Kunihiko Yoshiba

Niigata University, Niahi-niigata, Niigata, Japan

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Publications (49)100.26 Total impact

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    ABSTRACT: Introduction M2 (alternatively activated) macrophages are known to participate in wound healing and tissue repair. This study aimed to analyze the temporospatial changes in the distribution and density of M2 macrophage–associated molecule-expressing cells after pulpotomy with mineral trioxide aggregate (MTA) in rat molars to ascertain the role played by M2 macrophages in the healing of MTA-capped pulp tissue. Methods The maxillary first molars of 8-week-old Wistar rats were pulpotomized and capped with MTA. After 1–14 days, the teeth were examined after hematoxylin-eosin staining or immunoperoxidase staining of CD68 (a general macrophage marker) and M2 macrophage markers (CD163 and CD204). The density of positively stained cells was enumerated in the surface and inner regions (0–100 μm and 300–400 μm, respectively, from the wound surface). Results MTA capping initially caused mild inflammatory changes and the formation of a degenerative layer followed by progressive new matrix formation and calcified bridging. At 1–2 days, CD68-, CD163-, and CD204-positive cells started to accumulate beneath the degenerative layer, and the density of these cells was significantly higher in the surface region than in the inner region (P < .05). From 7 days onward, the 3 types of cells displayed an almost normal distribution beneath the newly formed dentinlike matrix. Conclusions After the pulpotomy of rat molars with MTA, M2 macrophage–associated molecule-expressing cells transiently accumulated beneath the degenerative layer under the MTA. This suggests that M2 macrophages participate in the initial phases of the healing of MTA-capped pulp tissue.
    Journal of Endodontics. 10/2014;
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    ABSTRACT: AimTo examine the temporospatial expression of dentin matrix protein 1 (DMP1; a non-collagenous protein involved in mineralized tissue formation), osteopontin (another non-collagenous protein detected during reparative dentinogenesis) and nestin (a marker of differentiating/differentiated odontoblasts), following direct pulp capping with calcium hydroxide in rat molars.MethodologyThe maxillary first molars of 8-week-old Wistar rats had their pulps exposed and capped with calcium hydroxide. The pulp-capped teethwere collected from 6 hours to 14 days postoperatively, and processed for immunohistochemistry for DMP1, osteopontin and nestin. Cell proliferation was monitored using 5-bromo-2’-deoxyuridine (BrdU) labeling.ResultsThe capped pulps initially exhibited superficial necrotic changes followed by the formation of new matrix and its mineralisation. DMP1-immunoreactivity was observed in the matrix beneath the necrotic layer from 6 hours onwards and present in the outer portion of the newly-formed mineralised matrix from 7 days onwards. Osteopontin displayed a similar expression pattern, although it occupied a narrower area than DMP1 at 6 and 12 hours. Nestin-immunoreactive cells appeared beneath the DMP1-immunoreactive area at 1 day, were distributed beneath the newly-formed matrix at 5 days, and exhibited odontoblast-like morphology by 14 days. BrdU-positive cells significantly increased at 2 and 3 days (P <0.05) and then decreased.Conclusions The deposition of DMP1 at -exposed pulp sites preceded the appearance of nestin-immunoreactive cells, active cell proliferation, and new matrix formation after pulp capping with calcium hydroxide in rat molars, suggesting that DMP1 acts as a trigger of pulp repair. The colocalisation of DMP1 and osteopontin suggests that these two proteins play complementary roles.This article is protected by copyright. All rights reserved.
    International Endodontic Journal 07/2014; · 2.05 Impact Factor
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    ABSTRACT: This study examined the protein and messenger RNA (mRNA) expression of molecules associated with M2 (wound healing) macrophages in mineral trioxide aggregate (MTA)-implanted rat subcutaneous tissue to elucidate the involvement of M2 macrophages in the connective tissue response to MTA. Silicone tubes containing freshly mixed MTA or a calcium hydroxide cement (Life; Kerr, Romulus, MI) were subcutaneously implanted into the backs of Wistar rats. Solid silicone rods implanted in different animals served as controls. The specimens were then double immunostained for ED1 (CD68, a general macrophage marker) and ED2 (CD163, an M2 macrophage marker). Immunostaining for CD34 (a marker for vascularization and wound healing) was also performed. Expression levels of CD34, CD163, and mannose receptor c type 1 (an M2 macrophage marker) mRNAs were determined with real-time polymerase chain reaction. MTA-implanted subcutaneous tissues showed significant increases in the density of ED1+ED2+ macrophages beneath the implantation site and expression levels of CD163 and MMR mRNAs compared with Life-implanted and control tissues. MTA-implanted subcutaneous tissues also showed a significant increase of CD34-immunostained areas and up-regulation of CD34 mRNAs compared with Life-implanted and control tissues. MTA implantation induced the accumulation of M2 macrophage marker (ED2)-expressing macrophages and enhanced the expression of M2 macrophage marker genes. MTA implantation also enhanced the expression of CD34, suggesting acceleration of the healing/tissue repair process. Taken together, biological connective tissue response to MTA may involve wound healing/tissue repair processes involving M2 macrophages.
    Journal of endodontics 03/2014; 40(3):379-83. · 2.95 Impact Factor
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    ABSTRACT: Introduction The prostaglandin transporter (Pgt) and multidrug resistance-associated protein (Mrp) 4 are membrane transport proteins that play crucial roles in the transmembrane uptake and/or efflux of prostaglandins (PGs). This study attempted to analyze the protein expression of Pgt and Mrp4 and their involvement in PGE2 efflux transport in lipopolysaccharide (LPS)-inflamed rat incisor pulp tissue. Methods Pulpitis was induced in the upper incisors of Wistar rats by treating them with LPS for 24 hours. The protein expression levels of Pgt, Mrp4, and microsomal PGE synthase (mPGES) were analyzed with immunofluorescent staining. The amount of PGE2 released from the inflamed pulp tissue in the presence or absence of dipyridamole (an Mrp4 inhibitor) was assessed by using an enzyme-linked immunosorbent assay. Results Double immunofluorescence staining revealed that the Pgt, Mrp4, and mPGES immunoreactivity co-localized in CD31-expressing endothelial cells. Moreover, the Mrp4 inhibitor caused a significant decrease in the amount of PGE2 released from the LPS-inflamed pulp (P < .01 at 24 hours). Conclusion Pgt, Mrp4, and mPGES expression was detected in the endothelial cells of normal and LPS-inflamed rat incisor pulp tissue, suggesting that these cells are associated with the biosynthesis and transmembrane transport of PGE2. The significant decrease in PGE2 release induced by the Mrp4 inhibitor suggests that Mrp4 contributes to the transport of PGE2 in the transmembrane efflux pathway.
    Journal of Endodontics. 01/2014;
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    ABSTRACT: Objectives: Recent studies have employed two markers, alpha-smooth muscle actin (α-SMA) and STRO-1, to detect cells with mesenchymal stem cell properties in dental pulp. In this work, the expression profiles of α-SMA and STRO-1 were explored in intact dental pulp, during the wound-healing process, as well as in tissue culture of adult human dental pulp. Methods: This research project was approved by the Niigata University Ethics Committee. Healthy pulps were mechanically exposed and capped with the clinically used materials MTA (ProRoot White MTA) or Ca(OH)2 to induce a mineralized barrier at the exposed surface. The teeth were extracted after 7-42 days and processed for immunohistochemical analysis using antibodies against α-SMA, STRO-1 and nestin (a neurogenic cytoskeletal protein expressed in odontoblasts). For pulp tissue culture, pulp was removed from intact teeth and sliced into 1.5mm-thick specimens. Results: In normal pulp, α-SMA was detected in vascular smooth muscle cells and pericytes. Double immunofluorescent staining with STRO-1 and α-SMA showed that STRO-1 was localized in vascular smooth muscle cells, pericytes and endothelial cells, in addition to nerve fibers. During the dental pulp healing, numerous α-SMA-positive cells emerged at the wound margin at 14 days, and the initially formed mineralized barrier was lined with α-SMA-positive cells similar in appearance to reparative odontoblasts, some of which co-expressed nestin. STRO-1 was abundant in nerve fibers. In the advanced stage of mineralized barrier formation at 42 days, cells lining the barrier were stained with nestin, and no staining of α-SMA was observed in those cells. Cultured dental pulp tissues demonstrated the appearance of α-SMA-positive fibroblasts stained with phosphorylated Smad2/3, the central mediators of TGF-β signaling, at 3 days. Conclusions: These results indicate that α-SMA-positive cells temporarily appear along the wound margin during the earlier phase of mineralized barrier formation and STRO-1 is confined in vascular and neuronal elements.
    IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
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    ABSTRACT: Objectives: Multidrug resistance associated protein (Mrp) 4 is a membrane transport protein that plays a crucial role in the transmembrane uptake and/or efflux of prostaglandins (PGs), and drugs such as nonsteroidal anti-inflammatory drugs. However, the role of this transporter in pulp inflammation is poorly understood. This study aimed to analyze the mRNA and protein expression of Mrp4 and its involvement in the PGE2 efflux transport in lipopolysaccharide (LPS)-inflamed rat incisor pulp. Methods: Pulpitis was induced in the upper incisor of Wistar rats by applying LPS for 6-24 h. The level of Mrp4 mRNA expression was determined in the LPS-inflamed and normal pulps by using real-time PCR. The protein expression for Mrp4 was analyzed with double immunofluorescence staining using an anti-Mrp4 antibody and CD31 (an endothelial cell marker). The amount of PGE2 released from inflamed pulp explants, in the presence or absence of dipyridamole (an Mrp4 inhibitor), was assessed by an enzyme-linked immunosorbent assay. Results: The level of Mrp4 mRNA expression in the inflamed pulps was significantly higher at 6-12 h after the LPS application, compared with that in the normal pulp (P < 0.01 at 6 h and P < 0.05 at 12 h). Double immunofluorescent staining revealed that Mrp4-immunoreactivity was colocalized in CD31-expressing endothelial cells. Moreover, the Mrp4 inhibitor caused a 51.8% decrease in the amount of PGE2 released from the LPS-inflamed pulps (P < 0.01 at 24h). Conclusions: In the LPS-inflamed pulp, Mrp4 mRNA was upregulated and immunolocalized in endothelial cells. The significant decrease of PGE2-release by the Mrp4 inhibitor suggests that this transporter contributes to the transport of PGE2 in the transmembrane efflux pathway.
    IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
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    ABSTRACT: Objective: Mineral trioxide aggregate (MTA) is a calcium silicate-based endodontic material currently used in a wide range of applications. MTA shows Ca release and surface apatite-like crystal deposition when immersed in PBS, suggesting its bioactivity. However, the structural changes of MTA in vivoremain unclear. The purpose of this study was to analyze ultrastructural and compositional changes of MTA when implanted in rat subcutaneous tissue. Methods: A mixture of white MTA (ProRoot MTA, Dentsply Tulsa Dental Products) and distilled water was filled in Teflon tubes, and then implanted into the subcutaneous tissue of 4-week-old male Wistar rats. After 1, 2 and 4 weeks, the distribution of Ca, P, Si, and Al were analyzed with a wavelength-dispersive X-ray spectroscopy electron probe microanalyzer (EPMA) along the cross-section of the specimens. SEM observation and chemical composition analysis of MTA surface were also conducted. In addition, ultra-thin sections of the connective tissue in contact with MTA were observed with TEM. Results: A layer of low Ca and high Si and Al concentrations was formed in the periphery of implanted MTA. This layer increased in thickness with time. The most superficial layer in contact with connective tissue showed high concentration of Ca and P. SEM observation of the surface of MTA revealed the formation of cubic-like or slate-like crystalline structures containing Ca and P. TEM analysis of the connective tissue revealed deposition of electron-dense needle-like structures along collagen fibrils. Conclusion: MTA implanted in rat subcutaneous tissue showed compositional changes and surface crystalline deposits containing Ca and P. MTA also induced needle-like crystal deposition in the surrounding connective tissue, suggesting a hard tissue-inducing property of MTA.
    IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
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    ABSTRACT: Objectives: Mineral trioxide aggregate (MTA) has been used for direct pulp capping because of its potential to induce hard tissue barrier formation, whereas pulp tissue responses to this material remain fully elucidated. This study aimed to analyze the temporal changes in the distribution of macrophage subpopulations in the rat molar pulp capped with MTA. Methods: Maxillary first molars of 8-week-old Wistar rats were exposed and capped with MTA. After 1-14 days, the teeth were processed for cryostat sections and subjected to either H-E staining or immunoperoxidase staining using monoclonal antibodies ED1 (pan-macrophage antibody), ED2 (anti-resident macrophages) and OX6 (anti-major histocompatibility complex class II molecules). Results: Histological changes of the MTA-capped pulp was characterized by (1) formation of a thin degenerative layer just beneath the pulp-capped site by day 2; (2) alignment of cylindrical cells, probably representing differentiating cells, beneath the degenerative layer on day 3; and (3) formation of a thin dentin-like matrix on day 7. A hard tissue barrier was clearly seen on day 14. ED1-positive and ED2-positive cells showed a marked accumulation beneath the degenerative layer by day 2. On day 3, ED1-positive cells were still concentrated in the vicinity of the degenerative layer whereas ED2-positive cells showed a reduced accumulation. OX6-positive cells showed a slight increase in density beneath the pulp-capped site by day 2. On day 7 and day 14, the three types of cells showed an almost normal distribution beneath the newly-formed dentin-like matrix. Conclusions: Macrophages showed a transient accumulation in the early stage of the reparative process of the MTA-capped pulp. Such a response may predominantly represent the accumulation of resident macrophages.
    IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
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    ABSTRACT: Small ubiquitin-related modifier (SUMO) conjugation (SUMOylation) is a post-translational modification involved in various cellular processes including the regulation of transcription factors. In this study, to analyze the involvement of SUMOylation in odontoblast differentiation, we examined the immunohistochemical localization of SUMO-1, SUMO-2/3, and Osterix during rat tooth development. At the bud and cap stages, localization of SUMOs and Osterix was hardly detected in the dental mesenchyme. At the bell stage, odontoblasts just beginning dentin matrix secretion and preodontoblasts near these odontoblasts showed intense immunoreactivity for these molecules. However, after the root-formation stage, these immunoreactivities in the odontoblasts decreased in intensity. Next, to examine whether the SUMOylation participates in dentin regeneration, we evaluated the distribution of SUMOs and Osterix in the dental pulp after cavity preparation. In the coronal pulp chamber of an untreated rat molar, odontoblasts and pulp cells showed no immunoreactivity. At 4 days after cavity preparation, positive cells for SUMOs and Osterix appeared on the surface of the dentin beneath the cavity. Odontoblast-like cells forming reparative dentin were immunopositive for SUMOs and Osterix at 1 week, whereas these immunoreactivities disappeared after 8 weeks. Additionally, we further analyzed the capacity of SUMO-1 to bind Osterix by performing an immunoprecipitation assay using C2C12 cells, and showed that Osterix could undergo SUMOylation. These results suggest that SUMOylation might regulate the transcriptional activity of Osterix in odontoblast lineage cells, and thus play important roles in odontoblast differentiation and regeneration.
    Histochemie 01/2013; · 2.61 Impact Factor
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    ABSTRACT: Major histocompatibility complex (MHC) class II molecule-expressing cells and macrophages play a pivotal role in mediating the host tissue response to biomaterials. This study investigated the responses of these cells to epoxy resin-based and 4-META-containing, methacrylate resin-based endodontic sealers (AH Plus and MetaSEAL respectively) in rat connective tissue. Silicone tubes loaded with one of the sealers or solid silicone rods (control) were subcutaneously implanted in male Wistar rats for three time periods of 7, 14, or 28 days. Tissue specimens were immunoperoxidase-stained for MHC class II molecules and CD68 (a general macrophage marker). Results showed that AH Plus-implanted tissue displayed significantly more MHC class II-positive cells than the control at 14 and 28 days, whereas MetaSEAL-implanted tissue showed significantly more CD68-positive cells than both AH Plus-implanted tissue and the control at all time periods. It was concluded that the epoxy resin-based sealer induced the infiltration of MHC class II molecule-expressing cells, whereas 4-META-containing, methacrylate resin-based sealer elicited macrophage infiltration.
    Dental Materials Journal 01/2013; 32(5):822-827. · 0.81 Impact Factor
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    ABSTRACT: Dental pulp is involved in the formation of bone-like tissue in response to external stimuli. However, the origin of osteoblast-like cells constructing this tissue and the mechanism of their induction remain unknown. We therefore evaluated pulp mineralization induced by transplantation of a green fluorescent protein (GFP)-labeled tooth into a GFP-negative hypodermis of host rats. Five days after the transplantation, the upper pulp cavity became necrotic; however, cell-rich hard tissue was observed adjacent to dentin at the root apex. At 10 days, woven bone-like tissue was formed apart from the dentin in the upper pulp. After 20 days, these hard tissues expanded and became histologically similar to bone. GFP immunoreactivity was detected in the hard tissue-forming cells within the root apex as well as in the upper pulp. Furthermore, immunohistochemical observation of α-smooth muscle actin, a marker for undifferentiated cells, showed a positive reaction in cells surrounding this bone-like tissue within the upper pulp but not in those within the root apex. Immunoreactivities of Smad4, Runx2, and Osterix were detected in the hard tissue-forming cells within both areas. These results collectively suggest that the dental pulp contains various types of osteoblast progenitors and that these cells might thus induce bone-like tissue in severely injured pulp.
    Journal of Histochemistry and Cytochemistry 08/2012; · 2.26 Impact Factor
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    ABSTRACT: This study aimed to examine the dynamics of odontoblast-lineage cells following cavity preparation with erbium:yttrium-aluminum-garnet (Er:YAG) laser in rat molars. Cavity preparation was made with Er:YAG laser in the mesial surface of the maxillary left first molar of 8-week-old Wistar rats. Contralateral first molar served as unirradiated control. Immediately, 6 and 12 h and 1, 2, 3, 5 and 7 days after the lasing (n = 5, each), specimens were collected and processed for immunohistochemistry for heat-shock protein (HSP)-25 and nestin as markers for odontoblast-lineage cells. Cell proliferation assay using bromodeoxyuridine (BrdU) labeling was also performed. Unirradiated teeth showed HSP-25- and nestin-immunoreactivity in odontoblasts. At 6-12 h after irradiation, the odontoblastic layer was disorganized and some of odontoblasts lost the immunoreactivity to HSP-25 and nestin. At 1-2 days, however, HSP-25- and nestin-immunoreactivities in the odontoblast layer showed a noticeable recovery, resulting in the rearrangement of odontoblast-like cells intensely immunoreactive to HSP-25 and nestin at 3-7 days. BrdU-positive cells showed a significant increase at 2 days (P < 0.05 vs. immediate previous time point; one-way analysis of variance and Scheffé post hoc test), peaked at 3 days and then decreased significantly (P < 0.05). It was concluded that under the present experimental condition in rat molars, cavity preparation with Er:YAG laser induced mild and reversible damage to odontoblasts. The reparative process was characterized by the rearrangement of HSP-25- and nestin-immunoreactive odontoblast-like cells, which took place subsequent to the odontoblastic layer disorganization with partial loss of these immunoreactivities.
    Odontology 06/2012; · 1.58 Impact Factor
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    ABSTRACT: Recent studies have employed two markers, alpha-smooth muscle actin (α-SMA) and STRO-1, to detect cells with mesenchymal stem cell properties in dental pulp. The present study aimed to explore the expression profile of α-SMA and STRO-1 in intact dental pulp as well as during wound healing in adult dental pulp tissue. Healthy pulps were mechanically exposed and capped with the clinically used materials MTA (ProRoot White MTA) or Ca(OH)₂ to induce a mineralized barrier at the exposed surface. After 7-42 days, the teeth were extracted and processed for immunohistochemical analysis using antibodies against α-SMA, STRO-1 and nestin (a neurogenic cytoskeletal protein expressed in odontoblasts). In normal pulp, α-SMA was detected in vascular smooth muscle cells and pericytes. Double immunofluorescent staining with STRO-1 and α-SMA showed that STRO-1 was localized in vascular smooth muscle cells, pericytes and endothelial cells, in addition to nerve fibers. During the process of dental pulp healing, numerous α-SMA-positive cells emerged at the wound margin at 14 days, and the initially formed mineralized barrier was lined with α-SMA-positive cells similar in appearance to reparative odontoblasts, some of which co-expressed nestin. STRO-1 was abundant in nerve fibers. In the advanced stage of mineralized barrier formation at 42 days, cells lining the barrier were stained with nestin, and no staining of α-SMA was detected in those cells. These observations indicate that α-SMA-positive cells temporarily appear along the wound margin during the earlier phase of mineralized barrier formation and STRO-1 is confined in vascular and neuronal elements.
    Histochemie 06/2012; 138(4):583-92. · 2.61 Impact Factor
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    ABSTRACT: Membrane transport proteins (transporters) play a crucial role in the transmembrane uptake and/or efflux of various compounds such as inorganic ions, endogenous bioactive substances such as prostaglandins (PGs), and drugs such as nonsteroidal anti-inflammatory drugs. This study aimed to analyze mRNA expression of selected transporters related to drug disposition and PG transport in normal and lipopolysaccharide (LPS)-inflamed rat incisor pulp. Pulp tissues were subjected to reverse transcription-polymerase chain reaction (PCR) detection for transporter isoforms belonging to organic anion transporting polypeptide (Oatp), organic anion transporter (Oat), organic cation transporter (Oct), multidrug resistance-associated protein (Mrp), and multidrug resistance protein (Mdr) families. The levels of mRNA expression for PG transporters (Oatp1a5, Oatp1b2, Oatp2a1, Oatp2b1, and Oatp3a1) were compared in normal and LPS-inflamed pulps by using real-time PCR. The pulp tissue expressed mRNAs for various transporters belonging to the Oatp, Oat, Oct, Mrp, and Mdr families. LPS inflammation caused significant up-regulation of Oatp2a1 (P < .01) and significant down-regulation of Oatp1a5, Oatp2b1 (P < .01), and Oatp3a1 (P < .05). Rat incisor dental pulp expressed mRNAs for various transporter isoforms. The levels of mRNA expression for PG transporters were significantly up-regulated or down-regulated in LPS-inflamed dental pulp.
    Journal of endodontics 05/2012; 38(5):648-52. · 2.95 Impact Factor
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    ABSTRACT: Angiogenic factors such as VEGFR2 (vascular endothelial cell growth factor receptor 2), Bcl-2 (a prosurvival and proangiogenic signaling molecule), and chemokine (C-X-C motif) ligand 1 (CXCL1) (a proangiogenic chemokine) may have critical roles in enhancing the establishment of apical periodontitis. To understand the role of these factors in the pathogenesis of apical periodontitis, we conducted immunohistochemical and molecular biological analysis. Apical periodontitis was induced in the lower first molars of Wistar rats by making unsealed pulp exposures. After, 14, 21, and 28 days, the molars were retrieved, embedded as frozen sample blocks, and cut in a cryostat. Normal lower first molars served as controls. Immunostaining for CD31 (a marker for endothelial cells), Bcl-2, and real-time polymerase chain reaction analysis of VEGFR2, Bcl-2, CXCL1, and CXCR2 messenger RNA were performed. In the real-time polymerase chain reaction analysis, messenger RNA was extracted from CD31-stained endothelial cells that were retrieved with laser capture microdissection. For statistical analysis of immunohistochemistry, the immunostained area was plotted, and pixel counts were determined. Then, the percentage of the immunostained area in the total area was calculated. The density of the CD31-stained area increased until 21 days after pulp exposure. On the other hand, Bcl-2-stained area showed the highest density at 14 days (active lesion expanding phase) and then decreased until 28 days (lesion stability phase). VEGFR2, Bcl-2, CXCL1, and CXCR2 messenger RNA expression in endothelial cells showed the highest levels at 14 days and then decreased until 28 days. The increase in microvascular density and the up-regulation of VEGFR2, Bcl-2, CXCL1, and CXCR2 messenger RNA expression in endothelial cells took place coincidently with the expanding phase of experimentally induced periapical lesions. These data suggest that these angiogenic factors play a role in the lesion development.
    Journal of endodontics 03/2012; 38(3):313-7. · 2.95 Impact Factor
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    ABSTRACT: The cells of the subodontoblastic cell-rich layer in dental pulp are speculated to contain odontoblast progenitor cells because of their positional relationship with odontoblasts as well as their high alkaline phosphatase (ALP) activity. However, it has yet to be determined whether these cells have the ability to differentiate into odontoblastic cells. In the present study, we firstly found that the majority of cells in the subodontoblastic layer expressed Thy-1, a cell-surface marker of stem and progenitor cells. Then, we evaluated the capacity of Thy-1 high- and low-expressing (Thy-1(high) and Thy-1(low)) cells separated from rat dental pulp cells by use of a fluorescence-activated cell sorter to differentiate into hard tissue-forming cells in vitro and in vivo. Following stimulation with bone morphogenetic protein-2, Thy-1(high) cells in vitro showed accelerated induction of ALP activity and formation of alizarin red-positive mineralized matrix compared with Thy-1(low) cells. Furthermore, subcutaneous implantation of Thy-1(high) cells efficiently induced the formation of bone-like matrix. These results collectively suggest that Thy-1-positive dental pulp cells localized in the subodontoblastic layer had the ability to differentiate into hard tissue-forming cells, and thus these cells may serve as a source of odontoblastic cells.
    Histochemie 02/2012; 137(6):733-42. · 2.61 Impact Factor
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    ABSTRACT: The degradation of fibrillins, the major constituents of microfibrils, is known to facilitate the release of active transforming growth factor-β (TGF-β), a signaling molecule contributing to mineralized tissue barrier formation in exposed dental pulps. To examine the involvement of fibrillins in the barrier formation, we examined the temporospatial expression of (1) genes and proteins of fibrillins and (2) factors possibly associated with fibrillin degradation and cytodifferentiation in exposed human pulps. Human pulp slice cultures were also examined for the role of fibrillins in mineralization. Clinically healthy pulps were mechanically exposed and capped with mineral trioxide aggregate. After 7 to 42 days, the teeth were processed for immunohistochemical and cytochemical staining of fibrillin-1, fibrillin-2, latent TGF-β-binding protein (LTBP)-1, matrix metalloproteinase-3 (MMP-3), alkaline phosphatase (ALP), and in situ hybridization of fibrillin-1. Pulp tissue slices cultured with β-glycerophosphate were analyzed for fibrillin-1, fibrillin-2, and ALP with the immunohistochemical/cytochemical staining and quantitative reverse-transcriptase polymerase chain reaction. Fibrillin-1-immunoreactivity was seen until 7 days but turned into undetectable since 14 days in the pulpal area just beneath the exposure site. MMP-3-immunoreaction was transiently detected at 14 days. At 42 days when the mineralized barrier was evident, fibrillin-1-immunoreactivity and fibrillin-1 expression remained down-regulated. Fibrillin-2, LTBP-1, and ALP were constantly detected in the fibrillin-1-undetectable area. Pulp slices cultured with β-glycerophosphate showed mineralization with up-regulation of ALP and down-regulation of fibrillin-1. Degradation and down-regulation of fibrillin-1 expression took place during the mineralized tissue barrier formation in exposed pulps in vivo and β-glycerophosphate-induced pulpal mineralization in vitro.
    Journal of endodontics 02/2012; 38(2):177-84. · 2.95 Impact Factor
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    ABSTRACT: To investigate subcutaneous tissue reactions to methacrylate resin-based root canal sealers by immunohistochemical assessment of inflammatory/immunocompetent cell infiltration. Silicone tubes containing freshly mixed Epiphany SE sealer, MetaSEAL, Super-Bond RC sealer, or a zinc oxide-eugenol sealer (Canals) were subcutaneously implanted into the backs of Wistar rats. Solid silicone rods implanted in different animals served as controls. After 7, 14 and 28 days, connective tissue surrounding the implants (n = 8, each) was processed for immunoperoxidase staining using OX6 (reactive to major histocompatibility complex class II molecules), ED1 (reactive to macrophages), and W3/13 (reactive primarily to neutrophils), and the number of positively stained cells within each field (1.2 × 0.8 mm) was enumerated. Statistical differences were analysed with Friedman's test and Scheffe's test (comparisons between test materials) or Mann-Whitney's U-test (test-control comparisons). Canals showed a significantly higher number of W3/13-positive cells (mostly neutrophils) than MetaSEAL at 28 days (P < 0.05). There were no significant differences in the numbers of OX6- or ED1-positive cells between each test material at any time point. Test-control comparisons revealed several significant differences for each antibody. This was most notable for ED1, where all the test materials at each time point, except for Epiphany SE at 28 days, showed significantly larger values than the corresponding controls. All the methacrylate resin-based sealers tested showed a similar level of inflammatory/immunocompetent cell infiltration. MetaSEAL induced less-intense neutrophil infiltration than Canals. Controls exhibited milder infiltration of inflammatory/immunocompetent cells compared with all the test materials.
    International Endodontic Journal 03/2011; 44(7):669-75. · 2.05 Impact Factor
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    ABSTRACT: Objectives: Dental pulp possesses a natural tissue repair potential. It has been well documented that the mineralized hard tissue is formed in the pulp cavity following replantation or transplantation of rat molars. However, the precise origin of the replacement population of progenitor cells remains unclear. In order to address cellular events during the pulp tissue repair and mineralization after tooth injury, we here investigated the behavior of dental pulp cells in an organ culture system of rat molar. Methods: Maxillary first molars of 4-week-old Wistar rats were extracted with forceps, and cultured on a membrane filter in DMEM containing 10% fetal calf serum. After 1, 2, 3, 5, and 7 days of culture, the teeth were fixed with 4% paraformaldehyde and processed for immunoperoxidase staining using antibodies against α-smooth muscle actin (α-SMA) and nestin (an intermediate filament expressed in differentiating and functional odontoblasts). Molars fixed immediately after extraction served as controls. Results: In the control teeth, roots were still developing and the apex remained widely open. α-SMA-immunoreactive cells were localized along the blood vessels. Odontoblasts were positive for nestin in the coronal pulp as well as in the root canal. In cultured teeth, odontoblasts and pulp cells of the coronal portion showed degenerative changes with the passage of time. In the root canal, nestin-immunoreactive odontoblasts were still found after 7 days of culture. From 2 days, α-SMA-immunoreactive fibroblastic cells appeared at the root apex, and they seemed to increase in number during the observation period. Conclusion: Upregulation of α-SMA was characteristically observed in fibroblastic cells of cultured rat molars. The α-SMA-immunoreactive cells may play a role in tissue repair of dental pulp. Supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Sciences.
    IADR General Session 2010; 07/2010
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    ABSTRACT: Regeneration of periodontal tissues, lost as a result of periodontal disease, is a key objective of periodontal treatment. Although several periodontal regeneration therapies have been devised, the origin of the undifferentiated cells that regenerate periodontal tissues remains unknown. Therefore, in the present study, to clarify the existence of osteoblast progenitor cells in the periodontal ligament, as well as to investigate the mechanism of alveolar bone regeneration without any effects from the original bone, we evaluated osteoblast differentiation induced by transplantation of GFP-transgenic rat molars into the subcutaneous tissues of wild-type rats. Ten days after transplantation, initial alveolar bone was formed apart from the cementum in the bifurcation region. After 20 days, this bone tissue had expanded to almost all of the bifurcation. GFP localization showed that the osteoblasts were derived from the transplant. Alpha-SMA- and BMP4-positive cells were observed near the root surface at 5 days after transplantation. With the progress of alveolar bone regeneration, osteoblasts expressing Runx2 and Osterix appeared in the bone-forming region. These results indicate that periodontal ligament tissue remaining on the root surface after a tooth extraction contains undifferentiated cells that have the ability to regenerate alveolar bone. The process of osteoblast differentiation in this model might be similar to that for normal alveolar bone formation. Thus, periodontal ligament cells might be useful for the regeneration of alveolar bone in tissue engineering applications.
    Journal of Oral Biosciences 01/2010; 52(2):72-80.

Publication Stats

578 Citations
100.26 Total Impact Points

Institutions

  • 1998–2014
    • Niigata University
      • • Division of Cariology, Operative Dentistry and Endodontics
      • • Department of Oral Health Science
      Niahi-niigata, Niigata, Japan
  • 2007–2013
    • Matsumoto Dental University
      • Department of Oral Histology
      Matsumoto, Nagano-ken, Japan
    • Shinshu University
      • Department of Dentistry and Oral Surgery
      Shonai, Nagano, Japan
  • 2002–2007
    • Niigata University of Pharmacy and Applied Life Sciences
      Niahi-niigata, Niigata, Japan