Kazuhiko Nakata

Aichi Gakuin University, Nagoya, Aichi, Japan

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Publications (31)74.52 Total impact

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    ABSTRACT: We previously established a method for the differentiation of induced pluripotent stem cells and embryonic stem cells into α2 integrin-positive odontoblast-like cells. We also reported that Wnt5 in response to interleukin (IL)-1β induces matrix metalloproteinase (MMP)-3-regulated cell proliferation in these cells. Our findings suggest that MMP-3 plays a potentially unique physiological role in the generation of odontoblast-like cells under an inflammatory state. Here, we examined whether up-regulation of autophagy-related gene (Atg) 5 by IL-1β was mediated by Wnt5 signaling, thus leading to increased proliferation of odontoblast-like cells. IL-1β increased the mRNA and protein levels of Atg5, microtubule-associated protein 1 light chain (LC3, a mammalian homolog of yeast Atg8) and Atg12. Treatment with siRNAs against Atg5, but not LC3 and Atg12, suppressed the IL-1β-induced increase in MMP-3 expression and cell proliferation. Our siRNA analyses combined with western blot analysis revealed a unique sequential cascade involving Atg5, Wnt5a and MMP-3, which resulted in the potent increase in odontoblastic cell proliferation. These results demonstrate the unique involvement of Atg5 in IL-1β-induced proliferation of embryonic stem cell-derived odontoblast-like cells.
    PLoS ONE 03/2015; in press. · 3.53 Impact Factor
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    ABSTRACT: Inorganic polyphosphate [Poly(P)] may represent a physiological source of phosphate and has the ability to induce bone differentiation in osteoblasts. We previously reported that cytokine-induced matrix metalloproteinase (MMP)-3 accelerates the proliferation of purified odontoblast-like cells. In this study, MMP-3 small interfering RNA (siRNA) was transfected into odontoblast-like cells derived from induced pluripotent stem cells to investigate whether MMP-3 activity is induced by Poly(P) and/or is associated with cell proliferation and differentiation into odontoblast-like cells. Treatment with Poly(P) led to an increase in both cell proliferation and additional odontoblastic differentiation. Poly(P)-treated cells showed a small but significant increase in dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1) mRNA expression, which are markers of mature odontoblasts. The cells also acquired additional odontoblast-specific properties including adoption of an odontoblastic phenotype typified by high alkaline phosphatase (ALP) activity and a calcification capacity. In addition, Poly(P) induced expression of MMP-3 mRNA and protein, and increased MMP-3 activity. MMP-3 siRNA-mediated disruption of the expression of these effectors potently suppressed the expression of odontoblastic biomarkers ALP, DSPP, and DMP-1, and blocked calcification. Interestingly, upon siRNA-mediated silencing of MMP-3, we noted a potent and significant decrease in cell proliferation. Using specific siRNAs, we revealed that a unique signaling cascade, Poly(P)→MMP-3→DSPP and/or DMP-1, was intimately involved in the proliferation of odontoblast-like cells.
    Experimental Cell Research 01/2015; in press. DOI:10.1016/j.yexcr.2015.01.007 · 3.37 Impact Factor
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    ABSTRACT: The aim of this study was to examine cerebral blood volume dynamics during volitional swallowing using multi-channel functional near-infrared spectroscopy (fNIRS) to understand the basic cortical activation patterns. Fifteen volunteers (age, 26.5±1.3 years, mean±SD) performed volitional swallowing of a 5-ml bolus of water as a task. A 52-channel fNIRS system was used for measuring oxy-Hb levels. We determined the oxy-Hb concentration changes in each channel by calculating the differences between rest and task oxy-Hb levels. Differences in rest and task data were assessed using a paired-t test (p< 0.05). A significant increase in oxy-Hb was found in 21 channels. The cortical regions that exhibited increased oxy-Hb concentration included the bilateral precentral gyrus, postcentral gyrus, inferior frontal gyrus, superior temporal gyrus, middle temporal gyrus, and supramarginal gyrus. These data provide the first description of cortical activation patterns during volitional swallowing using fNIRS, which will be useful for the evaluation of dysphasia and the effects of the rehabilitation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.
    Neuroscience Letters 12/2014; 588. DOI:10.1016/j.neulet.2014.12.034 · 2.06 Impact Factor
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    ABSTRACT: The C-shaped root canal constitutes an unusual root morphology that can be found primarily in mandibular second permanent molars. Due to the complexity of their structure, C-shaped root canal systems may complicate endodontic interventions. A thorough understanding of root canal morphology is therefore imperative for proper diagnosis and successful treatment. This review aims to summarize current knowledge regarding C-shaped roots and root canals, from basic morphology to advanced endodontic procedures. To this end, a systematic search was conducted using the MEDLINE, BIOSIS, Cochrane Library, EMBASE, Google Scholar, Web of Science, PLoS, and BioMed Central databases, and many rarely-cited articles were included. Furthermore, four interactive 3D models of extracted teeth are introduced that will allow for a better understanding of the complex C-shaped root canal morphology. In addition, the present publication includes an embedded best-practice video showing an exemplary root canal procedure on a tooth with a pronounced C-shaped root canal. The survey of this unusual structure concludes with a number of suggestions concerning future research efforts. This article is protected by copyright. All rights reserved.
    International Endodontic Journal 09/2014; 47:1012-1033. DOI:10.1111/iej.12256 · 2.27 Impact Factor
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    ABSTRACT: A pro-inflammatory cytokine mixture (CM: interleukin (IL)-1β, tumor necrosis factor-α and interferon-γ) and IL-1β-induced matrix metalloproteinase (MMP)-3 activity have been shown to increase the proliferation of rat dental pulp cells and murine stem cell-derived odontoblast-like cells. This suggests that MMP-3 may regulate wound healing and regeneration in the odontoblast-rich dental pulp. Here, we determined whether these results can be extrapolated to human dental pulp by investigating the effects of CM-induced MMP-3 up-regulation on the proliferation and apoptosis of purified odontoblast-like cells derived from human skeletal muscle stem cells. We used siRNA to specifically reduce MMP-3 expression. We found that CM treatment increased MMP-3 mRNA and protein levels as well as MMP-3 activity. Cell proliferation was also markedly increased, with no changes in apoptosis, upon treatment with CM and following the application of exogenous MMP-3. Endogenous tissue inhibitors of metalloproteinases were constitutively expressed during all experiments and unaffected by MMP-3. Although treatment with MMP-3 siRNA suppressed cell proliferation, it also unexpectedly increased apoptosis. This siRNA-mediated increase in apoptosis could be reversed by exogenous MMP-3. These results demonstrate that cytokine-induced MMP-3 activity regulates cell proliferation and suppresses apoptosis in human odontoblast-like cells.
    Experimental Cell Research 09/2014; in press. DOI:10.1016/j.yexcr.2014.09.015 · 3.37 Impact Factor
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    ABSTRACT: We previously reported that interleukin 1β acts via matrix metalloproteinase (MMP)-3 to regulate cell proliferation and suppress apoptosis in α2 integrin-positive odontoblast-like cells differentiated from mouse embryonic stem (ES) cells. Here we characterize the signal cascade underpinning odontoblastic differentiation in mouse ES cells. The expression of α2 integrin, extracellular matrix metalloproteinase inducer (Emmprin), and MMP-3 mRNA and protein were all potently increased during odontoblastic differentiation. Small interfering RNA (siRNA) disruption of the expression of these effectors potently suppressed the expression of the odontoblastic biomarkers dentin sialophosphoprotein, dentin matrix protein-1 and alkaline phosphatase, and blocked odontoblast calcification. Our siRNA, western blot and blocking antibody analyses revealed a unique sequential cascade involving α2 integrin, Emmprin and MMP-3 that drives ES cell differentiation into odontoblasts. This cascade requires the interaction between α2 integrin and Emmprin and is potentiated by exogenous MMP-3. Finally, although odontoblast-like cells potently express α2, α6, αV, β1, and β3 integrins, we confirmed that β1 integrin acts as the trigger for ES cell differentiation, apparently in complex with α2 integrin. These results demonstrate a unique and unanticipated role for an α2 integrin-, Emmprin-, and MMP-3-mediated signaling cascade in driving mouse ES cell differentiation into odontoblast-like cells.
    Experimental Cell Research 08/2014; in press(1). DOI:10.1016/j.yexcr.2014.08.004 · 3.37 Impact Factor
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    ABSTRACT: We previously established a method for differentiating induced pluripotent stem cells and embryonic stem (ES) cells into α2 integrin-positive odontoblast-like cells. We also reported that interleukin (IL)-1β induces matrix metalloproteinase (MMP)-3-regulated cell proliferation and suppresses apoptosis in these cells, suggesting that MMP-3 plays a potentially unique physiological role in the regeneration of odontoblast-like cells. Here, we examined whether up-regulation of MMP-3 activity by IL-1β was mediated by Wnt signaling and led to increased proliferation of odontoblast-like cells. IL-1β increased mRNA and protein levels of Wnt5a, Wnt5b and the Wnt receptor Lrp5. Exogenous Wnt5a and Wnt5b were found to increase MMP-3 mRNA, protein and activity, and interestingly the rate of proliferation in these cells. Treatment with siRNAs against Wnt5a, Wnt5b and Lrp5 suppressed the IL-1β-induced increase in MMP-3 expression and suppressed cell proliferation, an effect rescued by application of exogenous Wnt5. These results demonstrate the sequential involvement of Wnt5, Lrp5 and MMP-3 in effecting IL-1β-induced proliferation of ES cell-derived odontoblast-like cells.
    Experimental Cell Research 06/2014; in press(1). DOI:10.1016/j.yexcr.2014.05.006 · 3.37 Impact Factor
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    ABSTRACT: We reported previously that matrix metalloproteinase (MMP)-13 accelerates bone remodeling in oral periradicular lesions, and indicated a potentially unique role for MMP-13 in wound healing and regeneration of alveolar bone. The ADAM (a disintegrin and metalloproteinase) family is a set of multifunctional cell surface and secreted glycoproteins, of which ADAM-28 has been localized in bone and bone-like tissues. In this study, we show that interleukin (IL)-1β induces the expression of MMP-13 and ADAM-28 in homogeneous α7 integrin-positive human skeletal muscle stem cell (α7+hSMSC)-derived osteoblast-like (α7+hSMSC-OB) cells, and promotes proliferation while inhibiting apoptosis in these cells. At higher concentrations, however, IL-1β failed to induce the expression of these genes and caused an increase in apoptosis. We further employed ADAM-28 small interfering RNA (siRNA) to investigate whether IL-1β–induced MMP-13 expression is linked to this IL-1β–mediated changes in cell proliferation and apoptosis. Silencing ADAM-28 expression potently suppressed IL-1β–induced MMP-13 expression and activity, decreased cell proliferation and increased apoptosis in α7+hSMSC-OB cells. In contrast, MMP-13 siRNA had no effect on ADAM-28 expression, suggesting ADAM-28 regulates MMP-13. Exogenous MMP-13 induced α7+hSMSC-OB cell proliferation and could rescue ADAM-28 siRNA–induced apoptosis, and we found that proMMP-13 is partially cleaved into its active form by ADAM-28 in vitro. Overall, our results suggest that IL-1β–induced MMP-13 expression and changes in cell proliferation and apoptosis in α7+hSMSC-OB cells are regulated by ADAM-28.
    Experimental Cell Research 04/2014; DOI:10.1016/j.yexcr.2014.02.018 · 3.37 Impact Factor
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    ABSTRACT: Skeletal muscle stem cells represent an abundant source of autologous cells with potential for regenerative medicine that can be directed to differentiate into multiple lineages including osteoblasts and adipocytes. In the current study, we found that α7+ integrin-positive human skeletal muscle stem cells (α7+hSMSC) could differentiate into the odontoblast lineage under specific inductive conditions in response to bone morphogenetic protein-4 (BMP-4). Cell aggregates of FACS harvested α7+hSMSC were treated in suspension with retinoic acid (RA) followed by culture on a gelatin scaffold (GS) in the presence of BMP-4. Following this protocol, α7+hSMSC were induced to down regulate myogenic genes (MyoD and α7 integrin) and upregulate odontogenic markers including dentin sialophosphoprotein (DSPP), matrix metalloproteinase-20 (MMP-20) (Enamelysin), dentin sialoprotein (DSP) and alkaline phosphatase (ALP), but not osteoblastic genes (osteopontin and osteocalcin). Following RA and GS/BMP-4 treatment there was a coordinated switch in the integrin expression profile that paralleled odontoblastic differentiation where α1β1 integrin was strongly upregulated with the attenuation of muscle-specific α7β1 integrin expression. Interestingly, using siRNA knockdown strategies revealed that the differentiation-related expression of the α1 integrin receptor positively regulates the expression of the odontoblastic markers DSPP and MMP-20. These results strongly suggest that the differentiation of α7+hSMSC along the odontogenic lineage is dependent on the concurrent expression of α1 integrin.
    Journal of Biological Chemistry 04/2014; 289(20). DOI:10.1074/jbc.M113.526772 · 4.60 Impact Factor
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    ABSTRACT: Granulocyte-colony stimulating factor (G-CSF) has been shown to have combinatorial trophic effects with dental pulp stem cells for pulp regeneration. The aim of this investigation is to examine the effects of basic fibroblast growth factor (bFGF) in vitro and in vivo compared with those of G-CSF and to assess the potential utility of bFGF as an alternative to G-CSF for pulp regeneration. Five different types of cells were examined in the in vitro effects of bFGF on cell migration, proliferation, anti-apoptosis, neurite outgrowth, angiogenesis and odontogenesis compared with those of G-CSF. The in vivo regenerative potential of pulp tissue including vasculogenesis and odontoblastic differentiation was also compared using an ectopic tooth transplantation model. bFGF was similar to G-CSF in high migration, proliferation and anti-apoptotic effects and angiogenic and neurite outgrowth stimulatory activities in vitro. There was no significant difference between bFGF and G-CSF in the regenerative potential in vivo. The potential utility of bFGF for pulp regeneration is demonstrated as a homing/migration factor similar to the influence of G-CSF. This article is protected by copyright. All rights reserved.
    Oral Diseases 02/2014; DOI:10.1111/odi.12227 · 2.40 Impact Factor
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    ABSTRACT: The age-associated decline in the regenerative abilities of mesenchymal stem cells (MSCs) may be due to age-related changes in reduction in number, intrinsic properties of MSCs and extrinsic factors of the extracellular environment (the stem cell niche). The effect of age on the efficacy of MSCs transplantation on regeneration, however, has not been clearly demonstrated due to variable methods of isolation of MSCs and variations in stem cell populations. In this study, dental pulp stem cells (DPSCs) subsets were isolated from young and aged dog teeth based on their migratory response to granulocyte-colony stimulating factor (G-CSF) (MDPSCs). In order to study the age-associated changes, their biological properties and stability were compared and the regenerative potential was examined in a pulpectomized tooth model in aged dogs. MDPSCs from aged dogs were efficiently enriched in stem cells, expressing trophic factors with high proliferation, migration and anti-apoptotic effects as in MDPSCs from young dogs. However, pulp regeneration was retarded 120 days after autologous transplantation of aged MDPSCs. We further demonstrated that isolated periodontal ligament stem cells (PDLSCs) from aged dogs, representative of migrating stem cells from outside of the tooth compartment to regenerate pulp tissue, had lower proliferation, migration and anti-apoptotic abilities. These results therefore provide a better understanding of the mechanisms involved in the age-dependent decline in pulp regeneration, which are attributed to a decrease in the regenerative potential of resident stem cells.
    Experimental gerontology 01/2014; DOI:10.1016/j.exger.2014.01.020 · 3.34 Impact Factor
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    ABSTRACT: We previously showed that the stromelysin-1 (matrix metalloproteinase 3) may have a putative role in dental pulp wound healing. However, its mechanism of action in wound repair has not been characterized. This study used stromelysin small interfering RNA transfected into rat dental pulp fibroblast-like cells (DPFCs) to investigate whether stromelysin-1 activity is induced by cytokines (interleukin-1 beta, tumor necrosis factor alpha, and interferon gamma) and/or is associated with cell proliferation and apoptosis. We used reverse-transcription polymerase chain reaction, Western blotting, stromelysin-1 activity, WST-1 proliferation, and DNA fragmentation enzyme-linked immunosorbent assays to evaluate the siRNA-mediated down-regulation of stromelysin-1 expression and activity and changes in the proliferative/apoptotic responses associated with this reduced expression. Low concentrations of proinflammatory cytokines induced the expression of stromelysin-1 messenger RNA and protein and increased stromelysin-1 activity and cell proliferation, but they had no effect on apoptosis. When stromelysin-1 expression was silenced using stromelysin-1 siRNA, we noted a potent and significant suppression of cytokine-induced stromelysin-1 expression and activity, decreased cell proliferation, and increased apoptosis. Finally, exogenous stromelysin-1 was found to induce cell proliferation in DPFCs. Our results showed that proinflammatory cytokine-induced stromelysin-1 regulates cell proliferation and anti-apoptosis in DPFCs in addition to their better documented destructive role in inflammation.
    Journal of endodontics 01/2014; 40(1):89-94. DOI:10.1016/j.joen.2013.09.029 · 2.95 Impact Factor
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    ABSTRACT: We previously reported that matrix metalloproteinase (MMP)-3 accelerates wound healing following dental pulp injury. In addition, we reported that a proinflammatory cytokine mixture (tumor necrosis factor-α, interleukin (IL)-1β and interferon-γ) induced MMP-3 activity in odontoblast-like cells derived from mouse embryonic stem (ES) cells, suggesting that MMP-3 plays a potential unique physiological role in wound healing and regeneration of dental pulp in odontoblast-like cells. In this study, we tested the hypothesis that upregulation of MMP-3 activity by IL-1β promotes proliferation and apoptosis of purified odontoblast-like cells derived from induced pluripotent stem (iPS) and ES cells. Each odontoblast-like cell was isolated and incubated with different concentrations of IL-1β. MMP-3 mRNA and protein expression were assessed using RT-PCR and western blotting, respectively. MMP-3 activity was measured using immunoprecipitation and a fluorescence substrate. Cell proliferation and apoptosis were determined using ELISA for BrdU and DNA fragmentation, respectively. siRNA was used to reduce MMP-3 transcripts in these cells. Treatment with IL-1β increased MMP-3 mRNA and protein levels, and MMP-3 activity in odontoblast-like cells. Cell proliferation was found to markedly increase with no changes in apoptosis. Endogenous tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 were constitutively expressed during all experiments. The exocytosis inhibitor, Exo1, potently suppressed the appearance of MMP-3 in the conditioned medium. Treatment with siRNA against MMP-3 suppressed an IL-1β-induced increase in MMP-3 expression and activity, and also suppressed cell proliferation, but unexpectedly increased apoptosis in these cells (P<0.05). Exogenous MMP-3 was found to induce cell proliferation in odontoblast-like cells derived from iPS cells and ES cells. This siRNA-mediated increase in apoptosis could be reversed with exogenous MMP-3 stimulation (P<0.05). Taken together, IL-1β induced MMP-3-regulated cell proliferation and suppressed apoptosis in odontoblast-like cells derived from iPS and ES cells.
    PLoS ONE 12/2013; 8(12):e83563. DOI:10.1371/journal.pone.0083563 · 3.53 Impact Factor
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    ABSTRACT: We previously reported that matrix metalloproteinase-3 accelerates wound healing following dental pulp injury. In the present study, we tested the hypothesis that induction of matrix metalloproteinase-3 activity by interleukin-1β would promote proliferation and apoptosis of dental pulp cells. Dental pulp cells were isolated from rat incisors and subjected to interleukin-1β. Matrix metalloproteinase-3 mRNA and protein expression were assessed using reverse transcription-polymerase chain reaction and western blotting, respectively. Matrix metalloproteinase-3 activity was measured using fluorescence. Dental pulp cell proliferation and apoptosis were determined using enzyme-linked immunosorbent assays for BrdU and DNA fragmentation, respectively. siRNA was used to reduce matrix metalloproteinase-3 transcripts in these cells. Treatment with interleukin-1β increased matrix metalloproteinase-3 mRNA and protein levels as well as its activity in dental pulp cells. Cell proliferation was also markedly increased, with no changes in apoptosis observed. Treatment with siRNA against matrix metalloproteinase-3 potently suppressed this interleukin-1β-induced increase in matrix metalloproteinase-3 expression and activity, and also suppressed cell proliferation but unexpectedly increased apoptosis in these cells (P<0.05). This siRNA-mediated increase in apoptosis could be reversed with exogenous matrix metalloproteinase-3 stimulation (P<0.05). Interleukin-1β induces matrix metalloproteinase-3-regulated cell proliferation and suppresses apoptosis in dental pulp cells. This article is protected by copyright. All rights reserved.
    Oral Diseases 12/2013; DOI:10.1111/odi.12219 · 2.40 Impact Factor
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    ABSTRACT: Methods for differentiating induced pluripotent stem (iPS) cells into odontoblasts generally require epithelial-mesenchymal interactions. Here, we sought to characterize the cells produced by a 'hanging drop' technique for differentiating mouse iPS cells into odontoblast-like cells that requires no such interaction. Cells were cultured by the hanging drop method on a collagen type-I (Col-I) scaffold (CS) combined with bone morphogenetic protein (BMP)-4 (CS/BMP-4) without an epithelial-mesenchymal interaction. We evaluated the expression of odontoblast-related mRNA and protein, and the proliferation rate of these cells using reverse-transcription polymerase chain reaction, immunofluorescence staining, and BrdU cell proliferation enzyme-linked immunosorbent assay, respectively. The differentiated cells strongly expressed the mRNA for dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (Dmp-1), which are markers of mature odontoblasts. Osteopontin and osteocalcin were not expressed in the differentiated cells, demonstrating that the differentiated iPS cells bore little resemblance to osteoblasts. Instead, they acquired odontoblast-specific properties, including the adoption of an odontoblastic phenotype, typified by high alkaline phosphatase (ALP) activity and calcification capacity. The cell-surface expression of proteins such as integrins α2, α6, αV and αVβ3 was rapidly up-regulated. Interestingly, antibodies and siRNAs against integrin α2 suppressed the expression of DSPP and Dmp-1, reduced the activity of ALP and blocked calcification, suggesting that integrin α2 in iPS cells mediates their differentiation into odontoblast-like cells. The adhesion of these cells to fibronectin and Col-I, and their migration on these substrata, was significantly increased following differentiation into odontoblast-like cells. Thus, we have demonstrated that integrin α2 is involved in the differentiation of mouse iPS cells into odontoblast-like cells using the hanging drop culture method, and that these cells have the appropriate physiological and functional characteristics to act as odontoblasts in tissue engineering and regenerative therapies for the treatment of dentin and/or dental pulp damage.
    PLoS ONE 11/2013; 8(11):e80026. DOI:10.1371/journal.pone.0080026 · 3.53 Impact Factor
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    ABSTRACT: Human dental pulp stem cells (DPSCs) contain subsets of progenitor/stem cells with high angiogenic, neurogenic and regenerative potential useful for cell therapy. It is essential to develop a safe and efficacious method to isolate the clinical-grade DPSCs subsets from a small amount of pulp tissue without using conventional flow cytometry. Thus, a method for isolation of DPSCs subsets based on their migratory response to optimized concentration of 100 ng/ml of granulocyte-colony stimulating factor (G-CSF) was determined in this study. The DPSCs mobilized by G-CSF (MDPSCs) were enriched for CD105, C-X-C chemokine receptor type 4 (CXCR-4) and G-CSF receptor (G-CSFR) positive cells, demonstrating stem cell properties including high proliferation rate and stability. The absence of abnormalities/aberrations in karyotype and lack of tumor formation after transplantation in an immunodeficient mouse were demonstrated. The conditioned medium of MDPSCs exhibited anti-apoptotic activity, enhanced migration and immunomodulatory properties. Furthermore, transplantation of MDPSCs accelerated vasculogenesis in an ischemic hindlimb model and augmented regenerated pulp tissue in an ectopic tooth root model compared to that of colony-derived DPSCs, indicating higher regenerative potential of MDPSCs. In conclusion, this isolation method for DPSCs subsets is safe and efficacious, having utility for potential clinical applications to autologous cell transplantation.
    Biomaterials 08/2013; DOI:10.1016/j.biomaterials.2013.08.011 · 8.31 Impact Factor
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    ABSTRACT: Matrix metalloproteinase (MMP)-3 expression increases after pulpectomy and accelerates angiogenesis in rat dental pulp by an uncharacterised mechanism. Odontoblasts, a major component of dental pulp, could represent a therapeutic target. We investigated whether MMP-3 activity is induced by cytokines and/or is associated with cell proliferation and apoptosis in embryonic stem cell-derived odontoblast-like cells. We used reverse transcriptase polymerase chain reaction, western blotting, an MMP-3 activity assay, a BrdU-cell proliferation enzyme-linked immunosorbent assay and DNA fragmentation analysis to evaluate siRNA-mediated downregulation of MMP-3 expression and activity, and any changes in the proliferative and apoptotic responses associated with this reduced expression. Pro-inflammatory cytokines (interleukin-1β, tumour necrosis factor-α and interferon-γ, at relatively low concentrations) induced MMP-3 mRNA and protein expression, and increased MMP-3 activity and cell proliferation, but not apoptosis. MMP-3 silencing produced a potent and significant suppression of cytokine-induced MMP-3 expression and activity, decreased cell proliferation and increased apoptosis. These effects were rescued by application of exogenous MMP-3. Our results suggest that pro-inflammatory cytokines induce MMP-3-regulated cell proliferation and anti-apoptosis effects in odontoblast-like cells derived from embryonic stem cells, in addition to their well-documented destructive role in inflammation.
    Oral Diseases 07/2013; DOI:10.1111/odi.12165 · 2.40 Impact Factor
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    ABSTRACT: OBJECTIVES: We examined whether mouse embryonic stem (ES) cells can differentiate into odontoblast-like cells without epithelial-mesenchymal interaction. MATERIALS AND METHODS: Cells were cultured by the 'hanging drop' method using a collagen type-I scaffold (CS) combined with bone morphogenetic protein (BMP)-4 (CS/BMP-4). Expression of odontoblast-related mRNA and protein, and cell proliferation were performed by reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence staining and WST-1 assay, respectively. RESULTS: Cells potently expressed odontoblast-related cell marker mRNAs following induction of odontoblastic differentiation. Dentin sialophosphoprotein, a marker of mature odontoblasts, was strongly expressed in differentiated ES cells. The cells also acquired an odontoblast-like functional phenotype, as evidenced by the appearance of alkaline phosphatase activity and calcification. The cell-surface expression of α2, α6, αV and αVβ3 integrin proteins was rapidly upregulated in differentiated cells. Finally, anti-α2 integrin antibody suppressed the expression of odontoblastic markers in cells grown using this culture system, suggesting that α2 integrin expression in ES cells triggers their differentiation into odontoblast-like cells. CONCLUSIONS: Mouse ES cells cultured by the 'hanging drop' method are able to differentiate into cells with odontoblast-specific physiological functions and cell-surface integrin protein expression.
    Oral Diseases 05/2013; DOI:10.1111/odi.12134 · 2.40 Impact Factor
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    ABSTRACT: A disintegrin and metalloproteinases (ADAMs) are a family of transmembrane proteins that share a common domain structure. However, little is known about the possible involvement of ADAM-8 and ADAM-17 in the development of periradicular lesions. Here we demonstrated the expression of ADAM-8 and ADAM-17 in rat periradicular lesions. We induced experimentally periradicular lesions in rats. The animals were killed at 0, 2, 4, 6, and 8 weeks after pulp exposure. The left molars underwent immunofluorescence analysis for both ADAMs and for neutrophil elastase, and right molars were used for reverse transcription-polymerase chain reaction analysis of ADAM-8 and ADAM-17. The areas of these lesions were measured histometrically, and the numbers of all antigen-positive cells in the periapical portion were counted per unit area. The area of the periradicular lesions gradually expanded from 0 to 4 weeks, showing a large increase from week 2 to week 4. Both ADAM-8-positive and ADAM-17-positive cells gradually increased in number from 0 to 4 weeks and then decreased from 4 to 8 weeks. There were more ADAM-17-expressing cells than ADAM-8-expressing ones at all experimental periods except at 4 weeks. The highest expression of ADAM-8 mRNA was observed at 4 weeks, and there were significant differences between 0 and 2 weeks and between 4 and 6 weeks. The expression of ADAM-17 mRNA increased from 0 to 4 weeks and subsequently decreased from 4 to 8 weeks, with a significant difference between 4 and 6 weeks. Our results suggest that ADAM-8 and ADAM-17 may be related to the development of rat periradicular lesions.
    Journal of endodontics 05/2013; 39(5):638-42. DOI:10.1016/j.joen.2012.12.015 · 2.95 Impact Factor

Publication Stats

237 Citations
74.52 Total Impact Points

Institutions

  • 2002–2015
    • Aichi Gakuin University
      • Department of Endodontics
      Nagoya, Aichi, Japan
  • 2014
    • National Center for Geriatrics and Gerontology
      • Center for Development of Advanced Medicine for Dental and Oral Diseases
      Ōfu, Aichi, Japan