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Lipin2 is a phosphatidate phosphatase that plays critical roles in fat homeostasis. Alterations in Lpin2, which encodes lipin2, cause the autoinflammatory bone disorder Majeed syndrome. Lipin2 limits lipopolysaccharide (LPS)‐induced inflammatory responses in macrophages. However, little is known about the precise molecular mechanisms underlying its anti‐inflammatory function. In this study, we attempted to elucidate the molecular link between the loss of lipin2 function and autoinflammatory bone disorder. Using a Lpin2 knockout murine macrophage cell line, we showed that lipin2 deficiency enhances innate immune responses to LPS stimulation through excessive activation of the NF‐κB signaling pathway, partly because of TAK1 signaling upregulation. Lipin2 depletion also enhanced RANKL‐mediated osteoclastogenesis and osteoclastic resorption activity accompanied by NFATc1 dephosphorylation and increased nuclear accumulation. These results suggest that lipin2 suppresses the development of autoinflammatory bone disorder by fine‐tuning proinflammatory responses and osteoclastogenesis in macrophages. Therefore, this study provides insights into the molecular pathogenesis of monogenic autoinflammatory bone disorders and presents a potential therapeutic intervention.

DOI： 10.3390/ijms22062893

Language：English  

A subpopulation of mesenchymal stem cells, developmentally derived from multipotent neural crest cells that form multiple facial tissues, resides within the dental pulp of human teeth. These stem cells show high proliferative capacity in vitro and are multipotent, including adipogenic, myogenic, osteogenic, chondrogenic, and neurogenic potential. Teeth containing viable cells are harvested via minimally invasive procedures, based on various clinical diagnoses, but then usually discarded as medical waste, indicating the relatively low ethical considerations to reuse these cells for medical applications. Previous studies have demonstrated that stem cells derived from healthy subjects are an excellent source for cell-based medicine, tissue regeneration, and bioengineering. Furthermore, stem cells donated by patients affected by genetic disorders can serve as in vitro models of disease-specific genetic variants, indicating additional applications of these stem cells with high plasticity. This review discusses the benefits, limitations, and perspectives of patient-derived dental pulp stem cells as alternatives that may complement other excellent, yet incomplete stem cell models, such as induced pluripotent stem cells, together with our recent data.

DOI： 10.3390/ijms22052269

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© 2020, The Author(s). Space flight produces an extreme environment with unique stressors, but little is known about how our body responds to these stresses. While there are many intractable limitations for in-flight space research, some can be overcome by utilizing gene knockout-disease model mice. Here, we report how deletion of Nrf2, a master regulator of stress defense pathways, affects the health of mice transported for a stay in the International Space Station (ISS). After 31 days in the ISS, all flight mice returned safely to Earth. Transcriptome and metabolome analyses revealed that the stresses of space travel evoked ageing-like changes of plasma metabolites and activated the Nrf2 signaling pathway. Especially, Nrf2 was found to be important for maintaining homeostasis of white adipose tissues. This study opens approaches for future space research utilizing murine gene knockout-disease models, and provides insights into mitigating space-induced stresses that limit the further exploration of space by humans.

DOI： 10.1038/s42003-020-01227-2

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Dental enamel, the hardest tissue in the human body, is derived from dental epithelial cell ameloblast-secreted enamel matrices. Enamel mineralization occurs in a strictly synchronized manner along with ameloblast maturation in association with ion transport and pH balance, and any disruption of these processes results in enamel hypomineralization. G protein-coupled receptors (GPCRs) function as transducers of external signals by activating associated G proteins and regulate cellular physiology. Tissue-specific GPCRs play important roles in organ development, although their activities in tooth development remain poorly understood. The present results show that the adhesion GPCR Gpr115 (Adgrf4) is highly and preferentially expressed in mature ameloblasts and plays a crucial role during enamel mineralization. To investigate the in vivo function of Gpr115, knockout (Gpr115-KO) mice were created and found to develop hypomineralized enamel, with a larger acidic area because of the dysregulation of ion composition. Transcriptomic analysis also revealed that deletion of Gpr115 disrupted pH homeostasis and ion transport processes in enamel formation. In addition, in vitro analyses using the dental epithelial cell line cervical loop-derived dental epithelial (CLDE) cell demonstrated that Gpr115 is indispensable for the expression of carbonic anhydrase 6 (Car6), which has a critical role in enamel mineralization. Furthermore, an acidic condition induced Car6 expression under the regulation of Gpr115 in CLDE cells. Thus, we concluded that Gpr115 plays an important role in enamel mineralization via regulation of Car6 expression in ameloblasts. The present findings indicate a novel function of Gpr115 in ectodermal organ development and clarify the molecular mechanism of enamel formation.

DOI： 10.1074/jbc.RA120.014281

Language：Others  

Dental enamel is hardest tissue in the body and is produced by dental epithelial cells residing in the tooth. Their cell fates are tightly controlled by transcriptional programs that are facilitated by fate determining transcription factors and chromatin regulators. Understanding the transcriptional program controlling dental cell fate is critical for our efforts to build and repair teeth. In this review, we describe the current understanding of these regulators essential for regeneration of dental epithelial stem cells and progeny, which are identified through transgenic mouse models. We first describe the development and morphogenesis of mouse dental epithelium in which different subpopulations of epithelia such as ameloblasts contribute to enamel formation. Then, we describe the function of critical factors in stem cells or progeny to drive enamel lineages. We also show that gene mutations of these factors are associated with dental anomalies in craniofacial diseases in humans. We also describe the function of the master regulators to govern dental lineages, in which the genetic removal of each factor switches dental cell fate to that generating hair. The distinct and related mechanisms responsible for the lineage plasticity are discussed. This knowledge will lead us to develop a potential tool for bioengineering new teeth.

DOI： 10.3390/ijms21238952

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© 2020 Federation of American Societies for Experimental Biology Proteasome inhibitors exert an anabolic effect on bone formation with elevated levels of osteoblast markers. These findings suggest the important role of the proteasomal degradation of osteogenic regulators, while the underlying molecular mechanisms are not fully understood. Here, we report that the proteasome inhibitors bortezomib and ixazomib markedly increased protein levels of the osteoblastic key transcription factor osterix/Sp7 (Osx). Furthermore, we revealed that Osx was targeted by p38 and Fbw7 for proteasomal degradation. Mechanistically, p38-mediated Osx phosphorylation at S73/77 facilitated Fbw7 interaction to trigger subsequent Osx ubiquitination. Consistent with these findings, p38 knockdown or pharmacological p38 inhibition resulted in Osx protein stabilization. Treatment with p38 inhibitors following osteogenic stimulation efficiently induced osteoblast differentiation through Osx stabilization. Conversely, pretreatment of p38 inhibitor followed by osteogenic challenge impaired osteoblastogenesis via suppressing Osx expression, suggesting that p38 exerts dual but opposite effects in the regulation of Osx level to fine-tune its activity during osteoblast differentiation. Furthermore, Fbw7-depleted human mesenchymal stem cells and primary mouse calvarial cells resulted in increased osteogenic capacity. Together, our findings unveil the molecular mechanisms underlying the Osx protein stability control and suggest that targeting the Osx degradation pathway could help enhance efficient osteogenesis and bone matrix regeneration.

DOI： 10.1096/fj.202001340R

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There is growing evidence showing that tight junctions play an important role in developing enamel. Claudins are one of the main components of tight junctions and may have pivotal functions in modulating various cellular events, such as regulating cell differentiation and proliferation. Mutations in CLDN10 of humans are associated with HELIX syndrome and cause enamel defects. However, current knowledge regarding the expression patterns of claudins and the function of Cldn10 during tooth development remains fragmented. In this study, we aimed to analyze the expression patterns of claudin family members during tooth development and to investigate the role of Cldn10 in amelogenesis. Using cap analysis gene expression of developing mouse tooth germs compared with that of the whole body, we found that Cldn1 and Cldn10 were highly expressed in the tooth. Furthermore, single-cell RNA-sequence analysis using 7-day postnatal Krt14-RFP mouse incisors revealed Cldn1 and Cldn10 exhibited distinct expression patterns. Cldn10 has two isoforms, Cldn10a and Cldn10b, but only Cldn10b was expressed in the tooth. Immunostaining of developing tooth germs revealed claudin-10 was highly expressed in the inner enamel epithelium and stratum intermedium. We also found that overexpression of Cldn10 in the dental epithelial cell line, SF2, induced alkaline phosphatase (Alpl) expression, a marker of maturated stratum intermedium. Our findings suggest that Cldn10 may be a novel stratum intermedium marker and might play a role in cytodifferentiation of stratum intermedium.

DOI： 10.3389/fcell.2020.595593

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

Recently, it was suggested that the nitrite (NO2-) produced from NO3- by oral bacteria might contribute to oral and general health. Therefore, we aimed to clarify the detailed information about the bacterial NO2-production in the oral biofilm. Dental plaque and tongue-coating samples were collected, then the NO2-producing activity was measured. Furthermore, the composition of the NO2--producing bacterial population were identified using the Griess reagent-containing agar overlay method and molecular biological method. NO2--producing activity per mg wet weight varied among individuals but was higher in dental plaque. Additionally, anaerobic bacteria exhibited higher numbers of NO2--producing bacteria, except in the adults' dental plaque. The proportion of NO2--producing bacteria also varied among individuals, but a positive correlation was found between NO2--producing activity and the number of NO2--producing bacteria, especially in dental plaque. Overall, the major NO2--producing bacteria were identified as Actinomyces, Schaalia, Veillonella and Neisseria. Furthermore, Rothia was specifically detected in the tongue coatings of children. These results suggest that dental plaque has higher NO2--producing activity and that this activity depends not on the presence of specific bacteria or the bacterial compositions, but on the number of NO2--producing bacteria, although interindividual differences were detected.

DOI： 10.1038/s41598-020-73479-1

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© 2020 The Author(s) The transcription factor Sox21 is expressed in the epithelium of developing teeth. The present study aimed to determine the role of Sox21 in tooth development. We found that disruption of Sox21 caused severe enamel hypoplasia, regional osteoporosis, and ectopic hair formation in the gingiva in Sox21 knockout incisors. Differentiation markers were lost in ameloblasts, which formed hair follicles expressing hair keratins. Molecular analysis and chromatin immunoprecipitation sequencing indicated that Sox21 regulated Anapc10, which recognizes substrates for ubiquitination-mediated degradation, and determined dental-epithelial versus hair follicle cell fate. Disruption of either Sox21 or Anapc10 induced Smad3 expression, accelerated TGF-β1-induced promotion of epithelial-to-mesenchymal transition (EMT), and resulted in E-cadherin degradation via Skp2. We conclude that Sox21 disruption in the dental epithelium leads to the formation of a unique microenvironment promoting hair formation and that Sox21 controls dental epithelial differentiation and enamel formation by inhibiting EMT via Anapc10.

DOI： 10.1016/j.isci.2020.101329

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© 2020 Elsevier Inc. Lipin-2 is a phosphatidate phosphatase with key roles in regulating lipid storage and energy homeostasis. LPIN2-genetic deficiency is associated with an autoinflammatory disorder, underscoring its critical role in innate immune signaling; however, the regulatory mechanisms underlying protein stability remain unknown. Here, we demonstrate that Lipin-2 interacts with β-TRCP, a substrate receptor subunit of the SCFβ-TRCP E3 ligase, and undergoes ubiquitination and proteasomal degradation. β-TRCP-knockout in RAW264.7 macrophages resulted in Lipin-2 accumulation, leading to the suppression of LPS-induced MAPK activation and subsequent proinflammatory gene expression. Consistent with this, treatment with MLN4924, a Cullin-neddylation inhibitor that suppresses SCF E3 activity, increased Lipin-2 protein and concomitantly decreased Il1b expression. These findings suggested that β-TRCP-mediated Lipin-2 degradation affects macrophage-elicited proinflammatory responses and could lead to new therapeutic approaches to treat inflammatory diseases.

DOI： 10.1016/j.bbrc.2020.01.119

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© 2020 Elsevier Inc. Metatropic dysplasia (MD) is a congenital skeletal dysplasia characterized by severe platyspondyly and dumbbell-like long-bone deformities. These skeletal phenotypes are predominantly caused by autosomal dominant gain-of-function (GOF) mutations in transient receptor potential vanilloid 4 (TRPV4), which encodes a nonselective Ca2+-permeable cation channel. Previous studies have shown that constitutive TRPV4 channel activation leads to irregular chondrogenic proliferation and differentiation, and thus to the disorganized endochondral ossification seen in MD. Therefore, the present study investigated the role of TRPV4 in osteoblast differentiation and MD pathogenesis. Specifically, the behavior of osteoblasts differentiated from patient-derived dental pulp stem cells carrying a heterozygous single base TRPV4 mutation, c.1855C > T (p.L619F) was compared to that of osteoblasts differentiated from isogenic control cells (in which the mutation was corrected using the CRISPR/Cas9 system). The mutant osteoblasts exhibited enhanced calcification (indicated by intense Alizarin Red S staining), increased intracellular Ca2+ levels, strongly upregulated runt-related transcription factor 2 and osteocalcin expression, and increased expression and nuclear translocation of nuclear factor-activated T cell c1 (NFATc1) compared to control cells. These results suggest that the analyzed TRPV4 GOF mutation disrupts osteoblastic differentiation and induces MD-associated disorganized endochondral ossification by increasing Ca2+/NFATc1 pathway activity. Thus, inhibiting the NFATc1 pathway may be a promising potential therapeutic strategy to attenuate skeletal deformities in MD.

DOI： 10.1016/j.bbrc.2019.12.123

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

Epithelial-mesenchymal interaction has critical roles for organ development including teeth, during which epithelial thickening and mesenchymal condensation are initiated by precise regulation of the signaling pathway. In teeth, neural crest-derived mesenchymal cells expressed PDGF receptors migrate and become condensed toward invaginated epithelium. To identify the molecular mechanism of this interaction, we explored the specific transcriptional start sites (TSSs) of tooth organs using cap analysis of gene expression (CAGE). We identified a tooth specific TSS detected in the chromosome 15qD1 region, which codes microRNA-875 (mir875). MiR875-5p is specifically expressed in dental mesenchyme during the early stage of tooth development. Furthermore, PRRX1/2 binds to the mir875 promoter region and enhances the expression of mir875. To assess the role of miR875-5p in dental mesenchyme, we transfected mimic miR875-5p into mouse dental pulp (mDP) cells, which showed that cell migration toward dental epithelial cells was significantly induced by miR875-5p via the PDGF signaling pathway. Those results also demonstrated that miR875-5p induces cell migration by inhibiting PTEN and STAT1, which are regulated by miR875-5p as part of post-transcriptional regulation. Together, our findings indicate that tooth specific miR875-5p has important roles in cell condensation of mesenchymal cells around invaginated dental epithelium and induction of epithelial-mesenchymal interaction.

DOI： 10.1038/s41598-020-61693-w

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© 2019, The Author(s). Pannexin 3 (Panx3) is a regulator of bone formation. Panx3 forms three distinct functional channels: hemichannels, gap junctions, and endoplasmic reticulum (ER) Ca2+ channels. However, the gating mechanisms of the Panx3 channels remain unclear. Here, we show that the Panx3 ER Ca2+ channel is modulated by phosphorylation of the serine 68 residue (Ser68) to promote osteoblast differentiation. Among the 17 candidate phosphorylation sites identified, the mutation of Ser68 to Ala (Ser68Ala) was sufficient to inhibit Panx3-mediated osteoblast differentiation via reduction of Osterix and ALP expression. Using a Ser68 phospho-specific antibody (P-Panx3) revealed Panx3 was phosphorylated in prehypertrophic, hypertrophic chondrocytes, and bone areas of the newborn growth plate. In osteogenic C2C12 cells, P-Panx3 was located on the ER membranes. Importantly, the Ser68Ala mutation only affected Panx3 ER Ca2+ channel function. Ser68 on Panx3 was phosphorylated by ATP stimulation and PI3K/Akt signaling. Finally, real-time FRET imaging and ratio analysis revealed that the Panx3 channel conformation was sensitive to ATP. Together, the phosphorylation of Panx3 at Ser68 is an essential step controlling the gating of the Panx3 ER Ca2+ channel to promote osteogenesis.

DOI： 10.1038/s41598-019-55371-9

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

The transcription factor AmeloD stimulates epithelial cell motility essential for tooth morphology
The development of ectodermal organs, such as teeth, requires epithelial-mesenchymal interactions. Basic-helix-loop-helix (bHLH) transcription factors regulate various aspects of tissue development, and we have previously identified a bHLH transcription factor, AmeloD, from a tooth germ cDNA library. Here, we provide both in vitro and in vivo evidence that AmeloD is important in tooth development. We created AmeloD knockout (KO) mice to identify the in vivo functions of AmeloD that are critical for tooth morphogenesis. We found that AmeloD KO mice developed enamel hypoplasia and small teeth because of increased expression of E-cadherin in inner enamel epithelial (IEE) cells and it may cause inhibition of the cellmigration. We used the CLDE dental epithelial cell line to conduct further mechanistic analyses to determine if AmeloD overexpression in CLDE cells suppresses E-cadherin expression and promotes cell migration. Knockout of epiprofin (Epfn), another transcription factor required for tooth morphogenesis and development, and analysis of AmeloD expression and deletion revealed that AmeloD also contributed to multiple tooth formation in Epfn KO mice by promoting the invasion of dental epithelial cells into the mesenchymal region. Thus, AmeloD appears to play an important role in tooth morphogenesis by modulating E-cadherin and dental epithelial-mesenchymal interactions. These findings provide detailed insights into the mechanism of ectodermal organ development.

DOI： 10.1074/jbc.RA118.005298

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Basic-helix-loop-helix (bHLH) transcription factors play an important role in various organs’ development; however, a tooth-specific bHLH factor has not been reported. In this study, we identified a novel tooth-specific bHLH transcription factor, which we named AmeloD, by screening a tooth germ complementary DNA (cDNA) library using a yeast 2-hybrid system. AmeloD was mapped onto the mouse chromosome 1q32. Phylogenetic analysis showed that AmeloD belongs to the achaete-scute complex-like (ASCL) gene family and is a homologue of ASCL5. AmeloD was uniquely expressed in the inner enamel epithelium (IEE), but its expression was suppressed after IEE cell differentiation into ameloblasts. Furthermore, AmeloD expression showed an inverse expression pattern with the epithelial cell-specific cell–cell adhesion molecule E-cadherin in the dental epithelium. Overexpression of AmeloD in dental epithelial cell line CLDE cells resulted in E-cadherin suppression. We found that AmeloD bound to E-box cis-regulatory elements in the proximal promoter region of the E-cadherin gene. These results reveal that AmeloD functions as a suppressor of E-cadherin transcription in IEE cells. Our study demonstrated that AmeloD is a novel tooth-specific bHLH transcription factor that may regulate tooth development through the suppression of E-cadherin in IEE cells.

DOI： 10.1177/0022034518808254

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© 2018 Wiley Periodicals, Inc. Background: A biotooth is defined as a complete living tooth, made in laboratory cultures from a spontaneous interplay between epithelial and mesenchymal cell-based frontal systems. A good solution to these problems is to use induced pluripotent stem cells (iPSCs). However, no one has yet formulated culture conditions that effectively differentiate iPSCs into dental epithelial and dental mesenchymal cells phenotypes analogous to those present in tooth development. Results: Here, we tried to induce differentiation methods for dental epithelial cells (DEC) and dental mesenchymal cells from iPSCs. For the DEC differentiation, the conditional media of SF2 DEC was adjusted to embryoid body. Moreover, we now report on a new cultivation protocol, supported by transwell membrane cell culture that make it possible to differentiate iPSCs into dental epithelial and mesenchymal cells with abilities to initiate the first stages in de novo tooth formation. Conclusions: Implementation of technical modifications to the protocol that maximize the number and rate of iPSC differentiation, into mesenchymal and epithelial cell layers, will be the next step toward growing an anatomically accurate biomimetic tooth organ. Developmental Dynamics 248:129–139, 2019. © 2018 Wiley Periodicals, Inc.

DOI： 10.1002/dvdy.24663

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The transcription factor NKX2-3 mediates p21 expression and ectodysplasin-A signaling in the enamel knot for cusp formation in tooth development
Tooth morphogenesis is initiated by reciprocal interactions between the ectoderm and neural crest-derived mesenchyme. During tooth development, tooth cusps are regulated by precise control of proliferation of cell clusters, termed enamel knots, that are present among dental epithelial cells. The interaction of ectodysplasin-A (EDA) with its receptor, EDAR, plays a critical role in cusp formation by these enamel knots, and mutations of these genes is a cause of ectodermal dysplasia. It has also been reported that deficiency in , encoding a member of the NK2 homeobox family of transcription factors, leads to cusp absence in affected teeth. However, the molecular role of NKX2-3 in tooth morphogenesis is not clearly understood. Using gene microarray analysis in mouse embryos, we found that is highly expressed during tooth development and increased during the tooth morphogenesis, especially during cusp formation. We also demonstrate that NKX2-3 is a target molecule of EDA and critical for expression of the cell cycle regulator p21 in the enamel knot. Moreover, NKX2-3 activated the bone morphogenetic protein (BMP) signaling pathway by up-regulating expression levels of and in dental epithelium and decreased the expression of the dental epithelial stem cell marker SRY box 2 (SOX2). Together, our results indicate that EDA/NKX2-3 signaling is essential for enamel knot formation during tooth morphogenesis in mice.

DOI： 10.1074/jbc.RA118.003373

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

© 2017 The Author(s). The extracellular environment regulates the dynamic behaviors of cells. However, the effects of hydrostatic pressure (HP) on cell fate determination of mesenchymal stem cells (MSCs) are not clearly understood. Here, we established a cell culture chamber to control HP. Using this system, we found that the promotion of osteogenic differentiation by HP is depend on bone morphogenetic protein 2 (BMP2) expression regulated by Piezo type mechanosensitive ion channel component 1 (PIEZO1) in MSCs. The PIEZO1 was expressed and induced after HP loading in primary MSCs and MSC lines, UE7T-13 and SDP11. HP and Yoda1, an activator of PIEZO1, promoted BMP2 expression and osteoblast differentiation, whereas inhibits adipocyte differentiation. Conversely, PIEZO1 inhibition reduced osteoblast differentiation and BMP2 expression. Furthermore, Blocking of BMP2 function by noggin inhibits HP induced osteogenic maker genes expression. In addition, in an in vivo model of medaka with HP loading, HP promoted caudal fin ray development whereas inhibition of piezo1 using GsMTx4 suppressed its development. Thus, our results suggested that PIEZO1 is responsible for HP and could functions as a factor for cell fate determination of MSCs by regulating BMP2 expression.

DOI： 10.1038/s41598-017-18089-0

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© 2017 Elsevier Inc. Hajdu-Cheney syndrome (HCS), a rare autosomal disorder caused by heterozygous mutations in NOTCH2, is clinically characterized by acro-osteolysis, severe osteoporosis, short stature, neurological symptoms, cardiovascular defects, and polycystic kidneys. Recent studies identified that aberrant NOTCH2 signaling and consequent osteoclast hyperactivity are closely associated with the bone-related disorder pathogenesis, but the exact molecular mechanisms remain unclear. Here, we demonstrate that sustained osteoclast activity is largely due to accumulation of NOTCH2 carrying a truncated C terminus that escapes FBW7-mediated ubiquitination and degradation. Mice with osteoclast-specific Fbw7 ablation revealed osteoporotic phenotypes reminiscent of HCS, due to elevated Notch2 signaling. Importantly, administration of Notch inhibitors in Fbw7 conditional knockout mice alleviated progressive bone resorption. These findings highlight the molecular basis of HCS pathogenesis and provide clinical insights into potential targeted therapeutic strategies for skeletal disorders associated with the aberrant FBW7/NOTCH2 pathway as observed in patients with HCS. Fukushima et al. demonstrated that the sustained osteoclast activity in Hajdu-Cheney syndrome (HCS) is largely due to elevated protein abundance of the C terminus truncating NOTCH2 mutant that escapes FBW7-mediated ubiquitination and proteolysis, suggesting that the FBW7/NOTCH2 signaling pathway is a potential therapeutic target for osteolytic bone disorders, including HCS.

DOI： 10.1016/j.molcel.2017.10.018

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Tooth enamel is mineralized through the differentiation of multiple dental epithelia including ameloblasts and the stratum intermedium (SI), and this differentiation is controlled by several signaling pathways. Previously, we demonstrated that the transcriptional coactivator Mediator 1 (MED1) plays a critical role in enamel formation. For instance, conditional ablation of Med1 in dental epithelia causes functional changes in incisor-specific dental epithelial stem cells, resulting in mineralization defects in the adult incisors. However, the molecular mechanism by which Med1 deficiency causes these abnormalities is not clear. Here, we demonstrated that Med1 ablation causes early SI differentiation defects resulting in enamel hypoplasia of the Med1-deficient molars. Med1 deletion prevented Notch1-mediated differentiation of the SI cells resulting in decreased alkaline phosphatase (ALPL), which is essential for mineralization. However, it does not affect the ability of ameloblasts to produce enamel matrix proteins. Using the dental epithelial SF2 cell line, we demonstrated that MED1 directly activates transcription of the Alpl gene through the stimulation of Notch1 signaling by forming a complex with cleaved Notch1–RBP-Jk on the Alpl promoter. These results suggest that MED1 may be essential for enamel matrix mineralization by serving as a coactivator for Notch1 signaling regulating transcription of the Alpl gene.

DOI： 10.1074/jbc.M117.780866

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

© This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. 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 upregulation 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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© 2016 American Society for Bone and Mineral Research The synchronization of cell proliferation and cytodifferentiation between dental epithelial and mesenchymal cells is required for the morphogenesis of teeth with the correct functional shapes and optimum sizes. Epiprofin (Epfn), a transcription factor belonging to the Sp family, regulates dental epithelial cell proliferation and is essential for ameloblast and odontoblast differentiation. Epfn deficiency results in the lack of enamel and ironically the formation of extra teeth. We investigated the mechanism underlying the functions of Epfn in tooth development through the creation of transgenic mice expressing Epfn under the control of an epithelial cell-specific K5 promoter (K5-Epfn). We found that these K5-Epfn mice developed abnormally shaped incisors and molars and formed fewer molars in the mandible. Remarkably, ameloblasts differentiated ectopically and enamel was formed on the lingual side of the K5-Epfn incisors. By contrast, ameloblasts and enamel were found only on the labial side in wild-type mice, as Follistatin (Fst) expressed in the lingual side inhibits BMP4 signaling necessary for ameloblast differentiation. We showed that Epfn transfection into the dental epithelial cell line SF2 abrogated the inhibitory activity of Fst and promoted ameloblast differentiation of SF2 cells. We found that Epfn induced FGF9 in dental epithelial cells and this dental epithelial cell-derived FGF9 promoted dental mesenchymal cell proliferation via the FGF receptor 1c (FGFR1c). Taken together, these results suggest that Epfn preserves the balance between cell proliferation and cytodifferentiation in dental epithelial and mesenchymal cells during normal tooth development and morphogenesis. © 2016 American Society for Bone and Mineral Research.

DOI： 10.1002/jbmr.3024

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© The Author(s) 2017. Whole-organ regeneration has great potential for the replacement of dysfunctional organs through the reconstruction of a fully functional bioengineered organ using three-dimensional cell manipulation in vitro. Recently, many basic studies of whole-tooth replacement using three-dimensional cell manipulation have been conducted in a mouse model. Further evidence of the practical application to human medicine is required to demonstrate tooth restoration by reconstructing bioengineered tooth germ using a postnatal large-animal model. Herein, we demonstrate functional tooth restoration through the autologous transplantation of bioengineered tooth germ in a postnatal canine model. The bioengineered tooth, which was reconstructed using permanent tooth germ cells, erupted into the jawbone after autologous transplantation and achieved physiological function equivalent to that of a natural tooth. This study represents a substantial advancement in whole-organ replacement therapy through the transplantation of bioengineered organ germ as a practical model for future clinical regenerative medicine.

DOI： 10.1038/srep44522

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© 2017 The Author(s). Tooth development is initiated by epithelial-mesenchymal interactions via basement membrane (BM) and growth factors. In the present study, we found that nephronectin (Npnt), a component of the BM, is highly expressed in the developing tooth. Npnt localizes in the BM on the buccal side of the tooth germ and shows an expression pattern opposite that of the dental epithelial stem cell marker Sox2. To identify the roles of Npnt during tooth development, we performed knockdown and overexpression experiments using ex vivo organ and dental epithelial cell cultures. Our findings showed that loss of Npnt induced ectopic Sox2-positive cells and reduced tooth germ size. Over expression of Npnt showed increased proliferation, whereas the number of Sox2-positive cells was decreased in dental epithelial cells. Npnt contains 5 EGF-like repeat domains, as well as an RGD sequence and MAM domain. We found that the EGF-like repeats are critical for Sox2 expression and cell proliferation. Furthermore, Npnt activated the EGF receptor (EGFR) via the EGF-like repeat domains and induced the PI3K-Akt signaling pathway. Our results indicate that Npnt plays a critical scaffold role in dental epithelial stem cell differentiation and proliferation, and regulates Sox2 expression during tooth development.

DOI： 10.1038/srep45181

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The SCFβ^-TRCP E3 ubiquitin ligase complex targets Lipin1 for ubiquitination and degradation to promote hepatic lipogenesis
© 2017 The Authors, some rights reserved. The SCFβ-TRCP E3 ubiquitin ligase complex plays pivotal roles in normal cellular physiology and in pathophysiological conditions. Identification of β-transducin repeat-containing protein (β-TRCP) substrates is therefore critical to understand SCFβ-TRCP biology and function. We used a β-TRCP-phosphodegron motif-specific antibody in a β-TRCP substrate screen coupled with tandem mass spectrometry and identified multiple β-TRCP substrates. One of these substrates was Lipin1, an enzyme and suppressor of the family of sterol regulatory element-binding protein (SREBP) transcription factors, which activate genes encoding lipogenic factors. We showed that SCFβ-TRCP specifically interacted with and promoted the polyubiquitination of Lipin1 in a manner that required phosphorylation of Lipin1 by mechanistic target of rapamycin 1 (mTORC1) and casein kinase I (CKI). β-TRCP depletion in HepG2 hepatocellular carcinoma cells resulted in increased Lipin1 protein abundance, suppression of SREBP-dependent gene expression, and attenuation of triglyceride synthesis. Moreover, β-TRCP1 knockout mice showed increased Lipin1 protein abundance and were protected from hepatic steatosis induced by a high-fat diet. Together, these data reveal a critical physiological function of β-TRCP in regulating hepatic lipid metabolic homeostasis in part through modulating Lipin1 stability.

DOI： 10.1126/scisignal.aah4117

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

© The Author(s) 2016. Tooth crown morphogenesis is tightly regulated by the proliferation and differentiation of dental epithelial cells. Globoside (Gb4), a globo-series glycosphingolipid, is highly expressed during embryogenesis as well as organogenesis, including tooth development. We previously reported that Gb4 is dominantly expressed in the neutral lipid fraction of dental epithelial cells. However, because its functional role in tooth development remains unknown, we investigated the involvement of Gb4 in dental epithelial cell differentiation. The expression of Gb4 was detected in ameloblasts of postnatal mouse molars and incisors. A cell culture analysis using HAT-7 cells, a rat-derived dental epithelial cell line, revealed that Gb4 did not promote dental epithelial cell proliferation. Interestingly, exogenous administration of Gb4 enhanced the gene expression of enamel extracellular matrix proteins such as ameloblastin, amelogenin, and enamelin in dental epithelial cells as well as in developing tooth germs. Gb4 also induced the expression of TrkB, one of the key receptors required for ameloblast induction in dental epithelial cells. In contrast, Gb4 downregulated the expression of p75, a receptor for neurotrophins (including neurotrophin-4) and a marker of undifferentiated dental epithelial cells. In addition, we found that exogenous administration of Gb4 to dental epithelial cells stimulated the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase signalling pathways. Furthermore, Gb4 induced the expression of epiprofin and Runx2, the positive regulators for ameloblastin gene transcription. Thus, our results suggest that Gb4 contributes to promoting the differentiation of dental epithelial cells into ameloblasts.

DOI： 10.1038/ijos.2016.35

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© 2016 American Society for Bone and Mineral Research. Matrix molecules such as the enamel-related calcium-binding phosphoprotein ameloblastin (AMBN) are expressed in multiple tissues, including teeth, bones, and cartilage. Here we have asked whether AMBN is of functional importance for timely long bone development and, if so, how it exerts its function related to osteogenesis. Adolescent AMBN-deficient mice (AMBNΔ5-6) suffered from a 33% to 38% reduction in femur length and an 8.4% shorter trunk spinal column when compared with WT controls, whereas there was no difference between adult animals. On a cellular level, AMBN truncation resulted in a shortened growth plate and a 41% to 49% reduction in the number of proliferating tibia chondrocytes and osteoblasts. Bone marrow stromal cells (BMSCs) isolated from AMBN mutant mice displayed defects in proliferation and differentiation potential as well as cytoskeleton organization. Osteogenesis-related growth factors, such as insulin-like growth factor 1 (IGF1) and BMP7, were also significantly (46% to 73%) reduced in AMBN-deficient BMSCs. Addition of exogenous AMBN restored cytoskeleton structures in AMBN mutant BMSCs and resulted in a dramatic 400% to 600% increase in BMP2, BMP7, and Col1A expression. Block of RhoA diminished the effect of AMBN on osteogenic growth factor and matrix protein gene expression. Addition of exogenous BMP7 and IGF1 rescued the proliferation and differentiation potential of AMBN-deficient BMSCs. Confirming the effects of AMBN on long bone growth, back-crossing of mutant mice with full-length AMBN overexpressors resulted in a complete rescue of AMBNΔ5-6 bone defects. Together, these data indicate that AMBN affects extracellular matrix production and cell adhesion properties in the long bone growth plate, resulting in altered cytoskeletal dynamics, increased osteogenesis-related gene expression, as well as osteoblast and chondrocyte proliferation. We propose that AMBN facilitates rapid long bone growth and an important growth spurt during the skeletogenesis of adolescent tooth-bearing vertebrates.

DOI： 10.1002/jbmr.2788

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

© 2016 International Society of Matrix Biology. The extracellular matrix (ECM) provides structural support, cell migration anchorage, cell differentiation cues, and fine-tuned cell proliferation signals during all stages of bone fracture healing, including cartilaginous callus formation, callus remodeling, and bony bridging of the fracture gap. In the present study we have defined the role of the extracellular matrix protein ameloblastin (AMBN) in fracture resistance and fracture healing of mouse long bones. To this end, long bones from WT and AMBNδ5-6 truncation model mice were subjected to biomechanical analysis, fracture healing assays, and stem cell colony formation comparisons. The effect of exogenous AMBN addition to fracture sites was also determined. Our data indicate that lack of a functional AMBN in the bone matrix resulted in 31% decreased femur bone mass and 40% reduced energy to failure. On a cellular level, AMBN function inhibition diminished the proliferative capacity of fracture repair callus cells, as evidenced by a 58% reduction in PCNA and a 40% reduction in Cyclin D1 gene expression, as well as PCNA immunohistochemistry. In terms of fracture healing, AMBN truncation was associated with an enhanced and prolonged chondrogenic phase, resulting in delayed mineralized tissue gene expression and delayed ossification of the fracture repair callus. Underscoring a role of AMBN in fracture healing, there was a 6.9-fold increase in AMBN expression at the fracture site one week after fracture, and distinct AMBN immunolabeling in the fracture gap. Finally, application of exogenous AMBN protein to bone fracture sites accelerated callus formation and bone fracture healing (33% increase in bone volume and 19% increase in bone mineral density), validating the findings of our AMBN loss of function studies. Together, these data demonstrate the functional importance of the AMBN extracellular matrix protein in bone fracture prevention and rapid fracture healing.

DOI： 10.1016/j.matbio.2016.02.007

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

Bone morphogenetic proteins (BMPs) regulate hard tissue formation, including bone and tooth. Growth differentiation factor 5 (GDF5), a known BMP, is expressed in cartilage and regulates chondrogenesis, and mutations have been shown to cause osteoarthritis. Notably, GDF5 is also expressed in periodontal ligament tissue; however, its role during tooth development is unclear. Here, we used cell culture and in vivo analyses to determine the role of GDF5 during tooth development. GDF5 and its associated BMP receptors are expressed at the protein and mRNA levels during postnatal tooth development, particularly at a stage associated with enamel formation. Furthermore, whereas BMP2 was observed to induce evidently the differentiation of enamel-forming ameloblasts, excess GDF5 induce mildly this differentiation. A mouse model harbouring a mutation in GDF5 (W408R) showed enhanced enamel formation in both the incisors and molars, but not in the tooth roots. Overexpression of the W408R GDF5 mutant protein was shown to induce BMP2-mediated mRNA expression of enamel matrix proteins and downstream phosphorylation of Smad1/5/8. These results suggest that mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-signalling.

DOI： 10.1038/srep23670

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

Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Tooth morphogenesis is initiated by reciprocal interactions between the ectoderm and neural crest-derived mesenchyme, and the Wnt signaling pathway is involved in this process. We found that Plakophilin (PKP)1, which is associated with diseases such as ectodermal dysplasia/skin fragility syndrome, was highly expressed in teeth and skin, and was upregulated during tooth development. We hypothesized that PKP1 regulates Wnt signaling via its armadillo repeat domain in a manner similar to β-catenin. To determine its role in tooth development, we performed Pkp1 knockdown experiments using ex vivo organ cultures and cell cultures. Loss of Pkp1 reduced the size of tooth germs and inhibited dental epithelial cell proliferation, which was stimulated by Wnt3a. Furthermore, transfected PKP1-emerald green fluorescent protein was translocated from the plasma membrane to the nucleus upon stimulation with Wnt3a and LiCl, which required the PKP1 N terminus (amino acids 161 to 270). Localization of PKP1, which is known as an adhesion-related desmosome component, shifted to the plasma membrane during ameloblast differentiation. In addition, Pkp1 knockdown disrupted the localization of Zona occludens 1 in tight junctions and inhibited ameloblast differentiation; the two proteins were shown to directly interact by immunoprecipitation. These results implicate the participation of PKP1 in early tooth morphogenesis as an effector of canonical Wnt signaling that controls ameloblast differentiation via regulation of the cell adhesion complex.

DOI： 10.1371/journal.pone.0152206

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© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Cell-cell interaction via the gap junction regulates cell growth and differentiation, leading to formation of organs of appropriate size and quality. To determine the role of connexin43 in salivary gland development, we analyzed its expression in developing submandibular glands (SMGs). Connexin43 (Cx43) was found to be expressed in salivary gland epithelium. In ex vivo organ cultures of SMGs, addition of the gap junctional inhibitors 18α-glycyrrhetinic acid (18α-GA) and oleamide inhibited SMG branching morphogenesis, suggesting that gap junctional communication contributes to salivary gland development. In Cx43-/- salivary glands, submandibular and sublingual gland size was reduced as compared with those from heterozygotes. The expression of Pdgfa, Pdgfb, Fgf7, and Fgf10, which induced branching of SMGs in Cx43-/- samples, were not changed as compared with those from heterozygotes. Furthermore, the blocking peptide for the hemichannel and gap junction channel showed inhibition of terminal bud branching. FGF10 induced branching morphogenesis, while it did not rescue the Cx43-/- phenotype, thus Cx43 may regulate FGF10 signaling during salivary gland development. FGF10 is expressed in salivary gland mesenchyme and regulates epithelial proliferation, and was shown to induce ERK1/2 phosphorylation in salivary epithelial cells, while ERK1/2 phosphorylation in HSY cells was dramatically inhibited by 18α-GA, a Cx43 peptide or siRNA. On the other hand, PDGF-AA and PDGF-BB separately induced ERK1/2 phosphorylation in primary cultured salivary mesenchymal cells regardless of the presence of 18α-GA. Together, our results suggest that Cx43 regulates FGF10-induced ERK1/2 phosphorylation in salivary epithelium but not in mesenchyme during the process of SMG branching morphogenesis.

DOI： 10.1074/jbc.M115.674663

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

Interaction between fibronectin and β1integrin is essential for tooth development
© 2015 Saito et al. The dental epithelium and extracellular matrix interact to ensure that cell growth and differentiation lead to the formation of teeth of appropriate size and quality. To determine the role of fibronectin in differentiation of the dental epithelium and tooth formation, we analyzed its expression in developing incisors. Fibronectin mRNA was expressed during the presecretory stage in developing dental epithelium, decreased in the secretory and early maturation stages, and then reappeared during the late maturation stage. The binding of dental epithelial cells derived from postnatal day-1 molars to a fibronectin-coated dish was inhibited by the RGD but not RAD peptide, and by a β1 integrin-neutralizing antibody, suggesting that fi-bronectin-β1 integrin interactions contribute to dental epithelial-cell binding. Because fibronectin and β1 integrin are highly expressed in the dental mesenchyme, it is difficult to determine precisely how their interactions influence dental epithelial differentiation in vivo. Therefore, we analyzed β1 integrin conditional knockout mice (Intβ 1^lox-/lox-/K14-Cre) and found that they exhibited partial enamel hypoplasia, and delayed eruption of molars and differentiation of ameloblasts, but not of odontoblasts. Furthermore, a cyst-like structure was observed during late ameloblast maturation. Dental epithelial cells from knockout mice did not bind to fibronectin, and induction of ameloblastin expression in these cells by neurotrophic factor-4 was inhibited by treatment with RGD peptide or a fibronectin siRNA, suggesting that the epithelial interaction between fibronectin and β1 integrin is important for ameloblast differentiation and enamel formation.

DOI： 10.1371/journal.pone.0121667

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

© 2014 Wiley Periodicals, Inc. Hedgehog (Hh) family members are involved in multiple cellular processes including proliferation, migration, differentiation, and cell fate determination. Recently, the novel Hh agonists Hh-Ag 1.3 and 1.7 were identified in a high-throughput screening of small molecule compounds that activate the expression of Gli1, a target of Hh signaling. This study demonstrates that Hh-Ag 1.3 and 1.7 strongly activate the expression of endogenous Gli1 and promote osteoblast differentiation in the mesenchymal stem cell line C3H10T1/2. Both compounds stimulated alkaline phosphatase activity in a dose-dependent manner, and induced osteoblast marker gene expression in C3H10T1/2 cells, which indicated that they had acquired an osteoblast identity. Of the markers, the expression of osterix/Sp7, a downstream target of runt-related transcription factor (Runx)2, was induced by Hh-Ag 1.7, which also rescued the osteoblast differentiation defect of RD-127, a mesenchymal cell line from Runx2-deficient mice. Hh-Ags also activated canonical Wnt signaling and synergized with low doses of BMP-2 to enhance osteoblastic potential. Thus, Hh-Ag 1.7 could be useful for bone healing in individuals with abnormalities in osteogenesis, such as osteoporosis patients and the elderly, and can contribute to the development of novel therapeutics for the treatment of bone fractures and defects.

DOI： 10.1002/jcp.24823

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

Canonical Wnt signaling and BMP promote the proliferation and differentiation of osteoprogenitors, respectively. However, the regulatory mechanism involved in the transition from proliferation to differentiation is unclear. Here, we show that Panx3 (pannexin 3) plays a key role in this transition by inhibiting the proliferation and promoting the cell cycle exit. Using primary calvarial cells and explants, C3H10T1/2 cells, and C2C12 cells, we found that Panx3 expression inhibited cell growth, whereas the inhibition of endogenous Panx3 expression increased it. We also found that the Panx3 hemichannel inhibited cell growth by promoting β-catenin degradation through GSK3β activation. Additionally, the Panx3 hemichannel inhibited cyclin D1 transcription and Rb phosphorylation through reduced cAMP/PKA/CREB signaling. Furthermore, the Panx3 endoplasmic reticulum Ca2+ channel induced the transcription and phosphorylation of p21, through the calmodulin/Smad pathway, and resulted in the cell cycle exit. Our results reveal that Panx3 is a new regulator that promotes the switch from proliferation to differentiation of osteoprogenitors via multiple Panx3 signaling pathways. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M113.523241

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Pannexin 3 Inhibits Proliferation of Osteoprogenitor Cells by Regulating Wnt and p21 Signaling.
Canonical Wnt signaling and BMP promote the proliferation and differentiation of osteoprogenitors, respectively. However, the regulatory mechanism involved in the transition from proliferation to differentiation is unclear. Here, we show that Panx3 (pannexin 3) plays a key role in this transition by inhibiting the proliferation and promoting the cell cycle exit. Using primary calvarial cells and explants, C3H10T1/2 cells, and C2C12 cells, we found that Panx3 expression inhibited cell growth, whereas the inhibition of endogenous Panx3 expression increased it. We also found that the Panx3 hemichannel inhibited cell growth by promoting -catenin degradation through GSK3 activation. Additionally, the Panx3 hemichannel inhibited cyclin D1 transcription and Rb phosphorylation through reduced cAMP/PKA/CREB signaling. Furthermore, the Panx3 endoplasmic reticulum Ca(2+) channel induced the transcription and phosphorylation of p21, through the calmodulin/Smad pathway, and resulted in the cell cycle exit. Our results reveal that Panx3 is a new regulator that promotes the switch from proliferation to differentiation of osteoprogenitors via multiple Panx3 signaling pathways.

DOI： 10.1074/jbc.M113.523241

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

Similar to embryonic stem cells, induced pluripotent stem (iPS) cells can differentiate into various cell types upon appropriate induction, and thus, may be valuable cell sources for regenerative medicine. However, iPS cells have not been reported to differentiate into odontogenic cells for tooth regeneration. Here we demonstrated that neural crest-like cells (NCLC) derived from mouse iPS cells have the potential to differentiate into odontogenic mesenchymal cells. We developed an efficient culture protocol to induce the differentiation of mouse iPS cells into NCLC. We confirmed that the cells exhibited neural crest (NC) cell markers as evidenced by immunocytochemistry, flow cytometry, and real-time reverse transcription-polymerase chain reaction. Further, in recombination cultures of NCLC and mouse dental epithelium, NCLC exhibited a gene expression pattern involving dental mesenchymal cells. Some NCLC also expressed dentin sialoprotein. Conditioned medium of mouse dental epithelium cultures further enhanced the differentiation of NCLC into odontoblasts. These results suggest that iPS cells are useful cell sources for tooth regeneration and tooth development studies. © Copyright 2012, Mary Ann Liebert, Inc.

DOI： 10.1089/scd.2011.0210

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Epithelial-mesenchymal interactions regulate the growth and morphogenesis of ectodermal organs such as teeth. Dental pulp stem cells (DPSCs) are a part of dental mesenchyme, derived from the cranial neural crest, and differentiate into dentin forming odontoblasts. However, the interactions between DPSCs and epithelium have not been clearly elucidated. In this study, we established a mouse dental pulp stem cell line (SP) comprised of enriched side population cells that displayed a multipotent capacity to differentiate into odontogenic, osteogenic, adipogenic, and neurogenic cells. We also analyzed the interactions between SP cells and cells from the rat dental epithelial SF2 line. When cultured with SF2 cells, SP cells differentiated into odontoblasts that expressed dentin sialophosphoprotein. This differentiation was regulated by BMP2 and BMP4, and inhibited by the BMP antagonist Noggin.Wealso found that mouse iPS cells cultured with mitomycin C-treated SF2-24 cells displayed an epithelial cell-like morphology. Those cells expressed the epithelial cell markers p63 and cytokeratin-14, and the ameloblast markers ameloblastin and enamelin, whereas they did not express the endodermal cell marker Gata6 or mesodermal cell marker brachyury. This is the first report of differentiation of iPS cells into ameloblasts via interactions with dental epithelium. Co-culturing with dental epithelial cells appears to induce stem cell differentiation that favors an odontogenic cell fate, which may be a useful approach for tooth bioengineering strategies.

DOI： 10.1074/jbc.M111.285874

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

Impaired hippocampal long-term potentiation and failure of learning in 1,4-N-acetylgalactosaminyltransferase gene transgenic mice
Gangliosides (sialic acid-containing glycosphingolipids) play important roles in many physiological functions, including synaptic plasticity in the hippocampus, which is considered as a cellular mechanism of learning and memory. In the present study, three types of synaptic plasticity, long-term potentiation (LTP), long-term depression (LTD) and reversal of LTP (depotentiation, DP), in the field excitatory post-synaptic potential in CA1 hippocampal neurons and learning behavior were examined in 1,4-N- acetylgalactosaminyltransferase (1,4 GalNAc-T; GM2/GD2 synthase) gene transgenic (TG) mice, which showed a marked decrease in b-pathway gangliosides (GQ1b, GT1b and GD1b) in the brain and isolated hippocampus compared with wild-type (WT) mice. The magnitude of the LTP induced by tetanus (100 pulses at 100 Hz) in TG mice was significantly smaller than that in control WT mice, whereas there was no difference in the magnitude of the LTD induced by three short trains of low-frequency stimulation (LFS) (200 pulses at 1 Hz) at 20 min intervals between the two groups of mice. The reduction in the LTP produced by delivering three trains of LFS (200 pulses at 1 Hz, 20 min intervals) was significantly greater in the TG mice than in the WT mice. Learning was impaired in the four-pellet taking test (4PTT) in TG mice, with no significant difference in daily activity or activity during the 4PTT between TG and WT mice. These results suggest that the overexpression of 1,4 GalNAc-T resulted in altered synaptic plasticity of LTP and DP in hippocampal CA1 neurons and learning in the 4PTT, and this is attributable to the shift from b-pathway gangliosides to a-pathway gangliosides. © The Author 2011.

DOI： 10.1093/glycob/cwr090

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The pannexin proteins represent a new gap junction family. However, the cellular functions of pannexins remain largely unknown. Here, we demonstrate that pannexin 3 (Panx3) promotes differentiation of osteoblasts and ex vivo growth of metatarsals. Panx3 expression was induced during osteogenic differentiation of C2C12 cells and primary calvarial cells, and suppression of this endogenous expression inhibited differentiation. Panx3 functioned as a unique Ca 2+ channel in the endoplasmic reticulum (ER), which was activated by purinergic receptor/phosphoinositide 3-kinase (PI3K)/Akt signaling, followed by activation of calmodulin signaling for differentiation. Panx3 also formed hemichannels that allowed release of ATP into the extracellular space and activation of purinergic receptors with the subsequent activation of PI3K-Akt signaling. Panx3 also formed gap junctions and propagated Ca 2+ waves between cells. Blocking the Panx3 Ca 2+ channel and gap junction activities inhibited osteoblast differentiation. Thus, Panx3 appears to be a new regulator that promotes osteoblast differentiation by functioning as an ER Ca 2+ channel and a hemichannel, and by forming gap junctions.

DOI： 10.1083/jcb.201101050

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

Objective: The purpose of this study was to elucidate the effects of platelet-derived growth factors (PDGFs) during tooth development, as well as the mechanisms underlying the interactions of growth factors with PDGF signalling during odontogenesis. Design: We used an ex vivo tooth germ organ culture system and two dental cell lines, SF2 cells and mDP cells, as models of odontogenesis. AG17, a tyrosine kinase inhibitor, was utilised for blocking PDGF receptor signalling. To analyse the expressions of PDGFs, reverse transcriptase (RT)-PCR and immunohistochemistry were performed. Proliferation was examined using a BrdU incorporation assay for the organ cultures and a cell counting kit for the cell lines. The expressions of Fgf2 and ameloblastin were analysed by real-time RT-PCR. Results: The PDGF ligands PDGF-A and PDGF-B, and their receptors, PDGFRα and PDGFRβ, were expressed throughout the initial stages of tooth development. In the tooth germ organ cultures, PDGF-AA, but not PDGF-BB, accelerated cusp formation. Conversely, AG17 suppressed both growth and cusp formation of tooth germs. Exogenous PDGF-BB promoted mDP cell proliferation. Furthermore, PDGF-AA decreased Fgf2 expression and increased that of ameloblastin, a marker of differentiated ameloblasts. Conclusion: Our results indicate that PDGFs are involved in initial tooth development and regulate tooth size and shape, as well as ameloblast differentiation. © 2010 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.archoralbio.2010.03.011

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Pannexin 3 (Panx3) is a new member of the gap junction pannexin family, but its expression profiles and physiological function are not yet clear. We demonstrate in this study that Panx3 is expressed in cartilage and regulates chondrocyte proliferation and differentiation. Panx3 mRNA was expressed in the prehypertrophic zone in the developing growth plate and was induced during the differentiation of chondrogenic ATDC5 and N1511 cells. Panx3-transfected ATDC5 and N1511 cells promoted chondrogenic differentiation, but the suppression of endogenous Panx3 inhibited differentiation of ATDC5 cells and primary chondrocytes. Panx3-transfected ATDC5 cells reduced parathyroid hormone-induced cell proliferation and promoted the release of ATP into the extracellular space, possibly by action of Panx3 as a hemichannel. Panx3 expression in ATDC5 cells reduced intracellular cAMP levels and the activation of cAMP-response element-binding, a protein kinase A downstream effector. These Panx3 activities were blocked by anti-Panx3 antibody. Our results suggest that Panx3 functions to switch the chondrocyte cell fate from proliferation to differentiation by regulating the intracellular ATP/cAMP levels.

DOI： 10.1074/jbc.M110.127027

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

AMBN (ameloblastin) is an enamel matrix protein that regulates cell adhesion, proliferation, and differentiation of ameloblasts. In AMBN-deficient mice, ameloblasts are detached from the enamel matrix, continue to proliferate, and form a multiple cell layer; often, odontogenic tumors develop in the maxilla with age. However, the mechanism of AMBN functions in these biological processes remains unclear. By using recombinant AMBN proteins, we found that AMBN had heparin binding domains at the C-terminal half and that these domains were critical for AMBN binding to dental epithelial cells. Overexpression of full-length AMBN protein inhibited proliferation of human ameloblastoma AM-1 cells, but overexpression of heparin binding domain-deficient AMBN protein had no inhibitory effect. In full-length AMBN-overexpressing AM-1 cells, the expression of Msx2, which is involved in the dental epithelial progenitor phenotype, was decreased, whereas the expression of cell proliferation inhibitors p21 and p27 was increased. We also found that the expression of enamelin, a marker of differentiated ameloblasts, was induced, suggesting that AMBN promotes odontogenic tumor differentiation. Thus, our results suggest that AMBN promotes cell binding through the heparin binding sites and plays an important role in preventing odontogenic tumor development by suppressing cell proliferation and maintaining differentiation phenotype through Msx2, p21, and p27.

DOI： 10.1074/jbc.M109.033464

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

Objective: Down syndrome, a frequently encountered genetic disorder, is usually associated with medical problems related to infectious disease, such as periodontal diseases and prolonged wound healing. Although affected individuals are considered to have clinical problems related to high interferon (IFN) sensitivity, the molecular mechanisms of IFN activities are not completely understood. Design: Down syndrome derived fibroblasts, Detroit 539 (D1) and Hs 52.Sk (D2) cells, were used. To analyse the expressions of interferon (IFN) receptors and downstream of IFN-γ, western blotting was performed. Cell proliferation was determined by counting cells following trypan blue staining. Media levels of IL-1β, TNF-α, and IL-6 were quantified using ELISA. Results: IFN-γ receptor 2 and IFN-α receptor 1, but not IFN-γ receptor 1, were highly expressed in D1 and D2 cells, as compared to the control fibroblast cells. Cell proliferation by D1 and D2 cells was lower than that by the control fibroblasts, further, IFN-γ had a greater effect to inhibit cell proliferation by D1 and D2 cells. In addition, IFN-γ treatment increased the phosphorylation of STAT1 and MAPK in D1 cells as compared to normal fibroblasts. Also, the presence of exogenous IFN-γ in the growth medium significantly induced IL-6, but not IL-1β or TNF-α, in D1 and D2 cells. Conclusion: Taken together, our results are consistent with hypersensitive reactions to IFN-γ seen in patients with Down syndrome and may provide useful information to elucidate the mechanisms of IFN-γ activities in those individuals. © 2009 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.archoralbio.2009.07.009

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A coordinated reciprocal interaction between epithelium and mesenchyme is involved in salivary gland morphogenesis. The submandibular glands (SMGs) of Wnt1-Cre/R26R mice have been shown positive for mesenchyme, whereas the epithelium is β-galactosidase-negative, indicating that most mesenchymal cells are derived from cranial neural crest cells. Platelet-derived growth factor (PDGF) receptor α is one of the markers of neural crest-derived cells. In this study, we analyzed the roles of PDGFs and their receptors in the morphogenesis of mouse SMGs. PDGF-A was shown to be expressed in SMG epithelium, whereas PDGF-B, PDGFRα, and PDGFRβ were expressed in mesenchyme. Exogenous PDGF-AA and -BB in SMG organ cultures demonstrated increased levels of branching and epithelial proliferation, although their receptors were found to be expressed in mesenchyme. In contrast, short interfering RNA for Pdgfa and -b as well as neutralizing antibodies for PDGF-AB and -BB showed decreased branching. PDGF-AA induced the expression of the fibroblast growth factor genes Fgf3 and -7, and PDGF-BB induced the expression of Fgf1, -3, -7, and -10, whereas short interfering RNA for Pdgfa and Pdgfb inhibited the expression of Fgf3, -7, and -10, indicating that PDGFs regulate Fgf gene expression in SMG mesenchyme. The PDGF receptor inhibitor AG-17 inhibited PDGF-induced branching, whereas exogenous FGF7 and -10 fully recovered. Together, these results indicate that fibroblast growth factors function downstream of PDGF signaling, which regulates Fgf expression in neural crest-derived mesenchymal cells and SMG branching morphogenesis. Thus, PDGF signaling is a possible mechanism involved in the interaction between epithelial and neural crest-derived mesenchyme. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M710308200

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Gangliosides, sialic acid-containing glycosphingolipids, have been considered to play roles as neurotrophic factors. Exogenous gangliosides added to the culture medium of neuronal cells or injected in artificially injured sites of nerve tissues actually showed neurotrophic factor-like effects such as neurite extension and alleviation of nerve tissue deterioration. In this study, neuroregeneration in the mutant mice lacking complex gangliosides was examined. To determine whether the nervous system maintains regenerative activity in the long-term absence of complex gangliosides, we analyzed hypoglossal nerve regeneration after axotomy in the mutant mice of GM2/GD2 synthase. These mice exhibited marked impairment of regenerative activity both in the number of surviving neurons and in the number of peroxidase-positive neurons. Moreover, reduced levels of gene expression of neurotrophic factors and their receptors including CNTF, p75 NTR, TrkB, and others in hypoglossal neurons were observed in real-time reverse transcription-polymerase chain reaction combined with laser capture microdissection, suggesting that these molecules are, at least partly, involved in the regeneration of lesioned nerves and that their expression levels are precisely controlled in the presence of intact expression of complex gangliosides. The Author 2008. Published by Oxford University Press. All rights reserved.

DOI： 10.1093/glycob/cwn032

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

Neurotrophic factors play an important role in the development and maintenance of not only neural but also nonneural tissues. Several neurotrophic factors are expressed in dental tissues, but their role in tooth development is not clear. Here, we report that neurotrophic factor neurotrophin (NT)-4 promotes differentiation of dental epithelial cells and enhances the expression of enamel matrix genes. Dental epithelial cells from 3-day-old mice expressed NT-4 and three variants of TrkB receptors for neurotrophins (full-length TrkB-FL and truncated TrkB-T1 and -T2). Dental epithelial cell line HAT-7 expressed these genes, similar to those in dental epithelial cells. We found that NT-4 reduced HAT-7 cell proliferation and induced the expression of enamel matrix genes, such as ameloblastin (Ambn). Transfection of HAT-7 cells with the TrkB-FL expression construct enhanced the NT-4-mediated induction of Ambn expression. This enhancement was blocked by K252a, an inhibitor for Trk tyrosine kinases. Phosphorylation of ERK1/2, a downstream molecule of TrkB, was induced in HAT-7 cells upon NT-4 treatment. TrkB-FL but not TrkB-T1 transfection increased the phosphorylation level of ERK1/2 in NT-4-treated HAT-7 cells. These results suggest that NT-4 induced Ambn expression via the TrkB-MAPK pathway. The p75 inhibitor TAT-pep5 decreased NT-4-mediated induction of the expression of Ambn, TrkB-FL, and TrkB-T1, suggesting that both high affinity and low affinity neurotrophin receptors were required for NT-4 activity. We found that NT-4-null mice developed a thin enamel layer and had a decrease in Ambn expression. Our results suggest that NT-4 regulates proliferation and differentiation of the dental epithelium and promotes production of the enamel matrix.

DOI： 10.1074/jbc.M704913200

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In tooth morphogenesis, the dental epithelium and mesenchyme interact reciprocally for growth and differentiation to form the proper number and shapes of teeth. We previously identified epiprofin (Epfn), a gene preferentially expressed in dental epithelia, differentiated ameloblasts, and certain ectodermal organs. To identify the role of Epfn in tooth development, we created Epfn-deficient mice (Epfn-/-). Epfn-/- mice developed an excess number of teeth, enamel deficiency, defects in cusp and root formation, and abnormal dentin structure. Mutant tooth germs formed multiple dental epithelial buds into the mesenchyme. In Epfn-/- molars, rapid proliferation and differentiation of the inner dental epithelium were inhibited, and the dental epithelium retained the progenitor phenotype. Formation of the enamel knot, a signaling center for cusps, whose cells differentiate from the dental epithelium, was also inhibited. However, multiple premature nonproliferating enamel knot-like structures were formed ectopically. These dental epithelial abnormalities were accompanied by dysregulation of Lef-1, which is required for the normal transition from the bud to cap stage. Transfection of an Epfn vector promoted dental epithelial cell differentiation into ameloblasts and activated promoter activity of the enamel matrix ameloblastin gene. Our results suggest that in Epfn-deficient teeth, ectopic nonproliferating regions likely bud off from the self-renewable dental epithelium, form multiple branches, and eventually develop into supernumerary teeth. Thus, Epfn has multiple functions for cell fate determination of the dental epithelium by regulating both proliferation and differentiation, preventing continuous tooth budding and generation.

DOI： 10.1074/jbc.M708388200

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

We identified a new extracellular protein, TM14, by differential hybridization using mouse tooth germ cDNA microarrays. TM14 cDNA encodes 440 amino acids containing a signal peptide. The protein contains 3 EGF modules at the center, a C-terminal domain homologous to the fibulin module, and a unique Sushi domain at the N terminus. In situ hybridization revealed that TM14 mRNA was expressed by preodontoblasts and odontoblasts in developing teeth. TM14 mRNA was also expressed in cartilage, hair follicles, and extraembryonic tissues of the placenta. Immunostaining revealed that TM14 was localized at the apical pericellular regions of preodontoblasts. When the dentin matrix was fully formed and dentin mineralization occurred, TM14 was present in the predentin matrix and along the dentinal tubules. We found that the recombinant TM14 protein was glycosylated with N-linked oligosaccharides and interacted with heparin, fibronectin, fibulin-1, and dentin sialophosphoprotein. We also found that TM14 preferentially bound dental mesenchyme cells and odontoblasts but not dental epithelial cells or nondental cells such as HeLa, COS7, or NIH3T3 cells. Heparin, EDTA, and anti-integrin β1 antibody inhibited TM14 binding to dental mesenchyme cells, suggesting that both a heparan sulfate-containing cell surface receptor and an integrin are involved in TM14 cell binding. Our findings indicate that TM14 is a cell adhesion molecule that interacts with extracellular matrix molecules in teeth and suggest that TM14 plays important roles in both the differentiation and maintenance of odontoblasts as well as in dentin formation. Because of its protein characteristics, TM14 can be classified as a new member of the fibulin family: fibulin-7.

DOI： 10.1074/jbc.M705847200

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

Amelogenin is a novel enamel matrix protein. Knockout mice showed enhanced osteoclast formation and resorption of tooth cementum. This study investigated the effects of amelogenin on osteoclastogenesis. In co-cultures with calvaria osteoblasts and purified bone marrow cells, amelogenin inhibited osteoclastogenesis dramatically. Furthermore, amelogenin inhibited the expression of receptor activator of nuclear factor κB ligand (RANKL), macrophage-colony stimulating factor (M-CSF) and fibronectin in osteoblasts, while RANKL expression was induced by fibronectin and inhibited by treatment with fibronectin small interfering RNA. These results suggest that the inhibitory effects of amelogenin on osteoclastogenesis lead to downregulation of RANKL, M-CSF and fibronectin production in osteoblasts. © 2006 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.archoralbio.2006.09.016

Language：English   Publishing type：Research paper (other academic)  

During tooth development, dental epithelial cells interact with extracellular matrix components, such as the basement membrane and enamel matrix. Ameloblastin, an enamel matrix protein, plays a crucial role in maintaining the ameloblast differentiation state and is essential for enamel formation. Ameloblastin-null mice developed severe enamel hypoplasia. In mutant mice, dental epithelial cells started to differentiate into ameloblasts, but ameloblasts soon lost cell polarity, proliferated, and formed multiple cell layers, indicative of some aspects of preameloblast phenotypes. In addition, the expression of amelogenin, another component of the enamel matrix, was specifically reduced in mutant ameloblasts. More than 20% of amelobastin-null mice developed odontogenic tumors. We also found that recombinant ameloblastin specifically bound to ameloblasts and inhibited proliferation of dental epithelial cells. These results suggest that ameloblastin is an important regulator to maintain the differentiation state of ameloblasts. Copyright © 2005 S. Karger AG.

DOI： 10.1159/000091380

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

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 α5 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 α5 (Lama5) in tooth development using laminin α5-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 α5, the basement membrane in the inner dental epithelium becomes discontinuous. In normal mice, integrin α6β4, a receptor for laminin α5, is strongly localized at the basal layer of the epithelium, whereas in mutant mice, integrin α6β4 is expressed around the cell surface. In primary dental epithelium culture, laminin-10/11 promotes cell growth, spreading, and filopodia-like microspike formation. This promotion is inhibited by anti-integrin α6 and β4 antibodies and by phosphatidylinositol 3-kinase inhibitors and dominant negative Rho-GTPase family proteins Cdc42 and Rac. In organ culture, anti-integrin α6 antibody and wortmannin reduce tooth germ size and shape. Our studies demonstrate that laminin α5 is required for the proliferation and polarity of basal epithelial cells and suggest that the interaction between laminin-10/11-integrin α6β4 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

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

Language：English  

Mineralized tissues are unique in that they use proteins to attract and organize calcium and phosphate ions into a structured mineral phase, thus precise knowledge of the expression and extracellular distribution of matrix proteins is very important to understand their function. Tooth development is regulated by sequential and reciprocal interactions between neural crest-derived mesenchymal cells and the oral environment. However, the precise molecular mechanisms that mediate interactions between epithelium and mesenchymal cells are not clear, although basement membrane (BM) components have been shown to play important roles in these regulatory events. In addition, the extracellular matrix layer, whose main components are laminin, collagen IV, nidogen, and sulfated proteoglycan, and the BM layer are both considered to be involved with cell proliferation and differentiation. During tooth morphogenesis, extracellular matrices are dramatically changed. Further, the BM components, laminin and collagen IV support dental epithelium; however, in the late stage, they begin the processes of enamel matrix secretion and calcification, after which the BM structure between the dental epithelium and mesenchyme disappears. In addition, tooth abnormalities associated with several kinds of human diseases that cause mutations in the extracellular matrix, as well as the molecular mechanisms of the basement membrane and enamel matrix during tooth morphogenesis, are not clearly understood. In our review, we discuss the role of the extracellular matrix, with focus on the BM and enamel matrix during tooth morphogenesis. Copyright © Taylor & Francis Inc.

DOI： 10.1080/03008200500344017

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

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. © 2004 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.archoralbio.2004.09.014

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

alpha2,8-Sialyltransferase (alpha2,8S-T, GD3 synthase) has been reported to be involved in the enhanced cell proliferation of malignant tumors. Using a cloned cDNA of alpha2,8S-T, transfectant cells were established and the effects of the gene expression on the cell phenotypes were analyzed. In contrast with PC12 cells, in which we reported marked growth enhancement based on the transfection of alpha2,8S-T, Swiss3T3 cells showed no enhancement in either cell proliferation or phosphorylation of MAP kinases after the transfection of alpha2,8S-T when treated with platelet-derived growth factor. Correspondingly, the receptor for platelet-derived growth factor also showed no increased phosphorylation upon the factor stimulation. However, in the wound-healing scratching assay, the Swiss3T3 transfectant cells demonstrated increased mobility as the PC12 transfectant cells. These results suggest that the enhancing effects of alpha2,8S-T on the proliferation and mobility are differential depending on the cell types, and ganglioside-associating molecules in the individual cell types need to be investigated.

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

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

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

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

Tooth morphogenesis results from reciprocal interactions between oral epithelium and ectomesenchyme culminating in the formation of mineralized tissues, enamel, and dentin. During this process, epithelial cells differentiate into enamel-secreting ameloblasts. Ameloblastin, an enamel matrix protein, is expressed by differentiating ameloblasts. Here, we report the creation of ameloblastin-null mice, which developed severe enamel hypoplasia. In mutant tooth, the dental epithelium differentiated into enamel-secreting ameloblasts, but the cells were detached from the matrix and subsequently lost cell polarity, resumed proliferation, and formed multicell layers. Expression of Msx2, p27, and p75 were deregulated in mutant ameloblasts, the phenotypes of which were reversed to undifferentiated epithelium. We found that recombinant ameloblastin adhered specifically to ameloblasts and inhibited cell proliferation. The mutant mice developed an odontogenic tumor of dental epithelium origin. Thus, ameloblastin is a cell adhesion molecule essential for amelogenesis, and it plays a role in maintaining the differentiation state of secretory stage ameloblasts by binding to ameloblasts and inhibiting proliferation.

DOI： 10.1083/jcb.200409077

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Ganglioside GM1 has been considered to have a neurotrophic factor-like activity. To analyze the effects of endogenously generated GM1, the rat pheochromocytoma cell line PC12 was transfected with the GM1/GD1b/GA1 synthase gene and showed increased expression levels of GM1. To our surprise, GM1 +-transfectant cells (GM1+ cells) showed no neurite formation after stimulation with nerve growth factor (NGF). Autophosphorylation of NGF receptor TrkA and activation of ERK1/2 after NGF treatment were scarcely detected in GM1+ cells. Binding of 125I-NGF to PC12 cells was almost equivalent between GM1+ cells and controls. However, dimer formation of TrkA upon NGF treatment was markedly suppressed in GM1+ cells in both cross-linking analysis with Bis(sulfosuccinimidyl)suberate 3 and 125I-NGF binding assay. The sucrose density gradient fractionation of the cell lysate revealed that TrkA primarily located in the lipid raft fraction moved to the non-raft fraction in GM1+ cells. p75NTR and Ras also moved from the raft to non-raft fraction in GM1+ cells, whereas flotillin and GM1 persistently resided in the lipid raft. TrkA kinase activity was differentially regulated when GM1 was added to the kinase assay system in vitro, suggesting suppressive/enhancing effects of GM1 on NGF signals based on the concentration. Measurement of fluorescence recovery after photobleaching revealed that the membrane fluidity was reduced in GM1 + cells. These results suggested that overexpressed GM1 suppresses the differentiation signals mediated by NGF/TrkA by modulating the properties of the lipid raft and the intracellular localization of NGF receptors and relevant signaling molecules.

DOI： 10.1074/jbc.M403816200

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Bacterial infection sometimes impairs bone metabolism. In this study, we infected the osteoblastic cell line MC3T3-E1 with Mycobacterium bovis bacillus Calmette-Guérin (BCG) and identified genes that were up-regulated in the BCG-infected cells by the suppression subtractive hybridization method. A gene encoding 4-1BB (CD137), a member of the tumor necrosis factor-α receptor family, was found to be one of the up-regulated genes. Up-regulation of 4-1BB was also observed by infection with Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus, and by treatment with lipopolysaccharides and heat-killed BCG. Bone marrow cells and the macrophage-like cell lines J774 and RAW264.7 were found to express 4-1BB ligand (4-1BBL). Recombinant 4-1BB (r4-1BB) that was immobilized on culture plates strongly inhibited macrophage colony stimulating factor (M-CSF)/receptor activator of nuclear factor-κB ligand (RANKL)-induced in vitro osteoclast formation from bone marrow cells. Anti-4-1BBL antibody also inhibited osteoclast formation to a lesser extent, indicating involvement of reverse signaling through 4-1BBL during inhibition of osteoclast formation. A casein kinase I (CKI) inhibitor markedly suppressed the inhibitory effect of r4-1BB on M-CSF/ RANKL-induced osteoclast formation, suggesting that CKI might be involved in 4-1BB/4-1BBL reverse signaling. r4-1BB showed no effects on M-CSF- or RANKL-induced phosphorylation of I-κB, ERK1/2, p38, or JNK, whereas RANKL-induced phosphorylation of Akt, a downstream target of phosphatidylinositol 3-kinase (PI3K), was completely abolished by r4-1BB, suggesting that 4-1BB/4-1BBL reverse signaling may interfere with PI3K/Akt pathway. r4-1BB also abolished RANKL-mediated induction of nuclear factor of activated T cells-2. This study may elucidate a novel role of 4-1BB in cell metabolism, especially osteoclastogenesis.

DOI： 10.1074/jbc.M303791200

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Laminin α2 is subunit of laminin-2 (α2β1γ1), which is a major component of the muscle basement membrane. Although the laminin α2 chain is expressed in the early stage of dental mesenchyme development and localized in the tooth germ basement membrane, its expression pattern in the late stage of tooth germ development and molecular roles are not clearly understood. We analyzed the role of laminin α2 in tooth development by using targeted mice with a disrupted lama2 gene. Laminin α2 is expressed in dental mesenchymal cells, especially in odontoblasts and during the maturation stage of ameloblasts, but not in the pre-secretory or secretory stages of ameloblasts. Lama2 mutant mice have thin dentin and a widely opened dentinal tube, as compared with wild-type and heterozygote mice, which is similar to the phenotype of dentinogenesis imperfecta. During dentin formation, the expression of dentin sialoprotein, a marker of odontoblast differentiation, was found to be decreased in odontoblasts from mutant mice. Furthermore, in primary cultures of dental mesenchymal cells, dentin matrix protein, and dentin sialophosphoprotein, mRNA expression was increased in laminin-2 coated dishes but not in those coated with other matrices, fibronectin, or type I collagen. Our results suggest that laminin α2 is essential for odontoblast differentiation and regulates the expression of dentin matrix proteins.

DOI： 10.1074/jbc.M310013200

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

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

The polymorphic carbohydrate structures of gangliosides play regulatory roles. In particular, b-series gangliosides, all of which contain α-2,8 sialic acids, have been considered to be critical in various biological events such as adhesion, toxin binding, neurite extension, cell growth, and apoptosis. To clarify the physiological functions of b-series gangliosides in vivo, we have established a gene knockout mouse of GD3 synthase. Although all b-series structures were deleted in the mutant mice, they showed an almost complete nervous tissue morphology with no apparent abnormal behavior. Moreover, no differences in Fas-mediated apoptotic reaction of lymphocytes between wild type and the mutant mice were detected. However, the mutant mice exhibited clearly reduced regeneration of axotomized hypoglossal nerves compared with the wild type, suggesting that b-series gangliosides are more important in the repair rather than in the differentiation of the nervous system and apoptotic process induced via Fas.

DOI： 10.1074/jbc.C100395200

Language：English   Publisher：Journal of Cellular Physiology  

Enhanced osteoclastogenesis causes bone fragility, osteoporosis, and an increased risk of fractures. Recent studies have suggested a possible correlation between osteoporosis and the pathological features of Parkinson's disease (PD). To establish a molecular link between these conditions, we focused on the physiological function of the PD-related protein ubiquitin carboxy-terminal hydrolase L1 (UCHL1) in bone remodeling. To this end, we investigated the role of UCHL1 in regulating osteoclast differentiation in Uchl1 spontaneous mutant gad mice. We found that gad-mouse-derived osteoclast progenitors exhibit enhanced osteoclast differentiation. Likewise, CRISPR-mediated Uchl1 knockout in mouse macrophage-derived preosteoclast RAW-D cells increased RANKL-dependent osteoclastogenesis. Supporting this observation, these Uchl1-depleted cells showed elevated expression of osteoclast marker genes. To uncover the molecular mechanisms by which the loss of Uchl1 enhances osteoclast differentiation, we screened for UCHL1-interacting proteins in RAW-D preosteoclast cells and identified AKT1 as a potential UCHL1-regulated protein. UCHL1 depletion in preosteoclasts led to increased Thr308/Ser473 phosphorylation of AKT1. Furthermore, ectopic expression of UCHL1 decreased the K63-linked polyubiquitination of AKT1. These findings suggest that UCHL1 is critical in partially suppressing osteoclastogenesis through modulating AKT signaling.

DOI： 10.1002/jcp.70032

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Publishing type：Research paper (scientific journal)   Publisher：Pediatric Dental Journal  

Objectives: Dental anomalies are often associated with inherited diseases and are frequently seen in the field of pediatric dentistry. Dentin disorders are caused by genetic mutations of genes expressed in dental mesenchyme and these genetic mutations also cause skeletal dysplasia. However, only little genes are identified as the responsible gene of dentin disorders and therefore the genetic mechanism is poorly understood. Here, we identified the dental mesenchymal cell-specific genes using single-cell RNA-sequence (scRNA-seq) to clarify the pathogenesis of dentin disorders. Furthermore, we examined the association of inherited disease with dentin or skeletal abnormality using human disease database. Materials and methods: scRNA-seq were performed using post-natal day (P) seven mice incisor. Total 6260 cells of scRNA-seq dataset was obtained. Top 20 differentially expressed genes (DEGs) in odontoblast cluster and dental pulp cluster were identified. Inherited diseases of DEGs and their Clinical Synopsis were examined using Online Mendelian Inheritance in Man (OMIM). Results: The prevalence of inherited disease was 17/40 genes (42.5 %) and dental anomalies-associated inherited diseases were 12/35 diseases (34.3 %); 2.8 % in enamel abnormality, 14.3 % in dentin abnormality, and 17.1 % in other abnormality. The prevalence of dentin abnormality was 33.3 % in odontoblast and 7.7 % in dental pulp-associated diseases. The prevalence of skeletal abnormality in identified inherited diseases was 85.7 %. Conclusion: The DEGs in dental mesenchymal cells were highly associated with dentin abnormality and skeletal abnormality. Identification of dental mesenchymal cell specific genes using scRNA-seq may uncover the novel genetic mechanism of dentin disorders.

DOI： 10.1016/j.pdj.2024.100337

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

Epiprofin (Epfn), an Sp/KLF family transcription factor that regulates cell proliferation and determines cell fates, is essential for normal skin, hair follicle, and tooth development. We found that Epfn was expressed in parathyroid glands, and Epfn-knockout mice displayed elevated serum parathyroid hormone (PTH) concentrations, decreased bone volume, and intracranial ectopic calcification. To investigate the role of Epfn in the regulation of PTH expression, parathyroid gland explant and parathyroid cell line culture methods were used. Epfn expression was found to be upregulated in response to an increase in extracellular calcium concentration, whereas PTH expression was downregulated, thus demonstrating an inverse correlation. Forced expression of Epfn inhibited PTH gene expression and PTH promoter reporter activity in parathyroid cells. In addition, with a high extracellular calcium concentration, Epfn silencing in cultured parathyroid glands failed to block PTH gene expression. ChIP-qPCR analysis also revealed Epfn binding in the proximal region of the PTH promoter, which was accelerated in the presence of a high concentration of calcium ions. The results from our in vitro and ex vivo analyses suggest that Epfn is a newly identified negative regulator of PTH transcription by regulating the proximal PTH promoter. Furthermore, the expression of Epfn was significantly reduced in parathyroid adenomas of primary hyperparathyroidism patients. The identification of Epfn as a potential therapeutic target for the control of PTH production in hyperparathyroidism patients opens new avenues for targeted treatment approaches.

DOI： 10.1111/febs.70085

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Publisher：Materials Chemistry and Physics  

The authors regret to replace the original CRediT authorship contribution statement. Xiao Sun: Formal analysis, Data curation, Conceptualization. Yu Zhang: Funding acquisition, Formal analysis, Conceptualization. Satoshi Fukumoto: Formal analysis, Data curation, Conceptualization. Keiji Masuda: Funding acquisition, Data curation, Conceptualization. Ning Dong: Investigation, Data curation, Conceptualization. With the following Xiao Sun: Formal analysis, Data curation, Conceptualization. Yu Zhang: Formal analysis, Conceptualization. Satoshi Fukumoto: Formal analysis, Data curation, Conceptualization. Keiji Masuda: Data curation, Conceptualization. Ning Dong: Funding acquisition, Investigation, Data curation, Conceptualization. The authors would like to apologise for any inconvenience caused.

DOI： 10.1016/j.matchemphys.2024.130087

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Publisher：Materials Chemistry and Physics  

This study aims to investigate the adaptive strategies employed by dental pulp stem cells (DPSCs) in response to oral disease conditions, focusing on their survival and proliferation. DPSCs possess regenerative potential and play a crucial role in the repair and regeneration of dental tissues. The study explores the coping mechanisms of DPSCs under oral disease conditions using a chitosan-based biomaterial reinforced with calcium zirconium nanoparticles (CZNP). The investigation assesses the viability and proliferation of DPSCs in the presence of oral disease conditions and evaluates the performance of the chitosan-CZNP biomaterial. The study presents the characterization and performance evaluation of drug-loaded biomaterials with varying weight percentages of CZNP. Parameters such as porosity, drug release, fracture toughness, tensile strength, degradation rate, and apatite formation are examined. The results demonstrate that increasing the weight percentage of CZNP leads to higher porosity and drug release while maintaining or enhancing fracture toughness, tensile strength, and apatite formation. Notably, the biomaterial containing 7.5 wt% CZNP exhibits the highest drug release, fracture toughness, and apatite formation. These findings suggest that the chitosan-CZNP biomaterial effectively supports the survival and proliferation of DPSCs under oral disease conditions. An artificial neural network (ANN) is employed to predict the properties of samples with different weight percentages and apatite formation. The ANN successfully estimates the degradation rate, tensile strength, fracture toughness, drug release, and porosity of the samples. This research enhances our understanding of the coping mechanisms of DPSCs and highlights the potential of chitosan-CZNP biomaterials in oral tissue engineering and regenerative medicine. Further investigations are warranted to explore the long-term effects and clinical applications of these biomaterials in the treatment of oral diseases.

DOI： 10.1016/j.matchemphys.2024.129610

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Cellular Physiology  

Keratins are typical intermediate filament proteins of the epithelium that exhibit highly specific expression patterns related to the epithelial type and stage of cellular differentiation. They are important for cytoplasmic stability and epithelial integrity and are involved in various intracellular signaling pathways. Several keratins are associated with enamel formation. However, information on their expression patterns during tooth development remains lacking. In this study, we analyzed the spatiotemporal expression of keratin family members during tooth development using single-cell RNA-sequencing (scRNA-seq) and microarray analysis. scRNA-seq datasets from postnatal Day 1 mouse molars revealed that several keratins are highly expressed in the dental epithelium, indicating the involvement of keratin family members in cellular functions. Among various keratins, keratin 5 (Krt5), keratin 14 (Krt14), and keratin 17 (Krt17) are highly expressed in the tooth germ; KRT17 is specifically expressed in the stratum intermedium (SI) and stellate reticulum (SR). Depletion of Krt17 did not affect cell proliferation in the dental epithelial cell line SF2 but suppressed their differentiation ability. These results suggest that Krt17 is essential for SI cell differentiation. Furthermore, scRNA-seq results indicated that Krt5, Krt14, and Krt17 exhibited distinct expression patterns in ameloblast, SI, and SR cells. Our findings contribute to the elucidation of novel mechanisms underlying tooth development.

DOI： 10.1002/jcp.31387

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

Tooth development is a complex process involving various signaling pathways and genes. Recent findings suggest that ion channels and transporters, including the S100 family of calcium-binding proteins, may be involved in tooth formation. However, our knowledge in this regard is limited. Therefore, this study aimed to investigate the expression of S100 family members and their functions during tooth formation. Tooth germs were extracted from the embryonic and post-natal mice and the expression of S100a6 was examined. Additionally, the effects of S100a6 knockdown and calcium treatment on S100a6 expression and the proliferation of SF2 cells were examined. Microarrays and single-cell RNA-sequencing indicated that S100a6 was highly expressed in ameloblasts. Immunostaining of mouse tooth germs showed that S100a6 was expressed in ameloblasts but not in the undifferentiated dental epithelium. Additionally, S100a6 was localized to the calcification-forming side in enamel-forming ameloblasts. Moreover, siRNA-mediated S100a6 knockdown in ameloblasts reduced intracellular calcium concentration and the expression of ameloblast marker genes, indicating that S100a6 is associated with ameloblast differentiation. Furthermore, S100a6 knockdown inhibited the ERK/PI3K signaling pathway, suppressed ameloblast proliferation, and promoted the differentiation of the dental epithelium toward epidermal lineage. Conclusively, S100a6 knockdown in the dental epithelium suppresses cell proliferation via calcium and intracellular signaling and promotes differentiation of the dental epithelium toward the epidermal lineage.

DOI： 10.1096/fj.202302412RR

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Language：Japanese   Publisher：(公社)日本小児歯科学会  

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Cells sense and respond to extracellular mechanical stress through mechanotransduction receptors and ion channels, which regulate cellular behaviors such as cell proliferation and differentiation. Among them, PIEZO1, piezo-type mechanosensitive ion channel component 1, has recently been highlighted as a mechanosensitive ion channel in various cell types including mesenchymal stem cells. We previously reported that PIEZO1 is essential for ERK1/2 phosphorylation and osteoblast differentiation in bone marrow-derived mesenchymal stem cells (BMSCs), induced by hydrostatic pressure loading and treatment with the PIEZO1-specific activator Yoda1. However, the molecular mechanism underlying how PIEZO1 induces mechanotransduction remains unclear. In this study, we investigated that the role of the C-terminus in regulating extracellular Ca2+ influx and activating the ERK1/2 signaling pathway. We observed the activation of Fluo-4 AM in the Yoda1-stimulated human BMSC line UE7T-13, but not in a calcium-depleted cell culture medium. Similarly, Western blotting analysis revealed that Yoda1 treatment induced ERK1/2 phosphorylation, but this induction was not observed in calcium-depleted cell culture medium. To investigate the functional role of the C-terminus of PIEZO1, we generated HEK293 cells stably expressing the full-length mouse PIEZO1 (PIEZO1-FL) and a deletion-type PIEZO1 lacking the C-terminal intracellular region containing the R-Ras-binding domain (PIEZO1-ΔR-Ras). We found that Yoda1 treatment predominantly activated Flou-4 AM and ERK1/2 in PIEZO1-FL-trasfected cells but neither in PIEZO1-ΔR-Ras-transfected cells nor control cells. Our results indicate that the C-terminus of PIEZO1, which contains the R-Ras binding domain, plays an essential role in Ca2+ influx and activation of the ERK1/2 signaling pathway, suggesting that this domain is crucial for the mechanotransduction of osteoblastic differentiation in BMSCs.

DOI： 10.1016/j.bbrc.2023.09.080

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

<jats:title>Abstract</jats:title><jats:p>Mutations in the gene encoding the transient receptor potential vanilloid member 4 (TRPV4), a Ca<jats:sup>2+</jats:sup> permeable nonselective cation channel, cause TRPV4‐related disorders. TRPV4 is widely expressed in the brain; however, the pathogenesis underlying TRPV4‐mediated Ca<jats:sup>2+</jats:sup> deregulation in neurodevelopment remains unresolved and an effective therapeutic strategy remains to be established. These issues were addressed by isolating mutant dental pulp stem cells from a tooth donated by a child diagnosed with metatropic dysplasia with neurodevelopmental comorbidities caused by a gain‐of‐function TRPV4 mutation, c.1855C &gt; T (p.L619F). The mutation was repaired using CRISPR/Cas9 to generate corrected isogenic stem cells. These stem cells were differentiated into dopaminergic neurons and the pharmacological effects of folic acid were examined. In mutant neurons, constitutively elevated cytosolic Ca<jats:sup>2+</jats:sup> augmented AKT‐mediated α‐synuclein (α‐syn) induction, resulting in mitochondrial Ca<jats:sup>2+</jats:sup> accumulation and dysfunction. The TRPV4 antagonist, AKT inhibitor, or α‐syn knockdown, normalizes the mitochondrial Ca<jats:sup>2+</jats:sup> levels in mutant neurons, suggesting the importance of mutant TRPV4/Ca<jats:sup>2+</jats:sup>/AKT‐induced α‐syn in mitochondrial Ca<jats:sup>2+</jats:sup> accumulation. Folic acid was effective in normalizing mitochondrial Ca<jats:sup>2+</jats:sup> levels via the transcriptional repression of α‐syn and improving mitochondrial reactive oxygen species levels, adenosine triphosphate synthesis, and neurite outgrowth of mutant neurons. This study provides new insights into the neuropathological mechanisms underlying TRPV4‐related disorders and related therapeutic strategies.</jats:p>

DOI： 10.1096/fba.2023-00057

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Biochemical and Biophysical Research Communications  

Murine tooth germ development proceeds in continuous sequential steps with reciprocal interactions between the odontogenic epithelium and the adjacent mesenchyme, and several growth factor signaling pathways and their activation are required for tooth germ development. The expression of ADP-ribosylation factor (Arf)-like 4c (Arl4c) has been shown to induce cell proliferation, and is thereby involved in epithelial morphogenesis and tumorigenesis. In contrast, the other functions of Arl4c (in addition to cellular growth) are largely unknown. Although we recently demonstrated the involvement of the upregulated expression of Arl4c in the proliferation of ameloblastomas, which have the same origin as odontogenic epithelium, its effect on tooth germ development remains unclear. In the present study, single-cell RNA sequencing (scRNA-seq) analysis revealed that the expression of Arl4c, among 17 members of the Arf-family, was specifically detected in odontogenic epithelial cells, such as those of the stratum intermedium, stellate reticulum and outer enamel epithelium, of postnatal day 1 (P1) mouse molars. scRNA-seq analysis also demonstrated the higher expression of Arl4c in non-ameloblast and inner enamel epithelium, which include immature cells, of P7 mouse incisors. In the mouse tooth germ rudiment culture, treatment with SecinH3 (an inhibitor of the ARNO/Arf6 pathway) reduced the size, width and cusp height of the tooth germ and the thickness of the eosinophilic layer, which would involve the synthesis of dentin and enamel matrix organization. In addition, loss-of-function experiments using siRNAs and shRNA revealed that the expression of Arl4c was involved in cell proliferation and osteoblastic cytodifferentiation in odontogenic epithelial cells. Finally, RNA-seq analysis with a gene set enrichment analysis (GSEA) and Gene Ontology (GO) analysis showed that osteoblastic differentiation-related gene sets and/or GO terms were downregulated in shArl4c-expressing odontogenic epithelial cells. These results suggest that the Arl4c-ARNO/Arf6 pathway axis contributes to tooth germ development through osteoblastic/ameloblastic differentiation.

DOI： 10.1016/j.bbrc.2023.09.014

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Biochemical and Biophysical Research Communications  

Melatonin entrainment of suprachiasmatic nucleus-regulating circadian rhythms is mediated by MT1 and MT2 receptors. Melatonin also has neuroprotective and mitochondrial activating effects, suggesting it may affect neurodevelopment. We studied melatonin's pharmacological effects on autism spectrum disorder (ASD) neuropathology. Deciduous tooth-derived stem cells from children with ASD were used to model neurodevelopmental defects and differentiated into dopaminergic neurons (ASD-DNs) with or without melatonin. Without melatonin, ASD-DNs had reduced neurite outgrowth, mitochondrial dysfunction, lower mitochondrial Ca²⁺ levels, and Ca²⁺ accumulation in the endoplasmic reticulum (ER) compared to control DNs from typically developing children-derived stem cells. Melatonin enhanced IP3-dependent Ca²⁺ release from ER to mitochondria, improving mitochondrial function and neurite outgrowth in ASD-DNs. Luzindole, an MT1/MT2 antagonist, blocked these effects. Thus, melatonin supplementation may improve dopaminergic system development in ASD by modulating mitochondrial Ca²⁺ homeostasis via MT1/MT2 receptors.

DOI： 10.1016/j.bbrc.2023.09.050

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

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Abstract

Postnatal cell fate is postulated to be primarily determined by the local tissue microenvironment. Here, we find that Mediator 1 (Med1) dependent epigenetic mechanisms dictate tissue-specific lineage commitment and progression of dental epithelia. Deletion of Med1, a key component of the Mediator complex linking enhancer activities to gene transcription, provokes a tissue extrinsic lineage shift, causing hair generation in incisors. Med1 deficiency gives rise to unusual hair growth via primitive cellular aggregates. Mechanistically, we find that MED1 establishes super-enhancers that control enamel lineage transcription factors in dental stem cells and their progenies. However, Med1 deficiency reshapes the enhancer landscape and causes a switch from the dental transcriptional program towards hair and epidermis on incisors in vivo, and in dental epithelial stem cells in vitro. Med1 loss also provokes an increase in the number and size of enhancers. Interestingly, control dental epithelia already exhibit enhancers for hair and epidermal key transcription factors; these transform into super-enhancers upon Med1 loss suggesting that these epigenetic mechanisms cause the shift towards epidermal and hair lineages. Thus, we propose a role for Med1 in safeguarding lineage specific enhancers, highlight the central role of enhancer accessibility in lineage reprogramming and provide insights into ectodermal regeneration.

DOI： 10.1038/s42003-023-05105-5

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Other Link： https://www.nature.com/articles/s42003-023-05105-5

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Language：Japanese   Publisher：東北大学歯学会  

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Pediatric Dental Journal  

Purpose: This study was conducted to explore the possibility of detecting the presence of impacted teeth in the upper midline using facial morphometric measurements. Materials and Methods: The study included 260 children, aged 2–13 years, who visited our university hospital. To measure facial morphology, facial photographs were taken along with a ruler, and the correlation between each facial measurement point and the presence of mesiodens was statistically analyzed. Results: Each measurement point increased in a time-dependent manner and there was a correlation between age and distance. In contrast, the ratio of the inner-canthal distance to the outer-canthal distance was not associated with age. Moreover, an increase in this ratio suggested the possible presence of mesiodens. Since age is associated with the presence of mesiodens within the inner canthal distance, a multivariate analysis that considered the effects of age and sex was performed and showed that the inner canthus/outer canthus ratio was 0.45. When the odds ratio of the presence of mesiodens in the group of less than 1.0 was set to 1.0, the odds ratio of the group of 0.45 or more was 5.36. Conclusion: The presence of mesiodens can be predicted by measuring the ratio of the inner canthal distance to the outer canthal distance.

DOI： 10.1016/j.pdj.2023.04.001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Biochemical and Biophysical Research Communications  

Iroquois homeobox (Irx) genes are TALE-class homeobox genes that are evolutionarily conserved across species and have multiple critical cellular functions in fundamental tissue development processes. Previous studies have shown that Irxs genes are expressed during tooth development. However, the precise roles of genes in teeth remain unclear. Here, we demonstrated for the first time that Irx3 is an essential molecule for the proliferation and differentiation of odontoblasts. Using cDNA synthesized from postnatal day 1 (P1) tooth germs, we examined the expression of all Irx genes (Irx1-Irx6) by RT-PCR and found that all genes except Irx4 were expressed in the tooth tissue. Irx1-Irx3 a were expressed in the dental epithelial cell line M3H1 cells, while Irx3 and Irx5 were expressed in the dental mesenchymal cell line mDP cells. Only Irx3 was expressed in both undifferentiated cell lines. Immunostaining also revealed the presence of IRX3 in the dental epithelial cells and mesenchymal condensation. Inhibition of endogenous Irx3 by siRNA blocks the proliferation and differentiation of mDP cells. Wnt3a, Wnt5a, and Bmp4 are factors involved in odontoblast differentiation and were highly expressed in mDP cells by quantitative PCR analysis. Interestingly, the expression of Wnt5a (but not Wnt3a or Bmp4) was suppressed by Irx3 siRNA. These results suggest that Irx3 plays an essential role in part through the regulation of Wnt5a expression during odontoblast proliferation and differentiation.

DOI： 10.1016/j.bbrc.2023.02.004

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

Enamel is formed by the repetitive secretion of a tooth-specific extracellular matrix and its decomposition. Calcification of the enamel matrix via hydroxyapatite (HAP) maturation requires pH cycling to be tightly regulated through the neutralization of protons released during HAP synthesis. We found that Gpr115, which responds to changes in extracellular pH, plays an important role in enamel formation. Gpr115-deficient mice show partial enamel hypomineralization, suggesting that other pH-responsive molecules may be involved. In this study, we focused on the role of Gpr111/Adgrf2, a duplicate gene of Gpr115, in tooth development. Gpr111 was highly expressed in mature ameloblasts. Gpr111-KO mice showed enamel hypomineralization. Dysplasia of enamel rods and high carbon content seen in Gpr111-deficient mice suggested the presence of residual enamel matrices in enamel. Depletion of Gpr111 in dental epithelial cells induced the expression of ameloblast-specific protease, kallikrein-related peptidase 4 (Klk4), suggesting that Gpr111 may act as a suppressor of Klk4 expression. Moreover, reduction of extracellular pH to 6.8 suppressed the expression of Gpr111, while the converse increased Klk4 expression. Such induction of Klk4 was synergistically enhanced by Gpr111 knockdown, suggesting that proper enamel mineralization may be linked to the modulation of Klk4 expression by Gpr111. Furthermore, our in vitro suppression of Gpr111 and Gpr115 expression indicated that their suppressive effect on calcification was additive. These results suggest that both Gpr111 and Gpr115 respond to extracellular pH, contribute to the expression of proteolytic enzymes, and regulate the pH cycle, thereby playing important roles in enamel formation.

DOI： 10.1096/fj.202202053R

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Language：Japanese   Publisher：(公社)日本小児歯科学会  

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Language：Japanese   Publisher：(公社)日本小児歯科学会  

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Objectives: Dental anomalies show various symptoms and some of them are accompanied with inherited diseases. However, only a few of responsible genes of dental anomalies are identified. In this study, we aimed to establish a novel strategy for identification of responsible genes of dental anomalies using integration of single-cell RNA-sequence (scRNA-seq) and Online Mendelian Inheritance in Man (OMIM). Materials and methods: Single cells were isolated from mandibular incisor of post-natal day (P) seven mice and scRNA-seq were performed. Top 20 differentially expressed genes between clusters were identified and used for further analyses. Inherited diseases of differentially expressed genes and their Clinical Synopsis were examined using OMIM. Results: The prevalence of inherited disease was 43/80 genes (53.8%) and inherited diseases which associate with dental anomalies were 34/96 diseases (35.4%); 14.6% in enamel abnormality, 4.1% in dentin abnormality, and 16.7% in other abnormality. The prevalence of enamel abnormality was the highest in ameloblast, while that of other abnormality was high in non-ameloblast cell types. Chromosomal mapping of differentially expressed genes indicated that chromosome 4 has “hotspots” of dental anomalies-associated genes. Conclusion: The differentially expressed genes in dental epithelial cells were responsible for inherited disease which shows dental anomalies. The strategy employed in this study will contribute to identify the responsible gene for dental anomalies.

DOI： 10.1016/j.pdj.2023.03.004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Biological Chemistry  

Lipid rafts are membrane microdomains rich in cholesterol, sphingolipids, glycosylphosphatidylinositol-anchored proteins (GPI-APs), and receptors. These lipid raft components are localized at the plasma membrane and are essential for signal transmission and organogenesis. However, few reports have been published on the specific effects of lipid rafts on tooth development. Using microarray and single-cell RNA sequencing methods, we found that a GPI-AP, lymphocyte antigen-6 (Ly6)/Plaur domain-containing 1 (Lypd1), was specifically expressed in preodontoblasts. Depletion of Lypd1 in tooth germ using an ex vivo organ culture system and in mouse dental pulp (mDP) cells resulted in the inhibition of odontoblast differentiation. Activation of bone morphogenetic protein (BMP) signaling by BMP2 treatment in mDP cells promoted odontoblast differentiation via phosphorylation of Smad1/5/8, while this BMP2-mediated odontoblast differentiation was inhibited by depletion of Lypd1. Furthermore, we created a deletion construct of the C-terminus containing the omega site in LYPD1; this site is necessary for localizing GPI-APs to the plasma membrane and lipid rafts. We identified that this site is essential for odontoblast differentiation and morphological change of mDP cells. These findings demonstrated that LYPD1 is a novel marker of preodontoblasts in the developing tooth; additionally, they suggest that LYPD1 is important for tooth development and that it plays a pivotal role in odontoblast differentiation by regulating Smad1/5/8 phosphorylation through its effect as a GPI-AP in lipid rafts.

DOI： 10.1016/j.jbc.2023.104638

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Recent advances in regenerative technology have made the regeneration of various organs using pluripotent stem cells possible. However, a simpler screening method for evaluating regenerated organs is required to apply this technology to clinical regenerative medicine in the future. We have developed a simple evaluation method using a mouse tooth germ culture model of organs formed by epithelial–mesenchymal interactions. In this study, we successfully established a simple method that controls tissue development in a temperature-dependent manner using a mouse tooth germ ex vivo culture model. We observed that the development of the cultured tooth germ could be delayed by low-temperature culture and resumed by the subsequent culture at 37 °C. Furthermore, the optimal temperature for the long-term preservation of tooth germ was 25 °C, a subnormothermic temperature that maintains the expression of stem cell markers. We also found that subnormothermic temperature induces the expression of cold shock proteins, such as cold-inducible RNA-binding protein, RNA-binding motif protein 3, and serine and arginine rich splicing factor 5. This study provides a simple screening method to help establish the development of regenerative tissue technology using a tooth organ culture model. Our findings may be potentially useful for making advances in the field of regenerative medicine.

DOI： 10.1038/s41598-023-29629-2

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Language：Japanese   Publisher：(公社)日本小児歯科学会  

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Enamel is the highly mineralized outer layer of teeth; the cells responsible for enamel formation are ameloblasts. Local hypoxia and hypoxia inducible factor (HIF) in embryonic tissues are important to promote normal organogenesis. However, hypoxic state in tooth germs and the roles of HIF in ameloblast differentiation have not been understood. The aim of this study is to clarify the role of HIF in ameloblast differentiation during tooth germ development. We found that tooth germs were under hypoxia and HIF-1α and HIF-2α were expressed in tooth germs in embryonic mice. Then, we used HIF inhibitors to evaluate the function of HIF during tooth germ development. The HIF-2α inhibitor significantly decreased the size of tooth germs in organ culture, while the HIF-1α inhibitor did not apparently affect the size of tooth germs. The HIF-2α inhibitor enhanced the expression of amelogenin, a marker of ameloblast differentiation, in the tooth germs in organ culture and rat dental epithelial SF2 cells. Moreover, we found that the HIF-2α inhibitor-stimulating amelogenin expression was regulated by hes-related family basic helix-loop-helix transcription factor with YRPW motif 2(Hey2) in SF2 cells. These findings suggest that the HIF-2α–Hey2 axis plays an important role in ameloblast differentiation during tooth germ development.

DOI： 10.1177/00220345221111971

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チャンバー内細胞培養系を用いて歯原性間葉系細胞のサブセットが歯原性上皮細胞からエナメル芽細胞への分化に重要であることを発見した。歯髄細胞からGSK3β阻害剤BIOを用いて歯髄幹細胞様細胞を誘導し、歯髄幹細胞により誘導されるエナメル芽細胞分化のメカニズムについて検討した。BIOで誘導された歯髄細胞は間葉系幹細胞マーカーOct3/4およびBcrp1を高発現した。BIOで誘導された人工歯髄幹細胞を用いて、歯髄幹細胞に発現するエナメル芽細胞分化に必要な分子を同定した。歯原性上皮細胞との相互作用を介して歯髄幹細胞でPanx3発現が誘導された。さらにPanx3発現細胞でATP遊離が増加した。ATP刺激は歯原性上皮細胞に受容された。以上の結果から、歯髄幹細胞に発現するPanx3がエナメル芽細胞分化に重要で、Panx3によるATP遊離が上皮間葉系相互作用に寄与すると考えられた。

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Oral Biosciences  

OBJECTIVES: Epithelial-mesenchymal interactions are extremely important in tooth development and essential for ameloblast differentiation, especially during tooth formation. We aimed to identify the type of mesenchymal cells important in ameloblast differentiation. METHODS: We used two types of cell culture systems with chambers and found that a subset of dental mesenchymal cells is important for the differentiation of dental epithelial cells into ameloblasts. Among odontogenic mesenchymal cells, dental pulp stem cell-like cells induced the expression of ameloblast differentiation markers. Therefore, we induced dental pulp stem cell-like cells from dental pulp stem cells using the small molecule compound BIO (a GSK-3 inhibitor IX) to clarify the mechanism involved in inducing ameloblast differentiation of dental pulp stem cells. RESULTS: The BIO-induced dental pulp cells promoted the expression of mesenchymal stem cell markers Oct3/4 and Bcrp1. Furthermore, we used artificial dental pulp stem cells induced by BIO to identify the molecules expressed in dental pulp stem cells required for ameloblast differentiation. Panx3 expression was induced in the dental pulp stem cell through interaction with the dental epithelial cells. In addition, ATP release from cells increased in Panx3-expressing cells. We also confirmed that ATP stimulation is accepted in dental epithelial cells. CONCLUSIONS: These results showed that the Panx3 expressed in dental pulp stem cells is important for ameloblast differentiation and that ATP release by Panx3 may play a role in epithelial-mesenchymal interaction.

DOI： 10.1016/j.job.2022.10.002

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

OBJECTIVES: To investigate the detailed ultrastructural patterns of dental abnormalities affected by Axenfeld-Rieger syndrome (ARS) with a heterozygous microdeletion involving paired-like homeodomain 2 (PITX2) and explored the underlying molecular mechanisms driving enamel defects. SUBJECTS AND METHODS: Sanger sequencing, genomic quantitative PCR analysis, and chromosomal microarray analysis were used to screen the disease-causing mutation in one ARS proband. An exfoliated tooth from an ARS patient was analyzed with scanning electron microscopy and micro-computerized tomography. A stable Pitx2 knockdown cell line was generated to simulate PITX2 haploinsufficiency. Cell proliferation and ameloblast differentiation were analyzed, and the role of the Wnt/β-catenin pathway in proliferation of ameloblast precursor cells was investigated. RESULTS: An approximately 0.216 Mb novel deletion encompassing PITX2 was identified. The affected tooth displayed a thinner and broken layer of enamel and abnormal enamel biomineralization. PITX2 downregulation inhibited the proliferation and differentiation of inner enamel epithelial cells, and LiCl stimulation partially reversed the proliferation ability after Pitx2 knockdown. CONCLUSIONS: Enamel formation is disturbed in some patients with ARS. Pitx2 knockdown can influence the proliferation and ameloblast differentiation of inner enamel epithelial cells, and PITX2 may regulate cell proliferation via Wnt/β-catenin signaling pathway.

DOI： 10.1111/odi.14315

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

Mitochondrial fission factor (MFF) is an adapter that targets dynamin-related protein 1 from the cytosol to the mitochondria for fission. Loss-of-function MFF mutations cause encephalopathy due to defective mitochondrial and peroxisomal fission 2 (EMPF2). To elucidate the molecular mechanisms that were involved, we analyzed the functional effects of MFF depletion in deciduous teeth-derived dental pulp stem cells differentiating into dopaminergic neurons (DNs). When treated with MFF-targeting small interfering RNA, DNs showed impaired neurite outgrowth and reduced mitochondrial signals in neurites harboring elongated mitochondria. MFF silencing also caused mitochondrial Ca2+ accumulation through accelerated Ca2+ influx from the endoplasmic reticulum (ER) via the inositol 1,4,5-trisphosphate receptor. Mitochondrial Ca2+ overload led DNs to produce excessive reactive oxygen species (ROS), and downregulated peroxisome proliferator-activated receptor-gamma co-activator-1 alpha (PGC-1α). MFF was co-immunoprecipitated with voltage-dependent anion channel 1, an essential component of the ER-mitochondrial Ca2+ transport system. Folic acid supplementation normalized ROS levels, PGC-1α mediated mitochondrial biogenesis, and neurite outgrowth in MFF depleted DNs, without affecting their mitochondrial morphology or Ca2+ levels. We propose that MFF negatively regulates the mitochondrial Ca2+ influx from the ER. MFF-insufficiency recapitulated the EMPF2 neuropathology with increased oxidative stress and suppressed mitochondrial biogenesis. ROS and mitochondrial biogenesis might be potential therapeutic targets for EMPF2.

DOI： 10.3390/antiox11071361

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Recently, the development of dental materials has increased the availability of various hyperesthesia desensitizers. However, there are no studies on the duration of retreatment in terms of adherence rates. Thus, the adhesion rates of resin-based desensitizers were investigated. We used a conventional desensitizer and a recently developed desensitizer containing calcium salt of 4-methacryloxyethyl trimellitic acid (C-MET) and 10-methacryloyloxydecyl dihydrogen calcium phosphate (MDCP). These colored agents were applied to the surfaces of premolars and molars, and the area was measured from weekly oral photographs. Areas were statistically analyzed and mean values were calculated using 95% confidence intervals. A p-value of <0.05 was considered statistically significant. These rates were significantly higher on the buccal side of the maxilla and lower on the lingual side of the maxilla. In addition, the desensitizer containing C-MET and MDCP displayed significantly higher adhesion rates. It is suggested that this will require monthly follow-ups and reevaluation because both agents cause less than 10% adherence and there is almost no sealing effect after 4 weeks. In addition, the significantly higher adhesion rate of the desensitizer containing C-MET and MDCP indicated that the novel monomer contributed to the improvement in the adhesion ability.

DOI： 10.3390/ma15155172

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Down syndrome (DS) is one of the common genetic disorders caused by the trisomy of human chromosome 21 (HSA21). Mitochondrial dysfunction and redox imbalance play important roles in DS pathology, and altered dopaminergic regulation has been demonstrated in the brain of individuals with DS. However, the pathological association of these elements is not yet fully understood. In this study, we analyzed dopaminergic neurons (DNs) differentiated from deciduous teeth-derived stem cells of children with DS or healthy control children. As previously observed in the analysis of a single case of DS, compared to controls, patient-derived DNs (DS-DNs) displayed shorter neurite outgrowth and fewer branches, as well as downregulated vesicular monoamine transporter 2 and upregulated dopamine transporter 1, both of which are key regulators of dopamine homeostasis in DNs. In agreement with these expression profiles, DS-DNs accumulated dopamine intracellularly and had increased levels of cellular and mitochondrial reactive oxygen species (ROS). DS-DNs showed downregulation of non-canonical Notch ligand, delta-like 1, which may contribute to dopamine accumulation and increased ROS levels through DAT1 upregulation. Furthermore, DS-DNs showed mitochondrial dysfunction in consistent with lower expression of peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) and upregulation of a HSA21-encoded negative regulator of PGC-1α, nuclear receptor-interacting protein 1. These results suggest that dysregulated dopamine homeostasis may participate in oxidative stress and mitochondrial dysfunction of the dopaminergic system in DS.

DOI： 10.1096/fba.2021-00086

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Organ development is dictated by the regulation of genes preferentially expressed in tissues or cell types. Gene expression profiling and identification of specific genes in organs can provide insights into organogenesis. Therefore, genome-wide analysis is a powerful tool for clarifying the mechanisms of development during organogenesis as well as tooth development. Single-cell RNA sequencing (scRNA-seq) is a suitable tool for unraveling the gene expression profile of dental cells. Using scRNA-seq, we can obtain a large pool of information on gene expression; however, identification of functional genes, which are key molecules for tooth development, via this approach remains challenging. In the present study, we performed cap analysis of gene expression sequence (CAGE-seq) using mouse tooth germ to identify the genes preferentially expressed in teeth. The CAGE-seq counts short reads at the 5′-end of transcripts; therefore, this method can quantify the amount of transcripts without bias related to the transcript length. We hypothesized that this CAGE data set would be of great help for further understanding a gene expression profile through scRNA-seq. We aimed to identify the important genes involved in tooth development via bioinformatics analyses, using a combination of scRNA-seq and CAGE-seq. We obtained the scRNA-seq data set of 12,212 cells from postnatal day 1 mouse molars and the CAGE-seq data set from postnatal day 1 molars. scRNA-seq analysis revealed the spatiotemporal expression of cell type–specific genes, and CAGE-seq helped determine whether these genes are preferentially expressed in tooth or ubiquitously. Furthermore, we identified candidate genes as novel tooth-enriched and dental cell type–specific markers. Our results show that the integration of scRNA-seq and CAGE-seq highlights the genes important for tooth development among numerous gene expression profiles. These findings should contribute to resolving the mechanism of tooth development and establishing the basis for tooth regeneration in the future.

DOI： 10.1177/00220345211049785

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Development of chemotherapy has led to a high survival rate of cancer patients; however, the severe side effects of anticancer drugs, including organ hypoplasia, persist. To assume the side effect of anticancer drugs, we established a new ex vivo screening model and described a method for suppressing side effects. Cyclophosphamide (CPA) is a commonly used anticancer drug and causes severe side effects in developing organs with intensive proliferation, including the teeth and hair. Using the organ culture model, we found that treatment with CPA disturbed the growth of tooth germs by inducing DNA damage, apoptosis and suppressing cellular proliferation and differentiation. Furthermore, low temperature suppressed CPA-mediated inhibition of organ development. Our ex vivo and in vitro analysis revealed that low temperature impeded Rb phosphorylation and caused cell cycle arrest at the G1 phase during CPA treatment. This can prevent the CPA-mediated cell damage of DNA replication caused by the cross-linking reaction of CPA. Our findings suggest that the side effects of anticancer drugs on organ development can be avoided by maintaining the internal environment under low temperature.

DOI： 10.1038/s41598-022-06945-7

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Tissue-specific basic helix-loop-helix (bHLH) transcription factors play an important role in cellular differentiation. We recently identified AmeloD as a tooth-specific bHLH transcription factor. However, the role of AmeloD in cellular differentiation has not been investigated. The aim of this study was to elucidate the role of AmeloD in dental epithelial cell differentiation. We found that AmeloD-knockout (AmeloD-KO) mice developed an abnormal structure and altered ion composition of enamel in molars, suggesting that AmeloD-KO mice developed enamel hypoplasia. In molars of AmeloD-KO mice, the transcription factor Sox21 encoding SRY-Box transcription factor 21 and ameloblast differentiation marker genes were significantly downregulated. Furthermore, overexpression of AmeloD in the dental epithelial cell line M3H1 upregulated Sox21 and ameloblast differentiation marker genes, indicating that AmeloD is critical for ameloblast differentiation. Our study demonstrated that AmeloD is an important transcription factor in amelogenesis for promoting ameloblast differentiation. This study provides new insights into the mechanisms of amelogenesis.

DOI： 10.1002/jcp.30639

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Cell- and tissue-specific extracellular matrix (ECM) composition plays an important role in organ development, including teeth, by regulating cell behaviors, such as cell proliferation and differentiation. Here, we demonstrate for the first time that von Willebrand factor D and epidermal growth factor (EGF) domains (Vwde), a previously uncharacterized ECM protein, is specifically expressed in teeth and regulates cell proliferation and differentiation in inner enamel epithelial cells (IEEs) and enamel formation. We identified the Vwde as a novel ECM protein through bioinformatics using the NCBI expressed sequence tag database for mice. Vwde complementary DNA encodes 1773 amino acids containing a signal peptide, a von Willebrand factor type D domain, and tandem calcium-binding EGF-like domains. Real-time polymerase chain reaction demonstrated that Vwde is highly expressed in tooth tissue but not in other tissues including the brain, lung, heart, liver, kidney, and bone. In situ hybridization revealed that the IEEs expressed Vwde messenger RNA in developing teeth. Immunostaining showed that VWDE was localized at the proximal and the distal ends of the pericellular regions of the IEEs. Vwde was induced during the differentiation of mouse dental epithelium-derived M3H1 cells. Vwde-transfected M3H1 cells secreted VWDE protein into the culture medium and inhibited cell proliferation, whereas ameloblastic differentiation was promoted. Furthermore, Vwde increased the phosphorylation of extracellular signal-regulated kinase 1/2 and protein kinase B and strongly induced the expression of the intercellular junction protein, N-cadherin (Ncad). Interestingly, the suppression of endogenous Vwde inhibited the expression of Ncad. Finally, we created Vwde-knockout mice using the CRISPR-Cas9 system. Vwde-null mice showed low mineral density, rough surface, and cracks in the enamel, indicating the enamel hypoplasia phenotype. Our findings suggest that Vwde assembling the matrix underneath the IEEs is essential for Ncad expression and enamel formation.

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DOI： 10.3390/children8121189

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DOI： 10.3390/children8121189

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DOI： 10.3389/fcell.2020.595593

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DOI： 10.3390/ijms21238952

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Direct reprogramming, also known as a trans-differentiation, is a technique to allow mature cells to be converted into other types of cells without inducing a pluripotent stage. It has been suggested as a major strategy to acquire the desired type of cells in cell-based therapies to repair damaged tissues. Studies related to switching the fate of cells through epigenetic modification have been progressing and they can bypass safety issues raised by the virus-based transfection methods. In this study, a protocol was established to directly convert fully differentiated fibroblasts into diverse mesenchymal-lineage cells, such as osteoblasts, adipocytes, chondrocytes, and ectodermal cells, including neurons, by means of DNA demethylation, immediately followed by culturing in various differentiating media. First, 24 h exposure of 5-azacytidine (5-aza-CN), a well-characterized DNA methyl transferase inhibitor, to NIH-3T3 murine fibroblast cells induced the expression of stem-cell markers, that is, increasing cell plasticity. Next, 5-aza-CN treated fibroblasts were cultured in osteogenic, adipogenic, chondrogenic, and neurogenic media with or without bone morphogenetic protein 2 for a designated period. Differentiation of each desired type of cell was verified by quantitative reverse transcriptase-polymerase chain reaction/ western blot assays for appropriate marker expression and by various staining methods, such as alkaline phosphatase/alizarin red S/oil red O/alcian blue. These proposed procedures allowed easier acquisition of the desired cells without any transgenic modification, using direct reprogramming technology, and thus may help make it more available in the clinical fields of regenerative medicine.

DOI： 10.4196/kjpp.2020.24.6.463

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DOI： 10.1038/s41598-020-73479-1

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一般社団法人日本障害者歯科学会は、平成20年に日本障害者歯科学会指導歯科衛生士(以下:指導DH)、日本歯科衛生士会認定衛生士(認定分野B:障害者歯科、以下:認定DH)制度を導入した。本制度の下、障害者歯科診療のための歯科衛生士養成の認定DH研修ガイドライン(以下:研修ガイドライン)が作成され、指導DHは本研修ガイドラインに沿って認定DH等の育成を行うことが望ましいとされている。しかし、歯科衛生士(以下:DH)の障害者歯科研修あるいは指導等において、本ガイドラインに提示されている項目の達成状況を検討した報告はない。そこで本研究は、研修ガイドラインに準じたアンケート調査を行うことで、障害者歯科に携わるDHのガイドライン項目の達成状況を把握することを目的とした。調査は、指導DH、認定DH、および認定資格を有していないDH(以下:一般DH)を対象とした。本調査において、指導DH、認定DH、一般DHの各研修ガイドラインの項目の達成状況の評価に特徴が認められた。研修ガイドラインの項目によっては、臨床経験だけでなく、時代背景や教育課程の違いが関与していることが示唆された。これらの結果より、DH育成において研修ガイドラインの項目ごとにその達成状況を調査分析することは、今後のDH教育に必要とされる項目の検討や、臨床現場で働くそれぞれの立場に応じたDHに対する研修項目の拡充を検討する際に活用できると考えられた。(著者抄録)

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An amendment to this paper has been published and can be accessed via a link at the top of the paper.

DOI： 10.1038/s42003-020-01292-7

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DOI： 10.1038/s42003-020-01227-2

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DOI： 10.1074/jbc.RA120.014281

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DOI： 10.1096/fj.202001340R

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DOI： 10.3389/fcell.2020.00841

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DOI： 10.1016/j.isci.2020.101329

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Metatropic dysplasia (MD) is a congenital skeletal dysplasia characterized by severe platyspondyly and dumbbell-like long-bone deformities. These skeletal phenotypes are predominantly caused by autosomal dominant gain-of-function (GOF) mutations in transient receptor potential vanilloid 4 (TRPV4), which encodes a nonselective Ca2+-permeable cation channel. Previous studies have shown that constitutive TRPV4 channel activation leads to irregular chondrogenic proliferation and differentiation, and thus to the disorganized endochondral ossification seen in MD. Therefore, the present study investigated the role of TRPV4 in osteoblast differentiation and MD pathogenesis. Specifically, the behavior of osteoblasts differentiated from patient-derived dental pulp stem cells carrying a heterozygous single base TRPV4 mutation, c.1855C > T (p.L619F) was compared to that of osteoblasts differentiated from isogenic control cells (in which the mutation was corrected using the CRISPR/Cas9 system). The mutant osteoblasts exhibited enhanced calcification (indicated by intense Alizarin Red S staining), increased intracellular Ca2+ levels, strongly upregulated runt-related transcription factor 2 and osteocalcin expression, and increased expression and nuclear translocation of nuclear factor-activated T cell c1 (NFATc1) compared to control cells. These results suggest that the analyzed TRPV4 GOF mutation disrupts osteoblastic differentiation and induces MD-associated disorganized endochondral ossification by increasing Ca2+/NFATc1 pathway activity. Thus, inhibiting the NFATc1 pathway may be a promising potential therapeutic strategy to attenuate skeletal deformities in MD.

DOI： 10.1016/j.bbrc.2019.12.123

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Epithelial-mesenchymal interaction has critical roles for organ development including teeth, during which epithelial thickening and mesenchymal condensation are initiated by precise regulation of the signaling pathway. In teeth, neural crest-derived mesenchymal cells expressed PDGF receptors migrate and become condensed toward invaginated epithelium. To identify the molecular mechanism of this interaction, we explored the specific transcriptional start sites (TSSs) of tooth organs using cap analysis of gene expression (CAGE). We identified a tooth specific TSS detected in the chromosome 15qD1 region, which codes microRNA-875 (mir875). MiR875-5p is specifically expressed in dental mesenchyme during the early stage of tooth development. Furthermore, PRRX1/2 binds to the mir875 promoter region and enhances the expression of mir875. To assess the role of miR875-5p in dental mesenchyme, we transfected mimic miR875-5p into mouse dental pulp (mDP) cells, which showed that cell migration toward dental epithelial cells was significantly induced by miR875-5p via the PDGF signaling pathway. Those results also demonstrated that miR875-5p induces cell migration by inhibiting PTEN and STAT1, which are regulated by miR875-5p as part of post-transcriptional regulation. Together, our findings indicate that tooth specific miR875-5p has important roles in cell condensation of mesenchymal cells around invaginated dental epithelium and induction of epithelial-mesenchymal interaction.

DOI： 10.1038/s41598-020-61693-w

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Many genes encoding growth factors, receptors, and transcription factors are induced by the epithelial-mesenchymal interaction during tooth development. Recently, numerous functions of microRNAs (miRNAs) are reportedly involved in organogenesis and disease. miRNAs regulate gene expression by inhibiting translation and destabilizing mRNAs. However, the expression and function of miRNAs in tooth development remain poorly understood. This study aimed to analyze the expression of miRNAs produced during tooth development using a microarray system to clarify the role of miRNAs in dental development. miR-1 showed a unique expression pattern in the developing tooth. miR-1 expression in the tooth germ peaked on embryonic day 16.5, decreasing gradually on postnatal days 1 and 3. An in situ hybridization assay revealed that miR-1 is expressed at the cervical loop of the dental epithelium. The expression of miR-1 and connexin (Cx) 43, a target of miR-1, were inversely correlated both in vitro and in vivo. Knockdown of miR-1 induced the expression of Cx43 in dental epithelial cells. Interestingly, cells with miR-1 downregulation proliferated slower than the control cells. Immunocytochemistry revealed that Cx43 in cells with miR-1 knockdown formed both cell-cell gap junctions and hemichannels at the plasma membrane. Furthermore, the rate of ATP release was higher in cells with miR-1 knockdown than in control cells. Furthermore, Cx43 downregulation in developing molars was observed in Epiprofin-knockout mice, along with the induction of miR-1 expression. These results suggest that the expression pattern of Cx43 is modulated by miR-1 to control cell proliferation activity during dental epithelial cell differentiation.

DOI： 10.3389/fcell.2020.00156

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Lipin-2 is a phosphatidate phosphatase with key roles in regulating lipid storage and energy homeostasis. LPIN2-genetic deficiency is associated with an autoinflammatory disorder, underscoring its critical role in innate immune signaling; however, the regulatory mechanisms underlying protein stability remain unknown. Here, we demonstrate that Lipin-2 interacts with β-TRCP, a substrate receptor subunit of the SCFβ-TRCP E3 ligase, and undergoes ubiquitination and proteasomal degradation. β-TRCP-knockout in RAW264.7 macrophages resulted in Lipin-2 accumulation, leading to the suppression of LPS-induced MAPK activation and subsequent proinflammatory gene expression. Consistent with this, treatment with MLN4924, a Cullin-neddylation inhibitor that suppresses SCF E3 activity, increased Lipin-2 protein and concomitantly decreased Il1b expression. These findings suggested that β-TRCP-mediated Lipin-2 degradation affects macrophage-elicited proinflammatory responses and could lead to new therapeutic approaches to treat inflammatory diseases.

DOI： 10.1016/j.bbrc.2020.01.119

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Pannexin 3 (Panx3) is a regulator of bone formation. Panx3 forms three distinct functional channels: hemichannels, gap junctions, and endoplasmic reticulum (ER) Ca2+ channels. However, the gating mechanisms of the Panx3 channels remain unclear. Here, we show that the Panx3 ER Ca2+ channel is modulated by phosphorylation of the serine 68 residue (Ser68) to promote osteoblast differentiation. Among the 17 candidate phosphorylation sites identified, the mutation of Ser68 to Ala (Ser68Ala) was sufficient to inhibit Panx3-mediated osteoblast differentiation via reduction of Osterix and ALP expression. Using a Ser68 phospho-specific antibody (P-Panx3) revealed Panx3 was phosphorylated in prehypertrophic, hypertrophic chondrocytes, and bone areas of the newborn growth plate. In osteogenic C2C12 cells, P-Panx3 was located on the ER membranes. Importantly, the Ser68Ala mutation only affected Panx3 ER Ca2+ channel function. Ser68 on Panx3 was phosphorylated by ATP stimulation and PI3K/Akt signaling. Finally, real-time FRET imaging and ratio analysis revealed that the Panx3 channel conformation was sensitive to ATP. Together, the phosphorylation of Panx3 at Ser68 is an essential step controlling the gating of the Panx3 ER Ca2+ channel to promote osteogenesis.

DOI： 10.1038/s41598-019-55371-9

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Signal transmission from the mechanical forces to the various intracellular activities is a fundamental process during tissue development. Despite their critical role, the mechanism of mechanical forces in the biological process is poorly understood. In this study, we demonstrated that in the response to hydrostatic pressure (HP), the piezo type mechanosensitive ion channel component 1 (PIEZO1) is a primary mechanosensing receptor for odontoblast differentiation through coordination of the WNT expression and ciliogenesis. In stem cells from human exfoliated deciduous teeth (SHED), HP significantly promoted calcium deposition as well as the expression of odontogenic marker genes, PANX3 and DSPP, and WNT related-genes including WNT5b and WNT16, whereas HP inhibited cell proliferation and enhanced primary cilia expression. WNT signaling inhibitor XAV939 and primary cilia inhibitor chloral hydrate blocked the HP-induced calcium deposition. The PIEZO1 activator Yoda1 inhibited cell proliferation but induced ciliogenesis and WNT16 expression. Interestingly, HP and Yoda1 promoted nuclear translocation of RUNX2, whereas siRNA-mediated silencing of PIEZO1 decreased HP-induced nuclear translocation of RUNX2. Taken together, these results suggest that PIEZO1 functions as a mechanotransducer that connects HP signal to the intracellular signalings during odontoblast differentiation.

DOI： 10.1038/s41598-019-51381-9

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Background: Sublingual immunotherapy (SLIT) is used for the treatment of type 1 allergies, such as allergic rhinitis. SLIT leads to tolerance against allergens possibly via the redirection of allergen-specific T helper 2 cells to T helper 1 cells and the generation of peripheral regulatory T (Treg) cells. However, the detailed mechanisms remain unclear. Systemic tolerance to orally administered antigens (oral tolerance) has been extensively investigated. Recent studies have recognized the central role of Treg cells and classical dendritic cells (cDCs) in oral tolerance development.

Highlight: This review focuses on recent advances in the understanding of the underlying mechanisms of SLIT compared with those of oral tolerance. The sublingual administration of soluble protein antigens has been reported to induce antigen-specific Treg cells in oral mucosa-draining submandibular lymph nodes in mice. The generation of Treg cells is critical for SLIT efficacy because the transfer of SLIT-induced Treg cells confers tolerance against the antigens. A large number of oral cDCs with the CD103-CD11b+ phenotype exert retinoic acid-producing activity and convert naïve CD4+ T cells into Foxp3+ Treg cells in vitro in a transforming growth factor-β-dependent and retinoic acid-dependent manner. Oral CD103-CD11b+ cDCs transport sublingual antigens to submandibular lymph nodes and induce antigen-specific Treg cells. Sublingual antigens enter the mucosa most likely by crossing the sublingual ductal epithelium and are captured by oral antigen-presenting cells, especially macrophages.

DOI： 10.1016/j.job.2019.02.001

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Wound healing is a dynamic process that involves highly coordinated cellular events, including proliferation and migration. Oral gingival fibroblasts serve a central role in maintaining oral mucosa homeostasis, and their functions include the coordination of physiological tissue repair. Recently, surface pre‑reacted glass‑ionomer (S‑PRG) fillers have been widely applied in the field of dental materials for the prevention of dental caries, due to an excellent ability to release fluoride (F). In addition to F, S‑PRG fillers are known to release several types of ions, including aluminum (Al), boron (B), sodium (Na), silicon (Si) and strontium (Sr). However, the influence of these ions on gingival fibroblasts remains unknown. The aim of the present study was to examine the effect of various concentrations of an S‑PRG filler eluate on the growth and migration of gingival fibroblasts. The human gingival fibroblast cell line HGF‑1 was treated with various dilutions of an eluent solution of S‑PRG, which contained 32.0 ppm Al, 1,488.6 ppm B, 505.0 ppm Na, 12.9 ppm Si, 156.5 ppm Sr and 136.5 ppm F. Treatment with eluate at a dilution of 1:10,000 was observed to significantly promote the migration of HGF‑1 cells. In addition, the current study evaluated the mechanism underlying the mediated cell migration by the S‑PRG solution and revealed that it activated the phosphorylation of extracellular signal‑regulated kinase 1/2 (ERK1/2), but not of p38. Furthermore, treatment with a MEK inhibitor blocked the cell migration induced by the solution. Taken together, these results suggest that S‑PRG fillers can stimulate HGF‑1 cell migration via the ERK1/2 signaling pathway, indicating that a dental material containing this type of filler is useful for oral mucosa homeostasis and wound healing.

DOI： 10.3892/mmr.2019.10141

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© 2018 The Author(s). Background: Molar incisor hypomineralization (MIH) frequently occurs in children worldwide. However, MIH prevalence throughout Japan has not yet been investigated. The purpose of this study was to clarify MIH prevalence rates and to consider potential regional differences throughout Japan. Methods: A total of 4496 children aged 7-9 years throughout Japan were evaluated in this study. MIH prevalence rates among children were evaluated in eight regions throughout Japan. A child's residence was defined as the mother's residence during pregnancy. The localization of demarcated opacities and enamel breakdown was recorded on a standard code form using a guided record chart. Logistic regression analysis was used to evaluate whether MIH prevalence rates differed among age groups, sex, and regions. Results: The overall prevalence of MIH in Japan was 19.8%. The prevalence of MIH was 14.0% in the Hokkaido region, 11.7% in the Tohoku region, 18.5% in the Kanto Shin-Etsu region, 19.3% in the Tokai Hokuriku region, 22.3% in the Kinki region, 19.8% in the Chugoku region, 28.1% in the Shikoku region, and 25.3% in the Kyushu region. These regional differences were statistically significant. Moreover, MIH prevalence rates decreased with age. No significant sex differences in MIH prevalence rates were demonstrated. Conclusions: To our knowledge, this is the first MIH study carried out in several regions throughout Japan. Regional differences existed in MIH prevalence rates; particularly, MIH occurred more frequently in children residing in southwestern areas than those in northeastern areas of Japan.

DOI： 10.1186/s12199-018-0748-6

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ラット歯原性上皮細胞の分化とマイグレーションにおけるカルシウム応答の解析

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© 2016 The Pharmaceutical Society of Japan. Pain is sensed, transmitted, and modified by a variety of mediators and receptors. Histamine is a wellknown mediator of pain. In addition to their anti-histaminic effects, the classical, or 1st generation, anti-histamines (1st AHs) possess, to various degrees, anti-muscarinic, anti-serotonergic, anti-adrenergic, and other pharmacologic effects. Although there have been attempts to use 1st AHs as analgesics and/or analgesic adjuvants, the advent of non-steroidal anti-inflammatory drugs (NSAIDs) discouraged such trials. We previously reported that in patients with temporomandibular disorders, osteoporosis, and/or osteoarthritis, the analgesic effects of certain 1st AHs (chlorpheniramine and diphenhydramine) are superior to those of the NSAIDs flurbiprofen and indomethacin. Here, we compared analgesic effects among 1st AHs and NSAIDs against responses shown by mice to intraperitoneally injected 0.7% acetic acid. Since 1st AHs are water soluble, we selected water-soluble NSAIDs. For direct comparison, drugs were intravenously injected 30 min before the above tests. Histamine-H1-receptor-deficient (H1R-KO) mice were used for evaluating H1-receptor-independent effects. The tested 1st AHs (especially cyproheptadine) displayed or tended to display analgesic effects comparable to those of NSAIDs in normal and H1R-KO mice. Our data suggest that the anti-serotonergic and/or anti-adrenergic effects of 1st AHs make important contributions to their analgesic effects. Moreover, combination of a 1st AH with an NSAID (cyclooxygenase-1 inhibitor) produced remarkably potent analgesic effects. We propose that a 1st AH, by itself or in combination with a cyclooxygenase-1 inhibitor, should undergo testing to evaluate its usefulness in analgesia.

DOI： 10.1248/bpb.b15-00755

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白血病治療を目指したヒト乳歯歯髄細胞から人工骨髄誘導法の開発

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Although some studies have reported that breast-feeding and pacifier use influence finger-sucking, few have demonstrated whether the age at cessation of breast-feeding or pacifier use and persistent finger-sucking are related. Therefore, the purpose of this study was to examine whether the age at cessation of breast-feeding and pacifier use influenced persistent finger-sucking. Methods: A cross-sectional study of 555 36- to 47-month-olds was conducted in Nagasaki, Japan, using a questionnaire. Using the optimal cutoff point in a receiver-operating characteristic curve, the age was estimated at which cessation of pacifier use and breast-feeding had the most significant effect on persistent finger-sucking, and the estimated ages were assessed by multiple logistic regression analysis, incorporating all the questions in the questionnaire as independent variables. Results: The odds ratios for persistent finger-sucking when breast-feeding was stopped at an age younger than 12 months old or when pacifier use was stopped at an age younger than 14 months old were 3.77 (95 percent confidence interval (CI)=1.97-7.22) and 8.62 (95 percent CI=2.56-29.04), respectively. Conclusions: Cessation of breast-feeding before 12 months old or pacifier use before 14 months old was associated with persistent finger-sucking.

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Active metabolite vitamin D3, 1α,25-dihydroxyvitamin D3, is a pleiotropic factor and exhibits various physiological functions, including immunomodulating activities. In this study, the possible regulation of innate immune responses of human monocytic cells by a vitamin D3 analog was examined. Human monocytic THP-1 cells were pre-treated with OCT, vitamin D3 analog, 1α,25-dihydroxy-22-oxavitamin D3, followed by stimulation with various chemically synthesized Toll-like receptors (TLR) and NOD1 and NOD2 ligands. OCT-treated cells produced more IL-8 than non-treated cells upon stimulation with various chemically-synthesized ligands: TLR2-agonistic lipopeptide (FSL-1), TLR3-agonistic poly I:C, TLR4-agonistic lipid A (E. coli-type LA-15-PP), NOD1-agonistic FK565 and NOD2-agonistic muramyldipeptide (MDP). Among the ligands, MDP was the highest inducer of IL-8 production in OCT-treated THP-1 cells, and IL-8 production increased depending on the treatment time until 72 h. OCT up-regulated the expression of NOD2 in THP-1 cells, and OCT-treated cells exhibited higher activation of p38, JNK and ERK in the MAPK pathway, IκBα in the NF-κB pathway, and TAK1 upstream in response to MDP than non-treated cells. Analysis using siRNA against NOD2 and inhibitors of specific signal molecules indicated that the existence of NOD2 and activation of the above signaling molecules are required for enhanced production of IL-8 in OCT-treated THP-1 cells. These findings suggested that NOD2, NF-κB and MAPK pathways are involved in the activity of OCT to augment the response of human monocytic cells to MDP. © 2012 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.intimp.2012.10.027

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An organ culture system to model the physiological calcification process was designed using rat embryonic calvaria as a device for analyzing its mechanism. Standardized calvarial explants were dissected from rat embryos aged 18 and 20 days (E18 and E20) and cultured for 1, 3 and 5 days. The calcium content of the cultured explants was quantified by atomic absorption spectrophotometry. Equivalent explants were fixed, embedded in paraffin, sectioned and stained with von Kossa stain combined with hematoxylin-eosin or processed for energy-dispersive X-ray spectroscopy to determine the concentrations of calcium, phosphorus and carbon in the tissue. The total calcium content increased significantly in E18 and E20 cultured calvaria (E18cc and E20cc) over 5 days of culture. All cultured calvaria were von Kossa-positive, whereas the staining was intensified, and sound osteoblasts and osteocytes were observed in the bone matrix only in E18cc during the 5-day culture period. Concentrations of calcium and carbon increased significantly in E18cc over 5 days, whereas E20 showed little increase. Physiological calcification proceeded in E18cc, but not in E20cc. These results indicate that the organ culture system using E18 calvaria is useful for modeling the physiological calcification process in vitro. © The Author 2011. Published by Oxford University Press [on behalf of Japanese Society of Microscopy]. All rights reserved.

DOI： 10.1093/jmicro/dfr068

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少子超高齢社会では、国や社会制度は高齢者主体になりがちである。しかし、歯科疾患では、小児期の健康状態がその後の成人期、高齢期に大きく影響を与える。従って小児における口腔保健と歯科医療は非常に重要である。今回、平成22年度の保険点数改定結果および「混合歯列期歯周組織検査」に関するアンケート調査を実施した。対象は一般社団法人日本小児歯科学会の役員130名で、回答のあった58名(44.6%)について検討した結果、以下の結論を得た。1.保険改定結果については、非常に良かった3.5%、まあまあ良かった39.6%、かなり悪くなった5.2%、やや悪くなった17.2%、変化がない34.5%であった。2.本年度と昨年度との総点数の比較では、4月は1.7%上昇、5月は0.6%減少、6月も0.7%減少していた。3.点数増加の原因として、初診料・再診料の増点を92.9%、6歳未満の加算の復活を88.9%、自治体における乳幼児医療費助成の拡大を48.0%が回答していた。4.点数減少の原因として、子どもの受診が減少を55.6%、齲蝕の減少を52%が回答していた。また、混合歯列期歯周組織検査の導入は、39.1%が点数減少の原因として認め、60.9%は認めていなかった。5.歯周組織検査の実施割合は、P混検は4月30.7%、5月35.7%、6月35.2%であった。P基検は、4月31.4%、5月29.1%、6月29.8%であった。全く算定していないが、4月37.9%、5月35.2%、6月35.0%と3分の1以上に認められた。6.歯周組織検査に必要な時間は、P混検が約7.3分、P基検が約7.2分、P精検が約13.3分であった。7.成人の歯周病予防にも乳歯および混合歯列期の歯周組織検査の普及が必要であり、乳歯列期および混合歯列期の検査方法および検査用紙を考案した。(著者抄録)

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インテグリン-フィラミンA複合体による歯の形態形成の分子制御

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象牙芽細胞分化におけるギャップ結合分子の発現および機能解析

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神経栄養因子による歯原性上皮細胞の増殖、分化制御機構の解明

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歯科再生医療に歯の発生生物学はどのように貢献してきたか、そして今後どのように貢献できるか 歯原性上皮の分化の分子制御メカニズム

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First Introduction of Low aged Patients to Dental Treatment in the Clinic of Pediatric Dental Specialists
In order to investigate the actual conditions of the first introduction of low aged patients to dental treatment, a questionnaire survey was conducted among 117 of pediatric dental specialists who belongto the Japanese Society of Pediatric Dentistry. 56 specialists (47.9&#37;) answered the questionnaire and the following results were obtained through analysis of the data of 434 low aged patients.1)The main reasons of the selection of the dental clinic were ①because it was a pediatric dental specialists clinic (33.2&#37;), ②referal from another clinic (32.3&#37;), ③introduction from friends or family members (31.3&#37;). These results indicated the important role of the pediatric dental specialist.2)Parents accepted the crying of the child patient to some extent (50.7&#37;) and hoped that the child patients would be treated even though they may cry (44.7&#37;). 4.6&#37; of the parents did not want their child to be treated when they cry.3)69.8&#37; of the parents hoped to stay in the treatment room from the beginning to the end of the treatment.4)Reaction of the child patients was categorized "restrained because the patient moves (34.6&#37;)","well behaved (31.6&#37;)", "cooperative even though the patient may cry or move (19.1&#37;)" and "treated without restrain even though the patient cries and moves (14.7&#37;)".5)The pediatric dental specialists reacted mostly (71.9&#37;) by "practicing the treatment following the planned treatment schedule with necessary reaction to the patients".6)The numbers of the staff necessary for the treatment decreased in proportion to the age of the patient with an average of 2.7.7)The average treatment time was the longest for the four years old patients (42.8 min) and the average for all age groups was 38.6 min. The treatment time for the patients who were restrained because of crying and moving was longer than the patients who behaved well.8)The parents highly evaluated the treatment and the explanation by the pediatric specialists, which indicated the satisfaction of the parents whose child had treatment by the pediatric specialists.

DOI： 10.11411/jspd.48.3_397

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First Introduction of Low aged Patients to Dental Treatment in the Clinic of Pediatric Dental Specialists
In order to investigate the actual conditions of the first introduction of low aged patients to dental treatment, a questionnaire survey was conducted among 117 of pediatric dental specialists who belongto the Japanese Society of Pediatric Dentistry. 56 specialists (47.9&#37;) answered the questionnaire and the following results were obtained through analysis of the data of 434 low aged patients.1)The main reasons of the selection of the dental clinic were ①because it was a pediatric dental specialists clinic (33.2&#37;), ②referal from another clinic (32.3&#37;), ③introduction from friends or family members (31.3&#37;). These results indicated the important role of the pediatric dental specialist.2)Parents accepted the crying of the child patient to some extent (50.7&#37;) and hoped that the child patients would be treated even though they may cry (44.7&#37;). 4.6&#37; of the parents did not want their child to be treated when they cry.3)69.8&#37; of the parents hoped to stay in the treatment room from the beginning to the end of the treatment.4)Reaction of the child patients was categorized "restrained because the patient moves (34.6&#37;)","well behaved (31.6&#37;)", "cooperative even though the patient may cry or move (19.1&#37;)" and "treated without restrain even though the patient cries and moves (14.7&#37;)".5)The pediatric dental specialists reacted mostly (71.9&#37;) by "practicing the treatment following the planned treatment schedule with necessary reaction to the patients".6)The numbers of the staff necessary for the treatment decreased in proportion to the age of the patient with an average of 2.7.7)The average treatment time was the longest for the four years old patients (42.8 min) and the average for all age groups was 38.6 min. The treatment time for the patients who were restrained because of crying and moving was longer than the patients who behaved well.8)The parents highly evaluated the treatment and the explanation by the pediatric specialists, which indicated the satisfaction of the parents whose child had treatment by the pediatric specialists.

DOI： 10.11411/jspd.48.3_397

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エピプロフィンによる歯原性上皮細胞の増殖と分化の制御機構

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唾液腺分岐におけるギャップ結合の役割解明と再生への応用

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Maxillary Canine Replantation in the Patient with Eruption Disturbance due to Odontoma
We report a clinical case of the replantation of impacted maxillary left canine due to an odontoma.The patient had received dental management since infancy in our hospital. Delayed eruption of maxillary left central and lateral incisors had been noted. From the ages of 9 years and 3 months to 11 years and 5 months, retractions of these incisors were performed using the multi-bracket method. At the age of 12 years and 7 months, due to prolonged retention of the maxillary left primary canine,X-ray photographic examination was conducted. A well-delineated radiopaque image was observed near the root apex of the left primary canine. The cusp of impacted maxillary left canine was present near the root apex of lateral incisor. The retraction of maxillary left canine seemed to have difficulty because of its almost completed root formation and the odontoma. At the age of 12 years and 10 months, the odontoma was surgically removed, and the impacted canine was extracted and replanted into the extraction socket of primary canine. Two months after the replantation, endodontic treatment was started. At the age of 14 years and 8 months, restoration with resin-veneered crown was performed.A long single root of the replanted tooth might be advantageous for replantation. Also, in this case,the removed socket of the odontoma was an appropriate size for the replantaion space of impacted tooth. Further, proper aseptic treatment could be done because the replantation tooth was completely impacted. We considered that these situations might contribute to the success of replantation.

DOI： 10.11411/jspd.48.1_101

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顎顔面発生研究の新規展開 若手研究者の発想とねらい 上皮器官形成における転写因子エピプロフィンの役割

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Objectives: Connexin43 (Cx43) is one of the proteins associated with gap junction. Connexin43 knockout mice die after birth owing to hypoplastic lungs. The purpose of this study was to analyze the hypoplastic lung of Cx43 knockout mice to clarify the role of the Cx43 during lung development. Methods: Adult hetero Cx43 mice were mated. Newborn mice were divided into the following groups: wild, hetero, and knockout. Total RNA was extracted from the right lung, and the left lung was fixed for immunohistochemical staining. The mRNA expression of surfactant protein C, aquaporin-5, and α-smooth muscle actin were analyzed by reverse transcriptase polymerase chain reaction. H&E and immunohistochemical staining for those markers were performed. Results: The mRNA expression of aquaporin-5, surfactant protein C, and α-smooth muscle actin was significantly lower in knockout mice than that in the wild and hetero mice. H&E staining in the knockout mice showed narrow airspaces and thicker interalveolar septae. Immunohistochemical staining in all markers showed the formation of alveoli to be delayed in the knockout mice. Conclusion: Based on these findings, Cx43 is closely related to alveolar and vascular formation during lung development. © 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.jpedsurg.2009.07.070

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Dental epithelium proliferation and differentiation regulated by ameloblastin.

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P561T heterozygous missense mutation in the growth hormone receptor (GHR) is a candidate genetic polymorphism (single-nucleotide polymorphism) for human mandibular growth. The purpose of this study was to assess whether this mutation affects mandibular growth during early childhood. The difference in mandibular growth between P561T heterozygous and wild-type individuals was analysed by cephalometric measurements during childhood. The subjects included 33 children with mandibular protrusion (aged 3-12 years, 16 males and 17 females) and 27 normal children (aged 3-13 years, 14 males and 13 females). Genomic DNA extracted from buccal epithelial cells was genotyped for the P561T heterozygous mutation with a molecular analysis (polymerase chain reaction - restriction fragment length polymorphism method). Two of the patients with normal occlusion and five with mandibular protrusion were heterozygous for the mutation. Chi-square analysis showed that the frequency of this mutation did not differ statistically between the normal and mandibular protrusion subjects. Multilevel model analysis of the 101 cephalograms showed that the mutation reduced the linear measurements of the mandible. These findings suggest that P561T heterozygous mutation affects mandibular growth during early childhood, and this mutation in the GHR gene is hypothesized to function as an inhibitory factor in the process of mandibular growth.

DOI： 10.1093/ejo/cjp017

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エナメル芽細胞分化過程における細胞間結合の役割

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象牙芽細胞の分化に関わる新奇ギャップ結合分子の同定

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歯原性上皮細胞におけるエピプロフィンの機能解析

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The purpose of this case report is to describe the treatment outcome of a 4-year-old girl with a dentigerous cyst on the primary mandibular right second premolar of her caries-free mouth. Her primary predecessor was extracted and the lesion decompressed by using a removable appliance with a resin projection. The succeeding premolar had not developed further 8 months later, however, and had to be extracted. Crown formation of the succeeding premolar might have been interrupted by the presence of the cyst. To our knowledge, this is the earliest reported asymptomatic dentigerous cyst in the mandibular premolar region with a vital primary second molar.

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歯と歯周組織、骨の再生 エナメル質形成の分子制御機構

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Platelet-derived growth factors play an important role in early tooth germ development

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Platelet-derived growth factors play an important role in early tooth germ development

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The Differences Between Right and Left in the Formation and Eruption of the Permanent First Molar
We surveyed the progression of permanent molar eruption to analyze its difference between the right and left side of the jaw. A total of 476 children were selected among children who visited our clinic, based on specific criteria for this study. Results 1. The tooth germ of the first molar started moving for eruption at 3-4 years old intramandible, and at 4-5 years intra maxilla. Then the first molar was going to erupt and reached the occlusal plane at 7 years old in mandible, and at 8-9 years old in maxilla although there were individual variations. 2. The cases with differential progression between right and left sides in first molar eruption occupied 13.2&#37;. Unexpectedly, however, the cases with such difference in first molar formation occupied only 7.4&#37;. This suggests that tooth eruption is only partially correlated with its formation and, therefore,additional factors might play a role in tooth eruption, which remains to be elucidated in this study. 3. In most 6-year old children, the root of mandibular first molar completed up to 2/3 of the mature root length, while 1/2 in maxilla. Individual variations were also seen in maxilla. 4. In some cases with the delayed eruption of first molar, the second molars were also delayed to erupt. These first and second molars were not always side by side. We clinically encounter many cases with a differential progress between the right and left sides in permanent molar eruption. It is not feasible to diagnose these cases as physiological variations or abnormal eruptions. The data presented here will give helpful information to determine whether any treatments are required for these cases.

DOI： 10.11411/jspd.47.1_73

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The present status and Problems in Pediatric Dental care : Results of a questionnaire investigation answered by pediatric dental specialists
In order to know the present status and problems in pediatric dental care, a questionnaire was answered by 956 pediatric dental specialists who belong to the Japanese Society of Pediatric Dentistry. Summarizing the 455 collected answers, the findings on pediatric dental care of the pediatric dental specialists were as follows. 1. 60&#37; of the pediatric dental specialists estimated that the role of the pediatric dentistry will increase in bringing up the morphology and function of the mouth and teeth of children. 2. Pediatric dental specialists were worrying about the future of pediatric dental care in Japan because the present dental reward system is heading for a structure that is economically difficult for dental offices that focus on pediatric dentistry. 3. Although 70&#37; of the pediatric dental specialists expressed the necessity of a dental office that specializes only in pediatric dentistry, they were thinking it necessary to practice orthodontic and general dentistry at the same time from an economic view point. 4. 90 &#37; of the pediatric dental specialists desire that they can treat patients on both dental treatments covered by the Japanese health insurance and occlusal guidance treatments that are not covered by the insurance, at the same time in order to reduce the frequency and fee of the dental treatment.

DOI： 10.11411/jspd.47.1_51

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The Differences Between Right and Left in the Formation and Eruption of the Permanent First Molar
We surveyed the progression of permanent molar eruption to analyze its difference between the right and left side of the jaw. A total of 476 children were selected among children who visited our clinic, based on specific criteria for this study. Results 1. The tooth germ of the first molar started moving for eruption at 3-4 years old intramandible, and at 4-5 years intra maxilla. Then the first molar was going to erupt and reached the occlusal plane at 7 years old in mandible, and at 8-9 years old in maxilla although there were individual variations. 2. The cases with differential progression between right and left sides in first molar eruption occupied 13.2&#37;. Unexpectedly, however, the cases with such difference in first molar formation occupied only 7.4&#37;. This suggests that tooth eruption is only partially correlated with its formation and, therefore,additional factors might play a role in tooth eruption, which remains to be elucidated in this study. 3. In most 6-year old children, the root of mandibular first molar completed up to 2/3 of the mature root length, while 1/2 in maxilla. Individual variations were also seen in maxilla. 4. In some cases with the delayed eruption of first molar, the second molars were also delayed to erupt. These first and second molars were not always side by side. We clinically encounter many cases with a differential progress between the right and left sides in permanent molar eruption. It is not feasible to diagnose these cases as physiological variations or abnormal eruptions. The data presented here will give helpful information to determine whether any treatments are required for these cases.

DOI： 10.11411/jspd.47.1_73

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Current State of Education of Pediatric Dentistry in Dental Schools in Japan
To survey the current state of the education of Pediatric Dentistry in our country, a questionnaire investigation was done to all Dental Colleges and School of Dentistry in Universities. The questionnaire consisted of three fields, such as lecture of Pediatric Dentistry, basic laboratory practice and basic clinical practice. According to the questionnaire, the following things were confirmed. All Universities still have the syllabus concerning Pediatric Dentistry, except for one University. The average hours of lecture was 55 hours. The University with the longest course hours was 6 times higher than the shortest University. The average hours of basic laboratory practice was 35 hours. The private Universities were significantly longer than national and public Universities. While, the period of basic clinical practice was similar in all Universities, however it started 6 months earlier in private Universities compared with national and public Universities. Moreover, there were differences in intervention levels of students to the treatment of the patients and evaluation procedures of basic clinical practice in Universities. In the present study, it was confirmed that the education of Pediatric Dentistry varied considerably between Universities. Especially, the average hours of lecture and basic laboratory practice were longer in private Universities compared to national and public Universities. Furthermore, basic clinical practice starts significantly earlier in private Universities.

DOI： 10.11411/jspd1963.46.5_517

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Current State of Education of Pediatric Dentistry in Dental Schools in Japan
To survey the current state of the education of Pediatric Dentistry in our country, a questionnaire investigation was done to all Dental Colleges and School of Dentistry in Universities. The questionnaire consisted of three fields, such as lecture of Pediatric Dentistry, basic laboratory practice and basic clinical practice. According to the questionnaire, the following things were confirmed. All Universities still have the syllabus concerning Pediatric Dentistry, except for one University. The average hours of lecture was 55 hours. The University with the longest course hours was 6 times higher than the shortest University. The average hours of basic laboratory practice was 35 hours. The private Universities were significantly longer than national and public Universities. While, the period of basic clinical practice was similar in all Universities, however it started 6 months earlier in private Universities compared with national and public Universities. Moreover, there were differences in intervention levels of students to the treatment of the patients and evaluation procedures of basic clinical practice in Universities. In the present study, it was confirmed that the education of Pediatric Dentistry varied considerably between Universities. Especially, the average hours of lecture and basic laboratory practice were longer in private Universities compared to national and public Universities. Furthermore, basic clinical practice starts significantly earlier in private Universities.

DOI： 10.11411/jspd1963.46.5_517

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新奇細胞-細胞間蛋白質Pannexin3(Panx3)による軟骨細胞の増殖と分化の制御機構の解明

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象牙芽細胞と骨芽細胞の違いを考える 象牙芽細胞特異的分子の同定と分化制御

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We investigated here whether adiponectin can exhibit an inhibitory effect on tumor necrosis factor-alpha (TNF-α)- and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis by using RAW264 cell D clone with a high efficiency to form osteoclasts. Globular adiponectin (gAd) strongly inhibited TNF-α/RANKL-induced differentiation of osteoclasts by interfering with TNF receptor-associated factor 6 production and calcium signaling; consequently, the induction of nuclear factor of activated T cells c1 (NFATc1) was strongly inhibited. Moreover, we observed that inhibition of AMP-activated protein kinase abrogated gAd inhibition for TNF-α/RANKL-induced NFATc1 expression. Our data suggest that adiponectin acts as a potent regulator of bone resorption observed in diseases associated with cytokine activation. © 2008 Federation of European Biochemical Societies.

DOI： 10.1016/j.febslet.2007.12.037

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In congenital diaphragmatic hernia (CDH), both mortality and morbidity are mainly caused by pulmonary hypoplasia and persistent pulmonary hypertension. Insulin-like growth factors (IGFs) are one of the growth factors that may play an important role in the fetal lung development. Elucidating the roles of these growth factors regarding fetal lung development would thus provide new insight regarding the optimal therapy for CDH patients. The aim of this study is to investigate the role of IGFs in the fetal lung development. The mRNA expression of IGFs and its receptors was analyzed by real-time RT-PCR from embryonic day (E) 11.5 to E18.5 mice. In addition, the lungs dissected from the E17.5 mice were divided into the following three groups; lungs cultured only in the serum-free medium (group I n = 5), lungs cultured in medium containing either IGF-I (group II n = 5), or IGF-II (group III n = 5). All cultures were investigated by immunohistochemistry, using the antibodies of thyroid transcription factor (TTF)-1, prosurfactant protein (proSp)-C, alpha smooth muscle actin (α-SMA), and anti-proliferating cell nuclear antigen (PCNA). The mRNA expression level of both IGF-I and IGF-II was higher during the earlier stage than that of later stage. In contrast, the mRNA expression of both IGF-I receptor (IGF-IR) and IGF-II receptor (IGF-IIR) was higher from the E17.5 to E18.5 than that at any other stage. The number of positive cells for TTF-1, proSp-C, α-SMA and PCNA increased more in both groups II and III than in group I. Based on our findings, IGFs are suggested to induce alveolar and vascular maturation in the late stages of fetal lung development. Therefore, the administration of IGFs to the fetal CDH lung may thus be able to effectively improve the symptoms of hypoplastic lung. © 2007 Springer-Verlag.

DOI： 10.1007/s00383-007-1977-8

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Clinical　Statistic Study of Surgical Procedures for Outpatient in Our Dental Hospital

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Study of Relationship between Dental Caries Susceptibility and Milk Suckling Style in New Born Babies Based on Dental Health Examination at Eighteen Months of Age

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Study of Relationship between Dental Caries Susceptibility and Milk Suckling Style in New Born Babies Based on Dental Health Examination at Eighteen Months of Age

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BACKGROUND: Cleft lip with or without cleft palate is the most common congenital anomaly in the craniofacial region. Knowledge of the molecular mechanisms behind normal lip fusion can contribute to better intervention and improved functional clinical outcome. Transforming growth factor-β3 (TGF-β3) has been implicated in lip morphogenesis. Therefore, we hypothesized that TGF-β3 functions during lip fusion through regulation of angiogenesis and mesenchymal cell cycle progression during early developmental stages. METHODS: To test this hypothesis we used the CL/Fraser mouse model, which has a high incidence of cleft lip. Lips isolated from embryonic day (ED) 11.5 mouse embryos were allowed to develop in serum-free organ cultures in the presence or absence of TGF-β3. The lips that developed in these cultures fused in 2 days. RESULTS: During normal development, we detected positive immunoreactions for TGF-β3 at the site of fusion. We also detected mesenchymal cells that were immunopositive for Flk-1 and CD31, which are markers for endothelial cell precursors. Exogenous TGF-β3 accelerated lip fusion in culture. This enhancement was associated with an increase in the number of capillary blood vessels in the lips cultured in the presence of TGF-β3, in comparison with controls. In tandem, TGF-β3 increased the level of expression of both Flk-1 and CD31. Our data suggest that an elevated level of TGF-β3 may promote angiogenesis in developing lips that is mediated by increased Flk-1 and CD31 expression. We also detected increased cyclin D1 expression (a marker for cell proliferation) in the presence of TGF-β3, which suggests that TGF-β3 promoted cell proliferation. CONCLUSIONS: TGF-β3 promoted cell proliferation and angiogenesis in lip mesenchymal tissues. These events led to enhanced lip fusion in the presence of TGF-β3. © 2005 Wiley-Liss, Inc.

DOI： 10.1002/bdra.20191

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Molecular Mechanisum of Tooth Morphogenesis Regulated by Extracellular Matrix
Tooth regeneration is one of the final goal of dental science. However, it is difficult to form proper tooth containing enamel, dentin and cementum. In this study, we focused on the function of extrecellular matrix, especially enamel matrix protein, which is essential for the enamel formation. Ameloblastin is one of the enamel matrix protein secreted by ameloblasts. This involved in the determination of cell polarity, regulation of proliferation and secretion of amelogenin. In ameloblastin null-mutants, severe enamel hypoplasia was observed. This phenotype was similar to human amelogenesis imperfecta. Furteher, the mutant mice developed an odontogenic tumor of dental epithelium origin. Thus, ameloblastin is one of the affected gene of amelogenesis imperfecta, and may inhibit tumor growth of odontogenic tumor as a therapeutic agents.

DOI： 10.11411/jspd1963.43.3_380

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Several members of the transforming growth factor (TGF)-β superfamily are expressed in developing teeth from the initiation stage through adulthood. Of those, TGF-β1 regulates odontoblast differentiation and dentin extracellular matrix synthesis. However, the molecular mechanism of TGF-β3 in dental pulp cells is not clearly understood. In the present study, beads soaked with human recombinant TGF-β3 induced ectopic mineralization in dental pulp from fetal mouse tooth germ samples, which increased in a dose-dependent manner. Further, TGF-β3 promoted mRNA expression, and increased protein levels of osteocalcin (OCN) and type I collagen (COL I) in dental pulp cells. We also observed that the expression of dentin sialophosphoprotein and dentin matrix protein 1 was induced by TGF-β3 in primary cultured dental pulp cells, however, not in calvaria osteoblasts, whereas OCN, osteopontin and osteonectin expression was increased after treatment with TGF-β3 in both dental pulp cells and calvaria osteoblasts. Dentin sialoprotein was also partially detected in the vicinity of TGF-β3 soaked beads in vivo. These results indicate for the first time that TGF-β3 induces ectopic mineralization through upregulation of OCN and COL I expression in dental pulp cells, and may regulate the differentiation of dental pulp stem cells to odontoblasts.

DOI： 10.1111/j.1440-169x.2005.00790.x

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A Follow Up Survey of the Effect of Oral Health Care Guidance for Disabled Children
Preventive dentistry is particularly important for disabled children. Tooth brushing at home is therefore, essential. However, most of disabled children tend not to be cooperative with respect to tooth brushing at home. Brushing in home would be thereby a great burden on their parents. We have carried out a project of oral health care consisting of oral examination, instruction in tooth brushing, and individual consultation at day care facilities for disabled children twice a year for 17 years. There are two keynotes in this project, that is forming a habit of tooth brushing at home and having a family dentist. To investigate the effect of our oral health care instruction, we made a follow-up survey by sending out a questionnaire through the mail to 144 guardians who had been given oral health care instructions when they went to the day care facility and whose children were within 3 years after leaving the facility. The contents of questionnaire included whether the children brushed teeth every day and the degree of cooperation during tooth brushing, and whether they had a family dentist or not. As the results, the ratio of children who brushed their teeth every day after leaving the facility was 86&#37;, which was almost the same ratio as that during the period of time the facility was visited. In addition, the degree of cooperation during brushing tended to improve after leaving the facility. The ratio of those who had a family dentist after leaving the facility was 81 &#37;, which increased from 57&#37; before leaving the facility. It was therefore concluded that our oral health care instruction from early childhood was effective to some extent. We would like to examine the project refer to the results of this study. Based on the present results, it was found that the habit of tooth brushing was firmly established at the time of leaving the facility, and that the number of people having a family dentist had increased, the significance of oral health instruction.

DOI： 10.11411/jspd1963.43.1_41

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A Follow Up Survey of the Effect of Oral Health Care Guidance for Disabled Children
Preventive dentistry is particularly important for disabled children. Tooth brushing at home is therefore, essential. However, most of disabled children tend not to be cooperative with respect to tooth brushing at home. Brushing in home would be thereby a great burden on their parents. We have carried out a project of oral health care consisting of oral examination, instruction in tooth brushing, and individual consultation at day care facilities for disabled children twice a year for 17 years. There are two keynotes in this project, that is forming a habit of tooth brushing at home and having a family dentist. To investigate the effect of our oral health care instruction, we made a follow-up survey by sending out a questionnaire through the mail to 144 guardians who had been given oral health care instructions when they went to the day care facility and whose children were within 3 years after leaving the facility. The contents of questionnaire included whether the children brushed teeth every day and the degree of cooperation during tooth brushing, and whether they had a family dentist or not. As the results, the ratio of children who brushed their teeth every day after leaving the facility was 86&#37;, which was almost the same ratio as that during the period of time the facility was visited. In addition, the degree of cooperation during brushing tended to improve after leaving the facility. The ratio of those who had a family dentist after leaving the facility was 81 &#37;, which increased from 57&#37; before leaving the facility. It was therefore concluded that our oral health care instruction from early childhood was effective to some extent. We would like to examine the project refer to the results of this study. Based on the present results, it was found that the habit of tooth brushing was firmly established at the time of leaving the facility, and that the number of people having a family dentist had increased, the significance of oral health instruction.

DOI： 10.11411/jspd1963.43.1_41

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Molecular mechanism of tissue morphogenesis via laminin-integrin interaction

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Molecular mechanism of tissue morphogenesis via laminin-integrin interaction

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DOI： 10.11411/jspd1963.41.2_461

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DOI： 10.11411/jspd1963.41.2_377

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DOI： 10.11411/jspd1963.41.2_324

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DOI： 10.11411/jspd1963.41.2_460

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DOI： 10.11411/jspd1963.41.2_324

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Cleft palate- and lip-related gene expression analyzed by cDNA microarray hybridization

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

出力可変型キセノン照射器の光重合型レジンの物理的性質に及ぼす影響
This study was undertaken to investigate the effect of the Xenon-lamp curing unit (Flipo) on the elemental properties of light curing resin composite by comparison with a halogen-lamp curing unit (XL). 1) The shear bond strengths of resin composite to bovine dentin using each curing unit were measured. The adhesive system and resin composite used in this study were Clearfil Liner Bond II® and Clearfil AP-X® (shades: A 2 and A 4). The irradiation time of XL was 40 seconds as the control, and that of Flipo was 5 seconds (stepped output of 2 seconds at 50&#37; maximal output followed by full output for 3 seconds). The result of the test showed that the shear bond strength produced by Flipo was about equal to that by XL for shade A 2, but for shade A 4 that produced by Flipo was significantly lower than that by XL (p<0.01). Using XL showed high bond strengths independent of shade. 2) The knoop hardness of the resin composite was measured for XL for 40 seconds, Flipo for 5 seconds and Flipo for 10 seconds (2 seconds at 50&#37; maximal output followed by full output for 8 seconds)using Clearfil AP-X® (shades: A 2 and A 4). The result showed that when the shade was A 2, the knoop hardness produced by Flipo for 5 seconds was about equal to that by XL for 40 seconds, and that of Flipo for 10 seconds was significantly higher than those two groups (p<0.01). For shade of A 4, the knoop hardness produced by Flipo for 10 seconds was about equal to that of XL for 40seconds, and that of Flipo for 5 seconds was significantly lower than those two(p<0.01). 3) The dye penetration test was performed for cavities prepared on extracted human premolar and filled with resin under the light curing condition of XL for 40 seconds, Flipo for 5 seconds (two stepped output) and Flipo for 5 seconds of exposure at 100&#37; intensity to evaluate marginal sealing and wall adaptation. The class V cavity with the base at depth of 1 mm in the dentin was prepared on the buccal surface. The group of XL for 40 seconds showed the best marginal sealing and wall adaptation, but when using Flipo at 100&#37; intensity for 5 seconds, the dye penetration score was higher and both the incidence and width of contraction gap were significantly increased. The results of the present experiment proved that using the Xenon-lamp curing unit (Flipo) is effective for hardening a resin composite of shade A 2 less than 2.0 mm-deep with 1/6 of irradiation time needed for the halogen-lamp curing unit (XL). However, if the cavity is deeper than 2.0 mm, sufficient polymerization may not be obtained by using Flipo for 5 seconds, especially for shade A 4, and moreover, using Flipo for 5 seconds at 100&#37; intensity may degrade the marginal seal effectiveness and wall adaptation.

DOI： 10.11411/jspd1963.39.1_146

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

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

Language：Japanese  

DOI： 10.11411/jspd1963.37.2_283

Language：Japanese  

Inhibitory Effect of Dentin Permeability Using Collagen Solution
The permeability characteristics of dentin are important factors which influence the transmission of variable stimuli to the pulp. The permeability of dentin is of crucial importance in obtaining good dentin bonding. We have, therefore, investigated to evaluate the potential value of the type I collagen to coat the acid-etched and opened dentinal tubules and to inhibit the permeation of dentinal fluid in vitro study. The collagen solutions used in this study were prepared by mixing Cellmatrix type I -A®, PBS and 0.08 mM NaOH containing 200 mM HEPES at a final pH 7.4. The bovine dentin surfaces were treated with 10&#37; phosphoric acid for 30 seconds to remove the smear layer and open the dentinal tubules, followed by application of the collagen solution at 37°C for 60 seconds. The microstructures of the dentin surfaces were observed using a scanning electron microscopy (SEM). The permeability of the dentin with or without the collagen application was evaluated under the simulated pulpal pressure of about 25 mmHg. A intrapulpal fluid in this study was reproduced by using 1&#37; bovine serum albumin/PBS, and the value of BSA permeated on the dentin surface was quantified by the Lowly method. As a result, the following findings were obtained. 1. SEM observation of the dentin surface treated with 10&#37; phosphoric acid followed by the application of 0.3&#37; type I collagen solution at 37°C for 60 seconds showed that the dentinal tubules were covered with a layer of membranous structure. However it was also observed that partially opened dentinal tubules still remained on the treated dentin surface. 2. Under the simulated pulpal pressure of about 25 mmHg, compared with the dentin treated with phosphoric acid only, the permeability of the dentin treated with 10&#37; phosphoric acid followed by collagen solution application was significantly inhibited. As a result of this basic study, we observed the ability of the collagen application to the dentin surface to seal the dentinal tubules and inhibit the permeation of dentinal fluid.

DOI： 10.11411/jspd1963.36.4_660

Language：Japanese  

Inhibitory Effect of Dentin Permeability Using Collagen Solution
The permeability characteristics of dentin are important factors which influence the transmission of variable stimuli to the pulp. The permeability of dentin is of crucial importance in obtaining good dentin bonding. We have, therefore, investigated to evaluate the potential value of the type I collagen to coat the acid-etched and opened dentinal tubules and to inhibit the permeation of dentinal fluid in vitro study. The collagen solutions used in this study were prepared by mixing Cellmatrix type I -A®, PBS and 0.08 mM NaOH containing 200 mM HEPES at a final pH 7.4. The bovine dentin surfaces were treated with 10&#37; phosphoric acid for 30 seconds to remove the smear layer and open the dentinal tubules, followed by application of the collagen solution at 37°C for 60 seconds. The microstructures of the dentin surfaces were observed using a scanning electron microscopy (SEM). The permeability of the dentin with or without the collagen application was evaluated under the simulated pulpal pressure of about 25 mmHg. A intrapulpal fluid in this study was reproduced by using 1&#37; bovine serum albumin/PBS, and the value of BSA permeated on the dentin surface was quantified by the Lowly method. As a result, the following findings were obtained. 1. SEM observation of the dentin surface treated with 10&#37; phosphoric acid followed by the application of 0.3&#37; type I collagen solution at 37°C for 60 seconds showed that the dentinal tubules were covered with a layer of membranous structure. However it was also observed that partially opened dentinal tubules still remained on the treated dentin surface. 2. Under the simulated pulpal pressure of about 25 mmHg, compared with the dentin treated with phosphoric acid only, the permeability of the dentin treated with 10&#37; phosphoric acid followed by collagen solution application was significantly inhibited. As a result of this basic study, we observed the ability of the collagen application to the dentin surface to seal the dentinal tubules and inhibit the permeation of dentinal fluid.

DOI： 10.11411/jspd1963.36.4_660

Language：Japanese  

Use of CELAY^【○!R】 System for Onlay and Inlay Fabrication for Primary Teeth : A Two Year Follow Up

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Histo-Pathological Changes in Dental Pulp Capped with 4-META/MMA-TBB Resin (Report 1)
The purpose of this study was to evaluate histopathologically the 4-META/MMA-TBB resin as a direct pulp capping material. Fifty-three Class V or Class I deep cavity were prepared in four beagle dogs under general anesthesia. Without local anesthesia, cavity floors were intentionally perforated to expose the pulp. Sunmedical Co.4-META/MMA-TBB resin "Orthomite Super Bond" was directly applied to the exposed pulp (SB group). As the control, Neo Dental Chemical Products Ca(OH)₂ medicament "Calvital" was used as a direct pulp capping agent (CV group). Both in the SB groups and in the CV groups, the cavities were lined with "Orthomite Super Bond" and filled with the Kuraray Co. composite resin "Clearfil Photo Anterior" after etching and bonding. After 14 and 30 days, the histo-pathological responses of the pulp were analyzed. The number of the cases in each of the groups was: SB-14 days (17 cases), SB-30 days (14 cases), CV-14 days (13 cases) and CV-30 days (9 cases). 1 ) Inflammatory cells infiltrated into the pulp: 4 cases (23.5&#37;) in the SB-14 day group,8 cases (57.1&#37;) in the SB-30 day group,5 cases (38.5&#37;) in the CV-14 day group and 2 cases (22.2&#37;) in the CV-30 day group. 2 ) Partial necrosis of the pulp was observed in the 2 cases (14.3&#37;) in the SB-30 day group. Complete necrosis of the pulp was observed in the 2 cases (15.4&#37;) in the CV-14 day group and in the 2 cases (22.2&#37;) in the CV-30 day group. 3 ) Circulatory disturbances in the remaining pulp: 10 cases (58.8&#37;) in the SB-14 day group,6cases (42.9&#37;) in the SB-30 day group,6 cases (46.2&#37;) in the CV-14 day group and 4 cases (44.4&#37;) in the CV-30 day group. 4 ) Macrophage migration aroud the interface between pulp and capping materials: 8 cases (47.1&#37;)in the SB-14 day group,10 cases (71.4&#37;) in the SB-30 day group,7 cases (53.8&#37;) in the CV-14day group and 4 cases (44.4&#37;) in the CV-30 day group. 5 ) Dentin bridge formation: 12 cases (70.6&#37;) in the SB-14 day group, none in the SB-30 day group,1 case (7.7&#37;) in the CV-14 day group and 3 cases (33.3&#37;) in the CV-30 day group. 6 ) In all of the 4 groups, the SB-14 day group showed the best histo-pathological results, however, the results of the SB-30 day group was poor.

DOI： 10.11411/jspd1963.35.3_401

Language：Japanese  

Histo-Pathological Changes in Dental Pulp Capped with 4-META/MMA-TBB Resin (Report 1)
The purpose of this study was to evaluate histopathologically the 4-META/MMA-TBB resin as a direct pulp capping material. Fifty-three Class V or Class I deep cavity were prepared in four beagle dogs under general anesthesia. Without local anesthesia, cavity floors were intentionally perforated to expose the pulp. Sunmedical Co.4-META/MMA-TBB resin "Orthomite Super Bond" was directly applied to the exposed pulp (SB group). As the control, Neo Dental Chemical Products Ca(OH)₂ medicament "Calvital" was used as a direct pulp capping agent (CV group). Both in the SB groups and in the CV groups, the cavities were lined with "Orthomite Super Bond" and filled with the Kuraray Co. composite resin "Clearfil Photo Anterior" after etching and bonding. After 14 and 30 days, the histo-pathological responses of the pulp were analyzed. The number of the cases in each of the groups was: SB-14 days (17 cases), SB-30 days (14 cases), CV-14 days (13 cases) and CV-30 days (9 cases). 1 ) Inflammatory cells infiltrated into the pulp: 4 cases (23.5&#37;) in the SB-14 day group,8 cases (57.1&#37;) in the SB-30 day group,5 cases (38.5&#37;) in the CV-14 day group and 2 cases (22.2&#37;) in the CV-30 day group. 2 ) Partial necrosis of the pulp was observed in the 2 cases (14.3&#37;) in the SB-30 day group. Complete necrosis of the pulp was observed in the 2 cases (15.4&#37;) in the CV-14 day group and in the 2 cases (22.2&#37;) in the CV-30 day group. 3 ) Circulatory disturbances in the remaining pulp: 10 cases (58.8&#37;) in the SB-14 day group,6cases (42.9&#37;) in the SB-30 day group,6 cases (46.2&#37;) in the CV-14 day group and 4 cases (44.4&#37;) in the CV-30 day group. 4 ) Macrophage migration aroud the interface between pulp and capping materials: 8 cases (47.1&#37;)in the SB-14 day group,10 cases (71.4&#37;) in the SB-30 day group,7 cases (53.8&#37;) in the CV-14day group and 4 cases (44.4&#37;) in the CV-30 day group. 5 ) Dentin bridge formation: 12 cases (70.6&#37;) in the SB-14 day group, none in the SB-30 day group,1 case (7.7&#37;) in the CV-14 day group and 3 cases (33.3&#37;) in the CV-30 day group. 6 ) In all of the 4 groups, the SB-14 day group showed the best histo-pathological results, however, the results of the SB-30 day group was poor.

DOI： 10.11411/jspd1963.35.3_401

Language：Japanese  

Histological study of accessory canals in the pulpal chamber floor : Measurement of dog accessory canals

Language：Japanese  

Histological study of accessory canals in the pulpal chamber floor : Measurement of dog accessory canals

Language：Japanese  

Clinical Study on the Restoration of Primary Teeth : Comparison with 1984 and 1994
This report investigated 1,466 primary-tooth restorations in 374 children, who attended the Nagasaki University Dental Clinic of Pediatric Dentistry in 1994. Data resulting from the investigation were compared with those obtained in 1984. Results and Conclusions 1. Of all the restorations performed in 1984,37.5% were composite resin fillings; 28.8% were stainlesssteel crowns; 16.6% were metal inlays; 11.3% were amalgam fillings. On the other hand, of all the restorations performed in 1994,60.3% were composite resin fillings; 21.0% were metal inlays; 14.1% were stainless-steel crowns. 2. In 1984, of primary molars with decayed proximal surfaces,75.4% were restored with metal inlays,14.3% were restored with composite resin fillings and 10.0% were restored with amalgam fillings. In 1994, of the decayed proximal surfaces of primary molars,51.0% were restored with metal inlays and 47.5% were restored with composite resin fillings. 3. Both in 1984 and in 1994, composite resin fillings were used preferentially for primary anterior teeth in which caries extended only into the enamel or only into the dentin, and for primary molars in which caries extended only into the enamel. In 1994, composite resin fillings were used preferentially for primary anterior teeth in which caries extended into the pulp and for primary molars in which caries extended only into the dentin, too. 4. Compared with the results in 1984, in 1994, the percentages of the composite resin fillings increased both for primary anterior teeth and for primary molars. In 1994, no amalgam filling was used and glass-ionomer cement fillings were used as a new method.

DOI： 10.11411/jspd1963.34.1_214

Language：Japanese  

DOI： 10.11411/jspd1963.33.2_312

Language：Japanese  

DOI： 10.11411/jspd1963.33.2_312

Presentation type：Symposium, workshop panel (public)  

Venue：Zurich   Country：Switzerland  

Presentation type：Oral presentation (general)  

Venue：西鉄グランドホテル（福岡市）   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：まつもと市民芸術館（松本市）   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：まつもと市民芸術館（松本市）   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：まつもと市民芸術館（松本市）   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：まつもと市民芸術館（松本市）  

Presentation type：Oral presentation (general)  

Venue：まつもと市民芸術館（松本市）   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：まつもと市民芸術館（松本市）   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：Brisbane   Country：Australia  

Presentation type：Oral presentation (general)  

Venue：九州大学（福岡市）   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：福岡市（九州大学）   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：鶴見大学（横浜市）   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：鹿児島市   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：New Orleans   Country：United States  

Presentation type：Symposium, workshop panel (public)  

Venue：New Orleans   Country：United States  

Presentation type：Symposium, workshop panel (public)  

Venue：東京   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：東京   Country：Japan  

Presentation type：Symposium, workshop panel (public)