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Language：English   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：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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：(公社)日本小児歯科学会  

象牙質疾患の病因を解明するため、単一細胞RNAシークエンシング法を用いて歯間葉細胞に特異的な遺伝子を同定し、象牙質異常または骨格異常がみられる遺伝性疾患との関連性について検討した。生後7日のマウスの切歯を検体として単一細胞RNAシークエンシング法を実施し、総計で細胞6260個分のデータセットを得た。そのデータから象牙芽細胞のクラスタと歯髄クラスタにおける発現変動遺伝子(DEG)、上位20種ずつを確定した。そして遺伝性疾患データベースのOnline Mendelian Inheritance(OMIM)を使い、上記のDEGが有する意義について検討した。遺伝性疾患の原因遺伝子であったDEGは40種中17種(42.5%)に上った。象牙芽細胞および歯髄間葉に関する遺伝性疾患35疾患のうち、歯奇形を伴っていた遺伝性疾患は12疾患(34.3%)あった。象牙芽細胞関連疾患のうち象牙質異常を伴う疾患であった率は33.3%、歯髄関連疾患での同率は7.7%であった。同定された遺伝性疾患のうち骨格異常を伴う疾患であったものは85.7%に達した。歯間葉細胞にみられるDEGは、象牙質異常および骨格異常と高度に関連していた。

Language：English   Publishing type：Research paper (scientific journal)   Publisher：(一社)日本小児内分泌学会  

DOI： 10.1297/cpe.2024-0024

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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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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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：(公社)日本小児歯科学会  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：(公社)日本小児歯科学会  

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：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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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：(一社)歯科基礎医学会  

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：Materials  

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

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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Other Link： http://journals.sagepub.com/doi/full-xml/10.1177/00220345211049785

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

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.

DOI： 10.1002/jcp.30667

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/jcp.30667

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

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

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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DOI： 10.3390/children8121189

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Connexin 43 (Cx43) is an integral membrane protein that forms gap junction channels. These channels mediate intercellular transport and intracellular signaling to regulate organogenesis. The human disease oculodentodigital dysplasia (ODDD) is caused by mutations in Cx43 and is characterized by skeletal, ocular, and dental abnormalities including amelogenesis imperfecta. To clarify the role of Cx43 in amelogenesis, we examined the expression and function of Cx43 in tooth development. Single-cell RNA-seq analysis and immunostaining showed that Cx43 is highly expressed in pre-secretory ameloblasts, differentiated ameloblasts, and odontoblasts. Further, we investigated the pathogenic mechanisms of ODDD by analyzing Cx43-null mice. These mice developed abnormal teeth with multiple dental epithelium layers. The expression of enamel matrix proteins such as ameloblastin (Ambn), which is critical for enamel formation, was significantly reduced in Cx43-null mice. TGF-β1 induces Ambn transcription in dental epithelial cells. The induction of Ambn expression by TGF-β1 depends on the density of the cultured cells. Cell culture at low densities reduces cell-cell contact and reduces the effect of TGF-β1 on Ambn induction. When cell density was high, Ambn expression by TGF-β1 was enhanced. This induction was inhibited by the gap junction inhibitors, oleamide, and 18α-grycyrrhizic acid and was also inhibited in cells expressing Cx43 mutations (R76S and R202H). TGF-β1-mediated phosphorylation and nuclear translocation of ERK1/2, but not Smad2/3, were suppressed by gap junction inhibitors. Cx43 gap junction activity is required for TGF-β1-mediated Runx2 phosphorylation through ERK1/2, which forms complexes with Smad2/3. In addition to its gap junction activity, Cx43 may also function as a Ca2+ channel that regulates slow Ca2+ influx and ERK1/2 phosphorylation. TGF-β1 transiently increases intracellular calcium levels, and the increase in intracellular calcium over a short period was not related to the expression level of Cx43. However, long-term intracellular calcium elevation was enhanced in cells overexpressing Cx43. Our results suggest that Cx43 regulates intercellular communication through gap junction activity by modulating TGF-β1-mediated ERK signaling and enamel formation.

DOI： 10.3389/fphys.2021.748574

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The epithelial-mesenchymal interactions are essential for the initiation and regulation of the development of teeth. Following the initiation of tooth development, numerous growth factors are secreted by the dental epithelium and mesenchyme that play critical roles in cellular differentiation. During tooth morphogenesis, the dental epithelial stem cells differentiate into several cell types, including inner enamel epithelial cells, which then differentiate into enamel matrix-secreting ameloblasts. Recently, we reported that the novel basic-helix-loop-helix transcription factor, AmeloD, is actively engaged in the development of teeth as a regulator of dental epithelial cell motility. However, the gene regulation mechanism of AmeloD is still unknown. In this study, we aimed to uncover the mechanisms regulating AmeloD expression during tooth development. By screening growth factors that are important in the early stages of tooth formation, we found that TGF-β1 induced AmeloD expression and ameloblast differentiation in the dental epithelial cell line, SF2. TGF-β1 phosphorylated ERK1/2 and Smad2/3 to induce AmeloD expression, whereas treatment with the MEK inhibitor, U0126, inhibited AmeloD induction. Promoter analysis of AmeloD revealed that the proximal promoter of AmeloD showed high activity in dental epithelial cell lines, which was enhanced following TGF-β1 stimulation. These results suggested that TGF-β1 activates AmeloD transcription via ERK1/2 phosphorylation. Our findings provide new insights into the mechanisms that govern tooth development.

DOI： 10.1002/jcp.30389

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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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DOI： 10.1016/j.pdj.2020.08.001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Biochemistry & Molecular Biology (ASBMB)  

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, though their activities in tooth development remains poorly understood. The present results show that the adhesion-GPCR <italic>Gpr115</italic> (<italic>Adgrf4</italic>) is highly and preferentially expressed in mature ameloblasts and plays a crucial role during enamel mineralization. To investigate the in vivo function of <italic>Gpr115</italic>, knockout (<italic>Gpr115</italic>-KO) mice were created and found to develop hypo-mineralized enamel, with a larger acidic area due to the dysregulation of ion composition. Transcriptomic analysis also revealed that deletion of <italic>Gpr115</italic> disrupted pH homeostasis and ion transport processes in enamel formation. In addition, in vitroanalyses using the dental epithelial cell line Cervical Loop-Derived Dental Epithelial (CLDE) cell demonstrated that <italic>Gpr115</italic> is indispensable for the expression of carbonic anhydrase 6 (<italic>Car6</italic>), which has a critical role in enamel mineralization. Furthermore, an acidic condition induced Car6 expression under the regulation of <italic>Gpr115</italic> in CLDE cells. Thus, we concluded that <italic>Gpr115</italic> plays an important role in enamel mineralization via regulation of Car6 expression in ameloblasts. The present findings indicate a novel function of <italic>Gpr115</italic> in ectodermal organ development and clarify the molecular mechanism of enamel formation.

DOI： 10.1074/jbc.ra120.014281

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DOI： 10.1016/j.isci.2020.101329

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© Copyright © 2020 Nakamura, Iwamoto, Nakamura, Shindo, Saito, Yamada, Yamada, Fukumoto and Nakamura. 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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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

<title>Abstract</title>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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Other Link： http://www.nature.com/articles/s41598-020-61693-w

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

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

PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：(一社)歯科基礎医学会  

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

DOI： 10.3389/fdmed.2026.1744911

PubMed

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

Aim: Fixed-appliance therapy during occlusal guidance in children increases the risk of enamel demineralization, often presenting as white spot lesions (WSLs). This study evaluated the clinical performance of a hydroxyapatite-inducing coating (Bio Coat Ca) for preventing new WSLs and improving pre-existing hypomineralization in pediatric patients. Methods: This study evaluated 18 polycarbonate brackets bonded in three children undergoing occlusal guidance therapy. Bio Coat Ca was applied to labial enamel surfaces prior to bonding with Super Bond. Clinical outcomes included bracket retention, incidence of new WSLs, and changes in pre-existing hypomineralized enamel. Standardized intraoral photographs were analyzed using ImageJ to quantify lesion area ratios before and after treatment. Bracket survival was evaluated using Kaplan–Meier analysis. Results: Of the 18 polycarbonate brackets evaluated, with five bond failures within 377 days (overall retention 72.2 %). This retention rate is comparable to previously reported outcomes for polycarbonate brackets, indicating that Bio Coat Ca does not compromise bonding performance. No new WSLs were observed in any case. Pre-existing hypomineralized areas decreased markedly, with lesion area ratios reduced to 11.76 %, 11.83 %, and 0.34 % in the three cases, respectively. Conclusion: A single application of a calcium-releasing bioactive coating prior to bracket bonding was associated with the absence of new WSLs and the improvement of pre-existing enamel hypomineralization in children, without compromising bracket adhesion. This approach may be particularly beneficial for pediatric patients with limited cooperation or special health care needs.

DOI： 10.1016/j.pdj.2025.100368

Scopus

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

Background: Oral care is essential for preventing ventilator-associated pneumonia (VAP) in intensive care units (ICUs) patients. Patients in pediatric ICUs (PICUs) require specialized oral management due to anatomical and developmental factors, yet detailed reports are limited. Case presentation: We report a 9-year-old girl with chronic heart failure who developed ventilator-associated gingivitis (VAG) and other oral complications during long-term mechanical ventilation. Oral management was conducted collaboratively by pediatric dentists, dental hygienists, nurses, and physicians. Conclusion: This case underscores the importance of early, continuous oral care and interdisciplinary collaboration in managing oral complications and supporting overall health in ventilated PICUs patients.

DOI： 10.1016/j.pdj.2025.100358

Scopus

Authorship：Last author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：(一社)日本障害者歯科学会  

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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Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2020&ichushi_jid=J02489&link_issn=&doc_id=20201110230001&doc_link_id=10.14958%2Fjjsdh.41.277&url=https%3A%2F%2Fdoi.org%2F10.14958%2Fjjsdh.41.277&type=J-STAGE&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00007_3.gif

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

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

DOI： 10.3389/fcell.2020.00841

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Responsible for pages：76-85   Language：Japanese   Book type：General book, introductory book for general audience

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Total pages：179   Responsible for pages：86-102  

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Responsible for pages：1161-1164   Language：Japanese   Book type：General book, introductory book for general audience

Responsible for pages：101-104   Language：Japanese   Book type：General book, introductory book for general audience

Responsible for pages：51-60   Language：Japanese   Book type：General book, introductory book for general audience

Event date： 2025.9

Language：Japanese   Presentation type：Poster presentation  

Venue：鹿児島   Country：Japan  

Event date： 2025.5

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：新潟県　新潟市   Country：Japan  

Event date： 2025.5

Language：Japanese   Presentation type：Poster presentation  

Venue：新潟県　新潟市   Country：Japan  

Event date： 2024.11

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Venue：福岡県　小倉   Country：Japan  

Event date： 2024.11

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Venue：福岡県　小倉   Country：Japan  

Event date： 2024.11

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Venue：福岡県　小倉   Country：Japan  

Event date： 2024.10 - 2024.11

Language：English   Presentation type：Oral presentation (general)  

Venue：TAI Bangkok   Country：Thailand  

Event date： 2024.10 - 2024.11

Language：English   Presentation type：Poster presentation  

Venue：Tai Bangkok   Country：Thailand  

Event date： 2024.10 - 2024.11

Language：English   Presentation type：Poster presentation  

Venue：Tai Bangkok   Country：Thailand  

Event date： 2024.10 - 2024.11

Language：English   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：Tai Bangkok   Country：Thailand  

Event date： 2024.10

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：東京都　府中市   Country：Japan  

Event date： 2024.5

Language：English   Presentation type：Poster presentation  

Venue：神奈川県横須賀市   Country：Japan  

Event date： 2023.11

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Venue：北海道　札幌市   Country：Japan  

Event date： 2023.10

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Venue：福岡市   Country：Japan  

Event date： 2023.10

Language：English   Presentation type：Poster presentation  

Venue：福島県郡山   Country：Japan  

Event date： 2023.9

Language：Japanese   Presentation type：Poster presentation  

Venue：東京   Country：Japan  

Event date： 2023.5

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：長崎県　長崎市   Country：Japan  

Event date： 2023.5

Language：Japanese   Presentation type：Poster presentation  

Venue：長崎県　長崎市   Country：Japan  

Event date： 2022.11

Language：Japanese   Presentation type：Poster presentation  

Venue：北海道　札幌市   Country：Japan  

Event date： 2022.11

Language：Japanese   Presentation type：Poster presentation  

Venue：北海道　札幌市   Country：Japan  

Event date： 2022.11

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Event date： 2022.11

Language：Japanese   Presentation type：Poster presentation  

Venue：福岡県　福岡市   Country：Japan  

Event date： 2022.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：岡山県　倉敷市   Country：Japan  

Event date： 2022.10

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Event date： 2022.10

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：秋田県　秋田市（web開催）   Country：Japan  

Event date： 2022.10

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Event date： 2022.9

Language：Japanese   Presentation type：Poster presentation  

Venue：徳島   Country：Japan  

Event date： 2022.5

Language：Japanese   Presentation type：Poster presentation  

Venue：千葉県　千葉市   Country：Japan  

Event date： 2022.5

Language：Japanese   Presentation type：Poster presentation  

Venue：千葉県　千葉市（web開催）   Country：Japan  

Event date： 2022.5

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：千葉県　千葉市   Country：Japan  

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Event date： 2022.5

Language：Japanese   Presentation type：Poster presentation  

Venue：千葉市（web開催）   Country：Japan  

Event date： 2021.11

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：宮城県　仙台市   Country：Japan  

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Event date： 2021.10

Language：Japanese   Presentation type：Poster presentation  

Venue：岩手県　盛岡市   Country：Japan  

Event date： 2021.10

Language：Japanese   Presentation type：Poster presentation  

Venue：岩手県　盛岡市   Country：Japan  

Event date： 2021.10

Language：Japanese   Presentation type：Poster presentation  

Venue：岩手県　盛岡市   Country：Japan  

Event date： 2021.9

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

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Event date： 2021.3

Presentation type：Symposium, workshop panel (nominated)  

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Event date： 2021.2

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：宮城県　仙台市（オンライン）   Country：Japan  

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Event date： 2021.2

Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Event date： 2021.2

Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Event date： 2020.11

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：福岡県　福岡市   Country：Japan  

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Event date： 2020.5

Language：Japanese   Presentation type：Poster presentation  

Venue：沖縄県　宜野湾市（web開催）   Country：Japan  

Event date： 2020.5

Language：Japanese   Presentation type：Poster presentation  

Venue：沖縄県　宜野湾市（web開催）   Country：Japan  

Language：Japanese  

Language：Japanese   Presentation type：Poster presentation  

Language：Japanese   Presentation type：Poster presentation  

Language：Japanese   Presentation type：Poster presentation  

Language：Japanese  

Language：Japanese  

J-GLOBAL

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Language：Japanese   Publisher：秋田県小児保健会  

Language：Japanese   Publisher：(株)デンタルダイヤモンド社  

Authorship：Corresponding author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Language：Japanese   Publisher：医歯薬出版(株)  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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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：Japanese   Publisher：(公社)日本小児歯科学会  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Language：Japanese   Publishing type：Research paper, summary (national, other academic conference)   Publisher：(公社)日本小児歯科学会  

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Audience：Teachers,　Governmental agency

Type：Lecture

主に保育士を対象とした講演会
〜福岡っ子の健やかな発育を育む生活習慣〜　

Audience：Scientific

Type：Lecture

Audience：Teachers,　Governmental agency

Type：Lecture

主に保育士を対象とした講演会
〜福岡っ子の健やかな発育を育む生活習慣〜　

間食指導ガイド、指しゃぶり指導ガイド作成

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宮城県幼稚園・保育園歯科保健推進委員

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新生東北大学小児歯科

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診療行為に関連した死亡の調査分析モデル事業 臨床立会医

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