Authorship：Lead author,　Corresponding author   Language：English  

BACKGROUND: The Journal of Oral Biosciences is devoted to advancing and disseminating fundamental knowledge concerning every aspect of oral biosciences. HIGHLIGHT: This review features review articles in the fields of "Bone Cell Biology," "Tooth Development & Regeneration," "Tooth Bleaching," "Adipokines," "Milk Thistle," "Epithelial-Mesenchymal Transition," "Periodontitis," "Diagnosis," "Salivary Glands," "Tooth Root," "Exosome," "New Perspectives of Tooth Identification," "Dental Pulp," and "Saliva" in addition to the review articles by the winner of the "Lion Dental Research Award" ("Plastic changes in nociceptive pathways contributing to persistent orofacial pain") presented by the Japanese Association for Oral Biology. CONCLUSION: The review articles in the Journal of Oral Biosciences have inspired its readers to broaden their knowledge about various aspects of oral biosciences. The current editorial review introduces these exciting review articles.

DOI： 10.1016/j.job.2023.01.008

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Various growth and transcription factors are involved in tooth development and developmental abnormalities; however, the protein dynamics do not always match the mRNA expression level. Using a proteomic approach, this study comprehensively analyzed protein expression in epithelial and mesenchymal tissues of the tooth germ during development. First molar tooth germs from embryonic day 14 and 16 Crlj:CD1 (ICR) mouse embryos were collected and separated into epithelial and mesenchymal tissues by laser microdissection. Mass spectrometry of the resulting proteins was carried out, and three types of highly expressed proteins [ATP synthase subunit beta (ATP5B), receptor of activated protein C kinase 1 (RACK1), and calreticulin (CALR)] were selected for immunohistochemical analysis. The expression profiles of these proteins were subsequently evaluated during all stages of amelogenesis using the continuously growing incisors of 3-week-old male ICR mice. Interestingly, these three proteins were specifically expressed depending on the stage of amelogenesis. RACK1 was highly expressed in dental epithelial and mesenchymal tissues during the proliferation and differentiation stages of odontogenesis, except for the pigmentation stage, whereas ATP5B and CALR immunoreactivity was weak in the enamel organ during the early stages, but became intense during the maturation and pigmentation stages, although the timing of the increased protein expression was different between the two. Overall, RACK1 plays an important role in maintaining the cell proliferation and differentiation in the apical end of incisors. In contrast, ATP5B and CALR are involved in the transport of minerals and the removal of organic materials as well as matrix deposition for CALR.

DOI： 10.1007/s10266-023-00790-4

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In our previous study we established an animal model for immediately placed implants using mice and clarified that there were no significant differences in the chronological healing process at the bone-implant interface between immediately and delayed placed implants blasted with hydroxyapatite (HA)/β-tricalcium phosphate (β-TCP) (ratio 1:4). This study aimed to analyze the effects of HA/β-TCP on osseointegration at the bone-implant interface after immediately placed implants in the maxillae of 4-week-old mice. Right maxillary first molars were extracted and cavities were prepared with a drill and titanium implants, blasted with or without HA/β-TCP, were placed. The fixation was followed-up at 1, 5, 7, 14, and 28 days after implantation, and the decalcified samples were embedded in paraffin and prepared sections were processed for immunohistochemistry using anti-osteopontin (OPN) and Ki67 antibodies, and tartrate-resistant acid phosphatase histochemistry. The undecalcified sample elements were quantitatively analyzed by an electron probe microanalyzer. Bone formation occurred on the preexisting bone surface (indirect osteogenesis) and on the implant surface (direct osteogenesis), indicating that osseointegration was achieved until 4 weeks post-operation in both of the groups. In the non-blasted group, the OPN immunoreactivity at the bone-implant interface was significantly decreased compared with the blasted group at week 2 and 4, as well as the rate of direct osteogenesis at week 4. These results suggest that the lack of HA/β-TCP on the implant surface affects the OPN immunoreactivity on the bone-implant interface, resulting in decreased direct osteogenesis following immediately placed titanium implants.

DOI： 10.3390/ijms24043124

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OBJECTIVES: Shortening the root of a mouse molar prior to tooth replantation results in early revascularization in the pulp cavity and activation of the dental pulp quiescent stem cells. This study aimed to validate the effects of pulp chamber floor perforation on pulpal healing after tooth replantation as a strategy to promote early revascularization into the pulp. METHODS: The maxillary first molars of three-week-old Crlj:CD1 mice were extracted and repositioned into the original socket: the left teeth were immediately replanted (control group: CG), whereas the floor of the pulp chamber of the right teeth was perforated with a tungsten carbide bur before tooth replantation (experimental group: EG). The samples were collected from three days to eight weeks postoperatively. In addition to the TUNEL assay, immunohistochemistry for Nestin, CK14, and Ki-67 was conducted. RESULTS: In the EG, early revascularization occurred with a decrease in apoptosis and an increase in cell proliferation, facilitating pulpal healing, compared with the CG. The rate of Nestin-positive perimeter in the distal root significantly increased on days 5 and 14 and the amount of Nestin-positive hard tissue increased on day 14. However, on day 7, the number of epithelial cell rests of Malassez in the EG significantly decreased, making the EG susceptible to ankylosis at the floor. CONCLUSIONS: Intentionally perforating the floor of the pulp chamber provides a route for early revascularization, resulting in better pulpal healing after tooth replantation.

DOI： 10.1016/j.job.2023.01.007

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The layered structures of facial muscles and their topographical relationship with facial fasciae are still not fully understood. This study aimed to clarify the layered structures of facial muscles and fasciae in the temporal-malar-mandible-neck region. Thirty-four human cadavers were examined during gross anatomy courses at Niigata University (2017-2020). The face was composed of 3-layered (deep, middle, and superficial) fasciae and 4-layered facial muscles (first superficial, second superficial, third, and fourth muscle layers) according to the attachment of muscles and their topographical relationship with the fasciae. The deep fascia covered the temporal and masseter muscles. The parotid gland and facial nerves were enveloped in the middle fascia. The superficial fascia was continuous with the second superficial muscle layer. The connection between fourth and superficial muscles was at the malar and buccal areas, where the platysma blended with the masseter and the plural muscles blended with the buccinator. Our findings suggest that cooperation between the 4-layered structure of the facial muscles surrounding the apertures of the eyes and mouth and the superficial fascia enables humans to produce complex facial expressions. Furthermore, the spread of inflammation in the face may be owing to the layered facial muscles and fasciae, as these layered structures separate tissues into multiple compartments.

DOI： 10.1097/SCS.0000000000008700

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BACKGROUND: Tooth identification is important not only for anatomists and anthropologists but also for dental practitioners and dental students studying dental anatomy courses. This review paper provides an overview of the significance of tooth identification focusing on the morphological and developmental background. HIGHLIGHT: The process of tooth identification comprises five steps of distinction: (1) between deciduous and permanent teeth; (2) between tooth classes; (3) between maxillary and mandibular teeth; (4) within the same tooth class; and (5) between the left and right sides of a tooth. According to Mühlreiter's features, the mesial half is more developed than the distal half, and the curvature feature is associated with the configuration of the dental arch. Each step of tooth identification refers to effective traits and characteristics. The possibility that systemic conditions affect dental morphology should be considered. Tooth identification is occasionally difficult owing to individual variations (size and shape, supernumerary tubercles, root fusion) and sex-based differences. A tooth type error within the same class is the most frequent error in tooth identification, followed by a left or right side error. CONCLUSION: To understand tooth identification, it is necessary to have comprehensive knowledge of dental morphology. A broad education with regard to tooth evolution and comparative odontology, as well as a thorough understanding of the morphology and function of teeth, which play a crucial role in sustaining life as organs of mastication, is essential.

DOI： 10.1016/j.job.2022.05.004

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Chondroitin sulfate proteoglycan (CSPG), one of the major extracellular matrices, plays an important part in organogenesis. Its core protein and chondroitin sulfate (CS) chain have a specific biological function. To elucidate the role of CS in the developmental and healing process of the dental pulp, we performed an experimental tooth replantation in CS N-acethylgalactosaminyltransferase-1 (T1) gene knockout (KO) mice. We also performed cell proliferation assay and qRT-PCR analysis for the WT and T1KO primary dental pulp cells using T1-siRNA technique and external CS. During tooth development, CS was diffusely expressed in the dental papilla, and with dental pulp maturation, CS disappeared from the differentiated areas, including the odontoblasts. In fully developed molars, CS was restricted to the root apex region colocalizing with Gli1-positive cells. In the healing process after tooth replantation, CD31-positive cells accumulated in the CS-positive stroma in WT molars. In T1KO molars, the appearance of Ki67- and Gli1-positive cells in the dental pulp was significantly fewer than in WT molars in the early healing stage, and collagen I-positive reparative dentin formation was not obvious in T1KO mice. In primary culture experiments, siRNA knockdown of T1 gene significantly suppressed cell proliferation in WT dental pulp cells, and the mRNA expression of cyclin D1 and CD31 was significantly upregulated by external CS in T1KO dental pulp cells. These results suggest that CS is involved in the cell proliferation and functional differentiation of dental pulp constituent cells, including vascular cells, in the healing process of dental pulp tissue after tooth injury.

DOI： 10.1007/s00441-022-03575-3

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BACKGROUND: Regenerative medicine has emerged as a multidisciplinary field with the promising potential of renewing tissues and organs. The main types of adult stem cells used in clinical trials are hematopoietic and mesenchymal stem cells (MSCs). Stem cells are defined as self-renewing clonogenic progenitor cells that can generate one or more types of specialized cells. HIGHLIGHT: MSCs form adipose, cartilage, and bone tissue. Their protective and regenerative effects, such as mitogenic, anti-apoptotic, anti-inflammatory, and angiogenic effects, are mediated through paracrine and endocrine mechanisms. Dental pulp is a valuable source of stem cells because the collection of dental pulp for stem cell isolation is non-invasive, in contrast to conventional sources, such as bone marrow and adipose tissue. Teeth are an excellent source of dental pulp stem cells (DPSCs) for therapeutic procedures and they can be easily obtained after tooth extraction or the shedding of deciduous teeth. Thus, there is increased interest in optimizing and establishing standard procedures for obtaining DPSCs; preserving well-defined DPSC cultures for specific applications; and increasing the efficiency, reproducibility, and safety of the clinical use of DPSCs. CONCLUSION: This review comprehensively describes the biological characteristics and origins of DPSCs, their identification and harvesting, key aspects related to their characterization, their multilineage differentiation potential, current clinical applications, and their potential use in regenerative medicine for future dental and medical applications.

DOI： 10.1016/j.job.2022.01.002

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BACKGROUND: The Journal of Oral Biosciences is devoted to advancing and disseminating fundamental knowledge concerning every aspect of oral biosciences. HIGHLIGHT: This review features review articles in the fields of "Extracellular Vesicles," "Propolis," "Odontogenic Tumors," "Periodontitis," "Periodontium," "Flavonoids," "Lactoferrin," "Dental Plaque," "Anatomy," "Induced Pluripotent Stem Cells," "Bone Cell Biology," "Dysgeusia," "Dental Caries," and "Dental Pulp Cavity," in addition to the review article by the winners of the "Lion Award" ("Sox9 function in salivary gland development") presented by the Japanese Association for Oral Biology. CONCLUSION: These reviews in the Journal of Oral Biosciences have inspired its readers to broaden their knowledge regarding various aspects of oral biosciences. The current editorial review introduces these exciting review articles.

DOI： 10.1016/j.job.2022.02.001

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OBJECTIVES: Original odontoblasts and regenerated odontoblast-like cells (OBLCs) may differently regulate Nestin expression. This study aimed to investigate the role of the subodontoblastic layer (SOBL) using green fluorescent protein (GFP) reactivity in the process of OBLC differentiation after tooth drilling in Nestin-enhanced GFP transgenic mice. METHODS: A groove-shaped cavity was prepared on the mesial surface of the maxillary first molars of 5- or 6-week-old mice under deep anesthesia. Immunohistochemical staining for Nestin and GFP and Nestin in situ hybridization were conducted on the sections obtained at 1-14 days postoperative. RESULTS: Odontoblasts showed intense endogenous Nestin protein and mRNA expression, whereas the coronal SOBL cells showed a Nestin-GFP-positive reaction in the control groups. The injured odontoblasts had significantly decreased Nestin immunoreactivity as well as decreased expression of Nestin mRNA 1-2 days after the injury; subsequently, newly differentiated OBLCs were arranged along the pulp-dentin border, with significantly increased Nestin expression as well as increased expression of Nestin mRNA on days 3-5 to form reparative dentin. Nestin-GFP-positive cells at the pulp-dentin border significantly increased in number on days 1 and 2. GFP(+)/Nestin(+) and GFP(-)/Nestin(+) cells were intermingled in the newly differentiated OBLCs. CONCLUSIONS: The commitment of Nestin-GFP-positive cells into Nestin-positive OBLCs suggests that the restriction of endogenous Nestin protein and mRNA expression in the static SOBL cells was removed by exogenous stimuli, resulting in their migration along the pulp-dentin border and their differentiation into OBLCs.

DOI： 10.1016/j.job.2022.01.001

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After dental implantation, osteopontin (OPN) is deposited on the hydroxyapatite (HA) blasted implant surface followed by direct osteogenesis, which is significantly disturbed in Opn-knockout (KO) mice. However, whether applying OPN on the implant surface promotes direct osteogenesis remains unclarified. This study analyzed the effects of various OPN modified protein/peptides coatings on the healing patterns of the bone-implant interface after immediately placed implantation in the maxilla of four-week-old Opn-KO and wild-type (WT) mice (n = 96). The decalcified samples were processed for immunohistochemistry for OPN and Ki67 and tartrate-resistant acid phosphatase histochemistry. In the WT mice, the proliferative activity in the HA binding peptide-OPN mimic peptide fusion coated group was significantly higher than that in the control group from day 3 to week 1, and the rates of OPN deposition and direct osteogenesis around the implant surface significantly increased in the recombinant-mouse-OPN (rOPN) group compared to the Gly-Arg-Gly-Asp-Ser peptide group in week 2. The rOPN group achieved the same rates of direct osteogenesis and osseointegration as those in the control group in a half period (week 2). None of the implant surfaces could rescue the direct osteogenesis in the healing process in the Opn-KO mice. These results suggest that the rOPN coated implant enhances direct osteogenesis during osseointegration following implantation.

DOI： 10.3390/ijms23031039

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INTRODUCTION: The role of osteopontin (OPN) following severe injury remains to be elucidated, especially its relationship with type I collagen (encoded by the Col1a1 gene) secretion by newly-differentiated odontoblast-like cells (OBLCs). In this study, we examined the role of OPN in the process of reparative dentin formation with a focus on reinnervation and revascularization after tooth replantation in Opn knockout (KO) and wild-type (WT) mice. METHODS: Maxillary first molars of 2- and 3-week-old-Opn KO and WT mice (Opn KO 2W, Opn KO 3W, WT 2W, and WT 3W groups) were replanted, followed by fixation 3-56 days after operation. Following micro-computed tomography analysis, the decalcified samples were processed for immunohistochemistry for Ki67, Nestin, PGP 9.5, and CD31 and in situ hybridization for Col1a1. RESULTS: An intense inflammatory reaction occurred to disrupt pulpal healing in the replanted teeth of the Opn KO 3W group, whereas dental pulp achieved healing in the Opn KO 2W and WT groups. The tertiary dentin in the Opn KO 3W group was significantly decreased in area compared with the Opn KO 2W and WT groups, with a significantly low percentage of Nestin-positive, newly-differentiated OBLCs during postoperative days 7-14. In the Opn KO 3W group, the blood vessels were significantly decreased in area and pulp healing was disturbed with a failure of pulpal revascularization and reinnervation. CONCLUSIONS: OPN is necessary for proper reinnervation and revascularization to deposit reparative dentin following severe injury within the dental pulp of erupted teeth with advanced root development.

DOI： 10.1016/j.reth.2022.09.011

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BACKGROUND: There is no available data on the occurrence rate of a converged alveolar canal, the detailed three-dimensional (3D) courses of alveolar canals/grooves (ACGs), or the contribution of each superior alveolar nerve to each area in the maxilla. This study aimed to clarify the 3D courses of ACGs, the relationship between ACGs and superior alveolar nerves, and the contribution of posterior superior alveolar nerves (PSANs) using computed tomography (CT) with histological analysis. METHODS: During the gross anatomy course at Niigata University, we investigated nine human cadavers. RESULTS: All anterior and posterior ACGs converged into the common alveolar canal, which contained blood vessels and several nerve bundles surrounded by perineurium, located at the nasal floor near the pyriform aperture. Histometrical analysis clarified that 16.3% of the nerve bundles in this canal were derived from PSANs, and 67% of the bundles were dispersed while they coursed down to the nasal floor. There seems to be no relationship between the density of nerve bundles in the canal and the number of remaining anterior teeth. CONCLUSIONS: Data obtained from observing the detailed 3D courses of anterior and posterior ACGs, and their relationship with superior alveolar nerves, suggest that PSANs partially contribute to the nociception of the anterior teeth.

DOI： 10.1016/j.aanat.2021.151784

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BACKGROUND/AIM: Root length is a critical factor for dental pulp regeneration following tooth replantation. The aim of this study was to analyze the effects of reducing the root length by apicoectomy on the pulp healing process using a model for tooth replantation. MATERIAL AND METHODS: After extraction of the upper first molars (M1) of 3-week-old mice, the roots from the experimental group (EG) were shortened to half to two-thirds of their length before replantation, whereas in the control group (CG) the extracted teeth were immediately repositioned into their alveolar sockets. To determine the effects of root resection on the survival of inherent pulp cells, this study included tooth transplantation with root resection using wild-type (WT) and green fluorescent protein (GFP) transgenic mice. The M1 of GFP transgenic mice were transplanted into the alveolar socket of the M1 of WT mice. The roots of the right M1 were shortened (EG), whereas the left M1 remained untreated (CG). RESULTS: Apoptotic cells in the EG significantly decreased in number compared with the CG at day 3. Cell proliferative activity in the EG was significantly higher than that in the CG in the root pulp during days 3-5, and nestin-positive odontoblast-like cells began to arrange themselves along the pulp-dentin border in the cusp area at day 5 in the EG but not in the CG. At week 2, tertiary dentin had formed throughout the pulp in the EG, whereas the combined tissue of dentin and bone occupied the pulp space in 60% of the CG. Root resection also positively affected the survival of inherent pulp cells to differentiate into odontoblast-like cells as demonstrated by transplantation using GFP transgenic mice. CONCLUSIONS: Reducing the root length accelerated pulp regeneration following tooth replantation due to the better environment for revascularization.

DOI： 10.1111/edt.12679

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There is little anatomical evidence about the venous plexus in the floor of the oral cavity, although venous injury can elicit late postoperative bleeding after oral surgery and it is difficult to identify the exact location of such an injury. The aim of this study was to assess the relative risk for venous injury during surgery. We investigated the course patterns of the venous plexus in the floor of the oral cavity and analyzed their relationships to those of the arteries using 23 human cadavers (41 halves) in the anatomy course at Niigata University during 2016-2018. The venous plexus in the floor of the oral cavity comprised the perforating submental vein, the vena comitans of the hypoglossal nerve, the vena comitans of the submandibular duct, the vena comitans of the lingual nerve, the sublingual vein, and the deep lingual vein. Individual variations of this plexus include duplications or absences of some veins. There is a high incidence of a submental branch running above the mylohyoid or perforating submental artery in the sublingual fossa among individuals with the perforating submental vein piercing the mylohyoid muscle, whereas the sublingual artery has a high incidence there when there is no perforating submental vein. The course patterns of arteries in the floor of the oral cavity can be predicted by estimating the course patterns of the submental veins. The course patterns of the submental veins or veins associated with the nerves and submandibular duct need to be carefully considered during surgery.

DOI： 10.1002/ca.23738

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Amelogenesis consists of secretory, transition, maturation, and post-maturation stages, and the morphological changes of ameloblasts at each stage are closely related to their function. p130 Crk-associated substrate (Cas) is a scaffold protein that modulates essential cellular processes, including cell adhesion, cytoskeletal changes, and polarization. The expression of p130Cas was observed from the secretory stage to the maturation stage in ameloblasts. Epithelial cell-specific p130Cas-deficient (p130CasΔepi-) mice exhibited enamel hypomineralization with chalk-like white mandibular incisors in young mice and attrition in aged mouse molars. A micro-computed tomography analysis and Vickers micro-hardness testing showed thinner enamel, lower enamel mineral density and hardness in p130CasΔepi- mice in comparison to p130Casflox/flox mice. Scanning electron microscopy, and an energy dispersive X-ray spectroscopy analysis indicated the disturbance of the enamel rod structure and lower Ca and P contents in p130CasΔepi- mice, respectively. The disorganized arrangement of ameloblasts, especially in the maturation stage, was observed in p130CasΔepi- mice. Furthermore, expression levels of enamel matrix proteins, such as amelogenin and ameloblastin in the secretory stage, and functional markers, such as alkaline phosphatase and iron accumulation, and Na+/Ca2++K+-exchanger in the maturation stage were reduced in p130CasΔepi- mice. These findings suggest that p130Cas plays important roles in amelogenesis (197 words).

DOI： 10.1016/j.bone.2021.116210

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Fluoride can alter the formation of mineralized tissues, including enamel, dentin, and bone. Dentin fluorosis occurs in tandem with enamel fluorosis. However, the pathogenesis of dentin fluorosis and its mechanisms are poorly understood. In this study, we report the effects of fluoride on the initiation of dentin matrix formation and odontoblast function. Mice from two enamel fluorosis susceptible strains (A/J and C57BL/6J) were given either 0 or 50 ppm fluoride in drinking water for 4 weeks. In both mouse strains, there was no overall change in dentin thickness, but fluoride treatment resulted in a significant increase in the thickness of the predentin layer. The lightly mineralized layer (LL), which lies at the border between predentin and fully mineralized dentin and is associated with dentin phosphoprotein (DPP), was absent in fluoride exposed mice. Consistent with a possible reduction of DPP, fluoride-treated mice showed reduced immunostaining for dentin sialoprotein (DSP). Fluoride reduced RUNX2, the transcription regulator of dentin sialophosphoprotein (DSPP), that is cleaved to form both DPP and DSP. In fluoride-treated mouse odontoblasts, the effect of fluoride was further seen in the upstream of RUNX2 as the reduced nuclear translocation of β-catenin and phosphorylated p65/NFκB. In vitro, MD10-F2 pre-odontoblast cells showed inhibition of the Dspp mRNA level in the presence of 10 μM fluoride, and qPCR analysis showed a significantly downregulated level of mRNAs for RUNX2, β-catenin, and Wnt10b. These findings indicate that in mice, systemic exposure to excess fluoride resulted in reduced Wnt/β-catenin signaling in differentiating odontoblasts to downregulate DSPP production via RUNX2.

DOI： 10.1007/s12011-020-02434-y

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The dental pulp is a soft connective tissue of ectomesenchymal origin that harbors distinct cell populations, capable of interacting with each other to maintain the vitality of the tooth. After tooth injuries, a sequence of complex biological events takes place in the pulpal tissue to restore its homeostasis. The pulpal response begins with establishing an inflammatory reaction that leads to the formation of a matrix of reactionary or reparative dentin, according to the nature of the exogenous stimuli. Using several in vivo designs, antigen-presenting cells, including macrophages and dendritic cells (DCs), are identified in the pulpal tissue before tertiary dentin deposition under the afflicted area. However, the precise nature of this phenomenon and its relationship to inherent pulp cells are not yet clarified. This literature review aims to discuss the role of pulpal DCs and their relationship to progenitor/stem cells, odontoblasts or odontoblast-like cells, and other immunocompetent cells during physiological and pathological dentinogenesis. The concept of "dentin-pulp immunology" is proposed for understanding the crosstalk among these cell types after tooth injuries, and the possibility of immune-based therapies is introduced to accelerate pulpal healing after exogenous stimuli.

DOI： 10.3390/jcm10153348

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This study aimed to clarify the effect of mineral trioxide aggregate (MTA) on pulp healing in the infected pulp by direct pulp capping (DPC). Thirty-six male ICR mice were divided into infected and uninfected groups. The pulp tissue was exposed to the oral flora for 24 h after pulp exposure in the infected group, or not exposed in the uninfected group, followed by sealing with MTA, calcium hydroxide cement (CH), or no DPC. Pulpal healing process was analyzed by hematoxylin-eosin staining and immunohistochemistry for nestin and Ki67. The active cell proliferation occurred on 1 week in the both MTA and CH groups, followed by the differentiation of odontoblast-like cells on 2 weeks in the MTA group, whereas their differentiation were not facilitated in the CH group. MTA is suggested to be a useful material for DPC with the infected and uninfected pulp tissue.

DOI： 10.4012/dmj.2020-393

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BACKGROUND: The Journal of Oral Biosciences is devoted to the advancement and dissemination of fundamental knowledge concerning every aspect of oral biosciences. HIGHLIGHT: This review featured the review articles in the fields of "Microbiology," "Palate," "Stem Cells," "Mucosal Diseases," "Bone Cell Biology," "MicroRNAs," "TRPV1 Cation Channels," and "Interleukins" in addition to the review article by prize-winners of the "Rising Members Award" ("DKK3 expression and function in head and neck squamous cell carcinoma and other cancers"), presented by the Japanese Association for Oral Biology. CONCLUSION: These reviews in the Journal of Oral Biosciences have inspired the readers of the journal to broaden their knowledge regarding the various aspects of oral biosciences. The current editorial review introduces these exciting review articles.

DOI： 10.1016/j.job.2021.02.001

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© 2020 The Japanese Society for Regenerative Medicine Introduction: Responses of oral-microflora-exposed dental pulp to a triple antibiotic paste (TAP), a mixture of ciprofloxacin, metronidazole, and minocycline in ointment with macrogol and propylene glycol, remain to be fully clarified at the cellular level. This study aimed to elucidate responses of oral-microflora-exposed dental pulp to capping with TAP in mouse molars. Methods: A cavity was prepared on the first molars of 6-week-old mice to expose the dental pulp for 24 h. The exposed pulp was capped with TAP (TAP group) or calcium hydroxide cement (CH group), in addition to the combination of macrogol (M) and propylene glycol (P) (MP, control group), followed by a glass ionomer cement filling. The samples were collected at intervals of 1, 2, and 3 weeks, and immunohistochemistry for nestin and Ki-67 and deoxyuride-5′-triphosphate biotin nick end labeling (TUNEL) assay were performed in addition to quantitative real-time polymerase chain reaction (qRT-PCR) analyses. Results: The highest occurrence rate of pulp necrosis was found in the control group followed by the CH group at Weeks 2 and 3, whereas the highest occurrence rate of healed areas in the dental pulp was observed in the TAP group at each time point. Tertiary dentin formation was first observed in the dental pulp of the TAP group at Week 2. In contrast, bone-like and/or fibrous tissues were frequently observed in the CH group. qRT-PCR analyses clarified that TAP activated the stem and dendritic cells at Weeks 1 and 2, respectively. Conclusions: The use of TAP as a pulp-capping agent improved the healing process of oral-microflora-exposed dental pulp in mouse molars.

DOI： 10.1016/j.reth.2020.10.001

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Glucose is an essential source of energy for mammalian cells and is transported into the cells by glucose transporters. There are 2 types of glucose transporters: one is a passive glucose transporter, GLUT ( SLC2A), and the other is a sodium-dependent active glucose transporter, SGLT ( SLC5A). We previously reported that the expression of GLUTs during tooth development is precisely and spatiotemporally controlled and that the glucose uptake mediated by GLUT1 plays a crucial role in early tooth morphogenesis and tooth size determination. This study aimed to clarify the localization and roles of SGLT1 and SGLT2 in murine ameloblast differentiation by using immunohistochemistry, immunoelectron microscopy, an in vitro tooth organ culture experiment, and in vivo administration of an inhibitor of SGLT1/2, phloridzin. SGLT1, which has high affinity with glucose, was immunolocalized in the early secretory ameloblasts and the ruffle-ended ameloblasts in the maturation stage. However, SGLT2, which has high glucose transport capacity, was observed in the stratum intermedium, papillary layer, and ameloblasts at the maturation stage and colocalized with Na⁺-K⁺-ATPase. The inhibition of SGLT1/2 by phloridzin in the tooth germs induced the disturbance of ameloblast differentiation and enamel matrix formation both in vitro (organ culture) and in vivo (mouse model). The expression of SGLT1 and SGLT2 was significantly upregulated in hypoxic conditions in the ameloblast-lineage cells. These findings suggest that the active glucose uptake mediated by SGLT1 and SGLT2 is strictly regulated and dependent on the intra- and extracellular microenvironments during tooth morphogenesis and that the appropriate passive and active glucose transport is an essential event in amelogenesis.

DOI： 10.1177/0022034520916130

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Other Link： http://journals.sagepub.com/doi/full-xml/10.1177/0022034520916130

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BACKGROUND: Although numerous reports have demonstrated that the junctional epithelium (JE) is derived from the reduced enamel epithelium (REE), the fate of the REE-derived JE remains controversial. The present study elucidated the fate of the REE-derived JE and the cell dynamics of stem/progenitor cells in the JE following tooth eruption. METHODS: Mandibular first molar germs (embryonic days 15 to postnatal 1-day-old) were transplanted into the socket of 2-week-old mice after extraction of the upper first molars of B6 wild-type (WT) and green fluorescent protein (GFP) transgenic mice. After analysis by µ-CT, paraffin sections were processed for immunohistochemistry for Nestin, Ki67 and GFP. We also performed chasing analysis using BrdU-administered TetOP-H2B-GFP mice. RESULTS: Amelogenesis progressed normally in the cervical areas, and the structure of the JE was like that in normal tooth development. The JE was GFP-negative in transplantations using GFP transgenic mice as the host, and the oral epithelium (OE) showed a positive reaction. By contrast, the JE remained GFP-positive throughout the experimental period in transplantations using GFP transgenic mice as the donor. Chasing analysis revealed that H2B-GFP- and 5-Bromo-2'-deoxyuridine (BrdU)-labeled cells in the basal side of the JE translocated to oral side of the JE as the chasing time increased. Some label-retaining cells remained at the supra-basal cell layer in the JE. CONCLUSION: These results suggest that REE-derived cell niche in the JE is maintained for a long time following tooth eruption. Therefore, the JE may be available as the source of the odontogenic epithelium.

DOI： 10.1002/JPER.19-0269

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

Background: Journal of Oral Biosciences is devoted to the advancement and dissemination of fundamental knowledge concerning every aspect of oral biosciences.Highlight: This review features review articles in the fields of "Bone Cell Biology," "Microbiology," "Oral Heath," "Biocompatible Materials," "Mouth Neoplasm," and "Biological Evolution" in addition to the review articles by winners of the Lion Dental Research Award ("Role of nicotinic acetylcholine receptors for modulation of microcircuits in the agranular insular cortex" and "Phospholipase C-related catalytically inactive protein: A novel signaling molecule for modulating fat metabolism and energy expenditure") and the Rising Members Award ("Pain mechanism of oral ulcerative mucositis and the therapeutic traditional herbal medicine hangeshashinto," "Mechanisms underlying the induction of regulatory T cells by sublingual immunotherapy," and "Regulation of osteoclast function via Rho-Pkn3-c-Src pathways"), presented by the Japanese Association for Oral Biology.Conclusion: These reviews in the Journal of Oral Biosciences have inspired the readers of the journal to broaden their knowledge regarding various aspects of oral biosciences. The current editorial review introduces these exciting review articles. (C) 2020 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.job.2020.02.001

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INTRODUCTION: Although dentin matrix protein 1 (DMP1) and osteopontin (OPN) act as substrates and signaling molecules for odontoblastlike cell differentiation after tooth injury, the mutual interaction between these proteins in the mechanism of odontoblastlike cell differentiation remains to be clarified. This study aimed to elucidate the role of DMP1 and OPN in regulating odontoblastlike cell differentiation after tooth injury. METHODS: A groove-shaped cavity was prepared on the mesial surface of the upper first molars in wild-type and Opn knockout (KO) mice. The demineralized paraffin sections were processed for immunohistochemistry for nestin and DMP1 and in situ hybridization for Dmp1. For the in vitro assay, the experiments of organ culture for evaluating dentin-pulp complex regeneration using small interfering RNA treatment were performed. RESULTS: Once preexisting odontoblasts died, nestin-positive newly differentiated odontoblastlike cells were arranged along the pulp-dentin border and began to express DMP1/Dmp1. In Opn KO mice, the expression of DMP1/Dmp1 was up-regulated compared with that of wild-type mice. The in vitro assay showed that the gene suppression of Dmp1 by small interfering RNA showed a tendency to decrease the differentiation rate of odontoblastlike cells from 70.1% to 52.2% in wild-type teeth. In addition, the suppression of Dmp1 in Opn KO teeth tended to lead to the inhibition of odontoblastlike cell differentiation. CONCLUSIONS: These results suggest that the expression of Dmp1 is up-regulated in Opn KO mice both in vivo and in vitro, and DMP1 compensates for the lack of OPN in regulating odontoblastlike cell differentiation after tooth injury.

DOI： 10.1016/j.joen.2019.10.002

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Glycogen is the stored form of glucose and plays a major role in energy metabolism. Recently, it has become clear that enzymatically synthesized glycogen (ESG) has biological functions, such as the macrophage-stimulating activity. This study aimed to evaluate the effect of ESG on osteogenesis. MC3T3-E1 cells were cultured with ESG, and their cell proliferative activity and osteoblast differentiation were measured. An in vivo study was conducted in which ESG pellets with BMP-2 were grafted into mouse calvarial defects and histomorphometrically analyzed for the new bone formation. To confirm the effect of ESG on bone growth in vivo, ESG was orally administered to pregnant mice and the femurs of their pups were examined. We observed that ESG stimulated cell proliferation and enhanced messenger RNA expression of osteocalcin and osteopontin in MC3T3-E1 cells. ESG was taken up by the cells associated with GLUT-1 and activated the Akt/GSK-3β pathway. In vivo, the new bone formation in the calvarial defect was significantly accelerated by ESG and the maternal administration of ESG promoted fetal bone growth. In conclusion, ESG stimulates cell proliferation and differentiation of preosteoblasts via the activation of Akt/GSK-3β signaling and promotes new bone formation in vivo, suggesting that ESG could be a useful stimulant for osteogenesis.

DOI： 10.1002/jcp.28039

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OBJECTIVES: Continuously growing rodent incisors have an apically located epithelial stem cell compartment, known as an "apical bud" (AB). Few studies have described the morphological features of ABs and stem cell niches in continuously growing premolars/molars. We attempted to clarify the relationship between the three-dimensional configuration of ABs and the stem cell niches in guinea pig cheek teeth. METHODS: We perfusion-fixed four-week-old guinea pigs, then decalcified their premolars/molars to produce serial paraffin sections, which we immunostained for Sox2. We reconstructed the serial sections using image processing and analysis software. We processed undecalcified samples for scanning electron microscopy by KOH digestion. RESULTS: Two types of epithelia with M and Δ shapes surrounded the S-shaped dental papilla in the apical region of the premolars/molars, and there were three Sox2-positive epithelial bulges above the M- and Δ-shaped epithelia. Sox2-positive epithelial stem cell niches were restricted to the apical side, and cell proliferation and differentiation immediately proceeded in the crown-analogue dentin. The Sox2-positive epithelial stem cell niches were sparsely distributed and extended to the occlusal side. We also detected continuously proliferating cells in the cervical loop and Hertwig's epithelial root sheath of the root-analogue dentin. CONCLUSIONS: Our findings suggest that guinea pig cheek teeth have three ABs, and the complex configuration of these types of teeth may be attributed to the prompt formation of crown-analogue dentin followed by the long-term formation of root-analogue dentin.

DOI： 10.1016/j.job.2019.01.002

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Background: The Journal of Oral Biosciences is devoted to the advancement and dissemination of fundamental knowledge regarding every aspect of oral biosciences.Highlight: This editorial review features summaries of review articles in the fields of "Bone Biology," "Epigenomics," "Periodontium," and "Amelogenesis" in addition to review articles by winners of the Lion Dental Research Award ("Role of non-canonical Wnt signaling pathways in bone resorption," "Mechanisms of orofacial sensory processing in the rat insular cortex," and "Analysis of the mechanism in salivary gland development using gene database") and the Rising Members Award ("Synergistic findings from microbiological and evolutional analyses of virulence factors among pathogenic streptococcal species" and "Free fatty acids may be involved in the pathogenesis of oral-related and cardiovascular diseases"), presented by the Japanese Association for Oral Biology.Conclusion: These reviews published in the Journal of Oral Biosciences have inspired the readers of the Journal to broaden their knowledge of various aspects in the oral biosciences. This editorial review summarizes these exciting articles. (C) 2019 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.job.2019.01.003

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Objectives: Although intracellular signaling pathways of insulin-like growth factor I (IGF-I) related to the proliferation of dental pulp cells have been investigated, the switching mechanism from cell proliferation to differentiation during odontogenesis remains elusive. This study aimed to elucidate the role of IGF binding protein (IGFBP) 3 and 5 in regulation of IGF-I during odontoblast differentiation in mouse incisors.Methods: The detailed expression patterns of IGF-I, IGF-I receptor (IGF-IR), IGFBP3, and IGFBP5 together with that of an odontoblast differentiation marker, nestin, were examined by immunohistochemistry and/or in situ hybridization using paraffinized sections of TetOP-H2B-GFP mouse incisors at postnatal 4 weeks.Results: Undifferentiated dental papilla cells and preodontoblasts (preOB) showed intense IGF-I- and IGF-IR alpha-positive reactions, and the expression was observed in differentiated odontoblasts, such as immature odontoblasts (iOB) and mature odontoblasts (mOB). IGFBP3/Igfbp3 was transiently expressed in preOB and early iOB, and the intensity of expression gradually reduced with the progression of odontoblast differentiation. In contrast, immunohistochemical analysis for IGFBP5 identified a positive reaction in the undifferentiated dental papilla cells and differentiated odontoblasts, and the expression of Igfbp5 was reduced in the differentiated odontoblasts.Conclusion: The present study demonstrated the expression patterns of IGF-I, IGF-IR, IGFBP3, and IGFBP5 during odontoblast differentiation in mouse incisors. These results suggested that IGFBP3 regulates the transition from the proliferative to differentiation stage by inhibiting the action of IGF-I on the proliferation of dental papilla cells, and that IGFBP5 plays an important role in the maintenance of the differentiated odontoblasts during tooth development. (C) 2019 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.job.2019.07.001

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Although insulin-like growth factor binding protein 5 (IGFBP5) may play a crucial role in activating the functions of periodontal and bone marrow stem cells, the factors responsible for regulating the maintenance of dental pulp stem cells (DPSCs) remain to be clarified. This study aimed to elucidate the role of IGFBP5 in maintaining pulpal homeostasis during tooth development and pulpal healing after tooth injury in doxycycline-inducible TetOP-histone 2B (H2B)-green fluorescent protein (GFP) transgenic mice (GFP expression was induced at E14.5 or E15.5) by using TUNEL assay, RT-PCR, in situ hybridization for Igfbp5, and immunohistochemistry for IGFBP5, Nestin, and GFP. To observe the pulpal response to exogenous stimuli, the roots of the maxillary first molars were resected, and the coronal portion was autografted into the sublingual region. Intense IGFBP5/Igfbp5 expression was observed in cells from the center of the pulp tissue and the subodontoblastic layer in developing teeth during postnatal Week 4. Intense H2B-GFP-expressing label-retaining cells (LRCs) were localized in the subodontoblastic layer in addition to the center of the pulp tissue, suggesting that slowly dividing cell populations reside in these areas. During postoperative days 3-7, the LRCs were maintained in the dental pulp, showed an IGFBP5-positve reaction in their nuclei, and lacked a TUNEL-positive reaction. In situ hybridization and RT-PCR analyses confirmed the expression of Igfbp5 in the dental pulp. These findings suggest that IGFBP5 play a pivotal role in regulating the survival and apoptosis of DPSCs during both tooth development and pulpal healing following tooth injury.

DOI： 10.1016/j.reth.2019.08.001

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

Objectives: Limited biological evidence exists regarding donor–host interaction in the periodontal tissue during allogenic tooth germ transplantation. This study aimed to clarify donor–host tissue interactions during periodontal tissue healing following tooth germ transplantation. Methods: This study compared the localization of putative stem cells in the periodontal ligament (PDL) by 5-bromo-2′-deoxyuridine (BrdU), Gli1, and periostin immunoreactions using pulse-chase paradigm (BrdU prenatal labeling: peritoneal pulse injections at embryonic days [E] 15–17) in TetOP–H2B–GFP mice (doxycycline administration at E14.5). The current study characterized periodontal tissue healing following allogenic tooth grafts in GFP-labeled donor or host and wild-type mice by pulse-chase paradigm and GFP, BrdU, Gli1, and periostin immunohistochemistry. Results: BrdU prenatal labeling demonstrated that dense label-retaining cells (BrdU–LRCs) disappeared from the PDL by postnatal week 2 (P2W). However, H2B–GFP–LRCs were localized in the PDL of TetOP–H2B–GFP mice during P3–8W, and Gli1-positive cells in the PDL increased at P2–3W, showing that H2B–GFP–LRCs in the PDL are derived from non-proliferating cells during E15–17. Transplanted molars formed cusps and roots and erupted into occlusion by two weeks postoperatively. The junctional epithelium and tooth-related zone of PDL were exclusively composed of donor cells, whereas the PDL alveolar-related zone was a hybrid structure of donor and host cells. Conclusions: The current tooth germ transplantation suggests that the PDL contains putative stem cells, which never proliferate during E15–17, and is composed of resident dental follicle-derived cells and other cell population.

DOI： 10.1016/j.job.2018.02.002

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Objectives: Limited biological evidence exists regarding donor host interaction in the periodontal tissue during allogenic tooth germ transplantation. This study aimed to clarify donor host tissue interactions during periodontal tissue healing following tooth germ transplantation.Methods: This study compared the localization of putative stem cells in the periodontal ligament (PDL) by 5-bromo-2'-deoxyuridine (BrdU), Gli1, and periostin immunoreactions using pulse-chase paradigm (BrdU prenatal labeling: peritoneal pulse injections at embryonic days [E] 15-17) in TetOP-H2B-GFP mice (doxycycline administration at E14.5). The current study characterized periodontal tissue healing following allogenic tooth grafts in GFP-labeled donor or host and wild-type mice by pulse-chase paradigm and GFP, BrdU, Gli1, and periostin immunohistochemistry.Results: BrdU prenatal labeling demonstrated that dense label-retaining cells (BrdU-LRCs) disappeared from the PDL by postnatal week 2 (P2W). However, H2B-GFP-LRCs were localized in the PDL of TetOP- H2B GFP mice during P3-8W, and Glil-positive cells in the PDL increased at P2-3W, showing that H2B GFP-LRCs in the PDL are derived from non-proliferating cells during E15-17. Transplanted molars formed cusps and roots and erupted into occlusion by two weeks postoperatively. The junctional epithelium and tooth-related zone of PDL were exclusively composed of donor cells, whereas the PDL alveolar-related zone was a hybrid structure of donor and host cells.Conclusions: The current tooth germ transplantation suggests that the PDL contains putative stem cells, which never proliferate during E15-17, and is composed of resident dental follicle-derived cells and other cell population. (C) 2018 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.job.2018.02.002

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© 2018, International & American Associations for Dental Research 2018. Tooth enamel is manufactured by the inner enamel epithelium of the multilayered enamel organ. Msx2 loss-of-function mutation in a mouse model causes an abnormal accumulation of epithelial cells in the enamel organ, but the underlying mechanism by which Msx2 regulates amelogenesis is poorly understood. We therefore performed detailed histological and molecular analyses of Msx2 null mice. Msx2 null ameloblasts and stratum intermedium (SI) cells differentiated normally in the early stages of amelogenesis. However, during subsequent developmental stages, the outer enamel epithelium (OEE) became highly proliferative and transformed into a keratinized stratified squamous epithelium that ectopically expressed stratified squamous epithelium markers, including Heat shock protein 25, Loricrin, and Keratin 10. Moreover, expression of hair follicle–specific keratin genes such as Keratin 26 and Keratin 73 was upregulated in the enamel organ of Msx2 mutants. With the accumulation of keratin in the stellate reticulum (SR) region and subsequent odontogenic cyst formation, SI cells gradually lost the ability to differentiate, and the expression of Sox2 and Notch1 was downregulated, leading to ameloblast depolarization. As a consequence, the organization of the Msx2 mutant enamel organ became disturbed and enamel failed to form in the normal location. Instead, there was ectopic mineralization that likely occurred within the SR. In summary, we show that during amelogenesis, Msx2 executes a bipartite function, repressing the transformation of OEE into a keratinized stratified squamous epithelium while simultaneously promoting the development of a properly differentiated enamel organ competent for enamel formation.

DOI： 10.1177/0022034518777746

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

The Nestin gene encodes type VI intermediate filament and is known to be expressed in undifferentiated cells during neurogenesis and myogenesis. To regulate Nestin expression, the first or second intron enhancer is activated in a tissue-dependent manner, for example, the former in mesodermal cells and the latter in neural stem cells. Although Nestin has also been used as a differentiation marker for odontoblasts during tooth development, how Nestin expression is regulated in odontoblasts remains unclear. Therefore, this study aimed to compare the expression patterns of Nestin-GFP (green fluorescent protein) with that of endogenous Nestin in developing teeth of Nestin-EGFP (enhanced GFP) transgenic mice, in which the second intron enhancer is connected with the EGFP domain, at postnatal 7d, 3w, and 8w. Immunohistochemical and in situ hybridization analyses revealed that endogenous Nestin protein and Nestin mRNA were intensely expressed in differentiated odontoblasts, while GFP immunoreactivity, which reflects the activity of Nestin second intron enhancer-mediated transcription, was mainly observed in the subodontoblastic layer. These results indicate that the first intron enhancer may be activated in differentiated odontoblasts. Intriguingly, Nestin-GFP expression in the subodontoblastic layer was found to be restricted to the coronal pulp of molars, which is susceptible to tooth injuries. Because the subodontoblastic layer serves as a reservoir of newly differentiated odontoblast-like cells upon exogenous stimuli to dentin, our findings suggest that the original odontoblasts and regenerated odontoblast-like cells may differently regulate Nestin expression.

DOI： 10.1007/s00418-018-1651-3

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Objectives The mechanisms regulating positional and ultrastructural changes in peripheral pulp capillaries during dentinogenesis have yet to be fully elucidated. This study aimed to clarify the relationship between the spatiotemporal localization and ultrastructure of peripheral capillaries and the dentin deposition and mineralization rate. Methods Maxillary first molars of 5- to 100-day-old Wistar rats were collected and prepared for transmission electron microscopy. The animals who had received multiple injections of lead disodium ethylenediaminetetraacetic acid were analyzed for dentin deposition rate. Non-demineralized maxillae were examined with an electron probe micro analyzer. Results The dentin deposition rate at the coronal position where dentinogenesis began on day 5 reached a peak during days 10–30, gradually decreased, and ceased on day 60. The mineral content of enamel was low on day 10 (the secretory stage of amelogenesis), drastically increased on day 15 (the maturation stage of amelogenesis), and reached a maximum and persisted until day 30. The dentin underneath the maturing enamel exhibited a synchronous increase in mineral content. The distance between the mineralization front and peripheral capillaries remained almost constant, suggesting that the positional changes of capillaries into the odontoblast layer were not due to migration of capillaries but could be attributed to an increase in the height of the odontoblast layer. Endothelial fenestrations became prominent in the most active phase of dentin deposition and mineralization. Conclusions These results indicate that the positional and ultrastructural changes in peripheral capillaries correlate with the active phase of dentin deposition and mineralization.

DOI： 10.1016/j.job.2017.05.006

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

Background Recently, we demonstrated that a pulse of 5-bromo-2’-deoxyuridine (BrdU) given to prenatal animals discloses the existence of slow-cycling long-term label-retaining cells (LRCs), or putative adult stem/progenitor cells, which reside in the dental pulp. Using several tooth injury models such as cavity preparation, tooth replantation, tooth or tooth crown transplantation, and tooth germ transplantation, we have clarified the dynamics and differentiation capacity of LRCs postoperatively. Our recent studies have demonstrated that allogenic tooth transplantation may influence the maintenance of dental pulp stem/progenitor cells. Highlight Dense LRCs are competent to proliferate and differentiate into odontoblast-like cells after tooth injuries. In the case of tooth replantation and autogenic tooth transplantation, dense LRCs remain in the perivascular environment in the center of the dental pulp for a long period. In contrast, allograft LRCs disappear from this niche during postoperative weeks 2–4. The loss of LRCs, even in cases without immunological rejection, is attributed to the extensive apoptosis taking place in these cells, with the exception of newly differentiated odontoblast-like cells. Conclusion Host and recipient interactions that occur with allografts disturb the maintenance of putative stem/progenitor cells, resulting in the disappearance of these cell types.

DOI： 10.1016/j.job.2017.03.001

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Objectives The molecular mechanisms regulating pulpal responses to GaAlAs laser irradiation remain to be clarified. This study aimed to assess the feasibility of a mouse model for studying pulpal responses to GaAlAs laser irradiation. Methods Maxillary first molars of 5-week-old ICR mice were irradiated at an output power of 1.0 W for 180 s, and samples were collected at intervals of 0, 1, 3, 5, 7, 10, and 14 days. The demineralized paraffin sections were processed for hematoxylin and eosin staining, immunohistochemistry for nestin (a marker for odontoblast differentiation) and Ki67 (a marker for cell proliferation), and a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results The intense nestin immunoreactivity in the odontoblast layer of the mesial pulp was weakened immediately after irradiation and was almost lost on Days 1–3, although the extent of pulpal damage was variable among individual animals. At around Day 1, numerous TUNEL-positive cells appeared in the degenerative zone and gradually decreased in number by Day 14. Active cell proliferation occurred in the mesial pulp during Days 5–10. Nestin-positive odontoblast-like cells appeared along the pulp-dentin border by Day 10, resulting in tertiary dentin formation on Day 14. Conclusions The current output energy induced apoptosis in the affected dental pulp, followed by active cell proliferation, resulting in tertiary dentin formation. This is the first report regarding laser irradiation of teeth in an in vivo mouse model. This model could enable further understanding of the function of certain proteins, including transcriptional factors.

DOI： 10.1016/j.job.2016.10.002

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BACKGROUND: The role of osteopontin (OPN) in the process of achieving osseointegration following implantation remains to be clarified. PURPOSE: This study aimed to analyze the healing patterns of the bone-implant interface after immediate placement of implants in the maxillae of 4-week-old Opn-knockout (KO) and wild-type (WT) mice. MATERIALS AND METHODS: After maxillary first molars were extracted, cavities were prepared with a drill and titanium implants blasted with ceramic abrasives containing hydroxyapatite/β-tricalcium phosphate were placed. Following fixation at 3, 5, 7, and 28 days after implantation, the samples were analyzed using immunohistochemistry, in situ hybridization, and an electron probe micro analyzer. RESULTS: Two types of bone healing were observed in the process of achieving osseointegration: "direct osteogenesis," where bone formation occurs at the implant surface, and "indirect osteogenesis," where it does at the pre-existing damaged bone surface in the WT mice. Direct osteogenesis occurred after the recruitment of tartrate resistant acid phosphatase-positive cells and the deposition of OPN on the implant surface. In contrast, the rate of osseointegration or direct osteogenesis was significantly low, and cell proliferation was disturbed in the Opn-KO mice. CONCLUSIONS: These results suggest that Opn-deficiency disturbs direct osteogenesis to lead the delayed osseointegration after immediate placement of endosseous implants.

DOI： 10.1111/cid.12467

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The mechanisms regulating the maintenance of quiescent adult stem cells in teeth remain to be fully elucidated. Our aim is to clarify the relationship between BrdU label-retaining cells (LRCs) and sonic hedgehog (Shh) signaling in murine teeth. After prenatal BrdU labeling, mouse pups were analyzed during postnatal day 1 (P1) to week 5 (P5W). Paraffin sections were processed for immunohistochemistry for BrdU, Sox2, Gli1, Shh, Patched1 (Ptch1) and Ki67 and for in situ hybridization for Shh and Ptch1. Dense LRCs, Gli1-(+) cells and Ptch1-(+) cells were co-localized in the outer enamel epithelium of the apical bud and apical dental papilla of incisors. In developing molars, dense LRCs were numerous at P1 but then decreased in number over the course of odontogenesis and were maintained in the center of pulp tissue. Gli1-(+) cells were maintained in the pulp horn during the examined stages, while they increased in number and were maintained in the center of pulp tissue during P2-5W. Ptch1-(+) cells were localized in the pulp horn at P1 and increased in number in the center of the pulp after P3W. Shh mRNA was first expressed in the enamel epithelium and then shifted to odontoblasts and other pulp cells. Shh protein was distributed in the epithelial and mesenchymal tissues of incisors and molars. These findings suggest that quiescent dental stem cells are regulated by Shh signaling, and that Shh signaling plays a crucial role in the differentiation and integrity of odontoblasts during epithelial-mesenchymal interactions and dentinogenesis.

DOI： 10.1007/s00441-017-2632-x

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Osteopontin (OPN) is a highly phosphorylated glycoprotein that is a prominent component of the mineralized extracellular matrix of bone. The secretion of OPN by immunocompetent cells plays a role in the differentiation of odontoblast-like cells during pulpal healing following tooth transplantation. This study aimed to clarify the role of OPN during reparative dentinogenesis. A groove-shaped cavity was prepared on the mesial surface of the upper first molars of wild-type (WT) and Opn knockout (KO) mice, and the samples were collected at intervals of 1 to 14 d. The demineralized sections were processed for immunohistochemistry for Ki67, nestin, OPN, dentin sialoprotein (DSP), integrin a alpha(v)beta(3), and type I collagen; in situ hybridization for Opn, col1a1, and dentin sialophosphoprotein (Dspp); and apoptosis assay. For the loss and gain of function experiments, an in vitro culture assay for evaluating dentin-pulp complex regeneration was performed. On day 1 in WT mice, odontoblasts beneath the affected dentin lost nestin immunoreactivity. On day 3, the expression of Opn was recognized at the mesial dental pulp, and OPN was deposited along the predentin-dentin border. Nestin-positive newly differentiated odontoblast- like cells expressed both Dspp and col1a1 and showed positive immunoreactivity for integrin a alpha(v)beta(3), DSP, and type I collagen. Until day 14, reparative dentin formation continued next to the preexisting dentin at the mesial coronal pulp. In contrast, there was no reparative dentin in the Opn KO mice where nestin- and DSP-positive newly differentiated odontoblast- like cells lacked immunoreaction for type I collagen. The in vitro organ culture demonstrated that the administration of recombinant OPN rescued the type I collagen secretion by odontoblast- like cells in the Opn KO mice. The results suggested that the deposition of OPN at the calcification front is essential for the type I collagen secretion by newly differentiated odontoblast- like cells to form reparative dentin during pulpal healing following cavity preparation.

DOI： 10.1177/0022034516645333

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BACKGROUND: There are no available data on the healing process at the bone-implant interface after immediate implant placement. PURPOSE: This study aimed to establish an animal experimental model of titanium implants placed in mouse maxillae and compare the healing pattern of the bone-implant interface after immediate implant placement with that after delayed implant placement. MATERIALS AND METHODS: Maxillary first molars (M1) from 4-week-old mice were extracted and replaced with the implant following drilling (immediate-placement group). In contrast, M1 from 2-week-old mice were extracted, followed by drilling and implantation after 4 weeks (delayed-placement group). The decalcified samples at 0-28 days after implantation were processed by immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, and tartrate-resistant acid phosphatase histochemistry. The elements and bone volume of undecalcified samples were quantitatively analyzed by an electron probe microanalyzer. RESULTS: Osseointegration was completed by 28 days after the procedure in both groups. There were no differences in contact area, bone loss at the cervical area, or rate of calcification at the bone-implant interface between the two groups. CONCLUSIONS: This study found no significant differences in the chronological healing process at the bone-implant interface between the two groups at the cellular level.

DOI： 10.1111/cid.12280

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BACKGROUND: Bleeding in the floor of the mouth during implant surgery is attributed to arterial injuries in the sublingual space. PURPOSE: This study aimed to assess the relative hemorrhage risk during implant surgery. MATERIALS AND METHODS: We investigated the occurrence rate and diameter of submental and sublingual arteries with special reference to their relationship with the course patterns of these arteries using 26 human cadavers. RESULTS: Three types of arteries were distinguished: main duct (MD), mucoperiosteal branches (MB), and cortical branches (CB). The occurrence rate of MB and CB was significantly high at the central incisor region in the upper part of the mylohyoid muscle, whereas the diameter of the MB and CB was significantly smaller than the occurrence rate of MD at the incisor regions. The occurrence rate of MD in the submental artery was significantly higher at the lateral incisor, canine, and premolar regions in the lower parts, whereas the occurrence rate of MD was significantly lower at the second and third molars in the upper parts. CONCLUSION: The susceptibility of the submental artery to injury is suggested at the incisors, canine, premolars, and first molar regions during implant surgery.

DOI： 10.1111/cid.12365

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Cells alter their energy metabolism depending on the stage of differentiation or various environments. In the ameloblast differentiation of continuous growing mouse incisors, we found temporary glycogen storage in preameloblasts before the start of enamel matrix secretion and investigated the relationship between enamel matrix secretion and glycogen metabolism. Immunohistochemistry showed that in the transitional stage from preameloblasts to secretory ameloblasts, the glycogen synthase changed from the inactive form to the active form, the expression of glycogen phosphorylase increased, and further, the levels of IGF-1, IGF-1 receptor and activated Akt increased. These results suggested that the activation of Akt signaling via IGF is linked to the onset of both glycogen metabolism and enamel matrix deposition. In the experiments using organ culture and ameloblast cell line, the activation of Akt signaling by IGF-1 stimulated glycogen metabolism through the up-regulation of Glut-1,-4 and Gsk-3β and the dephosphorylation of glycogen synthase. Subsequently, they resulted in increased enamel matrix secretion. In contrast, some inhibitors of Akt signals and glycogen synthesis/degradation down-regulated enamel matrix secretion. Taking these findings together, glycogen metabolism via Akt signaling is an essential system for the secretion of enamel matrix in ameloblast differentiation.

DOI： 10.1016/j.mod.2016.01.002

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

There are no available detailed data on the three-dimensional courses of the human superior alveolar nerves and vessels. This study aimed to clarify the relationships of the maxillary sinus with the superior alveolar nerves and vessels using cone-beam computed tomography (CT) combined with μ-CT and histological analyses. Digital imaging and communication in medicine data obtained from the scanned heads/maxillae of cadavers used for undergraduate/postgraduate dissection practice and skulls using cone-beam CT were reconstructed into three-dimensional (3D) images using software. The 3D images were compared with μ-CT images and histological sections. Cone-beam CT clarified the relationships of the maxillary sinus with the superior alveolar canals/grooves. The main anterior superior alveolar canal/groove ran anteriorly through the upper part of the sinus and terminated at the bottom of the nasal cavity near the piriform aperture. The main middle alveolar canal ran downward from the upper part of the sinus to ultimately join the anterior one. The main posterior alveolar canal ran through the lateral lower part of the sinus and communicated with the anterior one. Histological analyses demonstrated the existence of nerves and vessels in these canals/grooves, and the quantities of these structures varied across each canal/groove. Furthermore, the superior dental nerve plexus exhibited a network that was located horizontally to the occlusal plane, although these nerve plexuses appeared to be the vertical network that is described in most textbooks. In conclusion, cone-beam CT is suggested to be a useful method for clarifying the superior alveolar canals/grooves including the nerves and vessels.

DOI： 10.1002/ar.23327

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Human molars exhibit a type of metameric variation, which is the difference in serially repeated morphology within an organism. Various theories have been proposed to explain how this variation is brought about in the molars. Actualistic data that support the theories, however, are still relatively scarce because of methodological limitations. Here we propose new methods to analyse detailed tooth crown morphologies. We applied morphometric mapping to the enamel-dentine junction of human maxillary molars and examined whether odontogenetic models were adaptable to human maxillary molars. Our results showed that the upper first molar is phenotypically distinct among the maxillary molars. The average shape of the upper first molar is characterized by four well-defined cusps and precipitous surface relief of the occlusal table. On the other hand, upper third molar is characterized by smooth surface relief of the occlusal table and shows greater shape variation and distinct distribution patterns in morphospace. The upper second molar represents an intermediate state between first and third molar. Size-related shape variation was investigated by the allometric vector analysis, and it appeared that human maxillary molars tend to converge toward the shape of the upper first molar as the size increases. Differences between the upper first molar and the upper second and third molar can thus be largely explained as an effect of allometry. Collectively, these results indicate that the observed pattern of metameric variation in human molars is consistent with odontogenetic models of molar row structure (inhibitory cascade model) and molar crown morphology (patterning cascade model). This study shows that morphometric mapping is a useful tool to visualize and quantify the morphological features of teeth, which can provide the basis for a better understanding of tooth evolution linking morphology and development.

DOI： 10.1111/joa.12482

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

Objectives: Our recent study showed that cavity preparation increases the number of proliferative cells in the dental pulp during postoperative days 2-5. This study aimed to clarify pulp vascular changes following cavity preparation. Methods: Groove-shaped cavities were prepared on the medial surfaces of the upper first molars of 100-day-old Wistar rats. The animals were perfusion-fixed and tissues were collected during postoperative days 1-30, with subsequent India ink perfusion, immunohistochemistry for type IV collagen, CD31, and protein gene product (PGP) 9.5 and scanning electron microscopy with KOH digestion. The untreated upper first molars were used as controls. Results: In the controls, blood vessels with large diameters were located in the center of the pulp tissue and ramified to make capillary networks and PGP9.5-positive nerves were extensively arborized to form the subodontoblastic nerve plexus beneath the odontoblast layer. Cavity preparation induced disturbance in injured odontoblasts and subodontoblastic capillaries and nerves. Blood vessel density and thickness subsequently increased in the center and periphery of the pulp tissues with the exception of the subodontoblastic capillary network during postoperative days 3-5. PGP9.5-positive nerves overlapped with CD31-positive blood vessels in the mesial coronal pulp. Until day 30, when the tertiary dentin formation was completed, the pulp blood vasculature showed the same distribution and morphological features as that of the controls. Conclusions: These results suggest that increased vascular flow under neuronal regulation plays an important role in cell proliferation in the dental pulp following cavity preparation.

DOI： 10.1016/j.job.2015.05.003

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Autologous tooth germ transplantation of immature teeth is an alternative method of tooth replacement that could be used instead of dental implants in younger patients. However, it is paramount that the dental pulp remain vital and that root formation continue in the transplanted location. The goal of this study is to characterize the healing of allogenic tooth grafts in an animal model using GFP-labeled donor or host postnatal mice. In addition, the putative stem cells were labeled before transplantation with a pulse-chase paradigm. Transplanted molars formed cusps and roots and erupted into occlusion by 2 wk postoperatively. Host label-retaining cells (LRCs) were maintained in the center of pulp tissue associating with blood vessels. Dual labeling showed that a proportion of LRCs were incorporated into the odontoblast layer. Host cells, including putative dendritic cells and the endothelium, also immigrated into the pulp tissue but did not contribute to the odontoblast layer. Therefore, LRCs or putative mesenchymal stem cells are retained in the transplanted pulps. Hertwig's epithelial root sheath remains vital, and epithelial LRCs are present in the donor cervical loops. Thus, the dynamic donor-host interaction occurred in the developing transplant, suggesting that these changes affect the characteristics of the dental pulp.

DOI： 10.1177/0022034514556536

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

OBJECTIVES: The boundary where inner and outer enamel epithelia meet is referred to as the cervical loop (CL) in molar tooth germs. In contrast, rodent incisors are continuously growing: the labial side of the teeth is covered with enamel (crown-analog), and the lingual side is covered with cementum (root-analog). These results in the appearance of CL in the frontal sections apart from the apical end. However, many researchers have used the term "labial CL" to indicate the apical end of incisors. DESIGN: This study investigated the gene expression patterns for the enamel knot signaling center in tooth morphogenesis to clarify the difference between "labial CL" and molar CL. We examined the three-dimensional expression patterns of markers for the enamel knot including fibroblast growth factor 4 (Fgf4), sonic hedgehog (Shh), Msx2, and P21 in frontal sections of murine mandibular incisors. RESULTS: Serial frontal sections from the apical end of the postnatal incisor clearly demonstrated the existence of enamel knot-like structures composed of supra-inner enamel epithelium and stellate reticulum in the "labial CL". This structure includes the expression of all examined markers for enamel knots such as Fgf4, Shh, Msx2, and P21. CONCLUSIONS: The molar tooth germ-like structure is maintained indefinitely in the "labial CL". Therefore, the "labial CL" is not equivalent to the molar CL. The existence of an EK-like structure in the apical end of incisors implies that the usage of "labial CL" may be inappropriate for indicating this region. The "apical bud" could be used as an alternative term.

DOI： 10.1016/j.archoralbio.2015.05.001

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

Objectives: Glucose uptake plays a crucial role in early tooth morphogenesis and size determination. Recently, enzymatically synthesized glycogen (ESG), with the characteristics of natural glycogen (a major storage form of glucose), has been developed. This study aimed to elucidate the effectiveness of ESG on the pulpal healing process following intentionally delayed tooth replantation in mice.Methods: The upper first molar was extracted, immersed in phosphate buffered saline (PBS) or ESG (5000 kDa) solution (1 mg/mL) for 60 min, and then replanted. Immunohistochemistry (for nestin, osteopontin, and TUNEL assay, and reverse transcription-polymerase chain reaction were performed at different time points.Results: Increased apoptosis occurred in the dental pulp of mice from both treatment groups at Day 7, followed by active cell proliferation at Day 14 and tertiary dentin and/or bone-like tissue deposition at Day 21, in the PBS group. In contrast, active cell proliferation and coronal immunoreaction for nestin occurred around Day 10, and hard tissue deposition were observed at Day 14, in the ESG group. The mRNA expression of genes encoding dentin sialophosphoprotein and nestin first reappeared in the ESG group at Day 5, while expression levels of alkaline phosphatase and osteopontin, as well as Crillc, tended to increase from Day 3 in both groups, and that of the stem cell marker, octamer-binding transcription factor Oct314, greatly enhanced at Day 1, particularly in the ESG group.Conclusions: ESG improved the pulpal healing process of extracted teeth following intentionally delayed replantation, although both ESG and PBS may induce the formation of bone-like tissue. (C) 2015 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.job.2015.01.003

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INTRODUCTION: This study analyzed the detailed biological events underlying pulpal dynamics evoked by 3Mix (the mixture of ciprofloxacin, metronidazole, and minocycline) solution after intentionally delayed tooth replantation because 3Mix improves pulpal healing after tooth injuries. METHODS: The maxillary first molars of 3-week-old mice were extracted and immersed in 3Mix solution for 30 minutes in comparison with phosphate buffered saline (PBS) alone. Cell proliferation, apoptosis, and differentiation were assessed in extracted/replanted teeth during days 0-14 using immunohistochemistry, apoptosis assay, and reverse-transcriptase polymerase chain reaction. RESULTS: 3Mix solution accelerated odontoblast differentiation in the coronal pulp on day 7 and tertiary dentin formation on day 14, whereas the regenerative process was delayed in the PBS group. Cell proliferation and apoptosis occurred in the pulp of the 3Mix group during days 5-7 and subsequently decreased from days 7-14. On day 5, dentin sialophosphoprotein and nestin were first recovered in the 3Mix group, whereas expression levels for alkaline phosphatase, osteopontin, and osteocalcin increased in the PBS group. The expression levels for octamer-binding factor 3/4A and 3/4B reached the maximum level on day 1 and were sharply decreased on day 3 in both groups. High expression levels of Cd11c were first observed in the 3Mix group on day 1 and later at days 5 and 7. CONCLUSIONS: The results suggest that the application of 3Mix may suppress osteoblast differentiation by the migration of dendritic cells to the injury site and via the activation of stem/progenitor cells, resulting in the acceleration of odontoblastlike cell differentiation.

DOI： 10.1016/j.joen.2014.05.005

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We have proposed the new hypothesis that dental pulp stem cells play crucial roles in the pulpal healing process following exogenous stimuli in cooperation with progenitors. This study aimed to establish an in vitro culture system for evaluating dentin-pulp complex regeneration with special reference to the differentiation capacity of slow-cycling long-term label-retaining cells (LRCs). Three intraperitoneal injections of BrdU were given to pregnant ICR mice to map LRCs in the mature tissues of born animals. The upper bilateral first molars of 3-week-old mice were extracted and divided into two pieces and cultured for 0, 1, 3, 5 and 7 days using the Trowel's method. We succeeded in establishing an in vitro culture system for evaluating dentin-pulp complex regeneration, where most odontoblasts were occasionally degenerated and lost nestin immunoreactivity because of the separation of cell bodies from cellular processes in the dentin matrix by the beginning of in vitro culture. Numerous dense LRCs mainly resided in the center of the dental pulp associating with blood vessels throughout the experimental periods. On postoperative days 1-3, the periphery of the pulp tissue including the odontoblast layer showed degenerative features. By Day 7, nestin-positive odontoblast-like cells were arranged along the pulp-dentin border and dense LRCs were committed in the odontoblast-like cells. These results suggest that dense LRCs in the center of the dental pulp associating with blood vessels were supposed to be dental pulp stem/progenitor cells possessing regenerative capacity for forming newly differentiated odontoblast-like cells.

DOI： 10.1007/s00418-014-1200-7

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Our recent study suggested that allogenic tooth transplantation may affect the maintenance of dental pulp stem/progenitor cells. This study aims to elucidate the influence of allograft on the maintenance of dental pulp stem/progenitor cells following tooth replantation and allo- or auto-genic tooth transplantation in mice using BrdU chasing, immunohistochemistry for BrdU, nestin and Ki67, in situ hybridization for Dspp, transmission electron microscopy and TUNEL assay. Following extraction of the maxillary first molar in BrdU-labeled animals, the tooth was immediately repositioned in the original socket, or the roots were resected and immediately allo- or auto-grafted into the sublingual region in non-labeled or the same animals. In the control group, two types of BrdU label-retaining cells (LRCs) were distributed throughout the dental pulp: those with dense or those with granular reaction for BrdU. In the replants and autogenic transplants, dense LRCs remained in the center of dental pulp associating with the perivascular environment throughout the experimental period and possessed a proliferative capacity and maintained the differentiation capacity into the odontoblast-like cells or fibroblasts. In contrast, LRCs disappeared in the center of the pulp tissue by postoperative week 4 in the allografts. The disappearance of LRCs was attributed to the extensive apoptosis occurring significantly in LRCs except for the newly-differentiated odontoblast-like cells even in cases without immunological rejection. The results suggest that the host and recipient interaction in the allografts disturbs the maintenance of dense LRCs, presumably stem/progenitor cells, resulting in the disappearance of these cell types.

DOI： 10.1007/s00441-014-1818-8

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DOI： 10.1111/J.1600-0501.2011.02348.X

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INTRODUCTION: The molecular mechanisms behind odontoblast differentiation remain obscure. Lymphoid enhancer-binding factor 1 (Lef1) is a transcription factor that mediates Wnt signaling and has been suggested to regulate dentin sialophosphoprotein (Dspp) expression in vitro. This study aimed to clarify their precise relationship in the process of odontoblast differentiation in vivo. METHODS: The detailed spatiotemporal expression patterns of Lef1 and Dspp together with other known and putative odontoblast differentiation markers such as P21 and heat-shock protein 25 (Hsp25) were examined by in situ hybridization and immunohistochemistry on paraffin sections of rat incisors and developing molars at postnatal days 1-100. To observe odontoblast regeneration following tooth injury, a cavity was prepared on the upper first molar of 10-week-old rats and the expressions of Lef1 and Dspp were investigated. RESULTS: Following undifferentiated state expressing none of these examined markers, preodontoblasts begun to express P21, Lef1 and Hsp25 according to their progress of differentiation, although Dspp was undetectable. Immature odontoblasts commenced transcribing Dspp simultaneously with dentin calcification. Lef1, Dspp and Hsp25 were co-expressed in mature odontoblasts. In contrast to continuously growing incisors, Lef1, Dspp and P21 were down-regulated in the resting odontoblasts in molars when primary dentin formation was completed. Remarkably, Lef1 expression also preceded Dspp expression in newly differentiated odontoblast-like cells during the pulpal healing process after tooth injury. CONCLUSIONS: Lef1 expression precedes Dspp expression without exception in both primary and reparative dentinogeneses. Our results suggest that Lef1 might play a key role in odontoblast differentiation through regulating Dspp expression.

DOI： 10.1016/j.joen.2012.12.016

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

Objective: A mixture of ciprofloxacin, metronidazole, and minocycline (3Mix) has been reported to be effective against oral bacteria from carious and endodontic lesions in vitro and in vivo. The objective of this study was to establish an animal model using mice for the application of 3Mix following intentionally delayed tooth replantation and to investigate the effects of 3Mix on the healing process of dental pulp and periodontal tissues. Methods: Upper first molars of ICR mice were extracted, immersed in 3Mix solution at different concentrations for 5-60 min with or without the use of a transfer solution (phosphate buffer solution (PBS)), in addition to transfer solution alone, and subsequently repositioned in the sockets. Immunohis-tochemistry for nestin and Ki-67, histochemistry for TRAP, and TUNEL assay were performed to assess pulpal healing during days 7-21. Results: Increased apoptosis was observed in the PBS group at week 1, followed by cell proliferation at week 2, and tertiary dentin and/or bone-like tissue formation at week 3. In contrast, nestin-positive, newly differentiated, odontoblast-like cells began to align along the pulp-dentin border following the appearance of Ki-67- and TUNEL-positive cells during weeks 1-2 in the 3Mix groups, suggesting that pulpal healing was accelerated. Severe root ankylosis was observed exclusively in the 3Mix groups. Rinsing with PBS before replantation partially rescued the viability of the periodontal ligament, but pulpal healing was delayed. Conclusions: The application of 3Mix promotes pulpal regeneration of intentionally delayed replanted teeth
however, its use may induce severe damage to periodontal tissues. © 2013 Japanese Association for Oral Biology.

DOI： 10.1016/j.job.2013.03.001

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INTRODUCTION: This study aimed at clarifying the dynamics of bromodeoxyuridine (BrdU) label-retaining cells (LRCs) and their relationship to cell proliferation and apoptosis during pulpal healing after cavity preparation in mice. METHODS: A groove-shaped cavity was prepared on the mesial cervical surface of the upper first molars, and the samples were collected at intervals of 12 hours-14 days. The demineralized paraffin sections were processed for immunohistochemistry for BrdU, nestin, and Ki-67 and apoptosis assay using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) and in situ hybridization for dentin sialophosphoprotein (Dspp). RESULTS: During hour 12-day 1, odontoblasts and subodontoblastic cells beneath the affected dentin showed degenerative features and TUNEL-positive reactions, and the expressions of nestin and Dspp were lost in the damaged odontoblasts. TUNEL-positive reactions were observed even in the center of the pulp tissue, whereas dense and granular LRCs remained there. On days 2-3, Ki-67-positive cells were significantly increased in number in the center of mesial dental pulp. During days 3-5, granular and few dense LRCs were committed into some (not all) nestin-positive newly differentiated odontoblast-like cells, and these differentiated cells began to express nestin and Dspp. Until day 14, tertiary dentin formation occurred next to the preexisting dentin at the mesial pulp floor in addition to the mesial coronal pulp. CONCLUSIONS: These results suggest that odontoblasts and subodontoblastic cells degenerate after tooth drilling, and, subsequently, dental pulp stem/progenitor cells actively proliferate and differentiate into new odontoblast-like cells.

DOI： 10.1016/j.joen.2013.06.017

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Differentiated odontoblasts could not be identified by one unique phenotypic marker, but the combination of expression of dentin phosphoprotein (Dpp), dentin sialoprotein (Dsp), dentin matrix protein 1 (Dmp1), and nestin may be valuable for the assessment of these cells. However, the findings using these proteins remain controversial. This study aimed to compare two odontoblast differentiation markers: nestin and Dsp in the process of dentinogenesis in mice. We performed immunohistochemistry and/or in situ hybridization technique for nestin and Dsp using 3-week-old incisors as well as postnatal 1-day- to 8-week-old molars. Preodontoblasts began to express nestin and Dsp proteins and Dsp mRNA, which increased in their intensity according to the progress of odontoblast differentiation in both incisors and developing molars. Nestin was consistently expressed in the differentiated odontoblasts even after the completion of dentin matrix deposition. The expression of Dsp mRNA coincided with the odontoblast secretory activity for dentin matrix deposition. In contrast, other pulpal cells, predentin matrix and dentinal tubules also showed a positive reaction for Dsp protein in addition to differentiated odontoblasts. In conclusion, nestin is valuable as a differentiation marker for odontoblasts, whereas Dsp mRNA is a functional marker for their secretory activity.

DOI： 10.2220/biomedres.33.119

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Human dental pulp contains adult stem cells. Our recent study demonstrated the localization of putative dental pulp stem/progenitor cells in the rat developing molar by chasing 5-bromo-2'-deoxyuridine (BrdU)-labeling. However, there are no available data on the localization of putative dental pulp stem/progenitor cells in the mouse molar. This study focuses on the mapping of putative dental pulp stem/progenitor cells in addition to the relationship between cell proliferation and differentiation in the developing molar using BrdU-labeling. Numerous proliferating cells appeared in the tooth germ and the most active cell proliferation in the mesenchymal cells occurred in the prenatal stages, especially on embryonic Day 15 (E15). Cell proliferation in the pulp tissue dramatically decreased in number by postnatal Day 3 (P3) when nestin-positive odontoblasts were arranged in the cusped areas and disappeared after postnatal Week 1 (P1W). Root dental papilla included numerous proliferating cells during P5 to P2W. Three to four intraperitoneal injections of BrdU were given to pregnant ICR mice and revealed slow-cycling long-term label-retaining cells (LRCs) in the mature tissues of postnatal animals. Numerous dense LRCs postnatally decreased in number and reached a plateau after P1W when they mainly resided in the center of the dental pulp, associating with blood vessels. Furthermore, numerous dense LRCs co-expressed mesenchymal stem cell markers such as STRO-1 and CD146. Thus, dense LRCs in mature pulp tissues were believed to be dental pulp stem/progenitor cells harboring in the perivascular niche surrounding the endothelium.

DOI： 10.1007/s00441-012-1347-2

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

Glucose is an essential source of energy for body metabolism and is transported into cells by glucose transporters (GLUTs). Well-characterized class I GLUT is subdivided into GLUTs1-4, which are selectively expressed depending on tissue glucose requirements. However, there is no available data on the role of GLUTs during tooth development. This study aims to clarify the functional significance of class I GLUT during murine tooth development using immunohistochemistry and an in vitro organ culture experiment with an inhibitor of GLUTs1/2, phloretin, and Glut1 and Glut2 short interfering RNA (siRNA). An intense GLUT1-immunoreaction was localized in the enamel organ of bud-stage molar tooth germs, where the active cell proliferation occurred. By the bell stage, the expression of GLUT1 in the dental epithelium was dramatically decreased in intensity, and subsequently began to appear in the stratum intermedium at the late bell stage. On the other hand, GLUT2-immunoreactivity was weakly observed in the whole tooth germs throughout all stages. The inhibition of GLUTs1/2 by phloretin in the bud-stage tooth germs induced the disturbance of primary enamel knot formation, resulting in the developmental arrest of the explants and the squamous metaplasia of dental epithelial cells. Furthermore, the inhibition of GLUTs1/2 in cap-to-bell-stage tooth germs reduced tooth size in a dose dependent manner. These findings suggest that the expression of GLUT1 and GLUT2 in the dental epithelial and mesenchymal cells seems to be precisely and spatiotemporally controlled, and the glucose uptake mediated by GLUT1 plays a crucial role in the early tooth morphogenesis and tooth size determination.

DOI： 10.1016/j.ydbio.2011.12.020

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Publishing type：Research paper (scientific journal)   Publisher：Stem Cell, Regenerative Medicine and Cancer  

Human tooth-related diseases often occur throughout life. The tooth is a complex organ, which includes different types of mineralized tissues. Although this complex structure provides it with hardness and durability, the tooth components are occasionally vulnerable to damage such as mechanical trauma, bacterial infections, and dental procedures as well as congenital defects. Once the tooth suffers loss, it is difficult to expect complete regeneration of the injured tissue, unlike other tissues such as hair and bone. However, the tooth components have some limited regenerative capacity except for the enamel regeneration. Tooth development proceeds through sequential and reciprocal interactions between the oral epithelium and the cranial neural crest-derived mesenchyme. Human dental tissues harbor stem/progenitor cells in the dental pulp, periodontal ligament, and dental follicle, which are responsible for tooth and associated tissue development or regeneration. Stem cell biology provides us with informative insights for the improvement of regenerative methods for tooth structures. Regenerative medicine using dental stem cells could become a feasible tool for novel approaches to treat dental diseases such as caries, endodontitis, periodontitis, and pathological defects of the maxillofacial bones and teeth. This chapter focuses on the different sources of dental stem cells that have been used for producing tooth components or a whole tooth. The improvement in tissue engineering approaches for dental tissue regeneration using dental stem cells is promising, and remains one of the greatest challenges in the dental field in the near future. © 2011 by Nova Science Publishers, Inc. All rights reserved.

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INTRODUCTION: This study aimed to clarify pulpal responses to gallium-aluminum-arsenide (GaAlAs) laser irradiation. METHODS: Maxillary first molars of 8-week-old rats were irradiated at an output power of 0.5 or 1.5 W for 180 seconds, and the samples were collected at intervals of 0 to 14 days. The demineralized paraffin sections were processed for immunohistochemistry for heat-shock protein (HSP)-25 and nestin in addition to cell proliferation assay using bromodeoxyuridine (BrdU) labeling and apoptosis assay using deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL). RESULTS: Intense HSP-25 and nestin immunoreactivities in the odontoblast layer were weakened immediately after 0.5-W irradiation and recovered on day 1, resulting in slight tertiary dentin formation by day 14. On the contrary, 1.5-W irradiation immediately induced the loss of HSP-25 and nestin-immunoreactivities in the odontoblast layer. On day 1, numerous TUNEL-positive cells appeared in a degenerative zone that was surrounded by intense HSP-25 immunoreactivity. BrdU-positive cells occurred within the intensely HSP-25-immunopositive areas during days 2 through 5, whereas TUNEL-positive cells gradually decreased in number by day 5. HSP-25- and nestin-positive odontoblast-like cells were arranged along the pulp-dentin border by day 7, resulting in remarkable tertiary dentin formation on day 14. CONCLUSIONS: The output energy determined pulpal healing patterns after GaAlAs laser irradiation; the higher energy induced the apoptosis in the affected dental pulp including odontoblasts followed by active cell proliferation in the intense HSP-25-immunoreactive areas surrounding the degenerative tissue, resulting in abundant tertiary dentin formation. Thus, the optimal GaAlAs laser irradiation elicited intentional tertiary dentin formation in the dental pulp.

DOI： 10.1016/j.joen.2011.05.020

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Our recent study has demonstrated the localization of putative dental pulp stem cells in the developing molar by chasing 5-bromo-2'-deoxyuridine (BrdU)-labeling. However, their differentiation capacity subsequent to the tooth transplantation remains to be elucidated. This study aims to clarify the differentiation capacity of BrdU label-retaining dental pulp cells and their relationship to cell proliferation and apoptosis during pulpal healing following allogenic transplantation in mice. Following extraction of the mouse molar in BrdU-labeled animals, the roots and pulp floor were resected and immediately allo-grafted into the sublingual region in non-labeled animals, and vice versa. In the labeled transplants, label-retaining cells (LRCs) were increased in number and committed in nestin-positive newly differentiated odontoblast-like cells, whereas they were not committed in osteoblast-like cells. In the labeled host, on the contrary, LRCs were committed in neither odontoblast- nor osteoblast-like cells, although they were transiently increased in number and finally disappeared in the pulp tissue of the transplants. Interestingly, numerous apoptotic cells appeared in the pulp tissue including LRCs during the experimental period. These results suggest that transplanted LRCs maintain their proliferative and differentiation capacity in spite of extensive apoptosis occurring in the transplant, whereas transiently increased host-derived LRCs finally disappear in the pulp chamber following apoptosis.

DOI： 10.2220/biomedres.32.247

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Dental pulp elaborates both bone and dentin under pathological conditions such as tooth replantation/transplantation. This study aims to clarify the expression of granulocyte macrophage colony-stimulating factor (GM-CSF) and osteopontin (OPN) in the process of reparative dentin formation by allogenic tooth transplantation using in situ hybridization for OPN and immunohistochemistry for GM-CSF and OPN at both levels of light and electron microscopes. Following the extraction of the mouse molar, the roots and pulp floor were resected and immediately allografted into the sublingual region. On days 1 to 3, immunocompetent cells such as macrophages and dendritic cells expressed both GM-CSF and OPN, and some of them were arranged along the pulp-dentin border and extended their cellular processes into the dentinal tubules. On days 5 to 7, tubular dentin formation commenced next to the preexisting dentin at the pulp horn where nestin-positive odontoblast-like cells were arranged. Until day 14, bone-like tissue formation occurred in the pulp chamber, where OPN-positive osteoblasts surrounded the bone matrix. These results suggest that the secretion of GM-CSF and OPN by immunocompetent cells such as macrophages and dendritic cells plays a role in the maturation of dendritic cells and the differentiation of odontoblasts, respectively, in the regenerated pulp tissue following tooth transplantation.

DOI： 10.1369/0022155411403314

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Recently, we demonstrated that a pulse of BrdU given to prenatal animals reveals the existence of slow-cycling long-term label-retaining cells (LRCs), putative adult stem or progenitor cells, which reside in the dental pulp. This study aims to clarify responses of LRCs to allogenic tooth transplantation into mouse maxilla using prenatal BrdU-labeling, in situ hybridization for osteopontin and periostin, and immunohistochemistry for BrdU, nestin, and osteopontin. The upper-right first molars were allografted in the original socket between BrdU-labeled and non-labeled mice or between GFP transgenic and wild-type mice. Tooth transplantation caused degeneration of the odontoblast layer, resulting in the disappearance of nestin-positive reactions in the dental pulp. On postoperative days 5-7, tertiary dentin formation commenced next to the preexisting dentin where nestin-positive odontoblast-like cells were arranged in the successful cases. In BrdU-labeled transplanted teeth, dense LRCs were maintained in the center of the dental pulp beneath the odontoblast-like cells including LRCs, whereas LRCs disappeared in the area surrounding the bone-like tissue. In contrast, LRCs were not recognized in the pulp chamber of non-labeled transplants through the experimental period. Tooth transplantation using GFP mice demonstrated that the donor cells constituted the dental pulp of the transplant except for endothelial cells and some migrated cells, and the periodontal tissue was replaced by host-derived cells except for epithelial cell rests of Malassez. These results suggest that the maintenance of BrdU label-retaining dental pulp cells play a role in the regeneration of odontoblast-like cells in the process of pulpal healing following tooth transplantation.

DOI： 10.1007/s00418-011-0868-1

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Publishing type：Research paper (scientific journal)   Publisher：International Society of Histology & Cytology  

α-tricalcium phosphate (a TCP) with the addition of antimicrobials such as ciprofloxacin, metronidazole, and cefaclor (3Mix) has been applied to sterilize the infected dentin and pulp in vivo. Both clinical and animal experiments have shown that 3Mix is effective for sterilizing infected tissues. However, the responses of the infected dental pulp to 3Mix remain to be fully determined at the cellular level. This study aims to clarify the responses of neural elements and immune cells to antimicrobials during the healing process of infected pulp using immunohistochemistry for protein gene product (PGP) 9.5 and class II major histocompatibility complex molecules using both light and electron microscopy. An artificial pulp exposure was prepared on the maxillary molar of 14-week-old rats and maintained without any treatment for 12 - 24 h. Subsequently, the exposed pulp was covered with αTCP or α TCP containing 3Mix, followed with glass ionomer cement. A pulp abscess lacking both dendritic cells and PGP 9.5-reactive nerve fibers was induced after pulp capping with α TCP; in contrast, numerous dendritic cells accumulated along the pulp-dentin border followed by the differentiation of odontoblast - like cells and matrix deposition after the application of α TCP containing 3Mix. PGP 9.5-reactive nerve fibers were also densely distributed and surrounded the accumulated dendritic cells in the medial dental pulp beneath α TCP containing 3Mix. The findings indicate that the application of α TCP containing 3Mix to the infected pulp induces an intense accumulation of dendritic cells, suggesting that these cells play crucial roles in the differentiation of odontoblast like cells under pathological conditions. © 2011, International Society of Histology and Cytology. All rights reserved.

DOI： 10.1679/aohc.73.165

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Recent studies have demonstrated that human dental pulp contains adult stem cells. A pulse of the thymidine analog BrdU given to young animals at the optimal time could clarify where slow-cycling long-term label-retaining cells (LRCs), putative adult stem cells, reside in the pulp tissue. This study focuses on the mapping of LRCs in growing teeth and their regenerative capacity after tooth injuries. Two to seven peritoneal injections of BrdU into pregnant Wistar rats revealed slow-cycling long-term dense LRCs in the mature tissues of born animals. Numerous dense LRCs were postnatally decreased in number and reached a plateau at 4 weeks after birth when they mainly resided in the center of the dental pulp, associating with blood vessels. Mature dental pulp cells were stained with Hoechst 33342 and sorted into (<0.76%) side population cells using FACS, which included dense LRCs. Some dense LRCs co-expressed mesenchymal stem cell markers such as STRO-1 or CD146. Tooth injuries caused degeneration of the odontoblast layer, and newly differentiated odontoblast-like cells contained LRCs. Thus, dense LRCs in mature pulp tissues were supposed to be dental pulp stem cells possessing regenerative capacity for forming newly differentiated odontoblast-like cells. The present study proposes the new hypothesis that both granular and dense LRCs are equipped in the dental pulp and that the dense LRCs with proliferative capacity play crucial roles in the pulpal healing process following exogenous stimuli in cooperation with the granular LRCs.

DOI： 10.1007/s00418-010-0727-5

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

Autogenic tooth transplantation is now a common procedure in dentistry for replacing a missing tooth. However, there are many difficulties in clinical application of allogenic tooth transplantation because of immunological rejection. This study aims to clarify pulpal regeneration following allogenic tooth transplantation into the mouse maxilla by immunohistochemistry for 5-bromo-2'-deoxyuridine (BrdU) and nestin, and by the histochemistry for tartrate-resistant acid phosphatase (TRAP). The upper right first molar (M1) of 2-week-old mice was extracted and allografted in the original socket in both the littermate and non-littermate after the extraction of M1. Tooth transplantation weakened the nestin-positive reactions in the pulp tissue that had shown immunoreactivity for nestin before operation. On postoperative Days 5-7, tertiary dentin formation commenced next to the preexisting dentin where nestin-positive odontoblast-like cells were arranged in all cases of the littermate group until Day 14, except for one case showing immunological rejection in the pulp chamber. In the non-littermate group, bone-like tissue formation occurred in the pulp chamber in addition to tertiary dentin formation until Day 14. The rate of tertiary dentin was 38%, and the rate of the mixed form of dentin and bone-like tissue formation was 23% (the remainder was immunological rejection). Interestingly, the periodontal tissue recovered even in the case of immunological rejection in which the pulp chamber was replaced by sparse connective tissue. These results suggest that the selection of littermate or non-littermate is decisive for the survival of odontoblast-lineage cells and that the immunological rejection does not influence the periodontal regeneration.

DOI： 10.1002/ar.20831

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

Tooth development is characterized by a series of reciprocal epithelial-mesenchymal interactions between the ectoderm and underlying neural crest-derived mesenchyme. Spatiotemporally regulated paracrine signal molecules of several conserved families referred to as developmental genetic toolkits, such as bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), sonic hedgehog (Shh), the tumor necrosis factor (TNF) family, and Wnts, play crucial roles in mediating these tissue interactions. However, the developmental biology of tooth root formation has not been fully studied, especially as to how Hertwig's epithelial root sheath (HERS) is formed, the functional significance of HERS, and the fate of HERS. Recently, several important papers concerning the developmental biology of HERS and tooth root formation have been published by Japanese and Korean research groups. The current special issue features review articles by these authors who introduce their recent data on the developmental biology of tooth root development clarified using their particular strategies. Furthermore, we discuss the perspective of regenerative science and medicine in the field of dentistry. Exact knowledge of the mechanisms of tooth root formation would provide useful information for future regenerative treatments and the application of tissue-engineered teeth in dentistry. Further studies are required to clarify the expression patterns of signal molecules during tooth root formation, and exact knowledge about odontogenic stem cells and improvement of tissue engineering techniques are needed for future regenerative dentistry.

DOI： 10.2330/joralbiosci.50.147

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Odontoblast-lineage cells acquire heat-shock protein (HSP)-25-immunoreactivity (IR) after they complete their cell division, suggesting that this protein acts as a switch between cell proliferation and differentiation during tooth development. However, there are few available data concerning the relationship between cell proliferation and differentiation following cavity preparation. The present study aims to clarify the expression of HSP-25 in the odontoblast-lineage cells with their proliferative activity after cavity preparation by immunocytochemistry for HSP-25 and cell proliferation assay using 5-bromo-2'-deoxyuridine (BrdU) labeling. In untreated control teeth, intense HSP-25-IR was found in odontoblasts and some subodontoblastic mesenchymal cells. Cavity preparation caused the destruction of odontoblasts and the disappearance of HSP-25-IR was conspicuous at the affected site, although some cells retained HSP-25-IR and subsequently most of them disappeared from the pulp-dentin border by postoperative day 1. Contrary, some subodontoblastic mesenchymal cells with weak HSP-25-IR began to take the place of degenerated cells, although no proliferative activity was recognizable in the dental pulp. Interestingly, proliferative cells in the dental pulp significantly increased in number on day 2 when the newly differentiating cells already arranged along the pulp-dentin border, and continued their proliferative activity in the wide range of the pulp tissue until day 5. These findings indicate that progenitor cells equipped in the subodontoblastic layer firstly migrate and differentiate into new odontoblast-like cells to compensate for the loss of the odontoblast layer, and subsequently the reorganization of dental pulp was completed by active proliferation of the mesenchymal cells occurring in a wide range of pulp tissue.

DOI： 10.1007/s00418-008-0438-3

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

Dental pulp elaborates both bone and dentin under pathological conditions such as tooth replantation/transplantation. This study aims to clarify the capability of dental pulp to elaborate bone tissue in addition to dentin by allogenic tooth transplantation using immunohistochemistry and histochemistry. After extraction of the molars of 3-week-old mice, the roots and pulp floor were resected and immediately allografted into the sublingual region in a littermate. In addition, we studied the contribution of donor and host cells to the regenerated pulp tissue using a combination of allogenic tooth transplantation and lacZ transgenic ROSA26 mice. On Days 5-7, tubular dentin formation started next to the preexisting dentin at the pulp horn where nestin-positive odontoblast-like cells were arranged. Until Day 14, bone-like tissue formation occurred in the pulp chamber, where intense tartrate-resistant acid phosphatase-positive cells appeared. Furthermore, allogenic transplantation using ROSA26 mice clearly showed that both donor and host cells differentiated into osteoblast-like cells with the assistance of osteoclast-lineage cells, whereas newly differentiated odontoblasts were exclusively derived from donor cells. These results suggest that the odontoblast and osteoblast lineage cells reside in the dental pulp and that both donor and host cells contribute to bone-like tissue formation in the regenerated pulp tissue.

DOI： 10.1369/jhc.2008.951558

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

Continuously growing rodent incisors have a special epithelial structure for maintaining adult stem cells that shows a bulbous epithelial protrusion at the apical end and is referred to as an "apical bud". Guinea pig cheek teeth (premolars and molars), also continuously growing teeth, have a complex crown shape consisting of plural cusps. The present study clarifies the existence of apical buds in guinea pig premolars/molars as it examines the relationship between the crown shape and the orientation of the apical buds by micro-computed tomography (micro-CT) and immunohistochemistry for 5-bromo-2'-deoxyuridine (BrdU). One premolar and three molar teeth in each side of the maxillae and mandibles assumed characteristic features: each horizontally-sectioned tooth showing a complex zigzag shape was composed of a core of dentin covered by a layer of enamel on all axial surfaces except the buccal of the uppers and the lingual of the lowers. Furthermore, four bulbous epithelial protrusions--including the stellate reticulum--were recognized in the apical end of each tooth, where slow-cycling long-term label-retaining cells resided 20 days after a peritoneal injection of BrdU. These data indicate that guinea pig premolars/molars have four apical buds where the epithelial adult stem cells reside. In contrast, rodent incisors, which show a single cone appearance, are covered by enamel on the labial side and possess only one apical bud. The results of this study suggest that plural apical buds, being arranged bucco-lingually and mesio-distally, produce the crown mold in a zigzag fashion.

DOI： 10.1679/aohc.71.317

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The mechanism regulating the divergent healing processes following tooth replantation is unclear. This study clarifies the relationship between the healing pattern, the time taken for tooth replantation, and the influence of occlusal force. We investigated the pulpal healing process after tooth replantation by immunohistochemistry for 5-bromo-2'-deoxyuridine and nestin and by histochemistry for tartrate-resistant acid phosphatase. The upper right first molar of 3-week-old mice was extracted and repositioned in the original socket immediately or 30 min to 6 h after the operation. We divided the animals into a non-occluded group in which the lower right first molar was extracted and an occluded group without extraction of the counterpart tooth. In control teeth (upper left first molar), the periphery of the coronal dental pulp showed intense nestin-positive reaction. Tooth replantation weakened the nestin-positive reaction in the pulp tissue. On postoperative days 5-7, tubular dentin formation commenced next to preexisting dentin in which nestin-positive odontoblast-like cells were arranged in successful cases. In other cases, bone-like tissue formation occurred in the pulp chamber until day 14. The ratio of tertiary dentin formation was significantly higher in the non-occluded group. The intentionally prolonged time for the completion of tooth replantation induced bone-like tissue formation, expanded inflammatory reaction, or fibrous tissue formation in pulp tissue. Thus, the lack of a proper oxygenated medium is probably decisive for the survival of odontoblast-lineage cells, and occlusal force during and/or after operation worsens the fate of these cells.

DOI： 10.1007/s00441-007-0424-4

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

A recent study provided genetic and morphological evidence that rat autosomal-recessive mutation, whitish chalk-like teeth (wct), induced tooth enamel defects resembling those of human amelogenesis imperfecta (AI). The wct locus maps to a specific interval of rat chromosome 14 corresponding to human chromosome 4q21 where the ameloblastin and enamelin genes exist, although these genes are not included in the wct locus. The effect of the wct gene mutation on the enamel matrix synthesis and calcification remains to be elucidated. This study clarifies how the wct gene mutation influences the synthesis of enamel matrix and its calcification by immunocytochemistry for amelogenin, ameloblastin and enamelin, and by electron probe micro-analysis (EPMA). The immunoreactivity for enamel proteins such as amelogenin, ameloblastin, and enamelin in the ameloblasts in the homozygous teeth was the same as that in the heterozygous teeth from secretory to transitional stages, although the homozygous ameloblasts became detached from the enamel matrix in the transitional stage. The flattened ameloblasts in the maturation stage of the homozygous samples contained enamel proteins in their cytoplasm. Thus, the wct mutation was found to prevent the morphological transition of ameloblasts from secretory to maturation stages without disturbing the synthesis of enamel matrix proteins, resulting in the hypo-mineralization of incisor enamel and cyst formation between the enamel organ and matrix. This mutation also prevents the transfer of iron into the enamel.

DOI： 10.1007/s00418-007-0297-3

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Our previous findings have demonstrated that the rat autosomal-recessive mutation, whitish chalk-like teeth (wct), induces enamel defects resembling those of human amelogenesis imperfecta (AI) in continuously growing incisor teeth. The present study clarifies the effect of the wct mutation on the morphogenesis and calcification of rat molar teeth. Formalin-fixed maxillae obtained from animals aged 4-30 days were examined by electron probe micro-analysis (EPMA) and by immunocytochemistry for amelogenin, ameloblastin, and enamelin. There were no distinct differences in the calcium and phosphorous contents and the amount of enamel between homozygous mutant and wild-type teeth during postnatal days 4-11. Although the mineral density in the enamel matrix considerably increased in the wild-type teeth until day 15, no changes occurred in mutant teeth during days 11-30. The immunoreactivity for enamel proteins in the secretory-stage ameloblasts in mutant teeth was similar to that in the wild-type teeth, and subsequently mutant maturation-stage ameloblasts became detached from the enamel surface, resulting in odontogenic cyst formation between the enamel organ and matrix until day 7 and the expansion of the cyst around the whole tooth crown on day 15. On day 30, the erupted mutant teeth presented morphological changes such as enamel destruction and tertiary dentin formation in addition to low mineral density in the enamel. Thus, the wct mutation prevents mineral transport without disturbing the synthesis of enamel proteins in molar teeth because of the absence of maturation-stage ameloblasts, in addition to the occurrence of odontogenic cysts.

DOI： 10.1007/s00441-007-0452-0

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

OBJECTIVES: The aim of this study is to clarify the functional significance of heat-shock protein (HSP)-25 during tooth development. DESIGN: We compared the expression of HSP-25 in the dental epithelial and mesenchymal cells with their proliferative activity during odontogenesis in rat molars on postnatal days 1-100 by immunohistochemistry using anti-HSP-25 and anti-5-bromo-2'-deoxyuridine (BrdU) for cell proliferation assay. RESULTS: On day 1, BrdU-immunoreactive cells were densely located in the inner enamel epithelium in the cervical loop and intercusped areas and the dental pulp adjacent to them, whereas HSP-25-immunoractivity (IR) was restricted to the cusped area where odontoblasts and ameloblasts had already differentiated. Subsequently, BrdU-IR shifted in the apical direction to be localized around Hertwig's epithelial root sheath during days 5-30, never overlapping with concomitantly apically-shifted HSP-25-IR. On days 60-100, BrdU-immunoreactive cells were hardly recognizable in the dental pulp, where HSP-25-IR was exclusively localized in the odontoblast layer. Furthermore, the odontoblast- and ameloblast-lineage cells exhibited two steps in the expression of HSP-25 throughout the postnatal stages: first, dental epithelial and pulpal mesenchymal cells showed a weak IR for HSP-25 after the cessation of their proliferative activity, and subsequently odontoblasts and ameloblasts consistently expressed an intense HSP-25-IR. CONCLUSION: Odontoblast- and ameloblast-lineage cells acquire HSP-25-IR after they complete their cell division, suggesting that this protein acts as a switch between cell proliferation and differentiation during tooth development. The consistent expression of HSP-25-IR in the formative cells may be involved in the maintenance of their functional integrity.

DOI： 10.1016/j.archoralbio.2005.09.007

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Under pathological conditions, dental pulp elaborates both bone and dentin matrix in which the contribution of periodontal tissue cannot be excluded. This study has aimed to clarify the capability of dental pulp to deposit bone matrix in an auto-graft experiment by using (1) immunohistochemistry for 5-bromo-2'-deoxyuridine (BrdU) and nestin and (2) histochemistry for tartrate-resistant acid phosphatase (TRAP). Following the extraction of the molars of 3-week-old mice, the roots and pulp floor were resected and immediately transplanted into the sublingual region. On Days 5-7, tubular dentin formation commenced next to the pre-existing dentin at the pulp horn in which nestin-positive odontoblast-like cells were arranged. Up until Day 14, bone-like tissue formation occurred in the pulp chamber in which intense TRAP-positive cells appeared. These results suggest that odontoblast- and osteoblast-lineage cells reside in the dental pulp. Overall, specific dental pulp regeneration should provide fundamental knowledge for the realization of human tooth regeneration in the near future.

DOI： 10.1007/s00441-006-0242-0

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

Odontoblast- and ameloblast-lineage cells acquire heat-shock protein (HSP)-25 immunoreactivity after they complete cell division during postnatal odontogenesis in rat molars. However, there are no data available concerning the relationship between the termination of cell proliferation and HSP-25 immunoreactivity during tooth morphogenesis. We compared the expression of HSP-25 in tooth germs with their proliferative activity in the rat prenatal to perinatal molar and postnatal incisor to clarify the functional significance of HSP-25 during tooth morphogenesis by immunohistochemistry using anti-HSP-25 and anti-Ki67/5-bromo-2'-deoxyuridine (BrdU). Numerous proliferating cells in developing molars were distributed throughout the tooth germ and HSP-25 immunoreactivity was recognizable in the dental epithelial and mesenchymal cells after they completed cell division. However, both cell proliferation and immunoreaction for HSP-25 are absent in the enamel knots. The distribution pattern of the proliferating cells in the incisors was basically identical to that in the prenatal molars except for the lack of non-proliferating secondary enamel knots and the sparse distribution of proliferating cells in the apical bud. Thus, HSP-25 protein is suggested to act as a switch between cell proliferation and terminal cyto-differentiation during odontogenesis.

DOI： 10.1111/j.1600-0722.2006.00362.x

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Dental pulp is assumed to possess the capacity to elaborate both bone and dentin matrix under the pathological conditions following tooth injury. This study was undertaken to clarify the mechanism inducing bone formation in the dental pulp by investigating the pulpal healing process, after tooth replantation, by micro-computed tomography (mu-CT), immunocytochemistry for heat-shock protein (HSP)-25 and cathepsin K (CK), and histochemistry for both alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP). Under deep anesthesia, the upper right first molar of 4-week-old Wistar rats was extracted and immediately repositioned in the original socket. In control teeth at this age, the periphery of the coronal dental pulp showed intense ALP-positive and HSP-25-positive reactions, whereas there were no TRAP-positive or CK-positive cells. Tooth replantation weakened or terminated ALP-positive and HSP-25-positive reactions in the pulp tissue at the initial stages. At 3-7 days after operation, the ALP-positive region recovered from the root apex to the coronal pulp followed by HSP-25-positive reactions in successful cases showing tertiary dentin formation. In other cases, TRAP-positive and CK-positive cells appeared in the pulp tissue of the replanted tooth at postoperative days 5-10 and remained associated with the bone tissue after 12-60 days. Immunoelectron microscopy clearly demonstrated that CK-positive osteoclast-lineage cells made contact with mesenchymal cells with prominent nucleoli and well-developed cell organelles. These data suggest that the appearance of TRAP-positive and CK-positive cells is involved in the induction of bone tissue formation in dental pulp.

DOI： 10.1007/s00441-005-0138-4

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

Rodent incisors are known to be continuously growing teeth that are maintained by both the cell-proliferation at the apical end and the attrition of the incisal edge. This type of tooth had a special epithelial structure for the maintenance of stem cells, showing the bulbous epithelial protrusion at the apical end. The morphological transition of the epithelial-mesenchymal compartment by serial transverse sections of the apical end toward the incisal direction is likely to reflect the development of the tooth germ in the prenatal stage. Based on the present histological and previous molecular biological studies, the special structure at the apical end is obviously different from the cervical loop giving rise to Hertwig's epithelial root sheath (HERS), in human, mouse and rat molar tooth germs. Hence, we propose a new concept that the eternal tooth bud producing various dental progeny is formed at the apical end of continuously growing teeth, and a new term "apical bud" for indicating this specialized epithelial structure. Furthermore, BrdU labelling analysis suggested that the guinea-pig molars, which were continuously growing teeth, also possessed plural specific proliferative regions and "apical bud" at the apical end.

DOI： 10.1016/j.archoralbio.2004.09.008

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Initial responses of odontoblasts and immunocompetent cells to cavity preparation by laser ablation were investigated in rat molars. In untreated control teeth, intense heat shock protein (Hsp) 25 immunoreactivity was found in the cell bodies of odontoblasts, whereas cells immunopositive for the class II major histocompatibility complex (MHC) antigen were predominantly located beneath the odontoblast layer in the dental pulp. Cavity preparation caused the destruction of the odontoblast layer and the shift of most class-II-MHC-positive cells from the pulp-dentin border toward the pulp core at the affected site. Twelve hours after cavity preparation, numerous class-II-MHC-positive cells appeared along the pulp-dentin border and extended their processes deep into the exposed dentinal tubules, but subsequently disappeared from the pulp-dentin border together with Hsp-25-immunopositive cells by 24 h after the operation. By 3-5 days postoperation, distinct abscess formation consisting of polymorphonuclear leukocytes was found in the dental pulp. The penetration of masses of oral bacteria was recognizable in the dentinal tubules beneath the prepared cavity. These findings indicate that cavity preparation by laser ablation induces remarkable inflammation by continuous bacterial infections via dentinal tubules in this experimental model, thereby delaying pulpal regeneration.

DOI： 10.1007/s00441-003-0840-z

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Adult stem cells have the capacity to self-renew and differentiate along multiple lineages in addition to contributing to ongoing tissue maintenance and regeneration after injury. They reside in specific locations called stem cell niches. In biology of the tooth, the discovery of dental epithelial stem cells in continuously growing teeth has been a recent breakthrough. The niche for the adult stem cells of these teeth is formed at the region of the apical end in tooth development. The region possesses a commonly specialized histological structure for the maintenance of adult stem cells and the production of various progenitor cells producing dental tissues. The molecular signals regulating the maintenance and cell fate decision of adult stem cells, such as Notch1, Lunatic fringe, fibroblast growth factor (FGF)-10, are expressed in the epithelial structure and the surrounding mesenchyme. Based on histological and molecular biological studies, we propose a new concept that the eternal tooth buds producing various dental progeny are formed at the apical end in the development of continuously growing teeth, and coin a new term of "apical bud" for indicating this specialized epithelial structure. Furthermore, the relationship between signaling centers and the expression of FGF-10 mRNA as the determinant of morphogenesis is discussed with an emphasis on tooth and limb development, taking note that the expression pattern of FGF-10 is an important key for understanding the mechanisms for the diversity of cusp patterns and between continuous and limited growth.

DOI： 10.1679/aohc.67.1

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DOI： 10.1016/j.job.2024.100604

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DOI： 10.3390/app142311358

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Synthetic oligodeoxynucleotides (ODNs) containing unmethylated cytosine-phosphate-guanine (CpG) motifs (CpG-ODNs) are ligand molecules for Toll-like receptor 9 (TLR9), which is expressed by odontoblasts in vitro and dental pulp cells. This study determined the effects of CpG-ODNs on pulpal immunomodulatory response and repair following injury. Briefly, the upper right first molars of three-week-old mice were extracted, immersed in Type A (D35) or B (K3) CpG-ODN solutions (0.1 or 0.8 mM) for 30 min, and then replanted. Pulpal healing and immunomodulatory activity were assessed by hematoxylin-eosin and AZAN staining, as well as immunohistochemistry. One week following the operation, inflammatory reactions occurred in all of the experimental groups; however, re-revascularization and newly formed hard tissue deposition were observed in the pulp chamber of all groups at week 2. A positive trend in the expression of immune cell markers was observed toward the CpG-ODN groups at 0.1 mM. Our data suggest that synthetic CpG-ODN solutions at low concentrations may evoke a long-lasting macrophage-TLR9-mediated pro-inflammatory, rather than anti-inflammatory, response in the dental pulp to modulate the repair process and hard tissue formation. Further studies are needed to determine the effects of current immunomodulatory agents in vitro and in vivo and develop treatment strategies for dental tissue regeneration.

DOI： 10.3390/biom14080931

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DOI： 10.1038/s12276-024-01264-5

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OBJECTIVES: Immunoglobulin (Ig)A nephropathy has been associated with oral infections such as periodontitis, but its pathogenesis is not fully understood; no treatments exist. This study analyzes the influence of IgA nephropathy, an autoimmune disease, on the pathogenesis of pulpitis and apical periodontitis. METHODS: Two groups of mice were used in pulp infection experiments: high serum IgA nephropathy model mice (HIGA) and control mice (BALB/c). Histologic analyses of the pulp and apical periodontal tissues were performed on days 3, 5, 7, 14, and 28 following oral bacterial infection. The dynamics of odontoblasts, apoptotic cells, and IgA expression were analyzed using anti-Nestin, TUNEL, and anti-IgA staining, respectively. RESULTS: Inflammatory cells infiltrated the exposed pulp at day three in both groups and by 14 days, these cells had infiltrated from the pulp to the apical periodontal tissue. The area of necrotic pulp tissue increased significantly in the control group at seven days. Odontoblasts decreased from day three onwards and disappeared by 28 days in both groups. The number of apoptotic cells in the pulp and apical periodontal tissues was significantly higher in the experimental group at day 28. The experimental group exhibited a significant increase in IgA production in the pulp after 14 days. Bone resorption in the apical periodontal tissue was significantly decreased in the experimental group at day 28. CONCLUSIONS: The results of this study suggest that IgA nephropathy may modulate the inflammatory response and sustain long-term biological defense responses in pulpitis and apical periodontitis in HIGA mice.

DOI： 10.1016/j.job.2023.11.003

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INTRODUCTION: Rheumatoid arthritis (RA) is an autoimmune disease that involves joint inflammation. Although periodontal disease reportedly contributes to RA onset, the associations of RA with pulpitis and apical periodontitis have not been described. The purpose of this study was to examine the effects of immune response disruption of RA for pulpitis and apical periodontitis with SKG mice. METHODS: SKG and BALB/c (control) mice were used to establish models of pulp infection. Histologic studies of pulp and apical periodontal tissue were performed at 3, 5, 7, 14, and 28 days; odontoblast dynamics were analyzed by antinestin staining, and apoptotic cells were examined by TdT-mediated digoxygenin (biotin)-dUTP nick end labeling staining. RESULTS: Inflammatory cell infiltration into the exposed pulp was observed at 3 days in the SKG and control group groups; the infiltration extended to the apical pulp area at 14 days after surgery. Inflammatory cell infiltration and bone resorption in the apical pulp area were observed from 14-28 days in the SKG and control groups; there were significant increases in inflammatory cell infiltration and bone resorption in the control group at 28 days. The numbers of apoptotic cells in pulp and apical periodontal tissue were higher in the SKG group than in the control group at 14 and 28 days. The number of odontoblasts decreased in the SKG and control groups until 14 days and then disappeared in the SKG and control groups at 28 days. CONCLUSIONS: This study suggested that immune response disruption in RA is involved in prolonging the inflammatory state of pulpitis and apical periodontitis.

DOI： 10.1016/j.joen.2023.08.003

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

The enamel knot (EK), located at the center of cap stage tooth germs, is a transitory cluster of nondividing epithelial cells. The EK acts as a signaling center that provides positional information for tooth morphogenesis and regulates the growth of tooth cusps. To identify species-specific cuspal patterns, this study analyzed the cellular mechanisms in the EK that were related to bone morphogenetic protein (Bmp), which plays a crucial role in cell proliferation and apoptosis. To understand the cellular mechanisms in the EK, the differences between 2 species showing different cuspal patterning, mouse (pointy bunodont cusp) and gerbil (flat lophodont cusp), were analyzed with quantitative reverse transcriptase polymerase chain reaction and immunofluorescent staining. Based on these, we performed protein-soaked bead implantation on tooth germs of the 2 different EK regions and compared the cellular behavior in the EKs of the 2 species. Many genes related with cell cycle, cell apoptosis, and cell proliferation were involved in BMP signaling in the EK during tooth development. A comparison of the cell proliferation and apoptosis associated with Bmp revealed distinctive patterns of the cellular mechanisms. Our findings indicate that the cellular mechanisms, such as cell proliferation and apoptosis, in the EK are related to Bmp4 and play an important role in tooth morphogenesis.

DOI： 10.1177/00220345231167769

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

Transient receptor potential melastatin 7 (TRPM7) is a unique ion channel connected to a kinase domain. We previously demonstrated that Trpm7 expression is high in mouse ameloblasts and odontoblasts, and that amelogenesis is impaired in TRPM7 kinase-dead mice. Here, we analyzed TRPM7 function during amelogenesis in Keratin 14-Cre;Trpm7fl/fl conditional knockout (cKO) mice and Trpm7 knockdown cell lines. cKO mice showed lesser tooth pigmentation than control mice and broken incisor tips. Enamel calcification and microhardness were lower in cKO mice. Electron probe microanalysis (EPMA) showed that the calcium and phosphorus contents in the enamel were lower in cKO mouse than in control mice. The ameloblast layer in cKO mice showed ameloblast dysplasia at the maturation stage. The morphological defects were observed in rat SF2 cells with Trpm7 knockdown. Compared with mock transfectants, the Trpm7 knockdown cell lines showed lower levels of calcification with Alizarin Red-positive staining and an impaired intercellular adhesion structures. These findings suggest that TRPM7 is a critical ion channel in enamel calcification for the effective morphogenesis of ameloblasts during amelogenesis.

DOI： 10.1111/eos.12920

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INTRODUCTION: Prostaglandin E2 (PGE2) exerts biological actions through its transport pathway involving intracellular synthesis, extracellular transport, and receptor binding. This study aimed to determine the localization of the components of the PGE2-transporting pathway in human dental pulp and explore the relevance of PGE2 receptors (EP2/EP4) to angiogenesis and dentinogenesis. METHODS: Protein localization of microsomal PGE2 synthase (mPGES), PGE2 transporters [(multidrug resistance-associated protein-4 (MRP4) and prostaglandin transporter (PGT)], and EP2/EP4 was analyzed using double immunofluorescence staining. Tooth slices from human third molars were cultured with or without butaprost (EP2 agonist) or rivenprost (EP4 agonist) for 1 week. Morphometric analysis of endothelial cell filopodia was performed to evaluate angiogenesis, and real-time polymerase chain reaction was performed to evaluate angiogenesis and odontoblast differentiation markers. RESULTS: MRP4 and PGT were colocalized with mPGES and EP2/EP4 in odontoblasts and endothelial cells. Furthermore, MRP4 was colocalized with mPGES and EP4 in human leukocyte antigen-DR-expressing dendritic cells. In the tooth slice culture, EP2/EP4 agonists induced significant increases in the number and length of filopodia and mRNA expression of angiogenesis markers (vascular endothelial growth factor and fibroblast growth factor-2) and odontoblast differentiation markers (dentin sialophosphoprotein and collagen type 1). CONCLUSIONS: PGE2-producing enzyme (mPGES), transporters (MRP4 and PGT), and PGE2-specific receptors (EP2/EP4) were immunolocalized in various cellular components of the human dental pulp. EP2/EP4 agonists promoted endothelial cell filopodia generation and upregulated angiogenesis- and odontoblast differentiation-related genes, suggesting that PGE2 binding to EP2/EP4 is associated with angiogenic and dentinogenic responses.

DOI： 10.1016/j.joen.2023.01.009

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Ascorbic acid (AA; vitamin C) plays a crucial role in the biosynthesis and secretion of collagen to produce the organic matrix of hard tissues. Nevertheless, the detailed mechanism by which AA induces reparative dentinogenesis is still unknown. This study aimed to investigate the pathway and function of AA during wound healing in a rat pulpotomy model. Sodium-dependent vitamin C transporter (SVCT) 2 and glucose transporter (GLUT) 1 were detected in odontoblasts, endothelial cells, and nerve fibers in normal pulp tissues. SVCT2 and GLUT1 were also expressed in odontoblast-like cells in pulpotomized tissues of Wistar rats, and immunopositive cells of SVCT2 were significantly increased at 5 days after pulpotomy (p < 0.05). By contrast, osteogenic disorder Shionogi (ODS) rats, which cannot generate AA, also expressed SVCT2 and GLUT1 in normal and wound healing conditions. However, in ODS rats, when compared with the AA-addition group, the formation of dentin bridges in the AA-loss group was not evident, a layer of osteopontin was significantly increased beneath the wound surface (p < 0.05), and alpha smooth muscle actin at the odontoblast-like cells observed along this layer was significantly increased (p < 0.05), but not Nestin. Moreover, the amounts of type 1 collagen generated in the reparative dentin and beneath the wound healing site were significantly diminished (p < 0.05). Macrophages expressing CD68 and CD206 increased beneath the wound site. Hence, AA may be involved in odontoblast-like cell differentiation and anti-inflammatory response during dental pulp wound healing. Our results provide new insights into the function of AA through SVCT2 and GLUT1 in reparative dentinogenesis and may help in developing new therapeutic targets for dental pulpal disease.

DOI： 10.1038/s41598-023-28197-9

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Taste receptor cells are taste bud epithelial cells that are dependent upon the innervating nerve for continuous renewal and are maintained by resident tissue stem/progenitor cells. Transection of the innervating nerve causes degeneration of taste buds and taste receptor cells. However, a subset of the taste receptor cells is maintained without nerve contact after glossopharyngeal nerve transection in the circumvallate papilla in adult mice. Here, we revealed that injury caused by glossopharyngeal nerve transection triggers the remaining differentiated K8-positive taste receptor cells to dedifferentiate and acquire transient progenitor cell-like states during regeneration. Dedifferentiated taste receptor cells proliferate, express progenitor cell markers (K14, Sox2, PCNA) and form organoids in vitro. These data indicate that differentiated taste receptor cells can enter the cell cycle, acquire stemness, and participate in taste bud regeneration. We propose that dedifferentiated taste receptor cells in combination with stem/progenitor cells enhance the regeneration of taste buds following nerve injury.

DOI： 10.1038/s12276-022-00924-8

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Maturation stage ameloblasts (M-ABs) are responsible for terminal enamel mineralization in teeth and undergo characteristic cyclic changes in both morphology and function between ruffle-ended ameloblasts (RA) and smooth-ended ameloblasts (SA). Energy metabolism has recently emerged as a potential regulator of cell differentiation and fate decisions; however, its implication in M-ABs remains unclear. To elucidate the relationship between M-ABs and energy metabolism, we examined the expression pattern of energy metabolic enzymes in M-ABs of mouse incisors. Further, using the HAT7 cell line with M-AB characteristics, we designed experiments to induce an energy metabolic shift by changes in oxygen concentration. We revealed that RA preferentially utilizes oxidative phosphorylation, whereas SA depends on glycolysis-dominant energy metabolism in mouse incisors. In HAT7 cells, hypoxia induced an energy metabolic shift toward a more glycolytic-dominant state, and the energy metabolic shift reduced alkaline phosphatase (ALP) activity and calcium transport and deposition with a change in calcium-related gene expression, implying a phenotype shift from RA to SA. Taken together, these results indicate that the energy metabolic state is an important determinant of the RA/SA phenotype in M-ABs. This study sheds light on the biological significance of energy metabolism in governing M-ABs, providing a novel molecular basis for understanding enamel mineralization and elucidating the pathogenesis of enamel hypomineralization.

DOI： 10.3389/fphys.2022.1062042

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Mesenchymal stromal cells (MSCs) differentiation into different lineages is precisely controlled by signaling pathways. Given that protein kinases play a crucial role in signal transduction, here we show that Microtubule Associated Serine/Threonine Kinase Family Member 4 (Mast4) serves as an important mediator of TGF-β and Wnt signal transduction in regulating chondro-osteogenic differentiation of MSCs. Suppression of Mast4 by TGF-β1 led to increased Sox9 stability by blocking Mast4-induced Sox9 serine 494 phosphorylation and subsequent proteasomal degradation, ultimately enhancing chondrogenesis of MSCs. On the other hand, Mast4 protein, which stability was enhanced by Wnt-mediated inhibition of GSK-3β and subsequent Smurf1 recruitment, promoted β-catenin nuclear localization and Runx2 activity, increasing osteogenesis of MSCs. Consistently, Mast4-/- mice demonstrated excessive cartilage synthesis, while exhibiting osteoporotic phenotype. Interestingly, Mast4 depletion in MSCs facilitated cartilage formation and regeneration in vivo. Altogether, our findings uncover essential roles of Mast4 in determining the fate of MSC development into cartilage or bone.

DOI： 10.1038/s41467-022-31697-3

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OBJECTIVES: Lysophosphatidic acid (LPA) is a potent bioactive phospholipid that exerts various functions upon binding to six known G protein-coupled receptors (LPA1-6); however; its role in a tooth remains unclear. This study aimed to explore the impact of the LPA/LPA receptor 6 (LPA6)/RhoA signaling axis on maturation stage ameloblasts (M-ABs), which are responsible for enamel mineralization. METHODS: The expression of LPA6 and LPA-producing synthetic enzymes during ameloblast differentiation was explored through immunobiological analysis of mouse incisors and molars. To elucidate the role of LPA6 in ameloblasts, incisors of LPA6 KO mice were analyzed. In vitro experiments using ameloblast cell lines were performed to validate the function of LPA-LPA6-RhoA signaling in ameloblasts. RESULTS: LPA6 and LPA-producing enzymes were strongly expressed in M-ABs. In LPA6 knockout mice, M-ABs exhibited abnormal morphology with the loss of cell polarity, and an abnormal enamel epithelium containing cyst-like structures was formed. Moreover, the expression of E-cadherin and zonula occludens-1 (ZO-1) significantly decreased in M-ABs. In vitro experiments demonstrated that LPA upregulated the expression of E-cadherin, ZO-1, and filamentous actin (F-actin) at the cellular membrane, whereas LPA6 knockdown decreased their expression and changed cell morphology. Furthermore, we showed that RhoA signaling mediates LPA-LPA6-induced junctional complexes. CONCLUSIONS: This study demonstrated that LPA-LPA6-RhoA signaling is essential for establishing proper cell morphology and polarity, via cell-cell junction and actin cytoskeleton expression and stability, of M-ABs. These results highlight the biological significance of bioactive lipids in a tooth, providing a novel molecular regulatory mechanism of ameloblasts.

DOI： 10.1016/j.job.2022.01.004

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Language：Japanese   Publisher：Japan Society for Laser Surgery and Medicine  

DOI： 10.2530/jslsm.jslsm-43_0015

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Other Link： https://search.jamas.or.jp/default/link?pub_year=2022&ichushi_jid=J01213&link_issn=&doc_id=20220727300007&doc_link_id=%2Fcd0rezae%2F2022%2F004302%2F008%2F0113-0119%26dl%3D0&url=https%3A%2F%2Fwww.medicalonline.jp%2Fjamas.php%3FGoodsID%3D%2Fcd0rezae%2F2022%2F004302%2F008%2F0113-0119%26dl%3D0&type=MedicalOnline&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00004_2.gif

DOI： 10.1101/2021.12.15.472878

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Tooth transplantation is one of the treatment options for extracted teeth that can be considered before dental implantation. Although the success rate of tooth transplantation is lower than that of implantation, tooth replantation and transplantation have the great advantage of using natural teeth. Tooth replantation might be considered a promising option in some cases. In present study, the expression patterns of revascularization and pulpal healing, which are the most important for the pulp viability, were analyzed after tooth replantation and allograft in mice. The inflammatory response and root dentin resorption were observed and not different between replantation and allograft in initiation of healing process. However, bonelike tissue formation, pulp revascularization and pulp healing were faster in replantation. The difference of healing patterns between tooth replantation and allograft found in present study will be helpful to select the treatment option and to understand healing mechanism.

DOI： 10.1016/j.bbrep.2021.100945

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

This study aimed to elucidate the role of the Sonic hedgehog (Shh)–Patched (Ptch)–Gli signaling pathway in maintaining dental epithelial and pulp stem/progenitor cells and regulating the function of odontoblasts. Doxycycline (dox)-inducible histone 2B (H2B)–green fluorescent protein (GFP) transgenic mice ingested dox at prenatal embryonic days 14.5 or 15.5 and their offspring were collected from postnatal day 1 (P1) to week 3 (P3W). Immunohistochemistry for Gli1, Ptch1, and Ptch2 and<italic>in situ</italic>hybridization for<italic>Shh</italic>and<italic>Ptch1</italic>were conducted. Mandibular incisors of postnatal day 2 H2B-GFP transgenic and wild-type mice were cultivated in a nutrient medium with Shh antibody for 4 days and subsequently processed for immunohistochemistry for Sox2. In molars, dense H2B-GFP-label-retaining cells (H2B-GFP-LRCs) were densely distributed throughout the dental pulp during P1 to postnatal week 2 (P2W) and decreased in number by postnatal P3W, whereas the number of dense H2B-GFP-LRCs in the subodontoblastic layer increased in number at P2W. Gli1⁺and Pthc1⁺cells were distributed throughout the enamel organ and dental pulp, including the odontoblast and subodontoblastic layers.<italic>Shh</italic>mRNA was expressed in the inner enamel epithelium and shifted into odontoblasts after dentin deposition.<italic>Ptch1</italic>mRNA was expressed in the inner enamel epithelium and cuspal pulpal tissue on P1 and decreased in intensity from postnatal week 1 to P3W. In incisors, the apical bud contained H2B-GFP-LRCs, Gli1⁺cells, and Ptch1⁺cells. The addition of Shh antibody to explants induced a decrease in the number of Sox2⁺cells due to the increase in apoptotic cells in the apical bud. Thus, the Shh–Ptch–Gli signaling pathway plays a role in maintaining quiescent adult stem cells and regulating the function of odontoblasts.

DOI： 10.3389/fdmed.2021.651334

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Stem cells are maintained in specific niches that strictly regulate their proliferation and differentiation for proper tissue regeneration and renewal. Molecular oxygen (O2) is an important component of the niche microenvironment, but little is known about how O2 governs epithelial stem cell (ESC) behavior. Here, we demonstrate that O2 plays a crucial role in regulating the proliferation of ESCs using the continuously growing mouse incisors. We have revealed that slow-cycling cells in the niche are maintained under relatively hypoxic conditions compared with actively proliferating cells, based on the blood vessel distribution and metabolic status. Mechanistically, we have demonstrated that, during hypoxia, HIF1α upregulation activates the RhoA signal, thereby promoting cortical actomyosin and stabilizing the adherens junction complex, including merlin. This leads to the cytoplasmic retention of YAP/TAZ to attenuate cell proliferation. These results shed light on the biological significance of blood-vessel geometry and the signaling mechanism through microenvironmental O2 to orchestrate ESC behavior, providing a novel molecular basis for the microenvironmental O2-mediated stem cell regulation during tissue development and renewal.

DOI： 10.1242/dev.194787

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BACKGROUND: Sulfated vizantin, a recently developed immunostimulant, has also been found to exert antibiofilm properties. It acts not as a bactericide, but as a detachment-promoting agent by reducing the biofilm structural stability. This study aimed to investigate the mechanism underlying this activity and its species specificity using two distinct ex vivo oral biofilm models derived from human saliva. RESULTS: The biofilm, composed mainly of the genus Streptococcus and containing 50 μM of sulfated vizantin, detached significantly from its basal surface with rotation at 500 rpm for only 15 s, even when 0.2% sucrose was supplied. Expression analyses for genes associated with biofilm formation and bacterial adhesion following identification of the Streptococcus species, revealed that a variety of Streptococcus species in a cariogenic biofilm showed downregulation of genes encoding glucosyltransferases involved in the biosynthesis of water-soluble glucan. The expression of some genes encoding surface proteins was also downregulated. Of the two quorum sensing systems involved in the genus Streptococcus, the expression of luxS in three species, Streptococcus oralis, Streptococcus gordonii, and Streptococcus mutans, was significantly downregulated in the presence of 50 μM sulfated vizantin. Biofilm detachment may be facilitated by the reduced structural stability due to these modulations. As a non-specific reaction, 50 μM sulfated vizantin decreased cell surface hydrophobicity by binding to the cell surface, resulting in reduced bacterial adherence. CONCLUSION: Sulfated vizantin may be a candidate for a new antibiofilm strategy targeting the biofilm matrix while preserving the resident microflora.

DOI： 10.1186/s12866-020-02033-w

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Nonsyndromic clefts of the lip and palate are common birth defects resulting from gene-gene and gene-environment interactions. Mutations in human MSX1 have been linked to orofacial clefting and we show here that Msx1 deficiency causes a growth defect of the medial nasal process (Mnp) in mouse embryos. Although this defect alone does not disrupt lip formation, Msx1-deficient embryos develop a cleft lip when the mother is transiently exposed to reduced oxygen levels or to phenytoin, a drug known to cause embryonic hypoxia. In the absence of interacting environmental factors, the Mnp growth defect caused by Msx1 deficiency is modified by a Pax9-dependent 'morphogenetic regulation', which modulates Mnp shape, rescues lip formation and involves a localized abrogation of Bmp4-mediated repression of Pax9 Analyses of GWAS data revealed a genome-wide significant association of a Gene Ontology morphogenesis term (including assigned roles for MSX1, MSX2, PAX9, BMP4 and GREM1) specifically for nonsyndromic cleft lip with cleft palate. Our data indicate that MSX1 mutations could increase the risk for cleft lip formation by interacting with an impaired morphogenetic regulation that adjusts Mnp shape, or through interactions that inhibit Mnp growth.

DOI： 10.1242/dev.189175

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BACKGROUND: The aim of this in vitro study was to examine the possible enhancement of the biofilm peeling effect of a sonic toothbrush following the use of an antimicrobial mouth rinse. METHODS: The biofilm at a noncontact site in the interdental area was treated by sound wave convection with the test solution or by immersion in the solution. The biofilm peeling effect was evaluated by determining the bacterial counts and performing morphological observations. A Streptococcus mutans biofilm was allowed to develop on composite resin discs by cultivation with stirring at 50 rpm for 72 h. The specimens were then placed in recesses located between plastic teeth and divided into an immersion group and a combination group. The immersion group was treated with phosphate buffer, chlorhexidine digluconate Peridex™ (CHX) mouth rinse or Listerine® Fresh Mint (EO) mouth rinse. The combination group was treated with CHX or EO and a sonic toothbrush. RESULTS: The biofilm thickness was reduced by approximately one-half compared with the control group. The combination treatment produced a 1 log reduction in the number of bacteria compared to the EO immersion treatment. No significant difference was observed in the biofilm peeling effect of the immersion group compared to the control group. CONCLUSIONS: The combined use of a sonic toothbrush and a mouth rinse enhanced the peeling of the biofilm that proliferates in places that are difficult to reach using mechanical stress.

DOI： 10.1186/s12903-020-01144-0

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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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

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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Porphyromonas gingivalis infection can lead to periodontitis and dysbiosis, which are known risk factors for rheumatoid arthritis (RA). We investigated whether P. gingivalis administration affected bone regeneration in mice with or without arthritis. We administered P. gingivalis to male DBA/1 J mice that were or were not sensitised to type II collagen-induced arthritis (CIA). All mice underwent drilling of bilateral femurs. We histologically evaluated new bone regeneration (bone volume of the defect [BVd]/tissue volume of the defect [TVd]) using micro-computed tomography (micro-CT), osteoclast number/bone area, and active osteoblast surface/bone surface (Ob.S/BS). We measured serum cytokine levels and bone mineral density of the proximal tibia using micro-CT. CIA resulted in significantly reduced bone regeneration (BVd/TVd) at all time-points, whereas P. gingivalis administration showed similar effects at 2 weeks postoperatively. CIA resulted in higher osteoclast number/bone area and lower Ob.S/BS at 2 and 3 weeks postoperatively, respectively. However, P. gingivalis administration resulted in lower Ob.S/BS only at 2 weeks postoperatively. During later-stage bone regeneration, CIA and P. gingivalis administration synergistically decreased BVd/TVd, increased serum tumour necrosis factor-α, and resulted in the lowest bone mineral density. Therefore, RA and dysbiosis could be risk factors for prolonged fracture healing.

DOI： 10.1038/s41598-019-56265-6

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INTRODUCTION: This study aimed to develop a technique to create sequential slices, allowing the fluorescent visualization of bacterial viability in all parts of an infected dentin. METHODS: Cylindrical dentin blocks were prepared from freshly extracted human teeth with a single-rooted canal. Each block was immersed in 5% sodium hypochlorite (NaOCl) and 17% EDTA for 5 minutes before being infected with Enterococcus faecalis. The bacteria were allowed to develop inside dentin specimens for 28 days under anaerobic conditions. The specimens were exposed in 2% NaOCl for either 2 minutes or 20 minutes at 20°C, 37°C, and 45°C, respectively. After staining with calcein AM (Thermo Fisher Scientific, Waltham, MA) and propidium iodide, the samples were cryoembedded, mounted on an adhesive film, and sectioned at a thickness of 10 μm along the running of the dentinal tubules. Stacks of fluorescent images were collected in the z dimension using confocal laser scanning microscopy, and the maximum affected distance from a root canal was measured from the 3-dimensional reconstructed image. The reliability of this technique was verified by comparison with a dye bleaching test. RESULTS: Horizontal sequential sections preserving 3-dimensional bacterial distribution and their viabilities could be made without decalcification. The treatment time contributed to the penetration of NaOCl into dentinal tubules, whereas temperature did not significantly affect the penetration. The judgment by confocal laser scanning microscopic analysis was consistent with that of a dye bleaching test. CONCLUSIONS: The horizontal sectioning method has the advantage of creating sequential sections, allowing information to be imaged at every portion.

DOI： 10.1016/j.joen.2019.06.004

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

OBJECTIVES: This study is aimed at evaluating the effect of a new glass ionomer cement (GIC) containing fluoro-zinc-silicate fillers on biofilm formation and ion incorporation. MATERIALS AND METHODS: Streptococcus mutans biofilms were developed on two GIC materials: Caredyne Restore (CD) and Fuji VII (FJ); and hydroxyapatite (HA) for 24 h at 37 °C using a flow cell system. The morphological structure and bacterial viability were analyzed using a confocal laser scanning microscopy. Bacterial adhesion during the initial 2 h was also assessed by viable cell counting. To study the ion incorporation, restored cavities prepared on the root surfaces of human incisors were subjected to the elemental mapping of the zinc and fluoride ions in the GIC-dentin interface using a wavelength-dispersive X-ray spectroscopy electron probe microanalyzer. RESULTS: Morphological observations revealed that biofilm formation in the CD group was remarkably inhibited compared with the HA and FJ groups, exhibiting sparse, thinner biofilm clusters. The microorganisms adhering to the CD group were significantly inhibited, revealing 2.9 ± 0.4 for CD, 4.9 ± 0.2 for FJ, and 5.4 ± 0.4 log colony-forming units (CFU) for HA. The CD zinc ion incorporation depth was 72.2 ± 8.0 μm. The fluoride penetration of CD was three times deeper than that of FJ; this difference was statistically significant (p < 0.05). CONCLUSIONS: Enhanced by the incorporation of zinc and fluoride ions, the new GIC inhibited biofilm formation by interfering with bacterial adhesion. CLINICAL RELEVANCE: A novel GIC comprised of fluoro-zinc-silicate fillers may improve clinical outcomes, such as root caries and minimally invasive dentistry.

DOI： 10.1007/s00784-019-02991-0

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Human tissue-specific stem cells (hTSCs), found throughout the body, can differentiate into several lineages under appropriate conditions in vitro and in vivo. By transfecting terminally differentiated cells with reprogramming factors, we previously produced induced TSCs from the pancreas and hepatocytes that exhibit additional properties than iPSCs, as exemplified by very low tumour formation after xenogenic transplantation. We hypothesised that hTSCs, being partially reprogrammed in a state just prior to iPSC transition, could be isolated from any terminally differentiated cell type through transient reprogramming factor overexpression. Cytochemical staining of human deciduous tooth-derived dental pulp cells (HDDPCs) and human skin-derived fibroblasts following transfection with Yamanaka's factors demonstrated increased ALP activity, a stem cell marker, three weeks after transfection albeit in a small percentage of clones. Repeated transfections (≤3) led to more efficient iPSC generation, with HDDPCs exhibiting greater multipotentiality at two weeks post-transfection than the parental intact HDDPCs. These results indicated the utility of iPSC technology to isolate TSCs from HDDPCs and fibroblasts. Generally, a step-wise loss of pluripotential phenotypes in ESCs/iPSCs occurs during their differentiation process. Our present findings suggest that the reverse phenomenon can also occur upon repeated introduction of reprogramming factors into differentiated cells such as HDDPCs and fibroblasts.

DOI： 10.1038/s41598-018-37291-2

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In this study, we attempted to localize the immunoreactivities of podoplanin/E11/gp38 and CD44, a counterpart possessing a high affinity to podoplanin/E11/gp38, as well as endomucin-immunoreactive blood vessels in the regions of odontoblast layers and the underlying sub-odontoblastic layers in murine tooth germs. Endomucin-reactive small blood vessels were scattered throughout the dental papillae of the tooth germs at postnatal day 1 but came to be localized close to the odontoblast/sub-odontoblastic layers until day 3. After postnatal day 5, small blood vessels were seen in odontoblast cell layers, while blood vessels with relatively larger diameters were seen forming in sub-odontoblastic layers. Immunoreactivities of podoplanin/E11/gp38 and CD44 were not detectable in the cells of dental papillae facing the inner enamel epithelium at postnatal day 1. However, at around postnatal days 3-5, podoplanin/E11/gp38 was localized in the odontoblast layer but not in the sub-odontoblastic layer, whereas CD44 was observed in the sub-odontoblastic layer but not in the odontoblast layer. The exclusive immunolocalization of podoplanin/E11/gp38 and CD44 in the odontoblast layers and sub-odontoblastic layers was seen after postnatal day 3 of the tooth germs, when the mesenchymal cells of dental papillae have already differentiated into mature odontoblasts at the cusp tip. Taken together, it seems likely that endomucin-reactive small blood vessels extended to the podoplanin/E11/gp38-positive odontoblast layers, whereas endomucin-reactive large blood vessels were already present in CD44-immmunopositive sub-odontoblastic layer, indicating the cellular regulation on the vascularization of endomucin-reactive endothelial cells during odontogenesis of the tooth germs.

DOI： 10.2220/biomedres.40.133

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Chondroitin sulfate (CS) proteoglycan is a major component of the extracellular matrix and plays an important part in organogenesis. To elucidate the roles of CS for craniofacial development, we analyzed the craniofacial morphology in CS N-acetylgalactosaminyltransferase-1 (T1) gene knockout (KO) mice. T1KO mice showed the impaired intramembranous ossification in the skull, and the final skull shape of adult mice included a shorter face, higher and broader calvaria. Some of T1KO mice exhibited severe facial developmental defect, such as eye defects and cleft lip and palate, causing embryonic lethality. At the postnatal stages, T1KO mice with severely reduced CS amounts showed malocclusion, general skeletal dysplasia and skin hyperextension, closely resembling Ehlers-Danlos syndrome-like connective tissue disorders. The production of collagen type 1 was significantly downregulated in T1KO mice, and the deposition of CS-binding molecules, Wnt3a, was decreased with CS in extracellular matrices. The collagen fibers were irregular and aggregated, and connective tissues were dysorganized in the skin and calvaria of T1KO mice. These results suggest that CS regulates the shape of the craniofacial skeleton by modulating connective tissue organization and that the remarkable reduction of CS induces hypoplasia of intramembranous ossification and cartilage anomaly, resulting in skeletal dysplasia.

DOI： 10.1038/s41598-018-35412-5

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

Background: The Journal of Oral Biosciences is devoted to the advancement and dissemination of fundamental knowledge concerning every aspect of oral biosciences.Highlight: This review article features the following topics that were presented in symposia held during the annual meeting of the Japanese Association for Oral Biology: "Multidimensional Bioimaging on Biofunction," "The Front Line of Research on Saliva and Salivary Gland," "Research on the Front Line in Recovery of Oral Sensory Function under Neuropathic Condition," "Mechanism for Generation and Transport of Intracellular Organelles," "Forefront of Research to Understand the Oral Micro-ecosystems," and "A New Horizon of Imaging for Bone Cells: How Far Can We Observe the Mechanisms Underlying Bone Biology?," in addition to review articles in the field of "Head and Neck Cancer," "Amelogenesis," "Biofilm," and "Dentin Sensitivity."Conclusion: These reviews in the Journal of Oral Biosciences have inspired its readers to broaden their knowledge regarding the various aspects of oral biosciences. The current editorial review introduces these exciting review articles. (C) 2018 Japanese Association for Oral Biology. Published by Elsevier B.V.

DOI： 10.1016/j.job.2017.12.001

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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：PERGAMON-ELSEVIER SCIENCE LTD  

OBJECTIVE: Lymphoid enhancer-binding factor-1 (LEF1) is a 48-kD nuclear protein that is expressed in pre-B and T cells. LEF1 is also an important member of the Wnt/β-catenin signaling pathway that plays important roles in the self-renewal and differentiation of embryonic stem cells. We speculated that LEF1 might function in the stem cells from human exfoliated deciduous teeth (SHED). In this study, we attempted to isolate such LEF1-positive cells from human deciduous dental pulp cells (HDDPCs) by genetic engineering technology, using the human LEF1 promoter. DESIGN: A piggyBac transposon plasmid (pTA-LEN) was introduced into HDDPCs, using the Neon® transfection system. After G418 selection, the emerging colonies were assessed for EGFP-derived fluorescence by fluorescence microscopy. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed using RNA isolated from these colonies to examine stem cell-specific transcript expression. Osteoblastic or neuronal differentiation was induced by cultivating the LEF1-positive cells with differentiation-inducing medium. RESULTS: RT-PCR analysis confirmed the expression of several stem cell markers, including OCT3/4, SOX2, REX1, and NANOG, in LEF1-positive HDDPCs, which could be differentiated into osteoblasts and neuronal cells. CONCLUSIONS: The isolated LEF1-positive HDDPCs exhibited the properties of stem cells, suggesting that LEF1 might serve as a marker for SHED.

DOI： 10.1016/j.archoralbio.2017.04.033

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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 {BV}  

Background: The Journal of Oral Biosciences is devoted to the advancement and dissemination of fundamental knowledge concerning every aspect of oral biosciences.Highlight: This review article features the following topics presented in symposia that were held during the annual meeting of the Japanese Association for Oral Biology: "Frontiers of oral physiology," "Genetic and epigenetic changes to determine development, differentiation, and carcinogenesis," "Analytical methods and interpretation of variation in tooth morphology," "Regulatory mechanisms of vertebrate developmental body plan revealed by live-imaging and mathematical analyses," "Studies on dentin sialophosphoprotein (DSPP) through morphological and functional perspectives," and "Oral biofilm and microbiome research".Conclusion: These published reviews in the Journal of Oral Biosciences have inspired the readers of the Journal to broaden their knowledge regarding the various aspects of Oral Biosciences. The current editorial review introduces these exciting review articles. (C) 2016 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.job.2016.12.001

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Highly coordinated regulation of cell proliferation and differentiation contributes to the formation of functionally shaped and sized teeth; however, the mechanism underlying the switch from cell cycle exit to cell differentiation during odontogenesis is poorly understood. Recently, we identified pannexin 3 (Panx3) as a member of the pannexin gap junction protein family from tooth germs. The expression of Panx3 was predominately localized in preodontoblasts that arise from dental papilla cells and can differentiate into dentin-secreting odontoblasts. Panx3 also co-localized with p21, a cyclin-dependent kinase inhibitor protein, in preodontoblasts. Panx3 was expressed in primary dental mesenchymal cells and in the mDP dental mesenchymal cell line. Both Panx3 and p21 were induced during the differentiation of mDP cells. Overexpression of Panx3 in mDP cells reduced cell proliferation via up-regulation of p21, but not of p27, and promoted the Bone morphogenetic protein 2 (BMP2)-induced phosphorylation of Smad1/5/8 and the expression of dentin sialophosphoprotein (Dspp), a marker of differentiated odontoblasts. Furthermore, Panx3 released intracellular ATP into the extracellular space through its hemichannel and induced the phosphorylation of AMP-activated protein kinase (AMPK). 5-Aminoimidazole-4-carboxamide-ribonucleoside (AICAR), an activator of AMPK, reduced mDP cell proliferation and induced p21 expression. Conversely, knockdown of endogenous Panx3 by siRNA inhibited AMPK phosphorylation, p21 expression, and the phosphorylation of Smad1/5/8 even in the presence of BMP2. Taken together, our results suggest that Panx3 modulates intracellular ATP levels, resulting in the inhibition of odontoblast proliferation through the AMPK/p21 signaling pathway and promotion of cell differentiation by the BMP/Smad signaling pathway.

DOI： 10.1371/journal.pone.0177557

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

The anatomic and functional combinations of cusps and lophs (ridges) define the tooth shape of rodent molars, which distinguishes species. The species-specific cusp patterns result from the spatiotemporal induction of enamel knots (EKs), which require precisely controlled cellular behavior to control the epithelial invagination. Despite the well-defined roles of EK in cusp patterning, the determinants of the ultimate cuspal shapes and involvement of epithelial cellular geometry are unknown. Using two typical tooth patterns, the lophodont in gerbils and the bunodont in mice, we showed that the cuspal shape is determined by the dental epithelium at the cap stage, whereas the cellular geometry in the inner dental epithelium (IDE) is correlated with the cuspal shape. Intriguingly, fine tuning Rac1 and RhoA interconvert cuspal shapes between two species by remolding the cellular geometry. Either inhibition of Rac1 or ectopic expression of RhoA could region-distinctively change the columnar shape of IDE cells in gerbils to drive invagination to produce cusps. Conversely, RhoA reduction in mice inhibited invagination and developed lophs. Furthermore, we found that Rac1 and RhoA modulate the choices of cuspal shape by coordinating adhesion junctions, actin distribution, and fibronectin localization to drive IDE invagination.

DOI： 10.1038/srep37828

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

Msh homeobox 1 (MSX1) encodes a transcription factor implicated in embryonic development of limbs and craniofacial tissues including bone and teeth. Although MSX1 regulates osteoblast differentiation in the cranial bone of young animal, little is known about the contribution of MSX1 to the osteogenic potential of human cells. In the present study, we investigate the role of MSX1 in osteogenic differentiation of human dental pulp stem cells isolated from deciduous teeth. When these cells were exposed to osteogenesis-induction medium, runt-related transcription factor-2 (RUNX2), bone morphogenetic protein-2 (BMP2), alkaline phosphatase (ALPL), and osteocalcin (OCN) mRNA levels, as well as alkaline phosphatase activity, increased on days 4-12, and thereafter the matrix was calcified on day 14. However, knockdown of MSX1 with small interfering RNA abolished the induction of the osteoblast-related gene expression, alkaline phosphatase activity, and calcification. Interestingly, DNA microarray and PCR analyses revealed that MSX1 knockdown induced the sterol regulatory element-binding protein 2 (SREBP2) transcriptional factor and its downstream target genes in the cholesterol synthesis pathway. Inhibition of cholesterol synthesis enhances osteoblast differentiation of various mesenchymal cells. Thus, MSX1 may downregulate the cholesterol synthesis-related genes to ensure osteoblast differentiation of human dental pulp stem cells.

DOI： 10.1155/2016/8035759

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

DOI： 10.1016/j.job.2015.12.001

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Type 1 diabetes occurs due to the autoimmune destruction of pancreatic beta-cells in islets. Transplantation of islets is a promising option for the treatment of patients with type 1 diabetes that experience hypoglycemic unawareness despite maximal care, but the present shortage of donor islets hampers such transplantation. Transplantation of insulin-producing cells derived from the patients themselves would be one of the most promising approaches to cure type 1 diabetes. Previously, we demonstrated that insulin-producing cells could be produced by transfecting murine pancreatic cells with Yamanaka's reprogramming factors. Non-obese diabetic (NOD) mice are naturally occurring mutant mice defective in insulin production due to autoimmune ablation of pancreatic beta-cells. In this study, we showed that glucose-sensitive insulin-producing cells are successfully generated by transfecting primary pancreatic cells from NOD mice (aged 6 months old) with a plasmid harboring the cDNAs for Oct-3/4, Sox2, Klf4, and c-Myc. Transfection was repeated 4 times in a 2 day-interval. Sixty-five days after final transfection, cobblestone-like colonies appeared. They proliferated in vitro and expressed pluripotency-related genes as well as Pdx1, a transcription factor specific to tissue-specific stem cells for the beta-cell lineage. Transplantation of these cells into nude mice failed to produce teratoma unlike induced pluripotent stem cells (iPSCs). Induction of these cells to the pancreatic beta-cell lineage demonstrated their capability to produce insulin in response to glucose. These findings suggest that functional pancreatic beta-cells can be produced from patients with type 1 diabetes. We call these resultant cells as "induced tissue-specific stem cells from the pancreas" (iTS-P) that could be valuable sources of safe and effective materials for cell-based therapy in type 1 diabetes.

DOI： 10.1371/journal.pone.0163580

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

An ideal antibiofilm strategy is to control both in the quality and quantity of biofilm while maintaining the benefits derived from resident microflora. Vizantin, a recently developed immunostimulating compound, has also been found to have antibiofilm property. This study evaluated the influence on biofilm formation of Streptococcus mutans in the presence of sulfated vizantin and biofilm development following bacterial adhesion on a hydroxyapatite disc coated with sulfated vizantin. Supplementation with sulfated vizantin up to 50 μM did not affect either bacterial growth or biofilm formation, whereas 50 μM sulfated vizantin caused the biofilm to readily detach from the surface. Sulfated vizantin at the concentration of 50 μM upregulated the expression of the gtfB and gtfC genes, but downregulated the expression of the gtfD gene, suggesting altered architecture in the biofilm. Biofilm development on the surface coated with sulfated vizantin was inhibited depending on the concentration, suggesting prevention from bacterial adhesion. Among eight genes related to bacterial adherence in S. mutans, expression of gtfB and gtfC was significantly upregulated, whereas the expression of gtfD, GbpA and GbpC was downregulated according to the concentration of vizantin, especially with 50 μM vizantin by 0.8-, 0.4-, and 0.4-fold, respectively. These findings suggest that sulfated vizantin may cause structural degradation as a result of changing gene regulation related to bacterial adhesion and glucan production of S. mutans.

DOI： 10.1016/j.bbrc.2016.10.021

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Language：English   Publisher：国際組織細胞学会  

光学・電子顕微鏡を用いて、蛋白質遺伝子産物(PGP)9.5とクラスII主要組織適合遺伝子複合体分子の免疫染色より、感染歯髄の治癒過程での抗生物質に対する神経系細胞と免疫細胞の応答を検討した。ラットモデルを用いた実験では、歯髄-象牙質境界に沿って多数の樹状細胞が集積し、次いで3Mix(シプロフロキサシン・メトロニダゾール・セファクロール)含有α-リン酸三カルシウム(αTCP)の適用後に象牙芽細胞様細胞の分化とマトリックス沈着が認められた。PGP9.5活性神経線維も、3Mix含有αTCPの下の歯髄中間に集積した樹状細胞の周辺に密集して分布していた。以上より、3Mix含有αTCPを感染した歯髄に適用すると、樹状細胞が一時的に誘導されると考えられ、これらの細胞は病的状態下の象牙芽細胞様細胞の分化に際して重要な役割を果たしていると考えられた。

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：ELSEVIER SCIENCE BV  

© 2015 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved. Background The Journal of Oral Biosciences is devoted to the advancement and dissemination of fundamental knowledge concerning every aspect of oral biosciences. Highlight This review article features the following topics: "Novel challenge for bone formation and bone resorption," "The front line of research on oral microbiota," "Clinical insight into the study of orofacial pain," "Carving a disease by omics," "The front line of bioimaging - a new light shining on oral biosciences," "Biodental engineering - integration of biology and material science," "Translational dental research over the CCN family," "Salivary glands," "Break the negative spiral consisting of periodontitis, diabetes, and Alzheimer disease: extending healthy life expectancy through oral health," "Immunology and oncology," "Oral microbiome and biofilm research: new concepts and new approaches," "Bone remodeling mechanisms of bone resorption and bone formation," and "The front line of oral biofilm research," in addition to review articles by invited authors in the field of microbiology. Conclusion These reviews published in the Journal of Oral Biosciences have inspired the readers of the journal to broaden their knowledge regarding various aspects of oral biosciences. The current editorial review introduces these exciting review articles.

DOI： 10.1016/j.job.2014.12.002

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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&lt;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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Chemical disinfection of oral biofilms often leaves biofilm structures intact. This study aimed to examine whether the residual structure promotes secondary bacterial adhesion. Streptococcus mutans biofilms generated on resin-composite disks in a rotating disc reactor were disinfected completely with 70% isopropyl alcohol, and were again cultured in the same reactor after resupplying with the same bacterial solution. Specimens were subjected to fluorescence confocal laser scanning microscopy, viable cell counts and PCR-Invader assay in order to observe and quantify secondarily adhered cells. Fluorescence microscopic analysis, particularly after longitudinal cryosectioning, demonstrated stratified patterns of viable cells on the disinfected biofilm structure. Viable cell counts of test specimens were significantly higher than those of controls, and increased according to the amount of residual structure and culture period. Linear regression analysis exhibited a high correlation between viable and total cell counts. It was concluded that disinfected biofilm structures favored secondary bacterial adhesion.

DOI： 10.1371/journal.pone.0116647

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DOI： 10.1016/j.job.2014.01.001

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

OBJECTIVE: The aim of this study was to determine whether different antiseptic mouthrinses show different penetration kinetics into Streptococcus mutans biofilms. MATERIALS AND METHODS: The biofilms, grown on glass-based dishes, were exposed to one of four mouthrinses containing chlorhexidine digluconate, essential oil, cetylpyridinium chloride, or isopropylmethylphenol. Then, penetration velocities were determined by monitoring fluorescence loss of calcein AM-stained biofilms with time-lapse confocal laser scanning microscopy. Bactericidal activity was assessed with fluorescent bacterial viable cell (Live/Dead) staining and viable cell counts. Bacterial detachment after the mouthrinse exposure was determined by measuring fluorescence reduction of SYTO9-stained biofilms. RESULTS: The essential oil-containing mouthrinse showed significantly faster penetration velocity than the other mouthrinses (ANCOVA and Bonferroni test, p < 0.05). However, even 5 min of exposure left the biofilm structure almost intact. After 30 s (consumer rinsing time) of exposure, the essential oil-containing mouthrinse showed the highest log reduction of viable cells (2.7 log CFU) measured by Live/Dead staining, and the mean reduction of total viable cells was 1.41 log CFU measured by viable cell count. CONCLUSIONS: The essential oil-containing mouthrinse showed the best penetration. Within 30 s of exposure, however, no mouthrinses injured all the microorganisms and all mouthrinses left the biofilm structure nearly intact. CLINICAL RELEVANCE: The mouthrinses tested showed different levels of biofilm penetration. The essential oil rinse was superior to other rinses by all three of the in vitro measurements performed.

DOI： 10.1007/s00784-013-1002-7

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Language：Japanese   Publisher：JAPANESE SOCIETY OF PERIODONTOLOGY  

The aims of this study were to evaluate the penetration ability and antimicrobial efficacy of an alcohol-free mouth rinse (Listerine^®Natural Care; N), newly developed to reduce the irritative property and improve the mouthfeel of Listerine^®, using artificially developed <i>Streptococcus mutans</i> biofilms. As test mouth rinses, Listerine^®Zero (Z), Listerine^®Fresh Mint (F) and an alcohol-free chlorhexidine gluconate-containing mouth rinse (CHG) were investigated. <i>S.mutans</i> biofilms were grown on glass-based dishes for 24 h under anaerobic conditions. The ability of the mouth rinses to penetrate these biofilms were analyzed by time-lapse monitoring of the fluorescence loss of the calcein-AM-stained biofilms by confocal laser scanning microscopy. The antimicrobial effects of the mouth rinses were evaluated by colony counting and Live/Dead staining observation. For each of the mouth rinses evaluated, a high correlation was observed between the time required to reach 50% of the initial fluorescence intensity and the biofilm thickness. The penetration velocity of N was similar to that of Z and F and significantly higher than that of CHG. For 30s exposure, the number of viable cells after exposure to N was similar to that after exposure to Z and F. Live/Dead staining observation showed that 99% of the cells were propidium iodide-positive after exposure to any of N, Z and F, being significantly higher than the percentage of propidium iodide-positive cells found after exposure to CHG. In the present study, it was concluded that the penetration ability and antimicrobial efficacy of Listerine^®Natural Care were similar to those of Listerine^®Zero and Listerine^®Fresh Mint, and significantly superior to those of the chlorhexidine gluconate-containing mouth rinse. Nihon Shishubyo Gakkai Kaishi (J Jpn Soc Periodontol) 56(3)：291-301, 2014.

DOI： 10.2329/perio.56.291

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Tooth crown patterning is governed by the growth and folding of the inner enamel epithelium (IEE) and the following enamel deposition forms outer enamel surface (OES). We hypothesized that overall dental crown shape and covariation structure are determined by processes that configurate shape at the enamel-dentine junction (EDJ), the developmental vestige of IEE. This this hypothesis was tested by comparing patterns of morphological variation between EDJ and OES in human permanent maxillary first molar (UM1) and deciduous second molar (um2). Using geometric morphometric methods, we described morphological variation and covariation between EDJ and OES, and evaluated the strength of two components of phenotypic variability, canalization and morphological integration, in addition to the relevant evolutionary flexibility, i.e. the ability to respond to selective pressure. The strength of covariation between EDJ and OES was greater in um2 than in UM1, and the way that multiple traits covary between EDJ and OES was different between these teeth. The variability analyses showed that EDJ had less shape variation and a higher level of morphological integration than OES, which indicated that canalization and morphological integration acted as developmental constraints. These tendencies were greater in UM1 than in um2. On the other hand, EDJ and OES had a comparable level of evolvability in these teeth. Amelogenesis could play a significant role in tooth shape and covariation structure, and its influence was not constant among teeth, which may be responsible for the differences in the rate and/or period of enamel formation.

DOI： 10.1111/joa.12180

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Rodent incisors maintain the ability to grow continuously and their labial dentin is covered with enamel. Bcl11b zinc-finger transcription factor is expressed in ameloblast progenitors in mouse incisors and its absence in Bcl11b(KO/KO) mice results in a defect in embryonic tooth development. However, the role of Bcl11b in incisor maintenance in adult tissue was not studied because of death at birth in Bcl11b(KO/KO) mice. Here, we examined compound heterozygous Bcl11b(S826G/KO) mice, one allele of which has an amino acid substitution of serine at position 826 for glycine, that exhibited hypoplastic maxillary incisors with lower concentrations of minerals at the enamel and the dentin, accompanying the maxillary bone hypoplasia. Histological examinations revealed hypoplasia of the labial cervical loop in incisors, shortening of the ameloblast progenitor region, and impairment in differentiation and proliferation of ameloblast-lineage cells. Interestingly, however, juvenile mice at 5days after birth did not show marked change in these phenotypes. These results suggest that attenuated Bcl11b activity impairs ameloblast progenitors and incisor maintenance. The number of BrdU label-reta

DOI： 10.1016/j.mod.2013.05.002

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これまで、成熟した口腔内バイオフィルムへの対応は、細菌をターゲットとしていかに迅速に殺菌するかが重要視されてきた。しかし、口腔内で使用される抗菌成分を用いた化学的コントロール法は、唾液による自浄作用や舌・口唇の動き、嚥下といった口腔機能の働きにより口腔内長時間保持が困難なため、バイオフィルムの厚みの増加とともに深層部への効果は減弱する。さらに、口腔内は常在菌が存在する特殊な器官であるため、バイオフィルム中の細菌が殺菌されたとしてもバイオフィルム構造が付着界面に残存した場合、新たなバイオフィルム形成の足場となる可能性がある。本研究では、Rotating Disc Reactorを用いて1日あるいは3日培養することにより作製したS.mutansバイオフィルムを殺菌後に対数増殖期の細菌培養液を灌流させ、死菌量の異なるバイオフィルム構造への二次付着量を比較検討した。その結果、殺菌処理後に残存するバイオフィルム構造に新たな浮遊細菌の付着が生じやすく、残存するバイオフィルム構造の量の増加とともに有意に付着細菌数も増加した。このことから、残存バイオフィルム構造が新たなバイオフィルム形成の起点となることが示唆され、殺菌効果だけに頼ったバイオフィルム制御戦略から、マトリックス結合を減弱・分散あるいは付着界面から剥離する戦略への転換が合理的と思われた。(著者抄録)

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DOI： 10.1016/j.job.2013.01.002

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

OBJECTIVE: Alveolar cancellous bone biopsy samples were extracted during dental implant preparation for investigating microstructural changes due to menopause and relationships between these changes and bone turnover markers. STUDY DESIGN: Subjects were 18 women receiving mandibular implants: premenopausal (n = 5), early postmenopausal (≤5 years; n = 3), and late postmenopausal (>5 years; n = 10). Bone turnover markers were measured and the samples analyzed using microscopic computerized tomography and 3-dimensional bone morphometry. RESULTS: The alveolar bone volume was significantly less in late postmenopausal women than in premenopausal ones. The trabeculae in early postmenopausal women were more separated and rod-like than in premenopausal ones (P < .05). Each alveolar bone parameter was significantly (P < .05) related to at least 1 bone turnover marker. CONCLUSIONS: Alveolar cancellous bone structure begins changing even in early postmenopausal women, and this structure varies in close relationship to bone turnover markers.

DOI： 10.1016/j.oooo.2011.10.028

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Language：Japanese   Publisher：The Japanese Society of Conservative Dentistry  

Purpose: Listerine mouthrinse has been reported to show a rapid membrane-damaging effect on biofilm bacteria. One drawback of Listerine, however, is that it may be irritative to oral mucosa due to the ethanol contained as a solvent. The aim of this study was to evaluate the penetration kinetics and antimicrobial effects of a new alcohol-free essential oil mouthwash (Listerine Zero), which has been developed to lower the irritative action of Listerine. Methods: Listerine Zero (Z), Listerine Fleshmint (F) and a chlorhexidine gluconate-containing mouthrinse (Peridex; P) were investigated as test mouthrinses, and a buffer was used as a negative control. Streptococcus mutans biofilms were grown on glass-based dishes for 24 h under anaerobic conditions. Penetration kinetics of the mouthrinses were analyzed with time-lapse confocal laser scanning microscopy where the fluorescence loss of calcein-AM-stained biofilms was monitored after exposure to mouthrinse. Antimicrobial effects of the mouthrinses were also compared using live/dead staining and plate counting. Results: The maximum biofilm thickness developed in this study was approximately 32 μm. The time required to reach 50 % of the initial fluorescence intensity (T50) and biofilm thickness exhibited a high correlation coefficient. P showed significantly smaller penetration velocity compared with the others, whereas there was no significant difference between Z and F. The live/dead staining analysis after 30 s exposure revealed that propidium iodide (a dead cell marker)-positive percentage was 99.8±0.1 % for Z and F and 41.4±5.9 % for P When viable cell counts after 30 s of mouthrinse exposure were determined by plate counting, all of the mouthrinses caused a significant reduction compared with the control. F and Z caused significantly smaller counts than P whereas there was no significant difference between Z and F Conclusion: Listerine Zero was as effective as Listerine Fleshmint in penetration property and antimicrobial effect against S. mutans biofilms of less than 32 μm thick. Both of these mouthrinses also showed significantly superior penetration property and antimicrobial effect than the chlorhexidine gluconate-containing mouthrinse.

DOI： 10.11471/shikahozon.56.105

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Signals of perlecan, an extracellular matrix molecule, which accumulates within the intercellular spaces of the stellate reticulum of the enamel organ, are mediated by at least two receptors, dystroglycan (DG) and integrin beta 1, in a case-dependent manner in various events in embryogenesis and pathogenesis. This study aims to understand the expression profiles of these two perlecan receptors at both protein and gene levels in murine enamel organ development. Before birth, alpha-DG was immunolocalized in stellate reticulum cells, in which perlecan was colocalized, while integrin beta 1 was mainly distributed in the peripheral enamel organ cells as well as the dental mesenchymal cells. On and after postnatal Day I, the expression of alpha-DG was dramatically decreased in the stellate reticulum, while integrin beta 1 was enhanced around blood vessels within the enamel organ. Furthermore, biosyntheses of alpha-DG and integrin beta 1 by dental epithelial and pulp mesenchymal cells were confirmed in vitro by using immunofluorescence and reverse-transcriptase polymerase chain reaction. The results suggest that DG is a perlecan receptor that specifically functions in the stellate reticulum of the embryonic stage, but that dental epithelial and mesenchymal cells are maturated by capturing perlecan signals differentially through integrin beta 1. (C) 2013 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.gep.2013.05.004

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The subject was a 53-year-old male. An alveolar bone sample was obtained from the site of the lower left first molar, before dental implant placement. Although the details of the trabecular structure were not visible with conventional computed tomography, micro-computed tomography (microCT) three-dimensional images of the alveolar bone biopsy sample showed several plate-like trabeculae extending from the lingual cortical bone. Histological observations of the bone sample revealed trabeculae, cuboidal osteoblasts, osteoclasts and hematopoietic cells existing in the bone tissue at the implantation site. Bone metabolic markers and calcaneal bone density were all within normal ranges, indicating no acceleration of the patient's bone metabolism. Using microCT, and histological and histomorphometrical techniques, a great deal of valuable information about the bone tissue was obtained from a biopsy sample extracted from the patient's planned implant site.

DOI： 10.2174/1874210601307010047

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Rodent incisors maintain the ability to grow continuously and their labial dentin is covered with enamel. Bcl11b zinc-finger transcription factor is expressed in ameloblast progenitors in mouse incisors and its absence in Bcl11b(KO/KO) mice results in a defect in embryonic tooth development. However, the role of Bcl11b in incisor maintenance in adult tissue was not studied because of death at birth in Bcl11b(KO/KO) mice. Here, we examined compound heterozygous Bcl11b(S826G/KO) mice, one allele of which has an amino acid substitution of serine at position 826 for glycine, that exhibited hypoplastic maxillary incisors with lower concentrations of minerals at the enamel and the dentin, accompanying the maxillary bone hypoplasia. Histological examinations revealed hypoplasia of the labial cervical loop in incisors, shortening of the ameloblast progenitor region, and impairment in differentiation and proliferation of ameloblast-lineage cells. Interestingly, however, juvenile mice at 5 days after birth did not show marked change in these phenotypes. These results suggest that attenuated Bcl11b activity impairs ameloblast progenitors and incisor maintenance. The number of BrdU label-retaining cells, putative stem cells, was lower in Bcl11b(S826G/KO) incisors, which suggests the incisor hypoplasia may be in part a result of the decreased number of stem cells. Interestingly, the level of Shh and FGF3 expressions, which are assumed to play key roles in the development and maintenance of ameloblasts and odontoblasts, was not decreased, though the expressed areas were more restricted in ameloblast progenitor and mesenchyme regions of Bcl11b(S826G/KO) incisors, respectively. Those data suggest that the incisor maintenance by Bcl11b is not directly related to the FGF epithelial-mesenchymal signaling loop including Shh but is intrinsic to ameloblast progenitors and possibly stem cells. (C) 2013 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.mod.2013.05.002

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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 Scheffé post hoc test), peaked at 3 days and then decreased significantly (P < 0.05). It was concluded that under the present experimental condition in rat molars, cavity preparation with Er:YAG laser induced mild and reversible damage to odontoblasts. The reparative process was characterized by the rearrangement of HSP-25- and nestin-immunoreactive odontoblast-like cells, which took place subsequent to the odontoblastic layer disorganization with partial loss of these immunoreactivities.

DOI： 10.1007/s10266-012-0078-x

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共焦点レーザー顕微鏡を用いた蛍光イメージングによるバイオフィルム中の細菌の生死判定法は、その三次元構造を破壊することなくバイオフィルム深層部を観察できるものの、染色時の一時点の情報しか得られない欠点があった。本研究では、Calcein-AM(CAM)染色法と共焦点レーザー顕微鏡を用いて、バイオフィルムの深層部に対する洗口液の膜傷害効果をリアルタイムに観察した。バイオフィルムの厚み(概算値)は最大で31.5μmであった。ガラスベースディッシュを用いて静置にて24時間嫌気培養することにより形成したStreptococcus mutansバイオフィルムにCAM染色を施した後、バイオフィルム底面の洗口液作用後の膜傷害効果を観察したところ、厚みの薄い部分から徐々に蛍光が消失した。50%および90%蛍光量減少までの時間(T50、T90)は、バイオフィルムの厚みと正の相関関係にあった。洗口液30秒作用後の生菌数は有意な減少は認められるものの、10^9cfu/ml以上が生存していた。CAM染色法は、膜傷害作用機序をもつ抗菌成分の浸透殺菌をリアルタイムに観察する方法として有用であると思われた。(著者抄録)

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DOI： 10.1016/j.job.2012.01.007

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

Oral biofilms, unlike those formed at most other sites in the human body, are unique because surgical intervention is usually unnecessary for their removal. The control of oral biofilms relies mainly on mechanical elimination. Chemical controls are used as alternative or adjunctive methods when elimination using dental instruments proves difficult. For many years, researches have focused on how microorganisms could be rapidly killed using high concentrations of antimicrobials or new antibacterial agents. New strategies that target the matrices of mature biofilms are needed because biofilm matrices inhibit the effect of antimicrobial agents through absorption and degradation. These strategies include the following possibilities: (1) use of antimicrobial agents with superior penetration properties and (2) degradation or detachment of matrix polymers. In order to investigate the penetration properties of antimicrobial agents, time-lapse microscopy can be used for noninvasive visualization of antimicrobial action inside models of oral biofilms. Factors that affect drug penetration properties include molecular weight, charge, hydrophilic-hydrophobic balance, and malabsorption/modification by the biofilm matrix. However, strategies that rely on antimicrobial agents have an important limitation. Even if the microorganisms are eradicated, their biofilm structure remains intact and may promote secondary biofilm and tartar formation. Therefore, future strategies should shift their focus to degradation and/or removal of matrix polymers. Some detachment-promoting agents have been reported. These agents have the potential to control biofilm accumulation effectively. Since the clinical efficacy remains unclear, and in addition to limitations of targeting a specific polysaccharide component and retention properties, further investigations are warranted. © 2012 Japanese Association for Oral Biology.

DOI： 10.1016/j.job.2012.09.002

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The root apex of the tooth elongates until the completion of root development. Although the signaling molecules inducing root elongation have been studied, the characteristic of the cells having the ability to maintain the root elongation remains unclear. This study aimed to investigate the characteristics of the cells involved in the root elongation. Octamer-binding factor 3/4 (Oct3/4) is known as one of the key regulators in maintaining the pluripotency and self-renewal properties of embryonic stem cells. Bmi1, the polycomb-group transcriptional repressor, has emerged as a key regulator in several cellular processes including stem cell self-renewal and cancer cell proliferation. At the beginning of root formation, ameloblasts expressed Oct3/4 in the nucleus, except in the apex of the cervical loop, in which Bmi1and cyclinD were expressed. At PN6, the expression of Oct3/4 in the ameloblasts shifted from the nucleus to the cytoplasm, whereas ameloblastin-negative Hertwig's epithelial root sheath (HERS) cells expressed Bmi1 and cyclinD. By PN10, the cells in the apex of HERS began to express Oct3/4 in their nucleus, whereas Bmi1 and cyclinD began to decrease in their expressions. The odontoblasts consistently expressed Oct3/4 in their cytoplasm. Our results suggest that (1) Oct3/4 creates the border between the ameloblasts from the proliferative region of HERS, (2) Bmi1-positive cells would be one of the candidates resulting in root elongation and (3) the Oct3/4 expression in the cytoplasm of odontoblasts may be related to maintain the odontoblastic characteristics.

DOI： 10.1007/s00441-011-1310-7

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Octamer-binding factor 3/4 (Oct3/4) is one of the key regulators maintaining the pluripotency and self-renewal in embryonic stem cells and is involved in the developmental events. However, the functional significance of Oct3/4 remains to be clarified during tooth morphogenesis. This study aimed to examine the functional role of Oct3/4 in mouse. During tooth morphogenesis (E11-E16.5), Oct3/4-positive cells, detected by nuclear immunoreaction, increased in number, and subsequently, their immunoreaction shifted from the nucleus to the cytoplasm at the stage of cell differentiation (E18.5). Quantitative real-time PCR clearly demonstrated the relationship between isoforms of Oct3/4 and the in vivo cellular localization of Oct3/4, suggesting that the Oct3/4 expressed in nucleus was Oct3/4A, whereas that expressed in the cytoplasm was Oct3/4B. RNAi knockdown of Oct3/4 induced apoptosis and arrested tooth morphogenesis. Our results suggest that (1) the increased number of Oct3/4-positive cells with nuclear immunoreaction correlate with active cell proliferation during tooth morphogenesis and (2) the shift of Oct3/4 from the nucleus to the cytoplasm plays a crucial role in cell differentiation.

DOI： 10.1007/s00418-011-0895-y

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OBJECTIVE: The effect of intermittent parathyroid hormone (PTH[1-34]) treatment on bone regeneration in a rat model of mandibular distraction was evaluated using microcomputed tomography. STUDY DESIGN: After a 5-day latency period, mandibles of 18 rats were distracted at 0.2 mm/12 hours for 10 days, and rats in the PTH and control groups received subcutaneous injections of PTH(1-34) at a dosage of 60 μg/kg body weight or a vehicle only, respectively, 3 times a week. The animals were humanely killed after 10 days of distraction and after 1 week and 3 weeks of consolidation. RESULTS: In reconstructed 3-dimensional images of the distracted mandible, mean bone volumes of the desired region of interest in the PTH group were significantly larger than those in the control group at all time points. CONCLUSIONS: Intermittent PTH(1-34) treatment enhances new bone formation during mandibular distraction in a rat model, and it may be effective for shortening the consolidation period.

DOI： 10.1016/j.oooo.2011.08.009

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Objectives: The aims of this study were to confirm the course of the anterior superior alveolar nerve (ASAN) canal in maxillary bone on CT images and to clarify the components of its contents to provide new evidence for neurovascularization of the anterior jaw bones. Methods: The heads and two jaw bone specimens (maxillae) of three formalin-perfused cadavers were examined. The ASAN canal course was verified on cone-beam computed tomography (CBCT) images of the heads. Subsequently, the canal structures branching from the inferior orbital canal were dissected macroanatomically and compared with the CBCT images. Microanatomically, the ASAN canal was visualized in two bone specimens from the infraorbital region using micro-computed tomography (micro-CT). To verify the micro-CT findings, each specimen was sectioned for comparison with the histological observations. Results: The gross anatomy revealed close correspondence between the course of the ASAN canal on CBCT images and that of the neurovascular bundle dissected from the canal structures branching from the inferior orbital canal. Microscopically, it was verified on micro-CT images that the ASAN canal contained neurovascular bundles including nerve bundles, arteries, and veins. Conclusions: We confirmed that the canal-like structure in the anterior maxillary bone on CT images is the ASAN canal. It should be noted that the ASAN canal is filled with neurovascular structures. The present findings may provide useful information for clinicians assessing potential risks prior to anterior jaw bone surgical procedures. © 2011 Japanese Society for Oral and Maxillofacial Radiology and Springer.

DOI： 10.1007/s11282-011-0067-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：日本歯科医学教育学会  

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Objectives The aims of this study were to confirm the course of the anterior superior alveolar nerve (ASAN) canal in maxillary bone on CT images and to clarify the components of its contents to provide new evidence for neurovascularization of the anterior jaw bones.
Methods The heads and two jaw bone specimens (maxillae) of three formalin-perfused cadavers were examined. The ASAN canal course was verified on cone-beam computed tomography (CBCT) images of the heads. Subsequently, the canal structures branching from the inferior orbital canal were dissected macroanatomically and compared with the CBCT images. Microanatomically, the ASAN canal was visualized in two bone specimens from the infraorbital region using micro-computed tomography (micro-CT). To verify the micro-CT findings, each specimen was sectioned for comparison with the histological observations.
Results The gross anatomy revealed close correspondence between the course of the ASAN canal on CBCT images and that of the neurovascular bundle dissected from the canal structures branching from the inferior orbital canal. Microscopically, it was verified on micro-CT images that the ASAN canal contained neurovascular bundles including nerve bundles, arteries, and veins.
Conclusions We confirmed that the canal-like structure in the anterior maxillary bone on CT images is the ASAN canal. It should be noted that the ASAN canal is filled with neurovascular structures. The present findings may provide useful information for clinicians assessing potential risks prior to anterior jaw bone surgical procedures.

DOI： 10.1007/s11282-011-0067-8

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Recent studies have demonstrated the existence of dental stem cells in the continuously growing tooth. However, much remains to be learned about the complex mechanism involving stem cells during tooth development. We determined the expression patterns of four stem cell markers ABCG2, Bmi-1, Oct-3/4, and Yap in the developing mouse incisors between embryonic day (E) 11 and postnatal day (PN) 20. ABCG2 was localized strongly in the perivascular region of the incisor mesenchyme from E11 to PN20, and in the odontoblasts from E18 to PN20. Bmi-1 was expressed in both the dental epithelium and mesenchyme from E11 to E14. The expression of Bmi-1 was noticeably reduced at E16, and was restricted to the apical bud from E16 to PN20. Oct-3/4 was localized in the nucleus of the cells in the superficial layer and stellate reticulum within the dental epithelium from Ell to E14 and in the apical bud from E16 to PN20. Meanwhile, once the ameloblasts and odontoblasts began to appear at El 6, they expressed Oct-3/4 in the cytoplasm. Yap was expressed in most of the basal cells of the incisor dental epithelium from Ell to E14, but was expressed mainly in the transit-amplifying (TA) cells within the basal cell layer from El 6 to PN20. The unique and overlapping expression patterns of ABCG2, Bmi-1, Oct-3/4, and Yap suggest the independent and interactive functions of the four stem cell markers in the developing mouse incisor. (C) 2010 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.gep.2010.11.001

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OBJECTIVE: The objective was to study the radiological and histologic changes in the mandibular cortices of ovariectomized monkeys. STUDY DESIGN: Twelve female, adult, Cynomolgus monkeys (Macaca fascicularis) were used. Under anesthesia, 1 group was bilaterally ovariectomized (OVX), and the other (control group) underwent sham surgery. Seventy-six weeks after surgery, the monkeys were humanely killed, their mandibles were excised, and their mandibular inferior cortices (MIC) and adjacent cortices were examined histologically and with panoramic radiographs and micro computed tomography. RESULTS: Striped shadows were seen on the endosteal side of the OVX cortices on panoramic radiographs. Histologic observation revealed many enlarged pores with eroded surfaces and calcein labeling (indicating osteon remodeling) in the OVX cortices. CONCLUSIONS: In the MIC and adjacent cortices of OVX monkeys, enlarged Haversian canals were seen and there were indications of a high rate of bone turnover. The enlarged Haversian canals resulted in striped shadows and unclear endosteal margins on radiographic images.

DOI： 10.1016/j.tripleo.2010.12.003

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We have previously demonstrated that tooth size is determined by dental mesenchymal factors. Exogenous bone morphogenetic protein (BMP)4, Noggin, fibroblast growth factor (FGF)3 and FGF10 have no effect on tooth size, despite the expressions of Bmp2, Bmp4, Fgf3, Fgf10 and Lef1 in the dental mesenchyme. Among the wingless (Wnt) genes that are differentially expressed during tooth development, only Wnt5a is expressed in the dental mesenchyme. The aims of the present study were to clarify the expression pattern of Wnt5a in developing tooth germs and the role of Wnt5a in the regulation of tooth size by treatment with exogenous WNT5A with/without an apoptosis inhibitor on in vitro tooth germs combined with transplantation into kidney capsules. Wnt5a was intensely expressed in both the dental epithelium and mesenchyme during embryonic days 14-17, overlapping partly with the expressions of both Shh and Bmp4. Moreover, WNT5A retarded the development of tooth germs by markedly inducing cell death in the non-dental epithelium and mesenchyme but not widely in the dental region, where the epithelial-mesenchymal gene interactions among Wnt5a, Fgf10, Bmp4 and Shh might partly rescue the cells from death in the WNT5A-treated tooth germ. Together, these results indicate that WNT5A-induced cell death inhibited the overall development of the tooth germ, resulting in smaller teeth with blunter cusps after tooth-germ transplantation. Thus, it is suggested that Wnt5a is involved in regulating cell death in non-dental regions, while in the dental region it acts as a regulator of other genes that rescue tooth germs from cell death.

DOI： 10.1007/s00441-011-1224-4

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Perlecan, a heparan sulfate proteoglycan, is enriched in the intercellular space of the enamel organ. To understand the role of perlecan in tooth morphogenesis, we used a keratin 5 promoter to generate transgenic (Tg) mice that over-express perlecan in epithelial cells, and examined their tooth germs at tissue and cellular levels. Immunohistochemistry showed that perlecan was more strongly expressed in the enamel organ cells of Tg mice than in wild-type mice. Histopathology showed wider intercellular spaces in the stellate reticulum of the Tg molars and loss of cellular polarity in the enamel organ, especially in its cervical region. Hertwig's epithelial root sheath (HERS) cells in Tg mice were irregularly aligned due to excessive deposits of perlecan along the inner, as well as on the outer sides of the HERS. Tg molars had dull-ended crowns and outward-curved tooth roots and their enamel was poorly crystallized, resulting in pronounced attrition of molar cusp areas. In Tg mice, expression of integrin β1 mRNA was remarkably higher at E18, while expression of bFGF, TGF-β1, DSPP and Shh was more elevated at P1. The overexpression of perlecan in the enamel organ resulted in irregular morphology of teeth, suggesting that the expression of perlecan regulates growth factor signaling in a stage-dependent manner during each step of the interaction between ameloblast-lineage cells and mesenchymal cells.

DOI： 10.1016/j.matbio.2011.08.001

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Language：Japanese   Publisher：The Japanese Society of Conservative Dentistry  

Irradiation of gallium-aluminum-arsenide (GaAlAs) semiconductor laser to rat molars is known to induce the formation of mineralized tissue in the pulp chamber. The purpose of this study was to analyze mRNA expression levels of selected non-collagenous proteins in GaAlAs laser-irradiated rat molars by means of reverse transcription-polymerase chain reaction (RT-PCR). The mesial surface of the upper right first molar of 8-week-old Wistar rats was irradiated by a pulsed GaAlAs semiconductor laser (Lightsurge 3000, Osada) at an output power of 1.5W for 60 seconds, 3 times. The first molars of unirradiated 8-wk-old rats were used as the control. After 1, 3 and 7 days, the first molars were extracted, and total RNAs were isolated from the coronal pulp. Then, expression levels of mRNAs for osteopontin, osteonectin, osteocalcin, dentin sialophosphoprotein, dentin matrix protein 1, and bone sialoprotein were analyzed by means of RT-PCR. The pulp reaction was examined histologically. mRNA expression levels of all the proteins were upregulated by 3 days, and the upregulation was maintained at 7 days. Histological observation revealed localized tissue necrosis in the mesial portion of the irradiated pulp at 1 and 3 days, whereas rearrangement of odontoblast-like cells and a small amount of newly formed calcified matrix were recognized at 7 days. In conclusion, it was demonstrated that GaAlAs laser irradiation to rat molars caused upregulation of mRNAs for non-collagenous proteins prior to the appearance of newly-generated mineralized tissue-forming cells.

DOI： 10.11471/shikahozon.53.495

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OBJECTIVE: The cellular events, underlying bone regeneration through rat mandibular distraction osteogenesis (DO) was examined using micro computerized tomography (microCT), histology, and histochemistry. STUDY DESIGN: After 5-day latency, mandibles were distracted at 0.2 mm/12 h for 10 days, and fixed at latency 5 days (L5D), distraction 3, 6, 10 days (D3D, D6D, D10D), and consolidation 1, 3, 6, 10 weeks (C1W, C3W, C6W, C10W). RESULTS: The microCT demonstrated radiopacity at the distraction gap (DG) during C1W, which was filled with new bone at C6W and C10W. At D3D, collagen fibers were aligned along the axis of the distraction vector. At D6D, alkaline phosphatase-positive osteoblasts and intramembranous ossification was observed. Collagen bundles became thicker with new bony trabeculae at D10D. Type II collagen-immunopositive areas first appeared at C1W. At C3W, cartilage tissue and endochondral ossification were found. By C6W, the entire DG had been bridged by new bone. The C10W specimens showed mature lamellar bone. CONCLUSION: Mandibular DO produces bone through both intramembranous and endochondral ossification.

DOI： 10.1016/j.tripleo.2008.06.030

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The purpose of this study was to investigate dentin-bridge formation in teeth following the transplantation of dental pulp-derived cells seeded on hydroxyapatite/tricalcium phosphate (HA/TCP) scaffolds. The dental pulp tissues were removed from the extracted first molar teeth of miniature pigs and single cell populations were subcultured. Second-passage cells that had alkaline phosphatase activity were combined with scaffolds. Cell-scaffold constructs were placed in contact with the exposed pulp tissue. The dimensions of the exposed pulp site were approximately 1-2.5 mm in diameter and 2-3 mm in depth from the tooth surface. After placing the constructs, the tooth was restored with composite resin. Six weeks after transplantation, hard tissue formation was observed on the pulp tissue in histology. Dentinal tubule-like structures were observed in most of the hard tissue generated, and columnar cells, which showed positive immunoreactions with dentin sialoprotein (DSP) and heat shock protein (HSP)-25, were aligned beneath the hard tissues. When only scaffolds were placed on the pulp tissues, particles of hard tissue were formed, however dentinal tubule-like structures and odontoblasts were not observed despite the formation of hard tissue. In conclusion, the implantation of dental pulp constructs into pulp exposed stimulates the formation of calcified dentin-like structures.

DOI： 10.18999/nagjms.71.1-2.51

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NAGOYA UNIV, SCHOOL MED  

The purpose of this study was to investigate dentin-bridge formation in teeth following the transplantation of dental pulp-derived cells seeded on hydroxyapatite/tricalcium phosphate (HA/TCP) scaffolds. The dental pulp tissues were removed from the extracted first molar teeth of miniature pigs and single cell populations were subcultured. Second-passage cells that had alkaline phosphatase activity were combined with scaffolds. Cell-scaffold constructs were placed in contact with the exposed pulp tissue. The dimensions of the exposed pulp site were approximately 1-2.5 mm in diameter and 2-3 mm in depth from the tooth surface. After placing the constructs, the tooth was restored with composite resin. Six weeks after transplantation, hard tissue formation was observed on the pulp tissue in histology. Dentinal tubule-like structures were observed in most of the hard tissue generated, and columnar cells, which showed positive immunoreactions with dentin sialoprotein (DSP) and heat shock protein (HSP)-25, were aligned beneath the hard tissues. When only scaffolds were placed on the pulp tissues, particles of hard tissue were formed, however dentinal tubule-like structures and odontoblasts were not observed despite the formation of hard tissue. In conclusion, the implantation of dental pulp constructs into pulp exposed stimulates the formation of calcified dentin-like structures.

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OBJECTIVE: The cellular events, underlying bone regeneration through rat mandibular distraction osteogenesis (DO) was examined using micro computerized tomography (microCT), histology, and histochemistry. STUDY DESIGN: After 5-day latency, mandibles were distracted at 0.2 mm/12 h for 10 days, and fixed at latency 5 days (L5D), distraction 3, 6, 10 days (D3D, D6D, D10D), and consolidation 1, 3, 6, 10 weeks (C1W, C3W, C6W, C10W). RESULTS: The microCT demonstrated radiopacity at the distraction gap (DG) during C1W, which was filled with new bone at C6W and C10W. At D3D, collagen fibers were aligned along the axis of the distraction vector. At D6D, alkaline phosphatase-positive osteoblasts and intramembranous ossification was observed. Collagen bundles became thicker with new bony trabeculae at D10D. Type II collagen-immunopositive areas first appeared at C1W. At C3W, cartilage tissue and endochondral ossification were found. By C6W, the entire DG had been bridged by new bone. The C10W specimens showed mature lamellar bone. CONCLUSION: Mandibular DO produces bone through both intramembranous and endochondral ossification.

DOI： 10.1016/j.tripleo.2008.06.030

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The tooth is one of the ectodermal organs that develop from epithelial-mesenchymal interactions during embryonic development. An understanding of the underlying molecular mechanisms would improve our knowledge of the growth factors that regulate cell proliferation and differentiation. One of the related aspects is mitogen-activated protein kinase (MAPK) signaling in tooth differentiation. The extracellular-signal regulated kinase (ERK)/mitogen-activated protein kinase kinase (MEK) cascade plays a pivotal role in many of the essential cellular processes underlying embryonic development, including responses to major developmental changes. However, the role of the ERK pathway in molar development is unclear. This study investigated epithelial patterning and tooth growth in the mouse embryo by monitoring ERK and fibroblast growth factor (FGF) signaling. ERK, MEK, and phosphatase and tensin homolog (PTEN) were activated at different levels and locations in the developing tooth at E13.5 to E16.5 and PN2. ERK was activated in the inner dental epithelium and cervical loop, while PTEN was activated in the outer dental epithelium. In addition, only ERK was activated in secretory ameloblast at PN2. To further define the pathways involving FGF and ERK, tooth germs were cultured in the presence of compounds to inhibit MAPK/ERK-mediated signaling. Western blot analysis indicated that pERK2 was strongly activated in the tooth germ. Moreover, the activation level of pERK1 was dramatically increased by exogenous FGF10 alone and by combined treatment with FGF10 and U0126. The reported results will improve our understanding of the unique developmental processes of the dental epithelium and tooth growth, and will help to elucidate the fundamental mechanisms of ERK signaling underlying tooth development.

DOI： 10.1002/jez.b.21309

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Teeth have been missing from Aves for almost 100 million years. However, it is believed that the avian oral epithelium retains the molecular signaling required to induce odontogenesis, and this has been widely examined using heterospecific recombinations with mouse dental mesenchyme. It has also been argued that teeth can form from the avian oral epithelium owing to contamination of the mouse mesenchyme with mouse dental epithelial cells. To investigate the possibility of tooth formation from chick oral epithelium and the characteristics of possible chick enamel, we applied LacZ transgenic mice during heterospecific recombination and examined the further tooth formation. Transmission electron microscopy was used to identify the two tissues during development after heterospecific recombination. No mixing was detected between chick oral epithelium and mouse dental mesenchyme after 2 days, and secretory ameloblasts with Tomes' processes were observed after 1 week. Teeth were formed after 3 weeks with a single cusp pattern, possibly determined by epithelial factors, which is similar to that of the avian tooth in the late Jurassic period. These recombinant teeth were smaller than mouse molars, whereas perfect structures of both ameloblasts and enamel showed histological characteristics similar to those of mice. Together these observations consistent with previous report that odontogenesis is initially directed by species-specific mesenchymal signals interplaying with common epithelial signals.

DOI： 10.1002/jez.b.21265

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

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

The klotho gene-deficient mouse is known as an animal model for an accelerated gerontic state, mimicking osteoporosis, skin atrophy, ectopic calcification, and gonadal dysplasia. To elucidate the influence of klotho deficiency on bone mineralization, we examined the ultrastructures of osteoblasts and bone matrices in addition to performing the elemental mapping of calcium, phosphorus, and magnesium in the bone. Under anesthesia, 4- and 5-week-old klotho-deficient mice (klotho(-/-)mice) and their wild-type littermates were perfused with either 4% paraformaldehyde for light microscopic observation or 4% paraformaldehyde and 0.0125% glutaraldehyde for electron microscopic observation. The femurs and tibiae were processed for both observations. Paraffin sections were subject to alkaline phosphatase and tartrate resistant acid phosphatase histochemistry. Semithin and ultrathin sections obtained from epoxy resin-embedded specimens were used for detecting mineralization - according to von Kossa's staining method - and for elemental mapping by electron probe micro-analyzer, respectively. Alkaline phosphatase-positive plump osteoblasts adjacent to the growth plate normally developed cell organelles in the klotho(-/-)metaphyses. This, however, contrasted with the flattened osteoblasts covering the metaphyseal trabeculae and accompanied by small tartrate resistant acid phosphatase-positive osteoclasts. The wild-type mice displayed the mineralized matrix at the zone of hypertrophic chondrocyte of the growth plate and well-mineralized metaphyseal trabeculae parallel to the longitudinal axis of the bone. Alternatively, the klotho(-/-)mice demonstrated a thick mineralized matrix from the proliferative zone of the growth plate as well as the large non-mineralized area in the metaphyseal trabeculae. Consistently, electron probe micro-analysis verified sporadic distributions of higher or lower concentrations of calcium and phosphorus in each trabecule of the klotho(-/-)mice. The distribution of magnesium, however, was almost uniform. Under transmission electron microscopy, osteoblasts on the metaphyseal trabeculae displayed less-developed cell organelles in the klotho(-/-)mice. Thus, the klotho deficiency appears not only to reduce osteoblastic population, but also to disturb bone mineralization.

DOI： 10.1111/j.1469-7580.2008.00859.x

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Klotho-deficient mice exhibit multiple pathological conditions resembling human aging. Our previous study showed alterations in the distribution of osteocytes and in the bone matrix synthesis in klotho-deficient mice. Although the bone and tooth share morphological features such as mineralization processes and components of the extracellular matrix, little information is available on how klotho deletion influences tooth formation. The present study aimed to elucidate the altered histology of incisors of klotho-deficient mice-comparing the findings with those from their wild-type littermates, by using immunohistochemistry for alkaline phosphatase (ALP), osteopontin, and dentin matrix protein-1 (DMP-1), terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) detection for apoptosis, and electron probe microanalyzer (EPMA) analysis on calcium (Ca), phosphate (P), and magnesium (Mg). Klotho-deficient incisors exhibited disturbed layers of odontoblasts, predentin, and dentin, resulting in an obscure dentin-predentinal border at the labial region. Several odontoblast-like cells without ALP activity were embedded in the labial dentin matrix, and immunopositivity for DMP-1 and osteopontin was discernible in the matrix surrounding these embedded odontoblast-like cells. TUNEL detection demonstrated an apoptotic reaction in the embedded odontoblast-like cells and pulpal cells in the klotho-deficient mice. EPMA revealed lower concentrations of Ca, P, and Mg in the klotho-deficient dentin, except for the dentin around abnormal odontoblast-like cells. These findings suggest the involvement of the klotho gene in dentinogenesis and its mineralization.

DOI： 10.1002/ar.20630

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Objective: Cortical activation is one of the procedures to accelerate tooth movement by manipulating the cortical bone. In this study, the effect of cortical activation on orthodontic tooth movement was investigated clinically and histologically in the surrounding bony tissue.
Materials and methods: In the lower and upper jaws of two beagle dogs, cortical activation was applied to the buccal and lingual side of the alveolar bone in the right jaw where 12 holes were made on each cortical plate 4 weeks after the extraction of all the second bicuspids while under deep anesthesia. All third bicuspids on both jaws were forced to move forward by a 150-g force using NiTi coil spring with/without guiding wire. The tooth movement was measured and the animals were killed after tooth movement.
Results: Rapid initial tooth movement was apparent after cortical activation. However, after 6 months of cortical activation, the cell number and cellular activity of the surrounding periodontal tissue were decreased.
Conclusions: This experiment showed that rapid initial tooth movement was apparent following the application of orthodontic force after cortical activation but the cellular activity and fibroblast structure were abnormal in the surrounding periodontal tissue.

DOI： 10.1111/j.1601-0825.2006.01286.x

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The enamel knot (EK), which is located in the center of bud and cap stage tooth germs, is a transitory cluster of non-dividing epithelial cells. The EK acts as a signaling center that provides positional information for tooth morphogenesis and regulates the growth of tooth cusps by inducing secondary EKs. The morphological, cellular, and molecular events leading to the relationship between the primary and secondary EKs have not been described clearly. This study investigated the relationship between the primary and secondary EKs in the maxillary and mandibular first molars of mice. The location of the primary EK and secondary EKs was investigated by chasing Fgf4 expression patterns in tooth germ at some intervals of in vitro culture, and the relationship between the primary EK and secondary EK was examined by tracing the primary EK cells in the E13.5 tooth germs which were frontally half sliced to expose the primary EK. After 48 hr, the primary EK cells in the sliced tooth germs were located on the buccal secondary EKs, which correspond to the future paracone in maxilla and protoconid in mandible. The Bmp4 expression in buccal part of the dental mesenchyme might be related with the lower growth in buccal epithelium than in lingual epithelium, and the Msx2 expressing area in epithelium was overlapped with the enamel cord (or septum) and cell dense area. The enamel cord might connect the primary EK with enamel navel to fix the location of the primary EK in the buccal side during the cap to bell stages. Overall, these results suggest that primary EK cells strictly contribute to form the paracone or protoconid, which are the main cusps of the tooth in the maxilla or mandible.

DOI： 10.1111/j.1432-0436.2006.00153.x

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OBJECTIVE: For the successful organ transplantation, immune rejection has to be considered. Autogenic transplantation of human teeth is generally carried out free of clinical difficulty because of the lack of immune reaction, whereas allogenic tooth transplantations easily induce host immune rejection to donor tissues. The aim of the present study was to evaluate the replacement of donor tissue by host cells after allogenic tooth transplantation
MATERIALS AND METHODS: First molars extracted from lacZ transgenic ROSA26 mice were transplanted into the alveolar socket and the tongue of host wildtype mice, where the first molar had existed.
RESULTS: Donor cells from lacZ transgenic mice were not detected in the periodontal ligament space, but rather in the pulp chamber of the donor tooth. Furthermore, if the pulp chamber was widely open to an affluent blood supply, odontoblasts and fibroblasts in the donor tissue survived in the dental pulp.
CONCLUSIONS: Our experimental models using lacZ transgenic ROSA26 mice clearly demonstrate that donor periodontal tissue cells are replaced by host cells and that periodontal tissue can regenerate after allogenic tooth transplantation. Furthermore, our models suggest that donor pulpal cells can survive if the vascular supply into the pulp chamber is sufficient.

DOI： 10.1111/j.1601-0825.2006.01213.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

Mouse, rat and human molars begin to form root after the completion of crown formation. In these teeth, fibroblast growth factor (Fgf) 10 disappears in the transitional stage from crown formation to root. By contrast, rodent incisors and vole molars demonstrate continuous growth, owing to the formation and maintenance of a stem cell compartment by the constant expression of Fgf10. To clarify the relationship between root formation and disappearance of Fgf10, we carried out two experiments for the loss and gain of Fgf10 function. First, we examined postnatal growth in the incisors of Fgf10-deficient mice, which have the defect of a dental epithelial stem cell compartment referred to as ;apical bud', after implantation under the kidney capsule. The growth at the labial side in the mutant mice mimics the development of limited-growth teeth. 5'-Bromo-2'-deoxyuridine (BrdU) labeling and cytokeratin (CK) 14 and Notch2 immunostaining suggested that the inhibition of inner enamel epithelium growth and the more-active proliferation of the outer enamel epithelium and/or stellate reticulum result in Hertwig's epithelial root sheath formation. Second, we examined the effects of Fgf10 overexpression in the transitional stage of molar germs, which led to the formation of apical bud involving in the inhibition of HERS formation. Taken together, these results suggest that the disappearance of Fgf10 signaling leads to the transition from crown to root formation, owing to the loss of a dental epithelial stem cell compartment.

DOI： 10.1242/dev.02307

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Intern. and American Associations for Dental Research  

Apoptosis plays important roles in various stages of organogenesis. In this study, we hypothesized that apoptosis would play an important role in tooth morphogenesis. We examined the role of apoptosis in early tooth development by using a caspase inhibitor, z-VAD-fmk, concomitant with in vitro organ culture and tooth germ transplantation into the kidney capsule. Inhibition of apoptosis at the early cap stage did not disrupt the cell proliferation level when compared with controls. However, the macroscopic morphology of mice molar teeth exhibited dramatic alterations after the inhibition of apoptosis. Crown height was reduced, and mesiodistal diameter was increased in a concentration-dependent manner with z-VAD-fmk treatment. Overall, apoptosis in the enamel knot would be necessary for the proper formation of molar teeth, including appropriate shape and size.

DOI： 10.1177/154405910608500610

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

The stratum intermedium develops as flattened cell layers on the proximal side of the ameloblast layer during tooth development. However, little information is available regarding the origin and the role. In this study, we indicate that some stratum intermedium cells originate from the inner enamel epithelium (IEE) in rat incisor organ cultures using DiI as a tracer. Immunohistochemical and in situ hybridization studies showed that the stratum intermedium cells express the Notch1 protein and Hes1 mRNAs, while the IEE and ameloblasts express the Jagged1. Further, we examined the role of Notch signaling using the dental epithelial cell line HAT-7. Recombinant Jagged1 protein enhanced the appearance of stratum intermedium cells in HAT-7 cultures and neutralization with an anti-Jagged1 antibody inhibited these effects. Additionally, overexpression of the Notch1 internal domain increased the number of stratum intermedium cells. We hypothesize that the stratum intermedium lineage differentiates from the ameloblast lineage via Notch signaling.

DOI： 10.1016/j.bbrc.2005.12.053

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The dentin-pulp complex is capable of repair after tooth injuries including dental procedures. However, few data are available concerning aged changes in pulpal reactions to such injuries. The present study aimed to clarify the capability of defense in aged pulp by investigating the responses of odontoblasts and cells positive for class II major histocompatibility complex (MHC) to cavity preparation in aged rat molars (300-360 days) and by comparing the results with those in young adult rats (100 days). In untreated control teeth, immunoreactivity for intense heat-shock protein (HSP)-25 and nestin was found in odontoblasts, whereas class-II-MHC-positive cells were densely distributed in the periphery of the pulp. Cavity preparation caused two types of pulpal reactions based on the different extent of damage in the aged rats. In the case of severe damage, destruction of the odontoblast layer was conspicuous at the affected site. By 12 h after cavity preparation, numerous class-II-MHC-positive cells appeared along the pulp-dentin border but subsequently disappeared together with HSP-25-immunopositive cells, and finally newly differentiated odontoblast-like cells took the place of the degenerated odontoblasts and acquired immunoreactivity for HSP-25 and nestin by postoperative day 3. In the case of mild damage, no remarkable changes occurred in odontoblasts after operation, and some survived through the experimental stages. These findings indicate that aged pulp tissue still possesses a defense capacity, and that a variety of reactions can occur depending on the difference in the status of dentinal tubules and/or odontoblast processes in individuals.

DOI： 10.1007/s00441-006-0230-4

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

Heat Shock Proteins (HSP) are molecular chaperones activated upon cellular stress/stimuli. HSP gene expression is regulated by Heat Shock Factors (HSF). We have recently demonstrated a functional role for heat shock factor-2 (HSF-2) in fibroblast growth factor-2 (FGF-2)-induced RANK ligand (RANKL), a critical osteoclastogenic factor expression on stromal/preosteoblast cells. In the present study, we show that FGF-2 treatment did not induce RANKL expression in HSF-2-/-stromal/preosteoblast cells. Interestingly, HSF-2 deficiency resulted in rapid induction of alkaline phosphatase (ALP) activity and osteocalcin mRNA expression in these cells. Furthermore, FGF-2 did not induce osteoclast formation in co-culture of normal mouse spleen cells and HSF-2-/-stromal/preosteoblast cells. Electron microscopy analysis demonstrated that osteoclasts from HSF-2-/-mice have poorly developed ruffled borders. These data further confirm that HSF-2 plays an important role in FGF-2-induced RANKL expression in stromal/preosteoblast cells. HSF-2 deficiency has pleotropic effects on gene expression during osteoblast differentiation and osteoclastogenesis in the bone microenvironment. Novel therapeutic agents that modulate HSF-2 activation may have therapeutic utility against increased levels of FGF-2 and bone destruction associated with pathologic conditions.

DOI： 10.1002/jcb.20737

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

Mice homozygous for klotho gene deletion are well established aging models as they mimic certain aspects of human senescence e.g. osteoporosis. Induced senescence may affect cellular functions and alter the histological properties of the extracellular matrices. The present study examined the histological and ultrastructural features of osteocytes and the surrounding bone matrix in klotho-deficient mice. As expected, osteoblasts showed a flattened shape with a weak immunoreactivity for alkaline phosphatase, and the bone matrix contained many empty osteocytic lacunae. The walls of both normal and empty lacunae were intensely immunopositive for osteopontin and dentin matrix protein-1, but featured an inconsistent immunoreactivity for osteocalcin and type I collagen. Not surprisingly, TUNEL-positivity, indicative of apoptosis, was found in many osteoblasts, osteocytes, and bone marrow cells of the klotho-deficient mice. In transmission electron microscopy, an amorphous matrix containing non-collagenous organic materials was recognizable around osteoblasts and in the osteocytic lacunae. Some osteoblasts on the bone surface featured these amorphous materials in vacuoles associated with their trans-Golgi network, indicating that, under klotho-deficient conditions, they synthesize and secrete the non-collagenous structures. Some osteocytes displayed pyknosis or degenerative traits. Thus, our findings provide histological evidence that klotho gene deletion influences the spatial distribution of osteocytes and the synthesis of bone matrix proteins in addition to the accelerated aging of bone cells.

DOI： 10.1679/aohc.68.371

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A rat mutant, whitish chalk-like teeth (wct), with white, chalk-like abnormal incisors, was discovered and morphologically and genetically characterized. The mutant rats showed tooth enamel defects that were similar to those of human amelogenesis imperfecta. The wct mutation was found to disturb the morphological transition of ameloblasts from secretory to maturation stages and to induce cyst formation. This mutation also disturbs the transfer of iron into the enamel, resulting in the whitish chalk-like incisors. A genetic linkage study indicated that the wct locus maps to a specific interval of rat chromosome 14 between D14Got13 and D14Wox2. Interestingly, the human chromosomal region orthologous to wct, a 5.5-Mb interval in human chromosome 4q21, is a critical region for the locus of human amelogenesis imperfecta AIH2. These results strongly suggest that this wct mutant is a useful model for the identification of genes responsible for amelogenesis imperfecta and molecular mechanisms of tooth development.

DOI： 10.1111/j.1600-0722.2005.00254.x

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

To elucidate precise mechanisms of appositional mineralization of bone, structural features of mineralizing collagen fibrils of the osteoid in normal and hypocalcaemic rats were examined in detail by transmission electron microscopy. Ultrathin sections of the osteoid of various types of bones of the rats fed with regular or normal calcium diet often displayed electrondense segments in the specific regions of the collagen fibrils located immediately adjacent to the mineralization front or to the mineralization nodules. Such dense segments appeared only after Ur-Pb staining and were more distinct in undecalcified specimens. Dense segments were undetectable in ultrathin sections picked up on ethylene glycol instead of water in the trough, even after Ur-Pb staining. Collagen fibrils in the widened osteoid of hypocalcaemic rats fed with calcium-free diet failed to show electron-dense segments. A careful comparison between the hydrously or anhydrously processed adjacent sections of a normal rat bone indicated a drastic dissolution of electron-dense material from the bone matrix near the mineralization front in hydrously processed sections and, thus, implicated the presence of labile mineral-matrix complexes in the recently mineralized bone matrix. Such labile sediments were readily dissociated within the ultrathin sections while the sections were floating on water and immediately adsorbed onto the pre-mineralizing collagen fibrils, where some conformational changes might have occurred. These data indicate that highly electron-dense segments appearing in the osteoidal collagen fibrils are a type of process-induced product, which indirectly represent possible structural alterations in the segmental portions of pre-mineralizing collagen fibrils in the osteoid of rat bones.

DOI： 10.1093/jmicro/dfh057

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The response of nerve fibres in the peri-implant epithelium to titanium implantation was investigated with an experimental model using rat maxilla and immunohistochemical techniques. The latter employed antibodies to protein gene product 9.5 (PGP9.5), and to calcitonin gene-related peptide (CGRP). In control rats without an implantation, a dense innervation of PGP9.5- and CGRP-positive nerve fibres was recognized throughout the junctional epithelium, as has been previously reported. A titanium-implantation induced a remarkable inflammatory reaction, as well as the destruction of covering epithelial cells. By 3-5 days post-implantation, inflammatory reaction showed a tendency to disappear, and the peri-implant epithelium showed proliferation and down-growth along the implant. At this stage, no nerve fibres were found around the peri-implant epithelium. At 10 days, a few nerve fibres reached the basal cell layers of the peri-implant epithelium, and entered it 15 days after implantation when the peri-implant epithelial cells showed morphological features roughly resembling those of normal junctional epithelial cells. At the complete osseointegration stage (days 20-30), the PGP9.5- and CGRP-positive nerve fibres, thin and beaded in appearance, were found distributed in the peri-implant epithelium. After 20 days, the numerical density of the intraepithelial nerves in the peri-implant epithelium appeared the same as, or less than, that in the normal junctional epithelium. These findings indicate that the peri-implant epithelium shows the same innervation as that in normal junctional epithelium, and that the intraepithelial nerve fibres in the peri-implant epithelium might have diverse functions, which have been suggested in the literature.

DOI： 10.1034/j.1600-0501.2003.140216.x

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The distribution of immunocompetent cells in the dentogingival junction of rat molars during root formation was investigated by immunocytochemistry using antibodies to class II major histocompatibility complex (MHC) molecules (OX6-antibody) and monocyte/macrophage lineage cells (ED1-antibody) as well as by histochemical reaction for periodic acid-Schiff (PAS). Two portions (the junctional epithelium in the mesial gingiva of the first molar, and the interdental gingiva between the first and second molars) were selected for observations. At the eruption stage of the first molar (16-18 days after birth), OX6-positive cells, dendritic or oval in shape, were abundantly distributed in the connective tissue between the oral epithelium and tooth germ. Positive cells with slender cell processes were also found beneath the ameloblast layer. At the commencement stage of the first molar occlusion (24-28 days after birth), numerous OX6-positive cells displaying a dendritic fashion existed preferentially in the mesial gingiva, but were fewer in the interdental gingiva. In contrast, the interdental gingiva showed a denser distribution of ED1-positive cells and PAS-reactive polymorphonuclear leukocytes (PMLs) than the mesial gingiva. At the completion stage of root formation (100-120 days after birth), the OX6-immunopositive cells invaded the deeper position of the mesial gingiva with the downgrowth of the epithelium; they had a considerably higher cell density compared with those in the interdental gingiva where PAS-reactive PMLs persisted. These findings indicated that the immunocompetent cells showed a region-specific distribution and cell density by their roles in immune response.

DOI： 10.1034/j.1600-0765.2003.01622.x

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

The regeneration process of the odontoblast cell layer incident to tooth injury, especially its relationship with immunocompetent cells in pulp healing, has not been fully understood. The purpose of the present study was to clarify this relationship between odontoblasts and immunocompetent cells in the process of pulp regeneration following cavity preparation in rat molars by immunocytochemistry for heat shock protein (Hsp) 25 as well as class II major histocompatibility complex (MHC) molecules. In untreated control teeth, intense Hsp 25-immunoreactivity was found in the cell bodies of odontoblasts and their processes within the predentin, whereas class II MHC-positive cells were predominantly located beneath the odontoblast cell layer. Cavity preparation caused the destruction of the odontoblast layer to form an edematous lesion and the shift of class II MHC-positive cells with the injured odontoblasts toward the pulp core at the affected site. Some damaged odontoblasts without apparent cytoplasmic processes, round in profile, retained the immunoreactivity for Hsp25, suggesting the survival of a part of the odontoblasts against artificial external stimuli. Twelve hours after cavity preparation, numerous class II MHC-positive cells appeared along the pulp-dentin border and extended their processes deep into the exposed dentinal tubules. By postoperative 72 hours, newly differentiated odontoblasts with Hsp 25-immunoreactivity were arranged at the pulp-dentin border, but the class II MHC-positive cells moved from the pulp-dentin border to the subodontoblastic layer. These findings indicate that the time course of changes in the expression of Hsp 25-immunoreactivity reflects the regeneration process of odontoblasts. The functional roles of Hsp 25-positive odontoblasts and immunocompetent cells such as class II MHC-positive cells in the process of pulp regeneration after cavity preparation are discussed in conjunction with our previous experimental data.

DOI： 10.1002/jemt.10289

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

Recent studies have established that heat shock proteins (HSPs) potentially play a role in immunosurveillance. The purpose of the present study was to clarify the relationship between the chronological changes of immunocompetent cells and the expression of HSP-25 in the process of pulpal regeneration after tooth injury in rat molars by immunocytochemistry for HSP-25 and class II major histocompatibility complex (MHC) antigen. In untreated control teeth, intense HSP-25 immunoreactivity was found in the cell bodies of odontoblasts. Both cavity preparation and tooth replantation caused the degeneration of the odontoblast layer to result in the loss of HSP-25 immunoreactions in the suffered dental pulp at the early stages after tooth injury. Numerous class II MHC-positive cells appeared along the pulp-dentin border and extended their cell processes into the dentinal tubules at 12-24 h after cavity preparation and 3 days after tooth replantation. Newly differentiated odontoblast-like cells with HSP-25 immunoreactivity were arranged at the pulp-dentin border and the class II MHC-positive cells retreated towards the subodontoblastic layer by post-operative days 3-5 after tooth injury. Thus, the common cellular events occur during pulpal regeneration following two different experimental injuries. These findings indicate that the time course of changes in the expression of HSP-25 immunoreactivity reflects the degeneration/regeneration process of odontoblasts and that the temporal appearance of the class II MHC-positive cells at the pulp-dentin border suggests their participation in odontoblast differentiation as well as in initial defence reactions during the pulpal regeneration process.

DOI： 10.1093/jmicro/52.6.581

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

Tissue responses to titanium implantation with two different surface conditions in our established implantation model in rat maxillae were investigated by light and transmission electron microscopy and by histochemistry for tartrate-resistant acid phosphatase (TRAPase) activity. Here we used two types of implants with different surface qualities: titanium implants sandblasted with Al2O3 (SA-group) and implants coated with hydroxyapatite (HA-group). In both groups, bone formation had begun by 5 days postimplantation when the inflammatory reaction had almost disappeared in the prepared bone cavity. In the SA-group, however, the bone formation process in the bone cavity was almost identical to that shown in our previous report using smooth surfaced implants (Futami et al. 2000): new bone formation, which occurred from the pre-existing bone toward the implant, was preceded by active bone resorption in the lateral area with a narrow gap, but not so in the base area with a wide gap. In the HA-group, direct bone formation from the implant toward the pre-existing bone was recognizable in both lateral and base areas. Many TRAPase-reactive cells were found near the implant surface. On the pre-existing bone, new bone formation occurred with bone resorption by typical osteoclasts. Osseointegration around the implants was achieved by postoperative day 28 in both SA- and HA-groups except for the lateral area, where the implant had been installed close to the cavity margin. These findings indicate that ossification around the titanium implants progresses in different patterns, probably dependent on surface properties and quality.

DOI： 10.1046/j.0905-7161.2003.00960.x

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

During tooth development, the expression of Msx2 mRNA is spatiotemporally shifted among the several components of the dental epithelium and mesenchyme. Recently, we demonstrated that Msx2-deficient mice display defective tooth, hair follicle, and mammary gland development, in addition to defects of skull ossification and persistent calvarial foramen. Although Msx2-deficient mice have shown the abnormalities of amelogenesis, the detailed phenotypes remain to be clarified. The present study analyzed the abnormalities of tooth development in Msx2-deficient mice. The phenotypes of defective teeth observed in the Msx2(-/-) mice were as follows. The enamel organ showed condensed features, preventing the vascular invasion there from inducing the TUNEL-negative degeneration of ameloblasts. Secretory ameloblasts represented abnormal features such as irregular-shaped Tomes's processes and numerous vacuoles in their distal cytoplasm. The enamel organ contained no TUNEL-positive cells, although the temporary appearance of TUNEL-positive cells occurred in the wild-type mice. Both crown and root in the Msx2(-/-) mice showed the irregular-shaped morphology, concomitant with the abnormal development of Hertwig's epithelial root sheath. The amelogenesis imperfecta was caused by the degeneration of ameloblasts and by the disappearance of the enamel-free area in the cusp area of molars. Our results provide evidence that Msx2 plays crucial roles in amelogenesis and the normal morphology of the tooth.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

The present immunocytochemical study reports on the expression of heat-shock protein (Hsp) 25 during odontogenesis in rat molars from postnatal 1 to 100 days. Hsp 25 immunoreactivity (IR) appeared in the immature dental mesenchymal cells and the differentiating and differentiated odontoblasts. At 30 days, the coronal odontoblasts retained intense Hsp25-IR, whereas the odontoblasts in the root and floor pulp were initially weak or negative but increased in IR in the later stages, indicating that the expression of Hsp 25 reflects the differentiation status of odontoblasts. During amelogenesis, the secretory ameloblasts were Hsp 25 immunopositive and the enamel free area (EFA) cells showed intense Hsp 25-IR when they developed a ruffled border. Ruffle-ended ameloblasts (RA) also consistently showed intense Hsp 25-IR, but smooth ended ameloblasts (SA) showed weak IR. These data suggest that Hsp 25 is related to the formation and maintenance of the ruffled border of RA and EFA cells.

DOI： 10.1080/03008200290000736

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

Responses of odontoblasts to cavity preparation in rat molars were investigated by immunocytochemistry for heat shock protein (Hsp) 25. In untreated control teeth, intense Hsp 25-immunoreactivity was found in the cell bodies of odontoblasts and their processes within the predentin. Confocal microscopy of Hsp 25-immunostained and rhodamine-labeled sections revealed that the immunoreactive odontoblasts were intensely labeled for phalloidin at the periphery of their cytoplasm and throughout their processes, but the reaction for phalloidin was limited within the inner half of the dentin. Cavity preparation caused an edematous reaction between the injured odontoblasts and predentin as well as a beaded swelling and successive destruction of the odontoblast processes. Immediately after cavity preparation, the odontoblasts beneath the edematous lesion showed an immunoreactivity for Hsp 25, which subsequently disappeared completely from the pulp-dentin border by 12 It after the operation. However, round cells without apparent cytoplasmic processes continued to be immunoreactive, suggesting the survival of a part of the odontoblasts against preparation stimuli. Numerous phalloidin-reactive but Hsp 25-immunonegative cells appeared along the pulp-dentin border and extended their processes deep into the exposed dentinal tubules, probably categorized in a lineage of immunocompetent cells. By postoperative 72 h, newly differentiated odontoblasts with Hsp 25-immunoreactivity were arranged at the pulp-dentin border. These findings indicate that the time course of changes in the expression of Hsp 25-immunoreactivity reflects the regeneration process of odontoblasts, and suggest that this protein is a useful marker substance for differentiated odontoblasts.

DOI： 10.1679/aohc.64.493

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

The postnatal expression of heat shock protein (Hsp) 25 during the amelogenesis of rat molars was investigated by immunocytochemistry and confocal microscopy. The localization pattern of Hsp 25-immunoreactivity in the inner enamel epithelium and ameloblast cell layer of the rat molars was almost identical to that in the rat incisors which we have previously reported: an intense Hsp 25-immunoreactivity, which first appeared in the preameloblasts, was recognized in secretory ameloblasts and ruffle-ended ameloblasts with stage-specific immunointensity. Confocal microscopy with Hsp 25-antibody and rhodamine-labeled phalloidin clearly demonstrated the co-localization of Hsp 25 and actin filaments in the ameloblast layer, supporting our hypothesis that this molecule might serve to reinforce the ameloblast layer during enamel formation as well as the formation and maintenance of the ruffled border in ruffle-ended ameloblasts. Interestingly, the enamel free area cells, which essentially lack the ability for enamel formation, showed the Hsp 25-immunoreactivity during 4-11 days when they developed a ruffled border, but decreased in that immunoreactivity after postnatal 15 days following apoptosis. Since Hsp 25 has been shown to be a specific inhibitor of apoptosis, the enamel-free area cells contribute to determine the outline of dentin at the cusped area. These data support our previous hypothesis on the diverse functions of Hsp 25 in amelogenesis.

DOI： 10.1679/aohc.64.369

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

The regeneration process of dental pulp following tooth replantation in rat molars was investigated by immuno cyto chemistry for heat shock protein (Hsp) 25 and protein gene product 9.5 (PGP 9.5). In control teeth at postnatal 4 weeks, the odontoblasts showed intense Hsp 25-immunoreactivity in the coronal dental pulp, but little or no immunoreactivity in the root and floor pulp. In contrast, the Hsp, 25-negative odontoblasts in the latter areas displayed immunoreactivity for PGP 9.5. Tooth replantation caused loss of Hsp 25- and PGP 9.5-immunoreactions in the dental pulp during postoperative days 1-3. At postoperative day 5, plump cells with clear nucleoli and several fine processes-presumably newly differentiated odontoblasts-at the pulp-dentin border became immunopositive for Hsp 25. These data suggest that the expression of Hsp 25- and PGP 9.5-immunoreactivity reflects the status of differentiation of the odontoblasts. Furthermore, some pulpal nerve fibers as well as the Schwann cells in the dental pulp, ordinarily negative in Hsp 25-immunoreaction, acquired their immunoreactivity by postoperative day 5, but lost it thereafter, suggesting the involvement of Hsp, 25 in the regeneration of pulpal nerve fibers. In the case of bone-like tissue formation in the pulp space, on the other hand, no Hsp 25-immunoreactive odontoblasts were recognized in the pulp-dentin border. Thus, the alignment of Hsp 25-immunopositive odontoblasts along the pulp-dentin border indicates a decisive factor for inducing the reparative dentin formation after tooth replantation.

DOI： 10.1679/aohc.64.425

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

The expression of immunoreactivities for superoxide dismutases (SODs), Mn-SOD and Cu/Zn-SOD. was immunohistochemically investigated in the lingual periodontal ligament and toe pads of adult rats. Immunocytochemistry for SODs revealed that the axon terminals of both the periodontal Ruffini endings and cutaneous Meissner's corpuscles showed mitochondrial Mn-SOD immunoreactivity, but not cytosolic Cu/Zn-SOD immunoreactivity, indicating Mn-SOD is a useful marker for identifying the mechanoreceptors. It is likely that Mn-SOD in the axon terminals of mechanoreceptors exerts protective action against nerve injury and neuronal death under severe conditions, serving to scavenge free radicals from the axon terminals. (C) 2001 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0006-8993(01)02458-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Society of Histological Documentation  

The regeneration process of dental pulp following tooth replantation in rat molars was investigated by immunocytochemistry for heat shock protein (Hsp) 25 and protein gene product 9.5 (PGP 9.5). In control teeth at postnatal 4 weeks, the odontoblasts showed intense Hsp 25-immunoreactivity in the coronal dental pulp, but little or no immunoreactivity in the root and floor pulp. In contrast, the Hsp 25-negative odontoblasts in the latter areas displayed immunoreactivity for PGP 9.5. Tooth replantation caused loss of Hsp 25- and PGP 9.5-immunoreactions in the dental pulp during postoperative days 1-3. At postoperative day 5, plump cells with clear nucleoli and several fine processes - presumably newly differentiated odontoblasts - at the pulp-dentin border became immunopositive for Hsp 25. These data suggest that the expression of Hsp 25- and PGP 9.5-immunoreactivity reflects the status of differentiation of the odontoblasts. Furthermore, some pulpal nerve fibers as well as the Schwann cells in the dental pulp, ordinarily negative in Hsp 25-immunoreaction, acquired their immunoreactivity by postoperative day 5, but lost it thereafter, suggesting the involvement of Hsp 25 in the regeneration of pulpal nerve fibers. In the case of bone-like tissue formation in the pulp space, on the other hand, no Hsp 25-immunoreactive odontoblasts were recognized in the pulp-dentin border. Thus, the alignment of Hsp 25-immunopositive odontoblasts along the pulpdentin border indicates a decisive factor for inducing the reparative dentin formation after tooth replantation.

DOI： 10.1679/aohc.64.425

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Society of Histological Documentation  

Responses of odontoblasts to cavity preparation in rat molars were investigated by immunocytochemistry for heat shock protein (Hsp) 25. In untreated control teeth, intense Hsp 25-immunoreactivity was found in the cell bodies of odontoblasts and their processes within the predentin. Confocal microscopy of Hsp 25-immunostained and rhodamine-labeled sections revealed that the immunoreactive odontoblasts were intensely labeled for phalloidin at the periphery of their cytoplasm and throughout their processes, but the reaction for phalloidin was limited within the inner half of the dentin. Cavity preparation caused an edematous reaction between the injured odontoblasts and predentin as well as a beaded swelling and successive destruction of the odontoblast processes. Immediately after cavity preparation, the odontoblasts beneath the edematous lesion showed an immunoreactivity for Hsp 25, which subsequently disappeared completely from the pulp-dentin border by 12 h after the operation. However, round cells without apparent cytoplasmic processes continued to be immunoreactive, suggesting the survival of a part of the odontoblasts against preparation stimuli. Numerous phalloidin-reactive but Hsp 25-immunonegative cells appeared along the pulp-dentin border and extended their processes deep into the exposed dentinal tubules, probably categorized in a lineage of immunocompetent cells. By postoperative 72 h, newly differentiated odontoblasts with Hsp 25-im-munoreactivity were arranged at the pulp-dentin border. These findings indicate that the time course of changes in the expression of Hsp 25-immunoreactivity reflects the regeneration process of odontoblasts, and suggest that this protein is a useful marker substance for differentiated odontoblasts.

DOI： 10.1679/aohc.64.493

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

The expression of heat shock protein (Hsp) 25 during odontogenesis in the dental pulp and enamel organ of rat incisors was investigated by immunocytochemistry and confocal microscopy. In the process of dentin formation, immature odontoblasts first exhibited Hsp 25-immunoreactivity, and increased in immunointensity with the advance of their differentiation. In the dental pulp, in contrast, intense immunoreaction in the mesenchymal cells became weak or negative in parallel with the progress of cell differentiation. The immunoreaction for Hsp 25 in the enamel organ revealed a characteristic stage-related alteration during amelogenesis. In secretory ameloblasts, the immunoreaction for Hsp 25 was found throughout their cell bodies, intense reactivity being located near the proximal and distal terminal webs. At the maturation stage, ruffle-ended ameloblasts (RA) consistently showed Hsp 25-immunoreactivity throughout the cell bodies, whereas smooth-ended ameloblasts (SA) lacking a ruffled border were weak in immunoreaction at the distal cytoplasm. Other cellular elements of the enamel organ were negative. The subcellular localization of Hsp 25-immunoreactivity in this study appeared essentially identical to that of actin filaments as demonstrated by confocal microscopy using rhodamine-labeled phalloidin. These immunocytochemical data suggest that the Hsp 25 molecule is involved in reinforcement of the cell layer following cell movement during odontogenesis and in the formation and maintenance of the ruffled border of RA.

DOI： 10.1679/aohc.63.381

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

The postnatal development of the terminal Schwann cell, an analogue of the lamellar cell in cutaneous sensory receptors, was examined by histochemistry for non-specific cholinesterase and immunohistochemistry for S-100 protein in the periodontal Ruffini endings of the rat incisor. Double immunohistochemistry for S-100 protein and protein gene product 9.5 (PGP 9.5) was also performed to examine the relationship between terminal Schwann cells and axons. Histochemistry for non-specific cholinesterase was able to demonstrate the age-related development of the terminal Schwann cells. the morphology and distribution of the developing terminal Schwann cells became almost identical to those in adults during postnatal days 15-18. Axons showing PGP 9.5-like immunoreactivity elongated and expanded after arrangement of terminal Schwann cells in the alveolus-related part. This suggests that the terminal Schwann cell is important in the development and maturation of the periodontal Ruffini endings. (C) 2000 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0006-8993(99)02463-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Society of Histological Documentation  

The expression of heat shock protein (Hsp) 25 during odontogenesis in the dental pulp and enamel organ of rat incisors was investigated by immunocytochemistry and confocal microscopy. In the process of dentin formation, immature odontoblasts first exhibited Hsp 25-immunoreactivity, and increased in immunointensity with the advance of their differentiation. In the dental pulp, in contrast, intense immunoreaction in the mesenchymal cells became weak or negative in parallel with the progress of cell differentiation. The immunoreaction for Hsp 25 in the enamel organ revealed a characteristic stage-related alteration during amelogenesis. In secretory ameloblasts, the immunoreaction for Hsp 25 was found throughout their cell bodies, intense reactivity being located near the proximal and distal terminal webs. At the maturation stage, ruffle-ended ameloblasts (RA) consistently showed Hsp 25-immunoreactivity throughout the cell bodies, whereas smooth-ended ameloblasts (SA) lacking a ruffled border were weak in immunoreaction at the distal cytoplasm. Other cellular elements of the enamel organ were negative. The subcellular localization of Hsp 25-immunoreactivity in this study appeared essentially identical to that of actin filaments as demonstrated by confocal microscopy using rhodamine-labeled phalloidin. These immunocytochemical data suggest that the Hsp 25 molecule is involved in reinforcement of the cell layer following cell movement during odontogenesis and in the formation and maintenance of the ruffled border of RA.

DOI： 10.1679/aohc.63.381

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

Responses of immunocompetent cells to tooth replantation during the regeneration process of the dental pulp in rat molars were investigated by immunocytochemistry using antibodies to class II major histocompatibility complex (MHC) molecules (OX6 antibody), monocyte/macrophage lineage cells (ED1 antibody) and protein gene product 9.5 (PGP 9.5), as well as by histochemical reaction for periodic acid-Schiff (PAS). Tooth replantation caused an increase in both the number of OX6- and ED1-positive cells and their immunointensity in the replanted pulp, but almost all PGP 9.5-immunoreactive nerves diminished in the initial stages. By postoperative day 3, many OX6- and ED1-immunopositive cells had accumulated along the pulp-dentin border to extend their cytoplasmic processes into the dentinal tubules in successful cases. Once reparative dentin formation had begun after postoperative day 7, OX6- and ED1-immmunopositive cells became scattered in the odontoblast layer, while reinnervation was found in the coronal pulp. The temporal appearance of these immunocompetent cells at the pulp-dentin border suggests their participation in odontoblast differentiation as well as in initial defense reactions during the pulpal regeneration process. On postoperative day 14, the replanted pulp showed three regeneration patterns: (1) reparative dentin, (2) bone-like tissue formation, and (3) an intermediate form between these. In all cases, PAS-reactive cells such as polymorphonuclear leukocytes (PML) and mesenchymal cells occurred in the pulp space. However, the prolonged stagnation of inflammatory cells was also discernible in the latter two cases. Thus, the findings on PAS reaction suggest that the migration of the dental follicle-derived cells into the pulp space and the subsequent total death of the proper pulpal cells are decisive factors for eliciting bone-like tissue formation in the replanted pulp.

DOI： 10.1007/s004410000263

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE AMERICA INC  

The composite structure of the mammalian skull, which forms predominantly via intramembranous ossification, requires precise pre- and post-natal growth regulation of individual calvarial elements. Disturbances of this process frequently cause severe clinical manifestations in humans. Enhanced DNA binding by a mutant MSX2 homeodomain results in a gain of function and produces craniosynostosis in humans(1,2). Here we show that Msx2-deficient mice have defects of skull ossification and persistent calvarial foramen, This phenotype results from defective proliferation of osteoprogenitors at the osteogenic front during calvarial morphogenesis, and closely resembles that associated with human MSX2 haploinsufficiency in parietal foramina(3) (PFM), Msx2(-/-) mice also have defects in endochondral bone formation. In the axial and appendicular skeleton, post-natal deficits in Pth/Pthrp receptor (Pthr) signalling and in expression of marker genes for bone differentiation indicate that Msx2 is required for both chondrogenesis and osteogenesis, Consistent with phenotypes associated with PFM, Msx2-mutant mice also display defective tooth, hair follicle and mammary gland development, and seizures, the latter accompanied by abnormal development of the cerebellum. Most Msx2-mutant phenotypes, including calvarial defects, are enhanced by genetic combination with Msx1 loss of function, indicating that Msx gene dosage can modify expression of the PFM phenotype. Our results provide a developmental basis for PFM and demonstrate that Msx2 is essential at multiple sites during organogenesis.

DOI： 10.1038/74231

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Academy of Periodontology  

Background: The detailed mechanism of osseointegration, the most appropriate implant-bone interface, remains unclear in jaw tissues at the ultrastructural level in contrast to the many reports using long bones. The present study reports on tissue response to titanium-implantation on an animal model using rat maxilla. Methods: Animals were sacrificed at 1 to 28 days post-implantation and prepared tissue specimens, freed from implants by a cryofracture technique, were processed for transmission electron microscopy and histochemistry for tartrate resistant acid phosphatase activity (TRAPase). Results: Different patterns in bone formation were recognized between lateral and base areas of implant cavities. In the lateral area with narrow gaps, bone deposition took place from the pre-existing bone towards the implant after active bone resorption by osteoclasts reactive to TRAPase. However, no distinct bone formation appeared in the lateral area where the implant had been installed close to the osteotomy margin. On the other hand, new bone formation was found at the base area without any apparent bone resorption. Interestingly, mononuclear cells reactive to TRAPase, presumably preosteoclasts, frequently occurred near preosteoblasts. Osseointegration around the implants was obtained in this model by 28 days post-implantation except for the lateral area with complete contact with implants, where the thin layer remained in contact with the implant surface. Conclusions: These findings indicate that ossification proceeds at different modes around the titanium implant in rat maxilla, depending on the nature of the recipient bones and the dimension of the gap between the implant and osteotomy margin.

DOI： 10.1902/jop.2000.71.2.287

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The distribution and ultrastructure of glycogen deposits were investigated in the murine tooth germ by histochemical periodic acid-Schiff (PAS) staining and transmission electron microscopy. Lower and upper first molars were examined in mouse embryos at embryonic days 11.5-17 (E11.5-E17) and in 2-day-old postnatal (P2) mice. The oral and dental epithelia and the mesenchymal cells were generally PAS-positive during tooth morphogenesis. PAS-negative cells were present at E13 in the distal tip of the tooth bud epithelium and in the contacting mesenchyme, and this complete lack of PAS reactivity continued in the dental papilla mesenchyme and inner enamel epithelium during the cap and bell stages. The lack of glycogen deposits in the interacting epithelium and mesenchyme during early morphogenesis may be associated with their demonstrated high signaling activities. Mesenchymal cells in the dental follicle consistently possessed small clusters or large pools of glycogen, which disappeared by P2. Since an intense PAS reaction was seen in mesenchymal cells at future bone sites, the glycogen in the dental follicle cells may be associated with their development into hard-tissue-forming cells. Ultrastructural observation of the enamel organ cells from the cap to early bell stages (E14-E15) revealed the occurrence of glycogen pools, which were associated with the Golgi apparatus and with vesicles having amorphous contents. Glycogen particles were also occasionally present inside vesicles or in the extracellular matrix. These may be associated with the exocytosis of glycosaminoglycan components into extracellular spaces and the formation of the stellate reticulum.

DOI： 10.1007/s004410051355

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

The postnatal expression of immunoreactivity for calretinin, one of the calcium binding proteins, and for protein gene product 9.5 (PGP 9.5), a general neuronal marker, was investigated in mechanoreceptive Ruffini endings in the periodontal ligament of the rat incisor. Age-related changes in the expression of these two proteins in periodontal nerves were further quantified with a computerized image analysis. At 1 day after birth, a few PGP 9.5-immunoreactive nerve fibers and a still smaller number of calretinin-positive fibers were found in the periodontal ligament: they were thin and beaded in appearance and no specialized nerve terminals were recognized, Tree-like terminals, reminiscent of immature Ruffini endings, were recognizable in 4-day-old rats by PGP 9.5-immunohistochemistry, while calretinin-immunostaining failed to reveal these specialized endings. At postnatal 7-11 days when PGP 9.5-immunostaining could demonstrate typical Ruffini endings, calretinin-immunopositive nerve fibers merely tapered off without forming the Ruffni type endings, A small number of Ruffini endings showing calretinin-immunoreactivity began to occur in the periodontal ligament at 24-26 days after birth when the occlusion of the first molars had been established. At the functional occlusion stage (60-80 days after birth), the Ruffini endings showing calretinin-immunoreactivity drastically increased in number and density, but less so than those positive for PGP 9.5-immunoreaction. The delayed expression of calretinin suggests that the function of the periodontal Ruffini endings is established after the completion of terminal formation because Ca2+, which binds to calcium binding proteins including calretinin with high affinity, plays an important role in mechano-electric transduction.

DOI： 10.1679/aohc.62.57

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

The distribution and ultrastructure of class II major histocompatibility complex (MHC)-positive cells were investigated in human dental pulp, employing immunohistochemistry using an anti-human leukocyte antigen (HLA)DR-monoclonal antibody. HLA-DR-immunopositive cells, appearing spindle-like or dendritic in profile, were densely distributed throughout the dental pulp. Under the electron microscope, these cells exhibited various sizes of vesicles containing clear or opaque contents, multivesicular bodies and characteristic fine tubulovesicular structures in their cytoplasm. Some reactive cells possessed coated pits and vesicles including electron-dense materials, indicating an active endocytosis. At the periphery of the pulp tissue, the HLA-DR-immunopositive cells were predominantly situated in the subodontoblastic layer, with some located in the odontoblast layer and/or predentin and extending their cytoplasmic processes into the dentinal tubules. Cell processes of these cells occasionally made contact with several odontoblast processes in the same way as the nerve fibers in the predentin. These cells never contained the typical phagosomes frequently observed in the HLA-DR-immunoreactive macrophages in the subodontoblastic layer and the pulp core. The results suggest that the HLA-DR-immunopositive cells in the odontoblast layer and/or predentin have some regulatory function on the odontoblasts under physiological conditions, in addition to their involvement in the initial defense reaction after tooth injury.

DOI： 10.1007/s004410051221

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Society of Histological Documentation  

The expression of heat shock protein 25 (Hsp 25) was investigated in the rat temporomandibular joint by immunocytochemistry combined with confocal and electron microscopy. Immunostaining with an antibody to Hsp 25 was able to demonstrate various cellular elements in the synovial membrane of the joint. Intense immunoreaction for Hsp 25 was recognized in certain cells comprising the synovial lining layer. Confocal microscopic observation revealed two characteristic profiles of the Hsp 25-positive cells with cytoplasmic processes: one extended thick and long processes towards the articular cavity, and the other prejected horizontally slender processes which covered the synovial membrane. Under the electron microscope, the immunoreactive synovial lining cells were characterized by a well-developed rough endoplasmic reticulum and secretory granules, suggesting that they can be categorized as fibroblastic type B cells. The covering by the cytoplasmic extensions was confirmed by immuno-electron microscopic observations. This cytoplasmic covering presumably performs a barrier function and expedites the effective secretion/resorption of synovial fluids. Since it has been proposed that Hsp 25 is associated with an estrogen receptor, the immunopositive synovial lining cells were considered estrogen-target cells. Immunoreactivity for Hsp 25 was also observed in the chondrocytes of the maturative and hypertrophic cell layers as well as in the cells of the articular disk. A suggestion was made that Hsp 25 might be involved in the inhibition of apoptosis of those cells.

DOI： 10.1679/aohc.62.483

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Language：Japanese   Publishing type：Research paper (scientific journal)  

The periodontal ligament has a rich sensory nerve supply which serves as a sensory apparatus in addition to tooth support. The periodontal ligament contains nociceptors and low-threshold mechanoreceptors. Stimuli applied to teeth evoke various oral reflexes, which make smooth mastication possible via the periodontal mechanoreceptors. Recent morphological and physiological studies have revealed that Ruffini endings, categorized as low-threshold slowly adapting type II (SA II), are essential mechanoreceptors in the periodontal ligament. The periodontal Ruffini endings are ultrastructurally characterized by expanded axon terminals filled with a number of mitochondria and terminal or lamellar Schwann cells. The axon terminals of the periodontal Ruffini endings have finger-like projections, i.e. axonal spines, extending into the surrounding tissue to detect the deformation of collagen fibers. As histochemical marker enzymes for the periodontal Ruffini endings, the axon terminals and terminal Schwann cells are reactive for cytochrome oxidase activity and both acid phosphatase activity and non-specific cholinesterase activity, respectively. Many experimental studies also have revealed that periodontal Ruffini endings have high potential for neuroplasticity, confirmed by intense immunoreactivity for p75NGFR and GAP-43. Mechanical stimuli due to tooth eruption and occlusion might be a prerequisite for the differentiation and maturation of the periodontal Ruffini endings. Further investigations are needed for clarifying the involvement of growth factors and the molecular mechanism of the development and regeneration processes of the Ruffini endings.

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

Tissue responses to experimentally induced actinomycotic lesions were investigated in mice by both light and transmission electron microscopy. Micro-organisms of Actinomyces israelii were entrapped in alginate gel and injected into the subcutaneous tissue over the periosteum of the mouse cranium. One day after the injection (initial stage), a non-stained amorphous structure was located in the core of the lesion, corresponding to the injected gel with bacteria. Numerous neutrophils surrounded the core region and phagocytized the injected complex actively. At days 3-7 (intermediate stage), the lesion became well developed. The core structure became eosinophilic and separated to form island-like structures. No lesion was recognized in the control group (gel without bacteria) until day 14. After 30 days (late stage), the lesions displayed more static features, similar to the "sulphur granules" characteristic of actinomycotic lesions. At the late stage, foamy cells increased in number and took the place of neutrophils in the alginate islands. By transmission electron microscopy these foamy cells were seen to be filled with lysosomal Vesicles containing electron-dense foreign material. Thus, these cells appeared to be macrophages that had phagocytized degenerated neutrophils containing bacteria. Along with the active phagocytosis by foamy cells that progressed in the late stage, a collagenous capsule became conspicuous and separated the lesion from the intact tissue. The bacteria remained in the gel islands until at least day 60, although they considerably decreased in number with time. Serum IgG antibody titres began to rise within 24 h of the injection: reached a peak concentration at day 14 and remained a significantly high (p &lt; 0.01, vs 0 time) until day 120. These results suggest that this animal model is useful for inducing experimental chronic infectious lesions. (C) 1998 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S0003-9969(98)00015-6

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

The three-dimensional architecture of enamel prisms was examined in cuspal enamel and compared with that in cervical enamel by light and electron microscopy as well as computer-assisted reconstruction using the developing enamel of several dog teeth.
Dog tooth enamel consists of two groups of alternately arranged enamel prisms oriented in opposite sideward directions basically forming thick horizontal rings, partly branching off from the stem. Along a 8-10 enamel prism-wide group, the enamel prisms emerge in parallel tilting uniformly to the same sideward direction. In cervices, groups of enamel prisms are arranged nearly in parallel displaying a regular arrangement of prisms. Approaching the cusp of tooth, the groups of enamel prisms fuse to a concentric cusp-centered arrangement and the prisms exhibit no periodic arrangement as shown in the cervical enamel.
It is suggested that the three-dimensional structure of enamel becomes complicated close to the cusp, contributing to the chewing stress of tooth. Anat. Rec. 252:355-368, 1998. (C) 1998 Wiley-Liss, Inc.

DOI： 10.1002/(SICI)1097-0185(199811)252:3<355::AID-AR3>3.0.CO;2-E

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Cytochrome oxidase (CO), one of the membrane-bound mitochondrial enzymes involved in oxidative phosphorylation, reflects the functional activity of mitochondria. Mitochondria in the enamel organ show drastic changes in localization during amelogenesis (Smith. INSERM, 1984;125:273-282). In understanding the functional aspects of the enamel organ, it is essential that one knows the exact CO activity in the respective mitochondria. The present study examines the CO activity of mitochondria in the enamel organ of rat incisors throughout the various stages of amelogenesis using light and transmission electron microscopy. CO activity was examined histochemically according to Seligman et al. (J. Cell. Biol., 1968;38:1-14) in decalcified sections of the upper and lower incisors of the rat. In the secretory stage, half of the mitochondria in the ameloblasts accumulated in the infranuclear region were reactive for CO. Both the population and CO activity of the infranuclear mitochondria of ameloblasts decreased significantly in the later stage where the enamel matrix secretion was almost complete. The CO-reactive mitochondria in the cells of the stratum intermedium (SI) gradually increased in number throughout the secretory stage. In the maturation stage, the ameloblasts contained intensively CO-reactive giant mitochondria in the proximal region and regular sized ones in the distal cytoplasm that were mostly devoid of detectable CO reactivity. The proportion of CO-reactive mitochondria in the supranuclear region and the population of mitochondria in the infranuclear regions of the smooth-ended ameloblasts were significantly higher as compared with the respective values in the ruffle-ended ameloblasts. In the late stages of enamel maturation, ameloblasts containing a large number of ferritin-filled pigment vesicles possessed numerous CO-reactive mitochondria between those vesicles in the supranuclear region, implicating an active role of the ameloblasts in iron transfer into the maturing enamel. The papillary layer cells possessed numerous intensively CO-reactive mitochondria throughout the maturation stage. A stage-related variation in the localization of CO-reactive mitochondria in the enamel organ of rat incisors was quantitatively demonstrated. It is conceivable that maturation stage ameloblasts form a functional unit with the papillary layer cells, and operate in energy-requiring events such as active ion transport to, and water and matrix protein removal from the maturating enamel. A sign of such functional integrity among the types of the enamel organ cells (ameloblasts, cells of SI, cells of stellate reticulum, and outer enamel epithelial cells) cannot be seen in the secretory stage. The secretory ameloblasts may function in matrix formation and calcium regulation in a less cooperative manner with the other cells of the enamel organ as compared to the maturation stage ameloblasts. Anat. Rec. 252:519-531, 1998. (C) 1998 Wiley-Liss, Inc.

DOI： 10.1002/(SICI)1097-0185(199812)252:4<519::AID-AR3>3.0.CO;2-I

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Society of Histological Documentation  

The distribution and ultrastructure of the class II major histocompatibility complex (MHC) antigen-expressing cells in the pulp tissue of human deciduous teeth during the process of physiological root resorption was surveyed by histochemical and immunocytochemical methods using an anti- human leukocyte antigen (HLA)-DR-monoclonal antibody. Dental pulp was found to contain numerous HLA-DR-positive cells of various shapes
those showing a dendritic appearance were located mainly in the periphery of the pulp tissue, associated closely with the odontoblasts. The immunopositive cells sometimes extended their cytoplasmic processes into the dentinal tubules and increased in number in the areas affected by dental caries, attrition or restorative procedures, implicating their role in immunosurveillance. The immunopositive cells were located consistently at the pulp-dentin border during the stage of active resorption, adjacent to the preodontoclasts or odontoclasts, and covered the exposed dentin surface after the detachment of the odontoclasts until the onset of cementum formation. These data suggest that the HLA-DR- immunopositive cells in the coronal pulp of human deciduous teeth play an inductive role in the differentiation, migration and/or activation of the odontoclasts and cementoblast-like cells during the stages of tooth resorption.

DOI： 10.1679/aohc.61.1

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Purpose. To clarify the acute effects of irradiation on the ultrastructural conformation of the dorsal mucosa of the rat tongue, with special reference to the changes in microvasculature. Methods. The proboscis of seventy rats were irradiated. The animals were then perfusion-fixed, followed by India ink-injection or resin casting at 3 to 7 days after irradiation. The bulk, frozen sections, or plastic embedded sections of the treated rat tongues were examined by light and electron microscopy. Results. In the dorsal epithelium of the rat tongue, multi-nucleated cells appeared in the basal layer at 3 days after irradiation. At day 5, the thickness of the epithelial layer and connective tissue papillae decreased dramatically, concomitant with the shortening of the capillary loops. At day 7, lingual papillae and connective tissue papillae disappeared, leaving dissociated epithelial cells and numerous neutrophils migrating throughout the tissue. Subepithelial blood vessels displayed drastic dilation with a number of neutrophils adhering to the endothelial surface, but without ultrastructural abnormalities in its cellular components. Conclusion. Early changes in the dorsal mucosa of the irradiated rat tongue were limited to the basal epithelial cells, leading to a total disruption of the epithelial layer. Atrophic changes of the capillary loops is due to the loss of the connective tissue papillae. Dilation and conformational changes of the subepithelial capillaries appear to result from the inflammatory reaction, taking place secondarily to the loss of the epithelial barrier of the irradiated tongue. A difference in radiosensitivity among the epithelial, endothelial, and mesenchymal cell components of the rat tongue in vivo is suggested.

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

Background: Developmental and chronological changes in the peripheral capillaries of the dental pulp and their relationship to odontoblasts during dentin formation has not been sufficiently detailed. This study aims to elucidate the morphological changes of the peripheral capillaries in relation to the life cycle of odontoblasts.
Methods: Peripheral capillaries of the dental pulp were examined in the labial region of rat incisors and in the crown region of rat molars by using light, transmission, and scanning electron microscopy.
Results: Before the start of dentin formation, continuous capillaries formed a coarse vascular network under the odontoblast layer. With the start of dentin deposition, capillaries began to invade into the odontoblast layer and finally located close to the predentin, where they formed a dense vascular network consisting of fenestrated capillaries. In the incisors, dentin was formed actively even near the incisal tip, and fenestrated capillaries continued to locate in the odontoblast layer. In the molars, however, the activity of dentin deposition gradually decreased with the advance of dentin formation, and the fenestrated capillaries altered to continuous capillaries and withdrew from the predentin border to the odontoblastic-pulpal border shortly before the cessation of active dentin deposition.
Conclusions: It is concluded that the changes in the peripheral capillaries are closely related to the secretory activity of the odontoblasts. To facilitate a rapid and sufficient supply of raw materials from the bloodstream to the calcifying front, peripheral capillaries first approach the odontoblasts, invade into the odontoblast layer close to the predentin with increases in density, and finally alter the endothelium from the continuous to the fenestrated type in compliance with the nutritional requirements of the odontoblasts, which lay down the dentin. When the activity of odontoblasts decreases, capillaries first alter the endothelium from the fenestrated to the continuous type, then retreat from the odontoblast layer, and finally locate below the odontoblast layer. (C) 1996 Wiley-Lies, Inc.

DOI： 10.1002/(SICI)1097-0185(199606)245:2<313::AID-AR14>3.0.CO;2-S

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Background: In order to clarify the regulatory factors that promote precipitation of enamel crystals in mammalian tooth germs, possible calcium binding domains were visualized at the epithelial-mesenchymal interface of rat incisor teeth by means of electron microscopy and X-ray microanalysis.
Methods: Adult rats were loaded with calcium (30 mM Ca) by vascular perfusion and further loaded through fixation and dehydration in the presence of high doses of calcium.
Results: Electron microscopy of anhydrously prepared Epon sections of the calcium-loaded rat incisors revealed numerous electron-dense granular deposits, enriched with calcium and phosphorus, scattering in the fibrous mantle dentin matrix and the intercellular spaces of the inner enamel epithelium, but not in the pulp tissues including the odontoblastic cells layer. The electron-dense deposits were specific for the enamel-related portion and were never shown to occur in the cementum-related portion. Proceeding incisally, dense deposits in both the mantle dentin matrix and presecretory ameloblast layer gradually converted to fine needlelike figures resembling the early enamel crystallites. Mineral deposits in experimental rats disappeared concomitant with the onset of normal mineralization of mantle dentin. There was no spatial correlation between the dense deposits and either stippled material or matrix vesicles.
Conclusions: These results indicate the presence of novel calcium-binding domains in the enamel-related portion of the epithelial-mesenchymal interface of rat incisors that form enamellike crystallites under calcium-loaded conditions. A contribution of these putative calcium-binding domains in the induction and spontaneous formation of enamel crystals is suggested. (C) 1996 Wiley-Liss, Inc.

DOI： 10.1002/(SICI)1097-0185(199606)245:2<174::AID-AR6>3.0.CO;2-X

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Immunocompetent cells in the enamel organ of rat incisors were examined immunohistochemically using OX6, ED1, and ED2 monoclonal antibodies known to recognize the Class II MHC molecules, a monocyte-macrophage lineage, and residential macrophages, respectively. The OX6 immunopositive cells (MHC cells) were located exclusively in the enamel maturation zone. MHC cells increased in number in the incisal direction and occasionally extended cytoplasmic processes deep into the ameloblast layer. Migration of MHC cells in the ameloblast layer were also encountered. MHC cells lacked phagolysosomes and could be distinguished from typical macrophages. ED2 immunopositive cells were not seen in the enamel organ. ED1 positive cells displayed identical localization to MHC cells except that some appeared in the transitional zone. MHC cells could not be seen in the enamel organ of rat molar tooth germs. Our data confirmed the presence of a large population of &quot;dendritic&quot; immunocompetent cells in the enamel organ of rat incisors and characterized the ultrastructural features of these cells. Biological significance of the immunocompetent cells in the enamel organ during amelogenesis needs to be clarified.

DOI： 10.1177/08959374960100022701

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

Responses of class II major histocompatibility complex (MHC) antigen-expressing cells to cavity preparation were investigated both in the human premolars and molars and the upper first molars of rats. In the human control teeth, class II MHC antigen-expressing cells were widely distributed throughout the dental pulp. The HLA-DR-immunopositive cells in the periphery of the pulp tissue showed dendritic appearance, and some of them being located in the predentin, extending their cytoplasmic processes into the dental tubules. In the control rat molars, OX6-immunopositive cells were located in the subodontoblastic layer and not found in the predentin. Many OX6-immunopositive cells accumulated along the pulp-dentin border 12-24 hours after cavity preparation and extended their cytoplasmic processes into the exposed dentinal tubules. In the human teeth, numerous neutrophil leucocytes migrated deep into the exposed dentinal tubules 24 hours after drilling, while most of the immunopositive cells were situated beneath the exudative lesion. The present results suggested the difference between class II MHC antigen-expressing cells in the human teeth and rat molars in chronological responses to foreign stimuli. It is also confirmed that numerous leucocytes migrate deep into the exposed dentinal tubules when destructive changes are induced in odontoblasts by cavity preparation in the human teeth.

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

Background: In order to clarify the regulatory factors that promote precipitation of enamel crystals in mammalian tooth germs, possible calcium binding domains were visualized at the epithelial-mesenchymal interface of rat incisor teeth by means of electron microscopy and X-ray microanalysis.
Methods: Adult rats were loaded with calcium (30 mM Ca) by vascular perfusion and further loaded through fixation and dehydration in the presence of high doses of calcium.
Results: Electron microscopy of anhydrously prepared Epon sections of the calcium-loaded rat incisors revealed numerous electron-dense granular deposits, enriched with calcium and phosphorus, scattering in the fibrous mantle dentin matrix and the intercellular spaces of the inner enamel epithelium, but not in the pulp tissues including the odontoblastic cells layer. The electron-dense deposits were specific for the enamel-related portion and were never shown to occur in the cementum-related portion. Proceeding incisally, dense deposits in both the mantle dentin matrix and presecretory ameloblast layer gradually converted to fine needlelike figures resembling the early enamel crystallites. Mineral deposits in experimental rats disappeared concomitant with the onset of normal mineralization of mantle dentin. There was no spatial correlation between the dense deposits and either stippled material or matrix vesicles.
Conclusions: These results indicate the presence of novel calcium-binding domains in the enamel-related portion of the epithelial-mesenchymal interface of rat incisors that form enamellike crystallites under calcium-loaded conditions. A contribution of these putative calcium-binding domains in the induction and spontaneous formation of enamel crystals is suggested. (C) 1996 Wiley-Liss, Inc.

DOI： 10.1002/(SICI)1097-0185(199606)245:2<174::AID-AR6>3.0.CO;2-X

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

Background: Developmental and chronological changes in the peripheral capillaries of the dental pulp and their relationship to odontoblasts during dentin formation has not been sufficiently detailed. This study aims to elucidate the morphological changes of the peripheral capillaries in relation to the life cycle of odontoblasts.
Methods: Peripheral capillaries of the dental pulp were examined in the labial region of rat incisors and in the crown region of rat molars by using light, transmission, and scanning electron microscopy.
Results: Before the start of dentin formation, continuous capillaries formed a coarse vascular network under the odontoblast layer. With the start of dentin deposition, capillaries began to invade into the odontoblast layer and finally located close to the predentin, where they formed a dense vascular network consisting of fenestrated capillaries. In the incisors, dentin was formed actively even near the incisal tip, and fenestrated capillaries continued to locate in the odontoblast layer. In the molars, however, the activity of dentin deposition gradually decreased with the advance of dentin formation, and the fenestrated capillaries altered to continuous capillaries and withdrew from the predentin border to the odontoblastic-pulpal border shortly before the cessation of active dentin deposition.
Conclusions: It is concluded that the changes in the peripheral capillaries are closely related to the secretory activity of the odontoblasts. To facilitate a rapid and sufficient supply of raw materials from the bloodstream to the calcifying front, peripheral capillaries first approach the odontoblasts, invade into the odontoblast layer close to the predentin with increases in density, and finally alter the endothelium from the continuous to the fenestrated type in compliance with the nutritional requirements of the odontoblasts, which lay down the dentin. When the activity of odontoblasts decreases, capillaries first alter the endothelium from the fenestrated to the continuous type, then retreat from the odontoblast layer, and finally locate below the odontoblast layer. (C) 1996 Wiley-Lies, Inc.

DOI： 10.1002/(SICI)1097-0185(199606)245:2<313::AID-AR14>3.0.CO;2-S

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：GORDON BREACH SCI PUBL LTD  

Responses of immunocompetent cells, especially class II major histocompatibility complex (MHC) antigen-expressing cells, were investigated after cavity preparation in the erupted upper first molar teeth of rats, by immunohistochemistry using OX6-monoclonal antibody.
In control teeth, OX6-immunopositive cells were predominantly located beneath the odontoblast layer in the dental pulp. Cavity preparation caused an acute edematous reaction between the injured odontoblasts and predentin, and most of OX6-immunopositive cells in the affected site shifted away from the pulp-dentin border. After 12-24 hours, many OX6-immunopositive cells accumulated along the pulp-dentin border and extended their cytoplasmic processes into the exposed dentinal tubules. After 72 hours, newly differentiated odontoblasts replaced the degenerated odontoblasts, and few OX6-immunopositive cells remained along the pulp-dentin border.
Our data suggest that some of the class II MHC antigen-expressing cells in the dental pulp participate in the initial defense reaction and presumably serve asa biological sensor for the external stimuli arriving through the exposed dentinal tubules.

DOI： 10.3109/03008209509013738

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Postnatal development of Ruffini endings was ultrastructuraally investigated in the upper incisors of the rat from 1 day to 60 days after birth by means of protein gene product 9. 5 (PGP 9. 5) immunocytochemistry. The immunostaining with PGP 9. 5 antibody clearly demonstrated chronological alterations of the distribution and ultrastructure of the Ruffini endings during postnatal development. At 1 day after birth, the PGP 9. 5 positive nerve terminals contained a few mitochondria and vesicles immunonegative for PGP 9. 5. Dendritic terminals appeared at 4 days after birth, with a small number of expanded or bulbous portions. These expanded portions possessed morphological features similar to those of the growth cone: several mitochondria and various kinds of vesicles. Typical Ruffini endings with dendritic ramification and expanded portions appeared 7–11 days after birth. At this stage, parts of the axon terminals extended through the slits of Schwann cell covering and formed finger‐like projections called axonal spines. These Ruffini endings increased dramatically in number after 24–26 days and were identical in density and morphology to those seen in adult rats. After the commencement of the occlusion between the incisors, the number of large mitochondria increased, in contrast to the decrease of the vesicles in the axon terminals. Moreover, the axonal spines increased both in number and in length. Thus, the periodontal nerve endings showed stage‐specific morphological features intimately related in timing to tooth eruption and occlusion. Functional stimuli possibly contribute to the final differentiation and maturation of the periodontal Ruffini endings. © 1995 Wiley‐Liss Inc. Copyright © 1995 Wiley‐Liss, Inc.

DOI： 10.1002/cne.903620409

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The relationship between odontoblasts and class II major histocompatibility complex (MHC) antigen-expressing cells in the process of dentinogenesis was studied in rat lower incisors, employing immuno-histochemistry using OX6-monoclonal antibody. The dental pulp contained numerous OX6-immunopositive cells that varied in morphology from dendritic to spindle under physiological conditions. Under the electron microscope, these immunopositive cells shared common cytoplasmic features, i.e., multivesicular bodies and characteristic fine tubulovesicular structures in their cytoplasm. At the early stage of dentinogenesis, OX6-immunopositive cells, presumably of the immature type, were located in the subodontoblastic layer. During active dentin formation, the 0X6-immunopositive cells increased in number and appeared in the odontoblast layer, associating intimately with fenestrated capillaries situated close to the predentin. These cells showed a dendritic appearance and possessed various sizes of multivesicular bodies and characteristic fine tubulovesicular structures, but never contained typical phagosomes. On the other hand, immunopositive macrophages characterized by typical phagosomes tended to occupy the central portion of the pulp. The results suggest that most, if not all, OX6-immunopositive cells situated deep in the odontoblast layer are dendritic cells playing a role in the defense system of the dental pulp against antigenic molecules arriving from the circulation via the fenestrated capillaries. The increasing number of OX6-immunopositive or immunonegative macrophages appearing near the incisai end of the tooth is thought to be involved in the elimination of degenerated odontoblasts. © 1994, International Society of Histology and Cytology. All rights reserved.

DOI： 10.1679/aohc.57.435

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Rapidly frozen and freeze-substituted submandibular glands of young female rats were embedded in Epon and processed for histochemical demonstration of calcium with the glyoxal bis (2-hydroxyanil) (GBHA) staining method. GBHA staining of thick Epon sections revealed discrete calcium reactions of moderate intensity in practically every secretory granule but not in other compartments of the acinar cells. the saliva in the excretory duct was also reactive with GBHA and showed a drastic decrease in staining intensity toward the distal segments of excretory ducts with larger diameters. in addition, the duct saliva contained numerous tiny particles that were highly GBHA reactive. Stromal cells and cells lining the excretory duct were totally free of reactions. in the acinar cells, X-ray analysis detected distinct peaks for calcium in secretory granules and smaller ones in the Golgi apparatus, while they were undetectable in the rough surfaced endoplasmic reticulum (RER), implicating post-RER calcium loading in the secretory pathway. Electron-dense deposits in the duct saliva showed distinct peaks both for calcium and phosphorus, though these appeared in the acinar secretory granules and other cytoplasmic regions lacked phosphorus. Our observations thus demonstrated physiologcal calcium in the intra- as well as extracellular compartments of the submandibular gland, and further confirmed drastic changes in chemical composition along the synthetic and secretory pathways of the saliva, by both histochemical and X-ray microanalytical methods. GBHA staining of calcium combined with X-ray microanalysis is useful for an evaluation of the physiology and histo-pathological changes of the salivary glands associated with initial phases of microliths as well as sialoliths formation. © 1993, International Society of Histology and Cytology. All rights reserved.

DOI： 10.1679/aohc.56.177

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DOI： 10.1679/AOHC.56.385

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

The relationship between odontoblasts and pulp capillaries in the process of dentinogenesis was studied in rat lower incisors, both on the labial and lingual sides, using light and transmission electron microscopy. The odontoblasts showed remarkable differences from the apical to the incisal end. Near the apical end of the tooth, "immature odontoblasts", which were thought to be involved in the formation of the mantle dentin, were arranged in a single layer, and continuous capillaries were located just beneath the odontoblasts. In the middle of the tooth, "mature odontoblasts" with highly developed cell organelles and notable processes formed a pseudostratified layer; fenestrated capillaries were found between these cells close to the predentin. The height of the odontoblast layer and the rate of dentin deposition on the labial (enamel-related) side was significantly greater than that on the lingual (cementum-related) side. Near the incisal end, cementum-related odontoblasts gradually decreased in height and number to become "post-odontoblasts" that produced atubular dentin; continuous capillaries were located subjacent to the post-odontoblasts. On the labial (enamel-related) side, however, odontoblasts retained their pseudostratification; fenestrated capillaries were still observed in the odontoblast layer. No atubular dentin was formed on the labial side.

DOI： 10.1007/BF00338053

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Language：Japanese  

生後30日から100日までのWistar系ラット上顎第一臼歯を観察の対象とし,加齢に伴う歯髄終末毛細血管の微細構造学的変化を透過電子顕微鏡で経時的に観察した.歯髄終末毛細血管は生後30日では象牙芽細胞層の内層にあり,象牙前質から約5 μmでterminal-wabの近傍に位置し,そのほとんどが広い有窓領域と多くの窓をもつ有窓性毛細血管で構成されている.これら終末毛細血管は生後60日になると次第に象牙前質から離れ始め,生後100日ではすべての毛細血管が象牙芽細胞層の下層に位置するようになる.有窓性毛細血管は生後40日以後ではほとんど観察されず,終末血管の大部分は連続性毛細血管で構成されるようになる.これらの変化のうち,終末毛細血管の位置的変化は象牙質形成と密接に関係していると思われるものの,有窓性毛細血管の出現時期は積極的な象牙質形成の時期とは必ずしも一致しておらず,むしろエナメル質の成熟期に対応している

DOI： 10.2330/joralbiosci1965.32.151

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Subepithelial blood vessels of the ral lingual papillae and their spatial relations to the connective tissue papillae and surface structures were demonstrated by light and scanning electron microscopy. In the rat. four types of papillae were distinguished on the dorsal surface of the tongue, i.e. the filiform, fungiform, foliate and circumvallate papillae. Vascular beds of various appearance were found in all four types of lingual papillae: a simple or twisted capillary loop in the filiform papilla: a basket- or petal-like network in the fungiform papilla: a ring-like network in the foliate papilla. and a conglomerated network surrounded by double heart-shaped capillary networks in the circumvallate papilla. These characteristic vascular beds corresponded to the shape of the connective tissue papillae and surface structures. The vascular bed beneath the gustatory epithelium in the fungiform, foliate and circumvallate papilla consisted of fine capillary networks next to the taste buds. © 1990 S. Karger AG, Basel.

DOI： 10.1159/000146823

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Subepithelial blood vessels of the rat lingual papillae and their spatial relations to the connective tissue papillae and surface structures were demonstrated by light and scanning electron microscopy. In the rat, four types of papillae were distinguished on the dorsal surface of the tongue, i.e. the filiform, fungiform, foliate and circumvallate papillae. Vascular beds of various appearance were found in all four types of lingual papillae: a simple or twisted capillary loop in the filiform papilla
a basket- or petal-like network in the fungiform papilla
a ring-like network in the foliate papilla, and a conglomerated network surrounded by double heart-shaped capillary networks in the circumvallate papilla. These characteristic vascular beds corresponded to the shape of the connective tissue papillae and surface structures. The vascular bed beneath the gustatory epithelium in the fungiform, foliate and circumvallate papilla consisted of fine capillary networks next to the taste buds.

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Responses of odontoblasts and pulp capillaries to cavity preparation were investigated in the upper first molar teeth of rats, using light and transmission electron microscopy. At 100 days of age, the blood vessels of the pulp formed a subodontoblastic network consisting of continuous capillaries at a short distance from the odontoblast layer. Cavity preparation caused the displacement of some odontoblasts into the dentinal tubules, while others were separated from the predentin by rapid inflammatory exudation after drilling. The subodontoblastic capillary network under the injured dentin was shifted inwards together with the separated odontoblasts. The endothelium of the shifted capillaries showed a remarkable increase of pinocytotic vesicles, an event thought to be closely related to the formation of the exudative lesion. By one day after cavity preparation, most of the damaged odontoblasts had degenerated. Many cells with high nucleus/cytoplasm (N/C) ratios and prominent nucleoli accumulated around the subodontoblastic capillaries, some of which had many endothelial fenestrae facing these cells. These cells were suggestive of newly differentiating odontoblasts receiving nutritional supply from the capillaries. Three days after cavity preparation, newly differentiating odontoblasts took the place of the degenerated odontoblasts. They began to produce reparative dentin by five days after cavity preparation. Capillaries were located beneath the newly differentiating odontoblasts, but endothelial fenestrae gradually decreased in number. During the active reparative dentin formation, capillaries remained closely beneath the new odontoblast layer. Although the rate of reparative dentin deposition was not significantly lower than that in the primary dentin formation, one could not recognize an invasion of capillaries into the odontoblast layer nor a remarkable increase of endothelial fenestrae, both of which are common in active primary dentin formation. The results suggest that the function of capillaries differs between primary and reparative dentin formation. © 1990, International Society of Histology and Cytology. All rights reserved.

DOI： 10.1679/aohc.53.423

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DOI： 10.2330/joralbiosci1965.32.151

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