Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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The periodontal ligament (PDL) contains mesenchymal stem cells (MSCs) that can differentiate into osteoblasts, cementoblasts, and fibroblasts. Nevertheless, the distribution and characteristics of these cells remain uncertain. Gli1, an essential hedgehog signaling transcription factor, functions in undifferentiated cells during embryogenesis. Therefore, in the present study, the differentiation ability of Gli1+ cells was examined using Gli1-CreERT2/ROSA26-loxP-stop-loxP-tdTomato (iGli1/Tomato) mice. In 4-wk-old iGli1/Tomato mice, Gli1/Tomato+ cells were only slightly detected in the PDL, around endomucin-expressing blood vessels. These cells had proliferated over time, localizing in the PDL as well as on the bone and cementum surfaces at day 28. However, in 8-wk-old iGli1/Tomato mice, Gli1/Tomato+ cells were quiescent, as most cells were not immunoreactive for Ki-67. These cells in 8-wk-old mice exhibited high colony-forming unit fibroblast activity and were capable of osteogenic, chondrogenic, and adipogenic differentiation in vitro. In addition, after transplantation of teeth of iGli1/Tomato mice into the hypodermis of wild-type mice, Tomato fluorescence indicating the progeny of Gli1+ cells was detected in the osteoblasts and osteocytes of the regenerated bone. These results demonstrate that Gli1+ cells in the PDL were MSCs and could contribute to the alveolar bone regeneration.

DOI： 10.1177/00220345221106921

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Laminin, a basement membrane heterotrimeric glycoprotein composed of α/β/γ subunits, has important tissue-specific functions in the control of cellular behavior. Our recent study showed the colocalization of CD163+ M2-like macrophages with Schwann cells in human dental pulp, leading us to hypothesize that the laminin isoform of Schwann cells is associated with CD163 expression. The present study investigated the distribution of laminin isoforms in human dental pulp and the underlying mechanisms that affect macrophage phenotypes. Immunofluorescence analysis indicated that blood vessels were exclusively positive for laminin α4 and α5, whereas laminin α2 was associated with Schwann cells. Unexpectedly, laminin α3/laminin-332 (α3β3γ2) was detected on lymphatic vessels. In intact and carious teeth, CD163+ cells were associated with laminin α2, whereas CD206 single-positive cells were present inside, outside, and along blood vessels. In vitro incubation of THP-1 macrophages in plates coated with laminin-211/511 or its functionally analogous E8 fragments of α-chain (E8-α) indicated that cell shapes differed between macrophages grown on laminin-211/E8-α2 and macrophages grown on laminin-511/E8-α5. Laminin-211/E8-α2-coated plates upregulated CD163 expression, compared with laminin-511/E8-α5-coated plates. Integrin α3- and integrin α6-neutralizing Abs altered the shape of THP-1 macrophages and upregulated mRNA levels of CD206 and CD163 in macrophages grown on laminin-511; the neutralizing Abs did not affect macrophages grown on laminin-211. These findings suggest that laminin isoforms differentially regulate macrophage behavior via distinct integrin-laminin affinities. Of note, laminin-332 is expressed by pulpal lymphatic vessels, the existence of which has been debated; laminin-211 might have a role in maintaining CD163 expression on macrophages.

DOI： 10.4049/immunohorizons.2100110

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Resin monomers and polymerisation initiators have been shown to be cytotoxic for pulp cells and to disturb odontoblast differentiation. This study aimed to compare the effect of a resin-modified calcium silicate cement (TheraCal LC; TC) and a resin-free calcium silicate cement (ProRoot MTA; PR) on pulpal healing after pulpotomy. Pulpotomy was performed on the maxillary first molars of 8-week-old rats using either PR or TC. After 1, 3, 7, 14 and 28 days, pulpal responses were assessed by micro-computed tomography, haematoxylin-eosin staining and immunostaining against CD68, which is a pan-macrophage marker. The results showed that pulpotomy with TC induced persistent infiltration of inflammatory cells, including CD68-positive macrophages, and delayed the formation of reparative dentin as compared with that with PR, although both materials allowed pulpal healing over the long term. Therefore, resin-modified TC was not as biocompatible nor bioinductive as resin-free PR when applied on the healthy pulp of rat molars.

DOI： 10.1111/aej.12568

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Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

This study compared the apatite-forming ability (AFA) levels of flowable and putty formulations of Nishika Canal Sealer BG Multi (F-NBG and P-NBG, respectively) and attempted to clarify the cause of differences in the AFA levels of F-NBG and P-NBG. NBG samples were aged in simulated body fluid (SBF) or 1-, 5-, or 10-g/L bovine serum albumin-containing SBF (BSA-SBF) and analyzed in terms of their ultrastructures, elemental compositions, and Raman spectra to identify apatite formation. The phosphate ion consumption rates of NBG samples in the media were evaluated as an indicator of apatite growth. The original elemental composition, calcium ion release, and alkalizing ability levels of F-NBG and P-NBG were also evaluated. Apparent apatite formation was detected on all NBG samples except F-NBG aged in 10-g/L BSA-SBF. P-NBG consumed phosphate ions faster than F-NBG. As-prepared P-NBG showed more silicon elements on its surface than as-prepared F-NBG. P-NBG released more calcium ions than F-NBG, although their alkalizing ability levels did not differ statistically. In conclusion, the AFA of P-NBG was greater than that of F-NBG, probably because of the greater ability of P-NBG to expose silanol groups on the surface and release calcium ions.

DOI： 10.3390/app11198969

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OBJECTIVE: Bioceramic-containing root canal sealers promote periapical healing via Ca2+ and OH- release and apatite formation on the surface. This study aimed to compare Ca2+ and OH- release and in vivo apatite formation of three bioceramic-containing root canal sealers: EndoSequence BC sealer (Endo-BC), MTA Fillapex (MTA-F), and Nishika Canal Sealer BG (N-BG). MATERIALS AND METHODS: Polytetrafluoroethylene tubes filled with sealers were immersed in distilled water for 6 and 12 h and for 1, 7, 14, and 28 days to measure Ca2+ and OH- release. Additionally, tubes filled with sealers were implanted in the backs of rats for 28 days, and in vivo apatite formation was analyzed using an electron probe microanalyzer. RESULTS: Endo-BC released significantly more Ca2+ than the other sealers at 6 and 12 h and 1 day. Ca2+ release was significantly lower from N-BG than from Endo-BC and MTA-F at 14 and 28 days. OH- release was significantly higher from Endo-BC than from the other sealers throughout the experiment, except at 1 day. OH- release was lower from N-BG than from MTA-F at 6 h and 7 days. Only Endo-BC implants exhibited apatite-like calcium-, phosphorus-, oxygen-, and carbon-rich spherulites and apatite layer-like calcium- and phosphorus-rich, but radiopaque element-free, surface regions. CONCLUSIONS: Ca2+ and OH- release is ranked as follows: Endo-BC > MTA-F > N-BG. Only Endo-BC demonstrated in vivo apatite formation. CLINICAL RELEVANCE: Endo-BC could promote faster periapical healing than MTA-F and N-BG.

DOI： 10.1007/s00784-021-04118-w

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When regenerative endodontic procedures (REPs) are performed on immature teeth diagnosed with pulp necrosis and apical periodontitis, various healing patterns occur. Furthermore, infected immature teeth with endodontic disorders often exhibit some remnant pulp and apical tissue. Therefore, this study investigated the impact of remnant healthy or fully functional pulp and apical tissue on healing patterns after REPs. Simulated REPs were performed on non-infected immature rat molars with different amounts of remnant pulp and apical tissue. Healing patterns in these teeth were assessed after 28 days. Teeth with 0.81-0.91 mm of remnant pulp healed with pulp-like tissue, dentin, and osteodentin-like dentin-associated mineralized tissue (OSD-DAMT); teeth with 0.60-0.63 mm of remnant pulp healed with pulp-like tissue and OSD-DAMT; teeth with 0.13-0.43 mm of remnant pulp healed with periodontal ligament (PDL)-like tissue, OSD-DAMT, and cementum-like dentin-associated mineralized tissue (CEM-DAMT); and teeth with disorganization of pulp and apical tissues at 0.15-0.38 mm beyond the root apex healed with PDL-like tissue, CEM-DAMT, and intracanal bone (IB). Loss of Hertwig's epithelial root sheath was observed with IB formation. These results showed that four distinct healing patterns occurred after REPs, depending on the preoperative amount of remnant healthy pulp and apical tissue.

DOI： 10.1038/s41598-020-78022-w

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Language：English   Publisher：(NPO)日本歯科保存学会  

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Language：Japanese   Publisher：(NPO)日本歯科保存学会  

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Macrophages are immune cells with high plasticity that perform many functions related to tissue injury and repair. They are generally categorized as 2 functional phenotypes: M1 (proinflammatory) and M2 (anti-inflammatory and prohealing). To investigate the role of macrophages in human dental pulp, we examined the localization and distributional alterations of macrophages in healthy dental pulp as well as during the reparative process of pulp capping with mineral trioxide aggregate (MTA) and in cariously inflamed pulp of adult human teeth. We also quantified the populations of M1/M2 macrophages in healthy dental pulp by flow cytometric analysis. CD68+CD86+ cells (M1 phenotype) and CD68+CD163+ cells (M2 phenotype) were 2.11% ± 0.50% and 44.99% ± 2.22%, respectively, of 2.96% ± 0.41% CD68+ cells (pan-macrophages) in whole healthy dental pulp. Interestingly, M2 phenotype macrophages were associated with Schwann cells in healthy pulp, during mineralized bridge formation, and in pulp with carious infections in vivo. Furthermore, the M2 macrophages associated with Schwann cells expressed brain-derived neurotrophic factor (BDNF) under all in vivo conditions. Moreover, we found that plasma cells expressed BDNF. Coculture of Schwann cells isolated from human dental pulp and human monocytic cell line THP-1 showed that Schwann cells induced M2 phenotypic polarization of THP-1 cell-derived macrophages. The THP-1 macrophages that maintained contact with Schwann cells were stimulated, leading to elongation of their cell shape and expression of M2 phenotype marker CD163 in cocultures. In summary, we revealed the spatiotemporal localization of macrophages and potent induction of the M2 phenotype by Schwann cells in human dental pulp. M2 macrophages protect neural elements, whereas M1 cells promote neuronal destruction. Therefore, suppressing the neurodestructive M1 phenotype and maintaining the neuroprotective M2 phenotype of macrophages by Schwann cells may be critical for development of effective treatment strategies to maintain the viability of highly innervated dental pulp.

DOI： 10.1177/0022034519894957

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OBJECTIVES: To determine glucose transporter 1 (GLUT1) and runt-related transcription factor 2 (RUNX2) expression during reparative dentinogenesis after pulpotomy with mineral trioxide aggregate (MTA) capping. SUBJECTS AND METHODS: Eight-week-old male Wistar rats were used. Pulp of the upper left first molar was exposed and capped with MTA. The upper right first molar of the same animal was used as a control. After collecting molars at various time points, GLUT1, RUNX2 and mammalian target of rapamycin (MTOR) were examined by immunohistochemistry. mRNA levels of Slc2a1 (encoding GLUT1), Runx2, Nestin and Mtor were determined by real-time PCR. RESULTS: Pulp exhibited progressive formation of reparative dentine lined with GLUT1- and MTOR-immunoreactive odontoblast-like cells at 5 days after pulpotomy. RUNX2 was detected in nuclei of most pulp tissue cells at day 5 after pulpotomy. Double immunofluorescence staining revealed GLUT1 immunoreactivity on odontoblast-like cells positive for Nestin or RUNX2, 5 days after pulpotomy. Slc2a1, Runx2, Nestin and Mtor mRNA levels were significantly upregulated on days 3-5 after pulpotomy. CONCLUSIONS: After rat molar pulpotomy, dental pulp induced formation of reparative dentine with colocalization of GLUT1 and Nestin or RUNX2. Moreover, mRNA levels of Slc2a1, Runx2, Nestin and Mtor were significantly upregulated in pulpotomized dental pulp.

DOI： 10.1111/odi.13230

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INTRODUCTION: Pulp capping materials allow healing of injured pulp with a layer of reparative dentin. Glucose is needed to cure the injured area. Glucose is transported by glucose transporter (Glut) 2 and Glut4, which are transmembrane proteins that act as gatekeepers. We hypothesized that the transport of glucose via Glut2/Glut4 might contribute to the production of a dentin bridge during wound healing. Therefore, we explored Glut2 and Glut4 expression during reparative dentinogenesis after mineral trioxide aggregate capping. METHODS: The upper left first molar of 8-week-old Wistar rats underwent pulpotomy with mineral trioxide aggregate. At 1, 3, 5, 7, and 14 days after treatment, localization and colocalization of Glut2, Glut4, nestin (odontoblast marker), and antiendothelial cell antigen 1 (RECA-1; endothelial cell marker) were analyzed with immunohistochemical staining. Messenger RNA expression levels of Slc2a2 (encoding Glut2), Slc2a4 (encoding Glut4), Igf-1r (encoding insulinlike growth factor 1 receptor), and nestin were analyzed in the extracted teeth using real-time polymerase chain reaction. RESULTS: Glut2 and Glut4 were localized within odontoblasts and endothelial cells in normal control teeth. Three days after pulpotomy, Glut2- and Glut4-positive cells were detected; 7 days after pulpotomy, immunoreactivity for Glut2 and Glut4 was confined to newly differentiated odontoblastlike cells arranged beneath reparative dentin. Messenger RNA expression levels of Slc2a2 and Slc2a4 were significantly up-regulated after pulpotomy. CONCLUSIONS: Glut2 and Glut4 regulate glucose transport during wound healing beneath the injured area. This may contribute to the development of new vital pulp therapy for patients with deep caries.

DOI： 10.1016/j.joen.2019.10.003

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AIM: To explore the expression profile of CD45+/pro-collagen I+ fibrocytes in intact dental pulps as well as during wound healing in adult dental pulp tissue. METHODOLOGY: A total of 16 healthy permanent teeth were obtained from young patients (18 to 25 years) undergoing orthodontic treatment. Routine pulp capping with mineral trioxide aggregate (MTA) was performed under local anaesthesia to induce a mineralized barrier at the exposed surface. Teeth were extracted from patients after 7, 14 and 35 days. Sections of the extracted teeth were prepared and stained for various markers using indirect immunofluorescence. Fibrocytes were counted, and the data were statistically evaluated using the Dunnett test. RESULTS: In uninflammed pulp tissue, a pro-collagen I-positive reaction was detected in odontoblasts, as well as in perivascular cells. Most of the CD45-positive cells were negative for pro-collagen I in normal pulp tissue, whereas CD45+/pro-collagen I+ fibrocytes were detected 7 days after injury. At day 14, fibrocytes were recognized under the fibrous matrix in contact with MTA and had infiltrated into regions of new capillary formation, where the fibrocytes were positively stained for vascular endothelial growth factor. By 35 days, fibrocytes were few, coincident with the formation of dentine bridges. The number of fibrocytes peaked 7 days post-injury and decreased at 14 days. CONCLUSIONS: The presence of fibrocytes in human pulp wound healing was observed. The spatiotemporal distribution of fibrocytes suggests that fibrocytes are involved in the early stages of pulp wound healing, specifically by contributing to new blood vessel formation.

DOI： 10.1111/iej.12940

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This study aimed to analyze the mRNA expression and protein localization of prostaglandin I2 (PGI2) synthase (PGIS), the PGI2 receptor (IP receptor) and transient receptor potential cation channel, subfamily V, member 1 (TRPV1) in force-stimulated rat molars, toward the elucidation of the PGI2-IP receptor-TRPV1 pathway that is in operation in the pulp and possibly associated with orthodontic pain and inflammation. Experimental force was applied to the maxillary first and second molars by inserting an elastic band between them for 6-72 h. PGIS, PTGIR (the IP receptor gene), and TRPV1 mRNA levels in the coronal pulp were analyzed with real-time PCR. PGIS, IP receptor, and TRPV1 proteins were immunostained. The force stimulation induced significant upregulation of PGIS at 6-24 h, and PTGIR and TRPV1 at 6 and 12 h in the pulp. PGIS was immunolocalized in odontoblasts and some fibroblasts in the force-stimulated pulp. The IP receptor and TRPV1 immunoreactivities were detected on odontoblasts and some nerve fibers. It was concluded that PGIS, PTGIR, and TRPV1 in rat molar pulp were significantly upregulated shortly after the force application, and that the IP receptor was co-expressed on TRPV1-expressing nerves and odontoblasts. These findings suggest that the PGI2-IP receptor-TRPV1 pathway is associated with the acute phase of force-induced pulp changes involving odontoblasts and nerves.

DOI： 10.1007/s10266-017-0309-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

AimTo evaluate the abilities of three calcium silicate-based pulp-capping materials (ProRoot MTA, TheraCal LC and a prototype tricalcium silicate cement) to produce apatite-like precipitates after being subcutaneously implanted into rats.
MethodologyPolytetrafluoroethylene tubes containing each material were subcutaneously implanted into the backs of Wistar rats. At 7, 14 and 28days post-implantation, the implants were removed together with the surrounding connective tissue, and fixed in 2.5% glutaraldehyde in cacodylate buffer. The chemical compositions of the surface precipitates formed on the implants were analysed with scanning electron microscopy-electron probe microanalysis (SEM-EPMA). The distributions of calcium (Ca) and phosphorus (P) at the material-tissue interface were also analysed with SEM-EPMA. Comparisons of the thicknesses of the Ca- and P-rich areas were performed using the Friedman test followed by Scheffe's test at a significant level of 5%.
ResultsAll three materials produced apatite-like surface precipitates containing Ca and P. For each material, elemental mapping detected a region of connective tissue in which the concentrations of Ca and P were higher than those in the surrounding connective tissue. The thickness of this Ca- and P-rich region exhibited the following pattern: ProRoot MTA > prototype tricalcium silicate cementTheraCal LC. ProRoot MTA had a significantly thicker layer of Ca and P than the other materials at all time-points (P<0.05), and a significant difference was detected between the prototype cement and TheraCal LC at 28days (P<0.05).
ConclusionAfter being subcutaneously implanted, all of the materials produced Ca- and P-containing surface precipitates and a Ca- and P-rich layer within the surrounding tissue. The thickness of the Ca- and P-rich layer of ProRoot MTA was significantly thicker than that of the other materials.

DOI： 10.1111/iej.12802

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Mineral trioxide aggregate (MTA) is a commonly used dental pulp-capping material with known effects in promoting reparative dentinogenesis. However, the mechanism by which MTA induces dentine repair remains unclear. The aim of the present study was to investigate the role of prostaglandin E2 (PGE2) in dentine repair by examining the localisation and mRNA expression levels of its transporter (Pgt) and two of its receptors (Ep2 and Ep4) in a rat model of pulpotomy with MTA capping. Ep2 expression was detected in odontoblasts, endothelial cells, and nerve fibres in normal and pulpotomised tissues, whereas Pgt and Ep4 were immunolocalised only in the odontoblasts. Moreover, mRNA expression of Slco2a1 (encoding Pgt), Ptger2 (encoding Ep2), and Ptger4 (encoding Ep4) was significantly upregulated in pulpotomised dental pulp and trigeminal ganglia after MTA capping. Our results provide insights into the functions of PGE2 via Pgt and Ep receptors in the healing dentine/pulp complex and may be helpful in developing new therapeutic targets for dental disease.

DOI： 10.1038/s41598-017-07167-y

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Introduction: Myofibroblasts express alpha smooth muscle actin (alpha-SMA) and play a critical role in wound healing. Myofibroblast differentiation is controlled by the joint actions of transforming growth factor beta 1 (TGF-beta 1) and the extradomain A fibronectin splice variant (EDA-FN). Currently, the contribution of myofi-broblasts to dental pulp healing is unknown. Therefore, we analyzed expressional characteristics of alpha-SMA positive cells and investigated TGF-beta 1, EDA-FN, and alpha-SMA expression levels after pulpotomy to better understand dental pulp healing. Methods: The maxillary first molars of 8-week-old Wistar rats were pulpotomized with mineral trioxide aggregate. After 1 to 14 days, localization and colocalization of alpha-SMA, rat endothelial cell antigen-1 (as a marker of endothelial cells), neuron-glial antigen 2 (as a marker of perivascular cells), prolyl-4-hydroxylase (P4H, as an additional marker of myofibroblasts), and EDA-FN were analyzed using immunohistochemistry and double immunofluorescence. Time-course changes in the messenger RNA expression levels of TGF-beta 1, EDA-FN, and alpha-SMA were evaluated using quantitative real-time polymerase chain reaction analysis. Results: Spindle-shaped alpha-SMA positive cells transiently appeared after pulpotomy. These cells initially emerged in the pulp core on day 3 and then accumulated at the wound site by day 5. These cells were isolated from rat endothelial cell antigen-1 positive cells and did not express neuron-glial antigen 2 but did express P4H. The messenger RNA levels of TGF-beta 1, EDA-FN, and alpha-SMA were significantly up-regulated after pulpotomy. EDA-FN and a-SMA were colocalized at the wound sites on day 5. Conclusions: In association with up regulation of TGF-beta 1 and EDA-FN expression, alpha-SMA and P4H double-positive cells accumulated at the wound sites after pulpotomy. This suggests that myofibroblasts participate in dental pulp healing.

DOI： 10.1016/j.joen.2017.02.018

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

ObjectivesGaAlAs lasers induce pulp mineralization by promoting reparative dentinogenesis. This study analyzed the expression of dentin matrix protein 1 (DMP1) and osteopontin in GaAlAs laser-irradiated rat molars, to examine the hypothesis that these proteins play a role in the laser-induced reparative dentinogenic process.
Materials and methodsThe mesial surfaces of the upper first molars of 8-week-old Wistar rats were irradiated with a pulsed GaAlAs laser. After 1-14days, mRNA expression of DMP1 and osteopontin in the coronal pulp was analyzed using real-time PCR. DMP1, osteopontin, and heat shock protein 25 (HSP25) were immunolocalized at 1-21days.
ResultsThe pulp exhibited a degenerative zone in its mesial portion on days 1-3, and progressive formation of reparative dentin lined with HSP25-immunoreactive odontoblast-like cells, from day 7 onwards. DMP1 and osteopontin mRNA expression were significantly upregulated on days 1-7 and 3-7, respectively. From day 7 onwards, DMP1 and osteopontin immunoreactivity colocalized along the boundary between the primary and reparative dentin.
ConclusionGaAlAs laser irradiation of rat molars induced upregulated DMP1 and osteopontin mRNA expression in the coronal pulp, followed by the formation of reparative dentin and the colocalization of DMP1 and osteopontin immunoreactivity at the site at which this tissue first appeared.

DOI： 10.1111/odi.12461

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SAGE PUBLICATIONS LTD  

Myofibroblasts and extracellular matrix are important components in wound healing. Alpha-smooth muscle actin (-SMA) is a marker of myofibroblasts. Fibrillin-1 is a major constituent of microfibrils and an extracellular-regulator of TGF-1, an important cytokine in the transdifferentiation of resident fibroblasts into myofibroblasts. To study the correlation between changes in fibrillin-1 expression and myofibroblast differentiation, we examined alterations in fibrillin-1 and -SMA expression in organotypic cultures of dental pulp in vitro. Extracted healthy human teeth were cut to 1-mm-thick slices and cultured for 7 days. In intact dental pulp, fibrillin-1 was broadly distributed, and -SMA was observed in pericytes and vascular smooth muscle cells. After 7 days of culture, immunostaining for fibrillin-1 became faint concomitant with a downregulation in its mRNA levels. Furthermore, fibroblasts, odontoblasts and Schwann cells were immunoreactive for -SMA with a significant increase in -SMA mRNA expression. Double immunofluorescence staining was positive for pSmad2/3, central mediators of TGF- signaling, and -SMA. The administration of inhibitors for extracellular matrix proteases recovered fibrillin-1 immunostaining; moreover, fibroblasts lost their immunoreactivity for -SMA along with a downregulation in -SMA mRNA. These findings suggest that the expression of -SMA is TGF-1 dependent, and fibrillin-1 degradation and downregulation might be implicated in the differentiation of myofibroblasts in dental pulp wound healing.

DOI： 10.1369/0022155415580622

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

AimTo examine the temporospatial expression of dentine matrix protein 1 (DMP1; a noncollagenous protein involved in mineralized tissue formation), osteopontin (another noncollagenous 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 teeth were collected from 6h to 14days postoperatively and processed for immunohistochemistry for DMP1, osteopontin and nestin. Cell proliferation was monitored using 5-bromo-2-deoxyuridine (BrdU) labelling.
ResultsThe capped pulps initially exhibited superficial necrotic changes followed by the formation of new matrix and its mineralization. DMP1 immunoreactivity was observed in the matrix beneath the necrotic layer from 6h onwards and present in the outer portion of the newly formed mineralized matrix from 7days onwards. Osteopontin displayed a similar expression pattern, although it occupied a narrower area than DMP1 at 6 and 12h. Nestin-immunoreactive cells appeared beneath the DMP1-immunoreactive area at 1day, were distributed beneath the newly formed matrix at 5days and exhibited odontoblast-like morphology by 14days. BrdU-positive cells significantly increased at 2 and 3days (P<0.05) and then decreased.
ConclusionsThe 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 colocalization of DMP1 and osteopontin suggests that these two proteins play complementary roles.

DOI： 10.1111/iej.12351

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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 hematoxylineosin staining or immunoperoxidase staining of CD68 (a general macrophage marker) and M2 macrophage markers (CD163 and CD204)1 The density of positively stained cells was enumerated in the surface and inner regions (0-100 mu m and 300-400 mu m, 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.

DOI： 10.1016/j.joen.2014.08.012

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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 PGE(2) efflux transport in lipopolysaccharide (LPS)-inflamed rat incisor pulp tissue. Methods: Pulpit's 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 PGE(2) 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 PGE(2) 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 PGE(2). The significant decrease in PGE(2) release induced by the Mrp4 inhibitor suggests that Mrp4 contributes to the transport of PGE(2) in the transmembrane efflux pathway.

DOI： 10.1016/j.joen.2013.12.024

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Introduction: 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. Methods: 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. Results: 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. Conclusions: 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.

DOI： 10.1016/j.joen.2013.11.011

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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.

DOI： 10.1007/s00418-013-1076-y

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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 Scheff, 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.

DOI： 10.1007/s10266-012-0078-x

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目的:細胞外基質fibrillin-1は,弾性線維の構成タンパクであると同時に,その分解によりtransforming growth factor-β(TGF-β)を遊離させることが知られている.また,著者らはfibrillin-1がヒト歯髄直接覆髄後の創傷治癒過程で発現を消失させることを見いだしている.そこで本研究では,TGF-β制御活性を有する細胞外基質decorinとfibronectin,および創傷治癒と組織線維化に関与するtenascin-Cが,ヒト歯髄直接覆髄後の創傷治癒過程でfibrillin-1様の局在変化を示すか否かを検索した.さらに,スライス培養したヒト歯髄を用いて,fibrillin-1の発現消失へのmatrix metalloproteinase(MMP)の関与の実態を検索した.材料と方法:矯正治療により抜歯予定のヒト健全歯をmineral trioxide aggregate(MTA)で直接覆髄した後,2週および6週後に抜歯し,fibrillin-1,fibronectin,decorinおよびtenascin-Cに対する免疫組織化学的染色を行った.また,健全歯髄を組織培養し,fibrillin-1 mRNA発現を定量RT-PCRで測定するとともに,MMP阻害剤であるNNGHを添加培養し,fibrillin-1タンパクの局在変化を免疫染色により解析した.結果:直接覆髄後2週および6週において,fibrillin-1に対する免疫陽性反応は,覆髄部直下の歯髄組織で局所的に消失していた.一方,fibronectin,decorinおよびtenascin-Cは同部で一様に陽性反応を示しており,明らかな局在変化は認められなかった.一方,培養歯髄組織ではfibrillin-1の染色性が減弱し,mRNAの発現も有意に低下したが,MMP-3 mRNA発現は有意に亢進した.NNGHを添加培養するとfibrillin-1の染色性が向上した.結論:検索対象とした4種のタンパクのなかでfibrillin-1のみ直接覆髄後の創傷治癒過程で局在変化を示した.Fibrillin-1免疫反応性の消失には,MMPsによるタンパクの分解に加えて遺伝子発現の下方制御も関与するものと考えられた.(著者抄録)

DOI： 10.11471/shikahozon.56.161

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Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2013&ichushi_jid=J01092&link_issn=&doc_id=20130712330001&doc_link_id=%2Fdo1conde%2F2013%2F005603%2F001%2F0161-0168%26dl%3D0&url=http%3A%2F%2Fwww.medicalonline.jp%2Fjamas.php%3FGoodsID%3D%2Fdo1conde%2F2013%2F005603%2F001%2F0161-0168%26dl%3D0&type=MedicalOnline&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00004_2.gif

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目的:Mineral trioxide aggregate(MTA)に対するマクロファージや樹状細胞の生体内での応答様式を追究するため,MTAを移植されたラット皮下結合組織における各種マクロファージ・樹状細胞関連分子の発現状況を,免疫組織化学的,もしくは定量的遺伝子発現解析により検討した.材料と方法:MTA(ProRoot MTA)および対照として水酸化カルシウム系覆髄材(Life)を被験材料とし,それぞれシリコンチューブへ充填後,Wistar系雄性ラットの背部皮下へ移植した.同一サイズのシリコンロッドをコントロール群とした.14日経過後に移植体周囲組織を採取し,ED1(CD68:抗マクロファージおよび樹状細胞)およびOX6(抗主要組織適合抗原クラス・分子)を用いて酵素抗体二重染色を施した後,ED1+/OX6+細胞およびED1+/OX6-細胞の密度を求めた.さらに,リアルタイムPCR法を用いてCD163(組織修復(M2)マクロファージのマーカー)およびCD34(血管内皮細胞,一部の真皮樹状細胞などが発現)のmRNA発現レベルを定量した.成績:MTA移植群では,ED1+/OX6+細胞はほかの全群と比較して,またED1+/OX6-細胞はLife移植群と比較して,有意に高い密度を示した(p<0.05).また,MTA移植群におけるCD163およびCD34 mRNA発現は,他群より有意に高レベルであった(p<0.05).結論:ラット皮下結合組織において,MTAはLifeと比較して有意に高密度のCD68陽性細胞亜群(ED1+/OX6+細胞およびED1+/OX6-細胞)の浸潤と,有意に高レベルのCD34,CD163 mRNA発現を誘導した.マクロファージの関与する創傷治癒・組織修復機構が,MTAに対する生体反応に関与する可能性が示唆された.(著者抄録)

DOI： 10.11471/shikahozon.56.9

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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.

DOI： 10.4012/dmj.2013-005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SAGE PUBLICATIONS LTD  

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 alpha-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. (J Histochem Cytochem 60:861-873, 2012)

DOI： 10.1369/0022155412459741

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Recent studies have employed two markers, alpha-smooth muscle actin (alpha-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 alpha-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 alpha-SMA, STRO-1 and nestin (a neurogenic cytoskeletal protein expressed in odontoblasts). In normal pulp, alpha-SMA was detected in vascular smooth muscle cells and pericytes. Double immunofluorescent staining with STRO-1 and alpha-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 alpha-SMA-positive cells emerged at the wound margin at 14 days, and the initially formed mineralized barrier was lined with alpha-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 alpha-SMA was detected in those cells. These observations indicate that alpha-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.

DOI： 10.1007/s00418-012-0978-4

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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.

DOI： 10.1007/s00418-012-0928-1

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Introduction: 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. Methods: 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. Results: 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). Conclusions: 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. (J Endod 2012;38:648-652)

DOI： 10.1016/j.joen.2012.02.012

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Introduction: Angiogenic factors such. as VEGFR2 (vascular endothelial cell growth factor receptor 2), BcI-2 (a prosurvival and proangiogenic signaling molecule), and chernokine (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. Methods: 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. lmmunostaining for CD31 (a marker for endothelial cells), BcI-2, and real-time polymerase chain reaction analysis of VEGFR2, BcI-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. Results: The density of the CD31-stained area increased until 21 days after pulp exposure. On the other hand, BcI-2 stained area showed the highest density at 14 days (active lesion expanding phase) and then decreased until 28 days (lesion stability phase). VEGFR2, BcI-2, CXCL1, and CXCR2 messenger RNA expression in endothelial cells showed the highest levels at 14 days and then decreased until 28 days. Conclusions: The increase in microvascular density and the upregulation of VEGFR2, BcI-2, CXCL1, and CXCR2 messenger RNA expression in endothelial cells took place coincidently with the expanding phase of experi- mentally induced periapical lesions. These data suggest that these angiogenic factors play a role in the lesion development. (J Endod 2012;38:313-317)

DOI： 10.1016/j.joen.2011.11.009

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Introduction: The degradation of fibrillins, the major constituents of microfibrils, is known to facilitate the release of active transforming growth factor-beta (TGF-beta), 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 cyto- differentiation in exposed human pulps. Human pulp slice cultures were also examined for the role of fibrillins in mineralization. Methods: 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-beta-binding protein (LTBP)-1, matrix metalloproteinase-3 (MMP-3), alkaline phosphatase (ALP), and in situ hybridization of fibrillin-1. Pulp tissue slices cultured with beta-glycero-phosphate were analyzed for fibrillin-1, fibrillin-2, and ALP with the immunohistochemical/cytochemical staining and quantitative reverse-transcriptase polymerase chain reaction. Results: 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 beta-glycerophosphate showed mineralization with up-regulation of ALP and down-regulation of fibrillin-1. Conclusions: Degradation and down-regulation of fibrillin-1 expression took place during the mineralized tissue barrier formation in exposed pulps in vivo and beta-glycerophosphate-induced pulpal mineralization in vitro. (J Endod 2012;38:177-184)

DOI： 10.1016/j.joen.2011.09.016

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Language：Japanese   Publisher：Japan Endodontic Association  

DOI： 10.20817/jeajournal.33.1_3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

P>Aim
To investigate subcutaneous tissue reactions to methacrylate resin-based root canal sealers by immunohistochemical assessment of inflammatory/immunocompetent cell infiltration.
Methodology
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 x 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).
Results
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.
Conclusions
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.

DOI： 10.1111/j.1365-2591.2011.01874.x

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ラット臼歯へGaAlAs半導体レーザーを照射した後に,歯髄腔内に硬組織形成が生じることが知られている.そこで本研究では,RT-PCR法を用いて同レーザー照射後のラット臼歯における各種非コラーゲンタンパクのmRNA発現を検索した.生後8週齢Wistar系雄性ラットの上顎第一臼歯近心に,半導体レーザー装置(オサダライトサージ3000)を用いて,出力1.5W,60秒3回の条件でレーザー照射を行った.なお,非照射の上顎臼歯を対照とした.照射1,3,7日後に抜歯を行い,歯冠部よりmRNAを抽出した後,RT-PCR法にてosteo-pontin,osteonectin,osteocalcin,dentin sialophosphoprotein,dentin matrix protein 1,およびbone sialoproteinのmRNA発現解析を行った.また,歯髄の反応を組織学的に観察した.その結果,すべてのタンパクのmRNA発現が3日後までに増加を示すとともに,この発現亢進は7日後も持続していることが明らかになった.組織学的には,1〜3日後までは歯髄の局所的壊死が照射部近傍に観察されたが,7日後には象牙芽細胞様細胞の再配列と少量の新生硬組織が認められた.以上より,半導体レーザー照射されたラット臼歯では,新生硬組織形成に先立ち各種非コラーゲンタンパクのmRNA発現レベルの上昇が生じることが確認された.(著者抄録)

DOI： 10.11471/shikahozon.53.495

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Association for Oral Biology  

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.

DOI： 10.2330/joralbiosci.52.72

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This study investigated the reparative process of mechanically exposed pulps capped with mineral trioxide aggregate (MTA). Maxillary first molars of 8-week-old rats were MTA-capped for 1-14 days, and 5-bromo-2'-deoxyuridine-labeled proliferating cells and immunoreactivity for nestin and osteopontin were analyzed. MTA capping caused mild necrotic changes followed by progressive new matrix formation and calcified bridging. Proliferating cells peaked at 3 days when matrix formation was inconspicuous. Nestin-expressing cells appeared at 3 days, were arranged beneath the newly formed matrix at 5 days, and showed odontoblast-like morphology by 14 days. Osteopontin immunoreactivity was detected just beneath the necrotic area after 1 day. These findings suggest that pulpal responses to MTA capping involve proliferation and migration of progenitors followed by their differentiation into odontoblast-like cells, a mechanism basically similar to those to calcium hydroxide. Osteopontin might play a triggering role in initiation of the pulpal reparative process.

DOI： 10.1016/j.joen.2008.03.021

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Regeneration of alveolar bone is essential for periodontal treatment. Recently, cell replacement therapy has been focused on periodontal disease, but the source of the cells that regenerate alveolar bone is still uncertain. Therefore, to clarify the source of these bone-regenerating cells, we transplanted GFP-transgenic rat molars into the subcutaneous tissues of wild-type rats. Five days after transplantation, the tooth was surrounded by connective tissue containing many blood vessels. At 10 days, bone-like tissue was formed in the connective tissue between the branches of the bifurcated root. This hard tissue expanded to almost all of this bifurcation area without osseous ankylosis after 20 days. All ostcoblast-like cells in the newly formed matrix were immunopositive for GFP. In addition, these cells and the peripheral cells of the matrix showed intense immunoreactivity for BMP4, Runx2, BSP, and OPN. These results demonstrate that periodontal ligament tissue contains osteoprogenitor cells that have the ability to regenerate alveolar bone. Our model suggests that these regeneration processes might be similar to normal alveolar bone formation. (c) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2007.09.054

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Matrix remodeling is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Periostin, originally identified in a mouse osteoblastic library, plays a role in cell adhesion and migration and in mechanical stress-induced matrix remodeling. In this study, we analyzed and compared the distribution patterns of TIMP-2 and periostin during mouse mandible development. Immunohistochemical staining for TIMP-2 and periostin was carried out on serial cryosections obtained from mice at embryonic days 13-16, postnatal day 2 (P2), P35, and 12 weeks of age. TIMP-2 and periostin exhibited a strikingly similar protein distribution during mandible development. From bud to early bell stages of molars, TIMP-2 and periostin were highly expressed on the lingual and anterior sides of the basement membrane and on the adjacent jaw mesenchyme. In pre- and postnatal incisors, the basement membrane of the apical loop and dental follicle was immunostained for TIMP-2 and periostin. At postnatal stages, TIMP-2 and periostin were prominently confined to the extracellular matrix (ECM) of gingival tissues, periodontal ligaments, and tendons (all recipients of mechanical strain). However, periostin was solely detected in the lower portion of the inner root sheath of hair follicles. Gingiva of P2 cultured in anti-TIMP-2 antibody-conditioned medium showed markedly reduced staining of periostin. We suggest that TIMP-2 and periostin are co-distributed on ECM exposed to mechanical forces and coordinately function as ECM modulators.

DOI： 10.1007/s00441-007-0439-x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

While mineralized tissue is formed in the pulp cavity after tooth replantation or transplantation, little is known of this hard tissue formation. Therefore, we conducted histological and immunohistochemical evaluations of hard tissue formed in the pulp of rat maxillary molars after tooth replantation. At S days after replantation, degenerated odontoblasts were lining the pulp cavity. At 14 days, dentin- or bone-like tissue was present in the pulp cavity. Immunore activity for osteopontin (OPN) and bone sialoprotein (BSP) was strong in the bone-like tissue, but weak in the dentin-like tissue. Conversely, dentin sialoprotein (DSP) was localized in the dentin-like tissue, but not in the bone-like tissue. Cells positive for BMP4, Smad4, Runx2, and Osterix were found around the blood vessels of the root apex at 5 days. At 14 days, these cells were also localized around the bone-like tissue. Cells expressing alpha-smooth muscle actin (SMA) were seen around the newly formed bone-like tissue, whereas no such cells were found around the newly formed dentin-like tissue. In an experiment involving the transplantation of a green fluorescent protein (GFP)-transgenic rat tooth into a wild-type rat tooth socket, GFP-positive cells were detected on the surface of the bone-like tissue and over all dentin-like tissue. These results indicate that the original pulp cells had the ability to differentiate into osteoblast-like cells as well as into odontoblast-like cells. (C) 2007 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.archoralbio.2007.04.015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT AMER ASSOC DENTAL RESEARCHI A D R/A A D R  

While dental pulp appears to be able to form mineralized matrices that do not always resemble dentin, the precise characteristics of the hard tissue and the mechanism of its induction remain unknown. Therefore, we evaluated hard tissue induced by transplantation of pulp into subcutaneous tissue. Seven days after transplantation, initial hard tissue was formed at the inner periphery of the pulp. After 14 days, this hard tissue expanded inwardly. Mineralized matrix was immunopositive for osteocalcin, osteopontin, and bone sialoprotein, but negative for dentin sialoprotein. Transplantation of GFP-labeled pulp into wild-type rats showed these formative cells to have been derived from the transplant. TEM observation revealed apatite crystals within necrotic cells and matrix vesicles at the initial stage of calcification. These results indicate that pulp cells possess the ability to form a bone- or cementum-like matrix. Calcification of the matrix may occur in necrotic cells and matrix vesicles, followed by collagenous calcification.

DOI： 10.1177/154405910708600515

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：HISTOCHEMICAL SOC INC  

The dental follicle contains mesenchymal cells that differentiate into osteoblasts, cementoblasts, and fibroblasts. However, the characteristics of these mesenchymal cells are still unknown. alpha-Smooth muscle actin (alpha-SMA) is known to localize in stem cells, and precursor cells of various tissues. In the present study, to characterize the undifferentiated cells in the dental follicle, immunohistochemical localization of a-SMA was examined during rat molar tooth development. Rat mandibles were collected at embryonic days (E) 15-20 and postnatal days (P) 7-28. Immunohistochemical stainings for alpha-SMA, periostin, Runt-related transcription factor-2 (Runx2), tissue nonspecific alkaline phosphatase (TNAP), and bone sialoprotein (BSP) were carried out using paraffin-embedded sections. a-SMA localization was hardly detected in the bud and cap stages. At the early bell stage, alpha-SMA-positive cells were visible in the dental follicle around the cervical loop. At the late bell to early root formation stage (P14), these cells were detected throughout the dental follicle, but they were confined to the apical root area at P28. Double immunostaining for alpha-SMA and periostin demonstrated that alpha-SMA-positive cells localized to the outer side of periostin-positive area. Runx2-positive cells were visible in the a-SMA-positive region. TNAP-positive cells in the dental follicle localized nearer to alveolar bone than Runx2-positive cells. BSP was detected in osteoblasts as well as in alveolar bone matrix. These results demonstrate that a-SMA-positive cells localize on the alveolar bone side of the dental follicle and may play a role in alveolar bone formation.

DOI： 10.1369/jhc.6A6980.2006

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Tissue inhibitors of metalloproteinases (TIMPs) possess multiple functions, in addition to their matrix metalloproteinase (MMP) inhibitory activity. The continuously growing incisor of mouse possesses a stem cell compartment at the apical end of the epithelium (the apical loop) and thus provides an excellent tool to analyze the mechanisms of organogenesis and cytodifferentiation. To understand the functions of TIMPs in tooth development, we have analyzed the gene expression and protein localization of TIMP-1, -2, and -3 during mouse incisor development, from embryonic day 13 (E13) to postnatal day 3 (P3). TIMP-1 was present on the basement membrane during early developmental stages. At P2, TIMP-1 was strongly detected along the apical loop, transiently disappeared from the basement membrane in the cytodifferentiation zone, and later reappeared at the distal end of functional ameloblasts. Expression of TIMP-2 protein was restricted to the outer part of the apical loop throughout the examined stages. At P2, TIMP-2 was present on the basement membrane at the outer part of the apical loop. The dental follicle also expressed Timp-2, and the corresponding protein was abundant within the extracellular matrix. Timp-3 mRNA was highly expressed in the mesenchyme surrounding the apical loop. During matrix formation, Timp-3 was expressed by subodontoblasts, and the protein was detected in this layer and between odontoblasts. Distinct temporal and spatial expression patterns of TIMPs suggest divergent functions of these factors in incisor organogenesis.

DOI： 10.1007/s00441-005-0123-y

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

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.

DOI： 10.1111/j.1600-0722.2006.00261.x

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To characterize osteodentin in the pre-functional rat incisor, we performed histological and histochemical evaluation of the anterior apex of the incisor in 3-day-old rats. The unerupted incisor was composed of osteodentin, with numerous cells present in the anterior apex. The osteodentin was immunopositive for osteocalcin, bone sialoprotein, and dentin sialoprotein, with an immunolocalization pattern similar to that of dentin. Back-scattered electron microanalysis (BSE) and electron probe microanalysis (EPMA) showed that osteodentin was not uniformly calcified. These results indicate that osteodentin in the rat incisor possesses dentin-like characteristics, and may be fragile in structure. © 2006, Japanese Association for Oral Biology. All rights reserved.

DOI： 10.2330/joralbiosci.48.132

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

The gene expression and protein distribution of matrix metalloproteinase (MMP) -2, -9, membrane type-1 MMP (MT1-MMP), as well as of TIMP-1, -2, and -3 were analyzed during mouse molar development. Immunohistochemical data demonstrated that all the MMPs investigated were expressed in the dental epithelium and mesenchyme. In contrast, gene and protein expression analysis for TIMPs showed that they were differentially expressed. TIMP-1 was expressed in the dental epithelium and mesenchyme between E13 and E16 and was transiently up-regulated at E14, the cap stage. TIMP-1 expression was also detected in differentiating odontoblasts. TIMP-2 RNA transcripts were found in the peridental and dental mesenchyme, odontoblasts, and ameloblasts. Protein analysis revealed high expression on the lingual side of the dental epithelium and underlying mesenchyme together with transient expression in the enamel knot at E14 and expression in the gingival tissue and enamel matrix postnatally. TIMP-3 RNA transcripts were found in discrete regions of the dental epithelium, including at high levels in the cervical loop at E16. Expression was also detected in preodontoblasts at E16 and transiently during ameloblast differentiation. Analysis of the protein distribution revealed a lower level of TIMP-3 on the lingual side of the dental epithelium at E14. MT1-MMP was expressed in the dental mesenchyme between E13 and E16, at relatively high levels in the cervical loop at E14, and in the odontoblasts and ameloblasts. The distinct temporospatial distribution patterns of the TIMPs suggest that these inhibitors play several intrinsic roles during tooth development. Developmental Dynamics 228.105-112, 2003. (C) 2003 Wiley-Liss, Inc.

DOI： 10.1002/dvdy.10352

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT AMER ASSOC DENTAL RESEARCHI A D R/A A D R  

Major histocompatibility complex (MHC) class 11 molecule-expressing cells are distributed in human dental pulp, and have been shown to accumulate beneath caries lesions. The responses of these cells and nerve fibers were analyzed under 5 different clinical conditions: shallow and deep experimental cavities, active and slow untreated caries, and treated caries. Under deep cavities, class 11 molecule-expressing dendritic cells displaced the injured odontoblasts during a period of one month, while such a response was not observed in shallow cavities and untreated or treated carious teeth. The class 11 molecules seen in the neural elements under active caries were no longer detectable in treated carious teeth. However, six months after treatment, clusters consisting of dendritic cells, Tlymphocytes, and nerve fibers still remained locally in the subodontoblastic area. These results indicate that dental pulps respond differently to cavity preparation and restoration between normal and caries conditions, and that immunoresponses persist for many months, even after caries treatment.

DOI： 10.1177/154405910308200604

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER-VERLAG  

While dental pulp undergoes calcification following tooth replantation or transplantation, we actually know little about these mechanisms. We therefore conducted histological and immunohistochemical evaluations of mineralized tissue that formed in the pulp of rat maxillary molar transplanted into abdominal subcutaneous tissue. One, 2, 3, and 4 weeks post-transplantation, the teeth were investigated immunohistochemically using antibodies to osteocalcin (OCN), osteopontin (OPN), bone sialoprotein (BSP), dentin sialoprotein (DSP), and tissue non-specific alkaline phosphatase (TNAP). In the 1st week after transplantation, cell-rich hard tissue was formed at the root apex. At 2 weeks, formations of hard tissue, with few cells in the root canals and bone-like tissue in the coronal pulp chamber, were noted. After 3 and 4 weeks, the amounts of these hard tissues were increased. The immunolocalization of OCN, OPN, and BSP was seen strongly in coronal and apical hard tissues, but weakly in the root hard tissue. Conversely, DSP localized in the root hard tissue, but not in other newly formed hard tissues. At I week, TNAP localized along the periphery of the apical hard tissue and the lower surfaces of root predentin. These results demonstrate that the newly formed hard tissues in the pulp cavity of subcutaneously transplanted molars could be classified into three types, suggesting that these might be formed by type-specific cells.

DOI： 10.1007/s00418-002-0478-z

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In the present undertaking, the distribution of odontoblast processes in human dentin was determined through the DiI carbocyanine dye fluorescent staining of the cell membrane, while F-actin was identified by rhodamine-phalloidin. Confocal laser scanning microscopy revealed intense labeling for both agents in inner dentin, while transmission electron microscopy (TEM) identified dentinal tubules including odontoblast processes in this area, each process being surrounded by a cell membrane and containing an abundance of filamentous structures. Electron-dense "lamina limitans" lined the dentinal tubules. Individual cell processes became narrower toward the middle area, and their overall numbers decreased as well under TEM. Labeling for F-actin was absent in both middle and outer dentin, while faint labeling for DiI was visible along the dentinal tubules as far as the dentino-enamel junction (DEJ), where it was also recognized within the tubules themselves. Under TEM, the dentinal tubules lined with electron-dense structures were, in fact, empty in the middle and outer dentin. Immediately below the DEJ, however, the tubules manifested dense concentrations of fine granular material. Our study, therefore, appears to suggest that odontoblast processes do not extend beyond the inner dentin of fully erupted human premolars.

DOI： 10.1007/s00418-002-0442-y

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL MUNKSGAARD  

While Er:YAG laser systems are in extensive use for caries removal and cavity preparation, the effects of such treatment on pulp tissue remain unclear. This study evaluates these systems using immunohistochemical methods and compares the results with information gained from treatment using conventional burs. Cervical cavities were prepared in the upper first molars of rats, using either an Er:YAG laser or a conventional tungsten-carbide bur. At intervals of 5 min, 6 h, 12 h, 1 d, 3 d and 7 d after cavity preparation, the teeth were processed for immunohistochemical analyses of tissue non-specific alkaline phosphatase, OX6-positive major histocompatibility complex class II antigen-expressing cells and PGP 9.5-immunoreactive nerve fibers. DNA fragmentation was detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. Tissue non-specific alkaline phosphatase was observed mainly in the subodontoblastic layer under the cavity lesion, from 5 min, in both groups. The inumunoreactivity was more pronounced in the laser group, but by 7 d no significant differences were recognizable. At 12 h, TUNEL-positive cells were detected around the odontoblastic layer in both groups. From 3 d to 7 d, a limited number of positive cells were still visible in the group that underwent standard treatment. Clear similarities in the distribution patterns of OX6-immunopositive cells and PGP 9.5-immunoreactive nerve fibers were also noted. From 12 h to 1 d, OX6-positive cells accumulated along the pulp-dentin border, extending their processes into the dentinal tubules. Numerous bead-like PGP 9.5-immunoreactive nerve fibers were observed under the odontoblastic layer at 7 d. These results demonstrated that there was no appreciable difference in the manner in which pulp tissue responded to treatment with either Er:YAG laser or a conventional drill. This would seem to indicate the usefulness of the Er:YAG laser system in the removal of caries and cavity preparation.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：QUINTESSENCE PUBLISHING CO INC  

It has been well-documented that the exposed dental pulp possesses the ability to form a hard tissue barrier (dentin bridge). However, the exact mechanisms by which pulp cells differentiate into odontoblasts in this process are still unknown. During primary dentinogenesis, odontoblasts express a number of mineral-related noncollagenous proteins, including bone sialoprotein (BSP), osteopontin (OPN), and dentin sialoprotein (DSP), a dentin-specific protein. The purpose of this study was to analyze immunohistochemically the expression of these molecules during reparative dentinogenesis in human teeth after pulp was capped with calcium hydroxide [Ca(OH)(2)]. At 14 days, a thin layer of fibrous matrix barrier was formed at the exposure site. An alignment of cells was observed adjacent to the barrier, and some cells were weakly immunopositive for BSP, OPN, and DSP. At 28 days, the hard tissue barrier was thickened and pulpally lined with elongated odontoblast-like cells that expressed BSP, OPN, and DSP. A number of globular structures intensely stained for BSP and OPN were observed between the cells. The superficial layer of the barrier was immunopositive for BSP and DSP, whereas OPN was found entirely in the matrix. These results suggest that BSP, OPN, and DSP are involved in reparative dentinogenesis after pulp capping with Ca(OH)(2) and that cells that form reparative dentin express an odontoblastic phenotype.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：QUINTESSENCE PUBLISHING CO INC  

The purpose of this study was to elucidate the pulpal calcification mechanism in the pulp cavity of subcutaneously transplanted rat molars. First and second maxillary molars of 4-week-old Wistar rats were extracted and immediately transplanted into abdominal subcutaneous connective tissue. At 1, 2, 3, and 4 weeks after transplantation, transplanted teeth were excised with the surrounding tissue, fixed, decalcified, embedded in paraffin, and sectioned. They were investigated by staining with hematoxylin-eosin and immunohistochemistry using antibodies to osteocalcin (OCN), osteopontin (OPN), bone sialoprotein (BSP), dentin sialoprotein (DSP), and tissue-nonspecific alkaline phosphatase (TNAP). One week after transplantation, coronal pulp tissue underwent degeneration, but blood vessels were noted to have entered the root canal from the apex. Some flat cells were found adjacent to the root predentin. Hard tissue including numerous cells was formed adjacent to the apical predentin toward the pulp cavity. At 2 weeks, blood vessels entered the pulp chamber, and bone-like tissue was formed apart from the predentin. In the root canal, hard tissue having few cells was formed adjacent to the predentin. After 3 weeks, the amounts of these hard tissues were significantly increased. In these hard tissues, OCN, OPN, and BSP were localized strongly in the coronal and apical hard tissues but weakly in the root hard tissue. On the other hand, DSP was localized in the root hard tissue but did not localize in the other newly formed hard tissues. TNAP localized strongly in the periphery of the apical hard tissue and the surface of the lower root predentin at I week, but uniform localization was observed in the soft tissue in the whole pulp cavity after 3 weeks. These findings demonstrate that the newly formed hard tissues in the pulp cavity of rat molars after transplantation into subcutaneous tissue could be classified into three types by histologic and immunohistochemical characteristics, and suggest that these might be formed by cells of three different types.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：UNIV BASQUE COUNTRY PRESS  

Rodent incisors are continuously growing teeth and enamel deposition is restricted to the labial side. In the present study, the expression of laminin-5 subunits (alpha 3, beta 3 and gamma 2) has been analyzed by in site hybridization in developing mouse lower incisors and compared to that reported in the molar. At the bud stage (E12), mRNAs for all subunits were detected in the whole epithelial thickening. At E14,when histogenesis had started,transcripts for a alpha 3 and gamma 2 subunits were restricted to the outer dental epithelium (ODE), whereas the beta 3 subunit was intensely expressed in the inner dental epithelium (IDE). A transient expression for alpha 3 subunit was seen in the enamel knot area and disappeared at E15. Subsequently, all laminin-5 subunit genes were re-expressed in differentiating ameloblasts on the labial side. Similar patterns of transcription were observed in incisor and molar, suggesting that the differential expression of laminin-5 subunits in the IDE might be involved in the histogenesis of the IDE and ameloblast differentiation. At E16.5, cells of the IDE at the anterior extremity of the incisor and in the anterior part of the lingual IDE expressed transcripts for a3 and beta 3 but not for gamma 2 subunit. Similar expression patterns were observed in the enamel-free areas of the E18 molar. This specific expression might thus be related to cells that do not differentiate as functional ameloblasts. Throughout incisor development, intense expression for all laminin-5 subunits was restricted to the labial side of the cervical loop. The asymmetrical expression of laminin-5 might be related to incisor morphogenesis and to the differences in histogenesis and cytodifferentiation of the IDE that exist in the labial versus lingual aspect of the cervical loop.

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Language：English   Publisher：BLACKWELL SCIENCE INC  

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT SOC HISTOLOGY & CYTOLOGY  

Nerve fibers and class II major histocompatibility complex (MHC) antigen-expressing dendritic cells have been known to gather in the dental pulp beneath carious lesions. Significant functional interactions presumably occur between the neural and immune elements. The present study analyzed the morphological relationship between class II-expressing cells and nerve fibers in fuman carious teeth, visualized by a HLA-DR monoclonal antibody and a protein gene product 9.5 (PGP 9.5) polyclonal antibody; a confocal laser scanning microscope (CLSM) and an electron microscope were used. In pulps affected by early caries, HLA-DR-positive dendritic cells aggregated mainly in the cell-free zone associated with bundles of PGP 9.5-immunoreactive nerve fibers, In pulps affected by advanced caries, the accumulated HLA-DR-positive cells and PGP 9.5-immunoreactive nerve fibers showed close association with each other especially beneath the odontoblast layer: the cells even embraced the nerve fibers. Intriguingly, class II molecules were recognized not only in dendritic cells but also in the Schwann cells of non-myelinated nerves in the pulp. Using immunoelectron microscopy, class II molecules were localized on the surface of the non-myelinating Schwann cells and also within some vesicles, whereas myelinating Schwann cells lacked this immunoreactivity. PGP 9.5-immunoreactive nerve fibers were also observed densely in the odontoblast layer, and CLSM revealed an intimate association of the nerve fibers and dendritic cells. The immunoreactivity for HLA-DR in Schwann cells depended upon the severity of the carious lesion. Class II-expressing Schwann cells are suggested to function as antigen-presenting cells in addition to dendritic cells.

DOI： 10.1679/aohc.61.343

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Laminin-5 is associated with several epithelial tissues and forms part of the anchoring filaments of hemidesmosomes. Recent data have shown that the expression of laminin-5 subunits is impaired in junctional epidermolysis bullosa (JEB), and, in these patients, enamel hypoplasia is commonly observed. Rodent incisors are continuously growing teeth with an asymmetry between their labial and lingual sides. Enamel matrix formation is restricted to the labial side. We have analyzed the changes in the expression and localization of laminin-5 subunits (alpha 3, beta 3, and gamma 2) in lower incisors of the mouse. The apical loop located at the end of the labial side contained stem cells and showed expression for all laminin-5 subunits. In the anterior direction, the inner dental epithelial cells (LDE) transiently lost the immunoreactivity for all subunits, whereas the transcripts for the beta 3 subunit remained in the IDE. All subunit mRNAs and proteins were expressed in ameloblasts facing predentine and also in secretory and maturation stage ameloblasts. Enamel matrix contained laminin-5. On the lingual side, the expression of laminin-5 subunits was continuous from the epithelial root sheath to the epithelial rests of Malassez in the periodontal ligament. These results suggest that spatial and temporal regulation of laminin-5 subunits correlates with the histogenesis of the dental organ, ameloblast differentiation, and enamel formation and also that laminin-5 plays a role in the adhesion between dental epithelial cells and the extracellular matrix (enamel or dentine) in areas where the dental basement membrane is absent.

DOI： 10.1007/s004410051044

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Tooth morphogenesis is regulated by epithelial-mesenchymal interactions mediated by the easement membrane (BM). Laminins are major glycoprotein components of the BMs, which are involved in several cellular activities. The expression and localization of the alpha 3, beta 3, and gamma 2 laminin-5 subunits have been analyzed by in situ hybridization and immunohistochemistry during mouse molar development. Initially (E12), mRNAs of all subunits were detected in the entire dental epithelium and the corresponding proteins were located in the BM. During cap formation (E13-14), transcripts for the alpha 3 and gamma 2 subunits were localized in the outer dental epithelium (ODE), whereas the beta 3 subunit mRNA was present in the inner dental epithelium (IDE). During the early bell stage (E16), immunoreactivity for all subunits disappeared from the BM along the IDE, although intense signals for beta 3 mRNA were detectable in cells of the IDE, Subsequently, when the dentinal matrix was secreted by odontoblasts (E18-19.5), mRNAs of all three subunits were re-expressed by ameloblasts, and the corresponding proteins were detected in ameloblasts and in the enamel matrix. Tissue recombination experiments demonstrated that when E16 IDE or ODE was associated with E18 dental papilla mesenchyme, immunostaining for all laminin-5 subunits disappeared from the BM, whereas when cultured with non-dental limb bud mesenchyme, they remained positive after 48 hr of culture. These results suggest that the temporospatial expression of laminin-5 subunits in tooth development, which appears to be differentially controlled by the dental mesenchyme, might be related to the enamel organ histo-morphogenesis and the ameloblast differentiation. (C) 1998 Wiley-Liss, Inc.

DOI： 10.1002/(SICI)1097-0177(199802)211:2<164::AID-AJA5>3.0.CO;2-F

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC DENTAL RESEARCH  

Class II major histocompatibility complex (MHC) antigen-expressing cells are generally associated with the early phase of the immune response. We have studied the distribution of class II-expressing cells in developing, normal, and carious human teeth to clarify when human pulp acquires an immunologic defense potential and how this reacts to dental caries. Antigen-expressing cells were identified immunohistochemically by means of HLA-DR monoclonal antibody. In the pulp of unerupted developing teeth, numerous HLA-DR-positive cells were distributed mainly in and around the odontoblast layer. In erupted teeth, HLA-DR-positive cells were located, for the most part, just beneath the odontoblast layer, with slender cytoplasmic processes extending into the layer. Superficial caries lesions caused an aggregation of HLA-DR-positive cells in dental pulp corresponding to the lesion. In teeth with deeper caries lesions, this aggregation of cells expanded to include the odontoblast layer. Also noted were HLA-DR-positive cells lying along the pulp-dentin border, with cytoplasmic processes projecting deep into the dentinal tubules, where they co-localized with odontoblast processes. These findings suggest that: (1) human dental pulp is equipped with immunologic defense potential prior to eruption; (2) in the initial stage of caries infection, an immunoresponse mediated by class-II-expressing cells is initiated in human dental pulp; and (3) HLA-DR-positive cells trespass deep into dentinal tubules as the caries lesion advances.

DOI： 10.1177/00220345960750081001

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Exposed dental pulp is known to possess the ability to form a hard-tissue barrier (dentin bridge). The exact mechanisms by which pulp cells differentiate into odontoblasts in this process are unknown. Fibronectin has been demonstrated to play a crucial role in odontoblast differentiation during tooth development. This study tested the hypothesis that fibronectin is involved in the initial stages of replacement odontoblast differentiation and reparative dentin formation. We observed its immunohistochemical localization during dentin bridge formation in human teeth, after pulp was capped with calcium hydroxide [Ca(OH)(2)]. One day after the capping, precipitation of crystalline structures was observed at the TEM level in association with cell debris at the interface between the superficial necrotic zone and underlying pulp tissue. This layer of dystrophic calcification showed positive reaction for fibronectin, and pulp cells appeared to be closely associated with this layer, seven to ten days post-operatively. At 14 days, an alignment of cells, some of which were elongated and odontoblast-like, was observed adjacent to the fibronectin-positive irregular matrix. Between the cells, corkscrew fiber-like fluorescence was visible. At 28 days, the irregular fibrous matrix was followed by the formation of tubular dentin-like matrix lined with odontoblast-like cells. Therefore, it would seem that fibronectin associated with the initially formed calcified layer might play a mediating role in the differentiation of pulp cells into odontoblasts during reparative dentinogenesis, after pulp was capped with Ca(OH)(2).

DOI： 10.1177/00220345960750081101

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

The immunolocalization of decorin was studied by confocal laser scanning microscopy and transmission electron microscopy. In the apical area of developing teeth, labelling for decorin was found in the dental papilla cells, preodontoblasts and also in the Hertwig's epithelial cells. Mantle dentine and the initial predentine were negative. In circumpulpal dentine, intense reactivity extended along the calcification front and dentinal tubules. Fluorescence was also evident in odontoblast cell bodies and their processes in predentine. None was perceived, however, in the predentinal matrix. Faint staining was observed on the calcified dentinal matrix. Immunoelectron microscopy revealed staining for decorin in collagen fibrils lining the predentine-dentine junction, and where arrays of labelled filaments were noted orthogonal to the collagen fibrils. Staining extending from the calcification front was observed in the matrix adjacent to the dentinal tubule. The decorin observed at the calcification front might regulate the mineralization of dentinal matrix. Copyright (C) 1996 Elsevier Science Ltd.

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The immune-localization of decorin in both normal and carious human teeth was studied using confocal laser scanning microscope and transmission electron microscope. In normal teeth, labeling for decorin was observed in odontoblast cell bodies and its processes located in predentin. No reaction was observed in predentinal matrix, however, fluorescence was intense along the calcification front and dentinal tubules. Odontoblasts under carious lesions reacted positively to decorin, while, the dentinal tubules affected by caries did not. Immunoelectron microscopy revealed staining for decorin in collagen fibrils lining the predentin-dentin junction. Decorin might regulate the mineralization of dentinal matrix.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Indirect immunofluorescence-based studies have shown similarities in the distribution patterns of fibronectin-positive fibrous structures and so-called von Korff fibres. The aim of the present study was to analyse the reactivity of fibronectin in the odontoblast layer of fully developed human teeth by means of immunoelectron microscopy. Between the odontoblasts, discrete and undulatory fibrillar fascicles with peroxidase labelling were observed. They seemed to be in contact with odontoblasts in some areas, while in others they appeared to be intervening between two neighbouring odontoblasts. Higher magnifications of the fibrillar material demonstrated axial periodic staining of about 70 nm. Peroxidase reaction of fibronectin was also recognized along the cell membrane of odontoblasts facing predentine. The fibronectin in fibrillar fascicles observed between odontoblasts would be held in place by the direct molecular interaction with collagen fibrils and contribute to the pulpward migration of these cells and maintenance of their specific morphology. At the distal end of odontoblasts, a tight seal would be maintained by means of odontoblast-fibronectin adhesion.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILLIAMS & WILKINS  

The effects of antibacterial drugs on bacterially contaminated dental pulps were investigated in monkeys. Class V buccal cavities with pulpal exposures were prepared and then left open to the oral environment for 24 h. The exposed pulps were capped with a-tricalcium phosphate (alpha-TCP) containing a mixture of antibacterial drugs. Either alpha-TCP or Ca(OH)(2) was used as a control. Pulpal responses were histologically evaluated after 4 wk. Those teeth capped with alpha-TCP alone showed total pulp necrosis and bacterial growth within the pulp chamber. By contrast, the pulps capped with alpha-TCP containing mixed antibacterial drugs remained almost normal without any necrotic layer, but showed persistent absorbing response to capping materials and no signs of hard tissue barrier formation. In teeth capped with Ca(OH)(2), a hard tissue barrier was formed below the exposure site, with a wide loss of pulp tissue. No inflammation was seen under the barrier. These results indicate that mixed antibacterial drugs added to alpha-TCP effectively disinfected pulpal lesions, without destroying any of the sound pulp tissue. However, hard tissue barrier formation was delayed by this mixture as compared with Ca(OH)(2).

DOI： 10.1016/S0099-2399(06)80551-0

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This study was performed to evaluate the pulpal response to alpha-tricalcium phosphate (alphaTCP) containing calcium hydroxide (Ca(OH)2). The dental pulps of monkeys were amputated and dressed with four agents: alphaTCP, alphaTCP containing 1% Ca(OH)2, alphaTCP containing 5% Ca(OH)2, and Ca(OH)2 being used as a control. The pulpal responses were histologically evaluated after 4 and 8 weeks. The pulp tissue treated with alphaTCP proliferated above the level of the original wound surface, and a thin layer of hard tissue barrier was formed directly against the capping agent. The barrier demonstrated atubular matrix lined with flattened or cuboidal cells, but occasionally appeared irregular in form. Ca(OH), dressing resulted in destruction of pulp tissue, with a thick hard tissue barrier being formed below the level of the exposure site. The barrier consisted coronally of osteodentin and pulpally of tubular dentin lined with odontoblast-like cells. By contrast, 1% Ca(OH), added to alphaTCP produced a slight proliferation of pulp tissue. An atubular matrix barrier, pulpally lined with cuboidal cells, formed above the exposure site. It was later followed by the formation of tubular matrix lined with columnar cells. Teeth treated with 5% Ca(OH), showed a thin necrotic layer and a thick barrier formation. The barrier was composed of tubular dentin-like tissue lined with odontoblast-like cells. It would appear that alphaTCP containing a small amount of Ca(OH)2 may be clinically useful as capping agent, as it induced consistent hard tissue formation, without excessive destruction of underlying pulp tissue.

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The distribution of fibronectin in dental pulp was studied in developing and developed human teeth by indirect immunofluorescence using a confocal laser scanning microscope. In the apical region of developing teeth, intense fluorescence was found along the basement membrane facing the mesenchyme of Hertwig's epithelial sheath and first-formed (mantle) predentine. With further elongation of odontoblasts, fibronectin was observed between the cells, appearing as corkscrew fibres passing from the pulp into predentine parallel to the long axis of the odontoblasts. In the coronal region of developing and developed teeth a similar distribution of fibronectin was observed in the odontoblast layer. At the border zone between odontoblasts and predentine the reaction was intense, but was weak in the predentine itself. In the calcified dentinal matrix it had disappeared completely, except for the area along the dentinal tubules. The results demonstrate that fibronectin is present in the odontoblast layer during all stages of dentinogenesis. Fibronectin-positive fibrous structures between odontoblasts probably correspond to von Korff fibres, and are closely related to odontoblast differentiation and dentinogenesis.

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Abstract This study was performed to evaluate the pulpal response to α‐tricalcium phosphate (αTCP) containing calcium hydroxide (Ca(OH)2). The dental pulps of monkeys were amputated and dressed with four agents: αTCP, αTCP containing 1% Ca(OH)2, αTCP containing 5% Ca(OH)2, and Ca(OH)2 being used as a control. The pulpal responses were histologically evaluated after 4 and 8 weeks. The pulp tissue treated with αTCP proliferated above the level of the original wound surface, and a thin layer of hard tissue barrier was formed directly against the capping agent. The barrier demonstrated atubular matrix lined with flattened or cuboidal cells, but occasionally appeared irregular in form. Ca(OH)2 dressing resulted in destruction of pulp tissue, with a thick hard tissue barrier being formed below the level of the exposure site. The barrier consisted coronally of osteodentin and pulpally of tubular dentin lined with odontoblast‐like cells. By contrast, 1% Ca(OH)2 added to αTCP produced a slight proliferation of pulp tissue. An atubular matrix barrier, pulpally lined with cuboidal cells, formed above the exposure site. It was later followed by the formation of tubular matrix lined with columnar cells. Teeth treated with 5% Ca(OH)2, showed a thin necrotic layer and a thick barrier formation. The barrier was composed of tubular dentin‐like tissue lined with odontoblast‐like cells. It would appear that αTCP containing a small amount of Ca(OH)2 may be clinically useful as a capping agent, as it induced consistent hard tissue formation, without excessive destruction of underlying pulp tissue. Copyright © 1994, Wiley Blackwell. All rights reserved

DOI： 10.1111/j.1600-9657.1994.tb00535.x

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根尖性歯周組織病変の難治化は、根尖部での複雑な解剖学的問題に加え、残存バイオフィルムによる細菌学的な影響もあるとされている。こうした様々な要因の改善策として外科的歯内療法が選択される。しかし、補綴処置を行う上で考慮する歯冠・歯根長比の問題で複数回に渡り歯根切断が行われることは稀である。今回は、歯根尖切除術を過去3度に渡って施行しても治癒しなかった症例に対して、マイクロスコープの使用下で再歯根尖切除術と根尖部の緊密な封鎖を目的としたmineral trioxide aggregate(以下MTA)による逆根管充填を含めた再外科的歯内療法が奏効した症例を報告する。患者は59歳の女性で、上顎右側中切歯および側切歯に対して打診痛、根尖部圧痛および瘻孔を認める慢性根尖膿瘍と診断し、最初に通常の感染根管治療を行った。その後、病変部の治癒が期待できなかったため歯根尖切除を行い、逆根管形成後にMTAで逆根管充填を行った。手術から1ヵ月後で瘻孔は消失し、さらに6ヵ月後のレントゲン写真では根尖部の透過像が消失しており、治癒傾向を認めた。(著者抄録)

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根尖性歯周組織病変の難治化は、根尖部での複雑な解剖学的問題に加え、残存バイオフィルムによる細菌学的な影響もあるとされている。こうした様々な要因の改善策として外科的歯内療法が選択される。しかし、補綴処置を行う上で考慮する歯冠・歯根長比の問題で複数回に渡り歯根切断が行われることは稀である。今回は、歯根尖切除術を過去3度に渡って施行しても治癒しなかった症例に対して、マイクロスコープの使用下で再歯根尖切除術と根尖部の緊密な封鎖を目的としたmineral trioxide aggregate(以下MTA)による逆根管充填を含めた再外科的歯内療法が奏効した症例を報告する。患者は59歳の女性で、上顎右側中切歯および側切歯に対して打診痛、根尖部圧痛および瘻孔を認める慢性根尖膿瘍と診断し、最初に通常の感染根管治療を行った。その後、病変部の治癒が期待できなかったため歯根尖切除を行い、逆根管形成後にMTAで逆根管充填を行った。手術から1ヵ月後で瘻孔は消失し、さらに6ヵ月後のレントゲン写真では根尖部の透過像が消失しており、治癒傾向を認めた。(著者抄録)

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目的:近年注目されている接着性レジンシーラーは根管壁象牙質への接着性が謳われる一方で,再根管治療時の除去が困難となることが懸念されている.そこで,比較的容易に除去可能な4-META含有レジンシーラー(メタシールSoft)が開発されたが,その生体親和性についての知見は十分とはいえない.本研究では,同シーラーをラット背部皮下に埋入後,局所に浸潤する炎症性細胞の分布密度を免疫組織化学的に定量することにより,生体親和性を評価した.材料と方法:被験レジン系シーラーとしてメタシールSoft,対照材料としてエポキシレジン系シーラー(AHプラス)および酸化亜鉛ユージノール系シーラー(キャナルス)を使用した.滅菌PTFEチューブに練和したシーラーを填入し,ただちに4週齢Wistar系雄性ラット(各観察期間ともn=5)の背部皮下組織内に埋入した.コントロールとしてシーラーを填入していない滅菌PTFEチューブを用いた.7,14,28日後に皮下組織を切り出し,4%パラホルムアルデヒド溶液で24時間浸漬固定し,凍結切片を作成後,H-E染色による組織学的観察を行うとともに,マクロファージ,好中球の局在についてそれぞれCD68,CD43を一次抗体として酵素抗体染色を行い観察した.さらに,チューブ開口部と接する組織内における陽性細胞の密度を求め,統計学的に分析した(一元配置分散分析およびBonferroni Dunn検定,α=0.05).結果:H-E染色では,すべての実験群において,7日経過例でチューブ開口部を中心とした炎症性細胞の浸潤が認められたが,時間の経過とともに炎症性細胞は減少し,線維組織による被包が観察された.メタシールSoft群ではシーラーの溢出がしばしば組織内に観察され,28日経過例においてもその残存が認められた.定量解析の結果,CD68陽性細胞については7日経過例でキャナルス群がコントロール群より有意に高い値を示したが,14,28日経過例においては各群間で有意差は認めなかった.CD43陽性細胞については,7,14日経過例でメタシールSoft群がキャナルス群より有意に低い値を示した.28日経過例では,各群間で有意差は認めなかった.結論:ラット皮下結合組織においては,4-META含有レジン系シーラーは酸化亜鉛ユージノール系シーラーと比較して,軽微な好中球浸潤を誘発することが示された.(著者抄録)

DOI： 10.11471/shikahozon.59.65

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tissue inhibitors of metalloproteinase(TIMP)-1、-2、-3の役割を解明する目的で、マウス切歯をモデルとして、その発生過程、細胞分化及び基質形成期におけるこれらの発現を検索した。マウス切歯形態形成、細胞分化、及び基質形成過程において、TIMP-1、-2、-3は時間的空間的にそれぞれ全く異なった発現パターンを示し、臼歯発生過程とも異なるものであった。歯胚上皮-間葉接合部に局在するTIMP-1はエナメル器のアポトーシスを回避している可能性が示唆された。基底膜に観察されるTIMP-2は、同様な機構で歯性上皮細胞の増殖を制御している可能性が示唆された。歯小嚢で発現されるTIMP-2はその組織化に関与していると思われた。現在のところ、象牙芽細胞下層におけるTIMP-3の役割は不明である。

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