dentin formed de novo. Some of the genes expressed include the dentin matrix proteins 1, 2, and 3, which are extracellular matrix molecules responsible for the ultimate formation of mineralized dentin. The biological response was also examined by histology and radiography and confirmed for mineral deposition by von Kossa staining. Thus, a transformed Odontoblast cell line was created with high proliferative capacity that might ultimately be used for the regeneration and repair of dentin in vivo.
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differentiation of embryonic mesenchymal cells to Odontoblast like cells by overexpression of dentin matrix protein 1
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Karthikeyan Narayanan, Amsaveni Ramachandran, Rampalli Srinivas, Bruce Quinn, Anne GeorgeAbstract:Abstract Cells of the craniofacial skeleton are derived from a common mesenchymal progenitor. The regulatory factors that control their differentiation into various cell lineages are unknown. To investigate the biological function of dentin matrix protein 1 (DMP1), an extracellular matrix gene involved in calcified tissue formation, stable transgenic cell lines and adenovirally infected cells overexpressing DMP1 were generated. The findings in this paper demonstrate that overexpression of DMP1 in pluripotent and mesenchyme-derived cells such as C3H10T1/2, MC3T3-E1, and RPC-C2A can induce these cells to differentiate and form functional Odontoblast-like cells. Functional differentiation of Odontoblasts requires unique sets of genes being turned on and off in a growth- and differentiation-specific manner. The genes studied include transcription factors like core binding factor 1 (Cbfa1), bone morphogenetic protein 2 (BMP2), and BMP4; early markers for extracellular matrix deposition like alkaline phosphatase (ALP), osteopontin, osteonectin, and osteocalcin; and late markers like DMP2 and dentin sialoprotein (DSP) that are expressed by terminally differentiated Odontoblasts and are responsible for the formation of tissue-specific dentin matrix. However, this differentiation pathway was limited to mesenchyme-derived cells only. Other cell lines tested by the adenoviral expression system failed to express Odontoblast-phenotypic specific genes. An in vitro mineralized nodule formation assay demonstrated that overexpressed cells could differentiate and form a mineralized matrix. Furthermore, we also demonstrate that phosphorylation of Cbfa1 (osteoblast-specific transcription factor) was not required for the expression of Odontoblast-specific genes, indicating the involvement of other unidentified Odontoblast-specific transcription factors or coactivators. Cell lines that differentiate into Odontoblast-like cells are useful tools for studying the mechanism involved in the terminal differentiation process of these postmitotic cells.
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in situ localization and chromosomal mapping of the ag1 dmp1 gene
Journal of Histochemistry and Cytochemistry, 1994Co-Authors: Anne George, Nancy A Jenkins, Debra J Gilbert, Neal G Copeland, Arthur VeisAbstract:Dentinogenesis is being used as a model for understanding the biomineralization process. The Odontoblasts synthesize a structural matrix comprised of Type I collagen fibrils which define the basic architecture of the tissue. The Odontoblasts also synthesize and deliver a number of dentin-specific acidic macromolecules into the extracellular compartment. These acidic macromolecules may be involved in regulating the ordered deposition of hydroxyapatite crystals within the matrix. AG1 is the first tooth-specific acidic macromolecule to have been cloned and sequenced. To identify which cells of the rat incisor pulp/Odontoblast complex were responsible for synthesis of AG1, in situ hybridization was used. Digoxigenin labeled sense and anti-sense AG1 riboprobes were prepared. The AG1 mRNA was found to be expressed in the mature secretory Odontoblasts. Neither pulp cells nor pre-Odontoblasts showed any staining with the anti-sense probes. Chromosomal localization studies placed the AG1 gene on mouse chromosome 5...
Rulang Jiang - One of the best experts on this subject based on the ideXlab platform.
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β catenin is required in Odontoblasts for tooth root formation
Journal of Dental Research, 2013Co-Authors: Seungo Ko, Xiao Yang, Rulang JiangAbstract:The tooth root is an important part of the tooth that works together with the surrounding periodontium to maintain the tooth in the alveolar socket. The root develops after crown morphogenesis. While the molecular and cellular mechanisms of early tooth development and crown morphogenesis have been extensively studied, little is known about the molecular mechanisms controlling tooth root formation. Here, we show that β-catenin is strongly expressed in Odontoblast-lineage cells and is required for root formation. Tissue-specific inactivation of β-catenin in developing Odontoblasts produced molars lacking roots and aberrantly thin incisors. At the beginning of root formation in the mutant molars, the cervical loop epithelium extended apically to form Hertwig’s epithelial root sheath (HERS), but root Odontoblast differentiation was disrupted and followed by the loss of some HERS inner layer cells. However, the outer layer of the HERS extended without the root, and the mutant molars finally erupted. The period...
