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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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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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Hypoxia is a state of inadequate supply of oxygen. Increasing evidence indicates that a hypoxic environment is strongly associated with abnormal organ development. Oxygen nanobubbles (ONBs) are newly developed nanomaterials that can deliver oxygen to developing tissues, including hypoxic cells. However, the mechanisms through which nanobubbles recover hypoxic tissues, such as developing tooth germs remain to be identified. In this study, tooth germs were cultured in various conditions: CO2 chamber, hypoxic chamber, and with 20% ONBs for 3 h. The target stages were at the cap stage (all soft tissue) and bell stage (hard tissue starts to form). Hypoxic tooth germs were recovered with 20% ONBs in the media, similar to the tooth germs incubated in a CO2 chamber (normoxic condition). The tooth germs under hypoxic conditions underwent apoptosis both at the cap and bell stages, and ONBs rescued the damaged tooth germs in both the cap and bell stages. Using kidney transplantation for hard tissue formation in vivo, amelogenesis and dentinogenesis imperfecta in hypoxic conditions at the bell stage were rescued with ONBs. Furthermore, glucose uptake by tooth germs was highly upregulated under hypoxic conditions, and was restored with ONBs to normoxia levels. Our findings indicate that the strategies to make use of ONBs for efficient oxygen targeted delivery can restore cellular processes, such as cell proliferation and apoptosis, glucose uptake, and hypomineralization in hypoxic environments.

DOI： 10.3389/fcell.2021.626224

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

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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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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Chondrogenesis is accompanied by not only cellular renovation, but also metabolic stress. Therefore, macroautophagy/autophagy is postulated to be involved in this process. Previous reports have shown that suppression of autophagy during chondrogenesis causes mild growth retardation. However, the role of autophagy in chondrocyte differentiation still largely remains unclear. Here, we show the important role of autophagy on chondrogenesis. The transition of mesenchymal cells to chondrocytes was severely impaired by ablation of Atg7, a gene essential for autophagy. Mice lacking Atg7 after the transition exhibited phenotypes severer than mutant mice in which Atg7 was removed before the transition. Atg7-deficient chondrocytes accumulated large numbers of glycogen granules, hardly proliferate and died specifically in the proliferative zone without any ER-stress signal. Our results suggest that the suppression of autophagy in prechondrogenic cells drives compensatory mechanism(s) that mitigate defective chondrogenesis, and that autophagy participates in glycogenolysis to supply glucose in avascular growth plates.Abbreviations: DDIT3/CHOP: DNA damage inducible transcript 3; ER: endoplasmic reticulum; NFE2L2/NRF2: nuclear factor, erythroid derived 2, like 2; SQSTM1/p62: sequestosome 1; STBD1: starch-binding domain-containing protein 1.

DOI： 10.1080/15548627.2019.1628541

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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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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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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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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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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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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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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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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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Brain-derived neurotrophic factor (BDNF), which is released due to nerve injury, is known to promote the natural healing of injured nerves. It is often observed that damage of mandibular canal induces local sclerotic changes in alveolar bone. We reported that peripheral nerve injury promotes the local production of BDNF; therefore, it was possible to hypothesize that peripheral nerve injury affects sclerotic changes in the alveolar bone. This study aimed to evaluate the effect of BDNF on osteogenesis using in vitro osteoblast-lineage cell culture and an in vivo rat osteotomy model. MC3T3-E1 cells were cultured with BDNF and were examined for cell proliferative activity, chemotaxis and mRNA expression levels of osteoblast differentiation markers. For in vivo study, inferior alveolar nerve (IAN) injury experiments and mandibular cortical osteotomy were performed using a rat model. In the osteotomy model, exogenous BDNF was applied to bone surfaces after corticotomy of the mandible, and we morphologically analyzed the new bone formation. As a result, mRNA expression of osteoblast differentiation marker, osteocalcin, was significantly increased by BDNF, although cell proliferation and migration were not affected. In the in vivo study, osteopontin-positive new bone formation was significantly accelerated in the BDNF-grafted groups, and active bone remodeling, involving trkB-positive osteoblasts and osteocytes, continued after 28 days. In conclusion, BDNF stimulated the differentiation of MC3T3-E1 cells and it promoted new bone formation and maturation. These results suggested that local BDNF produced by peripheral nerve injury contributes to accelerating sclerotic changes in the alveolar bone.

DOI： 10.1371/journal.pone.0169201

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

During tooth development, oral epithelial cells differentiate into ameloblasts in order to form the most mineralized tissue in the vertebrate body: enamel. During this process, ameloblasts directionally secrete enamel matrix proteins and morphologically change from low columnar cells to polarized tall columnar cells, both of which are essential for the proper formation of enamel. In this study, we elucidated the molecular mechanism that integrates ameloblast function and morphology. Immunohistochemistry revealed that the restricted expression of semaphorin 4D (Sema4D) and RhoA activation status are closely associated with ameloblast differentiation in mouse incisors. In addition, in vitro gain-of-function and loss-of-function experiments demonstrated that Sema4D acts upstream of RhoA to regulate cell polarity and amelogenin expression via the Plexin B1/Leukemia-associated RhoGEF (LARG) complex during ameloblast differentiation. Experiments in transgenic mice demonstrated that expression of a dominant-negative form of RhoA in dental epithelium hindered ameloblast differentiation and subsequent enamel formation, as well as perturbing the establishment of polarized cell morphology and vectorial amelogenin expression. Finally, we showed that spatially restricted Akt mediates between Sema4D-RhoA signaling and these downstream cellular events. Collectively, our results reveal a novel signaling network, the Sema4D-RhoA-Akt signal cascade, that coordinates cellular function and morphology and highlights the importance of specific spatiotemporally restricted components of a signaling pathway in the regulation of ameloblast differentiation. © 2016 American Society for Bone and Mineral Research.

DOI： 10.1002/jbmr.2876

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Language：Japanese   Publisher：(一社)歯科基礎医学会  

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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 αvβ3, 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 αvβ3, 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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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

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：Hindawi Limited  

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：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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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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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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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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The aim of this study was to demonstrate the presence of intraepithelial stroma represented by extracellular matrix (ECM) deposits in the junctional epithelium to clarify its function as a scaffold for leukocyte migration through epithelial cells. Twenty-three biopsy specimens from the gingiva including the junctional epithelium were examined to determine comparative protein and gene level expression profiles for keratin and ECM molecules between the junctional epithelium and the gingival epithelium using immunohistochemistry and in situ hybridization. Intraepithelial leukocyte types and frequencies were also determined and compared between the junctional and gingival epithelia. In the junctional epithelium, which was positive for keratin 19, perlecan was strongly deposited in intercellular space of the whole epithelial layer, while it was faintly positive around the parabasal layer of the gingival epithelium. Perlecan mRNA signals were enhanced to a greater degree in both epithelial and inflammatory cells within the junctional epithelium. In the junctional epithelium, greater numbers of neutrophils and macrophages were found as compared with the gingival epithelium. Our results showed that perlecan is the primary ECM molecule comprising intraepithelial stroma of the junctional epithelium, in which leukocytes may migrate on ECM scaffolds in intercellular space toward the surface of the gingival sulci or pockets.

DOI： 10.1007/s00418-014-1198-x

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

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 β1, 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, α-DG was immunolocalized in stellate reticulum cells, in which perlecan was colocalized, while integrin β1 was mainly distributed in the peripheral enamel organ cells as well as the dental mesenchymal cells. On and after postnatal Day 1, the expression of α-DG was dramatically decreased in the stellate reticulum, while integrin β1 was enhanced around blood vessels within the enamel organ. Furthermore, biosyntheses of α-DG and integrin β1 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 β1.

DOI： 10.1016/j.gep.2013.05.004

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

Immunohistochemical loss of keratin (K)13 is one of the most valuable diagnostic criteria for discriminating carcinoma in situ (CIS) from non-malignancies in the oral mucosa while K13 is stably immunolocalized in the prickle cells of normal oral epithelium. To elucidate the molecular mechanism for the loss of K13, we compared the immunohistochemical profiles for K13 and K16 which is not expressed in normal epithelia, but instead enhanced in CIS, with their mRNA levels by in-situ hybridization in formalin-fixed paraffin sections prepared from 23 CIS cases of the tongue, which were surgically removed. Reverse transcriptase-PCR was also performed using RNA samples extracted from laser-microdissected epithelial fragments of the serial paraffin sections in seven of the cases. Although more enhanced expression levels for K16 were confirmed at both the protein and gene levels in CIS in these seven cases, the loss of K13 was associated with repressed mRNA levels in four cases, but not in the other three cases. The results suggest that the loss of K13 is partly due to its gene repression, but may also be due to some unknown post-translational events.

DOI： 10.1038/modpathol.2011.218

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMEDICAL RESEARCH PRESS LTD  

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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Authorship：Lead author   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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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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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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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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OBJECTIVES: The deposition of perlecan, a heparan sulfate proteoglycan, is enhanced within oral carcinoma in situ (CIS) foci, while it dynamically switches from CIS foci to the stromal space in squamous cell carcinoma (SCC). Because α-dystroglycan and integrin β1 have been identified as two of the perlecan receptors, we wanted to determine their differential distributions before and after invasion of oral SCC. METHODS:  Eighty-two surgical tissue specimens of oral SCC containing different precancerous stages were examined by immunohistochemistry for perlecan, α-dystroglycan, integrin β1, and Ki-67. In addition, α-dystroglycan mRNA signals were localized by in situ hybridization. RESULTS: In normal epithelia, α-dystroglycan and integrin β1 were localized on the cell membrane of basal cells, while perlecan was faintly present in the intercellular spaces of parabasal cells. In epithelial dysplasia and CIS, α-dystroglycan and perlecan were well co-localized in the epithelial layer, especially in its lower half, and this co-localization was mostly overlapped with Ki-67-positive (+) cell zones. However, in SCC, α-dystroglycan was localized neither within carcinoma cell nests nor in the stroma, while perlecan disappeared from SCC foci but emerged in the stromal space, leaving integrin β1+ and Ki-67+ cells only to the periphery of SCC foci. α-Dystroglycan mRNA signals were basically identical to the α-dystroglycan protein localizations. CONCLUSION: The findings suggest that α-dystroglycan and integrin β1 act as perlecan receptors in oral precancerous lesions prior to invasion, and that the perlecan signals via the two different receptors function in cellular differentiation and proliferation of CIS cells, respectively.

DOI： 10.1111/j.1600-0714.2010.00990.x

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AIMS: One of the important histopathological characteristics of oral epithelial dysplasia is a two-phase appearance of rete processes, comprising an upper layer of keratinized cells and a lower layer of basaloid cells, and thereby creating a sharp contrast between these two separate cell populations. The aim of this study was to determine the cellular adhesion status of the basaloid cells. METHODS AND RESULTS: Immunohistochemistry for β-catenin, E-cadherin and their related molecules was carried out in surgical specimens of two-phase epithelial dysplasia of the oral mucosa. The lower-half basaloid cells and the upper keratinized cells were microdissected separately, and extracted DNA samples were subjected to methylation-specific polymerase chain reaction amplification for E-cadherin. β-Catenin was immunolocalized within the nuclei and cytoplasm of Ki67-positive lower-half basaloid cells, as well as on the cell membrane of upper parakeratotic cells. The basaloid cells of the lower-half were also positive for matrix metalloproteinase-7 and cyclin D1, β-catenin target gene products, α-dystroglycan, tenascin-C, and perlecan, but not for E-cadherin. The promoter region of the E-cadherin gene was hypermethylated. CONCLUSIONS: The solid proliferation of lower-half E-cadherin-free basaloid cells is enhanced by Wnt signalling cascades, as well as by the intraepithelial extracellular matrix or its bound growth factors.

DOI： 10.1111/j.1365-2559.2011.03929.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMEDICAL RESEARCH PRESS LTD  

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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AIMS: Intercellular deposition of perlecan, an extracellular matrix molecule, results in characteristic stellate reticulum-like structures in ameloblastomas. The aims of this study were to elucidate which types of perlecan receptors function within any particular type of tissue architecture of ameloblastoma. METHODS AND RESULTS: Protein and gene expression profiles for α-dystroglycan and integrin β1 were examined comparatively with those of their ligands in ameloblastoma using surgical specimens and cells in primary culture. In the follicular-type tumour cell foci, α-dystroglycan was localized uniformly over the stellate reticulum-like cells, while integrin β1 was restricted mainly to peripheral cells facing the stroma with the interface of the basement membrane, which was also rich in perlecan. In the plexiform-type, mRNA and protein signals for α-dystroglycan were enhanced in the periphery of tumour cell foci, especially in their invading fronts. Integrin β1 was also immunolocalized in the basal cell zone, which was considered to be the proliferation centre of ameloblastoma cells. Furthermore, biosynthesis of α-dystroglycan and integrin β1 by ameloblastoma cells was confirmed in vitro using immunofluorescence and reverse transcriptase-polymerase chain reaction. CONCLUSIONS: Ameloblastoma cells proliferate and are differentiated by capturing perlecan differentially with α-dystroglycan and integrin β1, respectively.

DOI： 10.1111/j.1365-2559.2010.03732.x

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

OBJECTIVE: Immunohistochemical and gene expression profiles of heparanase were determined in murine molar tooth germs from their embryonic to postnatal stages, paying special attention to neovascularization within the enamel organ, which is poorly vascularized before birth. DESIGN: Protein and gene expression profiles of heparanase, heparan sulfate (HS), vascular endothelial growth factor (VEGF), transforming growth factor-beta1 (TGF-beta1), and perlecan were comparatively examined by immunohistochemistry and in-situ hybridization, respectively, in mouse mandibular molar tooth germs from embryonic day 11.5 to postnatal day 6. At the same time, their mRNA expression levels were also confirmed by reverse transcriptase-polymerase chain reaction using laser-captured microdissection of enamel organ tissues. RESULTS: Stellate reticulum cells highly expressed perlecan but only slightly expressed heparanase and HS in their embryonic days. On and after postnatal day 1, the expressions of heparanase became dramatically higher in the stellate reticulum, while HS disappeared leaving the immunopositivity for perlecan core protein. Immunohistochemically, HS was enhanced around blood vessels which were newly formed after birth within the enamel organs, whose volume was also regressive. Similar expression patterns were obtained for VEGF and TGF-beta1. CONCLUSIONS: Such synchronized expression modes among the HS metabolism-related molecules suggested that heparanase plays an important role in degradation of HS chains, which is closely related to vascular penetration into the stellate reticulum, which may be one of the driving forces for the postnatal regression of the enamel organ.

DOI： 10.1016/j.archoralbio.2010.07.002

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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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To make reproducible diagnoses for oral carcinoma in situ (CIS), combined immunohistochemistry directed at the positioning of squamous cell proliferation (Ki-67) and differentiation (keratin (K) 13 and K19) was used, both of which support histological evaluations by providing biological evidence. Normal/hyperplastic epithelia was defined by K19+ cells only in the first basal layer, K13+ cells in the third basal and upper layers, and sporadic Ki-67+ cells in the second basal layer. These profiles indicated that a proliferating center of the oral epithelium is located in the parabasal cell layer, and K19 and K13 can be regarded as markers for basal and prickle cells, respectively. Epithelial dysplasia was characterized by irregular stratification of Ki-67+ cells and the absence of K19/K13 in proliferating cells. Irregular emerging of K19+ and K13+ cells in proliferating foci with unique stratification of atypical Ki-67+ cells indicated CIS. When the definition was applied, surgical margins in 172 recurrent cases were shown to contain CIS (39.4%) and squamous cell carcinoma (55.8%), indicating that the new diagnostic criteria for CIS reflected clinical behaviors of the cases. The results indicate that oral CIS contain more histological variations, especially those with definite keratinization, than what had been previously defined.

DOI： 10.1111/j.1440-1827.2009.02499.x

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Solitary fibrous tumor (SFT) is an uncommon spindle-cell neoplasm of mesenchymal origin. Because the pathogenetic background of SFT is still controversial, cytogenetic analysis could help in tumor diagnosis and prognosis. In this study, cultured SFT cells from a lower lip lesion that presented characteristic immunopositivity for CD34, vimentin, CD99, and BCL2 showed a unique cytogenetic finding: 46,XX,inv(2)(p21q35),t(3;12)(q25;q15). To our knowledge, this is the third report of cytogenetic result of a case involving the oral cavity. The SFT cells in culture that maintained their immunohistochemical expression of diagnostic molecules, showed unique chromosomal changes previously unreported when compared with already documented ones. Our data suggest that the complicated pathogenetic nature of SFT is possibly tumor- or organ-related.

DOI： 10.1016/j.cancergencyto.2009.05.003

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BACKGROUND: As one of the valuable tools for differential diagnoses of oral epithelial dysplasia, carcinoma in situ (CIS) and squamous cell carcinoma (SCC), we have proposed the immunohistochemistry for perlecan, a heparan sulfate proteoglycan (HSPG). As HSPGs have been shown to be extracellular docking molecules for matrix metalloproteinase (MMP) 7, our aim was to determine the expression mode of MMP-7 in these lesions for its possible diagnostic aid for oral borderline malignancies. METHODS: Twenty cases each of moderate dysplasia, CIS, SCC, and normal/hyperplastic/mild dysplastic epithelia of the tongue and buccal mucosa were immunohistochemically examined for MMP-1, -2 and -7 in reference to their perlecan immunolocalization. RESULTS: The expression of all three MMPs in the normal mucosal epithelium was restricted mainly to the parabasal layers. The most striking finding was strong expression of MMP-7 in epithelial dysplasia with a two-phase appearance: a clear demarcation of MMP-7-immunopositive (+) lower dysplastic/basaloid cells from non-positive upper keratinized cells. MMP-7+ cells were spread over the whole epithelial layer of CIS. In SCC, MMP-7 positivity was reduced from carcinoma cells but instead appeared in stromal cells. These expression profiles of MMP-7 resembled those of perlecan. MMP-1 and MMP-2 exhibited a similar but much weaker staining than MMP-7. CONCLUSION: These results suggest that the enhanced metabolism of perlecan associated with MMP-7 plays an important role in the cell proliferation of oral epithelia in their malignant transformation process, and that MMP-7 immunohistochemistry may be a valuable aid for identification of the cell proliferation center in oral CIS and dysplasia.

DOI： 10.1111/j.1600-0714.2009.00750.x

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BACKGROUND: The intraepithelial deposit of perlecan, a basement membrane-type heparan sulfate (HS) proteoglycan, has been demonstrated in neoplastic conditions such as salivary gland tumors, odontogenic tumors, and oral carcinoma in situ. Our aim was to determine whether perlecan turnover was enhanced in the lining cells of keratocystic odontogenic tumor (KCOT), which had been recently renamed from odontogenic keratocyst because of its accumulated evidence of neoplasm, as a possible background for neoplastic proliferation. METHODS: Ten surgical specimens from each of KCOT, dentigerous cyst, and radicular cyst were examined for the expressions of perlecan core protein, HS chains, heparanase, and Ki-67 by immunohistochemistry and in situ hybridization. RESULTS: In KCOT, perlecan core protein and HS chains were localized on the cell border from the parabasal to subkeratinized layers of the lining epithelium. Heparanase was localized in a similar fashion to those for perlecan and HS chains but was within the cytoplasm. mRNA signals for perlecan core protein and heparanase were mostly compatible with their protein signals. Ki-67-positive cells were localized mainly in the second basal cell layers with definitely higher labeling indices (approximately 31.3%, second layer). In contrast to KCOT, dentigerous cysts and radicular cysts had no perlecan, HS chains, and heparanase deposition in their linings with extremely lower Ki-67 indices (0.4-0.8%). CONCLUSION: The result suggests that the characteristic intra-lining-epithelial deposit of perlecan in KCOT, which has never been seen in other cystic jaw lesions, is a new evidence supporting the neoplastic nature of KCOT.

DOI： 10.1111/j.1600-0714.2007.00620.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

In tooth development, the oral ectoderm and mesenchyme coordinately and reciprocally interact through the basement membrane for their growth and differentiation to form the proper shape and size of the tooth. Laminin alpha 5 subunit-containing laminin-10/11 ( LM-511/521) is the major laminin in the tooth germ basement membrane. Here, we have examined the role of laminin alpha 5 ( Lama5) in tooth development using laminin alpha 5-null mouse primary dental epithelium and tooth germ organ cultures. Lama5-null mice develop a small tooth germ with defective cusp formation and have reduced proliferation of dental epithelium. Also, cell polarity and formation of the monolayer of the inner dental epithelium are disturbed. The enamel knot, a signaling center for tooth germ development, is defective, and there is a significant reduction of Shh and Fgf4 expression in the dental epithelium. In the absence of laminin alpha 5, the basement membrane in the inner dental epithelium becomes discontinuous. In normal mice, integrin alpha 6 beta 4, a receptor for laminin alpha 5, is strongly localized at the basal layer of the epithelium, whereas in mutant mice, integrin alpha 6 beta 4 is expressed around the cell surface. In primary dental epithelium culture, laminin-10/11 promotes cell growth, spreading, and filopodia-like micro-spike formation. This promotion is inhibited by anti-integrin alpha 6 and beta 4 antibodies and by phosphatidylinositol 3-kinase inhibitors and dominant negative Rho-GTPase family proteins Cdc42 and Rac. In organ culture, anti-integrin alpha 6 antibody and wortmannin reduce tooth germ size and shape. Our studies demonstrate that laminin alpha 5 is required for the proliferation and polarity of basal epithelial cells and suggest that the interaction between laminin-10/11-integrin alpha 6 beta 4 and the phosphatidylinositol 3-kinase-Cdc42/Rac pathways play an important role in determining the size and shape of tooth germ.

DOI： 10.1074/jbc.M509295200

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Perlecan, a heparan sulfate proteoglycan, is one of the major basement membrane macromolecules and plays an important role in cellular growth, differentiation, adhesion, and motility by its interaction with growth factors and cytokines. Recently, perlecan has been localized within the epithelial space in various pathophysiological conditions. As the intraepithelial accumulation of perlecan results in widening of the intercellular space, the most characteristic example of such phenomena is the stellate reticulum of the enamel organ of tooth germs, whose structure and function have been largely unknown. Stellate reticulum-like structures are also found in pathological conditions such as ameloblastoma or oral epithelial dysplasia. The biosynthesis of perlecan has been demonstrated in those epithelial cells, and such peculiar stellate reticulum appearances are shown to be due to intercellular deposits of overexpressed perlecan. However, when perlecan is transgened into mouse epithelial tissue using the keratin 5 promoter, the tooth cannot be formed normally. Thus, a constant overexpression of perlecan interferes with normal development. The time schedule of the intraepithelial expression of perlecan seems to be controlled critically in the process of odontogenesis. In this review article, we address the current views on the structure and function of perlecan acting in the intraepithelial stromal space, with special attention to its role in tooth morphogenesis. © 2006, Japanese Association for Oral Biology. All rights reserved.

DOI： 10.2330/joralbiosci.48.233

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BACKGROUND: Oral submucous fibrosis (OSF) is a chewing habit-related pre-cancerous condition of the oral mucosa affecting predominantly south Asians. It is histopathologically characterized by epithelial atrophy and fibrosis of the subepithelial connective tissue. Fibrosis extends all the way into the muscle layer, leading to difficulty in mouth opening. However, the dynamics of extracellular matrix (ECM) remodeling with OSF progression is largely unknown. METHODS: Forty biopsy specimens of OSF and 10 of normal buccal mucosa were examined for expression/deposition modes of eight ECM molecules by histochemistry, immunohistochemistry, and in situ hybridization. RESULTS: In the early stage of OSF, tenascin, perlecan, fibronectin, collagen type III were characteristically enhanced in the lamina propria and the submucosal layer. In the intermediate stage, the ECM molecules mentioned above and elastin were extensively and irregularly deposited around muscle fibers. In the advanced stage, such ECM depositions decreased and were entirely replaced with collagen type I only. Their gene expression levels varied with progression of fibrosis, but the mRNA signals were confirmed in fibroblasts in the submucosal fibrotic areas. CONCLUSIONS: The results indicate that the ECM remodeling steps in OSF are similar to each phase of usual granulation tissue formation. Restricted mouth opening may be a result of loss of variety of ECM molecules including elastin into the homogeneity of collagen type I replacing muscle fibers.

DOI： 10.1111/j.1600-0714.2005.00339.x

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To better understand the poorly vascularized background of the stroma of pleomorphic adenomas, we attempted to determine the expression of molecules related to blood vessels and hypoxic conditions in pleomorphic adenoma. Surgical specimens and tumor cells in primary culture of salivary pleomorphic adenomas were used for immunohistochemistry for CD31, vascular endothelial growth factor (VEGF) and its receptors Flk-1 and Flt-1, as well as for hypoxia markers, such as hypoxia-inducible factor-1alpha (HIF-1alpha) and lactate dehydrogenase-1 (LDH). At the same time, alternative splicing modes of the VEGF gene and expression levels of the HIF-1alpha gene were analyzed in surgical specimens by means of reverse-transcription polymerase chain reaction (RT-PCR) and direct sequencing of the PCR products. In addition to co-immunolocalization with CD31+ vascular endothelial cells, VEGF and its receptors were demonstrated in normal duct epithelial and myoepithelial cells as well as in tumor cells in ductal structures and in myxochondroid stromata. Immunolocalizations for HIF-1alpha and LDH were confirmed in the VEGF-positive area. Immunofluorescence signals for VEGF and others were confirmed in pleomorphic adenoma cells in culture. RT-PCR results showed that there were at least four splicing modes of the VEGF gene, among which VEGF(121) was most enhanced, and higher HIF-1alpha levels in pleomorphic adenomas. The results suggest that pleomorphic adenoma cells produce VEGF in several functional forms for their own proliferation or differentiation, and that the VEGF expression is controlled by hypoxic circumstances of poorly vascularized pleomorphic adenomas.

DOI： 10.1007/s00428-005-1219-1

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

The localization and biosynthesis of perlecan, a basement membrane-type heparan sulfate proteoglycan, were studied in developing tooth germs by using murine molars in neonatal and postnatal stages and primary cultured cells of the enamel organ and dental papilla to demonstrate the role of perlecan in normal odontogenesis. Perlecan was immunolocalized mainly in the intercellular spaces of the enamel organ as well as in the dental papilla/pulp or in the dental follicle. By in situ hybridization, mRNA signals for perlecan core protein were intensely demonstrated in the cytoplasm of stellate reticulum cells and in dental papilla/pulp cells, including odontoblasts and fibroblastic cells in the dental follicle. Furthermore, the in vitro biosyntheses of perlecan core protein by the enamel organ and dental papilla/pulp cells were confirmed by immunofluorescence, immunoprecipitation, and reverse transcriptase-polymerase chain reaction. The results indicate that perlecan is synthesized by the dental epithelial cells and is accumulated in their intercellular spaces to form the characteristic stellate reticulum, whose function is still unknown.

DOI： 10.1369/jhc.4A6479.2005

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GD3 synthase is one of the key enzymes involved with ganglioside synthesis, and its activity regulates the main profile of ganglioside expression. We analyzed the expression of the GD3 synthase gene in laser-dissected teeth germs using RT-PCR. The GD3 synthase gene was found expressed in brain, thymus, and tooth germ tissues, however, not in liver or skin specimens. Further, it was highly expressed during the early stage of tooth germ development (embryonic day 14.5), especially in dental epithelia, which gradually reduced in the molar site until postnatal day 7, whereas it was not in dental mesenchyme tissues. In addition, dental epithelial cells transiently transfected with the GD3 synthase gene showed enhanced proliferation. These results indicate that the GD3 synthase gene may be involved in early tooth development, particularly in the proliferation of dental epithelium.

DOI： 10.1016/j.archoralbio.2004.09.014

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BACKGROUND: One of the histologic characteristics of epithelial dysplasias of the oral mucosa is droplet-shaped rete processes resulting from a solid proliferation of basaloid cells. These basaloid cells are suddenly changed into an overlay of parakeratotic cells. However, it is unknown how this characteristic two-phase appearance is generated. METHODS: Formalin-fixed paraffin sections of the oral mucosal specimens with normal, hyperplastic, dysplastic epithelia and squamous cell carcinomas were examined for apoptosis by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method and for lymphoid cells by immunohistochemistry. RESULTS: Apoptotic cells were only located in the keratinized layer of normal/hyperplastic epithelia. However, in epithelial dysplasias, apoptotic cells were scattered in the middle or even in the lower parts of the epithelial layer with frequent vacuolation changes of epithelial cells. Within the epithelial layer of dysplasias, there were increased number of lymphocytes, which were immunopositive for CD45RO, CD8, and CD57- and CD68-immunopositive (+), S-100 protein-positive and major histocompatibility complex (MHC) class II-positive monocytic lineages. They increased in number with the severity of dysplastic degrees, and they were often located in the vicinity of apoptotic epithelial cells, but decreased in carcinomas in situ and invasive carcinomas, which contained fewer numbers of apoptotic figures. CONCLUSION: The findings indicate that intraepithelial infiltrations of both cytotoxic T cells and natural killer cells are closely related to the apoptotic phenomena of prickle cells, which may result in the characteristic 'two-phase appearance' of epithelial dysplasia.

DOI： 10.1111/j.1600-0714.2004.00283.x

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A case of angiolipoma occurring in the buccal mucosa of a 69-year-old male is described. The patient had noticed a painless mass in his buccal mucosa for 2 years. The surgically removed tumor, measuring 9 mm in diameter, was mainly located in the submucosal layer with focal expansion into the muscle layer. Histologically, the tumor was well-demarcated and composed of proliferations of mature fat cells and fibrous connective tissue containing many small blood vessels, which were evenly distributed. There was diffuse infiltration of a large number of mast cells, which were immunopositive for vascular endothelial growth factor (VEGF) especially around blood vessels, suggesting that VEGF produced by mast cells in angiolipomas plays an important role in the vascular proliferation in this particular tumor.

DOI： 10.1111/j.1600-0714.2004.00230.x

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BACKGROUND: Mucous retention cysts contain a mucous pool in the lumina, in which pure angiogenic processes are occasionally observed. By using this unique human material, our aim was to understand the in vivo angiogenic process. METHODS: Fifteen surgical tissue samples of mucous retention cysts of the lip were examined for expression of vascular endothelial markers and extracellular matrix molecules by immunohistochemistry and in situ hybridization (ISH). RESULTS: Endothelial cells forming new vascular channels showed immunopositivities for CD31, CD34, vascular endothelial growth factor (VEGF), and von Willebrand factor (vWF). These newly formed capillaries were surrounded by tenascin-positive matrices and further by a dense infiltration of CD68-positive cells with foamy to epitheloid appearances. Some of these cells were simultaneously positive for CD34, VEGF, and one of its receptors, Flk-1, and they showed definite mRNA as well as protein signals for tenascin. In addition, these cells often tended to be aligned, which suggested tubule formation. CONCLUSION: The results suggest that monocyte/macrophage lineage cells are a major source for endothelial cells at least in mucous retention cysts and that tenascin produced by those cells plays an important role in differentiation of endothelial cells.

DOI： 10.1111/j.1600-0714.2004.00282.x

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Osteoclasts in osteopetrotic (op/op) mice are substantially reduced by the absence of functional activities of macrophage colony-stimulating factor (M-CSF). However, it is known that osteoclasts appear in op/op skeletal bones with aging, although the molecular mechanism for this is unknown. In order to investigate osteoclastic recruitment in the jaw bones of op/op mice, osteoclastic distribution was analysed for 2 yr after birth by histochemistry for tartrate-resistant acid phosphatase activity and immunohistochemistry for cathepsin K. Osteoclasts in op/op mandibular bones decreased rapidly in number after birth and disappeared by 3 d, although there was no difference in the osteoclastic distribution between op/op and normal littermates at birth. At 2 wk, osteoclasts began to reappear around op/op tooth germs, where no apparent connective tissue layer intervened between tooth germs and bone trabeculae. They increased in number and were scattered over the mandible, reaching a maximum at 8 wk, when periodontal ligament-like structures were recognizable around incisor germs. Osteoclasts then again decreased gradually, and after 62 wk few osteoclasts were seen in op/op mandibular bones, whose marrow space disappeared. These findings suggest that osteoclasts are recruited in an M-CSF-independent manner in op/op mandibles, especially in areas around tooth germs.

DOI： 10.1111/j.0909-8836.2004.00109.x

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BACKGROUND: Intercellular deposition of perlecan, a major heparan sulfate proteoglycan (HSPG) of the basement membrane, is known to result in characteristic stellate reticulum-like structures in ameloblastomas or tooth germs. Although enlargement of the intercellular space is one of the histological characteristics of epithelial dysplasia of oral mucosa, the mode of expression of perlecan is poorly understood in these epithelial lesions. METHODS: Eighty-two biopsy specimens consisting of normal and hyperplastic epithelium, epithelial dysplasia, and squamous cell carcinomas were examined for both perlecan core protein and heparan sulfate (HS) chains by immunohistochemistry and in situ hybridization. RESULTS: In normal and hyperplastic epithelium, perlecan core protein and HS chains were localized in the cell border of parabasal cells and lower prickle cells, and HS chains were also found in basal cells. With an increase in the severity of epithelial dysplasia, the core protein was heavily and extensively deposited in the interepithelial space as well as in the cytoplasm of epithelial cells from the basal to the surface layers. Its gene expression was confirmed in the cells around the protein deposits. On the other hand, HS chains were enhanced in mild dysplasia, but decreased in moderate and severe dysplasias. In squamous cell carcinomas, either the core protein or HS chains were found scarcely in tumor cells but abundantly in the stromal space. CONCLUSIONS: The findings indicate that perlecan is localized in the intercellular space of the oral epithelia, and that it is over-expressed in dysplastic epithelial cells and is deposited in their interepithelial space, which results in the histology of reduction of cellular cohesion.

DOI： 10.1111/j.1600-0714.2004.00026.x

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Language：English   Publisher：Elsevier BV  

A case of solitary fibrous tumor of the Lip is described. A 65-year-old Japanese woman had a painless mass in her lower lip that gradually increased in size and finally ulcerated. Computed tomography revealed a well-demarcated submucosal mass. On the cut surface, the tumor was well-circumscribed, solid, and yellowish-white with small cystic spaces. Histopathologically, it was encapsulated and consisted of an interlacing proliferation of spindle-shaped cells immunopositive for CD34, vimentin, Bcl-2, and CD99, with scattered salivary glandular structures with irregular cellular arrangements. This is the first case report of solitary fibrous tumor of the lip with reactive hyperplasia of minor salivary gland components based on our review of the literature. © 2004 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.ooe.2004.06.002

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Language：English   Publisher：BLACKWELL MUNKSGAARD  

DOI： 10.1034/j.1600-0714.2003.t01-1-00162.x

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

We surveyed the expression of 557 cancer-related genes in 15 cases of well-differentiated OSCC by cDNA microarray analysis. To identify potential biomarkers for lymph node metastasis, all microarray data were compared by the Mann-Whitney test and the significance analysis of microarrays between OSCCs with and those without lymph node metastasis. The tissues of OSCCs with lymph node metastasis exhibited increased expression levels of MMP-1, MMP-3, uPA, integrin-alpha3, paxillin, tenascin C and IL-6 transcripts. All of these genes were included in common clusters on the Cluster/TreeView analysis, implying that functional gene, groups of proteolytic enzymes and integrin-related molecules are involved in cervical lymph node metastasis. The results of RTQ-PCR for differentially expressed genes were in accord with those of cDNA microarray analyses, suggesting that the data obtained by microarray gene expression analyses were valid. Consistent with cooperative expression patterns, immunohistochemical analyses demonstrated that products of MMP-1, MMP-3 and uPA were colocalized to components of the neoplastic stroma, particularly mononuclear inflammatory cells with well-developed eosinophilic cytoplasm. Our results suggest that expression levels of molecules involved in tissue remodeling and cell-ECM adhesion, especially MMP-1 and integrin-alpha3, can provide an accurate biomarker system for predicting the risk of cervical lymph node metastasis in OSCC. (C) 2003 Wiley-Liss, Inc.

DOI： 10.1002/ijc.11283

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

Lymphoepithelial lesions (LELs, or epi-myoepithelial islands) in lymphoepithelial sialadenitis (LESA, or benign lymphoepithelial lesion) of the salivary gland are known to be mainly composed of duct epithelial cells. However, other constituent cells are poorly characterized. Formalin-fixed paraffin sections obtained from six surgical specimens of LESA were examined using immunohistochemistry for cytoskeletal proteins, inflammatory cells, vascular endothelial cells, and extracellular matrix (ECM) molecules as well as by in situ hybridization for ECM molecules. In addition to keratin-immunopositive (+) duct-like epithelial cells, there were CD31/CD34+ vascular endothelial cells-which were either scattered in a singular fashion, in formed sheets, or in tubular structures-, CD20+ B lymphocytes, CD45RO+ T lymphocytes, and CD68 macrophages in the LELs. ECM molecules, such as heparan sulfate proteoglycan and tenascin, were immunolocalized in hyaline materials in the LELs. Their mRNAs were demonstrated mainly in endothelial cells and, to a lesser extent, in lympho-monocytic cells around hyaline materials, but were not as evident in epithelial constituent cells of LELs. The results indicate that endothelial cells as well as inflammatory cells are important constituents of the LELs, and the hyaline ECM cores mainly result from the intra-LEL angiogenesis by endothelial cells with the assistance of inflammatory cells. This intra-LEL vasculature seems to support regeneration and proliferation of salivary epithelial remnant cells.

DOI： 10.1007/s00428-003-0824-0

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

This report describes a rare case of hypoplastic primary incisor in which the pulp was exposed at the crown portion and covered by the gingiva in a 1-year-11-month-old boy. The patient was referred to us due to swelling of his labial cervical gingiva of the maxillary right primary central incisor, and on examination, extended to the hypoplastic labial surface. Radiographically, there was a round radiolucent area on the crown including the edge. Surgical removal of the swollen gingiva revealed a large defect of the labial aspect of the incisor, showing pulpal tissue inside. The tooth was treated by vital pulpotomy. Histopathologically, the removed gingival tissue contained many pieces of dysplastic tooth elements in the lamina propria portion which should have been connected to the exposed pulp. The findings suggested that pulp exposure resulted from focal dental hypoplasia not from resorption of the tooth.

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

Background: It is still controversial if atypical tumor cells scattered in salivary pleomorphic adenomas are precancerous and how carcinoma arises in pleomorphic adenomas.
Methods: We studied clinicopathologically the frequency and variation of cellular atypia among tumor cells and examined the expression status of p53 gene products as well as proliferating cell nuclear antigen (PCNA) in 101 surgical materials of pleomorphic adenomas.
Results: Histopathologically, atypical tumor cells were found in 51% of the cases examined. Their mode of distribution was classified into three groups: focal (six cases, 6%) which could be identified as focal carcinoma, measuring less than 1 mm in diameter; sporadic (15 cases, 15%) and singular (30 cases, 30%). These atypical cells were located mainly within sheet-like nests of tumor cells but not in chondroid or fibro-hyaline foci. Immunohistochemically, most of the atypical cells were positive for p53 gene products and PCNA.
Conclusion: The results indicated that atypical cells with p53 protein accumulation in their nuclei could be regarded as cells in a precancerous state not yet forming an apparent carcinomatous nest. Some cell population with these atypical cells are likely to form focal carcinomas and then to an apparent form of carcinoma in pleomorphic adenoma.

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

The localization and biosynthesis of basement membrane-type heparan sulfate proteoglycan (HSPG), known as perlecan, were studied in ameloblastomas using surgical tissue sections and cells in primary culture to demonstrate the existence of extracellular matrix (ECM) molecules in the intercellular space of epithelial tissue. HSPG was immunolocalized in the intercellular spaces of stellate reticulum-like cells and small vacuolar structures between basal cells in tumor cell nests as well as in myxofibrous stroma. By means of in-situ hybridization, mRNA signals for the HSPG core were intensely demonstrated in the cytoplasm of basal and parabasal cells of parenchyma. Furthermore, the in-vitro biosynthesis of HSPG core protein by ameloblastoma cells was confirmed using immunofluorescence, immunoprecipitation, and reverse-transcriptase polymerase chain reaction (RT-PCR). The results indicated that ameloblastoma cells synthesize HSPG and deposit it in their intercellular space. The intercellular HSPG might act as a carrier for transport of nutrients to tumor cells within ameloblastomatous foci.

DOI： 10.1007/s00428-001-0556-y

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

BACKGROUND: Odontoma-like structures are formed in the jaw bone of osteopetrotic (op/op) mice, which have a congenital deficiency in osteoclastic differentiation due to the absence of functional macrophage colony-stimulating factor (M-CSF). METHODS: To clarify the histopathogenesis of tooth malformation and odontoma-like structures, a 2-year postnatal process of development of the op/op mandibular incisor was examined radiologically and histologically. At the same time, extracellular matrix (ECM) remodeling around tooth germs was analyzed immunohistochemically. RESULTS: Abnormal forms of op/op tooth germ were noticeable even at 3 days after birth on a radiogram. Histologically, op/op mice were clearly distinguished by the disappearance of dental follicular space at 3 days. With aging, bone trabeculae, which were not remodeled, penetrated into op/op tooth germs and divided them into several daughter germs, which were recognized as odontomas. In mandibular incisor bodies, the immature ECM components, such as heparan sulfate proteoglycan and tenascin, were preserved diffusely in the dental papilla/pulp, which indicates that maturation of the stroma does not take place in op/op mandibular incisors. CONCLUSION: The observation suggests that the disturbed morphogenesis of op/op tooth germs is functionally explained by the disordered immunolocalization of ECM molecules, and that the dental follicular space is essential for normal tooth development because it prevents bone penetration into the tooth germs.

DOI： 10.1034/j.1600-0714.2002.00087.x

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

BACKGROUND: Incisor tooth germs of osteopetrotic (op/op) mice are known to fail to erupt, but form odontomas in their root apices instead, due to invasion of alveolar bone trabeculae into the tooth germs. The purpose of this study is to determine if the tooth developmental failures in op/op mice are intrinsic or secondarily arise as a result of the defective bone metabolism due to lack of macrophage colony-stimulating factor (M-CSF). METHODS: We isolated mandibular first molar tooth germs from normal and op/op mice and cultured them under conditions with or without bone tissues which had been formed around tooth germs. RESULTS: Tooth germs from normal mice, cultured for a week, showed almost the same developmental features as those of mice with the corresponding age. They were surrounded with dental follicular tissues and were never invaded by bone trabeculae. On the other hand, op/op tooth germs cultured in the presence of bone components were invaded by alveolar bone trabeculae around tooth germs in the same manner as shown in vivo. When cultured without bone, they developed without any interruptions. CONCLUSIONS: These findings indicated that op/op tooth germs had potential for normal development and that their abnormal development was a secondary phenomenon caused by lack of bone remodeling in the early phase of odontogenesis.

DOI： 10.1034/j.1600-0714.2002.00138.x

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

To investigate the molecular mechanism for enhanced fibrous stroma formation in intrahepatic cholangiocarcinoma (ICC), we surveyed the expression pattern of basement-membrane-type heparan sulfate proteoglycan (HSPG; also known as perlecan) at the core protein and the mRNA level in ICC as well as in other liver neoplasms and reactive hepatic diseases. Immunohistochemistry of paraffin-embedded liver sections with hyaluronidase pretreatment showed that HSPG was present in small amounts in normal liver around the bile ducts and the blood vessels within the portal area. There was no evident expression within the hepatic lobules, Intense immunoexpression of HSPG was seen in the tumor-specific fibro-myxoid stroma of ICC and metastatic liver cancer originating from the colon. However, tumor-specific stroma of hepatocellular carcinomas showed little or no expression of HSPG, At the mRNA level, signals for HSPG were found in tumor cells of cholangiocarcinoma and metastatic colonic carcinomas, and in myofibroblasts in the tumor fibro-myxoid-specific stroma, From immunoprecipitation and reverse transcription-polymerase chain reaction (RT-PCR) analyses, a cultured human intrahepatic cholangiocarcinoma cell line (CCKS1), was found to express high levels of HSPG core protein and mRNA, These findings suggest that biliary and metastatic colon carcinoma cells as well as stromal myofibroblasts have a potential for HSPG production. In order to investigate the growth, invasion and metastatic ability of ICC, further study of the 'self-made' stromal component of ICC may provide a new approach.

DOI： 10.1046/j.1440-1827.2001.01201.x

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

In order to determine the role of the basement membrane-type heparan sulfate proteoglycan (HSPG), known as perlecan, in the formation of the characteristic cribriform structures of salivary adenoid cystic carcinomas, the mode of expression of mRNA for the core protein of HSPG was investigated by using in situ hybridization (ISH) both in surgical specimens and in a cell system (ACC3) of adenoid cystic carcinomas. In the surgical specimens, the mRNA for the HSPG core was more intensely expressed in solid tumor cell nests, especially in smaller ones. Within the nests, the signals were detected almost exclusively in cuboidal cells forming small pseudocysts. In contrast, signals were absent in flat cells forming large pseudocysts or in carcinoma cell nests attaching to the peripheral nerves or blood vessels. In normal salivary gland tissues, myoepithelial cells expressed the mRNA at a high level, but acinar and duct epithelial cells did not. In the time-course experiment of ACC3 cells, signals for HSPG core increased with time and reached the maximum on day 4, decreasing thereafter in a culture condition in which cells reached confluence in a week. The results indicate that HSPG is biosynthesized by adenoid cystic carcinoma cells which are in the proliferation phase, and that tumor cells producing HSPG tend to form initial structures of stromal pseudocysts.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MOSBY-YEAR BOOK INC  

A case of melanotic neuroectodermal tumor of infancy occurring in the mandible is described. The patient was a 1-month-old boy with a rapidly growing tumor of the mandible. Computed tomography showed 2 well-defined osteolytic lesions in the right mandible. Histopathologic diagnosis of a biopsy specimen was melanotic neuroectodermal tumor of infancy. The tumor was excised with removal of the surrounding bone, but 1(1/2) months later it recurred, and segmental mandibulectomy and reconstruction of the defect with a titanium miniplate was performed. Retrospectively, evidence of recurrence was noted on computed tomography taken on the tenth postoperative day. The recurrence was caused by incomplete removal of the tumor. Histopathologically, the tumor cells of the recurrent lesion were dispersed extensively in the bone marrow, and bone remodeling was active. The surgical procedure may have stimulated tumor cell proliferation and reactive bone formation. The patient was followed for 2 years with no evidence of recurrence or metastasis.

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Language：Japanese   Publisher：新潟歯学会  

患者は男性3例,女性3例,年齢は33歳から64歳で全例が開業医からの紹介であった.異物について前歯科医師より告げられていたのは3例で,他は炎症症状などが出現し受診していた.迷入異物は根管充填剤2例,レジン1例,インプラント1例,エレベーターの破折片1例,アルジネート印象材1例で,迷入部位は上顎洞4例,下顎部2例で全例右側であった.全例で抗生剤等の投薬処置がなされており2例では異物の摘出が試みられていた.迷入より当科来院迄の期間は3日から6年と幅広く,初診時には全例に何らかの炎症症状が認められた.全例異物は外科的に摘出されたが,上顎洞根治術を施行したものが2例であった

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Language：Japanese   Publisher：Niigata University  

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Other Link： http://search.jamas.or.jp/link/ui/2006109734

Language：Japanese   Publisher：歯科基礎医学会  

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Language：Japanese   Publisher：The Japanese Society of Pediatric Dentistry  

DOI： 10.11411/jspd1963.42.2_190

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Language：English   Publisher：Japanese Association for Oral Biology  

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Language：English   Publisher：Japanese Society for Oral Pathology  

A 81-year-old female had suffered from a white lesion in the right lateral margin of the tongue for 10 years. The lesions was surgically removed and examined histopathologically. The surgical specimen showed small foci of squamous cell carcinoma invading up to 4 mm in the muscle layer with a diameter of less than 7 mm in the central portion. The carcinomatous foci were surrounded by epithelial dysplasia in various degrees with a dense lymphocytic infiltration in the lamina propriae. Some of the dysplasia parts just next to the carcinomatous foci contained obviously atypical cells without basal cell alignment but with an apparent keratinizing tendency, which could not be otherwise diagnosed as carcinoma in-situ. Based on this case report, a new concept of carcinoma in-situ of the oral mucosa was proposed, because the histology was different in terms of keratinization degree from so-called carcinoma in-situ as frequently seen in the cervix uteri, which were mainly composed of proliferation of basaloid cells.

DOI： 10.3353/omp.8.43

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Other Link： http://search.jamas.or.jp/link/ui/2004123235

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Other Link： http://search.jamas.or.jp/link/ui/2003003825

Language：English   Publisher：Japanese Association for Oral Biology  

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Other Link： http://search.jamas.or.jp/link/ui/2001034633

Language：Japanese   Publisher：Niigata University  

We report a rare case of a trichofolliculoma of the upper lip in a 48-year-old Japanese man. The tumor, measuring 12×11×11mm in size, was located in the dermis to subcutaneous layer around the mucocutaneous border of his upper lip. There was a small fistula-like pit in the skin covering the tumor. The patient had noticed the tumor for 15 years, during which it grew gradually with occasional excretion of white-colored cheese-like substance from the pit. Its clinical diagnosis was atheroma. Histopathologically, it consisted of a nodular proliferation of fibroblasts with fibrous connective tissues, within which there was a cystic space lined by skin with plentiful dermal appendages. This was the second case report of a labial trichofolliculoma from dental institutions.

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Other Link： http://search.jamas.or.jp/link/ui/2001031618

Language：Japanese   Publisher：(一社)日本病理学会  

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DOI： 10.3353/omp.4.79

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Language：Japanese   Presentation type：Oral presentation (general)  

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