担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Scientific Reports  

Alveolar bone loss caused by periodontal disease eventually leads to tooth loss. Periodontal ligament stem cells (PDLSCs) are the tissue-specific cells for maintaining and repairing the periodontal ligament, cementum, and alveolar bone. Here, we investigated the role of erythropoietin receptor (EPOR), which regulates the microenvironment-modulating function of mesenchymal stem cells, in PDLSC-based periodontal therapy. We isolated PDLSCs from patients with chronic periodontal disease and healthy donors, referred to as PD-PDLSCs and Cont-PDLSCs, respectively. PD-PDLSCs exhibited reduced potency of periodontal tissue regeneration and lower expression of EPOR compared to Cont-PDLSCs. EPOR-silencing suppressed the potency of Cont-PDLSCs mimicking PD-PDLSCs, whereas EPO-mediated EPOR activation rejuvenated the reduced potency of PD-PDLSCs. Furthermore, we locally transplanted EPOR-silenced and EPOR-activated PDLSCs into the gingiva around the teeth of ligament-induced periodontitis model mice and demonstrated that EPOR in PDLSCs participated in the regeneration of the periodontal ligament, cementum, and alveolar bone in the ligated teeth. The EPOR-mediated paracrine function of PDLSCs maintains periodontal immune suppression and bone metabolic balance via osteoclasts and osteoblasts in the periodontitis model mice. Taken together, these results suggest that EPOR signaling is crucial for PDLSC-based periodontal regeneration and paves the way for the development of novel options for periodontal therapy.

DOI： 10.1038/s41598-024-57361-y

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記述言語：その他   掲載種別：研究論文（学術雑誌）   出版者・発行元：Archives of Biochemistry and Biophysics  

The metastases of breast cancer to bone often cause osteolytic lesions not only by stimulating osteoclasts to resorb the bone but also by inhibiting osteoblasts from bone formation. Although tumor cell-derived extracellular vesicles (EVs) promote osteoclast differentiation and bone resorption, their roles in osteoblast differentiation and functions have not been elucidated. In this study, we investigated the effects of breast cancer cell-derived EVs on osteoblast differentiation and functions in vitro. We found that upon osteogenic induction, 4T1 bone metastatic mouse mammary tumor cell-derived EVs (4T1-EVs) were inhibited matrix mineralization of ST2 mouse bone marrow stromal cells. Temporal expression analysis of osteoblast marker genes, including runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase (Alp), collagen type I (Col1a1), bone sialoprotein (Bsp), and osteocalcin (Bglap) revealed that 4T1-EVs decreased their expression during the late stage of osteoblast differentiation. Elevated levels of c-Jun N-terminal kinase (JNK) phosphorylation, upon osteogenic induction, were diminished by 4T1-EVs, significantly. In contrast, the nullification of reduced JNK phosphorylation by anisomycin, a potent JNK activator, increased the expression levels of osteoblast differentiation markers. Overall, our data indicated that 4T1-EVs affect osteoblast maturation, at least partially, through the regulation of JNK activity, which provides novel insights into the pathological impact of osteolytic bone metastasis and the role of EVs in osteoblast differentiation.

DOI： 10.1016/j.abb.2023.109821

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Surgery Today  

Hirschsprung disease (HSCR) and its associated disorders (AD-HSCR) often result in severe hypoperistalsis caused by enteric neuropathy, mesenchymopathy, and myopathy. Notably, HSCR involving the small intestine, isolated hypoganglionosis, chronic idiopathic intestinal pseudo-obstruction, and megacystis-microcolon-intestinal hypoperistalsis syndrome carry a poor prognosis. Ultimately, small-bowel transplantation (SBTx) is necessary for refractory cases, but it is highly invasive and outcomes are less than optimal, despite advances in surgical techniques and management. Thus, regenerative therapy has come to light as a potential form of treatment involving regeneration of the enteric nervous system, mesenchyme, and smooth muscle in affected areas. We review the cutting-edge regenerative therapeutic approaches for managing HSCR and AD-HSCR, including the use of enteric nervous system progenitor cells, embryonic stem cells, induced pluripotent stem cells, and mesenchymal stem cells as cell sources, the recipient intestine's microenvironment, and transplantation methods. Perspectives on the future of these treatments are also discussed.

DOI： 10.1007/s00595-023-02741-6

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：一般社団法人　日本歯科理工学会  

Carbonate apatite (CO3Ap) is a major inorganic bone component and an effective bone substitute. To clarify the function of CO3Ap, we compared differences among CO3Ap, hydroxyapatite (HAp), and β-tricalcium phosphate (β-TCP) by focusing on mesenchymal stem cells (MSCs) that have a role in wound healing. For in vivo experiments, maxillary molars were removed and the bone substitute was inserted. MSC accumulation around extraction sockets was significantly promoted in CO3Ap and β-TCP groups. For in vitro experiments, MSCs were cultured with bone substitutes. The differentiation potential and amount of calcium deposition were significantly lower in CO3Ap and HAp groups than in the β-TCP group. Increases in insulin-like growth factor-I and vascular endothelial growth factor were found only in the CO3Ap group. CO3Ap-filled extraction sockets accumulated MSCs, and MSCs cultured in the presence of CO3Ap produced large amounts of growth factors. These results suggest that CO3Ap promotes healing of tooth extraction sockets.

DOI： 10.4012/dmj.2022-040

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担当区分：最終著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Frontiers in Endocrinology  

Systemic transplantation of mesenchymal stem cells (MSCs), such as bone marrow MSCs (BMMSCs) and stem cells from human exfoliated deciduous teeth (SHED), is considered a prominent treatment for osteopenia. However, the mechanism of action of the transplanted MSCs has been poorly elucidated. In the recipient target tissue, including bone and bone marrow, only a few donor MSCs can be detected, suggesting that the direct contribution of donor MSCs may not be expected for osteopenia treatment. Meanwhile, secretomes, especially contents within extracellular vesicles (EVs) released from donor MSCs (MSC-EVs), play key roles in the treatment of several diseases. In this context, administrated donor MSC-EVs may affect bone-forming function of recipient cells. In this review, we discuss how MSC-EVs contribute to bone recovery recipient tissue in osteopenia. We also summarize a novel mechanism of action of systemic administration of SHED-derived EVs (SHED-EVs) in osteopenia. We found that reduced telomerase activity in recipient BMMSCs caused the deficiency of microenvironmental modulating function, including bone and bone marrow-like niche formation and immunomodulation in estrogen-deficient osteopenia model mice. Systemic administration of SHED-EVs could exert therapeutic effects on bone reduction via recovering the telomerase activity, leading to the rejuvenation of the microenvironmental modulating function in recipient BMMSCs, as seen in systemic transplantation of SHED. RNase-preconditioned donor SHED-EVs diminished the therapeutic benefits of administrated SHED-EVs in the recipient osteopenia model mice. These facts suggest that MSC-EV therapy targets the recipient BMMSCs to rejuvenate the microenvironmental modulating function via telomerase activity, recovering bone density. We then introduce future challenges to develop the reproducible MSC-EV therapy in osteopenia.

DOI： 10.3389/fendo.2023.1151429

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記述言語：英語   出版者・発行元：Journal of Cellular Physiology  

Nuclear protein 1 (NUPR1) is a stress-induced protein activated by various stresses, such as inflammation and oxidative stress. We previously reported that Nupr1 deficiency increased bone volume by enhancing bone formation in 11-week-old mice. Analysis of differentially expressed genes between wild-type (WT) and Nupr1-knockout (Nupr1-KO) osteocytes revealed that high temperature requirement A 1 (HTRA1), a serine protease implicated in osteogenesis and transforming growth factor-β signaling was markedly downregulated in Nupr1-KO osteocytes. Nupr1 deficiency also markedly reduced HtrA1 expression, but enhanced SMAD1 signaling in in vitro-cultured primary osteoblasts. In contrast, Nupr1 overexpression enhanced HtrA1 expression in osteoblasts, suggesting that Nupr1 regulates HtrA1 expression, thereby suppressing osteoblastogenesis. Since HtrA1 is also involved in cellular senescence and age-related diseases, we analyzed aging-related bone loss in Nupr1-KO mice. Significant spine trabecular bone loss was noted in WT male and female mice during 6−19 months of age, whereas aging-related trabecular bone loss was attenuated, especially in Nupr1-KO male mice. Moreover, cellular senescence-related markers were upregulated in the osteocytes of 6−19-month-old WT male mice but markedly downregulated in the osteocytes of 19-month-old Nupr1-KO male mice. Oxidative stress-induced cellular senescence stimulated Nupr1 and HtrA1 expression in in vitro-cultured primary osteoblasts, and Nupr1 overexpression enhanced p16ink4a expression in osteoblasts. Finally, NUPR1 expression in osteocytes isolated from the bones of patients with osteoarthritis was correlated with age. Collectively, these results indicate that Nupr1 regulates HtrA1-mediated osteoblast differentiation and senescence. Our findings unveil a novel Nupr1/HtrA1 axis, which may play pivotal roles in bone formation and age-related bone loss.

DOI： 10.1002/jcp.30949

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記述言語：英語   出版者・発行元：(一社)日本歯科理工学会  

骨補填材としての炭酸アパタイト(炭酸Ap)をハイドロキシアパタイト(HAp)およびβリン酸三カルシウム(β-TCP)と比較し、間葉系幹細胞(MSC)に注目してそれらの間にみられる差異を検討した。6週齢の雄性ラット30匹を使用してin vivo実験を施行した。上顎の第1・2大臼歯を抜歯し、抜歯窩に上記3種の骨補填材のいずれかを移植した。その3日後と7日後に深麻酔死させ各種の評価に供した。その結果、炭酸Ap群とβ-TCP群では、抜歯窩付近のMSCの集積が有意に促進された。MSCを骨補填材の存在下で培養するin vitro実験も行ったところ、炭酸Ap群とHAp群では分化能とカルシウム沈着量がβ-TCP群よりも有意に低減していた。インスリン様成長因子I(IGF-I)と血管内皮細胞増殖因子(VEGF)が増加しているという結果は炭酸Ap群のみで認められた。炭酸Apを充填した抜歯窩にはMSCが集積し、炭酸Ap存在下で培養したMSCは成長因子類を大量に産生したことから、炭酸Apは抜歯窩の治癒を促進することが示唆された。

記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）  

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担当区分：最終著者,　責任著者   記述言語：その他   掲載種別：研究論文（学術雑誌）   出版者・発行元：Molecular Metabolism  

Objective: Chronic liver diseases often involve metabolic damage to the skeletal system. The underlying mechanism of bone loss in chronic liver diseases remains unclear, and appropriate therapeutic options, except for orthotopic liver transplantation, have proved insufficient for these patients. This study aimed to investigate the efficacy and mechanism of transplantation of immature hepatocyte-like cells converted from stem cells from human exfoliated deciduous teeth (SHED-Heps) in bone loss of chronic liver fibrosis. Methods: Mice that were chronically treated with CCl4 received SHED-Heps, and trabecular bone density, reactive oxygen species (ROS), and osteoclast activity were subsequently analyzed in vivo and in vitro. The effects of stanniocalcin 1 (STC1) knockdown in SHED-Heps were also evaluated in chronically CCl4 treated mice. Results: SHED-Hep transplantation (SHED-HepTx) improved trabecular bone loss and liver fibrosis in chronic CCl4-treated mice. SHED-HepTx reduced hepatic ROS production and interleukin 17 (Il-17) expression under chronic CCl4 damage. SHED-HepTx reduced the expression of both Il-17 and tumor necrosis factor receptor superfamily 11A (Tnfrsf11a) and ameliorated the imbalance of osteoclast and osteoblast activities in the bone marrow of CCl4-treated mice. Functional knockdown of STC1 in SHED-Heps attenuated the benefit of SHED-HepTx including anti-bone loss effect by suppressing osteoclast differentiation through TNFSF11–TNFRSF11A signaling and enhancing osteoblast differentiation in the bone marrow, as well as anti-fibrotic and anti-ROS effects in the CCl4-injured livers. Conclusions: These findings suggest that targeting hepatic ROS provides a novel approach to treat bone loss resulting from chronic liver diseases.

DOI： 10.1016/j.molmet.2022.101599

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Cancer Science  

Aberrant osteoclast formation and activation are the hallmarks of osteolytic metastasis. Extracellular vesicles (EVs), released from bone metastatic tumor cells, play a pivotal role in the progression of osteolytic lesions. However, the mechanisms through which tumor cell–derived EVs regulate osteoclast differentiation and function have not been fully elucidated. In this study, we found that 4T1 bone metastatic mouse mammary tumor cell–derived EVs (4T1-EVs) are taken up by mouse bone marrow macrophages to facilitate osteoclastogenesis. Furthermore, treatment of mature osteoclasts with 4T1-EVs promoted bone resorption, which was accompanied by enhanced survival of mature osteoclasts through the negative regulation of caspase-3. By comparing the miRNA content in 4T1-EVs with that in 67NR nonmetastatic mouse mammary tumor cell–derived EVs (67NR-EVs), miR-92a-3p was identified as one of the most enriched miRNAs in 4T1-EVs, and its transfer into mature osteoclasts significantly reduced apoptosis. Bioinformatic and Western blot analyses revealed that miR-92a-3p directly targeted phosphatase and tensin homolog (PTEN) in mature osteoclasts, resulting in increased levels of phospho-Akt. Our findings provide novel insights into the EV-mediated regulation of osteoclast survival through the transfer of miR-92a-3p, which enhances mature osteoclast survival via the Akt survival signaling pathway, thus promoting bone resorption.

DOI： 10.1111/cas.15557

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記述言語：英語   出版者・発行元：John Wiley & Sons Australia, Ltd  

骨転移乳癌細胞由来細胞外小胞(EV)が破骨細胞の分化や機能に及ぼす影響とそのメカニズムについて検討した。マウス乳癌細胞株4T1由来EV(4T1-EV)がマウス骨髄マクロファージに取り込まれ、破骨細胞形成を促進することを見出した。成熟破骨細胞を4T1-EVで処理すると、骨吸収が促進され、カスパーゼ3を負に制御して成熟破骨細胞の生存が促進された。4T1-EVと非転移性マウス乳癌細胞株67NR由来EV(67NR-EV)におけるmiRNAの内容を比較した。その結果、miR-92a-3pが4T1-EVに最も濃縮されたmiRNAの一つとして特定され、miR-92a-3pの成熟破骨細胞への移入によりアポトーシスが著しく抑制された。さらに、miR-92a-3pは成熟破骨細胞においてphosphatase and tensin homolog(PTEN)を直接標的とし、リン酸化Aktのレベルを増加させた。以上より、マウス乳癌細胞株4T1由来EVに存在するmiR-92a-3pは、Akt生存シグナル経路を介して成熟破骨細胞の生存を高め、骨吸収を促進させることが示された。

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：STAR Protocols  

Human dental pulp stem cell (hDPSCs)-based therapy is a feasible option for regenerative medicine, such as dental pulp regeneration. Here, we show the steps needed to colony-forming unit-fibroblasts (CFU-F)-based isolation, expansion, and cryopreservation of hDPSCs for manufacturing clinical-grade products under a xenogeneic-free/serum-free condition. We also demonstrate the characterization of hDPSCs by CFU-F, flow cytometric, and in vitro multipotent assays. For complete details on the use and execution of this protocol, please refer to Iwanaka et al. (2020).

DOI： 10.1016/j.xpro.2022.101386

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Scientific Reports  

Hirschsprung's disease is a congenital entero-neuropathy that causes chronic constipation and intestinal obstruction. New treatments for entero-neuropathy are needed because current surgical strategies have limitations5. Entero-neuropathy results from enteric nervous system dysfunction due to incomplete colonization of the distal intestine by neural crest-derived cells. Impaired cooperation between the enteric nervous system and intestinal pacemaker cells may also contribute to entero-neuropathy. Stem cell therapy to repair these multiple defects represents a novel treatment approach. Dental pulp stem cells derived from deciduous teeth (dDPSCs) are multipotent cranial neural crest-derived cells, but it remains unknown whether dDPSCs have potential as a new therapy for entero-neuropathy. Here we show that intravenous transplantation of dDPSCs into the Japanese Fancy-1 mouse, an established model of hypoganglionosis and entero-neuropathy, improves large intestinal structure and function and prolongs survival. Intravenously injected dDPSCs migrate to affected regions of the intestine through interactions between stromal cell-derived factor-1α and C-X-C chemokine receptor type-4. Transplanted dDPSCs differentiate into both pacemaker cells and enteric neurons in the proximal colon to improve electrical and peristaltic activity, in addition to their paracrine effects. Our findings indicate that transplanted dDPSCs can differentiate into different cell types to correct entero-neuropathy-associated defects.

DOI： 10.1038/s41598-022-10077-3

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：International Journal of Molecular Sciences  

Recent advances in mesenchymal stem/stromal cell (MSC) research have led us to consider the feasibility of MSC-based therapy for various diseases. Human dental pulp-derived MSCs (hDPSCs) have been identified in the dental pulp tissue of deciduous and permanent teeth, and they exhibit properties with self-renewal and in vitro multipotency. Interestingly, hDPSCs exhibit superior immunosuppressive functions toward immune cells, especially T lymphocytes, both in vitro and in vivo. Recently, hDPSCs have been shown to have potent immunomodulatory functions in treating systemic lupus erythematosus (SLE) in the SLE MRL/lpr mouse model. However, the mechanisms underlying the immunosuppressive efficacy of hDPSCs remain unknown. This review aims to introduce a new target of hDPSC-based therapy on the recipient niche function in SLE.

DOI： 10.3390/ijms23073479

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Osteoclasts are multinucleated cells formed through specific recognition and fusion of mononuclear osteoclast precursors derived from hematopoietic stem cells. Detailed cellular events concerning cell fusion in osteoclast differentiation remain ambiguous. Tunneling nanotubes (TNTs), actin-based membrane structures, play an important role in intercellular communication between cells. We have previously reported the presence of TNTs in the fusion process of osteoclastogenesis. Here we analyzed morphological details of TNTs using scanning electron microscopy. The osteoclast precursor cell line RAW-D was stimulated to form osteoclast-like cells, and morphological details in the appearance of TNTs were extensively analyzed. Osteoclast-like cells could be classified into three types; early osteoclast precursors, late osteoclast precursors, and multinucleated osteoclast-like cells based on the morphological characteristics. TNTs were frequently observed among these three types of cells. TNTs could be classified into thin, medium, and thick TNTs based on the diameter and length. The shapes of TNTs were dynamically changed from thin to thick. Among them, medium TNTs were often observed between two remote cells, in which side branches attached to the culture substrates and beaded bulge-like structures were often observed. Cell-cell interaction through TNTs contributed to cell migration and rapid transport of information between cells. TNTs were shown to be involved in cell-cell fusion between osteoclast precursors and multinucleated osteoclast-like cells, in which movement of membrane vesicles and nuclei was observed. Formation of TNTs was also confirmed in primary cultures of osteoclasts. Furthermore, we have successfully detected TNTs formed between osteoclasts observed in the bone destruction sites of arthritic rats. Thus, formation of TNTs may be important for the differentiation of osteoclasts both in vitro and in vivo. TNTs could be one target cellular structure for the regulation of osteoclast differentiation and function in bone diseases.

DOI： 10.1038/s41374-021-00656-9

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41374-021-00661-y

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Oral malignant melanoma, which frequently invades the hard palate or maxillary bone, is extremely rare and has a poor prognosis. Bone morphogenetic protein (BMP) is abundantly expressed in bone matrix and is highly expressed in malignant melanoma, inducing an aggressive phenotype. We examined the role of BMP signaling in the acquisition of an aggressive phenotype in melanoma cells in vitro and in vivo. In five cases, immunohistochemistry indicated the phosphorylation of Smad1/5 (p-Smad1/5) in the nuclei of melanoma cells. In the B16 mouse and A2058 human melanoma cell lines, BMP2, BMP4, or BMP7 induces morphological changes accompanied by the downregulation of E-cadherin, and the upregulation of N-cadherin and Snail, markers of epithelial-mesenchymal transition (EMT). BMP2 also stimulates cell invasion by increasing matrix metalloproteinase activity in B16 cells. These effects were canceled by the addition of LDN193189, a specific inhibitor of Smad1/5 signaling. In vivo, the injection of B16 cells expressing constitutively activated ALK3 enhanced zygoma destruction in comparison to empty B16 cells by increasing osteoclast numbers. These results suggest that the activation of BMP signaling induces EMT, thus driving the acquisition of an aggressive phenotype in malignant melanoma.

DOI： 10.1038/s41374-021-00661-y

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: Biliary atresia (BA) is a severe hepatobiliary disease in infants that ultimately results in hepatic failure; however, its pathological mechanism is poorly elucidated. Current surgical options, including Kasai hepatoportoenterostomy and orthotopic liver organ transplantations, are palliative; thus, innovation in BA therapy is urgent. METHODS: To examine whether BA-specific post-natal stem cells are feasible for autologous cell source for BA treatment, we isolated from human exfoliated deciduous teeth, namely BA-SHED, using a standard colony-forming unit fibroblast (CFU-F) method and compared characteristics as mesenchymal stem cells (MSCs) to healthy donor-derived control SHED, Cont-SHED. BA-SHED and Cont-SHED were intrasplenically transplanted into chronic carbon tetrachloride (CCl4)-induced liver fibrosis model mice, followed by the analysis of bile drainage function and donor integration in vivo. Immunohistochemical assay was examined for the regeneration of intrahepatic bile ducts in the recipient's liver using anti-human specific keratin 19 (KRT19) antibody. RESULTS: BA-SHED formed CFU-F, expressed MSC surface markers, and exhibited in vitro mesenchymal multipotency similar to Cont-SHED. BA-SHED showed less in vitro hepatogenic potency than Cont-SHED. Cont-SHED represented in vivo bile drainage function and KRT19-positive biliary regeneration in chronic carbon tetrachloride-induced liver fibrosis model mice. BA-SHED failed to show in vivo biliary potency and bile drainage function compared to Cont-SHED. CONCLUSION: These findings indicate that BA-SHED are not feasible source for BA treatment, because BA-SHED may epigenetically modify the underlying prenatal and perinatal BA environments. In conclusion, these findings suggest that BA-SHED-based studies may provide a platform for understanding the underlying molecular mechanisms of BA development and innovative novel modalities in BA research and treatment.

DOI： 10.1186/s13287-021-02652-8

リポジトリ公開URL： https://hdl.handle.net/2324/7172586

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： https://doi.org/10.1371/journal.pone.0259966

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41374-021-00633-2

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Adrenomedullin (ADM), a member of the calcitonin family of peptides, is a potent vasodilator and was shown to have the ability to modulate bone metabolism. We have previously found a unique cell surface antigen (Kat1 antigen) expressed in rat osteoclasts, which is involved in the functional regulation of the calcitonin receptor (CTR). Cross-linking of cell surface Kat1 antigen with anti-Kat1 antigen monoclonal antibody (mAbKat1) stimulated osteoclast formation only under conditions suppressed by calcitonin. Here, we found that ADM provoked a significant stimulation in osteoclastogenesis only in the presence of calcitonin; a similar biological effect was seen with mAbKat1 in the bone marrow culture system. This stimulatory effect on osteoclastogenesis mediated by ADM was abolished by the addition of mAbKat1. 125I-labeled rat ADM (125I-ADM)-binding experiments involving micro-autoradiographic studies demonstrated that mononuclear precursors of osteoclasts abundantly expressed ADM receptors, and the specific binding of 125I-ADM was markedly inhibited by the addition of mAbKat1, suggesting a close relationship between the Kat1 antigen and the functional ADM receptors expressed on cells in the osteoclast lineage. ADM receptors were also detected in the osteoclast progenitor cells in the late mitotic phase, in which only one daughter cell of the dividing cell express ADM receptors, suggesting the semiconservative cell division of the osteoclast progenitors in the initiation of osteoclastogenesis. Messenger RNAs for the receptor activity-modifying-protein 1 (RAMP1) and calcitonin receptor-like receptor (CRLR) were expressed in cells in the osteoclast lineage; however, the expression of RAMP2 or RAMP3 was not detected in these cells. It is suggested that the Kat1 antigen is involved in the functional ADM receptor distinct from the general ADM receptor, consisting of CRLR and RAMP2 or RAMP3. Modulation of osteoclastogenesis through functional ADM receptors abundantly expressed on mononuclear osteoclast precursors is supposed to be important in the fine regulation of osteoclast differentiation in a specific osteotrophic hormonal condition with a high level of calcitonin in blood.

DOI： 10.1038/s41374-021-00633-2

記述言語：日本語   掲載種別：研究論文（研究会，シンポジウム資料等）  

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Systemic transplantation of stem cells from human exfoliated deciduous teeth (SHED) is used to treat systemic lupus erythematosus (SLE)-like disorders in MRL/lpr mice. However, the mechanisms underlying the SHED-based therapy remain unclear. In this study, we hypothesized that trophic factors within SHED-releasing extracellular vesicles (SHED-EVs) ameliorate the SLE-like phenotypes in MRL/lpr mice. SHED-EVs were isolated from the culture supernatant of SHED. SHED-EVs were treated with or without RNase and systemically administered to MRL/lpr mice. Subsequently, recipient bone marrow mesenchymal stem cells (BMMSCs) isolated from SHED-EV-administered MRL/lpr mice were examined for the in vitro and in vivo activity of hematopoietic niche formation and immunoregulation. Furthermore, the recipient BMMSCs were secondarily transplanted into MRL/lpr mice. The systemic SHED-EV infusion ameliorated the SLE-like phenotypes in MRL/lpr mice and improved the functions of recipient BMMSCs by rescuing Tert mRNA-associated telomerase activity, hematopoietic niche formation, and immunoregulation. The secondary transplantation of recipient BMMSCs recovered the immune condition and renal functions of MRL/lpr mice. The RNase treatment depleted RNAs, such as microRNAs, within SHED-EVs, and the RNA-depleted SHED-EVs attenuated the benefits of SHED-EVs in MRL/lpr mice. Collectively, our findings suggest that SHED-secreted RNAs, such as microRNAs, play a crucial role in treating SLE by targeting the telomerase activity of recipient BMMSCs.

DOI： 10.4049/jimmunol.2001312

担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: Stem cells from human exfoliated deciduous teeth (SHED) have been reported to show the in vivo and in vitro hepatic differentiation, SHED-Heps; however, the cholangiogenic potency of SHED-Heps remains unclear. Here, we hypothesized that SHED-Heps contribute to the regeneration of intrahepatic bile duct system in chronic fibrotic liver. METHODS: SHED were induced into SHED-Heps under cytokine stimulation. SHED-Heps were intrasplenically transplanted into chronically CCl4-treated liver fibrosis model mice, followed by the analysis of donor integration and hepatobiliary metabolism in vivo. Immunohistochemical assay was examined for the regeneration of intrahepatic bile duct system in the recipient liver. Furthermore, SHED-Heps were induced under the stimulation of tumor necrosis factor alpha (TNFA). RESULTS: The intrasplenic transplantation of SHED-Heps into CCl4-treated mice showed that donor SHED-Heps behaved as human hepatocyte paraffin 1- and human albumin-expressing hepatocyte-like cells in situ and ameliorated CCl4-induced liver fibrosis. Of interest, the integrated SHED-Heps not only expressed biliary canaliculi ATP-binding cassette transporters including ABCB1, ABCB11, and ABCC2, but also recruited human keratin 19- (KRT19-) and KRT17-positive cells, which are considered donor-derived cholangiocytes, regenerating the intrahepatic bile duct system in the recipient liver. Furthermore, the stimulation of TNFA induced SHED-Heps into KRT7- and SRY-box 9-positive cells. CONCLUSIONS: Collectively, our findings demonstrate that infused SHED-Heps showed cholangiogenic ability under the stimulation of TNFA in CCl4-damaged livers, resulting in the regeneration of biliary canaliculi and interlobular bile ducts in chronic fibrotic liver. Thus, the present findings suggest that SHED-Heps may be a novel source for the treatment of cholangiopathy.

DOI： 10.1186/s13287-020-02113-8

記述言語：日本語  

記述言語：日本語  

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Background: Systemic transplantation of stem cells from human exfoliated deciduous teeth (SHED) recovers bone loss in animal models of osteoporosis; however, the mechanisms underlying this remain unclear. Here, we hypothesized that trophic factors within SHED-releasing extracellular vesicles (SHED-EVs) rescue osteoporotic phenotype. Methods: EVs were isolated from culture supernatant of SHED. SHED-EVs were treated with or without ribonuclease and systemically administrated into ovariectomized mice, followed by the function of recipient bone marrow mesenchymal stem cells (BMMSCs) including telomerase activity, osteoblast differentiation, and sepmaphorine-3A (SEMA3A) secretion. Subsequently, human BMMSCs were stimulated by SHED-EVs with or without ribonuclease treatment, and then human BMMSCs were examined regarding the function of telomerase activity, osteoblast differentiation, and SEMA3A secretion. Furthermore, SHED-EV-treated human BMMSCs were subcutaneously transplanted into the dorsal skin of immunocompromised mice with hydroxyapatite tricalcium phosphate (HA/TCP) careers and analyzed the de novo bone-forming ability. Results: We revealed that systemic SHED-EV-infusion recovered bone volume in ovariectomized mice and improved the function of recipient BMMSCs by rescuing the mRNA levels of Tert and telomerase activity, osteoblast differentiation, and SEMA3A secretion. Ribonuclease treatment depleted RNAs, including microRNAs, within SHED-EVs, and these RNA-depleted SHED-EVs attenuated SHED-EV-rescued function of recipient BMMSCs in the ovariectomized mice. These findings were supported by in vitro assays using human BMMSCs incubated with SHED-EVs. Conclusion: Collectively, our findings suggest that SHED-secreted RNAs, such as microRNAs, play a crucial role in treating postmenopausal osteoporosis by targeting the telomerase activity of recipient BMMSCs.

DOI： 10.1186/s13287-020-01818-0

担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Background: Human deciduous pulp stem cells (hDPSCs) have remarkable stem cell potency associated with cell proliferation, mesenchymal multipotency, and immunosuppressive function and have shown beneficial effects in a variety of animal disease models. Recent studies demonstrated that hDPSCs exhibited in vivo anti-fibrotic and anti-inflammatory action and in vivo hepatogenic-associated liver regeneration, suggesting that hDPSCs may offer a promising source with great clinical demand for treating liver diseases. However, how to manufacture ex vivo large-scale clinical-grade hDPSCs with the appropriate quality, safety, and preclinical efficacy assurances remains unclear. Methods: We isolated hDPSCs from human deciduous dental pulp tissues formed by the colony-forming unit-fibroblast (CFU-F) method and expanded them under a xenogeneic-free and serum-free (XF/SF) condition; hDPSC products were subsequently stored by two-step banking including a master cell bank (MCB) and a working cell bank (WCB). The final products were directly thawed hDPSCs from the WCB. We tested the safety and quality check, stem cell properties, and preclinical potentials of final hDPSC products and hDPSC products in the MCB and WCB. Results: We optimized manufacturing procedures to isolate and expand hDPSC products under a XF/SF culture condition and established the MCB and the WCB. The final hDPSC products and hDPSC products in the MCB and WCB were validated the safety and quality including population doubling ability, chromosome stability, microorganism safety, and stem cell properties including morphology, cell surface marker expression, and multipotency. We also evaluated the in vivo immunogenicity and tumorigenicity and validated in vivo therapeutic efficacy for liver regeneration in a CCl4-induced chronic liver fibrosis mouse model in the final hDPSC products and hDPSC products in the WCB. Conclusion: The manufacture and quality control results indicated that the present procedure could produce sufficient numbers of clinical-grade hDPSC products from a tiny deciduous dental pulp tissue to enhance clinical application of hDPSC products in chronic liver fibrosis.

DOI： 10.1186/s13287-020-01630-w

記述言語：英語   掲載種別：研究論文（学術雑誌）  

PURPOSE: Mesenchymal stem cell (MSC)-based cell therapies have emerged as a promising treatment option for various diseases. Due to the superior survival and higher differentiation efficiency, three-dimensional spheroid culture systems have been an important topic of MSC research. Stem cells from human exfoliated deciduous teeth (SHED) have been considered an ideal source of MSCs for regenerative medicine. Thus, in the present study, we introduce our newly developed method for fabricating SHED-based micro-hepatic tissues, and demonstrate the therapeutic effects of SHED-based micro-hepatic tissues in mouse disease models. METHODS: SHED-converted hepatocyte-like cells (SHED-HLCs) were used for fabricating spherical micro-hepatic tissues. The SHED-HLC-based spheroids were then transplanted both into the liver of mice with CCl4-induced chronic liver fibrosis and the kidney of factor VIII (F8)-knock-out mice. At 4 weeks after transplantation, the therapeutic efficacy was investigated. RESULTS: Intrahepatic transplantation of SHED-HLC-spheroids improved the liver dysfunction in association with anti-fibrosis effects in CCl4-treated mice. Transplanted SHED-converted cells were successfully engrafted in the recipient liver. Meanwhile, renal capsular transplantation of the SHED-HLC-spheroids significantly extended the bleeding time in F8-knock-out mice. CONCLUSIONS: These findings suggest that SHED-HLC-based micro-hepatic tissues might be a promising source for treating pediatric refractory diseases, including chronic liver fibrosis and hemophilia A.

DOI： 10.1007/s00383-019-04564-4

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Purpose: Mesenchymal stem cell (MSC)-based cell therapies have emerged as a promising treatment option for various diseases. Due to the superior survival and higher differentiation efficiency, three-dimensional spheroid culture systems have been an important topic of MSC research. Stem cells from human exfoliated deciduous teeth (SHED) have been considered an ideal source of MSCs for regenerative medicine. Thus, in the present study, we introduce our newly developed method for fabricating SHED-based micro-hepatic tissues, and demonstrate the therapeutic effects of SHED-based micro-hepatic tissues in mouse disease models. Methods: SHED-converted hepatocyte-like cells (SHED-HLCs) were used for fabricating spherical micro-hepatic tissues. The SHED-HLC-based spheroids were then transplanted both into the liver of mice with CCl₄-induced chronic liver fibrosis and the kidney of factor VIII (F8)-knock-out mice. At 4 weeks after transplantation, the therapeutic efficacy was investigated. Results: Intrahepatic transplantation of SHED-HLC-spheroids improved the liver dysfunction in association with anti-fibrosis effects in CCl₄-treated mice. Transplanted SHED-converted cells were successfully engrafted in the recipient liver. Meanwhile, renal capsular transplantation of the SHED-HLC-spheroids significantly extended the bleeding time in F8-knock-out mice. Conclusions: These findings suggest that SHED-HLC-based micro-hepatic tissues might be a promising source for treating pediatric refractory diseases, including chronic liver fibrosis and hemophilia A.

DOI： 10.1007/s00383-019-04564-4

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Wilson’s disease (WD) is an inherited metabolic disease arising from ATPase copper transporting beta gene (ATP7B) mutation. Orthotoropic liver transplantation is the only radical treatment of fulminant WD, although appropriate donors are lacking at the onset of emergency. Given the hepatogenic capacity and tissue-integration/reconstruction ability in the liver of stem cells from human exfoliated deciduous teeth (SHED), SHED have been proposed as a source for curing liver diseases. We hypothesized the therapeutic potential of SHED and SHED-converted hepatocyte-like- cells (SHED-Heps) for fulminant WD. SHED and SHED-Heps were transplanted into WD model Atp7b-mutated Long-Evans Cinnamon (LEC) rats received copper overloading to induce a lethal fulminant liver failure. Due to the superior copper tolerance via ATP7B, SHED-Hep transplantation gave more prolonged life-span of fulminant LEC rats than SHED transplantation. The integrated ATP7B-expressing SHED-Heps showed more therapeutic effects on to restoring the hepatic dysfunction and tissue damages in the recipient liver than the integrated naïve SHED without ATP7B expression. Moreover, SHED-Heps could reduce copper-induced oxidative stress via ATP7B- independent stanniocalcin 1 secretion in the fulminant LEC rats, suggesting a possible role for paracrine effect of the integrated SHED-Heps. Taken together, SHED-Heps offer a potential of functional restoring, bridging, and preventive approaches for treating fulminant WD.

DOI： 10.1038/s41598-018-38275-y

記述言語：その他   掲載種別：研究論文（学術雑誌）  

Stem cell therapy is an emerging treatment modality for various diseases. Because mesenchymal stem cells (MSCs) are known to accumulate at the site of damage, their possible clinical application has been investigated. MSCs are usually administered using intravenous injection, but this route carries a risk of pulmonary embolism. In contrast, topical injection of MSCs reportedly has an inferior therapeutic effect. We developed a remote administration method that uses collagen gel as a scaffold and investigated the effect of this scaffold on the retention of stemness, homing ability, and therapeutic effect using a mouse tooth extraction model. After verifying the retention of stemness of MSCs isolated from the bone marrow of donor mice in the scaffold, we administered MSCs subcutaneously into the back of the recipient mice with scaffold and observed the accumulation and the acceleration of healing of the extraction socket of the maxillary first molar. The MSCs cultured with scaffold retained stemness, the MSCs injected into back skin with scaffold successfully accumulated around the extraction socket, and socket healing was significantly enhanced. In conclusion, administration of MSCs with collagen scaffold at a remote site enhanced the lesion healing without the drawbacks of currently used administration methods.

DOI： 10.3390/app9224908

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Stem cell therapy is an emerging treatment modality for various diseases. Because mesenchymal stem cells (MSCs) are known to accumulate at the site of damage, their possible clinical application has been investigated. MSCs are usually administered using intravenous injection, but this route carries a risk of pulmonary embolism. In contrast, topical injection of MSCs reportedly has an inferior therapeutic effect. We developed a remote administration method that uses collagen gel as a scaffold and investigated the effect of this scaffold on the retention of stemness, homing ability, and therapeutic effect using a mouse tooth extraction model. After verifying the retention of stemness of MSCs isolated from the bone marrow of donor mice in the scaffold, we administered MSCs subcutaneously into the back of the recipient mice with scaffold and observed the accumulation and the acceleration of healing of the extraction socket of the maxillary first molar. The MSCs cultured with scaffold retained stemness, the MSCs injected into back skin with scaffold successfully accumulated around the extraction socket, and socket healing was significantly enhanced. In conclusion, administration of MSCs with collagen scaffold at a remote site enhanced the lesion healing without the drawbacks of currently used administration methods.

DOI： 10.3390/app9224908

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Bone remodeling is a continuous process characterized by highly coordinated cell-cell interactions in distinct multi-cellular units. Osteoclasts, which are specialized bone resorbing cells, play a central role in bone remodeling. Although the RANKL/RANK axis determines the gross number of osteoclasts present in bone tissue, detailed molecular events regulating bone remodeling related to osteoclast recruitment, initiation of bone remodeling, and coupling of bone resorption and bone formation are still ambiguous. We hypothesized that osteoblast-specific cell-surface molecules contribute to the molecular modulation of bone remodeling. Therefore, we searched for regulatory cell-surface molecules expressed on osteoblasts by use of B-cell hybridoma technology. We obtained a monoclonal antibody A7 (A7 MAb) highly specific to cells of osteoblast-lineage. Here we describe the expression pattern and possible role of A7 antigen specifically recognized by A7 MAb. In vitro, A7 antigen was expressed on cell-surface of osteoblasts and osteoblast-like bone marrow stromal cells. In vivo, A7 antigen was detected in a subset of bone surface osteoblasts and in osteocytes, with a typical cell membrane expression pattern. Tissue array analysis showed only a limited expression of A7 antigen in osteocytes close to the bone surface. Immunoblotting and immunoprecipitation analysis showed that A7 antigen is a lineage-specific cell-surface protein with an approximate molecular weight of 45 KDa. Cross-linking of cell-surface A7 antigen in cultures of osteoclastogenesis showed stimulation of osteoclast formation. Marked suppression of calcification in primary osteoblast cultures was observed when A7 antigen was cross-linked with anti-A7 antigen MAb, A7 MAb. These data suggest that A7 antigen regulates recruitment of osteoclasts and triggering of calcification. A7 antigen may be an important molecule involved in the precise regulation of bone remodeling.

DOI： 10.1038/s41374-018-0179-4

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Bone remodeling is a continuous process characterized by highly coordinated cell-cell interactions in distinct multi-cellular units. Osteoclasts, which are specialized bone resorbing cells, play a central role in bone remodeling. Although the RANKL/RANK axis determines the gross number of osteoclasts present in bone tissue, detailed molecular events regulating bone remodeling related to osteoclast recruitment, initiation of bone remodeling, and coupling of bone resorption and bone formation are still ambiguous. We hypothesized that osteoblast-specific cell-surface molecules contribute to the molecular modulation of bone remodeling. Therefore, we searched for regulatory cell-surface molecules expressed on osteoblasts by use of B-cell hybridoma technology. We obtained a monoclonal antibody A7 (A7 MAb) highly specific to cells of osteoblast-lineage. Here we describe the expression pattern and possible role of A7 antigen specifically recognized by A7 MAb. In vitro, A7 antigen was expressed on cell-surface of osteoblasts and osteoblast-like bone marrow stromal cells. In vivo, A7 antigen was detected in a subset of bone surface osteoblasts and in osteocytes, with a typical cell membrane expression pattern. Tissue array analysis showed only a limited expression of A7 antigen in osteocytes close to the bone surface. Immunoblotting and immunoprecipitation analysis showed that A7 antigen is a lineage-specific cell-surface protein with an approximate molecular weight of 45 KDa. Cross-linking of cell-surface A7 antigen in cultures of osteoclastogenesis showed stimulation of osteoclast formation. Marked suppression of calcification in primary osteoblast cultures was observed when A7 antigen was cross-linked with anti-A7 antigen MAb, A7 MAb. These data suggest that A7 antigen regulates recruitment of osteoclasts and triggering of calcification. A7 antigen may be an important molecule involved in the precise regulation of bone remodeling.

DOI： 10.1038/s41374-018-0179-4

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Introduction: Stem cells isolated from the root apical papilla of human teeth (stem cells from the apical papilla [SCAPs])are capable of forming tooth root dentin and are a feasible source for bioengineered tooth root regeneration. In this study, we examined the effect of acetylsalicylic acid (ASA)on odontogenic differentiation of SCAPs in vitro and in vivo. Methods: SCAPs were cultured under odontogenic conditions supplemented with or without ASA. ASA-treated SCAPs were also subcutaneously transplanted into immunocompromised mice. Results: ASA accelerates in vitro and in vivo odontogenic differentiation of SCAPs associated with down-regulation of runt-related nuclear factor 2 and up-regulation of specificity protein 7, nuclear factor I C, and dentin phosphoprotein. ASA up-regulated the phosphorylation of AKT in the odontogenic SCAPs. Of interest, pretreatments with phosphoinositide 3-kinase inhibitor LY294402 and small interfering RNA for AKT promoted ASA-induced in vitro and in vivo odontogenic differentiation of SCAPs. LY294402 and small interfering RNA for AKT also suppressed the ASA-induced expression of runt-related nuclear factor 2 and enhanced ASA-induced expression of specificity protein 7, nuclear factor I C, and dentin phosphoprotein in SCAPs. Conclusions: These findings suggest that a combination of ASA treatment and suppressive regulation of the phosphoinositide 3-kinase–AKT signaling pathway is a novel approach for SCAP-based tooth root regeneration.

DOI： 10.1016/j.joen.2019.01.016

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Introduction: Stem cells isolated from the root apical papilla of human teeth (stem cells from the apical papilla [SCAPs])are capable of forming tooth root dentin and are a feasible source for bioengineered tooth root regeneration. In this study, we examined the effect of acetylsalicylic acid (ASA)on odontogenic differentiation of SCAPs in vitro and in vivo. Methods: SCAPs were cultured under odontogenic conditions supplemented with or without ASA. ASA-treated SCAPs were also subcutaneously transplanted into immunocompromised mice. Results: ASA accelerates in vitro and in vivo odontogenic differentiation of SCAPs associated with down-regulation of runt-related nuclear factor 2 and up-regulation of specificity protein 7, nuclear factor I C, and dentin phosphoprotein. ASA up-regulated the phosphorylation of AKT in the odontogenic SCAPs. Of interest, pretreatments with phosphoinositide 3-kinase inhibitor LY294402 and small interfering RNA for AKT promoted ASA-induced in vitro and in vivo odontogenic differentiation of SCAPs. LY294402 and small interfering RNA for AKT also suppressed the ASA-induced expression of runt-related nuclear factor 2 and enhanced ASA-induced expression of specificity protein 7, nuclear factor I C, and dentin phosphoprotein in SCAPs. Conclusions: These findings suggest that a combination of ASA treatment and suppressive regulation of the phosphoinositide 3-kinase–AKT signaling pathway is a novel approach for SCAP-based tooth root regeneration.

DOI： 10.1016/j.joen.2019.01.016

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Stem cells from human exfoliated deciduous teeth (SHEDs), being a type of mesenchymal stem cell, are an ideal cell source for regenerative medicine. They have minimal risk of oncogenesis, high proliferative capacity, high multipotency, and immunosuppressive ability. Stem cell transplantation using SHED has been found to have an anti-fibrotic effect on liver fibrosis in mice. SHED transplantation and the bio 3D printer, which can create scaffold-free 3-D images of the liver and diaphragm, provide a new innovative treatment modality for intractable pediatric surgical diseases such as biliary atresia and diaphragmatic hernia.

DOI： 10.1007/s00595-019-01783-z

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Regenerative medicine using stem cells from human exfoliated deciduous teeth (SHED): a promising new treatment in pediatric surgery.
Stem cells from human exfoliated deciduous teeth (SHEDs), being a type of mesenchymal stem cell, are an ideal cell source for regenerative medicine. They have minimal risk of oncogenesis, high proliferative capacity, high multipotency, and immunosuppressive ability. Stem cell transplantation using SHED has been found to have an anti-fibrotic effect on liver fibrosis in mice. SHED transplantation and the bio 3D printer, which can create scaffold-free 3-D images of the liver and diaphragm, provide a new innovative treatment modality for intractable pediatric surgical diseases such as biliary atresia and diaphragmatic hernia.

DOI： 10.1007/s00595-019-01783-z

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Nitric oxide (NO) is thought to play a pivotal regulatory role in dental pulp tissues under both physiological and pathological conditions. However, little is known about the NO functions in dental pulp stem cells (DPSCs). We examined the direct actions of a spontaneous NO gas-releasing donor, NOC-18, on the odontogenic capacity of rat DPSCs (rDPSCs). In the presence of NOC-18, rDPSCs were transformed into odontoblast-like cells with long cytoplasmic processes and a polarized nucleus. NOC-18 treatment increased alkaline phosphatase activity and enhanced dentin-like mineralized tissue formation and the expression levels of several odontoblast-specific genes, such as runt related factor 2, dentin matrix protein 1 and dentin sialophosphoprotein, in rDPSCs. In contrast, carboxy-PTIO, a NO scavenger, completely suppressed the odontogenic capacity of rDPSCs. This NO-promoted odontogenic differentiation was activated by tumor necrosis factor-NF-κB axis in rDPSCs. Further in vivo study demonstrated that NOC-18-application in a tooth cavity accelerated tertiary dentin formation, which was associated with early nitrotyrosine expression in the dental pulp tissues beneath the cavity. Taken together, the present findings indicate that exogenous NO directly induces the odontogenic capacity of rDPSCs, suggesting that NO donors might offer a novel host DPSC-targeting alternative to current pulp capping agents in endodontics.

DOI： 10.1038/s41598-018-21183-6

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Suppression of AKT-mTOR signal pathway enhances osteogenic/dentinogenic capacity of stem cells from apical papilla.
BACKGROUND: Stem cells from apical papilla (SCAP) are a subpopulation of mesenchymal stem cells (MSCs) isolated from the apical papilla of the developing tooth root apex of human teeth. Because of their osteogenic/dentinogenic capacity, SCAP are considered as a source for bone and dentin regeneration. However, little is understood about the molecular mechanism of osteogenic/dentinogenic differentiation of SCAP. Phosphoinositide 3 kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) signal pathway participates in regulating the differentiation of various cell types, such as MSCs. In this study, we examined the role of the PI3K-AKT-mTOR signal pathway in the osteogenic/dentinogenic differentiation of SCAP. Moreover, we challenge to fabricate scaffold-free SCAP-based spheroidal calcified constructs. METHODS: SCAP were pretreated with or without small interfering RNA for AKT (AKT siRNA), PI3K inhibitor LY294402, and mTOR inhibitor rapamycin and were cultured under osteogenic/dentinogenic differentiation to examine in vitro and in vivo calcified tissue formation. Moreover, SCAP-based cell aggregates were pretreated with or without LY294402 and rapamycin. The cell aggregates were cultured under osteogenic/dentinogenic condition and were analyzed the calcification of the aggregates. RESULTS: Pretreatment with AKT siRNA, LY294402, and rapamycin enhances the in vitro and in vivo calcified tissue-forming capacity of SCAP. SCAP were fabricated as scaffold-free spheroids and were induced into forming calcified 3D constructs. The calcified density of the spheroidal constructs was enhanced when the spheroids were pretreated with LY294402 and rapamycin. CONCLUSIONS: Our findings indicate that the suppression of PI3K-AKT-mTOR signal pathway plays a role in not only enhancing the in vivo and in vitro osteogenic/dentinogenic differentiation of SCAP, but also promoting the calcification of scaffold-free SCAP-based calcified constructs. These findings suggest that a suppressive regulation of PI3K-AKT-mTOR signal pathway is a novel approach for SCAP-based bone and dentin regeneration.

DOI： 10.1186/s13287-018-1077-9

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Background: Stem cells from apical papilla (SCAP) are a subpopulation of mesenchymal stem cells (MSCs) isolated from the apical papilla of the developing tooth root apex of human teeth. Because of their osteogenic/dentinogenic capacity, SCAP are considered as a source for bone and dentin regeneration. However, little is understood about the molecular mechanism of osteogenic/dentinogenic differentiation of SCAP. Phosphoinositide 3 kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) signal pathway participates in regulating the differentiation of various cell types, such as MSCs. In this study, we examined the role of the PI3K-AKT-mTOR signal pathway in the osteogenic/dentinogenic differentiation of SCAP. Moreover, we challenge to fabricate scaffold-free SCAP-based spheroidal calcified constructs. Methods: SCAP were pretreated with or without small interfering RNA for AKT (AKT siRNA), PI3K inhibitor LY294402, and mTOR inhibitor rapamycin and were cultured under osteogenic/dentinogenic differentiation to examine in vitro and in vivo calcified tissue formation. Moreover, SCAP-based cell aggregates were pretreated with or without LY294402 and rapamycin. The cell aggregates were cultured under osteogenic/dentinogenic condition and were analyzed the calcification of the aggregates. Results: Pretreatment with AKT siRNA, LY294402, and rapamycin enhances the in vitro and in vivo calcified tissue-forming capacity of SCAP. SCAP were fabricated as scaffold-free spheroids and were induced into forming calcified 3D constructs. The calcified density of the spheroidal constructs was enhanced when the spheroids were pretreated with LY294402 and rapamycin. Conclusions: Our findings indicate that the suppression of PI3K-AKT-mTOR signal pathway plays a role in not only enhancing the in vivo and in vitro osteogenic/dentinogenic differentiation of SCAP, but also promoting the calcification of scaffold-free SCAP-based calcified constructs. These findings suggest that a suppressive regulation of PI3K-AKT-mTOR signal pathway is a novel approach for SCAP-based bone and dentin regeneration.

DOI： 10.1186/s13287-018-1077-9

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Background: Hyperbilirubinemia that occurs in pediatric liver diseases such as biliary atresia can result in the development of not only jaundice in the brain, eyes, and skin, but also tooth abnormalities including green pigmentation and dentin hypoplasia in the developing teeth. However, hyperbilirubinemia-induced tooth impairments remain after liver transplantation. No effective dental management to prevent hyperbilirubinemia-induced tooth impairments has been established. Methods: In this study, we focused on pamidronate, which is used to treat pediatric osteopenia, and investigated its effects on hyperbilirubinemia-induced tooth impairments. We cultured stem cells from human exfoliated deciduous teeth (SHED) under high and low concentrations of unconjugated bilirubin in the presence or absence of pamidronate. We then analyzed the effects of pamidronate on the cell death, associated signal pathways, and dentinogenic function in SHED. Results: We demonstrated that a high concentration of unconjugated bilirubin induced cell death in SHED via the mitochondrial pathway, and this was associated with the suppression of AKT and extracellular signal-related kinase 1 and 2 (ERK1/2) signal pathways and activation of the nuclear factor kappa B (NF-κB) signal pathway. The high concentration of unconjugated bilirubin impaired the in vitro and in vivo dentinogenic capacity of SHED, but not the low concentration. We then demonstrated that pamidronate decreased the bilirubin-induced cell death in SHED via the altered AKT, ERK1/2, and NF-κB signal pathways and recovered the bilirubin-impaired dentinogenic function of SHED. Conclusions: Our findings suggest that pamidronate may prevent tooth abnormalities in pediatric patients with hyperbilirubinemia.

DOI： 10.1186/s13287-018-1042-7

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Pamidronate decreases bilirubin-impaired cell death and improves dentinogenic dysfunction of stem cells from human deciduous teeth.
BACKGROUND: Hyperbilirubinemia that occurs in pediatric liver diseases such as biliary atresia can result in the development of not only jaundice in the brain, eyes, and skin, but also tooth abnormalities including green pigmentation and dentin hypoplasia in the developing teeth. However, hyperbilirubinemia-induced tooth impairments remain after liver transplantation. No effective dental management to prevent hyperbilirubinemia-induced tooth impairments has been established. METHODS: In this study, we focused on pamidronate, which is used to treat pediatric osteopenia, and investigated its effects on hyperbilirubinemia-induced tooth impairments. We cultured stem cells from human exfoliated deciduous teeth (SHED) under high and low concentrations of unconjugated bilirubin in the presence or absence of pamidronate. We then analyzed the effects of pamidronate on the cell death, associated signal pathways, and dentinogenic function in SHED. RESULTS: We demonstrated that a high concentration of unconjugated bilirubin induced cell death in SHED via the mitochondrial pathway, and this was associated with the suppression of AKT and extracellular signal-related kinase 1 and 2 (ERK1/2) signal pathways and activation of the nuclear factor kappa B (NF-κB) signal pathway. The high concentration of unconjugated bilirubin impaired the in vitro and in vivo dentinogenic capacity of SHED, but not the low concentration. We then demonstrated that pamidronate decreased the bilirubin-induced cell death in SHED via the altered AKT, ERK1/2, and NF-κB signal pathways and recovered the bilirubin-impaired dentinogenic function of SHED. CONCLUSIONS: Our findings suggest that pamidronate may prevent tooth abnormalities in pediatric patients with hyperbilirubinemia.

DOI： 10.1186/s13287-018-1042-7

記述言語：英語  

記述言語：その他  

Bilirubin reversibly affects cell death and odontogenic capacity in stem cells from human exfoliated deciduous teeth.

DOI： 10.1111/odi.12827

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Objective: Hyperbilirubinemia in patients with biliary atresia causes deciduous tooth injuries such as green pigmentation and dentin hypoplasia. In patients with biliary atresia who received liver transplantation, tooth structure appears to be recovered radiographically. Nevertheless, little is known about cellular mechanisms underlying bilirubin-induced damage and suppression of deciduous tooth formation. In this study, we examined the effects of bilirubin in stem cells from human exfoliated deciduous teeth (SHED) in vitro. Materials and Methods: SHED were cultured under exposure to excess of bilirubin and then interruption of bilirubin stimulation. Results: Bilirubin induced cell death and inhibited the odontogenic capacity of SHED by suppressing AKT and extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathways and enhancing nuclear factor kappa B p65 (NF-κB p65) pathway. The interruption of bilirubin stimulation reduced cell death and recovered the inhibited odontogenic capacity of bilirubin-damaged SHED. The bilirubin interruption also normalized the impaired AKT, ERK1/2, and NF-κB p65 signaling pathways. Conclusion: These findings suggest that tooth hypodontia in patients with hyperbilirubinemia might be due to bilirubin-induced cell death and dentinogenic dysfunction of odontogenic stem cells via AKT, ERK1/2, and NF-κB pathways and also suggested that bilirubin-induced impairments in odontogenic stem cells were reversible when bilirubin stimulation is interrupted.

DOI： 10.1111/odi.12827

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.33425/2639-9490.1018

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：日本語  

外科学の新知見(3)再生医療の最前線 ヒト脱落乳歯歯髄幹細胞を用いた腸管神経節細胞僅少症に対する腸管神経再生医療

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Exogenous nitric oxide stimulates the odontogenic differentiation of rat dental pulp stem cells.
Nitric oxide (NO) is thought to play a pivotal regulatory role in dental pulp tissues under both physiological and pathological conditions. However, little is known about the NO functions in dental pulp stem cells (DPSCs). We examined the direct actions of a spontaneous NO gas-releasing donor, NOC-18, on the odontogenic capacity of rat DPSCs (rDPSCs). In the presence of NOC-18, rDPSCs were transformed into odontoblast-like cells with long cytoplasmic processes and a polarized nucleus. NOC-18 treatment increased alkaline phosphatase activity and enhanced dentin-like mineralized tissue formation and the expression levels of several odontoblast-specific genes, such as runt related factor 2, dentin matrix protein 1 and dentin sialophosphoprotein, in rDPSCs. In contrast, carboxy-PTIO, a NO scavenger, completely suppressed the odontogenic capacity of rDPSCs. This NO-promoted odontogenic differentiation was activated by tumor necrosis factor-NF-κB axis in rDPSCs. Further in vivo study demonstrated that NOC-18-application in a tooth cavity accelerated tertiary dentin formation, which was associated with early nitrotyrosine expression in the dental pulp tissues beneath the cavity. Taken together, the present findings indicate that exogenous NO directly induces the odontogenic capacity of rDPSCs, suggesting that NO donors might offer a novel host DPSC-targeting alternative to current pulp capping agents in endodontics.

DOI： 10.1038/s41598-018-21183-6

記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: Multipotent mesenchymal stem cells (MSCs) are used clinically in regenerative medicine. Our previous report showed systemically injected MSCs improved peri-implant sealing and accelerated tissue healing. However, the risks of systemic MSC administration, including lung embolism, must be considered; therefore, their local application must be assessed for clinical safety and efficacy. We investigated differences in treatment effect between local and systemic MSC application using a rat oral implantation model. METHODS: Rat bone marrow-derived MSCs were isolated and culture-expanded. The rat's right maxillary first molars were extracted and replaced with experimental titanium implants. After 24 h, MSCs (1 × 106/ml) were systemically or locally injected into recipient rats via the tail vein (systemic group) or buccal subcutaneous tissue (local group), respectively. Rats treated in the absence of MSCs were included as a control (control group). The maxillary epithelium was assessed histologically after 4 weeks to evaluate laminin-332 (Ln-332) distribution and horseradish peroxidase invasion, as indicators of peri-implant epithelium (PIE) formation and PIE sealing to the implant surface, respectively. The effect of MSCs on rat oral epithelial cell (OEC) morphology was determined by coculture. RESULTS: Systemic group MSCs accumulated early at the peri-implant mucosa, while local group MSCs were observed in various organs prior to later accumulation around the implant surface. PIE formation and Ln-332-positive staining at the implant interface were enhanced in the systemic group compared with the local and control groups. Furthermore, OEC adherence on implants was reduced in high-density compared with low-density MSC cocultures. CONCLUSIONS: Local MSC injection was more ineffective than systemic MSC injection at enhancing PIE sealing around titanium implants. Thus, although local MSC administration has a wide range of applications, further investigations are needed to understand the exact cellular and molecular mechanisms of this approach prior to clinical use.

DOI： 10.1186/s40729-017-0112-4

記述言語：英語   掲載種別：研究論文（学術雑誌）  

As osteoclasts have the central roles in normal bone remodeling, it is ideal to regulate only the osteoclasts performing pathological bone destruction without affecting normal osteoclasts. Based on a hypothesis that pathological osteoclasts form under the pathological microenvironment of the bone tissues, we here set up optimum culture conditions to examine the entity of pathologically activated osteoclasts (PAOCs). Through searching various inflammatory cytokines and their combinations, we found the highest resorbing activity of osteoclasts when osteoclasts were formed in the presence of M-CSF, receptor activator of NF-κB ligand, and IL-1β. We have postulated that these osteoclasts are PAOCs. Analysis using confocal laser microscopy revealed that PAOCs showed extremely high proton secretion detected by the acid-sensitive fluorescence probe Rh-PM and bone resorption activity compared with normal osteoclasts. PAOCs showed unique morphology bearing high thickness and high motility with motile cellular processes in comparison with normal osteoclasts. We further examined the expression of Kindlin-3 and Talin-1, essential molecules for activating integrin b-chains. Although normal osteoclasts express high levels of Kindlin-3 and Talin-1, expression of these molecules was markedly suppressed in PAOCs, suggesting the abnormality in the adhesion property. When whole membrane surface of mature osteoclasts was biotinylated and analyzed, the IL-1β-induced cell surface protein was detected. PAOCs could form a subpopulation of osteo-clasts possibly different from normal osteoclasts. PAOC-specific molecules could be an ideal target for regulating pathological bone destruction.

DOI： 10.4049/jimmunol.1602035

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

IL-1β Induces Pathologically Activated Osteoclasts Bearing Extremely High Levels of Resorbing Activity: A Possible Pathological Subpopulation of Osteoclasts, Accompanied by Suppressed Expression of Kindlin-3 and Talin-1.
As osteoclasts have the central roles in normal bone remodeling, it is ideal to regulate only the osteoclasts performing pathological bone destruction without affecting normal osteoclasts. Based on a hypothesis that pathological osteoclasts form under the pathological microenvironment of the bone tissues, we here set up optimum culture conditions to examine the entity of pathologically activated osteoclasts (PAOCs). Through searching various inflammatory cytokines and their combinations, we found the highest resorbing activity of osteoclasts when osteoclasts were formed in the presence of M-CSF, receptor activator of NF-κB ligand, and IL-1β. We have postulated that these osteoclasts are PAOCs. Analysis using confocal laser microscopy revealed that PAOCs showed extremely high proton secretion detected by the acid-sensitive fluorescence probe Rh-PM and bone resorption activity compared with normal osteoclasts. PAOCs showed unique morphology bearing high thickness and high motility with motile cellular processes in comparison with normal osteoclasts. We further examined the expression of Kindlin-3 and Talin-1, essential molecules for activating integrin β-chains. Although normal osteoclasts express high levels of Kindlin-3 and Talin-1, expression of these molecules was markedly suppressed in PAOCs, suggesting the abnormality in the adhesion property. When whole membrane surface of mature osteoclasts was biotinylated and analyzed, the IL-1β-induced cell surface protein was detected. PAOCs could form a subpopulation of osteoclasts possibly different from normal osteoclasts. PAOC-specific molecules could be an ideal target for regulating pathological bone destruction.

DOI： 10.4049/jimmunol.1602035

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cell-based therapy has been proposed as an alternative to orthotopic liver transplantation. The novel transplantation of an in vitro-generated liver bud might have therapeutic potential. In vivo and ex vivo methods for growing a liver bud are essential for paving the way for the clinical translation of liver bud transplantation. We herein report a novel transplantation method for liver buds that are grown in vivo involving orthotopic transplantation on the transected parenchyma of the liver, which showed long engraftment and marked growth in comparison to heterotopic transplantation. Furthermore, this study demonstrates a method for rapidly fabricating scalable liver-like tissue by fusing hundreds of liver bud-like spheroids using a 3D bioprinter. Its system to fix the shape of the 3D tissue with the needle-array system enabled the fabrication of elaborate geometry and the immediate execution of culture circulation after 3D printing-thereby avoiding an ischemic environment ex vivo. The ex vivo-fabricated human liver-like tissue exhibited self-tissue organization ex vivo and engraftment on the liver of nude rats. These achievements conclusively show both in vivo and ex vivo methods for growing in vitro-generated liver buds. These methods provide a new approach for in vitro-generated liver organoids transplantation.

DOI： 10.1038/s41598-017-14542-2

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Laminin-332 (Lm-332), a major basement membrane protein, has been shown to provide a niche for some stem cells. Here, we found that Lm-332 was expressed in osteoblasts, and is implicated in the regulation of osteoclast differentiation. Immunofluorescence analysis of laminin-β3, a unique component of Lm-332, indicated specific expression of laminin-β3 in osteoblast-like cells localized on bone surface. RT-PCR analysis confirmed that α3, β3, and 2 chains of Lm-332 were all expressed in primary osteoblasts prepared from mouse calvaria. Lm-332 markedly inhibited osteoclastogenesis induced by receptor activator of nuclear factor kappa B (NF-B) ligand (RANKL) when bone marrow-derived macrophages (BMMs) were cultured on Lm-332-coated plates. Lm-332 also blocked RANKL-induced activation of mitogen-activated protein kinases (MAPKs) (ERK, JNK, and p38) and expression of NFATc1, c-Fos, and c-Jun. Lm-332 suppressed osteoclast differentiation while retaining macrophage phenotypes, including nonspecific esterase activity and gene expression of lysozyme and EGF-like module-containing mucin-like hormone receptor-like 1 (Emr1). Furthermore, the treatment of primary osteoblasts with osteoclastogenic factors dramatically suppressed expression of Lm-332. These findings suggest that Lm-332 produced by osteoblasts in bone tissues has a pivotal role in controlling normal bone remodeling through suppressing osteoclastogenesis.

DOI： 10.1038/labinvest.2017.55

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Laminin-332 (Lm-332), a major basement membrane protein, has been shown to provide a niche for some stem cells. Here, we found that Lm-332 was expressed in osteoblasts, and is implicated in the regulation of osteoclast differentiation. Immunofluorescence analysis of laminin-β3, a unique component of Lm-332, indicated specific expression of laminin-β3 in osteoblast-like cells localized on bone surface. RT-PCR analysis confirmed that α3, β3, and γ2 chains of Lm-332 were all expressed in primary osteoblasts prepared from mouse calvaria. Lm-332 markedly inhibited osteoclastogenesis induced by receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) when bone marrow-derived macrophages (BMMs) were cultured on Lm-332-coated plates. Lm-332 also blocked RANKL-induced activation of mitogen-activated protein kinases (MAPKs) (ERK, JNK, and p38) and expression of NFATc1, c-Fos, and c-Jun. Lm-332 suppressed osteoclast differentiation while retaining macrophage phenotypes, including nonspecific esterase activity and gene expression of lysozyme and EGF-like module-containing mucin-like hormone receptor-like 1 (Emr1). Furthermore, the treatment of primary osteoblasts with osteoclastogenic factors dramatically suppressed expression of Lm-332. These findings suggest that Lm-332 produced by osteoblasts in bone tissues has a pivotal role in controlling normal bone remodeling through suppressing osteoclastogenesis.

DOI： 10.1038/labinvest.2017.55

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cell-based therapy has been proposed as an alternative to orthotopic liver transplantation. The novel transplantation of an in vitro-generated liver bud might have therapeutic potential. In vivo and ex vivo methods for growing a liver bud are essential for paving the way for the clinical translation of liver bud transplantation. We herein report a novel transplantation method for liver buds that are grown in vivo involving orthotopic transplantation on the transected parenchyma of the liver, which showed long engraftment and marked growth in comparison to heterotopic transplantation. Furthermore, this study demonstrates a method for rapidly fabricating scalable liver-like tissue by fusing hundreds of liver bud-like spheroids using a 3D bioprinter. Its system to fix the shape of the 3D tissue with the needle-array system enabled the fabrication of elaborate geometry and the immediate execution of culture circulation after 3D printing-thereby avoiding an ischemic environment ex vivo. The ex vivo-fabricated human liver-like tissue exhibited self-tissue organization ex vivo and engraftment on the liver of nude rats. These achievements conclusively show both in vivo and ex vivo methods for growing in vitro-generated liver buds. These methods provide a new approach for in vitro-generated liver organoids transplantation.

DOI： 10.1038/s41598-017-14542-2

記述言語：日本語  

記述言語：日本語  

記述言語：日本語  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Background: Mesenchymal stem cells (MSCs) have been isolated from a variety of tissues, including bone marrow, adipose, and mucosa. MSCs have the capacity for self-renewal and differentiation. Reports have been published on the systemic administration of MSCs leading to functional improvements by engraftment and differentiation, thus providing a new strategy to regenerate damaged tissues. Recently, it has become clear that MSCs possess immunomodulatory properties and can therefore be used to treat diseases. However, the therapeutic effect mechanisms of MSCs are yet to be determined. Here, we investigated these mechanisms using a medication-related osteonecrosis of the jaw (MRONJ)-like mouse model. Methods: To generate MRONJ-like characteristics, mice received intravenous zoledronate and dexamethasone two times a week. At 1 week after intravenous injection, maxillary first molars were extracted, and at 1 week after tooth extraction, MSCs were isolated from the bone marrow of the mice femurs and tibias. To compare "diseased MSCs" from MRONJ-like mice (d-MSCs) with "control MSCs" from untreated mice (c-MSCs), the isolated MSCs were analyzed by differentiation and colony-forming unit-fibroblast (CFU-F) assays and systemic transplantation of either d-MSCs or c-MSCs into MRONJ-like mice. Furthermore, we observed the exchange of cell contents among d-MSCs and c-MSCs during coculture with all combinations of each MSC type. Results: d-MSCs were inferior to c-MSCs in differentiation and CFU-F assays. Moreover, the d-MSC-treated group did not show earlier healing in MRONJ-like mice. In cocultures with any combination, MSC pairs formed cell-cell contacts and exchanged cell contents. Interestingly, the exchange among c-MSCs and d-MSCs was more frequently observed than other pairs, and d-MSCs were distinguishable from c-MSCs. Conclusions: The interaction of c-MSCs and d-MSCs, including exchange of cell contents, contributes to the treatment potential of d-MSCs. This cellular behavior might be one therapeutic mechanism used by MSCs for MRONJ.

DOI： 10.1186/s13287-016-0367-3

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Therapeutic interactions between mesenchymal stem cells for healing medication-related osteonecrosis of the jaw.
BACKGROUND: Mesenchymal stem cells (MSCs) have been isolated from a variety of tissues, including bone marrow, adipose, and mucosa. MSCs have the capacity for self-renewal and differentiation. Reports have been published on the systemic administration of MSCs leading to functional improvements by engraftment and differentiation, thus providing a new strategy to regenerate damaged tissues. Recently, it has become clear that MSCs possess immunomodulatory properties and can therefore be used to treat diseases. However, the therapeutic effect mechanisms of MSCs are yet to be determined. Here, we investigated these mechanisms using a medication-related osteonecrosis of the jaw (MRONJ)-like mouse model. METHODS: To generate MRONJ-like characteristics, mice received intravenous zoledronate and dexamethasone two times a week. At 1 week after intravenous injection, maxillary first molars were extracted, and at 1 week after tooth extraction, MSCs were isolated from the bone marrow of the mice femurs and tibias. To compare "diseased MSCs" from MRONJ-like mice (d-MSCs) with "control MSCs" from untreated mice (c-MSCs), the isolated MSCs were analyzed by differentiation and colony-forming unit-fibroblast (CFU-F) assays and systemic transplantation of either d-MSCs or c-MSCs into MRONJ-like mice. Furthermore, we observed the exchange of cell contents among d-MSCs and c-MSCs during coculture with all combinations of each MSC type. RESULTS: d-MSCs were inferior to c-MSCs in differentiation and CFU-F assays. Moreover, the d-MSC-treated group did not show earlier healing in MRONJ-like mice. In cocultures with any combination, MSC pairs formed cell-cell contacts and exchanged cell contents. Interestingly, the exchange among c-MSCs and d-MSCs was more frequently observed than other pairs, and d-MSCs were distinguishable from c-MSCs. CONCLUSIONS: The interaction of c-MSCs and d-MSCs, including exchange of cell contents, contributes to the treatment potential of d-MSCs. This cellular behavior might be one therapeutic mechanism used by MSCs for MRONJ.

DOI： 10.1186/s13287-016-0367-3

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Clinically, irreversible pulpitis is treated by the complete removal of pulp tissue followed by replacement with artificial materials. There is considered to be a high potential for autologous transplantation of human dental pulp stem cells (DPSCs) in endodontic treatment. The usefulness of DPSCs isolated from healthy teeth is limited. However, DPSCs isolated from diseased teeth with irreversible pulpitis (IP-DPSCs) are considered to be suitable for dentin/pulp regeneration. In this study, we examined the stem cell potency of IP-DPSCs. In comparison with healthy DPSCs, IP-DPSCs expressed lower colony-forming capacity, population-doubling rate, cell proliferation, multipotency, in vivo dentin regeneration, and immunosuppressive activity, suggesting that intact IP-DPSCs may be inadequate for dentin/pulp regeneration. Therefore, we attempted to improve the impaired in vivo dentin regeneration and in vitro immunosuppressive functions of IP-DPSCs to enable dentin/pulp regeneration. Interferon gamma (IFN-γ) treatment enhanced in vivo dentin regeneration and in vitro T cell suppression of IP-DPSCs, whereas treatment with tumor necrosis factor alpha did not. Therefore, these findings suggest that IFN-γ may be a feasible modulator to improve the functions of impaired IP-DPSCs, suggesting that autologous transplantation of IFN-γ-accelerated IP-DPSCs might be a promising new therapeutic strategy for dentin/pulp tissue engineering in future endodontic treatment.

DOI： 10.1038/srep19286

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Clinically, irreversible pulpitis is treated by the complete removal of pulp tissue followed by replacement with artificial materials. There is considered to be a high potential for autologous transplantation of human dental pulp stem cells (DPSCs) in endodontic treatment. The usefulness of DPSCs isolated from healthy teeth is limited. However, DPSCs isolated from diseased teeth with irreversible pulpitis (IP-DPSCs) are considered to be suitable for dentin/pulp regeneration. In this study, we examined the stem cell potency of IP-DPSCs. In comparison with healthy DPSCs, IP-DPSCs expressed lower colony-forming capacity, population-doubling rate, cell proliferation, multipotency, in vivo dentin regeneration, and immunosuppressive activity, suggesting that intact IP-DPSCs may be inadequate for dentin/pulp regeneration. Therefore, we attempted to improve the impaired in vivo dentin regeneration and in vitro immunosuppressive functions of IP-DPSCs to enable dentin/pulp regeneration. Interferon gamma (IFN-γ) treatment enhanced in vivo dentin regeneration and in vitro T cell suppression of IP-DPSCs, whereas treatment with tumor necrosis factor alpha did not. Therefore, these findings suggest that IFN-γ may be a feasible modulator to improve the functions of impaired IP-DPSCs, suggesting that autologous transplantation of IFN-γ-accelerated IP-DPSCs might be a promising new therapeutic strategy for dentin/pulp tissue engineering in future endodontic treatment.

DOI： 10.1038/srep19286

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Introduction: Liver transplantation is a gold standard treatment for intractable liver diseases. Because of the shortage of donor organs, alternative therapies have been required. Due to their potential to differentiate into a variety of mature cells, stem cells are considered feasible cell sources for liver regeneration. Stem cells from human exfoliated deciduous teeth (SHED) exhibit hepatogenic capability in vitro. In this study, we investigated their in vivo capabilities of homing and hepatocyte differentiation and therapeutic efficacy for liver disorders in carbon tetrachloride (CCl₄)-induced liver fibrosis model mice. Methods: We transplanted SHED into CCl₄-induced liver fibrosis model mice through the spleen, and analyzed the in vivo homing and therapeutic effects by optical, biochemical, histological, immunological and molecular biological assays. We then sorted human leukocyte antigen-ABC (HLA-ABC)-positive cells from primary CCl4-damaged recipient livers, and analyzed their fusogenicity and hepatic characteristics by flow cytometric, genomic DNA, hepatocyte-specific gene assays. Furthermore, we examined the treatment effects of HLA-positive cells to a hepatic dysfunction by a secondary transplantation into CCl₄-treated mice. Results: Transplanted SHED homed to recipient livers, and expressed HLA-ABC, human hepatocyte specific antigen hepatocyte paraffin 1 and human albumin. SHED transplantation markedly recovered liver dysfunction and led to anti-fibrotic and anti-inflammatory effects in the recipient livers. SHED-derived HLA-ABC-positive cells that were sorted from the primary recipient liver tissues with CCl₄ damage did not fuse with the host mouse liver cells. Sorted HLA-positive cells not only expressed human hepatocyte-specific genes including albumin, cytochrome P450 1A1, fumarylacetoacetase, tyrosine aminotransferase, uridine 5′-diphospho-glucuronosyltransferase, transferrin and transthyretin, but also secreted human albumin, urea and blood urea nitrogen. Furthermore, SHED-derived HLA-ABC-positive cells were secondary transplanted into CCl₄-treated mice. The donor cells homed into secondary recipient livers, and expressed hepatocyte paraffin 1 and human albumin, as well as HLA-ABC. The secondary transplantation recovered a liver dysfunction in secondary recipients. Conclusions: This study indicates that transplanted SHED improve hepatic dysfunction and directly transform into hepatocytes without cell fusion in CCl₄-treated mice, suggesting that SHED may provide a feasible cell source for liver regeneration.

DOI： 10.1186/s13287-015-0154-6

記述言語：英語   掲載種別：研究論文（学術雑誌）  

In vivo hepatogenic capacity and therapeutic potential of stem cells from human exfoliated deciduous teeth in liver fibrosis in mice.
INTRODUCTION: Liver transplantation is a gold standard treatment for intractable liver diseases. Because of the shortage of donor organs, alternative therapies have been required. Due to their potential to differentiate into a variety of mature cells, stem cells are considered feasible cell sources for liver regeneration. Stem cells from human exfoliated deciduous teeth (SHED) exhibit hepatogenic capability in vitro. In this study, we investigated their in vivo capabilities of homing and hepatocyte differentiation and therapeutic efficacy for liver disorders in carbon tetrachloride (CCl4)-induced liver fibrosis model mice. METHODS: We transplanted SHED into CCl4-induced liver fibrosis model mice through the spleen, and analyzed the in vivo homing and therapeutic effects by optical, biochemical, histological, immunological and molecular biological assays. We then sorted human leukocyte antigen-ABC (HLA-ABC)-positive cells from primary CCl4-damaged recipient livers, and analyzed their fusogenicity and hepatic characteristics by flow cytometric, genomic DNA, hepatocyte-specific gene assays. Furthermore, we examined the treatment effects of HLA-positive cells to a hepatic dysfunction by a secondary transplantation into CCl4-treated mice. RESULTS: Transplanted SHED homed to recipient livers, and expressed HLA-ABC, human hepatocyte specific antigen hepatocyte paraffin 1 and human albumin. SHED transplantation markedly recovered liver dysfunction and led to anti-fibrotic and anti-inflammatory effects in the recipient livers. SHED-derived HLA-ABC-positive cells that were sorted from the primary recipient liver tissues with CCl4 damage did not fuse with the host mouse liver cells. Sorted HLA-positive cells not only expressed human hepatocyte-specific genes including albumin, cytochrome P450 1A1, fumarylacetoacetase, tyrosine aminotransferase, uridine 5'-diphospho-glucuronosyltransferase, transferrin and transthyretin, but also secreted human albumin, urea and blood urea nitrogen. Furthermore, SHED-derived HLA-ABC-positive cells were secondary transplanted into CCl4-treated mice. The donor cells homed into secondary recipient livers, and expressed hepatocyte paraffin 1 and human albumin, as well as HLA-ABC. The secondary transplantation recovered a liver dysfunction in secondary recipients. CONCLUSIONS: This study indicates that transplanted SHED improve hepatic dysfunction and directly transform into hepatocytes without cell fusion in CCl4-treated mice, suggesting that SHED may provide a feasible cell source for liver regeneration.

DOI： 10.1186/s13287-015-0154-6

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.stem.2015.04.016

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Transplantation of mesenchymal stem cells ameliorates secondary osteoporosis through interleukin-17-impaired functions of recipient bone marrow mesenchymal stem cells in MRL/lpr mice.
INTRODUCTION: Secondary osteoporosis is common in systemic lupus erythematosus and leads to a reduction in quality of life due to fragility fractures, even in patients with improvement of the primary disorder. Systemic transplantation of mesenchymal stem cells could ameliorate bone loss and autoimmune disorders in a MRL/lpr mouse systemic lupus erythematosus model, but the detailed therapeutic mechanism of bone regeneration is not fully understood. In this study, we transplanted human bone marrow mesenchymal stem cells (BMMSCs) and stem cells from exfoliated deciduous teeth (SHED) into MRL/lpr mice and explored their therapeutic mechanisms in secondary osteoporotic disorders of the systemic lupus erythematosus model mice. METHODS: The effects of systemic human mesenchymal stem cell transplantation on bone loss of MRL/lpr mice were analyzed in vivo and ex vivo. After systemic human mesenchymal stem cell transplantation, recipient BMMSC functions of MRL/lpr mice were assessed for aspects of stemness, osteogenesis and osteoclastogenesis, and a series of co-culture experiments under osteogenic or osteoclastogenic inductions were performed to examine the efficacy of interleukin (IL)-17-impaired recipient BMMSCs in the bone marrow of MRL/lpr mice. RESULTS: Systemic transplantation of human BMMSCs and SHED recovered the reduction in bone density and structure in MRL/lpr mice. To explore the mechanism, we found that impaired recipient BMMSCs mediated the negative bone metabolic turnover by enhanced osteoclastogenesis and suppressed osteoblastogenesis in secondary osteoporosis of MRL/lpr mice. Moreover, IL-17-dependent hyperimmune conditions in the recipient bone marrow of MRL/lpr mice damaged recipient BMMSCs to suppress osteoblast capacity and accelerate osteoclast induction. To overcome the abnormal bone metabolism, systemic transplantation of human BMMSCs and SHED into MRL/lpr mice improved the functionally impaired recipient BMMSCs through IL-17 suppression in the recipient bone marrow and then maintained a regular positive bone metabolism via the balance of osteoblasts and osteoclasts. CONCLUSIONS: These findings indicate that IL-17 and recipient BMMSCs might be a therapeutic target for secondary osteoporosis in systemic lupus erythematosus.

DOI： 10.1186/s13287-015-0091-4

記述言語：英語  

DOI： 10.1016/j.stem.2015.04.016

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Introduction: Secondary osteoporosis is common in systemic lupus erythematosus and leads to a reduction in quality of life due to fragility fractures, even in patients with improvement of the primary disorder. Systemic transplantation of mesenchymal stem cells could ameliorate bone loss and autoimmune disorders in a MRL/lpr mouse systemic lupus erythematosus model, but the detailed therapeutic mechanism of bone regeneration is not fully understood. In this study, we transplanted human bone marrow mesenchymal stem cells (BMMSCs) and stem cells from exfoliated deciduous teeth (SHED) into MRL/lpr mice and explored their therapeutic mechanisms in secondary osteoporotic disorders of the systemic lupus erythematosus model mice. Methods: The effects of systemic human mesenchymal stem cell transplantation on bone loss of MRL/lpr mice were analyzed in vivo and ex vivo. After systemic human mesenchymal stem cell transplantation, recipient BMMSC functions of MRL/lpr mice were assessed for aspects of stemness, osteogenesis and osteoclastogenesis, and a series of co-culture experiments under osteogenic or osteoclastogenic inductions were performed to examine the efficacy of interleukin (IL)-17-impaired recipient BMMSCs in the bone marrow of MRL/lpr mice. Results: Systemic transplantation of human BMMSCs and SHED recovered the reduction in bone density and structure in MRL/lpr mice. To explore the mechanism, we found that impaired recipient BMMSCs mediated the negative bone metabolic turnover by enhanced osteoclastogenesis and suppressed osteoblastogenesis in secondary osteoporosis of MRL/lpr mice. Moreover, IL-17-dependent hyperimmune conditions in the recipient bone marrow of MRL/lpr mice damaged recipient BMMSCs to suppress osteoblast capacity and accelerate osteoclast induction. To overcome the abnormal bone metabolism, systemic transplantation of human BMMSCs and SHED into MRL/lpr mice improved the functionally impaired recipient BMMSCs through IL-17 suppression in the recipient bone marrow and then maintained a regular positive bone metabolism via the balance of osteoblasts and osteoclasts. Conclusions: These findings indicate that IL-17 and recipient BMMSCs might be a therapeutic target for secondary osteoporosis in systemic lupus erythematosus.

DOI： 10.1186/s13287-015-0091-4

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Purpose: The aim of this study was to evaluate the efficacy of mesenchymal stem cells (MSCs) in a nitrofen-induced congenital diaphragmatic hernia (CDH) rat model. Methods: Pregnant rats were exposed to nitrofen on embryonic day 9.5 (E9.5). MSCs were isolated from the enhanced green fluorescent protein (eGFP) transgenic rat lungs. The MSCs were transplanted into the nitrofen-induced E12.5 rats via the uterine vein, and the E21 lung explants were harvested. The study animals were divided into three: the control group, the nitrofen-induced left CDH (CDH group), and the MSC-treated nitrofen-induced left CDH (MSC-treated CDH group). The specimens were morphologically analyzed using HE and immunohistochemical staining with proliferating cell nuclear antigen (PCNA), surfactant protein-C (SP-C), and α-smooth muscle actin. Results: The alveolar and medial walls of the pulmonary arteries were significantly thinner in the MSC-treated CDH group than in the CDH group. The alveolar air space areas were larger, while PCNA and the SP-C positive cells were significantly higher in the MSC-treated CDH group, than in the CDH group. MSC engraftment was identified on immunohistochemical staining of the GFP in the MSC-treated CDH group. Conclusions: MSC transplantation potentially promotes alveolar and pulmonary artery development, thereby reducing the severity of pulmonary hypoplasia.

DOI： 10.1007/s00383-014-3576-9

記述言語：日本語  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

PURPOSE: The aim of this study was to evaluate the efficacy of mesenchymal stem cells (MSCs) in a nitrofen-induced congenital diaphragmatic hernia (CDH) rat model. METHODS: Pregnant rats were exposed to nitrofen on embryonic day 9.5 (E9.5). MSCs were isolated from the enhanced green fluorescent protein (eGFP) transgenic rat lungs. The MSCs were transplanted into the nitrofen-induced E12.5 rats via the uterine vein, and the E21 lung explants were harvested. The study animals were divided into three: the control group, the nitrofen-induced left CDH (CDH group), and the MSC-treated nitrofen-induced left CDH (MSC-treated CDH group). The specimens were morphologically analyzed using HE and immunohistochemical staining with proliferating cell nuclear antigen (PCNA), surfactant protein-C (SP-C), and α-smooth muscle actin. RESULTS: The alveolar and medial walls of the pulmonary arteries were significantly thinner in the MSC-treated CDH group than in the CDH group. The alveolar air space areas were larger, while PCNA and the SP-C positive cells were significantly higher in the MSC-treated CDH group, than in the CDH group. MSC engraftment was identified on immunohistochemical staining of the GFP in the MSC-treated CDH group. CONCLUSIONS: MSC transplantation potentially promotes alveolar and pulmonary artery development, thereby reducing the severity of pulmonary hypoplasia.

DOI： 10.1007/s00383-014-3576-9

記述言語：日本語  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Mesenchymal stem cells (MSCs) have potential to differentiate into multiple cell lineages. Recently, it was shown that MSCs also have anti-inflammatory and immunomodulatory functions. In this report, we investigated the regulatory function of MSCs in the development of inflammatory bone destruction in rats with adjuvant-induced arthritis (AA rats). MSCs were isolated from rat bone marrow tissues, expanded in the presence of basic FGF, and intraperitoneally injected into AA rats. MSC administration significantly suppressed inflammatory parameters: swelling score, swelling width, and thickness of hind paw. Radiographic evaluation indicated that MSC significantly suppressed bone destruction. Histological analysis showed that administration of MSCs markedly suppressed osteoclastogenesis in AA rats. To further delineate their effects on osteoclastogenesis, MSCs were added to in vitro bone marrow cultures undergoing osteoclastogenesis. MSCs significantly suppressed osteoclastogenesis in this system. Chemokine receptor expression in MSCs was assessed by RT-PCR, and a chemotactic assay was performed using a transwell culture system. MSCs showed significant chemotaxis to MIP-1α (CCL3) and SDF-1α (CXCL12), chemokines preferentially expressed in the area of inflammatory bone destruction. Furthermore, MSCs expressed IL-10 and osteoprotegerin, cytokines that suppress osteoclastogenesis. These data suggest that recruitment of MSC to the area of bone destruction in AA rats could suppress inflammatory bone destruction and raise the possibility that MSCs may have potential for the treatment of inflammatory bone destruction in arthritis.

DOI： 10.1038/labinvest.2013.152

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Bone marrow mesenchymal stem cells (BMMSCs) are capable of differentiating into multiple cell types and regulating immune cell response. However, the mechanisms that govern the immunomodulatory properties of BMMSCs are still not fully elucidated. Here we show that telomerase-deficient BMMSCs lose their capacity to inhibit T cells and ameliorate the disease phenotype in systemic sclerosis mice. Restoration of telomerase activity by telomerase reverse transcriptase (TERT) transfection in TERT(-/-) BMMSCs rescues their immunomodulatory functions. Mechanistically, we reveal that TERT, combined with β-catenin and BRG1, serves as a transcriptional complex, which binds the FAS ligand (FASL) promoter to upregulate FASL expression, leading to an elevated immunomodulatory function. To test the translational value of these findings in the context of potential clinical therapy, we used aspirin treatment to upregulate telomerase activity in BMMSCs, and found a significant improvement in the immunomodulatory capacity of BMMSCs. Taken together, these findings identify a previously unrecognized role of TERT in improving the immunomodulatory capacity of BMMSCs, suggesting that aspirin treatment is a practical approach to significantly reduce cell dosage in BMMSC-based immunotherapies.

DOI： 10.1002/emmm.201303000

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Mesenchymal stem cells (MSCs) have potential to differentiate into multiple cell lineages. Recently, it was shown that MSCs also have anti-inflammatory and immunomodulatory functions. In this report, we investigated the regulatory function of MSCs in the development of inflammatory bone destruction in rats with adjuvant-induced arthritis (AA rats). MSCs were isolated from rat bone marrow tissues, expanded in the presence of basic FGF, and intraperitoneally injected into AA rats. MSC administration significantly suppressed inflammatory parameters: swelling score, swelling width, and thickness of hind paw. Radiographic evaluation indicated that MSC significantly suppressed bone destruction. Histological analysis showed that administration of MSCs markedly suppressed osteoclastogenesis in AA rats. To further delineate their effects on osteoclastogenesis, MSCs were added to in vitro bone marrow cultures undergoing osteoclastogenesis. MSCs significantly suppressed osteoclastogenesis in this system. Chemokine receptor expression in MSCs was assessed by RT-PCR, and a chemotactic assay was performed using a transwell culture system. MSCs showed significant chemotaxis to MIP-1 alpha (CCL3) and SDF-1 alpha (CXCL12), chemokines preferentially expressed in the area of inflammatory bone destruction. Furthermore, MSCs expressed IL-10 and osteoprotegerin, cytokines that suppress osteoclastogenesis. These data suggest that recruitment of MSC to the area of bone destruction in AA rats could suppress inflammatory bone destruction and raise the possibility that MSCs may have potential for the treatment of inflammatory bone destruction in arthritis.

DOI： 10.1038/labinvest.2013.152

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Objectives: The objective of this study was to investigate the effect of systemically transplanted mesenchymal stem cells (MSCs) on the peri-implant epithelial sealing around dental implants. Materials and Methods: MSCs were isolated from bone marrow of donor rats and expanded in culture. After recipient rats received experimental titanium dental implants in the bone sockets after extraction of maxillary right first molars, donor rat MSCs were intravenously transplanted into the recipient rats. Results: The injected MSCs were found in the oral mucosa surrounding the dental implants at 24 hours post- transplantation. MSC transplantation accelerated the formation of the peri-implant epithelium (PIE)-mediated mucosa sealing around the implants at an early stage after implantation. Subsequently, enhanced deposition of laminin-332 was found along the PIE-implant interface at 4 weeks after the replacement. We also observed enhanced attachment and proliferation of oral mucous epithelial cells. Conclusion: Systemically transplanted MSCs might play a critical role in reinforcing the epithelial sealing around dental implants.

DOI： 10.1371/journal.pone.0090681

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Bone marrow mesenchymal stem cells (BMMSCs) are capable of differentiating into multiple cell types and regulating immune cell response. However, the mechanisms that govern the immunomodulatory properties of BMMSCs are still not fully elucidated. Here we show that telomerase-deficient BMMSCs lose their capacity to inhibit T cells and ameliorate the disease phenotype in systemic sclerosis mice. Restoration of telomerase activity by telomerase reverse transcriptase (TERT) transfection in TERT^-/- BMMSCs rescues their immunomodulatory functions. Mechanistically, we reveal that TERT, combined with β-catenin and BRG1, serves as a transcriptional complex, which binds the FAS ligand (FASL) promoter to upregulate FASL expression, leading to an elevated immunomodulatory function. To test the translational value of these findings in the context of potential clinical therapy, we used aspirin treatment to upregulate telomerase activity in BMMSCs, and found a significant improvement in the immunomodulatory capacity of BMMSCs. Taken together, these findings identify a previously unrecognized role of TERT in improving the immunomodulatory capacity of BMMSCs, suggesting that aspirin treatment is a practical approach to significantly reduce cell dosage in BMMSC-based immunotherapies.

DOI： 10.1002/emmm.201303000

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Abnormal stem cell function makes a known contribution to many malignant tumors, but the role of stem cells in benign tumors is not well understood. Here, we show that ossifying fibroma (OF) contains a stem cell population that resembles mesenchymal stem cells (OFMSCs) and is capable of generating OF-like tumor xenografts. Mechanistically, OFMSCs show enhanced TGF-β signaling that induces aberrant proliferation and deficient osteogenesis via Notch and BMP signaling pathways, respectively. The elevated TGF-β activity is tightly regulated by JHDM1D-mediated epigenetic regulation of thrombospondin-1 (TSP1), forming a JHDM1D/TSP1/TGF-β/SMAD3 autocrine loop. Inhibition of TGF-β signaling in OFMSCs can rescue their abnormal osteogenic differentiation and elevated proliferation rate. Furthermore, chronic activation of TGF-β can convert normal MSCs into OF-like MSCs via establishment of this JHDM1D/TSP1/TGF-β/SMAD3 autocrine loop. These results reveal that epigenetic regulation of TGF-β signaling in MSCs governs the benign tumor phenotype in OF and highlight TGF-β signaling as a candidate therapeutic target.

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The role of phosphoinositide 3-kinase in adhesion of oral epithelial cells to titanium.
BACKGROUND: Oral epithelial cells (OECs) adhesion to titanium may improve the success rate of implant restoration. PURPOSE: We investigated the mechanism by which OECs adhere to titanium dental implants. MATERIALS AND METHODS: (1) After culturing rat OECs on titanium plates (Ti) or culture dishes in the presence or absence of a phosphoinositide 3-kinase (PI3K) activator or inhibitors and/or growth factors, and OEC morphology under these conditions were analyzed. (2) Right maxillary first molars were extracted and replaced with experimental implants. The rats were treated with or without growth factors. RESULTS: (1) Cell adherence was lower of OECs on Ti than in those on culture dishes, as were the levels of integrin β4 and the continuity of F-actin structures. After PI3K inhibition, markedly reducing adherence to both substrates. In contrast, PI3K activation with activator or insulin-like growth factor restored the OEC adherence and the expression of adhesion molecules on Ti to the levels seen in OECs cultured on dishes. Cell migration was inhibited by PI3K activation. (2) High expression of integrin β4 was observed in the peri-implant epithelia of PI3K-activated rats. CONCLUSION: These findings suggest that PI3K plays an important role in the adhesion of OECs to Ti.

DOI： 10.1016/j.archoralbio.2013.07.013

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Abnormal stem cell function makes a known contribution to many malignant tumors, but the role of stem cells in benign tumors is not well understood. Here, we show that ossifying fibroma (OF) contains a stem cell population that resembles mesenchymal stem cells (OFMSCs) and is capable of generating OF-like tumor xenografts. Mechanistically, OFMSCs show enhanced TGF-β signaling that induces aberrant proliferation and deficient osteogenesis via Notch and BMP signaling pathways, respectively. The elevated TGF-β activity is tightly regulated by JHDM1D-mediated epigenetic regulation of thrombospondin-1 (TSP1), forming a JHDM1D/TSP1/TGF-β/SMAD3 autocrine loop. Inhibition of TGF-β signaling in OFMSCs can rescue their abnormal osteogenic differentiation and elevated proliferation rate. Furthermore, chronic activation of TGF-β can convert normal MSCs into OF-like MSCs via establishment of this JHDM1D/TSP1/TGF-β/SMAD3 autocrine loop. These results reveal that epigenetic regulation of TGF-β signaling in MSCs governs the benign tumor phenotype in OF and highlight TGF-β signaling as a candidate therapeutic target.

DOI： 10.1016/j.stem.2013.08.010

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Improvement of oral epithelial adhesion to titanium (Ti) may significantly enhance the efficacy of dental implants. Here, we investigated whether insulin-like growth factor-1 (IGF-1) improved the sealing of the peri-implant epithelium (PIE) around the implant. Right maxillary first molars were extracted from rats and replaced with experimental implants. After 4 weeks of IGF-1 treatment, the implant-PIE interface exhibited a band of immunoreactive laminin-332 (Ln-5), similar to the tooth-junctional epithelium interface, that was partially absent in the untreated group. Immunoelectron microscopy showed a characteristic Ln-5-positive band including hemidesmosomes at both the apical and upper portions of the implant-PIE interface in the IGF-1-treated group. We also investigated the effects of IGF-1/PI3K inhibitors on the dynamics of rat oral epithelial cells (OECs) grown on Ti plates. In OECs cultured with IGF-1, adhesion protein expression increased, cell adherence to Ti plates was higher, and proliferation was faster, whereas migration and apoptosis were induced in the absence of IGF-1 or in the presence of both IGF-1 and a PI3K inhibitor. These data suggest that PI3K mediates the promotive effects of IGF-1, and that IGF-1 is effective at enhancing epithelial integration around Ti implants. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 101A:2896-2904, 2013.

DOI： 10.1002/jbm.a.34608

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Promotive effect of insulin-like growth factor-1 for epithelial sealing to titanium implants
Improvement of oral epithelial adhesion to titanium (Ti) may significantly enhance the efficacy of dental implants. Here, we investigated whether insulin-like growth factor-1 (IGF-1) improved the sealing of the peri-implant epithelium (PIE) around the implant. Right maxillary first molars were extracted from rats and replaced with experimental implants. After 4 weeks of IGF-1 treatment, the implant-PIE interface exhibited a band of immunoreactive laminin-332 (Ln-5), similar to the tooth-junctional epithelium interface, that was partially absent in the untreated group. Immunoelectron microscopy showed a characteristic Ln-5-positive band including hemidesmosomes at both the apical and upper portions of the implant-PIE interface in the IGF-1-treated group. We also investigated the effects of IGF-1/PI3K inhibitors on the dynamics of rat oral epithelial cells (OECs) grown on Ti plates. In OECs cultured with IGF-1, adhesion protein expression increased, cell adherence to Ti plates was higher, and proliferation was faster, whereas migration and apoptosis were induced in the absence of IGF-1 or in the presence of both IGF-1 and a PI3K inhibitor. These data suggest that PI3K mediates the promotive effects of IGF-1, and that IGF-1 is effective at enhancing epithelial integration around Ti implants.

DOI： 10.1002/jbm.a.34608

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Background: Oral epithelial cells (OECs) adhesion to titanium may improve the success rate of implant restoration. Purpose: We investigated the mechanism by which OECs adhere to titanium dental implants. Materials and methods: (1) After culturing rat OECs on titanium plates (Ti) or culture dishes in the presence or absence of a phosphoinositide 3-kinase (PI3K) activator or inhibitors and/or growth factors, and OEC morphology under these conditions were analyzed. (2) Right maxillary first molars were extracted and replaced with experimental implants. The rats were treated with or without growth factors. Results: (1) Cell adherence was lower of OECs on Ti than in those on culture dishes, as were the levels of integrin β4 and the continuity of F-actin structures. After PI3K inhibition, markedly reducing adherence to both substrates. In contrast, PI3K activation with activator or insulin-like growth factor restored the OEC adherence and the expression of adhesion molecules on Ti to the levels seen in OECs cultured on dishes. Cell migration was inhibited by PI3K activation. (2) High expression of integrin β4 was observed in the peri-implant epithelia of PI3K-activated rats. Conclusion: These findings suggest that PI3K plays an important role in the adhesion of OECs to Ti.

DOI： 10.1016/j.archoralbio.2013.07.013

記述言語：日本語  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

In orthodontic tooth movement (OTM), we should be concerned about external root resorption (ERR) as an undesirable iatrogenic problem, but its mechanisms are not fully understood. Since our previous epidemiologic studies found that patients with allergic diseases showed higher rates of ERR during orthodontic treatment, we explored the possible effect of allergic sensitization on ERR. In ovalbumin (OVA)-sensitized Brown-Norway rats, the amounts of ERR and OTM were greater than those in animals subjected to orthodontic force alone. The expression levels of RANKL and pro-inflammatory cytokines were increased in the periodontal tissues of sensitized rats with OTM, compared with control rats. Furthermore, leukotriene B4 (LTB4), a potent lipid mediator of allergic inflammation, and enzymes of the 5-lipoxygenase pathway, the biosynthetic pathway of leukotrienes, were also up-regulated. We found that low doses of aspirin suppressed ERR in allergen-sensitized rats, as well as the expressions of RANKL, pro-inflammatory cytokines, and LTB4. The present findings indicate that allergen sensitization has adverse effects on ERR under OTM, and that aspirin is a potential therapeutic agent for combating ERR.

DOI： 10.1177/0022034513488787

記述言語：その他  

Immune therapeutic potential of stem cells from human supernumerary teeth.

DOI： 10.1177/0022034513490732

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Discoveries of immunomodulatory functions in mesenchymal stem cells (MSCs) have suggested that they might have therapeutic utility in treating immune diseases. Recently, a novel MSC population was identified from dental pulp of human supernumerary teeth, and its multipotency characterized. Herein, we first examined the in vitro and in vivo immunomodulatory functions of human supernumerary tooth-derived stem cells (SNTSCs). SNTSCs suppressed not only the viability of T-cells, but also the differentiation of interleukin 17 (IL-17)-secreting helper T (Th17) -cells in in vitro co-culture experiments. In addition, systemic SNTSC transplantation ameliorated the shortened lifespan and elevated serum autoantibodies and nephritis-like renal dysfunction in systemic lupus erythematosus (SLE) model MRL/lpr mice. SNTSC transplantation also suppressed in vivo increased levels of peripheral Th17 cells and IL-17, as well as ex vivo differentiation of Th17 cells in MRL/lpr mice. Adoptive transfer experiments demonstrated that SNTSC-transplanted MRL/lpr mouse-derived T-cell-adopted immunocompromised mice showed a longer lifespan in comparison with non-transplanted MRL/lpr mouse-derived T-cell-adopted immunocompromised mice, indicating that SNTSC transplantation suppresses the hyper-immune condition of MRL/lpr mice through suppressing T-cells. Analysis of these data suggests that SNTSCs are a promising MSC source for cell-based therapy for immune diseases such as SLE.

DOI： 10.1177/0022034513490732

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Osteoclasts are the multinucleated giant cells formed by cell fusion of mononuclear osteoclast precursors. Despite the finding of several membrane proteins involving DC-STAMP as regulatory proteins required for fusion among osteoclast precursors, cellular and molecular events concerning this process are still ambiguous. Here we identified Tunneling Nanotubes (TNTs), long intercellular bridges with small diameters, as the essential cellular structure for intercellular communication among osteoclast precursors in prior to cell fusion. Formation of TNTs was highly associated with osteoclastogenesis and it was accompanied with the significant induction of the M-Sec gene, an essential gene for TNT formation. M-Sec gene expression was significantly upregulated by RANKL-treatment in osteoclast precursor cell line. Blockage of TNT formation by Latrunclin B or by M-Sec siRNA significantly suppressed osteoclastogenesis. We have detected the rapid intercellular transport of not only the membrane phospholipids labeled with DiI but also the DC-STAMP-GFP fusion protein through TNTs formed among osteoclast precursors during osteoclastogenesis. Transportation of such regulatory molecules through TNTs would be essential for the process of the specific cell fusion among osteoclast precursors. J. Cell. Biochem. 114: 1238-1247, 2013. © 2012 Wiley Periodicals, Inc.

DOI： 10.1002/jcb.24433

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Tunneling nanotube formation is essential for the regulation of osteoclastogenesis.
Osteoclasts are the multinucleated giant cells formed by cell fusion of mononuclear osteoclast precursors. Despite the finding of several membrane proteins involving DC-STAMP as regulatory proteins required for fusion among osteoclast precursors, cellular and molecular events concerning this process are still ambiguous. Here we identified Tunneling Nanotubes (TNTs), long intercellular bridges with small diameters, as the essential cellular structure for intercellular communication among osteoclast precursors in prior to cell fusion. Formation of TNTs was highly associated with osteoclastogenesis and it was accompanied with the significant induction of the M-Sec gene, an essential gene for TNT formation. M-Sec gene expression was significantly upregulated by RANKL-treatment in osteoclast precursor cell line. Blockage of TNT formation by Latrunclin B or by M-Sec siRNA significantly suppressed osteoclastogenesis. We have detected the rapid intercellular transport of not only the membrane phospholipids labeled with DiI but also the DC-STAMP-GFP fusion protein through TNTs formed among osteoclast precursors during osteoclastogenesis. Transportation of such regulatory molecules through TNTs would be essential for the process of the specific cell fusion among osteoclast precursors.

DOI： 10.1002/jcb.24433

記述言語：英語   掲載種別：研究論文（その他学術会議資料等）  

Integrin, a component of the hemidesmosome, plays a role for epithelial cell migration and adhesion. This study investigated the process of peri-implant epithelium (PIE) formation after implantation, and compared it to the process of oral mucosa healing after tooth extraction. At the healing site of extraction socket without implant, the original junctional epithelium (JE) had disappeared at week 2, and the oral epithelium (OE) with integrin-α3 positive basal cells extending from the sides of the wound, then joined in the middle of the extraction socket. On the other hand in implant group, newly formed epithelium with integrin-α3 positive cells from the OE extended apically 1 week after implantation. After 3 weeks, basal cells of the new epithelium consisted of those with integrin-α3 positive but β4 negative. Finally, after 4 weeks, integrin-β4 was expressed at the implant-PIE interface. These findings suggest that integrin α3β1 plays a role in cell migration during PIE formation from OE. Furthermore, after the completion of PIE constitution, integrin α6β4 contributes to the attachment to titanium.

DOI： 10.4028/www.scientific.net/KEM.529-530.407

記述言語：英語  

Integrin, a component of the hemidesmosome, plays a role for epithelial cell migration and adhesion. This study investigated the process of peri-implant epithelium (PIE) formation after implantation, and compared it to the process of oral mucosa healing after tooth extraction. At the healing site of extraction socket without implant, the original junctional epithelium (JE) had disappeared at week 2, and the oral epithelium (OE) with integrin-α3 positive basal cells extending from the sides of the wound, then joined in the middle of the extraction socket. On the other hand in implant group, newly formed epithelium with integrin-α3 positive cells from the OE extended apically 1 week after implantation. After 3 weeks, basal cells of the new epithelium consisted of those with integrin-α3 positive but β4 negative. Finally, after 4 weeks, integrin-β4 was expressed at the implant-PIE interface. These findings suggest that integrin α3β1 plays a role in cell migration during PIE formation from OE. Furthermore, after the completion of PIE constitution, integrin α6β4 contributes to the attachment to titanium. © (2013) Trans Tech Publications, Switzerland.

DOI： 10.4028/www.scientific.net/KEM.529-530.407

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Introduction. Bone marrow mesenchymal stem cells (BMMSCs) are a heterogeneous population of postnatal precursor cells with the capacity of adhering to culture dishes generating colony-forming unit-fibroblasts (CFU-F). Here we identify a new subset of BMMSCs that fail to adhere to plastic culture dishes and remain in culture suspension (S-BMMSCs). Methods. To catch S-BMMSCs, we used BMMSCs-produced extracellular cell matrix (ECM)-coated dishes. Isolated S-BMMSCs were analyzed by in vitro stem cell analysis approaches, including flow cytometry, inductive multiple differentiation, western blot and in vivo implantation to assess the bone regeneration ability of S-BMMSCs. Furthermore, we performed systemic S-BMMSCs transplantation to treat systemic lupus erythematosus (SLE)-like MRL/lpr mice. Results: S-BMMSCs are capable of adhering to ECM-coated dishes and showing mesenchymal stem cell characteristics with distinction from hematopoietic cells as evidenced by co-expression of CD73 or Oct-4 with CD34, forming a single colony cluster on ECM, and failure to differentiate into hematopoietic cell lineage. Moreover, we found that culture-expanded S-BMMSCs exhibited significantly increased immunomodulatory capacities in vitro and an efficacious treatment for SLE-like MRL/lpr mice by rebalancing regulatory T cells (Tregs) and T helper 17 cells (Th17) through high NO production. Conclusions: These data suggest that it is feasible to improve immunotherapy by identifying a new subset BMMSCs.

DOI： 10.1186/scrt131

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Human exfoliated deciduous teeth have been considered to be a promising source for regenerative therapy because they contain unique postnatal stem cells from human exfoliated deciduous teeth (SHED) with self-renewal capacity, multipotency and immunomodulatory function. However preservation technique of deciduous teeth has not been developed. This study aimed to evaluate that cryopreserved dental pulp tissues of human exfoliated deciduous teeth is a retrievable and practical SHED source for cell-based therapy. SHED isolated from the cryopreserved deciduous pulp tissues for over 2 years (25-30 months) (SHED-Cryo) owned similar stem cell properties including clonogenicity, self-renew, stem cell marker expression, multipotency, in vivo tissue regenerative capacity and in vitro immunomodulatory function to SHED isolated from the fresh tissues (SHED-Fresh). To examine the therapeutic efficacy of SHED-Cryo on immune diseases, SHED-Cryo were intravenously transplanted into systemic lupus erythematosus (SLE) model MRL/lpr mice. Systemic SHED-Cryo-transplantation improved SLE-like disorders including short lifespan, elevated autoantibody levels and nephritis-like renal dysfunction. SHED-Cryo amended increased interleukin 17-secreting helper T cells in MRL/lpr mice systemically and locally. SHED-Cryo-transplantation was also able to recover osteoporosis bone reduction in long bones of MRL/lpr mice. Furthermore, SHED-Cryo-mediated tissue engineering induced bone regeneration in critical calvarial bone-defect sites of immunocompromised mice. The therapeutic efficacy of SHED-Cryo transplantation on immune and skeletal disorders was similar to that of SHED-Fresh. These data suggest that cryopreservation of dental pulp tissues of deciduous teeth provide a suitable and desirable approach for stem cell-based immune therapy and tissue engineering in regenerative medicine. Citation: Ma L, Makino Y, Yamaza H, Akiyama K, Hoshino Y, et al. (2012) Cryopreserved Dental Pulp Tissues of Exfoliated Deciduous Teeth Is a Feasible Stem Cell Resource for Regenerative Medicine. PLoS ONE 7(12): e51777. doi:10.1371/journal.pone.0051777

DOI： 10.1371/journal.pone.0051777

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Human exfoliated deciduous teeth have been considered to be a promising source for regenerative therapy because they contain unique postnatal stem cells from human exfoliated deciduous teeth (SHED) with self-renewal capacity, multipotency and immunomodulatory function. However preservation technique of deciduous teeth has not been developed. This study aimed to evaluate that cryopreserved dental pulp tissues of human exfoliated deciduous teeth is a retrievable and practical SHED source for cell-based therapy. SHED isolated from the cryopreserved deciduous pulp tissues for over 2 years (25-30 months) (SHED-Cryo) owned similar stem cell properties including clonogenicity, self-renew, stem cell marker expression, multipotency, in vivo tissue regenerative capacity and in vitro immunomodulatory function to SHED isolated from the fresh tissues (SHED-Fresh). To examine the therapeutic efficacy of SHED-Cryo on immune diseases, SHED-Cryo were intravenously transplanted into systemic lupus erythematosus (SLE) model MRL/lpr mice. Systemic SHED-Cryo-transplantation improved SLE-like disorders including short lifespan, elevated autoantibody levels and nephritis-like renal dysfunction. SHED-Cryo amended increased interleukin 17-secreting helper T cells in MRL/lpr mice systemically and locally. SHED-Cryo-transplantation was also able to recover osteoporosis bone reduction in long bones of MRL/lpr mice. Furthermore, SHED-Cryo-mediated tissue engineering induced bone regeneration in critical calvarial bone-defect sites of immunocompromised mice. The therapeutic efficacy of SHED-Cryo transplantation on immune and skeletal disorders was similar to that of SHED-Fresh. These data suggest that cryopreservation of dental pulp tissues of deciduous teeth provide a suitable and desirable approach for stem cell-based immune therapy and tissue engineering in regenerative medicine.

DOI： 10.1371/journal.pone.0051777

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The clinical trial of allogenic mesenchymal stem cells (MSCs) transplantation for refractory SLE patients has shown significant safety and efficacy profiles. However, the optimum frequency of the MSCs transplantation (MSCT) is unknown. This study was undertaken to observe whether double transplantations of MSCs is superior to single transplantation. Fifty-eight refractory SLE patients were enrolled in this study, in which 30 were randomly given single MSCT, and the other 28 were given double MSCT. Patients were followed up for rates of survival, disease remission, and relapse, as well as transplantation-related adverse events. SLE disease activity index (SLEDAI) and serologic features were evaluated. Our results showed that no remarkable differences between single and double allogenic MSCT were found in terms of disease remission and relapse, amelioration of disease activity, and serum indexes in an SLE clinical trial with more than one year followup. This study demonstrated that single MSCs transplantation at the dose of one million MSCs per kilogram of body weight was sufficient to induce disease remission for refractory SLE patients.

DOI： 10.1155/2012/273291

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Systemic infusion of bone marrow mesenchymal stem cells (BMMSCs) yields therapeutic benefit for a variety of autoimmune diseases, but the underlying mechanisms are poorly understood. Here we show that in mice systemic infusion of BMMSCs induced transient T cell apoptosis via the FAS ligand (FASL)-dependent FAS pathway and could ameliorate disease phenotypes in fibrillin-1 mutated systemic sclerosis (SS) and dextran-sulfate-sodium-induced experimental colitis. FASL ^-/- BMMSCs did not induce T cell apoptosis in recipients, and could not ameliorate SS and colitis. Mechanistic analysis revealed that FAS-regulated monocyte chemotactic protein 1 (MCP-1) secretion by BMMSCs recruited T cells for FASL-mediated apoptosis. The apoptotic T cells subsequently triggered macrophages to produce high levels of TGFβ, which in turn led to the upregulation of CD4 ⁺CD25 ⁺Foxp3 ⁺ regulatory T cells and, ultimately, immune tolerance. These data therefore demonstrate a previously unrecognized mechanism underlying BMMSC-based immunotherapy involving coupling via FAS/FASL to induce T cell apoptosis.

DOI： 10.1016/j.stem.2012.03.007

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Systemic infusion of bone marrow mesenchymal stem cells (BMMSCs) yields therapeutic benefit for a variety of autoimmune diseases, but the underlying mechanisms are poorly understood. Here we show that in mice systemic infusion of BMMSCs induced transient T cell apoptosis via the FAS ligand (FASL)-dependent FAS pathway and could ameliorate disease phenotypes in fibrillin-1 mutated systemic sclerosis (SS) and dextran-sulfate-sodium-induced experimental colitis. FASL -/- BMMSCs did not induce T cell apoptosis in recipients, and could not ameliorate SS and colitis. Mechanistic analysis revealed that FAS-regulated monocyte chemotactic protein 1 (MCP-1) secretion by BMMSCs recruited T cells for FASL-mediated apoptosis. The apoptotic T cells subsequently triggered macrophages to produce high levels of TGFβ, which in turn led to the upregulation of CD4 +CD25 +Foxp3 + regulatory T cells and, ultimately, immune tolerance. These data therefore demonstrate a previously unrecognized mechanism underlying BMMSC-based immunotherapy involving coupling via FAS/FASL to induce T cell apoptosis. © 2012 Elsevier Inc.

DOI： 10.1016/j.stem.2012.03.007

記述言語：日本語  

間葉系幹細胞による炎症性骨破壊制御

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Although human orofacial bone-marrow-derived mesenchymal stem cells showed differentiation traits distinctly different from those of mesenchymal stem cells (MSCs) derived from long bone marrow (BMMSCs), mouse MSCs derived from orofacial bone have not been isolated due to technical difficulties, which in turn precludes the use of mouse models to study and cure orofacial diseases. In this study, we developed techniques to isolate and expand mouse orofacial bone/bone-marrow-derived MSCs (OMSCs) from mandibles and verified their MSC characteristics by single-colony formation, multi-lineage differentiation, and in vivo tissue regeneration. Activated T-lymphocytes impaired OMSCs via the Fas/Fas ligand pathway, as occurs in BMMSCs. Furthermore, we found that OMSCs are distinct from BMMSCs with respect to regulating T-lymphocyte survival and proliferation. Analysis of our data suggests that OMSCs are a unique population of MSCs and play an important role in systemic immunity. Abbreviations: BMMSC, bone marrow mesenchymal stem cell; HA/TCP, hydroxyapatite/tricalcium phosphate; OMSC, orofacial mesenchymal stem cell; OVX, ovariectomized.

DOI： 10.1177/0022034510387796

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Although human orofacial bone-marrow-derived mesenchymal stem cells showed differentiation traits distinctly different from those of mesenchymal stem cells (MSCs) derived from long bone marrow (BMMSCs), mouse MSCs derived from orofacial bone have not been isolated due to technical difficulties, which in turn precludes the use of mouse models to study and cure orofacial diseases. In this study, we developed techniques to isolate and expand mouse orofacial bone/bone-marrow-derived MSCs (OMSCs) from mandibles and verified their MSC characteristics by single-colony formation, multi-lineage differentiation, and in vivo tissue regeneration. Activated T-lymphocytes impaired OMSCs via the Fas/Fas ligand pathway, as occurs in BMMSCs. Furthermore, we found that OMSCs are distinct from BMMSCs with respect to regulating T-lymphocyte survival and proliferation. Analysis of our data suggests that OMSCs are a unique population of MSCs and play an important role in systemic immunity. Abbreviations: BMMSC, bone marrow mesenchymal stem cell; HA/TCP, hydroxyapatite/tricalcium phosphate; OMSC, orofacial mesenchymal stem cell; OVX, ovariectomized.

DOI： 10.1177/0022034510387796

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Introduction. Stem cells from human exfoliated deciduous teeth (SHED) have been identified as a population of postnatal stem cells capable of differentiating into osteogenic and odontogenic cells, adipogenic cells, and neural cells. Herein we have characterized mesenchymal stem cell properties of SHED in comparison to human bone marrow mesenchymal stem cells (BMMSCs). Methods. We used in vitro stem cell analysis approaches, including flow cytometry, inductive differentiation, telomerase activity, and Western blot analysis to assess multipotent differentiation of SHED and in vivo implantation to assess tissue regeneration of SHED. In addition, we utilized systemic SHED transplantation to treat systemic lupus erythematosus (SLE)-like MRL/lpr mice. Results: We found that SHED are capable of differentiating into osteogenic and adipogenic cells, expressing mesenchymal surface molecules (STRO-1, CD146, SSEA4, CD73, CD105, and CD166), and activating multiple signaling pathways, including TGF, ERK, Akt, Wnt, and PDGF. Recently, BMMSCs were shown to possess an immunomodulatory function that leads to successful therapies for immune diseases. We examined the immunomodulatory properties of SHED in comparison to BMMSCs and found that SHED had significant effects on inhibiting T helper 17 (Th17) cells in vitro. Moreover, we found that SHED transplantation is capable of effectively reversing SLE-associated disorders in MRL/lpr mice. At the cellular level, SHED transplantation elevated the ratio of regulatory T cells (Tregs) via Th17 cells. Conclusions: These data suggest that SHED are an accessible and feasible mesenchymal stem cell source for treating immune disorders like SLE.

DOI： 10.1186/scrt5

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Rodent incisors regenerate throughout the lifetime of the animal owing to the presence of epithelial and mesenchymal stem cells in the proximal region of the tooth. Enamel, the hardest component of the tooth, is continuously deposited by stem cell-derived ameloblasts exclusively on the labial, or outer, surface of the tooth. The epithelial stem cells that are the ameloblast progenitors reside in structures called cervical loops at the base of the incisors. Previous studies have suggested that FGF10, acting mainly through fibroblast growth factor receptor 2b (FGFR2b), is crucial for development of the epithelial stem cell population in mouse incisors. To explore the role of FGFR2b signaling during development and adult life, we used an rtTA transactivator/tetracycline promoter approach that allows inducible and reversible attenuation of FGFR2b signaling. Downregulation of FGFR2b signaling during embryonic stages led to abnormal development of the labial cervical loop and of the inner enamel epithelial layer. In addition, postnatal attenuation of signaling resulted in impaired incisor growth, characterized by failure of enamel formation and degradation of the incisors. At a cellular level, these changes were accompanied by decreased proliferation of the transitamplifying cells that are progenitors of the ameloblasts. Upon release of the signaling blockade, the incisors resumed growth and reformed an enamel layer, demonstrating that survival of the stem cells was not compromised by transient postnatal attenuation of FGFR2b signaling. Taken together, our results demonstrate that FGFR2b signaling regulates both the establishment of the incisor stem cell niches in the embryo and the regenerative capacity of incisors in the adult.

DOI： 10.1242/dev.051672

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Rodent incisors regenerate throughout the lifetime of the animal owing to the presence of epithelial and mesenchymal stem cells in the proximal region of the tooth. Enamel, the hardest component of the tooth, is continuously deposited by stem cell-derived ameloblasts exclusively on the labial, or outer, surface of the tooth. The epithelial stem cells that are the ameloblast progenitors reside in structures called cervical loops at the base of the incisors. Previous studies have suggested that FGF10, acting mainly through fibroblast growth factor receptor 2b (FGFR2b), is crucial for development of the epithelial stem cell population in mouse incisors. To explore the role of FGFR2b signaling during development and adult life, we used an rtTA transactivator/tetracycline promoter approach that allows inducible and reversible attenuation of FGFR2b signaling. Downregulation of FGFR2b signaling during embryonic stages led to abnormal development of the labial cervical loop and of the inner enamel epithelial layer. In addition, postnatal attenuation of signaling resulted in impaired incisor growth, characterized by failure of enamel formation and degradation of the incisors. At a cellular level, these changes were accompanied by decreased proliferation of the transitamplifying cells that are progenitors of the ameloblasts. Upon release of the signaling blockade, the incisors resumed growth and reformed an enamel layer, demonstrating that survival of the stem cells was not compromised by transient postnatal attenuation of FGFR2b signaling. Taken together, our results demonstrate that FGFR2b signaling regulates both the establishment of the incisor stem cell niches in the embryo and the regenerative capacity of incisors in the adult.

DOI： 10.1242/dev.051672

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Patients on high-dose bisphosphonate and immunosuppressive therapy have an increased risk of bisphosphonate-related osteonecrosis of the jaw (BRONJ); despite the disease severity, its pathophysiology remains unknown, and appropriate therapy is not established. Here we have developed a mouse model of BRONJ-like disease that recapitulates major clinical and radiographic manifestations of the human disease, including characteristic features of an open alveolar socket, exposed necrotic bone or sequestra, increased inflammatory infiltrates, osseous sclerosis, and radiopaque alveolar bone. We show that administration of zoledronate, a potent aminobisphosphonate, and dexamethasone, an immunosuppressant drug, causes BRONJ-like disease in mice in part by suppressing the adaptive regulatory T cells, Tregs, and activating the inflammatory T-helper-producing interleukin 17 cells, Th17. Most interestingly, we demonstrate that systemic infusion with mesenchymal stem cells (MSCs) prevents and cures BRONJ-like disease possibly via induction of peripheral tolerance, shown as an inhibition of Th17 and increase in Treg cells. The suppressed Tregs/Th17 ratio in zoledronate- and dexamethasone-treated mice is restored in mice undergoing salvage therapy with Tregs. These findings provide evidence of an immunity-based mechanism of BRONJ-like disease and support the rationale for in vivo immunomodulatory therapy using Tregs or MSCs to treat BRONJ.

DOI： 10.1002/jbmr.37

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Background: Potent stem/progenitor cells have been isolated from normal human dental pulps termed dental pulp stem cells (DPSCs). However, it is unknown whether these cells exist in inflamed pulps (IPs). Aims: To determine whether DPSCs can be identified and isolated from IPs
and if they can be successfully cultured, whether they retain tissue regeneration potential in vivo. Materials &amp
methods: DPSCs from freshly collected normal pulps (NPs) and IPs were characterized in vitro and their tissue regeneration potential tested using an in vivo study model. Results: The immunohistochemical analysis showed that IPs expressed higher levels of mesenchymal stem cell markers STRO-1, CD90, CD105 and CD146 compared with NPs (p &lt
0.05). Flow cytometry analysis showed that DPSCs from both NPs and IPs expressed moderate to high levels of CD146, stage-specific embryonic antigen-4, CD73 and CD166. Total population doubling of DPSCs-IPs (44.6 ± 2.9) was lower than that of DPSCs-NPs (58.9 ± 2.5) (p &lt
0.05), and DPSCs-IPs appeared to have a decreased osteo/dentinogenic potential compared with DPSCs-NPs based on the mineral deposition in cultures. Nonetheless, DPSCs-IPs formed pulp/dentin complexes similar to DPSCs-NPs when transplanted into immunocompromised mice. Conclusion: DPSCs-IPs can be isolated and their mesenchymal stem cell marker profiles are similar to those from NPs. Although some stem cell properties of DPSCs-IPs were altered, cells from some samples remained potent in tissue regeneration in vivo. © 2010 Future Medicine Ltd.

DOI： 10.2217/rme.10.30

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Background: Potent stem/progenitor cells have been isolated from normal human dental pulps termed dental pulp stem cells (DPSCs). However, it is unknown whether these cells exist in inflamed pulps (IPs). Aims: To determine whether DPSCs can be identified and isolated from IPs; and if they can be successfully cultured, whether they retain tissue regeneration potential in vivo. Materials & methods: DPSCs from freshly collected normal pulps (NPs) and IPs were characterized in vitro and their tissue regeneration potential tested using an in vivo study model. Results: The immunohistochemical analysis showed that IPs expressed higher levels of mesenchymal stem cell markers STRO-1, CD90, CD105 and CD146 compared with NPs (p < 0.05). Flow cytometry analysis showed that DPSCs from both NPs and IPs expressed moderate to high levels of CD146, stage-specific embryonic antigen-4, CD73 and CD166. Total population doubling of DPSCs-IPs (44.6 ± 2.9) was lower than that of DPSCs-NPs (58.9 ± 2.5) (p < 0.05), and DPSCs-IPs appeared to have a decreased osteo/dentinogenic potential compared with DPSCs-NPs based on the mineral deposition in cultures. Nonetheless, DPSCs-IPs formed pulp/dentin complexes similar to DPSCs-NPs when transplanted into immunocompromised mice. Conclusion: DPSCs-IPs can be isolated and their mesenchymal stem cell marker profiles are similar to those from NPs. Although some stem cell properties of DPSCs-IPs were altered, cells from some samples remained potent in tissue regeneration in vivo.

DOI： 10.2217/rme.10.30

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Skeletal muscles are formed from two cell lineages, myogenic and fibroblastic. Mesoderm-derived myogenic progenitors form muscle cells whereas fibroblastic cells give rise to the supportive connective tissue of skeletal muscles, such as the tendons and perimysium. It remains unknown how myogenic and fibroblastic cell-cell interactions affect cell fate determination and the organization of skeletal muscle. In the present study, we investigated the functional significance of cell-cell interactions in regulating skeletal muscle development. Our study shows that cranial neural crest (CNC) cells give rise to the fibroblastic cells of the tongue skeletal muscle in mice. Loss of Tgfbr2 in CNC cells (Wnt1-Cre;Tgfbr2^flox/flox) results in microglossia with reduced Scleraxis and Fgf10 expression as well as decreased myogenic cell proliferation, reduced cell number and disorganized tongue muscles. Furthermore, TGF-β2 beads induced the expression of Scleraxis in tongue explant cultures. The addition of FGF10 rescued the muscle cell number in Wnt1-Cre;Tgfbr2^flox/flox mice. Thus, TGF-β induced FGF10 signaling has a critical function in regulating tissue-tissue interaction during tongue skeletal muscle development.

DOI： 10.1016/j.ydbio.2010.02.030

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Skeletal muscles are formed from two cell lineages, myogenic and fibroblastic. Mesoderm-derived myogenic progenitors form muscle cells whereas fibroblastic cells give rise to the supportive connective tissue of skeletal muscles, such as the tendons and perimysium. It remains unknown how myogenic and fibroblastic cell-cell interactions affect cell fate determination and the organization of skeletal muscle. In the present study, we investigated the functional significance of cell-cell interactions in regulating skeletal muscle development. Our study shows that cranial neural crest (CNC) cells give rise to the fibroblastic cells of the tongue skeletal muscle in mice. Loss of Tgfbr2 in CNC cells (Wnt1-Cre;Tgfbr2(flox/flox)) results in microglossia with reduced Scleraxis and Fgf10 expression as well as decreased myogenic cell proliferation, reduced cell number and disorganized tongue muscles. Furthermore, TGF-beta 2 beads induced the expression of Scleraxis in tongue explant cultures. The addition of FGF10 rescued the muscle cell number in Wnt1-Cre;Tgfbr2(flox/flox) mice. Thus, TGF-beta induced FGF10 signaling has a critical function in regulating tissue-tissue interaction during tongue skeletal muscle development. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2010.02.030

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The ultimate goal of this study is to regenerate lost dental pulp and dentin via stem/progenitor cell-based approaches and tissue engineering technologies. In this study, we tested the possibility of regenerating vascularized human dental pulp in emptied root canal space and producing new dentin on existing dentinal walls using a stem/progenitor cell-mediated approach with a human root fragment and an immunocompromised mouse model. Stem/progenitor cells from apical papilla and dental pulp stem cells were isolated, characterized, seeded onto synthetic scaffolds consisting of poly-D,L-lactide/glycolide, inserted into the tooth fragments, and transplanted into mice. Our results showed that the root canal space was filled entirely by a pulp-like tissue with well-established vascularity. In addition, a continuous layer of dentin-like tissue was deposited onto the canal dentinal wall. This dentin-like structure appeared to be produced by a layer of newly formed odontoblast-like cells expressing dentin sialophosphoprotein, bone sialoprotein, alkaline phosphatase, and CD105. The cells in regenerated pulp-like tissue reacted positively to anti-human mitochondria antibodies, indicating their human origin. This study provides the first evidence showing that pulp-like tissue can be regenerated de novo in emptied root canal space by stem cells from apical papilla and dental pulp stem cells that give rise to odontoblast-like cells producing dentin-like tissue on existing dentinal walls.

DOI： 10.1089/ten.tea.2009.0518

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The ultimate goal of this study is to regenerate lost dental pulp and dentin via stem/progenitor cell-based approaches and tissue engineering technologies. In this study, we tested the possibility of regenerating vascularized human dental pulp in emptied root canal space and producing new dentin on existing dentinal walls using a stem/progenitor cell-mediated approach with a human root fragment and an immunocompromised mouse model. Stem/progenitor cells from apical papilla and dental pulp stem cells were isolated, characterized, seeded onto synthetic scaffolds consisting of poly-D,L-lactide/glycolide, inserted into the tooth fragments, and transplanted into mice. Our results showed that the root canal space was filled entirely by a pulp-like tissue with well-established vascularity. In addition, a continuous layer of dentin-like tissue was deposited onto the canal dentinal wall. This dentin-like structure appeared to be produced by a layer of newly formed odontoblast-like cells expressing dentin sialophosphoprotein, bone sialoprotein, alkaline phosphatase, and CD105. The cells in regenerated pulp-like tissue reacted positively to anti-human mitochondria antibodies, indicating their human origin. This study provides the first evidence showing that pulp-like tissue can be regenerated de novo in emptied root canal space by stem cells from apical papilla and dental pulp stem cells that give rise to odontoblast-like cells producing dentin-like tissue on existing dentinal walls.

DOI： 10.1089/ten.TEA.2009.0518

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Objective: Periodontal disease is an inflammatory disorder with widespread morbidities involving both oral and systemic health. The primary goal of periodontal treatment is the regeneration of the lost or diseased periodontium. In this study, we retrospectively examined feasibility and safety of reconstructing the periodontal intrabony defects with autologous periodontal ligament progenitor (PDLP) implantation in three patients. Materials and methods: In this retrospective pilot study, we treated 16 teeth with at least one deep intrabony defect of probing depth (PD) ≥ 6 mm with PDLP transplantation and evaluated clinical outcome measures in terms of probing depth, gingival recession and attachment gain for a duration of 32-72 months. Furthermore, we compare PDLPs with standard PDL stem cells (PDLSCs) and confirmed that PDLPs possessed progenitor characters. Results: Clinical examination indicated that transplantation of PDLPs may provide therapeutic benefit for the periodontal defects. All treated patients showed no adverse effects during the entire course of follow up. We also found that PDLPs were analogous to PDLSCs in terms of high proliferation, expression of mesenchymal surface molecules, multipotent differentiation, and in vivo tissue regain. However, PDLPs failed to express scleraxis, a marker of tendon, as seen in PDLSCs. Conclusions: This study demonstrated clinical and experimental evidences supporting a potential efficacy and safety of utilizing autologous PDL cells in the treatment of human periodontitis.

DOI： 10.1111/j.1601-0825.2009.01593.x

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Background: Alterations in the stem cell niche are likely to contribute to tumorigenesis; however, the concept of niche promoted benign tumor growth remains to be explored. Here we use keloid, an exuberant fibroproliferative dermal growth unique to human skin, as a model to characterize benign tumor-like stem cells and delineate the role of their "pathological" niche in the development of the benign tumor. Methods and Findings: Subclonal assay, flow cytometric and multipotent differentiation analyses demonstrate that keloid contains a new population of stem cells, named keloid derived precursor cells (KPCs), which exhibit clonogenicity, self-renewal, distinct embryonic and mesenchymal stem cell surface markers, and multipotent differentiation. KPCs display elevated telomerase activity and an inherently upregulated proliferation capability as compared to their peripheral normal skin counterparts. A robust elevation of IL-6 and IL-17 expression in keloid is confirmed by cytokine array, western blot and ELISA analyses. The altered biological functions are tightly regulated by the inflammatory niche mediated by an autocrine/paracrine cytokine IL-17/IL-6 axis. Utilizing KPCs transplanted subcutaneously in immunocompromised mice we generate for the first time a human keloid-like tumor model that is driven by the in vivo inflammatory niche and allows testing of the anti-tumor therapeutic effect of antibodies targeting distinct niche components, specifically IL-6 and IL-17. Conclusions/Significance: These findings support our hypothesis that the altered niche in keloids, predominantly inflammatory, contributes to the acquirement of a benign tumor-like stem cell phenotype of KPCs characterized by the uncontrolled self-renewal and increased proliferation, supporting the rationale for in vivo modification of the "pathological" stem cell niche as a novel therapy for keloid and other mesenchymal benign tumors.

DOI： 10.1371/journal.pone.0007798

記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: Alterations in the stem cell niche are likely to contribute to tumorigenesis; however, the concept of niche promoted benign tumor growth remains to be explored. Here we use keloid, an exuberant fibroproliferative dermal growth unique to human skin, as a model to characterize benign tumor-like stem cells and delineate the role of their "pathological" niche in the development of the benign tumor. METHODS AND FINDINGS: Subclonal assay, flow cytometric and multipotent differentiation analyses demonstrate that keloid contains a new population of stem cells, named keloid derived precursor cells (KPCs), which exhibit clonogenicity, self-renewal, distinct embryonic and mesenchymal stem cell surface markers, and multipotent differentiation. KPCs display elevated telomerase activity and an inherently upregulated proliferation capability as compared to their peripheral normal skin counterparts. A robust elevation of IL-6 and IL-17 expression in keloid is confirmed by cytokine array, western blot and ELISA analyses. The altered biological functions are tightly regulated by the inflammatory niche mediated by an autocrine/paracrine cytokine IL-17/IL-6 axis. Utilizing KPCs transplanted subcutaneously in immunocompromised mice we generate for the first time a human keloid-like tumor model that is driven by the in vivo inflammatory niche and allows testing of the anti-tumor therapeutic effect of antibodies targeting distinct niche components, specifically IL-6 and IL-17. CONCLUSIONS/SIGNIFICANCE: These findings support our hypothesis that the altered niche in keloids, predominantly inflammatory, contributes to the acquirement of a benign tumor-like stem cell phenotype of KPCs characterized by the uncontrolled self-renewal and increased proliferation, supporting the rationale for in vivo modification of the "pathological" stem cell niche as a novel therapy for keloid and other mesenchymal benign tumors.

DOI： 10.1371/journal.pone.0007798

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The oral mucosa is a highly specialised, stratified epithelium that confers protection from infection and physical, chemical and thermal stimuli. The non-keratinised junctional epithelium surrounds each tooth like a collar and is easily attacked by foreign substances from the oral sulcus. We found that TRPV2, a temperature-gated channel, is highly expressed in junctional epithelial cells, but not in oral sulcular epithelial cells or oral epithelial cells. Dual or triple immunolabelling with immunocompetent cell markers also revealed TRPV2 expression in Langerhans cells and in dendritic cells and macrophages. Electron microscopy disclosed TRPV2 immunoreactivity in the unmyelinated and thinly myelinated axons within the connective tissue underlying the epithelium. TRPV2 labelling was also observed in venule endothelial cells. The electron-dense immunoreaction in junctional epithelial cells, macrophages and neural axons occurred on the plasma membrane, on invaginations of the plasma membrane and in vesicular structures. Because TRPV2 has been shown to respond to temperature, hypotonicity and mechanical stimuli, gingival cells expressing TRPV2 may act as sensor cells, detecting changes in the physical and chemical environment, and may play a role in subsequent defence mechanisms.

DOI： 10.1007/s00418-009-0616-y

記述言語：英語   掲載種別：研究論文（学術雑誌）  

TRPV2 expression in rat oral mucosa.
The oral mucosa is a highly specialised, stratified epithelium that confers protection from infection and physical, chemical and thermal stimuli. The non-keratinised junctional epithelium surrounds each tooth like a collar and is easily attacked by foreign substances from the oral sulcus. We found that TRPV2, a temperature-gated channel, is highly expressed in junctional epithelial cells, but not in oral sulcular epithelial cells or oral epithelial cells. Dual or triple immunolabelling with immunocompetent cell markers also revealed TRPV2 expression in Langerhans cells and in dendritic cells and macrophages. Electron microscopy disclosed TRPV2 immunoreactivity in the unmyelinated and thinly myelinated axons within the connective tissue underlying the epithelium. TRPV2 labelling was also observed in venule endothelial cells. The electron-dense immunoreaction in junctional epithelial cells, macrophages and neural axons occurred on the plasma membrane, on invaginations of the plasma membrane and in vesicular structures. Because TRPV2 has been shown to respond to temperature, hypotonicity and mechanical stimuli, gingival cells expressing TRPV2 may act as sensor cells, detecting changes in the physical and chemical environment, and may play a role in subsequent defence mechanisms.

DOI： 10.1007/s00418-009-0616-y

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The BCL-6 co-repressor (BCOR) represses gene transcription by interacting with BCL-6 (Refs 1, 2). BCOR mutation is responsible for oculo-facio-cardio-dental (OFCD) syndrome, which is characterized by canine teeth with extremely long roots, congenital cataracts, craniofacial defects and congenital heart disease. Here we show that BCOR mutation increased the osteo-dentinogenic potential of mesenchymal stem cells (MSCs) isolated from a patient with OFCD, providing a molecular explanation for abnormal root growth. AP-2alpha was identified as a repressive target of BCOR, and BCOR mutation resulted in abnormal activation of AP-2alpha. Gain- and loss-of-function assays suggest that AP-2alpha is a key factor that mediates the increased osteo-dentinogenic capacity of MSCs. Moreover, we found that BCOR maintained tissue homeostasis and gene silencing through epigenetic mechanisms. BCOR mutation increased histone H3K4 and H3K36 methylation in MSCs, thereby reactivating transcription of silenced target genes. By studying a rare human genetic disease, we have unravelled an epigenetic mechanism for control of human adult stem cell function.

DOI： 10.1038/ncb1913

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The BCL-6 co-repressor (BCOR) represses gene transcription by interacting with BCL-6 (Refs 1, 2). BCOR mutation is responsible for oculo-facio-cardio-dental (OFCD) syndrome, which is characterized by canine teeth with extremely long roots, congenital cataracts, craniofacial defects and congenital heart disease. Here we show that BCOR mutation increased the osteo-dentinogenic potential of mesenchymal stem cells (MSCs) isolated from a patient with OFCD, providing a molecular explanation for abnormal root growth. AP-2α was identified as a repressive target of BCOR, and BCOR mutation resulted in abnormal activation of AP-2α. Gain- and loss-of-function assays suggest that AP-2α is a key factor that mediates the increased osteo-dentinogenic capacity of MSCs. Moreover, we found that BCOR maintained tissue homeostasis and gene silencing through epigenetic mechanisms. BCOR mutation increased histone H3K4 and H3K36 methylation in MSCs, thereby reactivating transcription of silenced target genes. By studying a rare human genetic disease, we have unravelled an epigenetic mechanism for control of human adult stem cell function.

DOI： 10.1038/ncb1913

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease that, despite the advances in immunosuppressive medical therapies, remains potentially fatal in some patients, especially in treatment-refractory patients. Here, we reported that impairment of bone marrow mesenchymal stem cells (BMMSCs) and their associated osteoblastic niche deficiency contribute in part to the pathogenesis of SLE-like disease in MRL/lpr mice. Interestingly, allogenic BMMSC transplantation (MSCT) is capable of reconstructing the bone marrow osteoblastic niche and more effectively reverses multiorgan dysfunction when compared with medical immunosuppression with cyclophosphamide (CTX). At the cellular level, MSCT, not CTX treatment, was capable to induce osteoblastic niche reconstruction, possibly contributing to the recovery of regulatory T-cells and reestablishment of the immune homeostasis. On the basis of the promising clinical outcomes in SLE mice, we treated four CTX/glucocorticoid treatment-refractory SLE patients using allogenic MSCT and showed a stable 12-18 months disease remission in all treated patients. The patients benefited an amelioration of disease activity, improvement in serologic markers and renal function. These early evidences suggest that allogenic MSCT may be a feasible and safe salvage therapy in refractory SLE patients.

DOI： 10.1002/stem.68

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease that, despite the advances in immunosuppressive medical therapies, remains potentially fatal in some patients, especially in treatment-refractory patients. Here, we reported that impairment of bone marrow mesenchymal stem cells (BMMSCs) and their associated osteoblastic niche deficiency contribute in part to the pathogenesis of SLE-like disease in MRL/lpr mice. Interestingly, allogenic BMMSC transplantation (MSCT) is capable of reconstructing the bone marrow osteoblastic niche and more effectively reverses multiorgan dysfunction when compared with medical immunosuppression with cyclophosphamide (CTX). At the cellular level, MSCT, not CTX treatment, was capable to induce osteoblastic niche reconstruction, possibly contributing to the recovery of regulatory T-cells and reestablishment of the immune homeostasis. On the basis of the promising clinical outcomes in SLE mice, we treated four CTX/glucocorticoid treatment-refractory SLE patients using allogenic MSCT and showed a stable 12-18 months disease remission in all treated patients. The patients benefited an amelioration of disease activity, improvement in serologic markers and renal function. These early evidences suggest that allogenic MSCT may be a feasible and safe salvage therapy in refractory SLE patients.

DOI： 10.1002/stem.68

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1111/j.1601-0825.2009.01564.x

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The vertebrate neural crest is a multipotent cell population that gives rise to a variety of different cell types. We have discovered that postmigratory cranial neural crest cells (CNCCs) maintain mesenchymal stem cell characteristics and show potential utility for the regeneration of craniofacial structures. We are able to induce the osteogenic differentiation of postmigratory CNCCs, and this differentiation is regulated by bone morphogenetic protein (BMP) and transforming growth factor-b signaling pathways. After transplantation into a host animal, postmigratory CNCCs form bone matrix. CNCC-formed bones are distinct from bones regenerated by bone marrow mesenchymal stem cells. In addition, CNCCs support tooth germ survival via BMP signaling in our CNCC-tooth germ cotransplantation system. Thus, we conclude that postmigratory CNCCs preserve stem cell features, contribute to craniofacial bone formation, and play a fundamental role in supporting tooth organ development. These findings reveal a novel function for postmigratory CNCCs in organ development, and demonstrate the utility of these CNCCs in regenerating craniofacial structures.

DOI： 10.1002/stem.2

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The vertebrate neural crest is a multipotent cell population that gives rise to a variety of different cell types. We have discovered that postmigratory cranial neural crest cells (CNCCs) maintain mesenchymal stem cell characteristics and show potential utility for the regeneration of craniofacial structures. We are able to induce the osteogenic differentiation of postmigratory CNCCs, and this differentiation is regulated by bone morphogenetic protein (BMP) and transforming growth factor-beta signaling pathways. After transplantation into a host animal, postmigratory CNCCs form bone matrix. CNCC-formed bones are distinct from bones regenerated by bone marrow mesenchymal stem cells. In addition, CNCCs support tooth germ survival via BMP signaling in our CNCC-tooth germ cotransplantation system. Thus, we conclude that postmigratory CNCCs preserve stem cell features, contribute to craniofacial bone formation, and play a fundamental role in supporting tooth organ development. These findings reveal a novel function for postmigratory CNCCs in organ development, and demonstrate the utility of these CNCCs in regenerating craniofacial structures.

DOI： 10.1002/stem.2

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Subcutaneous transplants of bone marrow mesenchymal stem cells (BMMSCs) are capable of generating ectopic bone and organizing functional hematopoietic marrow elements in animal models. Here we report that immunocompromised mice received subcutaneous BMMSC transplants using hydroxyapatite tricalcium phosphate as a carrier suppressed age- related degeneration in multiple organs and benefited an increase in life span extension compared with control litter- mates. The newly organized ectopic bone/ marrow system restores active hemato-poiesis via the erythropoietin receptor/ signal transducer and activator of transcription 5 (Stat5) pathway. Furthermore, the BMMSC recipient mice showed elevated level of Klotho and suppression of insulin-like growth factor I signaling, which may be the mechanism contributing to the alleviation of aging-like pheno-types and prolongation of life in the treated mice. This work reveals that erythropoietin receptor/Stat5 pathway contributes to BMMSC-organized ectopic hema-topoiesis, which may offer a treatment paradigm of reversing age-related degeneration of multiple organs in adult immunocompromised mice.

DOI： 10.1182/blood-2008-10-182246

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Subcutaneous transplants of bone marrow mesenchymal stem cells (BMMSCs) are capable of generating ectopic bone and organizing functional hematopoietic marrow elements in animal models. Here we report that immunocompromised mice received subcutaneous BMMSC transplants using hydroxyapatite tricalcium phosphate as a carrier suppressed age- related degeneration in multiple organs and benefited an increase in life span extension compared with control litter- mates. The newly organized ectopic bone/ marrow system restores active hemato-poiesis via the erythropoietin receptor/ signal transducer and activator of transcription 5 (Stat5) pathway. Furthermore, the BMMSC recipient mice showed elevated level of Klotho and suppression of insulin-like growth factor I signaling, which may be the mechanism contributing to the alleviation of aging-like pheno-types and prolongation of life in the treated mice. This work reveals that erythropoietin receptor/Stat5 pathway contributes to BMMSC-organized ectopic hema-topoiesis, which may offer a treatment paradigm of reversing age-related degeneration of multiple organs in adult immunocompromised mice. © 2009 by The American Society of Hematology.

DOI： 10.1182/blood-2008-10-182246

記述言語：英語   掲載種別：研究論文（学術雑誌）  

We examined the distribution of substance P and neurokinin-1 (NK1) receptors and substance-P-containing nerve fibers in the peri-implant mucosa around titanium dental implants in rats. Immunohistochemistry and immunocytochemistry revealed that substance-P-immunoreactive nerve fibers abundantly innervated the peri-implant epithelium (PIE) compared with other epithelia of the peri-implant mucosa. NK1 receptor mRNA and protein expression in the peri-implant mucosa were confirmed by reverse transcription with the polymerase chain reaction and immunoblotting. Immunoelectron microscopy revealed that NK1 receptor immunoreactivity was preferentially localized in peri-implant epithelial cells. NK1-receptor-positive products were found on the plasma membrane and in vesicles and granules in PIE cells. Neutrophils and intraepithelial nerve axons in the PIE were positive for the NK1 receptor. NK1 receptor immunoreactivity was also detected in endothelial cells, fibroblasts, and nerve fibers in the connective tissue beneath the PIE. These findings suggest that peri-implant tissue receives sensory information through regenerated nerves expressing substance P and the NK1 receptor. In the peri-implant mucosa, the substance P/NK1 receptor system may play a role in pain transmission, the endocytosis of neutrophils, the extravasation of crevicular fluid, and the migration of macrophages and neutrophils in response to neurogenic inflammation, as in healthy gingiva.

DOI： 10.1007/s00441-008-0720-7

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Distribution of substance P and neurokinin-1 receptors in the peri-implant epithelium around titanium dental implants in rats.
We examined the distribution of substance P and neurokinin-1 (NK1) receptors and substance-P-containing nerve fibers in the peri-implant mucosa around titanium dental implants in rats. Immunohistochemistry and immunocytochemistry revealed that substance-P-immunoreactive nerve fibers abundantly innervated the peri-implant epithelium (PIE) compared with other epithelia of the peri-implant mucosa. NK1 receptor mRNA and protein expression in the peri-implant mucosa were confirmed by reverse transcription with the polymerase chain reaction and immunoblotting. Immunoelectron microscopy revealed that NK1 receptor immunoreactivity was preferentially localized in peri-implant epithelial cells. NK1-receptor-positive products were found on the plasma membrane and in vesicles and granules in PIE cells. Neutrophils and intraepithelial nerve axons in the PIE were positive for the NK1 receptor. NK1 receptor immunoreactivity was also detected in endothelial cells, fibroblasts, and nerve fibers in the connective tissue beneath the PIE. These findings suggest that peri-implant tissue receives sensory information through regenerated nerves expressing substance P and the NK1 receptor. In the peri-implant mucosa, the substance P/NK1 receptor system may play a role in pain transmission, the endocytosis of neutrophils, the extravasation of crevicular fluid, and the migration of macrophages and neutrophils in response to neurogenic inflammation, as in healthy gingiva.

DOI： 10.1007/s00441-008-0720-7

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.2217/17460816.3.6.499

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Background: Osteoporosis is the most prevalent skeletal disorder, characterized by a low bone mineral density (BMD) and bone structural deterioration, leading to bone fragility fractures. Accelerated bone resorption by osteoclasts has been established as a principal mechanism in osteoporosis. However, recent experimental evidences suggest that inappropriate apoptosis of osteoblasts/osteocytes accounts for, at least in part, the imbalance in bone remodeling as occurs in osteoporosis. The aim of this study is to examine whether aspirin, which has been reported as an effective drug improving bone mineral density in human epidemiology studies, regulates the balance between bone resorption and bone formation at stem cell levels. Methods and Findings: We found that T cell-mediated bone marrow mesenchymal stem cell (BMMSC) impairment plays a crucial role in ovariectomized-induced osteoporosis. Ex vivo mechanistic studied revealed that T cell-mediated BMMSC impairment was mainly attributed to the apoptosis of BMMSCs via the Fas/Fas ligand pathway. To explore potential of using pharmacologic stem cell based intervention as an approach for osteoporosis treatment, we selected ovariectomy (OVX)-induced osteoporosis mouse model to examine feasibility and mechanism of aspirin-mediated therapy for osteoporosis. We found that aspirin can inhibit T cell activation and Fas ligand induced BMMSC apoptosis in vitro. Further, we revealed that aspirin increases osteogenesis of BMMSCs by aiming at telomerase activity and inhibits osteoclast activity in OVX mice, leading to ameliorating bone density. Conclusion: Our findings have revealed a novel osteoporosis mechanism in which activated T cells induce BMMSC apoptosis via Fas/Fas ligand pathway and suggested that pharmacologic stem cell based intervention by aspirin may be a new alternative in osteoporosis treatment including activated osteoblasts and inhibited osteoclasts.

DOI： 10.1371/journal.pone.0002615

記述言語：英語   掲載種別：研究論文（学術雑誌）  

OBJECTIVE: Stem cells from human exfoliated deciduous teeth (SHED) are a population of highly proliferative postnatal stem cells capable of differentiating into odontoblasts, adipocytes, neural cells, and osteo-inductive cells. To examine whether SHED-mediated bone regeneration can be utilized for therapeutic purposes, we used SHED to repair critical-size calvarial defects in immunocompromised mice.
MATERIALS AND METHODS: We generated calvarial defects and transplanted SHED with hydroxyapatite/tricalcium phosphate as a carrier into the defect areas.
RESULTS: SHED were able to repair the defects with substantial bone formation. Interestingly, SHED-mediated osteogenesis failed to recruit hematopoietic marrow elements that are commonly seen in bone marrow mesenchymal stem cell-generated bone. Furthermore, SHED were found to co-express mesenchymal stem cell marker, CC9/MUC18/CD146, with an array of growth factor receptors such as transforming growth factor beta receptor I and II, fibroblast growth factor receptor I and III, and vascular endothelial growth factor receptor I, implying their comprehensive differentiation potential.
CONCLUSIONS: Our data indicate that SHED, derived from neural crest cells, may select unique mechanisms to exert osteogenesis. SHED might be a suitable resource for orofacial bone regeneration.

DOI： 10.1111/j.1601-0825.2007.01396.x

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Background: Osteoporosis is the most prevalent skeletal disorder, characterized by a low bone mineral density (BMD) and bone structural deterioration, leading to bone fragility fractures. Accelerated bone resorption by osteoclasts has been established as a principal mechanism in osteoporosis. However, recent experimental evidences suggest that inappropriate apoptosis of osteoblasts/osteocytes accounts for, at least in part, the imbalance in bone remodeling as occurs in osteoporosis. The aim of this study is to examine whether aspirin, which has been reported as an effective drug improving bone mineral density in human epidemiology studies, regulates the balance between bone resorption and bone formation at stem cell levels. Methods and Findings: We found that T cell-mediated bone marrow mesenchymal stem cell (BMMSC) impairment plays a crucial role in ovariectomized-induced osteoporosis. Ex vivo mechanistic studied revealed that T cell-mediated BMMSC impairment was mainly attributed to the apoptosis of BMMSCs via the Fas/Fas ligand pathway. To explore potential of using pharmacologic stem cell based intervention as an approach for osteoporosis treatment, we selected ovariectomy (OVX)-induced osteoporosis mouse model to examine feasibility and mechanism of aspirin-mediated therapy for osteoporosis. We found that aspirin can inhibit T cell activation and Fas ligand induced BMMSC apoptosis in vitro. Further, we revealed that aspirin increases osteogenesis of BMMSCs by aiming at telomerase activity and inhibits osteoclast activity in OVX mice, leading to ameliorating bone density. Conclusion: Our findings have revealed a novel osteoporosis mechanism in which activated T cells induce BMMSC apoptosis via Fas/Fas ligand pathway and suggested that pharmacologic stem cell based intervention by aspirin may be a new alternative in osteoporosis treatment including activated osteoblasts and inhibited osteoclasts. © 2008 Yamaza et al.

DOI： 10.1371/journal.pone.0002615

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Objective: Stem cells from human exfoliated deciduous teeth (SHED) are a population of highly proliferative postnatal stem cells capable of differentiating into odontoblasts, adipocytes, neural cells, and osteo-inductive cells. To examine whether SHED-mediated bone regeneration can be utilized for therapeutic purposes, we used SHED to repair critical-size calvarial defects in immunocompromised mice. Materials and methods: We generated calvarial defects and transplanted SHED with hydroxyapatite/tricalcium phosphate as a carrier into the defect areas. Results: SHED were able to repair the defects with substantial bone formation. Interestingly, SHED-mediated osteogenesis failed to recruit hematopoietic marrow elements that are commonly seen in bone marrow mesenchymal stem cell-generated bone. Furthermore, SHED were found to co-express mesenchymal stem cell marker, CC9/MUC18/CD146, with an array of growth factor receptors such as transforming growth factor β receptor I and II, fibroblast growth factor receptor I and III, and vascular endothelial growth factor receptor I, implying their comprehensive differentiation potential. Conclusions: Our data indicate that SHED, derived from neural crest cells, may select unique mechanisms to exert osteogenesis. SHED might be a suitable resource for orofacial bone regeneration.

DOI： 10.1111/j.1601-0825.2007.01396.x

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Mesenchymal stem cells (MSCs) have been isolated from the pulp tissue of permanent teeth (dental pulp stem cells or DPSCs) and deciduous teeth (stem cells from human exfoliated deciduous teeth). We recently discovered another type of MSCs in the apical papilla of human immature permanent teeth termed stem cells from the apical papilla (SCAP). Here, we further characterized the apical papilla tissue and stem cell properties of SCAP using histologic, immunohistochemical, and immunocytofluorescent analyses. We found that the apical papilla is distinctive to the pulp in terms of containing less cellular and vascular components than those in the pulp. Cells in the apical papilla proliferated 2- to 3-fold greater than those in the pulp in organ cultures. Both SCAP and DPSCs were as potent in osteo/dentinogenic differentiation as MSCs from bone marrows, whereas they were weaker in adipogenic potential. The immunophenotype of SCAP is similar to that of DPSCs on the osteo/dentinogenic and growth factor receptor gene profiles. Double-staining experiments showed that STRO-1 coexpressed with dentinogenic markers such as bone sialophosphoprotein, osteocalcin, and growth factors FGFR1 and TGFβRI in cultured SCAP. Additionally, SCAP express a wide variety of neurogenic markers such as nestin and neurofilament M upon stimulation with a neurogenic medium. We conclude that SCAP are similar to DPSCs but a distinct source of potent dental stem/progenitor cells. Their implications in root development and apexogenesis are discussed.

DOI： 10.1016/j.joen.2007.11.021

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Mesenchymal stem cells (MSCs) have been isolated from the pulp tissue of permanent teeth (dental pulp stem cells or DPSCs) and deciduous teeth (stem cells from human exfoliated deciduous teeth). We recently discovered another type of MSCs in the apical papilla of human immature permanent teeth termed stem cells from the apical papilla (SCAP). Here, we further characterized the apical papilla tissue and stem cell properties of SCAP using histologic, immunohistochemical, and immunocytofluorescent analyses. We found that the apical papilla is distinctive to the pulp in terms of containing less cellular and vascular components than those in the pulp. Cells in the apical papilla proliferated 2- to 3-fold greater than those in the pulp in organ cultures. Both SCAP and DPSCs were as potent in osteo/dentinogenic differentiation as MSCs from bone marrows, whereas they were weaker in adipogenic potential. The immunophenotype of SCAP is similar to that of DPSCs on the osteo/dentinogenic and growth factor receptor gene profiles. Double-staining experiments showed that STRO-1 coexpressed with dentinogenic markers such as bone sialophosphoprotein, osteocalcin, and growth factors FGFR1 and TGFβRI in cultured SCAP. Additionally, SCAP express a wide variety of neurogenic markers such as nestin and neurofilament M upon stimulation with a neurogenic medium. We conclude that SCAP are similar to DPSCs but a distinct source of potent dental stem/progenitor cells. Their implications in root development and apexogenesis are discussed. © 2008 American Association of Endodontists.

DOI： 10.1016/j.joen.2007.11.021

記述言語：日本語  

P41. Cystatin C stimulates the osteogenic differentiation of human bone marrow mesenchymal stem cells(Poster Presentation,The 5^<th> Annual Meeting of Japanese Association of Regeneration Dentistry)

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cystatin C (CysC) is a natural cysteine proteinase inhibitor that suppresses the differentiation and bone-resorptive function of osteoclasts. By contrast, the effect of CysC on the differentiation and bone-formative function of osteoblasts has not been elucidated thoroughly. We examined the effects of CysC on mouse osteoblastic cells using in vitro cultures from bone marrow and calvaria and ex vivo calvarial cultures. CysC-stimulated cells showed increased alkaline phosphatase (ALP) activity, mineralization of the new bone matrix, and calvarial bone formation. The cells treated with CysC immunodepleted by anti-CysC antibody (iCysC) and a chemical papain-like cysteine proteinase inhibitor, E-64, did not induce mineralization. Elevated mRNA levels of bone morphogenetic protein (BMP)-2, the differentiation marker osteocalcin, and a master osteogenic transcription factor, Runx2, were observed in CysC-treated cells. These results suggest that CysC affects the BMP signaling cascades in osteoblastic cells and then promotes osteoblast differentiation, mineralization, and bone formation.

DOI： 10.1016/j.bbrc.2007.06.028

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cystatin C stimulates the differentiation of mouse osteoblastic cells and bone formation.
Cystatin C (CysC) is a natural cysteine proteinase inhibitor that suppresses the differentiation and bone-resorptive function of osteoclasts. By contrast, the effect of CysC on the differentiation and bone-formative function of osteoblasts has not been elucidated thoroughly. We examined the effects of CysC on mouse osteoblastic cells using in vitro cultures from bone marrow and calvaria and ex vivo calvarial cultures. CysC-stimulated cells showed increased alkaline phosphatase (ALP) activity, mineralization of the new bone matrix, and calvarial bone formation. The cells treated with CysC immunodepleted by anti-CysC antibody (iCysC) and a chemical papain-like cysteine proteinase inhibitor, E-64, did not induce mineralization. Elevated mRNA levels of bone morphogenetic protein (BMP)-2, the differentiation marker osteocalcin, and a master osteogenic transcription factor, Runx2, were observed in CysC-treated cells. These results suggest that CysC affects the BMP signaling cascades in osteoblastic cells and then promotes osteoblast differentiation, mineralization, and bone formation.

DOI： 10.1016/j.bbrc.2007.06.028

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Hertwig's epithelial root sheath (HERS) cells are a unique population of epithelial cells in the periodontal ligament compartment. To date, their functional role has not been fully elucidated. Our hypothesis was that HERS cells may be involved in regulating differentiation of periodontal ligament stem cells (PDLSCs) and forming cementum in vivo. In this study, we found that HERS cells may be capable of promoting PDLSC differentiation and undergoing epithelial-mesenchymal transition in vitro. Immunohisto-chemical staining, Western blot analysis, a transwell co-culture system, and in vivo transplantation were used to characterize the interplay between HERS cells and PDLSCs, as well as the epithelial-mesenchymal transition (EMT) of HERS cells. TGFβ1 was capable of inducing the epithelial-mesenchymal transition of HERS cells through activating the PI3K/AKT pathway. Furthermore, HERS cells were able to form cementum-like tissue when transplanted into immunocompromised mice. Abbreviations: bone marrow mesenchymal stem cell, BMMSC; bone sialoprotein, BSP; hydroxyapatite/tricalcium phosphate, HA/TCP; Hertwig's epithelial root sheath, HERS; osteocalcin, OCN; periodontal ligament, PDL; periodontal ligament stem cell, PDLSC; phosphatidylinositol 3-kinase, PI3K.

DOI： 10.1177/154405910708600703

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Hertwig's epithelial root sheath (HERS) cells are a unique population of epithelial cells in the periodontal ligament compartment. To date, their functional role has not been fully elucidated. Our hypothesis was that HERS cells may be involved in regulating differentiation of periodontal ligament stem cells (PDLSCs) and forming cementum in vivo. In this study, we found that HERS cells may be capable of promoting PDLSC differentiation and undergoing epithelial-mesenchymal transition in vitro. Immunohisto-chemical staining, Western blot analysis, a transwell co-culture system, and in vivo transplantation were used to characterize the interplay between HERS cells and PDLSCs, as well as the epithelial-mesenchymal transition (EMT) of HERS cells. TGFβ1 was capable of inducing the epithelial-mesenchymal transition of HERS cells through activating the PI3K/AKT pathway. Furthermore, HERS cells were able to form cementum-like tissue when transplanted into immunocompromised mice. Abbreviations: bone marrow mesenchymal stem cell, BMMSC
bone sialoprotein, BSP
hydroxyapatite/tricalcium phosphate, HA/TCP
Hertwig's epithelial root sheath, HERS
osteocalcin, OCN
periodontal ligament, PDL
periodontal ligament stem cell, PDLSC
phosphatidylinositol 3-kinase, PI3K.

DOI： 10.1177/154405910708600703

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Nitric oxide (NO) stimulates osteoblast differentiation, but whether NO contributes to odontoblast differentiation during dentin repair is unknown. By using reverse transcription/polymerase chain reaction and immunostaining, we investigated the gene expression and/or immunolocalization of endothelial NO synthase (eNOS), inducible NOS (iNOS), and nitrotyrosine (a biomarker for NO-derived peroxinitrite), and alkaline phosphatase (ALP) and osteocalcin (early and terminal differentiation markers of odontoblasts, respectively) in dental pulp tissue after rat tooth preparation. At the early stage (1-3 days) post-preparation, markedly increased expression of iNOS and nitrotyrosine was found in odontoblasts and pulp cells beneath the cavity, whereas eNOS expression was significantly decreased. ALP mRNA expression was significantly increased after 1 day but decreased after 3 days, whereas ALP activity was weak in the dentin-pulp interface under the cavity after 1 day but strong after 3 days. Osteocalcin mRNA expression was significantly increased at this stage. At 7 days post-preparation, tertiary dentin was formed under the cavity. All the molecules studied were expressed at control levels in odontoblasts/pulp cells beneath the cavity. These findings show that abundant NO is released from odontoblasts and pulp cells at an early stage after tooth preparation and indicate that, after tooth preparation, the up-regulation of iNOS and nitrotyrosine in odontoblasts is synchronized with increased cellular expression of ALP and osteocalcin. Therefore, the NO synthesized by iNOS after tooth preparation probably participates in regulating odontoblast differentiation during tertiary dentinogenesis.

DOI： 10.1007/s00441-005-0003-5

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Mesenchymal stem cells (MSCs) are able to differentiate into a variety of cell types, offering promising approaches for stem cell-mediated tissue regeneration. Here, we explored the potential of utilizing MSCs to reconstruct orofacial tissue, thereby altering the orofacial appearance. We demonstrated that bone marrow MSCs were capable of generating bone structures and bone-associated marrow elements on the surfaces of the orofacial bone. This resulted in significant recontouring of the facial appearance in mouse and swine. Notably, the newly formed bone and associated marrow tissues integrated with the surfaces of the recipient bones and re-established a functional bone marrow organ-like system. These data suggested that MSC-mediated tissue regeneration led to a body structure extension, with the re-establishment of all functional components necessary for maintaining the bone and associated marrow organ. In addition, we found that the subcutaneous transplantation of another population of MSCs, the human periodontal ligament stem cells (PDLSCs), could form substantial amounts of collagen fibers and improve facial wrinkles in mouse. By contrast, bone marrow MSCs failed to survive at 8 weeks post-transplantation under the conditions used for the PDLSC transplantation. This study suggested that the mutual interactions between donor MSCs and recipient microenvironment determine long-term outcome of the functional tissue regeneration. Disclosure of potential conflicts of interest is found at the end of this article.

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Sequential expression of endothelial nitric oxide synthase, inducible nitric oxide synthase, and nitrotyrosine in odontoblasts and pulp cells during dentin repair after tooth preparation in rat molars.
Nitric oxide (NO) stimulates osteoblast differentiation, but whether NO contributes to odontoblast differentiation during dentin repair is unknown. By using reverse transcription/polymerase chain reaction and immunostaining, we investigated the gene expression and/or immunolocalization of endothelial NO synthase (eNOS), inducible NOS (iNOS), and nitrotyrosine (a biomarker for NO-derived peroxinitrite), and alkaline phosphatase (ALP) and osteocalcin (early and terminal differentiation markers of odontoblasts, respectively) in dental pulp tissue after rat tooth preparation. At the early stage (1-3 days) post-preparation, markedly increased expression of iNOS and nitrotyrosine was found in odontoblasts and pulp cells beneath the cavity, whereas eNOS expression was significantly decreased. ALP mRNA expression was significantly increased after 1 day but decreased after 3 days, whereas ALP activity was weak in the dentin-pulp interface under the cavity after 1 day but strong after 3 days. Osteocalcin mRNA expression was significantly increased at this stage. At 7 days post-preparation, tertiary dentin was formed under the cavity. All the molecules studied were expressed at control levels in odontoblasts/pulp cells beneath the cavity. These findings show that abundant NO is released from odontoblasts and pulp cells at an early stage after tooth preparation and indicate that, after tooth preparation, the up-regulation of iNOS and nitrotyrosine in odontoblasts is synchronized with increased cellular expression of ALP and osteocalcin. Therefore, the NO synthesized by iNOS after tooth preparation probably participates in regulating odontoblast differentiation during tertiary dentinogenesis.

DOI： 10.1007/s00441-005-0003-5

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Mesenchymal stem cells (MSCs) are able to differentiate into a variety of cell types, offering promising approaches for stem cell-mediated tissue regeneration. Here, we explored the potential of utilizing MSCs to reconstruct orofacial tissue, thereby altering the orofacial appearance. We demonstrated that bone marrow MSCs were capable of generating bone structures and bone-associated marrow elements on the surfaces of the orofacial bone. This resulted in significant recontouring of the facial appearance in mouse and swine. Notably, the newly formed bone and associated marrow tissues integrated with the surfaces of the recipient bones and re-established a functional bone marrow organ-like system. These data suggested that MSC-mediated tissue regeneration led to a body structure extension, with the re-establishment of all functional components necessary for maintaining the bone and associated marrow organ. In addition, we found that the subcutaneous transplantation of another population of MSCs, the human periodontal ligament stem cells (PDLSCs), could form substantial amounts of collagen fibers and improve facial wrinkles in mouse. By contrast, bone marrow MSCs failed to survive at 8 weeks post-transplantation under the conditions used for the PDLSC transplantation. This study suggested that the mutual interactions between donor MSCs and recipient microenvironment determine long-term outcome of the functional tissue regeneration.

DOI： 10.1634/stemcells.2006-0576

記述言語：英語  

DOI： 10.1002/9780470167526.ch8

記述言語：英語  

DOI： 10.1002/9780470167526.ch8

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Mesenchymal stem cell-mediated tissue regeneration is a promising approach for regenerative medicine for a wide range of applications. Here we report a new population of stem cells isolated from the root apical papilla of human teeth (SCAP, stem cells from apical papilla). Using a minipig model, we transplanted both human SCAP and periodontal ligament stem cells (PDLSCs) to generate a root/periodontal complex capable of supporting a porcelain crown, resulting in normal tooth function. This work integrates a stem cell-mediated tissue regeneration strategy, engineered materials for structure, and current dental crown technologies. This hybridized tissue engineering approach led to recovery of tooth strength and appearance.

DOI： 10.1371/journal.pone.0000079

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Mesenchymal stem cell-mediated tissue regeneration is a promising approach for regenerative medicine for a wide range of applications. Here we report a new population of stem cells isolated from the root apical papilla of human teeth (SCAP, stem cells from apical papilla). Using a minipig model, we transplanted both human SCAP and periodontal ligament stem cells (PDLSCs) to generate a root/periodontal complex capable of supporting a porcelain crown, resulting in normal tooth function. This work integrates a stem cell-mediated tissue regeneration strategy, engineered materials for structure, and current dental crown technologies. This hybridized tissue engineering approach led to recovery of tooth strength and appearance. © 2006 Sonoyama et al.

DOI： 10.1371/journal.pone.0000079

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The craniofacial region contains many specified tissues including bone, cartilage, muscle, blood vessels and neurons. Defect or dysfunction of the craniofacial tissue after post-cancer ablative surgery, trauma, congenital malformations and progressive deforming skeletal diseases has a huge influence on the patient's life. Therefore, functional reconstruction of damaged tissues is highly expected. Bone marrow-derived mesenchymal stem cells (BMMSCs) are one of the most well characterized postnatal stem cell populations, and considered to be utilized for cell-based clinical therapies. Here, the current understanding and the potential applications in craniofacial tissue regeneration of BMMSCs are reviewed, and the current limitations and drawbacks are also discussed.

DOI： 10.1111/j.1601-0825.2006.01300.x

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The craniofacial region contains many specified tissues including bone, cartilage, muscle, blood vessels and neurons. Defect or dysfunction of the craniofacial tissue after post-cancer ablative surgery, trauma, congenital malformations and progressive deforming skeletal diseases has a huge influence on the patient's life. Therefore, functional reconstruction of damaged tissues is highly expected. Bone marrow-derived mesenchymal stem cells (BMMSCs) are one of the most well characterized postnatal stem cell populations, and considered to be utilized for cell-based clinical therapies. Here, the current understanding and the potential applications in craniofacial tissue regeneration of BMMSCs are reviewed, and the current limitations and drawbacks are also discussed.

DOI： 10.1111/j.1601-0825.2006.01300.x

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Experimentally, temporomandibular joint (TMJ) nerve units respond to capsaicin, which is used clinically to treat TMJ pain. However, the existence of capsaicin receptors in the TMJ has not previously been clearly demonstrated. Immunohistochemical analysis has revealed the presence of transient receptor potential vanilloid subtype 1 (TRPV1) expression in the nerves and synovial lining cells of the TMJ. TRPV1-immunoreactive nerves are distributed in the synovial membrane of the joint capsule and provide branches to the joint compartment. The disc periphery is supplied by TRPV1 nerves that are mostly associated with small arterioles, and occasional nerves penetrate to the synovial lining layer. Double immunofluorescence has shown that many TRPV1-immunoreactive nerves are labeled with neuropeptide calcitonin gene-related peptide, whereas few are labeled with IB4-lectin. The results provide evidence for the presence of TRPV1 in both nerves and synovial lining cells, which might thus be involved in the mechanism of nociception and inflammation in the TMJ.

DOI： 10.1007/s00441-006-0183-7

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Capsaicin receptor expression in the rat temporomandibular joint.
Experimentally, temporomandibular joint (TMJ) nerve units respond to capsaicin, which is used clinically to treat TMJ pain. However, the existence of capsaicin receptors in the TMJ has not previously been clearly demonstrated. Immunohistochemical analysis has revealed the presence of transient receptor potential vanilloid subtype 1 (TRPV1) expression in the nerves and synovial lining cells of the TMJ. TRPV1-immunoreactive nerves are distributed in the synovial membrane of the joint capsule and provide branches to the joint compartment. The disc periphery is supplied by TRPV1 nerves that are mostly associated with small arterioles, and occasional nerves penetrate to the synovial lining layer. Double immunofluorescence has shown that many TRPV1-immunoreactive nerves are labeled with neuropeptide calcitonin gene-related peptide, whereas few are labeled with IB4-lectin. The results provide evidence for the presence of TRPV1 in both nerves and synovial lining cells, which might thus be involved in the mechanism of nociception and inflammation in the TMJ.

DOI： 10.1007/s00441-006-0183-7

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The dentin-pulp complex is a peripheral end-organ supplied by dense sensory nerve fibers. Substance P, a representative neuropeptide widely distributed in the dental pulp, has been reported to play roles in pain transmission and the amplification of inflammation. We analyzed here the expression of the neurokinin 1 (NK1) receptor, preferentially activated by substance P, using immunocytochemistry in rat dental pulp at both the light and electron microscopic levels. Conspicuous NK1 receptor immunoreactivity was found in the odontoblasts; immunolabelings were present at their plasma membrane and endosomal structures, especially in their cytoplasmic processes. Immunoreactions for NK1 receptor were also detectable in a part of the nerve terminals associated with the cytoplasmic processes of the odontoblasts. Furthermore, the endothelial cells of capillaries and post-capillary venules and the fibroblasts were labeled with the NK1 receptor in the subodontoblast layer. These findings suggest that pulpal cells and nerve fibers are targets for substance P that mediate multiple functions, including a vasoactive function and the regulation of vascular permeability as well as the modulation of pain transmission.

DOI： 10.1679/aohc.68.259

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Immunocytochemical localization of the neurokinin 1 receptor in rat dental pulp.
The dentin-pulp complex is a peripheral end-organ supplied by dense sensory nerve fibers. Substance P, a representative neuropeptide widely distributed in the dental pulp, has been reported to play roles in pain transmission and the amplification of inflammation. We analyzed here the expression of the neurokinin 1 (NK1) receptor, preferentially activated by substance P, using immunocytochemistry in rat dental pulp at both the light and electron microscopic levels. Conspicuous NK1 receptor immunoreactivity was found in the odontoblasts; immunolabelings were present at their plasma membrane and endosomal structures, especially in their cytoplasmic processes. Immunoreactions for NK1 receptor were also detectable in a part of the nerve terminals associated with the cytoplasmic processes of the odontoblasts. Furthermore, the endothelial cells of capillaries and post-capillary venules and the fibroblasts were labeled with the NK1 receptor in the subodontoblast layer. These findings suggest that pulpal cells and nerve fibers are targets for substance P that mediate multiple functions, including a vasoactive function and the regulation of vascular permeability as well as the modulation of pain transmission.

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Laminin-5 (Ln-5) is an important molecule associated with epithelial cell adhesion and migration. In the gingiva around the tooth, Ln-5 localizes within basement membranes between the junctional epithelium (JE) and the tooth or connective tissue. Recently, we reported that in the oral mucosa around a dental implant, Ln-5 is expressed within the basement membranes at the implant-peri-implant epithelium (PIE) interface, and at the PIE-connective tissue interface. However, the ultrastructural localization of Ln-5 within or along the PIE has not yet been reported. Therefore, peri-implant oral mucosa was treated with anti-Ln-5 (γ2 chain) antibody and examined using immuno-electron microscopy. Ln-5 was localized in the cells of the innermost-third layer and basal layer of the PIE. A 100-nm-wide Ln-5-positive internal basal lamina (basement membrane) and hemidesmosomes as adhesion structures were formed at the apical portion of the implant-PIE interface. However, at the upper-middle portion of the interface, these adhesion structures were not observed. Furthermore, at the PIE-connective tissue interface, the Ln-5-positive external basal lamina (basement membrane) and hemidesmosomes were partially deficient. Judging from these findings, we concluded that Ln-5 contributes to the attachment of the PIE to the titanium surface, and that PIE attached to titanium at the apical portion of the dental implant-PIE interface.

DOI： 10.1016/j.biomaterials.2005.03.046

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Laminin-5 (Ln-5), a component of the basement membrane (BM), regulates epithelial cell migration and adhesion. This study used anti-Ln-5 (γ2chain) antibody to investigate the distribution of Ln-5 during the formation of peri-implant epithelium (PIE) in rats, and compared it to the distribution of Ln-5 during oral mucosa formation after tooth extraction. One day after extraction, the junctional epithelium (JE) had disappeared. After 3 days, new epithelium formed from the oral sulcular epithelium (OSE) and extended horizontally over the wound with Ln-5-positive cells at the leading edge. After 5 days, the epithelium extending from the OSE on each side of the wound joined and formed additional new epithelium. The new epithelium expressed Ln-5 in the BM. After 1-2 weeks, the oral epithelium (OE) extending from the sides of the wound joined in the center. Thereafter, OSE and new epithelium disappeared, and only OE remained covering the wound. Three days after implantation (titanium), no JE remained. New epithelium formed from the keratinized OSE extending apically with Ln-5-positive cells. After 1-2 weeks, the new epithelium became the PIE and spread further apically facing the implant surface. Ln-5 was expressed at the PIE-connective tissue interface, but not at the implant-PIE interface. Finally, after 4 weeks, Ln-5 was expressed at the implant-PIE interface, and the PIE was non-keratinized epithelium. These findings suggest that Ln-5 induces cell migration during PIE formation, and that PIE originates from OSE. Furthermore, they support the hypothesis that Ln-5 contributes to the attachment of PIE to titanium, regardless of the delay in the synthesis and deposition of Ln-5 at the titanium-PIE interface.

DOI： 10.1016/j.biomaterials.2004.05.033

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The junctional epithelium (JE) is a primary site of defense against periodontal pathogens. Cystatin C is an endogenous inhibitor of cysteine proteinases such as cathepsin B and also has antibacterial actions against periodontal pathogens. However, the distribution and role of cystatin C in JE have not been clarified. To investigate the function of cystatin C in the host defense at dentogingival junction, we examined the immunolocalization of cystatin C and cathepsin B in rat gingiva using light and electron microscopy. The JE (particularly the coronal portion) was immunopositive for cystatin C, and immunoelectron microscopy revealed that cystatin C was localized to the vesicular, granular, and vacuolar compartments of JE cells. The pattern of cathepsin B immunoreactivity in JE cells resembled that of cystatin C. Both cystatin C and cathepsin B appeared to be localized to endosomal/lysosomal compartments within JE cells. These findings suggest that cystatin C regulates cysteine proteinase activity and exerts antibacterial effects both in the lysosomal compartments of JE cells and in the intercellular spaces of the JE. Cystatin C is thus able to participate in host defense against periodontal pathogens at the dentogingival junction.

DOI： 10.1267/ahc.38.121

記述言語：英語   掲載種別：研究論文（学術雑誌）  

We examined, in rats, the expression of osteocalcin and Jun D in the early stage of reactionary dentin formation after tooth preparation and the accompanying morphological changes. Reverse transcription/polymerase chain reaction analysis revealed strong expression of osteocalcin mRNA in pulp tissue at 2 and 3 days post-preparation compared with that in control teeth. Light microscopy demonstrated that, at the dentin-pulp interface, damaged odontoblasts were detached from the dentin matrix immediately after preparation, with neutrophils lining the dental surface after 1 day. After 2-3 days, differentiated odontoblasts appeared at the interface. Reactionary dentin with tubular structures was formed under the cavity after 10 days. Immunoelectron microscopy showed that trace amounts of osteocalcin were expressed in odontoblasts at 2 days post-preparation, and abundant osteocalcin was found in the highly developed Golgi apparatus and granules at 3 days post-preparation. Osteocalcin was also found on type I collagen fibrils in newly formed predentin. The existing dentinal tubules were filled with osteocalcin-coated type I collagen fibrils. We observed, by immnohistochemistry, that Jun D was temporally expressed in the nuclei of the odontoblasts at 1 and 2 days post-preparation. However, no Jun D was found in the dental pulp cells at any other time or in control teeth. Thus, osteocalcin expression is correlated with reactionary dentin formation, and Jun D is associated with osteocalcin expression in odontoblasts. Osteocalcin may also serve as an obturator of the dentinal tubules to protect dental pulp vitality against external irritants after preparation.

DOI： 10.1007/s00441-004-1035-y

記述言語：英語   掲載種別：研究論文（学術雑誌）  

We examined, in rats, the expression of osteocalcin and Jun D in the early stage of reactionary dentin formation after tooth preparation and the accompanying morphological changes. Reverse transcription/ polymerase chain reaction analysis revealed strong expression of osteocalcin mRNA in pulp tissue at 2 and 3 days post-preparation compared with that in control teeth. Light microscopy demonstrated that, at the dentin pulp interface, damaged odontoblasts were detached from the dentin matrix immediately after preparation, with neutrophils lining the dental surface after 1 day. After 2-3 days, differentiated odontoblasts appeared at the interface. Reactionary dentin with tubular structures was formed under the cavity after 10 days. Immunoelectron microscopy showed that trace amounts of osteocalcin were expressed in odontoblasts at 2 days post-preparation, and abundant osteocalcin was found in the highly developed Golgi apparatus and granules at 3 days postpreparation. Osteocalcin was also found on type I collagen fibrils in newly formed predentin. The existing dentinal tubules were filled with osteocalcin-coated type I collagen fibrils. We observed, by immnohistochemistry, that Jun D was temporally expressed in the nuclei of the odontoblasts at 1 and 2 days post-preparation. However, no Jun D was found in the dental pulp cells at any other time or in control teeth. Thus, osteocalcin expression is correlated with reactionary dentin formation, and Jun D is associated with osteocalcin expression in odontoblasts. Osteocalcin may also serve as an obturator of the dentinal tubules to protect dental pulp vitality against external irritants after preparation.

DOI： 10.1007/s00441-004-1035-y

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Many materials with differing surfaces have been developed for clinical implant therapy in dentistry and orthopedics. We analyzed the quantity of new bone formed in vivo around calcium-immobilized titanium implants with surfaces modified using pamidronate (PAM), a nitrogen-containing bisphosphonate (N-BP), implants of pure titanium, and titanium implants immobilized with calcium ions. New bone formation was visualized using fluorescent labeling (calcein blue and alizarin complexone) with intravenous injection at 1 and 3 weeks after implantation. After 4 weeks, undecalcified sections were prepared, and new bone formation around the implants was examined by morphometry using confocal laser scanning microscopy images. After 1 week, more new bone formed around the PAM-immobilized implant than around the calcium-immobilized and pure titanium implants. This was also seen with the new bone formation after 3 weeks. After 4 weeks, significantly more new bones were formed around the BP-immobilized implant than around the calcium ion-implanted and pure titanium implants. The new N-BP-modified titanium surface stimulates new bone formation around the implant, which might contribute to the success of implant therapy.

DOI： 10.1016/j.biomaterials.2004.02.072

記述言語：日本語  

記述言語：日本語  

記述言語：日本語  

記述言語：日本語  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Synovial hyperplasia is a feature of degenerative temporomandibular joint (TMJ) disease. However, the mechanism by which hyperplasia progresses in the TMJ is unknown. Based on the hypothesis that the oxidative stress generated by mechanical loading causes degenerative changes in the TMJ synovium, we investigated the generation of the highly reactive species, peroxynitrite, and the occurrence of DNA damage in the synovium. After condylar hypermobility of rat TMJs, a marker of peroxynitrite, nitrotyrosine, was localized to the nuclei and cytoplasm of the synovial lining cells and fibroblasts in synovitis-induced TMJ. DNA single-strand breaks were found in the nuclei of the synovial cells only after enzyme treatment, whereas DNA double-strand breaks were not detected. These findings indicate that condylar hypermovement induces the proliferation of synovial cells, and suggest that oxidative stress leads to the progression of synovial hyperplasia via DNA damage of the synovial cells in TMJs after mechanical loading.

DOI： 10.1177/154405910408300807

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Synovial hyperplasia is a feature of degenerative temporomandibular joint (TMJ) disease. However, the mechanism by which hyperplasia progresses in the TMJ is unknown. Based on the hypothesis that the oxidative stress generated by mechanical loading causes degenerative changes in the TMJ synovium, we investigated the generation of the highly reactive species, peroxynitrite, and the occurrence of DNA damage in the synovium. After condylar hypermobility of rat TMJs, a marker of peroxynitrite, nitrotyrosine, was localized to the nuclei and cytoplasm of the synovial lining cells and fibroblasts in synovitis-induced TMJ. DNA single-strand breaks were found in the nuclei of the synovial cells only after enzyme treatment, whereas DNA double-strand breaks were not detected. These findings indicate that condylar hypermovement induces the proliferation of synovial cells, and suggest that oxidative stress leads to the progression of synovial hyperplasia via DNA damage of the synovial cells in TMJs after mechanical loading.

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The mechanism by which bone collagen and other organic components are degraded by the osteoclast during osteoclastic bone resorption was unclear until the 1980s. Studies conducted since the early 1990s have identified lysosomal proteases, mainly cathepsins that are active at low pH, involved in osteoclastic bone resorption. Several cathepsins, such as cathepsins C, D, B, E, G and L, were initially demonstrated to take part in the degradation of organic bone matrix in osteoclasts. Cathepsin K, which has high proteolytic activity and localizes primarily in osteoclasts, was discovered in 1995. This first tissue-specific cathepsin was associated with pycnodysostosis, a genetic disorder observable as an osteopetrotic phenotype in cathepsin K-deficient mice. Cystatin C, an endogenous inhibitor of cysteine proteases, regulates the activity of cathepsin K. However, detailed morphological observations suggest that the organic bone matrix is degraded by not only cathepsin K, but also by matrix metalloproteinases or other cathepsins. The osteoclast possesses a unique endocytotic/exocytotic structure and each cathepsin is specifically localized in the osteoclast, which implies that each cathepsin contributes cooperatively to the process of osteoclastic bone resorption. Further studies may clarify the regulation of cathepsin activities and the roles of cathepsins during bone remodelling.

DOI： 10.1093/jmicro/52.6.551

記述言語：日本語  

記述言語：英語  

Capsaicin, the pungent substance in hot peppers, evokes a sensation of burning pain by stimulating the vanilloid receptor 1 (VR1) on primary afferent neurons. Immunohistochemistry revealed that the taste papillae in the tongue and palate are richly innervated by VR1-immunoreactive nerve fibers. Furthermore, VR1 protein expression was seen in the epithelium facing the oral cavity, although taste cells seemed to be devoid of VR1. The most conspicuous VR1 expression was observed in the epithelial cells of the palatal rugae, although there were no VR1-immunoreactive nerves there. The finding that VR1 is expressed not only in primary afferents but also in oral epithelial cells suggests that it is of great importance in the perception of capsaicin, heat, and acid in the mouth. Since VR1 is known to play a key role in nociception and inflammatory pain, it may be a new target for the treatment of oral pain.

記述言語：英語   掲載種別：研究論文（その他学術会議資料等）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Capsaicin, the pungent substance in hot peppers, evokes a sensation of burning pain by stimulating the vanilloid receptor 1 (VR1) on primary afferent neurons. Immunohistochemistry revealed that the taste papillae in the tongue and palate are richly innervated by VR1-immunoreactive nerve fibers. Furthermore, VR1 protein expression was seen in the epithelium facing the oral cavity, although taste cells seemed to be devoid of VR1. The most conspicuous VR1 expression was observed in the epithelial cells of the palatal rugae, although there were no VR1-immunoreactive nerves there. The finding that VR1 is expressed not only in primary afferents but also in oral epithelial cells suggests that it is of great importance in the perception of capsaicin, heat, and acid in the mouth. Since VR1 is known to play a key role in nociception and inflammatory pain, it may be a new target for the treatment of oral pain.

DOI： 10.1177/154405910308200513

記述言語：英語   掲載種別：研究論文（学術雑誌）  

NF-κB plays a pivotal role in pathogenesis in general arthritis. However, the participation of NF-κB in inflammation of the temporomandibular joint (TMJ) is poorly understood. We examined NF-κB expression in rat TMJs with synovitis induced by condyle hypermobility. By immunohistochemistry, NF-κB immunoreactivity was found mainly in the cytoplasm, not the nucleus, of the synovial lining cells of induced-synovitis and control TMJs. Southwestern histochemistry, a new method for detecting transcription factors, showed greater NF-κB expression in the nucleus of the synovial lining cells in the hypertrophic synovium than in control synovium. Increased numbers of the synovial lining cells with immunoreactivity for inducible nitric oxide synthase (iNOS), which is transcriptionally regulated by NF-κB, were also seen in the inflamed synovium. These findings indicate that excess mechanical stress increases NF-κB activation in the TMJ and suggest that active NF-κB is involved in the progression of TMJ inflammation.

DOI： 10.1177/154405910308200307

記述言語：英語  

NF-kB Activation and iNOS Expression in the Synovial Membrane of Rat Temporomandibular Joints after Induced Synovitis

記述言語：英語   掲載種別：研究論文（学術雑誌）  

This review summarizes the biological properties of the junctional epithelium, focusing on its developmental aspects, wide intercellular spaces and desmosomes, dense granules, permeability barrier, phagocytotic activity, adhesive structures and nerve terminals. It also discusses the morphology and functions of long junctional epithelium and peri-implant epithelium. Junctional epithelium is derived from the reduced enamel epithelium during tooth development. Apoptosis occurs in the border between oral and reduced enamel epithelia during tooth eruption. Junctional epithelium expresses a cytokeratin-19 immunoreaction, suggesting that this protein is a consistent differentiation marker. Wide intercellular spaces, which contain neutrophils and nerve endings, are formed as there are fewer desmosomes than in the oral epithelium. Dense, membrane-bound granules in the epithelium might correspond with membrane-coating granules, as revealed by their shape, components and freeze-fracture images. Junctional epithelium with high permeability contains exogenously expressed α-defensins, while stratified epithelia contain endogenously expressed β-defensins. The phagocytotic activity in this epithelium remains unclear. Integrin-α6β4 and laminin-5 form a complex in the tooth surface internal basal lamina. Long junctional epithelium created experimentally attaches to the cementum surface by hemidesmosomes and basal lamina. The peri-implant epithelium differs in proliferation and in adhesive structure from the normal junctional epithelium. In conclusion, wide intercellular spaces and poorly developed desmosomes are closely correlated with a permeable nature. There is still uncertainty over the phagocytotic activity of the epithelium. Integrin-α6β4 and laminin-5 form a significant complex in the internal basal lamina. Junctional epithelium receives a rich sensory nerve and has a high rate of cell turnover. Long junctional epithelium can be produced rapidly during wound healing, due to high proliferative activity. Peri-implant epithelium might be a poorly adhered and permeable epithelium.

DOI： 10.1093/jmicro/52.6.627

記述言語：日本語  

記述言語：日本語  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Horseradish peroxidase (HRP) tracer was applied to the gingival sulcus of implants or natural teeth at 5, 25, or 50 mg/ml to investigate the sealing capacities of the peri-implant epithelium (PIE) and junctional epithelium (JE); the extent of HRP penetration was observed under electron microscopy. A Ti-6Al-4V implant was inserted either immediately (immediate implantation) or 2 weeks (delayed implantation) after extraction of the maxillary left first molar of rats. The JE of the right molar was used as a control. Although the whole PIE of undecalcified sections appeared to be attached to the implant surface, electron microscopically, the internal basement lamina (IBL) and hemidesmosomes were deficient in the coronal-middle region of the PIE. There were extensive extracellular deposits of HRP in the intercellular spaces between PIE cells, and HRP was blocked to some extent by the lamina lucida and lamina densa of the external basal lamina and basal cell junction. HRP was detected in the connective tissue under the PIE, but was not found in the connective tissue under the JE. Intracellularly, HRP was found in the vesicles and granules of PIE cells and JE cells. These were fewer in number in PIE cells than in JE cells. There were no differences between the findings for immediate and delayed implantation. The results indicate that a deficiency in the IBL permitted penetration of HRP from the gingival sulcus into the connective tissue under the PIE, and suggest that the endocytotic capacity of PIE cells is inferior to that of JE cells.

DOI： 10.1034/j.1600-0501.2002.130303.x

記述言語：英語  

Horseradish peroxidase (HRP) tracer was applied to the gingival sulcus of implants or natural teeth at 5, 25, or 50 mg/ml to investigate the sealing capacities of the periimplant epithelium (PIE) and junctional epithelium (JE); the extent of HRP penetration was observed under electron microscopy. A Ti-6Al-4V implant was inserted either immediately (immediate implantation) or 2 weeks (delayed implantation) after extraction of the maxillary left first molar of rats. The JE of the right molar was used as a control. Although the whole PIE of undecalcified sections appeared to be attached to the implant surface, electron microscopically, the internal basement lamina (IBL) and hemidesmosomes were deficient in the coronal-middle region of the PIE. There were extensive extracellular deposits of HRP in the intercellular spaces between PIE cells, and HRP was blocked to some extent by the lamina lucida and lamina densa of the external basal lamina and basal cell junction. HRP was detected in the connective tissue under the PIE, but was not found in the connective tissue under the JE. Intracellularly, HRP was found in the vesicles and granules of PIE cells and JE cells. These were fewer in number in PIE cells than in JE cells. There were no differences between the findings for immediate and delayed implantation. The results indicate that a deficiency in the IBL permitted penetration of HRP from the gingival sulcus into the connective tissue under the PIE, and suggest that the endocytotic capacity of PIE cells is inferior to that of JE cells.

DOI： 10.1034/j.1600-0501.2002.130303.x

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Nitric oxide (NO) is generated from L-arginine by NO synthase (NOS) and has multiple functions under both physiological and pathological conditions. One isoform of NOS, inducible NOS (iNOS) is expressed in the cells such as macrophages after induction with various cytokines or mechanical stress. In this study, we investigated the distribution of iNOS, interleukin-1β (IL-1β) and its receptor, the IL-1 receptor (IL-1R), in the synovial membrane of the temporomandibular joint (TMJ) of normal rats using immunolight and immunoelectron microscopy. By light microscopy, an immunopositive reaction for iNOS and IL-1β was found in the superficial cells of the synovial membrane of both the anterior and posterior portions of the articular disc. Immunoelectron microscopy revealed that iNOS-immunoreactive products were deposited in the cytoplasm and vesicles, and on the plasma membrane of type-A (macrophage-like) and B (fibroblast-like) cells of the superficial layer. IL-1R-positive products were found both on the plasma membrane and in the vesicles of type-A cells of the synovial lining, and were observed in macrophages in the sublining layer. These results reveal that iNOS and IL-1β localize to the synovial membrane of the rat TMJ under physiological conditions. Therefore, it is likely that autocrine/paracrine effects of IL-1β induce NO generation by iNOS via the IL-1R in type-A cells. It is considered that cytokine-induced NO may play an important role in the physiological maintenance, e.g. self-protection, by synovial lining cells of the synovial membrane in the TMJ.

DOI： 10.1267/ahc.35.11

記述言語：日本語  

顎関節炎モデルラットにおける反応性窒素酸化物の炎症増悪への関与

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Substance P (SP) is an important member of the tachykinin family of neuropeptides, which work as neurotransmitters or neuromodulators. Recent advances in the analysis of SP receptors, particularly the neurokinin-1 receptors (NK₁-Rs) that have high affinity for SP, have demonstrated that they are distributed not only in the cells of the neuronal or immune systems but also in peripheral cells. Therefore, the effect of SP and its cellular receptors is not limited to the nervous or immune systems, but is more extensive than previously appreciated. SP-like immunoreactive (SP-LI) axons have been localized in both bone and gingival tissue, and SP receptors are widely distributed in osteoclasts, osteoblasts, and junctional epithelial cells, as well as in neutrophils and endothelial cells. The distribution of SP-LI axons and SP receptors suggests that SP may directly modulate bone metabolism and gingival tissue functions through SP receptors.

DOI： 10.1007/s007950170001

記述言語：英語  

We compared the distribution of a cysteine proteinase inhibitor, cystatin C, with that of cathepsin K in osteoclasts of the mouse tibia by immunolight and immunoelectron microscopy. Light microscopically, strong immunoreactivity for cystatin C was found extracellularly along the resorption lacuna and intracellularly in the organelles of osteoclasts. In serial sections, various patterns of cystatin C and cathepsin K localization were seen, specifically: (1) some resorption lacuna were positive for both cystatin C and cathepsin K; (2) others were positive for either cystatin C or cathepsin K, but not both; and (3) some lacuna were negative for both. In osteoclasts, the localization of cystatin C was similar to that of cathepsin K. Furthermore, cystatin C immunoreactivity was detected in preosteoclasts and osteoblasts, whereas cathepsin K was seen only in preosteoclasts. Electron microscopically, cystatin C immunoreactive products were found in the rough endoplasmic reticulum (ER), Golgi apparatus, vesicles, granules, and vacuoles of osteoclasts. These cystatin C-positive vesicles had fused or were in the process of fusion with the ampullar vacuoles (extracellular spaces) containing cystatin C-positive, fragmented, fibril-like structures. The extracellular cystatin C was deposited on and between the cytoplasmic processes of ruffled borders, and on and between type I collagen fibrils. In the basolateral region of osteoclasts, cystatin C-positive vesicles and granules also fused with vacuoles that contained cystatin C-positive or negative fibril-like structures. These results indicate that osteoclasts not only synthesize and secrete cathepsin K from the ruffled border into the bone resorption lacunae, but also a cysteine proteinase inhibitor, cystatin C. Therefore, it is suggested that cystatin C regulates the degradation of bone matrix by cathepsin K, both extracellularly and intracellularly. (C) 2001 by Elsevier Science Inc. All rights reserved.

DOI： 10.1016/S8756-3282(01)00466-5

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The distribution and fine structure of nerve fibers containing neuropeptide Y (NPY), tyrosine hydroxylase (TH), and vasoactive intestinal polypeptide (VIP) in the temporomandibular joint were investigated by both the avidin-biotin complex method and an indirect immunofluorescence technique. The innervation pattern of NPY- and TH-positive fibers differed from that of VIP-positive fibers. Specifically, the former was distributed in both the superficial and deep sublining layers, while the latter was mostly located in the deep sublining layer. NPY- and TH-immunoreactive fibers were largely confined to vascular elements; occasional fibers were observed in the synovial lining layer close to the joint cavity. More nerves with NPY and TH immunoreactivity were observed close to the upper joint compartment than near the lower compartment NPY and TH immunoreactivity was dramatically reduced in the TMJ of superior cervical ganglionectomized animals, indicating the sympathetic origin of these nerves. NPY immunoreactivity was found only in unmyelinated axons, which were located in the adventitia and adventitia-medial border of arteries or arterioles. Occasionally, axons were near the joint cavity, in areas free of vascular structures. These observations show that abundant sympathetic nerves supply the temporomandibular joint of the rat and provide a morphological basis for the involvement of different neuropeptides in vascular regulatory and modulatory functions in physiological and pathophysiological conditions.

DOI： 10.1007/s004290100163

記述言語：英語  

The distribution and fine structure of nerve fibers containing neuropeptide Y (NPY), tyrosine hydroxylase (TH), and vasoactive intestinal polypeptide (VIP) in the temporomandibular joint were investigated by both the avidin-biotin complex method and an indirect immunofluorescence technique. The innervation pattern of NPY- and TH-positive fibers differed from that of VIP-positive fibers. Specifically, the former was distributed in both the superficial and deep sublining layers, while the latter was mostly located in the deep sublining layer. NPY- and TH-immunoreactive fibers were largely confined to vascular elements; occasional fibers were observed in the synovial lining layer close to the joint cavity. More nerves with NPY and TH immunoreactivity were observed close to the upper joint compartment than near the lower compartment NPY and TH immunoreactivity was dramatically reduced in the TMJ of superior cervical ganglionectomized animals, indicating the sympathetic origin of these nerves. NPY immunoreactivity was found only in unmyelinated axons, which were located in the adventitia and adventitia-medial border of arteries or arterioles. Occasionally, axons were near the joint cavity, in areas free of vascular structures. These observations show that abundant sympathetic nerves supply the temporomandibular joint of the rat and provide a morphological basis for the involvement of differ ent neuropeptides in vascular regulatory and modulatory functions in physiological and pathophysiological conditions.

DOI： 10.1007/s004290100163

記述言語：英語   掲載種別：研究論文（学術雑誌）  

This study demonstrated the simultaneous expression of cathepsin K (CK) mRNA by in situ hybridization and CK protein by immunoelectron microscopy in odontoclasts in mouse maxillae after experimental tooth movement. On the pressure side (the area under pressure during tooth movement), CK mRNA was detected in odontoclasts in resorption lacunae in the tooth root, in osteoclasts in bone resorption lacunae, and in fibroblasts in the periodontal ligament. Using electron microscopy, CK protein was detected at the apex of odontoclasts, intracellularly in vesicles and granules, and extracellularly in irregularly shaped vacuoles (extracellular spaces), on the plasma membrane of the ruffled border, and on and between typical striated type I collagen fibrils in the lacunae. These vesicles and granules appeared to fuse with irregular vacuoles containing CK-positive fragmented fibril-like structures close to the ruffled border. In the basolateral portion of odontoclasts, small amounts of CK-positive rough endoplasmic reticulum (ER) were found. CK-positive intracellular vacuoles (not extracellular spaces) also appeared to fuse with the vesicles and granules. However, these fused organelles rarely contained fragmented fibril-like structures. They are probably endolysosomes. The distribution of CK in odontoclasts was similar to that previously seen in osteoclasts. Furthermore, CK-positive fibril-like structures were found in the vacuoles of fibroblasts. These results indicated that during tooth movement CK is synthesized in odontoclasts on the pressure side and secreted into the tooth resorption lacunae. Therefore, CK may take part in the degradation of the dentin matrix (type I collagen fibrils and non-collagenous protein) of the tooth root, and in the subsequent intracellular degradation of endocytosed fragmented fibril-like structures in endolysosomes.

DOI： 10.1007/s004410000327

記述言語：日本語  

Immunohistochemical Localization of Osteoclast Differentiation Factor in Periradicular Tissue in Rat

記述言語：英語  

This study demonstrated the simultaneous expression of cathepsin K (CK) mRNA by in situ hybridization and CK protein by immunoelectron microscopy in odontoclasts in mouse maxillae after experimental tooth movement. On the pressure side (the area under pressure during tooth movement), CK mRNA was detected in odontoclasts in resorption lacunae in the tooth root, in osteoclasts in bone resorption lacunae, and in fibroblasts in the periodontal ligament. Using electron microscopy CK protein was detected at the apex of odontoclasts, intracellularly in vesicles and granules, and extracellularly in irregularly shaped vacuoles (extracellular spaces), on the plasma membrane of the ruffled border, and on and between typical striated type I collagen fibrils in the lacunae. These vesicles and granules appeared to fuse with irregular vacuoles containing CK-positive fragmented fibril-like structures close to the ruffled border. Fn the basolateral portion of odontoclasts, small amounts of CK-positive rough endoplasmic reticulum (ER) were found. CK-positive intracellular vacuoles (not extracellular spaces) also appeared to fuse with the vesicles and granules. However, these fused organelles rarely contained fragmented fibril-like structures. They are probably endolysosomes. The distribution of CK in odontoclasts was similar to that previously seen in osteoclasts. Furthermore, CK-positive fibril-like structures were found in the vacuoles of fibroblasts. These results indicated that during tooth movement CK is synthesized in odontoclasts on the pressure side and secreted into the tooth resorption lacunae. Therefore, CK may take part in the degradation of the dentin matrix (type I collagen fibrils and non-collagenous protein) of the tooth root, and in the subsequent intracellular degradation of endocytosed fragmented fibril-like structures in endolysosomes.

DOI： 10.1007/s004410000327

記述言語：英語   掲載種別：研究論文（学術雑誌）  

An epidemiologic examination was carried out to reveal the prevalence of the periodontal diseases and oral pigmentation in patients with Yusho in 2000. The results obtained were as follows. 1) 63 patients out of 69 patients with Yusho, who were measured periodontal pocket depth according to Ramfjord' methods, had at least one tooth with periodontal pocket deeper than 3 mm. Similarly, 188 teeth out of a total 285 examined teeth showed periodontal pocket with more than 3 mm depth. 2) In this examination, intraoral sinus tracts stoma were observed in 9 patients out of 70 patients. Radiographic examination and probing examination of pocket depth indicated that periapical lesions were involved in these intraoral sinus tract formation. 3) Oral pigmentation was observed in 46 out of 76 patients with Yusho. In this study, gingival pigmentation was most predominant among oral pigmentation. These results indicated that PCBs had yet affected the mechanism of oral pigmentation and metabolism of alveolar bone.

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Axons containing substance P (SP) serve bone tissue, however, the role of SP in bone metabolism, particularly on osteoclastic bone resorption, is unknown. Therefore, we examined the distribution of neurokinin 1-receptors (NK₁-R), which have a high affinity to SP, in rat osteoclasts in vivo, and investigated the effects of SP on osteoclast formation and osteoclastic bone resorption in vitro. Using electron microscopy, immunoreactive products of NK₁-R were seen in the plasma membrane and cytoplasm of osteoclasts, and were also observed in preosteoclast-like cells. Cell suspensions containing osteoclasts were prepared from neonatal rats and cultured on ivory slices. The addition of 10^-10-10^-6 M SP caused the number of mono- and multi-nucleated tartrate-resistant acid phosphatase positive (TRAP(+)) cells to increase. The increase in TRAP(+) cells with the addition of 10^-8 M SP was inhibited by treatment with the SP receptor antagonist. In cultures on glass coverslips, time-lapse studies show that SP induced cell spreading within 5 min and maintained the spreading. The number of resorption pits excavated by the osteoclasts and the resorption area per osteoclast increased in a 48-hour incubation with 10^-8 M SP. These results suggest that SP stimulates osteoclast formation and activates osteoclastic bone resorption through NK₁-R.

DOI： 10.1267/ahc.34.31

記述言語：英語   掲載種別：研究論文（学術雑誌）  

We compared the distribution of a cysteine proteinase inhibitor, cystatin C, with that of cathepsin K in osteoclasts of the mouse tibia by immunolight and immunoelectron microscopy. Light microscopically, strong immunoreactivity for cystatin C was found extracellularly along the resorption lacuna and intracellularly in the organelles of osteoclasts. In serial sections, various patterns of cystatin C and cathepsin K localization were seen, specifically: (1) some resorption lacuna were positive for both cystatin C and cathepsin K; (2) others were positive for either cystatin C or cathepsin K, but not both; and (3) some lacuna were negative for both. In osteoclasts, the localization of cystatin C was similar to that of cathepsin K. Furthermore, cystatin C immunoreactivity was detected in preosteoclasts and osteoblasts, whereas cathepsin K was seen only in preosteoclasts. Electron microscopically, cystatin C immunoreactive products were found in the rough endoplasmic reticulum (ER), Golgi apparatus, vesicles, granules, and vacuoles of osteoclasts. These cystatin C-positive vesicles had fused or were in the process of fusion with the ampullar vacuoles (extracellular spaces) containing cystatin C-positive, fragmented, fibril-like structures. The extracellular cystatin C was deposited on and between the cytoplasmic processes of ruffled borders, and on and between type I collagen fibrils. In the basolateral region of osteoclasts, cystatin C-positive vesicles and granules also fused with vacuoles that contained cystatin C-positive or negative fibril-like structures. These results indicate that osteoclasts not only synthesize and secrete cathepsin K from the ruffled border into the bone resorption lacunae, but also a cysteine proteinase inhibitor, cystatin C. Therefore, it is suggested that cystatin C regulates the degradation of bone matrix by cathepsin K, both extracellularly and intracellularly.

DOI： 10.1016/S8756-3282(01)00466-5

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Axons containing substance P (SP) serve bone tissue, however, the role of SP in bone metabolism, particularly on osteoclastic bone resorption, is unknown. Therefore, we examined the distribution of neurokinin 1-receptors (NK₁-R), which have a high affinity to SP, in rat osteoclasts in vivo, and investigated the effects of SP on osteoclast formation and osteoclastic bone resorption in vitro. Using electron microscopy, immunoreactive products of NK₁-R were seen in the plasma membrane and cytoplasm of osteoclasts, and were also observed in preosteoclast-like cells. Cell suspensions containing osteoclasts were prepared from neonatal rats and cultured on ivory slices. The addition of 10^-10-10^-6 M SP caused the number of mono- and multi-nucleated tartrate-resistant acid phosphatase positive (TRAP(+)) cells to increase. The increase in TRAP(+) cells with the addition of 10^-8 M SP was inhibited by treatment with the SP receptor antagonist. In cultures on glass coverslips, time-lapse studies show that SP induced cell spreading within 5 min and maintained the spreading. The number of resorption pits excavated by the osteoclasts and the resorption area per osteoclast increased in a 48-hour incubation with 10^-8 M SP. These results suggest that SP stimulates osteoclast formation and activates osteoclastic bone resorption through NK₁-R.

DOI： 10.1267/ahc.34.31

記述言語：日本語  

A Role of Phosphatydilinositol 3-Kinase (PI 3-Kinase) in Osteoclastic Bone Resorption

記述言語：英語  

Background: The role played by the internal basal lamina (IBL) and hemidesmosomes between an implant and the periimplant epithelium (PIE) in the adherence of the epithelium to the implant is controversial. This study used rat maxilla implantation models to clarify the ultrastructure of the PIE-implant interface.
Methods: Ti-6Al-4V implants were inserted either immediately or 2 weeks after the extraction of the upper left first molar of 6- or 4-week-old rats, respectively. The junctional epithelium (JE) of the upper right molars in the same animals was used as a control. Four weeks after implantation, the animals were sacrificed to prepare specimens for light and immunoelectron microscopy.
Results: Under light microscopy, the PIE appeared to attach to the implant surface. Ultrastructurally, IBL, consisting of the lamina densa and lamina lucida, and hemidesmosomes were formed only in the lower region, and rarely in the middle region, of the PIE-implant interface. In control teeth, the IBL and hemidesmosomes formed throughout the dento-JE interface. Laminin-1 was found in the IBL and also in the vesicles and vacuoles of the PIE and JE cells. Statistical analysis showed that there was also a significant difference in the amount of IBL between the PIE-implant and dento-JE interfaces.
Conclusions: PIE attached to the implant via hemidesmosomes and IBL in the lower region of the PIE-implant interface. Although PIE cells may secrete laminin-1, which contributes to epidermal cell adhesion, the PIE which attaches to implants only in the lower region of the interface is considered to be the poorly adhered epithelium.

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Background: The role played by the internal basal lamina (IBL) and hemidesmosomes between an implant and the periimplant epithelium (PIE) in the adherence of the epithelium to the implant is controversial. This study used rat maxilla implantation models to clarify the ultrastructure of the PIE-implant interface. Methods: Ti-6Al-4V implants were inserted either immediately or 2 weeks after the extraction of the upper left first molar of 6- or 4-week-old rats, respectively. The junctional epithelium (JE) of the upper right molars in the same animals was used as a control. Four weeks after implantation, the animals were sacrificed to prepare specimens for light and immunoelectron microscopy. Results: Under light microscopy, the PIE appeared to attach to the implant surface. Ultrastructurally, IBL, consisting of the lamina densa and lamina lucida, and hemidesmosomes were formed only in the lower region, and rarely in the middle region, of the PIE-implant interface. In control teeth, the IBL and hemidesmosomes formed throughout the dento-JE interface. Laminin-1 was found in the IBL and also in the vesicles and vacuoles of the PIE and JE cells. Statistical analysis showed that there was also a significant difference in the amount of IBL between the PIE-implant and dento-JE interfaces. Conclusions: PIE attached to the implant via hemidesmosomes and IBL in the lower region of the PIE-implant interface. Although PIE cells may secrete laminin-1, which contributes to epidermal cell adhesion, the PIE which attaches to implants only in the lower region of the interface is considered to be the poorly adhered epithelium.

DOI： 10.1902/jop.2000.71.6.961

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Background: The role played by the internal basal lamina (IBL) and hemidesmosomes between an implant and the periimplant epithelium (PIE) in the adherence of the epithelium to the implant is controversial. This study used rat maxilla implantation models to clarify the ultrastructure of the PIE-implant interface. Methods: Ti-6Al-4V implants were inserted either immediately or 2 weeks after the extraction of the upper left first molar of 6- or 4-week-old rats, respectively. The junctional epithelium (JE) of the upper right molars in the same animals was used as a control. Four weeks after implantation, the animals were sacrificed to prepare specimens for light and immunoelectron microscopy. Results: Under light microscopy, the PIE appeared to attach to the implant surface. Ultrastructurally, IBL, consisting of the lamina densa and lamina lucida, and hemidesmosomes were formed only in the lower region, and rarely in the middle region, of the PIE-implant interface. In control teeth, the IBL and hemidesmosomes formed throughout the dento-JE interface. Laminin-1 was found in the IBL and also in the vesicles and vacuoles of the PIE and JE cells. Statistical analysis showed that there was also a significant difference in the amount of IBL between the PIE-implant and dento-JE interfaces. Conclusions: PIE attached to the implant via hemidesmosomes and IBL in the lower region of the PIE-implant interface. Although PIE cells may secrete laminin-1, which contributes to epidermal cell adhesion, the PIE which attaches to implants only in the lower region of the interface is considered to be the poorly adhered epithelium.

DOI： 10.1902/jop.2000.71.6.961

記述言語：日本語  

インプラント周囲上皮とその界面における閉鎖性に関する研究

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The distributions of substance P (SP) and the neurokinin-1 receptor (NK₁-R), the receptor preferentially activated by SP, were examined in rat gingiva by immunocytochemical methods with light and electron microscopy. SP-immunoreactive nerve fibers were located preferentially in the junctional epithelium (JE) but few in the other oral and oral sulcular epithelia. NK₁-R immunoreactivity was found in the endothelial cells (capillaries and postcapillary venules underlying the JE). NK₁-R-labeled and -unlabeled unmyelinated nerve fibers were located close to the blood vessels and partially or completely covered by a Schwann cell sheath. In the JE, labeled naked axons without Schwann cell sheaths were observed. Neutrophils and macrophages in the connective tissue underlying the JE and in the JE were also labeled with NK₁-R. Furthermore, NK₁-R was found in the JE cells. Basically, immunoreaction products for NK₁-R were found throughout various cells (endothelial cells, neutrophils, and JE cells) at invaginations of the plasma membrane and in vesicular and granular structures that are probably endosomes and are found close to both the plasma membrane and the nucleus. This is a first report, demonstrating the presence of NK₁-R in the gingival tissue in the normal nonstimulated condition. Furthermore, it is thought that SP may modulate the permeability of blood vessels beneath the JE, the production of antimicrobial agents in neutrophils, and the proliferation and endocytotic ability of JE cells through NK₁-R.

DOI： 10.1007/s004410051349

記述言語：英語  

The distributions of substance P (SP) and the neurokinin-l receptor (NK1-R), the receptor preferentially activated by SP, were examined in rat gingiva by immunocytochemical methods with light and electron microscopy. SP-immunoreactive nerve fibers were located preferentially in the junctional epithelium (JE) but few in the other oral and oral sulcular epithelia. NK1-R immunoreactivity was found in the endothelial cells (capillaries and postcapillary venules underlying the JE). NK1-R-labeled and -unlabeled unmyelinated nerve fibers were located close to the blood vessels and partially or completely covered by a Schwann cell sheath. In the JE, labeled naked axons without Schwann cell sheaths were observed. Neutrophils and macrophages in the connective tissue underlying the JE and in the JE were also labeled with NK1-R. Furthermore, NK1-R was found in the JE cells. Basically, immunoreaction products for NK1-R were found throughout various cells (endothelial cells, neutrophils, and JE cells) at invaginations of the plasma membrane and in vesicular and granular structures that are probably endosomes and are found close to both the plasma membrane and the nucleus. This is a first report, demonstrating the presence of NK1-R in the gingival tissue in the normal nonstimulated condition. Furthermore, it is thought that SP may modulate the permeability of blood vessels beneath the JE, the production of antimicrobial agents in neutrophils, and the proliferation and endocytotic ability of JE cells through NK1-R.

DOI： 10.1007/s004410051349

記述言語：日本語  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The localization of cathepsin K protein in mouse osteoclasts was examined by immunolight and immunoelectron microscopy using the avidin-biotin-peroxidase complex method with anti-cathepsin K (mouse) antibody. With light microscopy, a strong immunoreaction for cathepsin K was found extracellularly along the bone and cartilage resorption lacunae and detected intracellularly in vesicles, granules, and vacuoles throughout the cytoplasm of multinuclear osteoclasts and chondroclasts attached to the surface of the bone or cartilage. Mononuclear cells, probably preosteoclasts, some distance from the bone also contained a few cathepsin K-positive vesicles and granules. Cathepsin K was sometimes found in the cisternal spaces of the rough endoplasmic reticulum and vesicles of the Golgi apparatus with electron microscopy of the basolateral region of the osteoclasts. Cathepsin K-positive vesicles and granules as lysosomal compartments were present in various stages of fusion with vacuoles as endosomal compartments that contained fragmented cathepsin K-negative fibril-like structures. Some of the vacuoles (endolysosomes), which seemed to be formed by this process of fusion, contained cathepsin K-positive vesicles and fibril-like structures that did not show the regular cross striation of type I collagen fibrils. In the apical region of the osteoclasts, cathepsin K-positive vesicles and pits had already fused with or were in the process of fusing with the ampullar extracellular spaces. There were large deposits of cathepsin K on fragmented fibril-like structures without regular cross striation in the extracellular spaces, as well as on and between the cytoplasmic processes of the ruffled border. There were also extensive deposits of cathepsin K on the type I collagen fibrils with cross striation in the bone resorption lacunae. Osteoblasts and osteocytes were negative for cathepsin K. In the immunocytochemical controls, no immunoreaction was found in the osteoclasts or preosteoclasts, or on the collagen fibrils in the resorption lacunae. The results indicate that cathepsin K is produced in mature osteoclasts attached to the bone and secreted into the bone resorption lacunae. The findings suggest that cathepsin K participates in the extracellular degradation of collagen fibrils in the resorption lacunae and in the subsequent degradation of the fragmented fibrils in the endolysosomes. It is also suggested that cathepsin K degrades the organic cartilage matrix. Copyright (C) 1998 Elsevier Science Inc.

DOI： 10.1016/S8756-3282(98)00138-0

記述言語：英語  

The localization of cathepsin K protein in mouse osteoclasts was examined by immunolight and immunoelectron microscopy using the avidin-biotin-peroxidase complex method with anti-cathepsin K (mouse) antibody. With light microscopy, a strong immunoreaction for cathepsin K was found extracellularly along the bone and cartilage resorption lacunae and detected intracellularly in vesicles, granules, and vacuoles throughout the cytoplasm of multinuclear osteoclasts and chondroclasts attached to the surface of the bone or cartilage. Mononuclear cells, probably preosteoclasts, some distance from the bone also contained a few cathepsiu K-positive vesicles and granules. Cathepsin K was sometimes found in the cisternal spaces of the rough endoplasmic reticulum and vesicles of the Golgi apparatus with electron microscopy of the basolateral region of the osteoclasts. Cathepsin K-positive vesicles and granules as lysosomal compartments were present in various stages of fusion with vacuoles as endosomal compartments that contained fragmented cathepsin K-negative fibril-like structures. Some of the vacuoles (endolysosomes), which seemed to be formed by this process of fusion, contained cathepsin K-positive vesicles and fibril-like structures that did not show the regular cross striation of type I collagen fibrils, In the apical region of the osteoclasts, cathepsin K-positive vesicles and pits had already fused with or were in the process of fusing with the ampullar extracellular spaces, There were large deposits of cathepsin K on fragmented fibril-like structures without regular cross striation in the extracellular spaces, as well as on and between the cytoplasmic processes of the ruffled border. There were also extensive deposits of cathepsin K on the type I collagen fibrils with cross striation in the bone resorption lacunae. Osteoblasts and osteocytes were negative for cathepsin K, In the immunocytochemical controls, no immunoreaction was found in the osteoclasts or preosteoclasts, or on the collagen fibrils in the resorption lacunae. The results indicate that cathepsin K is produced in mature osteoclasts attached to the bone and secreted into the bone resorption lacunae. The findings suggest that cathepsin K participates in the extracellular degradation of collagen fibrils in the resorption lacunae and in the subsequent degradation of the fragmented fibrils in the endolysosomes. It is also suggested that cathepsin K degrades the organic cartilage matrix. (Bone 23: 499-509; 1998) (C) 1998 by Elsevier Science Inc, All rights reserved.

DOI： 10.1016/S8756-3282(98)00138-0

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Substance P (SP) is a neuropeptide that is released from axons of sensory neurons and causes signal transduction through the activation of the neurokinin-1 receptor (NK1-R). The present study demonstrates the distribution of SP-like-immunoreactive (SP-LI) axons and the localization of NK1-Rs in rat bone tissue using the avidin-biotin-peroxidase complex method. Axons with SP-LI were commonly found near the trabecular bone in the temporal bone marrow, but they were only sparsely distributed in the mandible, femur, and tibia. Immunoreactivity for NK1-Rs was found on the plasma membrane and in the cytoplasm of the osteoclasts. In the osteoblasts and osteocytes, a small number of weak, punctate immunoreactive products of NK1-Rs were distributed close to the plasma membrane. At the electron-microscopic level, immunoreactivity for NK1-R was distributed mainly in the whole cytoplasm, except for the clear zone of the osteoclasts, and in pit-like structures along the plasma membrane. The NK1-R-immunoreactive structures in the cytoplasm were divided into two types of organelles, consisting of vesicular and vacuolar structures (probably transport vesicles and early endosomes). In the osteoblasts and osteocytes, the number of NK1-R-positive vesicular structures was fewer than in the osteoclasts. These results thus suggest that SP secreted by the sensory axons could directly modulate bone metabolism via NK1-Rs.

DOI： 10.1007/s004410051100

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Substance P (SP) is a neuropeptide that is released from axons of sensory neurons and causes signal transduction through the activation of the neurokinin-1 receptor (NK1-R). The present study demonstrates the distribution of SP-like-immunoreactive (SP-LI) axons and the localization of NK1-Rs in rat bone tissue using the avidin-biotin-peroxidase complex method. Axons with SP-LI were commonly found near the trabecular bone in the temporal bone marrow, but they were only sparsely distributed in the mandible, femur, and tibia. Immunoreactivity for NK1-Rs was found on the plasma membrane and in the cytoplasm of the osteoclasts. In the osteoblasts and osteocytes, a small number of weak, punctate immunoreactive products of NK1-Rs were distributed close to the plasma membrane. At the electron-microscopic level, immunoreactivity for NK1-R was distributed mainly in the whole cytoplasm, except for the clear zone of the osteoclasts, and in pit-like structures along the plasma membrane. The NK1-R-immunoreactive structures in the cytoplasm were divided into two types of organelles, consisting of vesicular and vacuolar structures (probably transport vesicles and early endosomes). In the osteoblasts and osteocytes, the number of NK1-R-positive vesicular structures was fewer than in the osteoclasts. These results thus suggest that SP secreted by the sensory axons could directly modulate bone metabolism via NK1-Rs.

DOI： 10.1007/s004410051100

記述言語：英語   掲載種別：研究論文（学術雑誌）  

We investigated the co-localization of lysosomal cathepsins B, H and L, and horseradish peroxidase (HRP) in junctional epithelial (JE) cells both as a fluid-phase endocytotic marker to demonstrate the fluid-phase endocytotic capacity of JE cells, and to understand the morphological relationships of the endocytosed foreign substances to lysosomal cathepsins in these cells. The diaminobenzidine (DAB) histochemical and cytochemical methods and immunohistochemical avidin-biotin-peroxidase complex and immunocytochemical post-embedding colloidal gold methods were used. Under light microscopy, DAB reaction products based on HRP were found in JE but were rare or absent in the oral sulcular epithelium and oral epithelium. Immunolabeling for cathepsins B and H was found in the granular structures of the cells, but no cathepsin L was identified. With electron microscopy, DAB reaction products, which indicated both HRP and the azurophil granules of neutrophils, were endocytosed into JE cells. Using a post-embedding technique, gold particles indicating HRP were present on the plasma membrane of JE cells, at the periphery of electronlucent vacuoles, and in the electrondense granules. Gold particles indicating cathepsin B or H were found in the electrondense granules. With different sizes of colloidal golds, the co-localization of cathepsin B or H with HRP was indicated only in the electrondense portion of the larger vacuoles consisting of electronlucent and -dense parts. This study provided the first morphological data which indicate that JE has a fluid phase endocytotic capacity, and which suggest that the lysosomal cathepsins B and H are involved in the intracellular degradation of foreign substances invading through the gingival sulcus in JE cells.

DOI： 10.1111/j.1600-0765.1997.tb00575.x

記述言語：日本語  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The localization of cathepsin L and rat salivary cystatin-3 (RSC-3) in rat osteoclasts (rat femoral and alveolar bones) treated with or without E- 64 (control) was examined immunocytochemically. In osteoclasts pretreated with E-64, immunoreactivity for cathepsin L was very weak extracellularly compared to that in the control osteoclasts. However, it was strong intracellularly. The localization of RSC-3 was unclear in the control osteoclasts, while in E-64 treated osteoclasts, both the clear zone and ruffled border areas showed a very strong immunoreaction. At the electron- microscopic level, in normal osteoclasts, numerous immunoreaction products for cathepsin L were found extracellularly in the bone matrix under the ruffled border, while few intracellular products were observed. In contrast, in the E-64-treated osteoclasts, only a few immunoreaction products were found extracellularly, while intracellularly cathepsin L was found in numerous endosome-lysosomal vacuoles. In the immunoreaction for RSC-3, the cytoplasm of the ruffled border was positive, and the tips of the RSC-3- positive ruffled border appeared to enter deeply into the bone matrix. Intracellularly, the granular reaction products of RSC-3 were found in the vacuoles (probably autophagolysosomes). Thus, in E-64-treated osteoclasts, inhibition of the extracellular release of cathepsin L was demonstrated. In addition, intralysosomal accumulation of RSC-3 and deep penetration of the RSC-3-positive ruffled border into the hone matrix were found. These findings suggest that RSC-3 is associated with the inhibition of cathepsin L in both the lysosomes (in the osteoclasts) and bone matrix.

DOI： 10.1016/S0003-9969(97)00003-4

記述言語：英語  

The localization of cathepsin L and rat salivary cystatin-3 (RSC-3) in rat osteoclasts (rat femoral and alveolar bones) treated with or without E-64 (control) was examined immunocytochemically. In osteoclasts pretreated with E-64, immunoreactivity for cathepsin L was very weak extracellularly compared to that in the control osteoclasts. However, it was strong intracellularly. The localization of RSC-3 was unclear in the control osteoclasts, while in E-64 treated osteoclasts, both the clear zone and ruffled border areas showed a very strong immunoreaction. At the electron-microscopic level, in normal osteoclasts, numerous immunoreaction products for cathepsin L were found extracellularly in the bone matrix under the ruffled border, while few intracellular products were observed. In contrast, in the E-64-treated osteoclasts, only a few immunoreaction products were found extracellularly, while intracellularly cathepsin L was found in numerous endosome-lysosomal vacuoles. In the immunoreaction for RSC-3, the cytoplasm of the ruffled border was positive, and the tips of the RSC-3-positive ruffled border appeared to enter deeply into the bone matrix. Intracellularly, the granular reaction products of RSC-3 were found in the vacuoles (probably autophagolysosomes). Thus, in E-64-treated osteoclasts, inhibition of the extracellular release of cathepsin L was demonstrated. In addition, intralysosomal accumulation of RSC-3 and deep penetration of the RSC-3-positive ruffled border into the bone matrix were found. These findings suggest that RSC-3 is associated with the inhibition of cathepsin L in both the lysosomes (in the osteoclasts) and bone matrix. (C) 1997 Elsevier Science Ltd.

DOI： 10.1016/S0003-9969(97)00003-4

記述言語：日本語  

A Study of the localization of cystein proteases and the intracellular digestion in the rat junctional epitherial cells : An immunocytochemical study of the localization of cathepsins B and H and Horseradish peroxidase as a tracer

記述言語：日本語  

A cytochemical study on the effect substance P topically applied on the junctional epithelium of rat gingiva

記述言語：日本語  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Nerve fibers with substance P-like immunoreactivity (SP-IR) in the junctional epithelium (JE) of 32-42-d-old rats were examined by both light and electron microscopy using the avidin-biotin-peroxidase complex method. The density of nerve fibers with SP-IR was highest in the middle portion of the JE; however, a few fibers were localized in the coronal portion of the JE and close to the enamel surface. Also, rich innervation was found especially in the basal cell layer of the JE. Unmyelinated axons with SP-IR in the connective tissue underlying the JE were enveloped by Schwann cells but lost their Schwann cell sheath almost completely in the JE. The axons often formed varicosities with SP-IR as terminals in various areas of the JE. The terminals contained numerous large granular vesicles, small clear vesicles and a few mitochondria, and were surrounded by the cytoplasmic processes of the junctional epithelial cells. These terminals were sometimes located close to neutrophils in the JE; the minimum gap distance between the terminals and the processes of junctional epithelial cells or neutrophils was about 20 nm. A few terminals with SP-IR came close to the enamel surface, and the minimal distance between the terminals and the enamel surface was about 5 μm. SP-IR in the nerve terminals in the JE fixed with 0.1% or 0.25% glutaraldehyde was distributed diffusely in the axoplasm or was confined to the granular vesicles. These findings show that substance P is contained in the large granular vesicles in the nerve terminals, and suggest that these terminals may function as modulators of junctional epithelial cells and neutrophils.

DOI： 10.1111/j.1600-0765.1996.tb00483.x

記述言語：英語  

Nerve fibers with substance P-like immunoreactivity (SP-IR) in the junctional epithelium (JE) of 32-42-d-old rats were examined by both light and electron microscopy using the avidin-biotin-peroxidase complex method. The density of nerve fibers with SP-IR was highest in the middle portion of the JE; however, a few fibers were localized in the coronal portion of the JE and close to the enamel surface. Also, rich innervation was found especially in the basal cell layer of the JE. Unmyelinated axons with SP-IR in the connective tissue underlying the JE were enveloped by Schwann cells but lost their Schwann cell sheath almost completely in the JE. The axons often formed varicosities with SP-IR as terminals in various areas of the JE. The terminals contained numerous large granular vesicles, small clear vesicles and a few mitochondria, and were surrounded by the cytoplasmic processes of the junctional epithelial cells. These terminals were sometimes located close to neutrophils in the JE; the minimum gap distance between the terminals and the processes of junctional epithelial cells or neutrophils was about 20 nm. A few terminals with SP-IR came close to the enamel surface, and the minimal distance between the terminals and the enamel surface was about 5 mu m. SP-IR in the nerve terminals in the JE fixed with 0.1% or 0.25% glutaraldehyde was distributed diffusely in the axoplasm or was confined to the granular vesicles. These findings show that substance P is contained in the large granular vesicles in the nerve terminals, and suggest that these terminals may function as modulators of junctional epithelial cells and neutrophils.

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Nerve fibres immunoreactive for substance P (SP) and calcitonin gene-related peptide (CGRP) were located preferentially at the base of the junctional epithelium. Occasional fibres were observed in close proximity to the subepithelial, small blood vessels. The vascular connective tissue papillae projecting into the epithelium were more densely surrounded by SP- and CGRP-immunoreactive fibres in the interdental col than in other regions of the gingiva. In some cases, hyperplasia of the junctional epithelium was noted in the interdental col where the connective tissue papillae were invaded by widened vessels, indicating severe irritation. SP- and CGRP-immunoreactive fibres around these papillae showed increases in their immunoreactivity and thickness, with some fibres terminating as large expansions. Double immunohistochemical staining revealed the co-existence of SP and CGRP in all nerve fibres within and under the junctional epithelium. Capsaicin pretreatment eliminated most of the immunoreactivity for both peptides. Intravenous infusion of capsaicin or SP caused increased permeability in vessels underlying the junctional epithelium, as indicated by Monastral blue labelling. Labelled vessels were arranged not only in a network extending under the epithelium but also in loops protruding into the connective tissue papillae. These labelled vessels were most abundant in the interdental col, where vascular loops with more complex configurations exhibited strong staining in their walls. In the case of hyperplasia of the junctional epithelium in the interdental col, widened vessels showing extensive labelling in their walls were observed. In capsaicin-pretreated animals, capsaicin-induced extravasation was abolished, while the effect of SP was still observed. These findings provide evidence that capsaicin-sensitive sensory nerves supplying the junctional epithelium are involved in neurogenic plasma extravasation in the rat gingiva. The enhancement of neurogenic plasma extravasation in the col may be a vascular response associated with a higher susceptibility of this region to gingival inflammation.

DOI： 10.1016/0003-9969(95)00060-3

記述言語：英語  

Nerve fibres immunoreactive for substance P (SP) and calcitonin gene-related peptide (CGRP) were located preferentially at the base of the junctional epithelium. Occasional fibres were observed in close proximity to the subepithelial, small blood vessels. The vascular connective tissue papillae projecting into the epithelium were more densely surrounded by SP- and CGRP-immunoreactive fibres in the interdental col than in other regions of the gingiva. In some cases, hyperplasia of the junctional epithelium was noted in the interdental col where the connective tissue papillae were invaded by widened vessels, indicating severe irritation. SP- and CGRP-immunoreactive fibres around these papillae showed increases in their immunoreactivity and thickness, with some fibres terminating as large expansions. Double immunohistochemical staining revealed the co-existence of SP and CGRP in all nerve fibres within and under the junctional epithelium. Capsaicin pretreatment eliminated most of the immunoreactivity for both peptides. Intravenous infusion of capsaicin or SP caused increased permeability in vessels underlying the junctional epithelium, as indicated by Monastral blue labelling. Labelled vessels were arranged not only in a network extending under the epithelium but also in loops protruding into the connective tissue papillae. These labelled vessels were most abundant in the interdental col, where vascular loops with more complex configurations exhibited strong staining in their walls. In the case of hyperplasia of the junctional epithelium in the interdental col, widened vessels showing extensive labelling in their walls were observed. In capsaicin-pretreated animals, capsaicin-induced extravasation was abolished, while the effect of SP was still observed. These findings provide evidence that capsaicin-sensitive sensory nerves supplying the junctional epithelium are involved in neurogenic plasma extravasation in the rat gingiva. The enhancement of neurogenic plasma extravasation in the col may be a vascular response associated with a higher susceptibility of this region to gingival inflammation.

記述言語：日本語  

Substance P or calcitonin gene-related peptide-immunoreactive nerve fibers in the junctional epithelium of rats : Confocal laser scanning microscopic and immuno-electronmicroscopic observation

記述言語：日本語  

記述言語：日本語  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The localization of cathepsin L or type I collagen in the osteoclasts (rat femur) treated with or without E-64 (control) was examined immunocytochemically to investigate how E-64 affects the osteoclasts. Using a light microscope in the E-64-treated osteoclasts, the immunoreactivity for cathepsin L was extracellularly very weak compared with that in the control osteoclasts, but was strong intracellularly. The intracellular immunoreactivity for type I collagen was found in the vacuoles in the E-64-treated osteoclasts but not in any vacuoles in the control osteoclasts. On the other hand, the extracellular immunoreactivity along the resorption lacunae of the E-64-treated osteoclasts was somewhat weaker than that of the control osteoclasts. Using electron microscopy in the E-64-treated osteoclasts, only a small number of extracellular immunoreaction products for cathepsin L were seen along the resorption lacunae. In addition, intracellular cathepsin L was deposited in the endosome-lysosomal vacuoles which were well-developed by E-64. Gold particles indicating type I collagen appeared on the bone matrix, and they were also detected in the vacuoles and vesicles in the E-64-treated osteoclasts. However, they were not detected in the organelles of the control osteoclasts. Thus, in the E-64-treated osteoclasts, the extracellular release of cathepsin L was insufficient to suppress the degradation of collagen. In addition, the undegraded collagen seemed to be endocytosed. The above findings thus suggest that bone resorption is inhibited by this incomplete degradation of collagen.

DOI： 10.1267/ahc.28.523

担当ページ：361-365   記述言語：日本語   著書種別：学術書

Temporomandibular Disorder Ultrastructure of synovium of temporomandibular joint

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国名：日本国  

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国名：日本国  

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国名：アメリカ合衆国  

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開催地：福岡   国名：日本国  

開催年月日： 2010年3月

会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：盛岡   国名：日本国  

開催年月日： 2010年2月

会議種別：口頭発表（招待・特別）  

開催地：福岡   国名：日本国  

開催年月日： 2009年10月

会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：Honolulu   国名：アメリカ合衆国  

開催年月日： 2008年11月

会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：Beijin   国名：中華人民共和国  

開催年月日： 2008年11月

会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：Beijin   国名：中華人民共和国  

開催年月日： 2008年11月

会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：Beijin   国名：中華人民共和国  

開催年月日： 2003年4月

会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：福岡   国名：日本国  

会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：福岡   国名：日本国  

会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：福岡   国名：日本国  

会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：大阪   国名：日本国  

会議種別：口頭発表（一般）  

開催地：北九州   国名：日本国  

会議種別：口頭発表（一般）  

開催地：北九州   国名：日本国  

会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：大宮   国名：日本国  

会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：北九州   国名：日本国  

会議種別：口頭発表（一般）  

開催地：福岡   国名：日本国  

会議種別：口頭発表（一般）  

開催地：福岡   国名：日本国  

会議種別：口頭発表（一般）  

開催地：鹿児島   国名：日本国  

会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：Vancouver   国名：カナダ  

会議種別：口頭発表（一般）  

開催地：福島   国名：日本国  

会議種別：口頭発表（一般）  

開催地：大阪   国名：日本国  

会議種別：口頭発表（一般）  

開催地：東京   国名：日本国  

会議種別：口頭発表（一般）  

開催地：京都   国名：日本国  

会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：Washigton D. C.   国名：アメリカ合衆国  

会議種別：口頭発表（一般）  

開催地：福岡   国名：日本国  

会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：東京   国名：日本国  

会議種別：口頭発表（一般）  

開催地：横須賀   国名：日本国  

会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：横須賀   国名：日本国  

会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：Kazusa   国名：日本国  

会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：神戸   国名：日本国  

会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：さいたま市   国名：日本国  

会議種別：口頭発表（一般）  

開催地：東京   国名：日本国  

会議種別：口頭発表（一般）  

開催地：北九州   国名：日本国  

会議種別：口頭発表（一般）  

開催地：東京   国名：日本国  

会議種別：口頭発表（一般）  

開催地：東京   国名：日本国  

会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：東京   国名：日本国  

会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：名古屋  

会議種別：口頭発表（一般）  

開催地：東京   国名：日本国  

会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：Osaka   国名：日本国  

会議種別：口頭発表（一般）  

開催地：福岡   国名：日本国  

会議種別：口頭発表（一般）  

開催地：福岡   国名：日本国  

会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：盛岡   国名：日本国  

会議種別：口頭発表（一般）  

開催地：盛岡   国名：日本国  

会議種別：口頭発表（一般）  

開催地：盛岡   国名：日本国